Srivastava 2016

Production, properties, and applications of endo-β-mannanases
Praveen Kumar Srivastava, Mukesh Kapoor
PII: S0734-9750(16)30136-7
DOI: doi:10.1016/j.biotechadv.2016.11.001
Reference: JBA 7082
To appear in: Biotechnology Advances
Received date: 28 December 2015

Revised date: 12 October 2016
Accepted date: 7 November 2016
Please cite this article as: Srivastava Praveen Kumar, Kapoor Mukesh, Produc-
tion, properties, and applications of endo-β-mannanases, Biotechnology Advances (2016),
doi:10.1016/j.biotechadv.2016.11.001
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Title: Production, properties, and applications of endo-β-mannanases
Authors: Praveen Kumar Srivastava and Mukesh Kapoor§*
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Addresses: Department of Protein Chemistry and Technology, CSIR-Central Food
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Technological Research Institute, Mysuru-570 020
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Academy of Scientific and Innovative Research (AcSIR), CSIR-CFTRI Campus,
Mysuru, India
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*Corresponding Author: E-mail: mkapoor@cftri.res.in; Phone: +91-821-2515331
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Abstract
The present review provides up-to-date information on the occurrence and
methodologies used for producing and purifying endo-β-mannanases and a
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comprehensive comparison of their biochemical properties. The amalgamation of
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biochemical, molecular and structural biology approaches which have been used for
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understanding endo-β-mannanase families, catalytic mechanism, substrate binding,
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non-catalytic modules, trans-glycosylation, and multi-functional enzyme complexes
has been given critical attention. A separate section entailing the state-of-the-art about
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thermostable endo-β-mannanases, which has emerged as an exciting field of both
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basic and applied research, is also deliberated. The remarkable progress made by
endo-β-mannanases in various industrial sectors like food, feed, detergents, biofuel

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and oil drilling is also emphasized.

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Key words: Endo-β-mannanase, Biochemical properties, Genetic engineering
Structural organization, Thermo-stability, Industrial application

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1. Introduction
Plant cell walls are macromolecular structures comprising of an intricate intermix of
cellulose and a matrix of polysaccharides, glycoproteins, proteoglycans, low-
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molecular-weight compounds, and ions (Willats et al., 2001). They represent an
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abundant, inexpensive, uniquely sustainable, and renewable resource that offers
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enormous potential in the production of numerous food and non-food consumer
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products such as prebiotics, fuels, organic chemicals, and polymeric materials (Broda,
1992).
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Cellulose, hemicellulose and lignin in an approximate ratio of 2:1:1 makes up
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to 90% of the plant cell wall (Van Zyl et al., 2010). Cellulose, the most abundant
biopolymer, comprises of a bundle of linear poly β-1,4-glucan chains organized by

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intra-chain and inter-chain H-bonding networks (Kuhad et al., 1997). Hemicelluloses

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comprise 25-30% of total wood dry weight, and are composed of more than 20
different monosaccharides in diverse combinations (Filho, 1998; Pérez et al., 2002).

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Xylans represent the pre-dominant hemicellulosic component in hardwood from

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angiosperms, while mannans represent the most abundant hemicelluloses in

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softwoods, and specialized structures like plant seeds (Beg et al., 2001; Pérez et al.,
2002).
The structural and heterogeneous nature of mannans requires close association
and synergy between a variety of main chain and side chain cleaving enzymes like
endo-β-mannanase (EC 3.2.1.78), exo-β-mannosidase (EC 3.2.1.25), β-glucosidase
(EC 3.2.1.21), acetyl mannan esterases (EC 3.1.1.6), and α-galactosidase (EC
3.2.1.22) for their biodegradation (Dhawan and Kaur, 2007; Malgas et al., 2015a,
2015b). Mannan-degrading enzyme systems can be found in several glycosyl
hydrolase families (like GH 1, GH 2, GH 3, GH 5, GH 26, GH 27, GH 113 etc.)
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(www.cazy.org). They contribute to 1) microbial metabolism by the generation of
energy equivalents (simpler monosaccharides/oligosaccharides) from substrate
hydrolysis, 2) in plant‘s growth, maturation, and ripening by the metabolism of cell
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wall mannans (Moreira and Filho, 2008).
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Mannanases obtained from various GH families are quite different with
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respect to their primary structure, but are similar in their spatial arrangement, (β/α)8-
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barrel protein fold and are grouped into GH-A clan (Hilge et al., 1998). They often
display modular architectures generally comprising of catalytic domain(s),
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carbohydrate binding module(s) (CBM), and additional functional domain(s) (Sunna,
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2010). X-ray crystallographic and site-directed mutagenesis studies from a wide range
of species elucidated that endo-β-mannanases 1) require at least five substrate binding

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sub-sites to carry out efficient catalysis of oligo- or polysaccharide substrates and 2)

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have an open cleft shaped active site with strictly conserved acid/base catalyst
(Glutamate at the beta-strand 4 end) and nucleophile catalyst (Glutamate at the beta-
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strand 7 end) which are 5.5 Ǻ apart (Hilge et al., 1998; Hogg et al., 2003; Yan et al.,
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2008; Tailford et al., 2009). Recently, these enzymes have gathered significant
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attention from both academia and industry due to their potential applications in
bioconversion of lignocellulosic residues, food, feed, detergents, oil drilling, and
pharmaceuticals (Chauhan et al., 2012; Malgas et al., 2015a).
The recent progress made in the occurrence, structure, properties and
industrial applications of β-mannan and endo-β-mannanases (GH 5, GH 26, and GH
113) are discussed in the present review. Structural determinants, families, reaction
mechanism of endo-β-mannanases and a holistic view of thermo-stable endo-β-
mannanases are also covered. However, the present review does not cover other
mannan degrading enzymes like exo-β-mannosidases, α-galactosidases and esterases
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etc. or plant polysaccharide-degrading enzymes and synergistic interactions thereof,
and thus, the reader is advised to refer to earlier reviews (Gilbert et al., 2008; Malgas
et al., 2015a; Chauhan and Gupta, 2016) for further details.
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2. β-Mannan: An important hemicellulose
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Mannans show a great deal of structural diversity and are sub-divided into four types
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viz. pure/linear mannan, glucomannan, galactomannan, and galactoglucomannan on
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the basis of their sugar backbone composition (Popa and Spiridon, 1998) (Fig. 1).
They perform a variety of functions in plants, like acting as storage polysaccharides
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especially in endosperm or vacuoles of different seeds and vegetative tissues (Meier
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and Reid, 1982), providing structural integrity to the cell wall by binding to cellulose
and in addition, they also function like signalling molecules in growth and
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development (Liepman et al., 2007). Currently, there is a great deal of interest in

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mannan-based polysaccharides as they provide multi-faceted benefits like immuno-
pharmacological, therapeutic, bio-medical, and prebiotic properties which find usage

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in food, feed, and pharmaceuticals sectors (Maier et al., 1993; Moreira and Filho,
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2008; Ferreira et al., 2012; Yamabhai et al., 2016).

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2.1. Structure, occurrence and properties of β-mannan
2.1.1. Linear mannan
Linear mannan consists of a linear chain of β-1,4 linked D-mannosyl residues. They
are the major structural components in softwoods, hardwoods and plant seeds
(Aspinall, 1959). A neutral and water-soluble polysaccharide isolated from the current
pseudobulbs of Onicidium was a linear β, 1-4 linked mannan (Wang et al., 2006). The
linear mannan in the seed endosperm of Phytelephas macrocarpa and seeds derived
from Umbelliferae species provided protection from mechanical damage (Reid and
Edward, 1995). In some algal species, pure mannans have been believed to replace
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cellulose as the main cell wall glycan and are called as α-cellulose (Fernández et al.,
2012). Sulphated mannans (carbohydrates: sulphate, 2.7:1) in genus Codium form an
amorphous central layer and act as an interphase region between the neutral and
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acidic layers (Fernández et al., 2012). Linear mannan act as a reserve material in
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families‘ viz. Apiaceae, Rubiaceae, and Asteraceae, while in Schizolobium
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parahybae, family Caesalpiniaceae it did not act as a reserve material (Petkowicz et
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al., 2007).
2.1.2. Galactomannan
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Galactomannans are composed of 1,4-linked β-D-mannopyranosyl residues
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substituted by single 1,6-linked α-D-galactopyranosyl groups at the C-6 position in
sugar residues along the polysaccharide chain (McCleary et al., 1985).

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Galactomannans in general, form highly viscous and stable aqueous solutions which
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are used as stiffeners and stabilizers in various food products (Srivastava and Kapoor,
2005). In the pharmaceutical industry, galactomannans are used as controlled drug

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release agents (Toti and Aminabhavi, 2004). The frequency of side group substitution
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varies in galactomannans. For example, galactomannans isolated from guar seeds

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(Cyamopsis tetragonolobus, average molecular weight 220 kDa), tara gum,
(Caesalpinea spinosa) and carob seeds (Ceratonia siliqua, average molecular weight
310 kDa) have mannose-to-galactose ratios of 2:1, 3:1, and 4:1, respectively. The
average degree of polymerization (DP) of guar gum, tara gum, and carob gum ranges
between 3000-5000 (McCleary et al., 1985; Mudgil et al 2012a, b; Sébastien et al.,
2014). Galactomannans represents the most abundant constituents of coffee beans.
The β-(1→4)-linked mannan chains in coffee are substituted at O-6 position with
single galactose residues approximately every 100 residues (Campos-Vega et al.,
2015). Simões et al. (2010) have shown that pattern of acetylation was quite different
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in galactomannans found in coffee residues and infusions. Recently, galactomannans
have been isolated and characterized from the seeds of Gleditsia triacanthos,
Caesalpiniapul cherrima and Adenanthera pavonina. Electron spray ionization-
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tandem mass spectrometry (ESI-MS/MS) analysis of G. tricanthos galactomannan
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indicated the presence of acetyl and pentose groups in addition to galactose
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substitution (Cerqueira et al., 2011). A galactomannan from the seeds of Cassia
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grandis had a constant mannose/galactose ratio of 2.44:1 (Albuquerque et al., 2014),
while galactomannan from the seeds of vinal (Prosopis ruscifolia) had
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mannose/galactose ratio of 1.6, with traces of arabinose and glucose residues (Busch
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et al., 2015). 3D-molecular modelling studies established that intra-molecular
interactions between hairy (substituted) and smooth (unsubstituted) regions in

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galactomannan from Sophora alopecuroides L. seeds are forming intra-molecular

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junction zones via hydrogen bonding in galactomannans with low mannose/galactose
ratios, e.g., 1.48-1.84 (Guo et al., 2016) (Fig. 2).

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2.1.3. Glucomannan
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Glucomannan (average DP >200) is characterized by the presence of β-1,4-linked D-

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mannosyl and D-glucosyl residues, generally in 3:1 ratio, and are frequently
acetylated at the C-2, C-3 or C-6 positions (Northcote, 1972). Acetylated
glucomannans constitute about 60-80% of Konjac tuber (Maeda et al., 1980), and are
also reported from Lupinus varius (Ishurd et al., 2006). Konjac glucomannan has been
well-exploited in food and pharmaceutical sectors for the management of weight and
prevention of chronic diseases (Ishurd et al., 2006; Liang et al., 2015; Shah et al.,
2015). All the major fractions of O-acetyl-glucomannan from Dendrobium officinale
shared same major structure (2, 3-O-acetyl-glucomannan) but had different
mannose/glucose ratio, molecular weight, and intrinsic viscosity (Xing et al., 2014).
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Amorphophallus oncophyllus glucomannan, when compared to commercial
glucomannan, showed higher solubility and degree of acetylation, but lower viscosity,
water holding capacity and DP (Harmayani et al., 2014). Glucomannan from Bletilla
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striata showed relative mole ratio of 2.4:1 mannose to glucose and molecular size of
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135 kDa (Wang et al., 2015).
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2.1.4. Galactoglucomannan
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Galactoglucomannans are characterized by the presence of β-1,4-linked D-mannosyl
and D-glucosyl residues substituted with α-1,6-linked galactosyl side group in 3:1:1
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molar ratio (Puls, 1993; Popa and Spiridon, 1998). Schröder et al. (2001) reported
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galactoglucomannan from ripe kiwifruit with 1:2:2 galactose–glucose–mannose ratio
and molecular weight in the range of 16–42 kDa. Water extracted fractions of
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rapeseed straw were rich in galactoglucomannan (Svärd et al., 2015). Acetylated

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galactoglucomannans are classified into two groups viz. galactose rich and acetyl poor
and galactose poor and acetyl rich (Willför et al., 2008). They form the predominant
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portions of hemicelluloses found in wood from gymnosperms like Norway spruce

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(Picea abies) (Ekholm et al., 2012), angiosperms like black berry (Rubus fruticosus)
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(Cartier et al., 1988), spruce (Wilför et al., 2008) and ferns (Bailey and Pain, 1971).
Galactoglucomannan oligosaccharides alleviated cadmium stress in Arabidopsis
thaliana (Kučerová et al., 2014), while galactoglucomannan from Dendrobium
huoshanense has been suggested as anti-fibrotic agent for the prevention of liver
injury and fibrosis (Pan et al., 2012). Galactoglucomannans are also used as raw
material for packaging films and as an oxygen barrier (Jansson et al., 2014; Kisonen
et al., 2015). Recently, spruce galactoglucomannan and its carboxymethyl derivatives
were found to inhibit lipid oxidation (Lehtonen et al., 2016). For more information
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about mannans, readers are advised to refer to earlier reviews (Dhawan and Kaur,
2007; Moreira and Filho, 2008).
3. Endo-β-mannanase production from microorganisms
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Mannanases are ubiquitous in nature and have been isolated from bacteria, fungi,
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actinomycetes, plants, and animals (Table 1). Submerged fermentation (SmF) and
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solid state fermentation (SSF) are used predominantly for producing microbial endo-
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β-mannanases (Dhawan and Kaur, 2007; Chauhan et al., 2012). Several research
groups have attempted to improve endo-β-mannanase production by process
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optimization (Chantorn et al., 2013; Vijayalaxmi et al., 2013; Yin et al., 2013;
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Chauhan et al., 2014a).
3.1. Submerged fermentation (SmF)

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SmF is the method of choice for producing endo-β-mannanase from microorganisms

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(Chauhan et al., 2012, 2014a; Srivastava and Kapoor, 2014). Wild-type microbial
endo-β-mannanases are extra-cellular, and their production is influenced by physico-

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chemical and nutritional parameters like incubation time, pH, temperature, N2 content,
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and C-source. Approaches like one-factor-at-a-time, statistical methods like Plackett

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burman, Box Behnken, and central composite design or their combinations have been
used to improve endo-β-mannanase production (Ozturk et al., 2010; Srivastava and
Kapoor, 2014) (Table 2).
3.2. Solid state fermentation (SSF)
SSF offers colossal benefits for microbial endo-β-mannanase bioprocesses which
include, increased process productivity, less waste-water production, eco-friendly
nature, a higher concentration of enzyme, simple equipments, and low levels of
catabolic repression (Beg et al., 2000; Srivastava and Kapoor, 2014; Kuhad and
Kapoor, 2015). However, problems associated with scale up, downstream processing,
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biomass estimation, and control over process parameters has negatively affected its
adoption at industrial scale. Only a few microorganisms, mostly fungi, can produce
endo-β-mannanase under SSF conditions (Barrios-González, 2012; Thomas et al.,
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2013) (Table 2).
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4. Heterologous cloning and expression of endo-β-mannanase gene
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A large number of endo-β-mannanases have been cloned and expressed in
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heterologous hosts (Table 3) in order to a) make them suitable for industrial
applications by increasing their thermal stability, protease resistance, and pH stability
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(Sunna, 2010; Li et al., 2013; Xu et al., 2013), b) understand structure-function
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relationships, c) delineate the role of particular amino acid residues (Yan et al., 2008),
d) increase production (Songsiriritthigul et al., 2010; Kaira et al., 2016; Srivastava et

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al., 2016), and e) purify enzyme in single step with high yield and specific activity
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(Huang et al., 2014).
Endo-β-mannanse gene from bacterial sources are usually overexpressed in

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Escherichia coli (Srivastava et al., 2016) but there are also reports of other
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heterologous hosts like Bacillus megaterium (Summpunn et al., 2011), Brevibacillus

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brevis (Zhou et al., 2013), Pichia pastoris (Qiao et al., 2010), and Kluyveromyces
cicerisporous (Pan et al., 2011). Fungal endo-β-mannanase genes are heterologously
expressed most commonly in P. pastoris but Aspergillus sp. (Van Zyl et al., 2010) has
also been reported to express endo-β-mannanase. The β-mannanase encoding man5A
gene from acidophilic Bispora sp. MEY-1 was heterologously overexpressed in seeds
of maize plant (Xu et al., 2013). The size of endo-β-mannanase gene reported from
prokaryotic and eukaryotic sources ranges between 978-2010 kb. The fully mature
recombinant endo-β-mannanase proteins contain anywhere between 326-669 amino
acid residues with molecular weight ranging from 31-65 kDa (Table 3).
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Highest enzyme titres obtained after cloning and expression of endo-β-
mannanase gene are reported from Aspergillus aculeatus (expressed in Yarrowia
lipolytica) 1575 IU/ml (Yang et al., 2009) and Bacillus sp. CFR1601 [expressed in
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Hi-Control E.coli BL21 (DE3)] 8,406 U/ml (Kaira et al., 2016). Recombinant endo-β-
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mannanase from Aspergillus usamii constituted more than 75% of the total protein
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secreted into the culture supernatant (Li et al., 2014). A β-mannanase gene from
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Aspergillus sulphureus was codon optimized and expressed in P. pastoris with a yield
of 3 mg/ml and 35% increase in enzyme activity (Chen et al., 2009). Similarly, β-
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mannanase gene from B. subtilis MA139 was codon optimized and resulting
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recombinant enzyme had higher enzyme activity (Qiao et al., 2010).
5. Purification of endo-β-mannanases
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Many protocols involving clarification, concentration/precipitation of enzyme

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followed by 2-6 chromatographic steps depending on the properties of endo-β-
mannanase(s) have been reported in the literature for the purification of endo-β-
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mannanases (Table 4).

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6. Structural determinants, families, and reaction mechanism of endo-β-

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mannanases
6.1. Endo-β-mannanase families
Endo-β-mannanases from various organisms have been classified in glycoside
hydrolase (GH) families 5, 26, and 113 on the basis of amino acid sequence similarity
(www.cazy.org). A recent study (Shimizu et al., 2015) identified a novel mannanase
from Aspergillus nidulans which had no amino acid sequence similarity with other 1,4
endo-β-mannanases or to any protein with a known function and had been placed in a
new glycoside hydrolase family 134. Endo-β-mannanases obtained from various GH
families are quite different with respect to their primary structure, however
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interestingly they are very similar in their spatial arrangement, (β/α)8-barrel protein
fold and are grouped into GH-A clan (Fig. 3). A) GH 5 endo-β-mannanases: They are
primarily produced by eukaryotic organisms like fungi, plants, and animals (Larsson
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et al., 2006; Van Zyl et al., 2010). However, few bacteria like Cellulosimicrobium sp.
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HY-13 (Kim et al., 2011a, 2011b), Vibrio sp. strain MA-138 (Tanaka et al., 2009),
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Thermotoga petrophila (Dos Santos et al., 2012), and Bacillus licheniformis (Ethier et
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al., 1998) have also been reported to produce GH5 endo-β-mannanase. B) GH 26
endo-β-mannanases: They are found predominantly in bacteria like Bacillus sp.
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CFR1601 (Srivastava et al., 2014, 2016), Bacillus subtilis WL-3 (Yoon et al., 2008),
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B. subtilis B 23 (Zhou et al., 2013), Clostridium cellulovorans (Jeon et al., 2011),
Paenibacillus polymyxa (Cho et al., 2006), Pantoea agglomerans (Wang et al., 2010),
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Pseudomonas fluorescens (Bolam et al., 1996), and Cellulomonas fimi (Hekmat et al.,
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2010). Presence of GH 5 (Ethier et al., 1998) and GH 26 (Songsiriritthigul et al.,
2010) mannanases have been reported from two different strains of B. licheniformis,
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whereas coprophilic ascomycete Podospora anserina has been reported to produce

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both GH 5 and GH 26 endo-β-mannanase (Couturier et al., 2013a, 2013b).

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6.2. Substrate binding sub-sites
Endo-β-mannanase requires at least five substrate binding sub-sites to carry out
efficient catalysis of oligo- or polysaccharide substrates. According to Davies et al.
(1997), these sub-sites are numbered as +4, +3, +2, +1, -1, -2, -3 etc. starting from the
non-reducing end-to-the-reducing end, with hydrolysis of the O-glycosidic bond
placed between -1 and +1 sub-sites. Though the site of cleavage is same (-1 and +1
sub-sites) but the sub-sites used for interaction with the glycoside substrate may
differ. For instance, for interacting with mannotriose, a GH 5 endo-β-mannanase from
Thermobifida fusca (Hilge et al., 1998) (Fig. 4) employs -2, -3, and -4 enzyme sub-
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sites, while a GH26 endo-β-mannanase from Cellvibrio japonicas (Hogg et al., 2003)
makes use of -1, -2, and -3 sub-sites. GH5 enzyme, BaMan5A, derived from Bacillus
agaradhaerens can accommodate glucose or mannose at both -2 and +1 subsites,
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while GH26 B. subtilis mannanase, BsMan26A, displays strong specificity only for
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mannose residues at its negative binding sites (Tailford et al., 2009). Therefore,
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BaMan5A was able to hydrolyze glucomannan because the polar residue at the -2
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sub-site can make productive contact with the substrate 2-OH group in either its axial
(as in mannose) or its equatorial (as in glucose) configuration. However, other distal
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sub-sites do not exploit the 2-OH group as a specificity determinant. The tighter
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mannose recognition at the -2 sub-site in BsMan26A is mediated by interactions of
polar residues with the axial 2-OH group of a 4C1 ground state mannoside and is well
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supported by previous studies on the C. japonicus GH26 mannanases CjMan26A

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(Hogg et al., 2003) and CjMan26C (Cartmell et al., 2008). The mutagenic variants of
CjMan26A, in which polar residues have been removed, do not distinguish between
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mannose and glucose at the -2 subsite. Arg-361, His-377, and Glu-121 made pivotal
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productive polar interactions with mannose at the -2 subsite, but arginine, through its
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multiple interactions with O2 and O3, made the most significant contribution towards
substrate binding (Tailford et al., 2009). GH26 endo-β-mannanases select mannose at
both the -1 and -2 sub-sites and preferentially attack homo-polymers of mannose like
mannooligosaccharides or soluble mannans such as galactomannans. However, GH5
mannanases appear to display relaxed specificity for glucose or mannose at the
majority of the sub-sites and thus are optimized for glucomannan attack which is
found in the cell walls of angiosperms (Tailford et al., 2009). Kumagai et al. (2015)
highlighted the captivating differences in the properties of GH5 mannanases from
Streptomyces thermolilacinus (StMan) and T. fusca (TfMan) with respect to substrate
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recognition. StMan and TfMan hydrolysed galactosylmannooligosaccharide (GGM5;
6III,6IV-α-D-galactosyl mannopentaose) to GGM3 and M2, and GGM4 and M1,
respectively (Fig. 5). Both loop 7 and 8 were found as key regions for determining
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substrate specificity.
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The presence of cellulose-specific non-catalytic carbohydrate binding modules
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in many of GH5 enzymes functionally corroborates plant cell wall as the prime target
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of GH 5 enzymes (Hogg et al., 2003; Tailford et al., 2009). Couturier et al. (2013b)
have recently carried out structural and biochemical analyses of glycoside hydrolase
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families 5 (PaMan5A) and 26 (PaMan26A) β-(1,4)-mannanases from coprophilic
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fungus P. anserina. They have reported that the P. anserina mannanase (PaMan26A)
does not seem to agree with the previous models suggested for GH26 endo-β-
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mannanases from B. subtilis and C. japonicus as it had atypical hydrolysis pattern

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with the release of mannotetrose and mannose from mannopentose resulting from a
predominant binding mode involving the -4 sub-site.

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It could be concluded that many characteristics of endo-β-mannanases with

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respect to substrate specificity are conserved, but there are certain subtle differences
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in the key regions which interact with the substrate and leads to heterogeneity in the
sub-sites used for interaction with the sugar moieties.
6.3. Reaction mechanism
Endo-β-mannanases carry out catalysis by the double displacement mechanism with
retention of configuration (Couturier et al., 2013a, 2013b). In the first stage of
reaction, a glycosyl-enzyme intermediate is formed by a combination of nucleophilic
attack of the anomeric carbon and protonation of the glycosidic oxygen. In the later
stage of the reaction, the enzyme-substrate complex is cleaved by water attacking the
anomeric carbon of glycosyl group and releasing the enzyme in its original state
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(Bolam et al., 1996). GH 26 family mannanase (Pseudomonas cellulosa Man26A)

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follows S5B2,5oS2 pyranoside ring inter-conversions by adopting B2,5
conformation in the transition state. The key specificity determinant in these
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pseudoaxial ring inter-conversions for the hydrolysis of β-mannosides is the O3 atom.
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The 1S5 conformation of mannose orients the susceptible glycosidic linkage into an
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axial position which simultaneously permits unhindered attack by nucleophilic
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glutamate at the opposite face of anomeric carbon (Ducros et al., 2002). Analysis of
computed Free Energy Surface of retaining β-D-mannosidases further corroborated
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this catalytic itinerary (Ardèvol et al., 2010). Similarly, other reports also support the
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fact that β-D-mannosidases catalyse the hydrolysis of sugar backbone via a boat like
conformation (Vincent et al., 2004; Tailford et al., 2008). However, other
glycosidases like cellulases or xylanases display 1S34H34C1 pseudo-rotational

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itinerary (Ducros et al., 2002; Vincent et al., 2004) (Fig. 6).
X-ray crystallographic and site directed mutagenesis studies of GH 26 endo-β-

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mannanase from B. subtilis z-2 (Yan et al., 2008), P. fluorescens ssp. (Bolam et al.,
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1996), P. cellulosa (Hogg et al., 2001), C. fimi (Hekmat et al., 2010),and C. japonicus
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(Le Nours et al., 2005; Tailford et al., 2009) elucidated an open cleft shaped active
site with strictly conserved acid/base catalyst (Glutamate at the beta-strand 4 end) and
nucleophile catalyst (Glutamate at the beta-strand 7 end) which are 5.5 Ǻ apart (Hogg
et al., 2001). The catalytic acid-base and nucleophile residues in endo-β-mannanases
from different organisms are summarized in Table 5.
6.4. Non-catalytic carbohydrate binding modules (CBMs): Implications on substrate
hydrolysis
Endo-β-mannanases like other glycosyl hydrolases often display modular
architectures generally comprising of catalytic domain(s), CBM, and additional
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functional domain(s). A CBM is defined as a contiguous amino acid sequence within
a carbohydrate-active enzyme with independent fold and function and have
carbohydrate-binding activity. Their major function is to bind the enzyme to the
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substrate and thereby enhance its catalytic activity, especially towards insoluble
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substrates. At present, 71,527 CBMs have been classified on the basis of amino acid
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sequence, binding specificity and structure into 74 families (www.cazy.org). On the
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basis of three dimensional structure, CBMs have been divided into seven different
‗fold families‘; with most CBMs belonging to the β-sandwich fold family (Hägglund
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et al., 2003; Hervé et al., 2010; Zhang et al., 2014).
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Mannanases from Trichoderma reesei and Agaricus bisporus have been
shown to comprise of catalytic domain (CD) 5 at the N-terminal and CBM of family 1
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at the C-terminal (Hägglund et al., 2003; Tang et al., 2001). Among bacteria,
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mannanases from T. petrophila (Da Silva et al., 2014), Caldocellulosiruptor
saccharolyticus (Morris et al., 1995), and Clostridium thermocellum ATCC 27405

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(Ghosh et al., 2013) have been shown to contain CBM of families 27 at the C-
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terminal, 27 at the central position, and 35 appended at the N-terminal, respectively.

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The full length (CtManf) endo-β-mannanase from C. thermocellum ATCC 27405
exhibited higher activity and hydrolyzing capacity at higher temperatures than its
truncated derivative (CtManT) containing only catalytic domain. These observations
were attributed to the appended N-terminal CtCBM35 domain 1) facilitating increased
catalysis by concentrating the catalytic module in the vicinity of the substrate and 2)
prolonging the binding of the substrate (Ghosh et al., 2014a).
The properties of the CBM encoded by T. reesei β-mannanase Man5A was
studied by constructing a mutant lacking the part encoding the CBM (Man5A∆CBM).
Both Man5A∆CBM and wild-type enzyme were able to hydrolyse mannopentaose,
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soluble locust bean gum galactomannan and insoluble ivory nut mannan in similar
fashion. However, Man5A∆CBM showed a significant decrease in hydrolysis of
mannan/cellulose complex when compared with wild-type protein (Hägglund et al.,
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2003).
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In Caldibacillus cellulovorans, putative ManA protein was encoded by
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complete manA ORF2 and had multi-domain structure comprising of four domains: a
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putative N-terminal domain (D1) of unknown function, an internal carbohydrate
binding domain (CBD) (D2), a β-mannanase catalytic domain (D3), and a C-terminal
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CBD (D4). These domains were linked to each other via proline-threonine-rich
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peptides (Sunna et al., 2000). Modular β-mannanase (Man5C) from Vibrio sp. strain
MA-138 consisted of a catalytic module connected through a linker region to a C-

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terminal CBM27. The presence of C-terminal CBM27 contributed to the binding of

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substrate to the modular enzyme and thereby increased the rate of depolymerisation of
soluble mannan. The truncated derivative without CBM27 exhibited 1.5–3.5-fold

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lower catalytic efficiency (kcat/Km) towards soluble mannans as compared to the full-
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length Man5C (Tanaka et al., 2009). The modular organization of PaMan26A shows
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a family 35CBM at its N-terminal end, a short linker (12 residues, R133 to N141)
region rich in proline residues and no glycosylation sites. Moreover, the linker was
tightly bound to the CD and made sure that the CBM and CD are in close association
with each other (Couturier et al., 2013a, 2013b).
Despite the ability of CBMs to increase the affinity of the modular enzyme
towards its substrate, few studies have shown a decrease in turnover number (kcat) and
the kinetic efficiency (kcat/Km) of truncated derivatives upon removal of CBMs
(Sunna, 2010; Wang et al., 2014) indicating that the catalytic process was less
efficient in their presence. Sterical hindrances in substrate catalysis imposed by CBMs
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and unproductive binding due to competition in between CBM and catalytic module
to soluble substrate have been proposed as the plausible reasons (Sunna, 2010).
Truncation of CBM (ThMan5A△CBM) from mannanase from Trichoderma
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harzianum improved its expression efficiency and properties of encoded enzyme. The
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recombinant enzyme (ThMan5A△CBM) produced 2,460 ± 45.1 U/ml of mannanase
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activity in the culture supernatant which was 2.3-fold higher than the full-length
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ThMan5A gene (Wang et al., 2014). As most of the CBM functional studies have
been carried out with purified substrates, the likelihood of them mimicking the
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molecular complexity of the natural hemicellulose present in plant cell walls are
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abysmally low.
7. Biochemical properties of endo-β-mannanases

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7.1. pH
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Fungal sources: Acidic pH optima has been reported for GH 5 endo-β-mannanases
from fungal sources like A. sulphureus (pH 2.4) (Chen et al., 2007), A. aculeatus (pH
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2.5) (Pham et al., 2010), Penicillium oxalicum GZ2 (Liao et al., 2014), and
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Trichoderma reesi (pH 3.5) (Eneyskaya et al., 2009). However, a novel endo-β-
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mannanase belonging to a new GH family 134 from A. nidulans was found to be
optimally active at near neutral pH (Shimizu et al., 2015). With respect to pH
stability, endo-β-mannanases obtained from many fungal sources retained appreciable
(80%) activity in acidic to neutral pH range (4-7). Bacterial sources: They are
reported to be maximally active at neutral to alkaline pH. For example, endo-β-
mannanase (GH 5) from Bacillus nealsonii PN11 (Chauhan et al., 2014b) and Bacillus
N16-5 (GH 26) (Lin et al., 2007) were optimally active at pH 8 and 9.6, respectively.
An endo-β-mannanase (GH 26) from Bacillus sp. CFR1601 was optimally active at
pH 7 (Srivastava and Kapoor, 2015). Bacterial mannanases showed significant
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stability in the pH range 6-9 (Table 6). Interestingly, endo-β-mannanase from
Acinetobacter sp. ST 1-1 retained more than 80% activity in a broad pH range 3-10
(Titapoka et al., 2008).
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7.2. Temperature
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Most endo-β-mannanases reported till date showed maximal activity in the
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temperature range of 40-650C (Table 6). However, GH 5 endo-β-mannanase from
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Cryptopygus antarcticus displayed maximum activity at 300C (Song et al., 2008),
while Thermotoga neapolitana 5068 and P. oxalicum GZ2 GH 5 endo-β-mannanases
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exhibited maximum activity at 920C (Duffaud et al., 1997) and 800C (Liao et al.,
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2014), respectively. Endo-β-mannanases from Bacillus N16-5 (GH 26) (Lin et al.,
2007), A. aculeatus (GH 5) (Pham et al., 2010), and Caldicellulosiruptor Rt 8B.4 (GH
26) (Sunna, 2010) displayed maximum activity at temperatures more than 700C.
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7.3. Molecular weight and isoelectric point
The molecular weight of endo-β-mannanases reported from various sources is within

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30-80 kDa range (Table 6). However, endo-β-mannanases belonging to GH 26 family

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from C. thermocellum (Ghosh et al., 2014a, 2014b) and unclassified family from
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Penicillium occitanis (Blibech et al., 2010) showed low molecular weight of 15kDa
and 18 kDa, respectively. Thermoanaerobacterium polysaccharolyticum produced a
high molecular weight (120 kDa) endo-β-mannanase (Cann et al., 1999), while C. fimi
produced three endo-β-mannanase having molecular weights of 30 kDa, 75 kDa and
100 kDa) (Stoll et al., 1999). The isoelectric point (pI) of most endo-β-mannanases is
reported in the range 4-8 (Table 6). In some cases, multiple forms of enzymes are
isoforms, produced from the same gene but differ due to subtle post-translational
modifications (Puchart et al., 2004) whereas in other organisms these enzymes are
encoded by different genes (Millward-Sadler et al., 1996).
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7.4. Kinetic parameters
The Km and Vmax of endo-β-mannanases are reported to be in the range of 0.0095-27.4
mg/ml and 29-16666.7 µmol/ml or mg/min, respectively. Locust bean gum and
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Konjac mannan are the most commonly used substrates for the estimation of kinetic
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parameters (Table 6).
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7.5. Effect of metal ions, surfactants, and chemical reagents
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Among additives, the effect of metal ions on endo-β-mannanases activity is quite
intriguing. For example, Fe3+ and Fe2+ inhibited activity of endo-β-mannanase from
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Paenibacillus cookie (Yin et al., 2012), whereas Fe3+ activated and Fe2+ had no effect
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on endo-β-mannanase from B. subtilis WY34 (Jiang et al., 2006). Similarly, Ag2+ also
showed differential effects wherein some cases it acted as activator while in other
cases as an inhibitor (Jiang et al., 2006; Luo et al., 2009; Yin et al., 2012). Co2+ and
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Hg2+ activated and inhibited many endo-β-mannanases, respectively (Jiang et al.,
2006; Luo et al., 2009; Kim et al., 2011b; Katrolia et al., 2012; Yin et al., 2012).
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Metal ion chelators (EDTA/EGTA/citrate) inhibited endo-β-mannanases from

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Cellulosimicrobium sp. strain HY-13 (Kim et al., 2011a), C. thermocellum (Ghosh et

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al., 2013), and P. cookie (Yin et al., 2012) suggesting the importance of metal ions
during catalytic reaction. However, endo-β-mannanase from Sphingomonas sp. JB13
(Zhou et al., 2012) was stimulated by EDTA.
Disulphide reducing agents (iodoacetamide/β-mercaptoethanol/dithiothrietol)
had no major effect on endo-β-mannanase activity from P. cookie (Yin et al., 2012)
and B. subtilis WY34 (Jiang et al., 2006), but inhibited endo-β-mannanase from B.
nealsonii PN11 (Chauhan et al., 2014b). However, in other cases they activated endo-
β-mannanases from Cellulosimicrobium sp. strain HY-13 (Kim et al., 2011a), Bispora
antennata CBS 126.38 (Liu et al., 2012), and Rhizomucor miehei (Katrolia et al.,
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2013). N-Bromosuccinimide, a tryptophan modifying agent, inhibited endo-β-
mannanases from Cellulosimicrobium sp. strain HY-13 (Kim et al., 2011a, 2011b)
and B. subtilis WY34 (Jiang et al., 2006). Alkylating agent N-ethylemaleimide
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slightly inhibited endo-β-mannanases from P. cookie (Yin et al., 2012).
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Non-ionic surfactants have mostly stimulated or had an insignificant effect on
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the activity of endo-β-mannanases (Kim et al., 2011a, 2011b; Chauhan et al., 2014b).
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However, ionic surfactants like SDS and CTAB led to a drastic reduction in endo-β-
mannanase activity (Jiang et al., 2006; Zhou et al., 2012; Chauhan et al., 2014b;
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Srivastava and Kapoor, 2015). In few studies, organic solvents have been shown to
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stimulate (Chauhan et al., 2014b) or inhibit ( Zhou et al., 2012; Chauhan et al., 2014b)
the activity of endo-β-mannanases.

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8. Transglycosylation activity
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The transglycosylation activity leads to the synthesis of new glycosides or
oligosaccharides which are longer than the original substrate. The glycone substrate,
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forming the glycosyl enzyme intermediate, serve as a donor and the incoming
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carbohydrate hydroxyl group act as an acceptor molecule rather than water. Synthesis
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of mannose-containing glycol-conjugates using traditional organic chemistry-based
approaches is laborious and time-consuming. Endo-β-mannanases because of their
retaining double displacement mechanism are able to perform the transglycosylation
reactions and thus offers an easy alternative. GH 5 or 113 family endo-β-mannanase
from P. anserina (GH 5) (Couturier et al., 2013b), A. nidulans FGSC A4 (GH 5)
(Dilokpimol et al., 2011), Mytilus edulis (GH 5) (Larsson et al., 2006), and
Alicyclobacillus acidocaldarius Tc-12-31(GH 113) (Zhang et al., 2008) were able to
catalyze transglycosylation reactions by producing longer manno-oligosaccharides of
DP 4–6 from donor manno-oligosaccharides. Rosengren et al. (2014) recently showed
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very high transglycosylation activity in A. nidulans GH 5 endo-β-mannanase.
Acidophilic β-mannanase from Gloeophyllum trabeum CBS900.73 was able to
produce mannooligosaccharides with higher degree of polymerization from
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mannotetrose by transglycosylation (Wang et al., 2016). There is no evidence at
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present for transglycosylation activity in GH 26 endo-β-mannanases.
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9. Mannanase as a part of multi-functional enzyme complex
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Many enzymes are secreted individually by microorganisms in extracellular medium
but a few forms a multi-enzyme complex (MEC). MEC gives a competitive edge to
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microorganisms in ecological terms by improving the substrate hydrolysis and energy
MA
generation. Cellulosome which coordinate the deconstruction of cellulose and
hemicellulose and play a major role in carbon turnover represents one of the finest
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examples of cell bound multienzyme complex (Fontes and Gilbert, 2010). The
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cellulosome of C. thermocellum, an anaerobic bacterium, showcases co-operative
interactions within an MEC for sustaining cell growth and survival. C. thermocellum
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does not utilize mannose and xylose but still produces hemicellulose degrading
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enzymes like xylanase and mannanases as part of cellulosome which is dominated by

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enzymes (endoglucanases, exoglucanases and β-glucosidases) active in the
degradation of cellulose. This paradox is explained by the pivotal role of xylanase and
mannanases in cleavage of mannan/xylan matrix surrounding cellulose thereby
facilitating the access to cellulose by cellulases leading to production of sugars which
can be assimilated by the microorganism (Halstead et al., 1999). A large gene cluster
for cellulosome from Clostridium cellulovorans comprises of various genes (cbpA-
exgS-engH-engK-hbpA-engL-manA-engM-engN; 22 kb) which include the scaffolding
protein cellulose binding protein A (cbpA), exoglucanase (exgS), several
endoglucanases (engH, engK, engL, engM, and engN) of family 9, mannanase
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(manA), hydrophobic protein (hbpA) containing a surface layer homology domain,
and a hydrophobic (or cohesin) domain (Tamaru et al., 2000). The β-mannanase
reported from anaerobic extreme thermophile Caldocellum saccharolyticum is also a
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part of a multi-domain enzyme. The amino-terminal catalytic domain has β-
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mannanase activity, while the carboxy-terminal domain acts as an endoglucanase
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(Gibbs et al., 1992). A multi-enzyme complex was reported from B. licheniformis
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SVD1 contained two endoglucanases, seven xylanases, two mannanases, and one
pectinase (Van Dyk et al., 2010). Cellulosomal complex of Clostridium cellulovorans
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also displayed endo-β-mannanase activity (Jeon et al., 2011). A fusion gene was
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isolated from a microbial metagenome which encodes an N-terminal endo-1,4-β-
mannanase and C-terminal 1,3-1,4-β-glucanase (Wong et al., 2013).

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10. Thermal stability of endo-β-mannanases: delineating the contributions of

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structural elements
A substantive amount of work has been carried out in the past several years to shed
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light on the molecular basis of endo-β-mannanase thermal stability by understanding

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its biochemical and structural properties especially from microbial sources like A.
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usamii (Tang et al., 2013) and Streptomyces (Kumagai et al., 2012, 2013a, 2013b). It
has been shown by several studies on full length endo-β-mannanase, its truncated
derivatives or mutants that several molecular interactions like disulphide bridges, H-
bonding, and structural features like metal binding site with affinity for bivalent metal
ions like Ca2+, Mg2+, Mn2+, Ni2+ and Zn2+, linker sequences rich in Thr/Ser, and
internal repeats play a significant role in endo-β-mannanase thermal stability (Yan et
al., 2008; Kumagai et al., 2012, 2013a, 2013b; Lu et al., 2014; Srivastava et al., 2016).
Recent observations have also suggested that CBMs also function in an
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unconventional manner towards endo-β-mannanase thermal stability (Sunna, 2010;
Dos Santos et al., 2012).
10.1. The role of metal binding site
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The thermal stability of wild-type and mutant endo-β-mannanases from Streptomyces
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thermoluteus (StManII) and Streptomyces lividans (SlMan) was enhanced (up to 170C
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of Tm value) by addition of Ca2+ and Mn2+. In the catalytic domain of these endo-β-
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mannanases, a bivalent ion-binding site which binds one ion per molecule of enzyme
was responsible for thermal stability. The bivalent ions (Mg2+, Ca2+, and Mn2+) shared
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the same single binding site. Moreover, presence of Ca2+ and Mn2+ also led to changes
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in the secondary structures of these endo-β-mannanases. Primary structure alignment
of many GH5 bacterial mannanases has revealed high conservation of residues at the
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calcium-binding site (Kumagai et al., 2012, 2013a, 2013b).

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In endo-β-mannanases like BCman from B. subtilis Z-2 (Yan et al., 2008) and
ManB-1601 from Bacillus sp. CFR1601 (Srivastava et al., 2014, 2016), presence of a
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metal-binding motif comprising of six-coordinate Zn2+ with the His(1)-Glu(336)-

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His(23) has been shown (Fig. 7). The metal-binding motif linked the flexible loops at
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the N-terminus and C-terminus and increased the rigidity of the protein eventually
leading to high thermal stability. Studies on ManB-1601 thermal stability elaborated
the quintessential function of metal ions as EDTA treatment of holoenzyme
promulgated the population of conformational state that unfolds at lower temperature
(Srivastava et al., 2014, 2016).
10.2. The role of non-catalytic carbohydrate binding module (CBMs)
CBMs facilitate not only non-catalytic disruption of polysaccharide but also provide
molecular flexibility between the structural domains and optimize the geometry
between domains with respect to high temperatures (Blake et al., 2006; Hervé et al.,
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2010). Deletion of CBM 27 domain from GH5 β-mannanase from T. petrophila led
to reduction in midpoint of thermal denaturation from 1000C to 880C but had no effect
on catalytic efficiency or substrate specificity (Dos Santos et al., 2012). Thermo-
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stability of GH5 β-mannanase (AuMan5A) derived from A. usamii YL-01-78 having
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only catalytic domain, has been improved by fusion with family 1 CBM of the T.
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reesei cellobiohydrolase I (TrCBH I). However, the fusion protein showed increased
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Km and slightly altered Vmax values (Tang et al., 2013). Lu et al. (2014) and Wang et
al. (2014) provided striking evidence on improvement of thermal stability of GH5 β-
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mannanase (Man5XZ3) from A. nidulans XZ3 and ThMan5A from T. harzianum
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MGQ2 by removal of CBM, respectively.
The positioning of CBM domain with respect to catalytic domain affects
thermal sensitivity. It was shown in CsMan26, a modular β-mannanase from

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Caldicellulosiruptor strain Rt8.B4, consisting of two N-terminal family-27 CBMs
followed by a family 35 CBM and a family 26 glycoside hydrolase catalytic module

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(Sunna, 2010).
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Linker sequences between catalytic domain and CBM also contribute towards
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thermo-stability of endo-β-mannanases. Removal of CBM1 (Man5ΔCBM) led to
improvement in thermo-stability of Man5XZ3, a multi-modular β-mannanase encoded
by A. nidulans XZ3, but removal of the linker region along with CBM1 (Man5ΔCL)
resulted in poor thermo-stability (Lu et al., 2014). These findings taken together with
the role of CBM‘s in substrate anchoring suggest that a) Presence of CBMs leads to
high efficiency of catalytic module towards the complex natural substrate like plant
cell wall by promoting molecular anchoring leading to close association and longer
contact with the substrate and b) CBMs work as thermo-stabilizing domains as
evident in many endo-β-mannanases but thermo-stabilising functions of CBM‘s still
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remains highly intriguing as there are reports of endo-β-mannanases wherein they do
not contribute towards thermo-stability. The latter observation also finds support from
CBM modules present in enzymes from mesophilic counterparts (Shoseyov et al.,
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2006).
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10.3. Role of disulphide bridge, salt bridge, and H-bond
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B. subtilis z-2 mannanase, BCman, showed a marked reduction in its thermal stability
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and loss of activity at 800C when the disulphide linkages were reduced with β-
mercaptoethanol. It has been postulated that conformational environment and solvent
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accessibility of disulphide bridges are the key requirements for the latter to act as
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stabilization factor in improving proteins thermo-stability (Yan et al., 2008).
However, no such role of disulphide bridge was evident in the thermal stability of
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endo-β-mannanase from Bacillus sp. CFR1601 (Srivastava et al., 2014).

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The average ion-pair interaction per residue and utilization of arginine in such
ion-pairs (forming multiple hydrogen bonds with acidic partner) along with a
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propensity to form salt bridges also helps in contributing towards thermo-stability of

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proteins. This has been shown for thermo-stable endo-β-mannanase (Q1.1) from
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Thermomonospora fusca which had 0.07 average ion pair interaction per residue.
Q1.1 and S. lividans endo-β-mannanases share high sequence similarity (54.0%).
However, Q1.1 with 10 additional salt bridges and four ion triplets (two of them
include an arginine forming hydrogen bond) has 270C higher temperature optima
(850C) than S. lividans endo-β-mannanases (580C) (Hilge et al., 1998).
11. Structure-based rational design and molecular evolution: Advent of endo-β-
mannanase with improved activity and stability
Endo-β-mannanase from P. agglomerans has been engineered using modified DNA
shuffling and site-directed mutagenesis. From a library of 19,700 clones, two mutant
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enzymes Gly267Ser and His134Arg/Phe141Leu displayed 1.14 and 3.3-fold increase
in catalytic efficiency (kcat/Km) towards locust bean gum, respectively. Enhancement
of polar contacts or positive charges by replacement of His 134 with either Arg or Lys
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by site-directed mutagenesis resulted in 2.81- and 2.75-fold increase, respectively in
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the catalytic efficiency of the enzyme and decrease in Km (Man26P > His134Lys
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>His134Arg) when compared with the native enzyme. The presence of weakly
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nucleophilic Ser (Gly267Ser) at 267 in place of Gly helped by forming new hydrogen
bonds which can interact with hydroxyl groups of the substrate and thereby facilitated
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the interaction of the enzyme with the substrate (Wang et al., 2013).
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In another study, site-directed mutagenesis has been employed to improve the
acid-stability of endo-β-mannanase from B. subtilis B10-02 by changing several

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surface-exposed amino acid residues to acidic or neutral ones. Among the mutations,
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replacement of His 54 residue with Asp shifted the stability and optimal activity from
pH 6.5 to 5.5 (Xu et al., 2013).

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With the rationale of improving hemicellulose hydrolysis, Guo et al. (2013)

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have constructed chimeras of xylanase and mannanase. The properties of xylanase

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and mannanase in chimera and their synergistic abilities in efficiently hydrolyzing
Luffa cylindrica fiber was shown to be influenced by the type of linker, the length of
linker, and by their order of integration. A structure-based rational design was also
employed to engineer GH-5 mannanase from Aspergillus niger BK01. Among the 23
mutants created, Tyr216Trp mutant showed an 18±2.7% improvement in its specific
activity as compared to the wild-type enzyme. The higher catalytic activity of this
mutant was attributed to 1) the extended aromatic ring of Trp leading to lower
substrate affinity by altering the interaction of polysaccharides, and 2) prevention of
the enzyme from retaining the substrate at the binding site (Huang et al., 2014). The
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Tm value and half-life of a mesophilic GH 5 β-mannanase from A. usamii was
improved by 12.10C and 48-fold, respectively by substitution of loop-structure using
rational design followed by megaprimer PCR (Dong et al., 2016).
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Couturier et al. (2013a) have employed a random mutagenesis strategy using
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error-prone PCR and two steps of high-throughput enzymatic screening to generate
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variants (10,800 mutants) of GH5 (PaMan5A) and GH26 (PaMan26A) endo-β-
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mannanases from ascomyceteous fungus P. anserina which displayed improved
activity (lowered Km and increased kcat/Km) towards galactomannan. One single
(PaMan5A-G311S), two double

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(PaMan5A-K139R/ Y223H, PaMan26A-
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P140L/D416G) and two triple mutants (PaMan5A-V256L/G276V/Q316H, PaMan5A-
W36R/I195T/V256A) were used for further characterization. Circular dichroism, pH

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and temperature profiles of these mutants were similar to those of respective parental
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enzymes. However, all the selected variants displayed significant improvement in
activity as compared to wild-type enzymes. PaMan26A-P140L/D416G double mutant

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having mutations in the linker region (P140L) and at the entrance of the active site
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(D416G) showed a 30% increase in kcat/Km compared to the parental enzyme. The
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reduced linker rigidity by virtue of first mutation (P140L) was given as the reason to
partially explain the increase in kcat/Km, while the lack of carboxylic side chain due to
second mutation (D416G) facilitated entry of substrate to the active site. Triple
mutant PaMan5A-W36R/I195T/V256A showed improved turnover towards
galactomannan and 1.8-fold higher catalytic efficiency. Single mutant PaMan5A-
G311S with almost unmodified structure except slight movement of the beta-strand 8
showed 8.2-fold increase in kcat/Km due to reduced Km possibly by the positioning of
W315 residue at the surface of enzyme by the interaction of hydroxyl group of serine
with nearby residues.
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12. Industrial applications of endo-β-mannanases
12.1. Functional food
Prebiotics and dietary fibres comprises of non-digestible oligosaccharides (NDO‘s)
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and polysaccharides whose chemical structure prevents them from being digested in
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mammalian small intestine due to the absence of required digestive enzymes.
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However, they are readily assimilated and metabolized by colonic probiotic
SC
microorganisms like Lactobacillus and Bifidobacteria. Fermentation of these
carbohydrates by probiotic strains is mediated through a pathway by glycolytic
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enzymes leading to the production of short chain fatty acids (SCFA) like acetic acid,
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propionic acid, and butyric acid. SCFA in turn impart various functional roles in the
body such as establishment of probiotic microbial community, growth inhibition of

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pathogenic bacteria, as an energy source (mainly by acetate) in human and animal

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muscle, kidney, heart, and brain, hypo-cholesterolemic (mainly by propionate) effects,
protection against inflammatory bowel disease, ulcerative colitis, and Crohn‘s disease
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(mainly by butyrate) (Li and Nie, 2015).

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Manno-oligosaccharides (MOS) represent an excellent prebiotic food

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ingredient. They are derived from the hydrolysis of mannan from guar gum, locust
bean gum or konjac gum. Several researchers have studied the potential of
mannanases from microorganisms like P. occitanis (locust bean gum) (Blibech et al.,
2011), B. subtilis CAe24 (copra meal) (Rungrassamee et al., 2014), Bacillus sp.
CFR1601 (guar gum, locust bean gum) (Srivastava and Kapoor, 2014; Srivastava et
al., 2015b) Bifidobacterium adolescentis (ivory nut mannan) (Kulcinskaja et al.,
2013), and Bifidobacterium animalis subsp. lactis Bl-04 (ivory nut mannan and locust
bean gum) (Morrill et al., 2015) in the production of MOS (Table 7).
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Guar gum (GG) is utilized in food products like dairy, soups, sauces, and
bakery as a source of dietary fibre and for hydration, thickening, and stabilizing
properties (Yoon et al., 2008). It also helps in reducing serum cholesterol and
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alleviating symptoms of irritable bowel syndrome. However, the high viscosity of GG
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hampers its incorporation in food products besides affecting protein efficacy and
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nutrient digestion. Partially hydrolysed guar gum (PHGG), which offers health
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benefits and stabilizing properties similar to GG, can be easily incorporated in food
products due to its high solubility and less viscosity and therefore, represents a
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feasible alternative to GG for the food industry. PHGG is also effective in treating
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irritable bowel syndrome (Yoon et al., 2008), and it has been shown recently that
PHGG could also serve as an ideal natural soluble fibre for delivering acute and long
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term satiety effects and can reduce energy intake from whole day snacking (Rao et al.,
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2015). The partially hydrolysed galactomannan from Caesalpinia pulcherrima
presented suitable properties to be added as an alternative dietary fibre source in a

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wide range of food products, particularly beverages (Buriti et al., 2014). PHGG is
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produced conventionally using physical (hydrothermal, ultra-sonication, and

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irradiation) and chemical (acid) methods which require high energy inputs and are
environment un-friendly (Gupta et al., 2015). Instead, low-cost and mild enzymatic
depolymerization of GG using endo-β-mannanases has been reported as a plausible
alternative (Srivastava and Kapoor, 2015).
12.2. Coffee and fruit juice clarification
Presence of mannans in coffee and fruit juice extracts leads to high viscosity, which
hampers their industrial processing and thus acts as a bottleneck in their effective
commercialization. Endo-β-mannanases from B. nealsonii PN11 (Chauhan et al.,
2014b), and Sclerotium rolfsii (Sachslehner et al., 2000) were successfully employed
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for reducing the dynamic viscosity of coffee extract. An immobilized mannanase was
shown to hydrolyse coffee galactomannan (Nicolas et al., 1998). Recently, application
of endo-β-mannanase from Lactobacillus plantarum M24 (Nadaroglu et al., 2015)
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and Bacillus pumilus (M27) (Adiguzel et al., 2014) was reported in clarification of
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peach and orange, apricot, grape, and apple juices, respectively.
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12.3. Feed sector
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Improved nutrient utilization, feed conversion efficacy, higher egg production,
improved growth performance and immunity, increased ileum protein digestibility,
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and reduced uric acid excretion were reported after usage of mannanases in poultry
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feed (Lee et al., 2003; Wu et al., 2005; Zou et al., 2006). Supplementation of multi-
enzyme complex (Natuzyme® and Hemicell®) which included endo-β-mannanase, to

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fish feed significantly improved growth performance and feed utilization in Caspian
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salmon (Salmotrutta caspius) (Ali Zamini et al., 2014). Supplementation of β-
mannanase (480U/kg dry matter) to feed rich in mannan (palm kernel meal, copra
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meal, soybean hull, etc.) stimulated growth, nitrogen utilization, and feed efficiency in
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growing Korean native goats (Lee et al., 2014).

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12.4. Detergent sector
Mannanases have also been used in detergent formulations for the removal of stains
containing mannan gums which otherwise are difficult to clean due to their ability to
get adsorbed onto cellulose fibres (Srivastava and Kapoor, 2014).
12.5. Saccharification and bioethanol
Mannan degrading enzymes can be employed synergistically for converting
lignocellulosic polysaccharides into fermentable sugars which can be subsequently
used for ethanolic fermentation (Galbe and Zacchi, 2002; Chandel et al., 2015).
Jørgensen et al. (2010) have shown the utilization of palm kernel cake (PKC) for the
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production of fermentable sugars by using a cocktail of xylanase and mannanase. A
crude solution of endo-β-mannanase and xylanase was shown to produce xylose and
reducing sugar from aqueous ammonia pre-treated corn cob powder (Zhang and Sang,
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2015). Supplementation of fungal β-mannanase and β-mannosidase to the
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Talaromyces cellulolyticus cellulase system ameliorated the hydrolysis of softwood
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(ball-milling-treated Douglas fir) (Inoue et al., 2015). P. anserina mannanases,
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PaMan5A (GH 5) and PaMan26A (GH 26), were cloned and expressed in Pichia
pastoris for their utilization in the enzymatic hydrolysis (saccharification) of
lignocellulosic biomass (spruce)

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by T. reesei. Results obtained showed
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supplementation of T. reesei industrial xylanase-rich enzyme cocktail (E508) with a
combination of PaMan5A and PaMan26A was better in releasing sugars (34%) as

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compared to PaMan5A (28%) or PaMan26A (17%) alone (Couturier et al., 2011).

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12.6. Oil drilling
In oil drilling operations, now-a-days, guar gum is being used to flood the well along
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with sand particles followed by pressurizing the bedrock until it fractures. To ease
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product flow, thinning of the polymer solution is carried out, and mannanases which
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can catalyse the hydrolysis of guar gum at elevated temperatures (>80°C) are proving
useful for this purpose. A β-mannanase from Enterobacter sp. strain N18 has been
used as a gel-breaker in place of chemical gel breakers for low-temperature oil wells
and other industrial fields (You et al., 2016).
13. Concluding remarks and future perspectives
Combinatorial and coherent methodologies like structure-based rational design,
directed evolution and fundamental biochemistry have resulted in the emergence of
endo-β-mannanases to suit the need of both academia and industry. Improvement in
scientific understanding by cutting-edge experimentation in the structure-activity
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relationship, catalytic mechanism, and factors involved in conferring stability to
enzymes would help to create more robust and active endo-β-mannanases so as to aid
hemicellulose hydrolysis under industrial conditions. Mining novel genes from
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mother-nature using metagenomic libraries and next generation sequencing platforms
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can also help to unravel powerful endo-β-mannanases with hitherto unknown
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properties.
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Acknowledgements
Authors thank Prof. Ram Rajashekharan, Director, CSIR-CFTRI, for constant
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encouragement. The support obtained under project MLP0116 is gratefully
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acknowledged. P.K.S thanks, University Grants Commission (UGC), New Delhi,
India, for Junior and Senior Research Fellowship.

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Legends to figures
Figure 1 Schematic representation of various types of β, 1-4
mannan/heteromannan.
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Figure 2 Molecular modelling of schematic structures of galactomannan (F25):
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(A) 2D model; (B) 3D model without energy minimization; (C) 3D
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model with energy minimization. Reprinted with permission from Guo
et al., 2016, Copyright© 2016 Elsevier.
Figure 3
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Spatial arrangement of endo-mannanase belonging to glycoside
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hydrolase family 5 (A) Schematic representation of the structure of T.
fusca β-mannanase. β Strands β1 and β0, which form the bottom of the
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barrel, are shown as blue arrows; all other β strands are shown as red
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arrows. Helices are depicted as green spirals. The figure was generated
using the program MOLSCRIPT. Reprinted with permission from

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Hilge et al., 1998, Copyright© 1998 Elsevier, and family 26 (B)
Structural model of ManB-1601 built using BCman (PDB: 2QHA

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chain 1) as template. Superimposition of three-dimensional structure of
ManB-1601 (blue) with BCman(red). The figure was generated using
PyMOL graphics (http://www.pymol.org). Reprinted with permission
from Srivastava et al., 2016, Copyright© 2016 Elsevier.
Figure 4 Schematic representation of the T. fusca mannanase–mannotriose
complex interactions. The positions of the four subsites are indicated
below. As the –1 mannosyl residue is not clearly visible in the
electrondensity map, it is depicted with dashed lines at the expected
position. The five water molecules are represented by spheres. The two
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water molecules in the –1 subsite have low B factors and may represent
the positions of HO–C(3) and HO–C(2). In the –3 subsite the double
conformation of the primary hydroxyl group is shown. Reprinted with
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permission from Hilge et al., 1998, Copyright© 1998 Elsevier.
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Figure 5 GGM5 hydrolysis by StMandC and TfMandC. (A) Schematic
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representation of GGM5 hydrolysis by mannanases. The parenthesis
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and numbers show the minus subsites in StMandC and TfMandC. The
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triangles show the cleavage site for StMandC (closed) and TfMandC
(open). Reprinted with permission from Kumagai et al., 2015,

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Copyright© 2015 John Wiley and Sons.
Figure 6 Conformational states of sugar during glycosylation step of cellulase

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(family-5 and -7 retaining cellulases) (Scheme 1) and mannanase
(Man26A E212A) (Scheme 2) hydrolysis. Reprinted with permission

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from Ducros et al., 2002, Copyright© 2002 John Wiley and Sons.
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Figure 7 Structural model of ManB-1601 built using BCman (PDB: 2QHA

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chain 1) as template. Metal binding site (in golden brown) in zinc-
coordination environment involving His (1), His (23), Glu (336)
residues is shown in stick representation. The figure was generated
using PyMOL graphics (http://www.pymol.org). Reprinted with
permission from Srivastava et al., 2016, Copyright© 2016 Elsevier.
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H OH H OH H OH
H OH
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HO HO HO O
HO O O O
O O O O
O
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HO H HO H H H
H H HO H H
HO
H H H H H H
H H
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Mannose Mannose Mannose Mannose
OH Linear mannan
Galactose HO
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H O
H
HO H
H OH
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H O H OH
H OH H H OH
HO HO HO O
HO O O O
O O O O
O
HO H HO H H H
H H HO H H
HO
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H H H H H H
H H
Mannose Mannose Mannose Mannose
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Galactomannan
H OH H OH H OH
H OH
H O HO
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O H O HO O
O O O O
HO O
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HO H HO HO
H OH H H H H
OH
H H H H H H
H H
Glucose Mannose Glucose Mannose
Glucomannan
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HO OH
Galactose H O
H
HO H
H OH
H
H OH H O H OH
H OH
H O HO O H O HO O
O O O O
HO HO HO O
H H H HO H
OH H OH H
H H H H H H
H H
Glucose Mannose Glucose Mannose
Galactoglucomannan
Figure 1
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Figure 2
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(A) (B)
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Figure 3
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Figure 4
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Figure 5
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Scheme 1. Substrate conformations along the glycosylation step of family-5 and -7 retaining cellulases: a) Michaelis complex
D
(1S3), b) transition state (4H3), c) covalent intermediate (4C1). Planes containing four atoms within the pyranoside
ring are indicated with gray solid and dashed lines. Reprinted with permission from Ducros et al. (2002). Copyright
TE
© 2002 John Wiley and Sons.

P
CE
Scheme 2. Substrate conformations along the glycosylation step of Man26A E212A. a) Michaelis complex (1S5), b)
AC
transition state (B2,5), c) covalent intermediate (OS2). Gray lines indicate planes containing four atoms within
the pyranoside ring. . Reprinted with permission from Ducros et al. (2002). Copyright © 2002 John Wiley and
Sons.
Figure 6
72
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RIP
SC
NU
MA
D
TE
P
CE
AC
Figure 7
73
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Table 1 Sources of mannanase§
Sources Reference
Bacteria
Bacillus licheniformis DSM13 Songsiriritthigul et al., 2010
T
Bacillus nealsonii PN-11 Chauhan et al., 2014a, 2014b
Bacillus subtilis B23 Zhou et al., 2011
IP
Bacillus pumilus GBSW19 Zang et al., 2015
R
Bacillus subtilis YH12 Liu et al., 2015
SC
Caldicellulosiruptor strain Rt8B.4 Sunna, 2010
Fungi
NU
Aspergillus niger BK01 Huang et al., 2014
Aspergillus niger strain BCC4525 Sornlake et al., 2013
Chrysonilia sitophila Gonçalves et al., 2012
MA
Neosartorya fischeri P1 Yang et al., 2015
Penicillium chrysogenum QML-2 Zhang and Sang, 2015

Penicillium occitanis Pol6 Blibech et al., 2010
D
Talaromyces leycettanus JCM12802 Wang et al., 2015

Other microorganisms
TE
Streptomyces sp. Shi et al., 2011
Symbiotic protist community in termite gut Tsukagoshi et al., 2014a, 2014b

P
(Reticulitermes speratus)
CE
Plants
Arabidopsis thaliana Wang et al., 2015
Germinating Coffea arabica grains Marraccini et al., 2001
AC
Germinating Lilium testaceum bulbs Wozniewski et al., 1992

Lactuca sativa Dulson & Bewley, 1989; Halmer, 1989
Oryza sativa L. cv. Taichung 65 Ren et al., 2007
Ripening fruits of tomato Sozzi et al., 1996; Wang et al., 2009

(Lycopersicon lycopersicum)
Trigonellafoenum-graecum Dirk et al., 1999
Animals
Antarctic springtail (Cryptopygus antarcticus) Kim et al., 2013; Song et al., 2008
Blue mussel (Mytilus edulis) Larsson et al., 2006; Xu et al., 2002
Common sea hare (Aplysia kurodai) Zahura et al., 2010
Edible snail (Helix lucorum) Flari et al., 1995
§ Only representative references published after 2010 are included with respect to microbial
sources. For more information, the reader is advised to refer to Chauhan et al., 2012, Dhawan
and Kaur, 2006, Zyl et al., 2010 and Moreira and Filho, 2008.
74
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Table 2 Optimized conditions for endo-mannanase production from

microorganisms under fermentative process§
Organism Incubation Incubation Initial SmF/ Agitation Reference

time Temperature pH SSF (rpm)
0
(h) ( C)
T
Bacillus 48 40 11 SmF 200 Vijayalaxmi et
IP
halodurans al., 2013
strain PPKS-
R
2
SC
Bacillus 96 37 8 SmF 150 Chauhan et al.,
nealsonii 2014a
NU
PN-11
Bacillus 36 50 - SmF 180 Summpunn et
MA
subtilis BCC al., 2011
41051
Bacillus sp. 24 45 6 SmF 200 Srivastava and
D
CFR1601 Kapoor, 2014

TE
Bacillus sp. 72 37 6 SSF - Srivastava and

CFR1601 Kapoor, 2013
P
Bacillus 96 50 - SmF 200 Jiang et al.,

CE
subtilis 2006
WY34
Aspergillus 96 32 - SSF - Wu et al., 2011
AC
niger LW-1
Aspergillus 48 30 5.5 SSF - Yin et al.,
niger SN-09. 2013
Penicillium 144 30 4.5 SSF - Zhang and
chrysogenum Sang, 2015
QML-2
Streptomyces 48 28 6.5 SmF 130 Pradeep et al.,
sp. CS428 2016
§ Only references published after 2010 or not covered in earlier reviews are
included. For earlier references, the reader is advised to refer to Chauhan et al.,
2012, Dhawan and Kaur, 2006, Zyl et al., 2010 and Moreira and Filho, 2008.
75
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Table 3 Heterologous cloning and expression of endo-mannanase gene from
various organisms§
T
Ge Endo-
Amino Molecu
IP
ne mannan
Source Heterolog acids lar
Vector size pI ase Reference
organism ous host (numb weight
(bp product
R
er) (kDa)
) ion
SC
GH 5 endo-mannanases
Bacteria
Bacillus E. coli pET- 113 377 45 NR NR Zang et al.,
(28)*
NU
pumilus BL21 30a- 4 2015
GBSW19 c(+)
Bacillus E. coli pH6E 208 694 76.089 NR NR Ethier et
stearothermop BL21 X3 5 al., 1998
MA
hilus
Bacillus E. coli pSMA 110 369 41 5.3 5.5 Chauhan et
nealsonii PN- DH10β RT 0 5 IU/ml al., 2015
11
D
Clostridium E. coli pET NR NR NR NR NR Malgas et

cellulovorans BL21(DE3 29b al., 2015b
TE
)
Fungi
P
Aspergillus P. pastoris pPICZ - - 56 - NR Dilokpimol

nidulans X33 α et al., 2011
CE
FGSC A4 A
Aspergillus P. pastoris pPICZ 115 383 37.5 4.1 29 U/ml Li et al.,
niger LW-1 GS115 α 2 (21)*[1 5 2012
7]#
AC
A
Chaetomium P. pastoris pPIC9 125 451 50 4.5 50030 Katrolia et
sp. CQ31 GS115 K 1 U/ml§ al., 2012
Neosartorya P. pastoris pPIC9 118 373 39.5 5.9 101.5 Yang et al.,
fischeri P1 GS115 7 3 U/ml 2015
Penicillium P. pastoris pPIC9 115 384 39 NR 2.5 g/l§ Cai et al.,
sp. C6 GS115 5 (26)* 2011
Penicillium P. pastoris pPICZ 138 426 45 5.0 NR Liao et al
oxalicum GZ- GS115 α 0 9 2015
2 A
Rhizomucor E. coli pET- 133 378 44 NR NR Katrolia et
miehei BL21 2 0 al., 2013
8
a
(
+
)
76
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Others
Cryptopygus E. coli pET- 114 382 39.5 5.9 436.6 Song et al.,
antarcticus strain 3 9 9 U/l 2008
Rosetta- 2
gami2 a
(DE3) (
T
+
IP
)
Haliotis discus - - 113 377 39.6 NR NR Ootsuka et
(17)*
R
hannai 4 al., 2006
Mytilus edulis P. pastoris pPICZ 110 368 40 6.8 900 Xu et al.,
SC
with a 4 5- mg/ml 2002
Saccharom 8.1
yces B 5
NU
cerevisiae
α-factor
GH 26 endo-mannanases
MA
Bacteria
Bacillus E. coli pFLA 108 336 45 NR 54,266 Songsiriritt
licheniformis BL21 G-CTS 0 U/l higul et al.,
DSM 13 2010
Bacillus sp. E. coli pUC18 299 997 130 NR NR Hatada et
D
strain HB101 4 al., 2005

TE
JAMB-750
Bacillus Brevibacill pHY- 110 NR 40 NR NR Zhou et al.,
subtilis B 23 us brevis p43 0 2013
P
Bacillus Bacillus pMA- 108 360 40.14 NR 220 Liu et al.,

subtilis YH12 subtilis 5 3 U/ml 2015
CE
strain
WB600
Bacillus sp. E. coli pRSE 108 336 40 5.0 666 Srivastava
AC
CFR1601 BL21(DE3 T-A 3 7 U/ml et al., 2016

)
Bacillus sp. E. coli BL pRSE - - - - 8406 Kaira et al.,
CFR1601 21(DE3)Hi T-A U/ml 2016
-control
cells
Bacillus Pichia pPIC9 108 362 41 5.8 5156.74 Li et al.,
subtilis WD23 pastoris K 9 1 2 U/ml 2015
GS115
Clostridium E. coli pET- 144 478 53 NR NR Ghosh et
thermocellum BL21(DE3 28a 9 al., 2013
ATCC 27405 ) (+)
Sphingobacter E. coli pET- 111 371 42.2 NR 65.3 Zhang et
ium sp. GN25 BL21 2 6 mg/l al., 2015
(DE3) 8
a
(
+
77
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)
Reticulitermes P. pastoris pPICZ 390 NR 40 NR 0.67 g/l Tsukagoshi
₸
speratus a et al.,
- 2014a,
A 2014b
Fungi
T
Aspergillus A. oryzae NR NR NR 35.2 NR NR Freiesleben
IP
nidulans et al., 2016
NR: Not reported; *Amino acids present for signal peptide; # Amino acids present for
R
pro-peptide; § high cell density fermentation; ₸ Symbiotic microflora of Reticulitermes
SC
speratus; § Only references published after 2010 or not covered in earlier reviews are
included. For earlier references, the reader is advised to refer to Chauhan et al., 2012,
NU
Dhawan and Kaur, 2006, Zyl et al., 2010 and Moreira and Filho, 2008.
MA
D
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CE
AC
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Table 4 Protocols employed for purifying wild-type endo-mannanases from

different organisms.
Specific
Purification Number Yield Purification
Organism activity Reference
protocol of steps (%) fold
(IU/mg)
T
Aplysia (NH4)2SO4(40- 4 3.3 14.5 27.4 Zahura et
IP
kurodai 60%), al., 2010
Toyopearl
Phenyl-650 M,
R
Toyopearl CM-
SC
650 M, and
Mono-S
Bacillus (NH4)2SO4(60– 3 8.92 38.96 2280.9 Chauhan
nealsonii 80%), Sephadex et al.,
NU
PN11 G-150, and 2014b
DEAE-
Cellulose
MA
Bacillus (NH4)2SO4(40- 3 20.3 5.4 8302.4 Jiang et
subtilis 80%), al., 2006
WY34 SuperdexTM75,
and
D
Q-Sepharose
fast flow
TE
Bacillus sp. (NH4)2SO4 (50– 4 21.3 50.7 10461.5 Srivastava

CFR1601 80%), DEAE- et al.,
Cellulose, 2016
P
DEAE-
CE
Sepharose and
Phenyl-
Sepharose
Coffea (NH4)2SO4 4 10 7000 1400 Marraccini
AC
arabica L. (35%), Phenyl- et al.,

Sepharose, 2001
Mono Q, and
Superdex 75 HR
10/30
Helix Hydroxyapetite, 3 13 26.4 29 Flari et al.,
lucorum L. DEAE- 1995
Sepharose, and
QA-Trisacryl
Paenibacillus (NH4)2SO4(40- 4 6.4 90.2 635.4 Yin et al.,
cookie 60%), 2012
DEAE-
Sepharose and
Sephacryl S-100
HR column
(two times)
Penicillium (NH4)2SO4 4 5.3 9.4 160 Kurakake
oxalicum SO (90%), et al.,
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Sephadex G-25, 2006

Super Q
Toyopearl, and
TSKgel
G3000SW
T
RIP
SC
NU
MA
D
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CE
AC
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Table 5 Positioning of catalytic acid base and nucleophile in mannanases

from different organisms.
Organism Catalytic acid Catalytic Reference

base residue nucleophile
T
residue
A symbiotic protist from termite Glu191 Glu288 Tsukagoshi et al.,
IP
gut 2014a, 2014b
R
Alicyclobacillus acidocaldarius Glu151 Glu231 Zhang et al., 2008
SC
Tc-12-31
Aplysia kurodai (sea hare) Glu162 Glu293 Mizutani et al.,
NU
2012
Bacillus subtilis z2 Glu167 Glu266 Yan et al., 2008
Bacillus nealsonii PN11 Glu152 Glu247 Chauhan et al.,
MA
2015
Cellulomonas fimi Glu175 Glu282 Hekmat et al.,
2010
D
Cellvibrio japonicas Glu212 Glu320 Tailford et al.,

TE
2009
Cellvibrio mixtus Glu429 Glu519 Dias et al., 2004
P
Chrysonilia sitophila Glu181 Glu301 Gonçalves et al.,

CE
2012
Lycopersicon esculentum Glu204 Glu318 Bourgault et al.,
AC
(Tomato) 2005
Penicillium sp. C6 Glu203 Glu314 Cai et al., 2011
Pseudomonas fluorescens ssp. Glu212 Glu320 Bolam et al.,
Cellulose 1996; Hogg et al.,
2001
Thermomonospora fusca Glu128 Glu225 Hilge et al., 1998
Trichoderma reesei Glu169 Glu276 Sabini et al., 2000
81
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Table 6 Biochemical properties of endo-mannanase from various sources§
Mole
cular
Source/Fa Temperatur Kinetic Refer
pH weig pI
mily e (0C) parameters ence
ht
T
(kDa)
Classified Opti Stab Opti Stab Km Vmax
IP
mum ility mum ility
GH 5
R
Bact
SC
eria
Bacil 8.8 5-10 65 t1/2 50 N 7.22 750 Chau
lus 3h at R (LBG) (LBG), han et
NU
neals 700C , 344 al.,
onii 11.59 (GG) 2014b
PN1 (GG), and
MA
1 and 256
18.73 (CM)
(CM) µmol/
mg/ml ml/min
Clost 5.5– 5.5– 40 90 47 N NR NR Malga
D
ridiu 7.0 7.0 min R s et

TE
m at al.,
cellul 370C 2015b
ovor , 30
P
ans min
at
CE
500C
Fung
i
AC
Gloe 2.5 2.0 600C 35- 38.6 5. 3.71 2525 Wang

ophyl to 650C kDa 1 mg/ml U/min/ et al.,
lum 10.0 3 (LBG) mg 2016
trabe
um
CBS
900.7
3
Neos 4 3.0– 80 t1/2 39.5 5. 0.83 1937.2 Yang
artor 7.0 6h at 9 mg/ml µmol/ et al.,
ya 600C 3 (LBG) min/m 2015
fisch g
eri (LBG)
P1
GH
26
Bact
82
ACCEPTED MANUSCRIPT
eria
Bacil 6 5-10 55 NR NR N 5 5000 Srivas
lus R mg/ml tava
sp. and
CFR Kapo
1601 or,
T
2015
IP
Dicty 5 4-7 80 16h 40 N NR NR Gibbs
oglo at R et al.,
R
mus 80° 1999
ther C
SC
moph and
ilum t1/2
Rt46 5.4
B.1 min
NU
at
90°
C
MA
Fung
i
Aspe 5.5 - - Tm 35.2 N NR NR Freies
rgillu at R leben
D
s 53◦C et al.,
nidul 2016
TE
ans
Retic 5 5.0– 40 80% 40 N NR NR Tsuka
uliter 6.5 < R goshi
P
mes 300C et al.,

CE
spera , 2014a
tus₸ 400C
activ
AC
ity
lost
Uncl
assifi
ed
Bacil 6.5 4.5– 55 95% 40.14 N 30 16666. Liu et
lus 7.5 activ R mg/ml 7 U/mg al.,
subtil ity at (LBG) (LBG) 2015
is 550C
YH1 , 3h
2
Paen 5 5-7 50 Stab 68 N 0.009 556 Yin et
ibaci le R 5 and and al.,
llus belo 0.013 484 2012
cooki w 6 mg/ U/min/
e 400C ml for mg, for
, t1/2 LBG LBG
30 and and
83
ACCEPTED MANUSCRIPT
min KGM, KGM,

at respec respect
620C tively ively
NR: Not reported; LBG: Locust bean gum; KGM: Konjac glucomannan; GG: Guar
gum; CM: Copra mannan; GH: Glycosyl hydrolase; ₸ Symbiotic protist of
T
Reticulitermes speratus, § only references published after 2012 or not covered in earlier
IP
reviews are included. For earlier references, the reader is advised to refer to Chauhan
et al., 2012, Dhawan and Kaur, 2006, Zyl et al., 2010 and Moreira and Filho, 2008.
R
SC
NU
MA
D
P TE
CE
AC
84
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Table 7 Production conditions, yield, type and prebiotic role of

mannooligosaccharides (MOS)
Organism Hydrolys Substra Yield Degree of Prebiotic role Reference

is te polymerizati s
condition on
T
s (DP)
Bacillus 550C, 6 h LBG 11% 2-5 Stimulated Chauhan
IP
nealsonii Mannobiose Lactobacillus et al.,
PN-11 , 23% casei and 2014b
R
Mannotrios inhibited
e, 20% Salmonella
SC
Mannotetro enterica
se, 18%
Mannopent
NU
ose
Bacillus 500C, LBG 1.19 mg/ml 1-6 NR Zang et
pumilus 24h,10 al., 2015
GBSW19 mg/ml,
MA
LBG 10
U/mg,
endo-
mannana
D
se
TE
Bacillus sp. pH 6, GG 14% 2 and 4 Lactobacillus Srivastav

CFR1601 500C, salivarius a and
300 min, and L. Kapoor,
10 IU/ml Plantarum 2014
P
Bacillus sp. pH 6, LBG 30% 2, 3 and 5 Several Srivastav

CE
CFR1601 500C, Lactobacilli a et al.,

270 min, 2015b
10 IU/ml
AC
Bacillus pH 6.5, LBG, 1–7 (LBG), NR Liu et al.,

subtilis 550C, 8h KGM 2–7 (KGM) 2015
YH12 and XG and 1–2
(XG)
Bifidobacteri pH 5.3, KGM, - 2, 3 and 4 - Kulcinsk
um 300C, carob, aja et al.
adolescentis 0.2-1 GG 2013
µM
enzyme
Bifidobacteri pH 6, LBG - 2, 3, 4, 5 - Morrill et
um animalis 370C, 5.3 and al., 2015
subsp. lactis nM INM
Bl-04 enzyme
Clostridium pH 6.9, Defatte 40% 2-6 Lactobacillus Ghosh et
thermocellu 600C, 10 d copra Mannobiose acidophilus al., 2014
m min meal , NRRL B-
18% 4496 and
Mannotrios Bifidobacteri
85
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e um infantis
Penicillium pH 4.0, LBG 33%*, 3-4 NR Blibech

occitanis 700C, 40%# et al.,
T
10h 2011
IP
Penicillium pH 5, GG NR 2-7 NR Kurakake
oxalicum SO 400C, et al.,
R
24h 2006
Trichoderma 48h KGM 50% 2-5 NR Mikkelso
SC
reesei n et al.,
2013
NR: Not reported; * Free enzyme, # Immobilized enzyme; LBG: Locust bean gum; KGM: Konjac
NU
glucomannan; XG: Xanthan gum, GG: Guar gum, INM: Ivory nut mannan
MA
D
P TE
CE
AC
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Highlights
 Production, purification, heterologous expression and biochemical
characteristics of endo-β-mannanases
T
 The molecular and structural features of endo-β-mannanases responsible for
IP
its activity and thermal stability
R
 The use of rational design and genetic engineering approaches for improving
SC
endo-β-mannanases
NU
 Biotechnological applications of endo-β-mannanases
MA
D
P TE
CE
AC
87

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Production, properties, and applications of endo-β-mannanases

Praveen Kumar Srivastava, Mukesh Kapoor

To appear in: Biotechnology Advances

Received date: 28 December 2015

Title: Production, properties, and applications of endo-β-mannanases

Authors: Praveen Kumar Srivastava and Mukesh Kapoor§*

The present review provides up-to-date information on the occurrence and

methodologies used for producing and purifying endo-β-mannanases and a

endo-β-mannanases in various industrial sectors like food, feed, detergents, biofuel

and oil drilling is also emphasized.

Key words: Endo-β-mannanase, Biochemical properties, Genetic engineering

Structural organization, Thermo-stability, Industrial application

Plant cell walls are macromolecular structures comprising of an intricate intermix of

cellulose and a matrix of polysaccharides, glycoproteins, proteoglycans, low-

biopolymer, comprises of a bundle of linear poly β-1,4-glucan chains organized by

intra-chain and inter-chain H-bonding networks (Kuhad et al., 1997). Hemicelluloses

different monosaccharides in diverse combinations (Filho, 1998; Pérez et al., 2002).

Xylans represent the pre-dominant hemicellulosic component in hardwood from

angiosperms, while mannans represent the most abundant hemicelluloses in

The structural and heterogeneous nature of mannans requires close association

endo-β-mannanase (EC 3.2.1.78), exo-β-mannosidase (EC 3.2.1.25), β-glucosidase

2015b). Mannan-degrading enzyme systems can be found in several glycosyl

hydrolase families (like GH 1, GH 2, GH 3, GH 5, GH 26, GH 27, GH 113 etc.)

(www.cazy.org). They contribute to 1) microbial metabolism by the generation of

energy equivalents (simpler monosaccharides/oligosaccharides) from substrate

hydrolysis, 2) in plant‘s growth, maturation, and ripening by the metabolism of cell

display modular architectures generally comprising of catalytic domain(s),

of species elucidated that endo-β-mannanases 1) require at least five substrate binding

sub-sites to carry out efficient catalysis of oligo- or polysaccharide substrates and 2)

bioconversion of lignocellulosic residues, food, feed, detergents, oil drilling, and

pharmaceuticals (Chauhan et al., 2012; Malgas et al., 2015a).

The recent progress made in the occurrence, structure, properties and

industrial applications of β-mannan and endo-β-mannanases (GH 5, GH 26, and GH

mechanism of endo-β-mannanases and a holistic view of thermo-stable endo-β-

mannan degrading enzymes like exo-β-mannosidases, α-galactosidases and esterases

etc. or plant polysaccharide-degrading enzymes and synergistic interactions thereof,

et al., 2015a; Chauhan and Gupta, 2016) for further details.

They perform a variety of functions in plants, like acting as storage polysaccharides

development (Liepman et al., 2007). Currently, there is a great deal of interest in

mannan-based polysaccharides as they provide multi-faceted benefits like immuno-

pharmacological, therapeutic, bio-medical, and prebiotic properties which find usage

2008; Ferreira et al., 2012; Yamabhai et al., 2016).

2.1. Structure, occurrence and properties of β-mannan

2.1.1. Linear mannan

2012). Sulphated mannans (carbohydrates: sulphate, 2.7:1) in genus Codium form an

sugar residues along the polysaccharide chain (McCleary et al., 1985).

2005). In the pharmaceutical industry, galactomannans are used as controlled drug

varies in galactomannans. For example, galactomannans isolated from guar seeds

(Cyamopsis tetragonolobus, average molecular weight 220 kDa), tara gum,

between 3000-5000 (McCleary et al., 1985; Mudgil et al 2012a, b; Sébastien et al.,

2014). Galactomannans represents the most abundant constituents of coffee beans.

single galactose residues approximately every 100 residues (Campos-Vega et al.,

in galactomannans found in coffee residues and infusions. Recently, galactomannans

Caesalpiniapul cherrima and Adenanthera pavonina. Electron spray ionization-

while galactomannan from the seeds of vinal (Prosopis ruscifolia) had

interactions between hairy (substituted) and smooth (unsubstituted) regions in

galactomannan from Sophora alopecuroides L. seeds are forming intra-molecular