Bioresource Technology 337 (2021) 125399

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Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Chemoenzymatic production of chitooligosaccharides employing ionic

liquids and Thermomyces lanuginosus chitinase
Manish Kumar a, Jogi Madhuprakash b, Venkatesh Balan c, Amit Kumar Singh d, V. Vivekanand e,
Nidhi Pareek a, *
a
Department of Microbiology, School of Life Sciences, Central University of Rajasthan, Ajmer 305817, Rajasthan, India
b
Department of Plant Sciences, School of Life Sciences, University of Hyderabad, Prof. CR Rao Road, Gachibowli, Hyderabad 500046, India
c
Department of Engineering Technology, College of Technology, University of Houston, Sugar Land, TX 77479, USA
d
Department of Mechanical Engineering, Malaviya National Institute of Technology, Jaipur 302017, Rajasthan, India
e
Centre for Energy and Environment, Malaviya National Institute of Technology, Jaipur 302017, Rajasthan, India

H I G H L I G H T S G R A P H I C A L A B S T R A C T

• Chemoenzymatic COS generation

employing ionic liquids and
T. lanuginosus chitinase.
• IL pretreated chitin appeared amor­
phous and could facilitate enzyme
accessibility.
• Optimization led to 1.5 and 1.3 fold
enhanced (GlcNAc)2 and (GlcNAc)3
production.
• 0.1 g of both (GlcNAc)2 and (GlcNAc)3
was purified from 1 g TBP pretreated
chitin.
• Proficient bio-catalytic valorization of
coastal residuals to chito-bioactives.

A R T I C L E I N F O A B S T R A C T

Keywords: The aim of this work was to study a two-step chemoenzymatic method for production of short chain chitooli­
Chitin gosaccharides. Chitin was chemically pretreated using sulphuric acid, sodium hydroxide and two different ionic
Chitooligosaccharides liquids, 1-Ethyl-3-methylimidazolium bromide and Trihexyltetradecylphosphonium bis(2,4,4-trimethylpentyl)
Chitinase
phosphinate under mild processing conditions. Pretreated chitin was further hydrolyzed employing purified
Pretreatment
Ionic liquid
chitinase from Thermomyces lanuginosus ITCC 8895. Trihexyltetradecylphosphonium bis(2,4,4-trimethylpentyl)
phosphinate treated chitin appeared amorphous and resulted in generation of 1.10 ± 0.89 mg ml− 1 of
(GlcNAc)2 and 1.07 ± 0.92 mg ml− 1 of (GlcNAc)3. Further derivation of optimum conditions through two-factor-
9 run experiments resulted in to 1.5 and 1.3 fold increments in (GlcNAc)2 and (GlcNAc)3 production, respec­
tively. 0.1 g of both (GlcNAc)2 and (GlcNAc)3 has been purified from the Trihexyltetradecylphosphonium bis
(2,4,4-trimethylpentyl)phosphinate pretreated chitin (1 g) employing cation exchange chromatography. The
present study will lay the foundation for development of a green sustainable solution for cost effective upcycling
of coastal residual resources to chito-bioactives.

* Corresponding author.
E-mail address: nidhipareek@curaj.ac.in (N. Pareek).

https://doi.org/10.1016/j.biortech.2021.125399
Received 22 March 2021; Received in revised form 5 June 2021; Accepted 8 June 2021
Available online 11 June 2021
0960-8524/© 2021 Elsevier Ltd. All rights reserved.
M. Kumar et al.
1. Introduction
Chitin-derived products are of immense significance in the current
scenario due to their vast biological applications to produce nano­
particles and nanofibers, and other applications in drug and gene de­
livery, nutrition fortification and wound dressing (Yang et al., 2017; Das
et al., 2015; Lodhi et al., 2014). The chitooligosaccharides (COS) are the
homo- or hetro-oligomers of N-acetyl-D-glucosamine (GlcNAc) and D-
glucosamine (GlcN) which are linked by β-1,4-glycosidic bonds. The
COS are derived from chitin or chitosan (CHS) with the degree of
polymerization (DP) ranging from 2 to 20 and can be prepared by
chemical or enzyme processing methods (Kaczmarek et al., 2019). The
value for COS is defined by its length and the degree of acetylation (DA)
(Singh et al., 2020; Liu et al., 2019; Zhao et al., 2019; Şenel, 2019). Amid
chitin and its derivatives, COS have attracted a wide range of application
including antimicrobial, antioxidant, gene therapy, immune cell prolif­
eration, and tumor growth inhibition due to its superior solubility, low
viscosity, and increased absorption properties in the intestine for anti-
obesity applications (Phil et al., 2018; Liaqat and Eltem, 2018).
The chemical production approaches are rapid and produce high
yield; however the hydrolysis reactions are difficult to control, resulting
in GlcNAc as a major product (Hamed et al., 2016). The enzymatic
methods of producing COS appeared favorable as they do not produce
any toxic byproducts benefiting the environment, high specificity so that
reaction can be controlled and hydrolysis reaction are carried at low
temperatures (Pan et al., 2019; Hamer et al., 2015). However, the
enzymatic approach still face some limitations such as low COS yield
and high cost of production (Kaczmarek et al., 2019; Jung and Park,
2014). The recent knowledge in the field of chitinolytic enzymes espe­
cially chitinase from different mesophilic and thermophilic bacteria and
fungi have given several options that will help to overcome some of the
limitations of enzymatic approaches and expected to play a major role in
the development of robust bioprocess (Kidibule et al., 2020; Hamid
et al., 2013). The production of COS is dependent on both the type of
chitinolytic enzymes used and recalcitrant nature of chitin (Kidibule
et al., 2020; 2018). Several biopolymers including proteins, cellulose,
hemicellulose and chitin have been successfully pretreated using
different chemical, physical and biological methods (Mukherjee et al.,
2020; Villa-Lerma et al., 2013; Guoxiang et al., 2010). Among these
methods, chemical pretreatment has been found to be cost effective and
are reported to increase the porosity and increase enzyme accessibility
so that chitinolytic enzymes could act more efficiently (Suryawanshi
et al., 2019; Li et al., 2019). Since harsh chemicals results in the for­
mation of undesirable toxic side products, several researchers are
exploring mild pretreatment chemical such as ionic liquids (ILs)
(Sánchez et al., 2018; Li et al., 2016). Recently, a combination of both
chemical and enzymatic processing called chemoenzymatic approaches
are getting popular for the development of cost effect method of pro­
ducing COS (Ogata, 2020; Harmsen et al., 2020; Queda et al., 2019).
ILs are organic salts that are liquid at a temperature below 100 ◦ C
(Lei et al., 2017). ILs contains cations such as imidazolium, pyridinium,
pyrrolidinium, or ammonium; organic anions like CH3COO‾ or inor­
ganic anions like Cl‾, Br‾, and BF4‾ (Chiappe and Pieraccini, 2005). ILs
have shown superior dissolution and processability behavior with bio­
macromolecules because of their high chemical and thermal stability,
high ionic conductivity, non-inflammability, and trivial vapor pressure
(Chiappe and Pieraccini, 2005). ILs can be recycled due to their low
volatility, which will reduce chemical pollution to the environment.
Though they do not contribute to air pollution due to negligible vapor
pressure ILs, their impact on aquatic and terrestrial eco-system is yet to
be assessed (Steudte, 2014). ILs have been well extensively explored for
the dissolution of lignocellulose to produce biofuels and biochemicals
(Usmani et al., 2020; Brandt et al., 2013). Same knowledge can be
applied to pretreat chitin (Shamshina and Berton, 2020; Husson et al.,
2017). Li et al. (2019) reported the generation of GlcNAc (175.62 mg)
and (GlcNAc)2 (341.70 mg) from 1 g of 1-ethyl-3lmethylimidazolium
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Bioresource Technology 337 (2021) 125399
acetate pretreated chitin hydrolysed using Streptomyces albolongus chi­
tinase. Notable COS production has been reported using chemical pre­
treatment of chitin followed by enzymatic hydrolysis (Husson et al.,
2017; Xu et al., 2020). However, limited literature is available on the
employment of ionic liquids for pretreatment of chitin for further
enzymatic hydrolysis. Moreover, scarce reports are available on the
utilization of thermostable chitinase for COS production. Although
conventional chemicals viz. acids, alkali have considered as effective
pretreatment agents for chitin disintegration; but low product quality
and environmental concerns boost the search for contemporary agents
viz. ILs. The reusablility of ILs could be crutial in economic prospective
for the development of an efficient process for COS production.Thus, the
present study explores the impact of traditional harsh chemicals (H2SO4
and NaOH) and ILs pretreatment of chitin followed by hydrolysis using
purified chitinase from thermophilic fungus, T. lanuginosus (Kumar
et al., 2018) for COS production. The two ILs 1-Ethyl-3-methylimidazo­
lium bromide (EMB) and Trihexyltetradecylphosphonium bis(2,4,4-
trimethylpentyl)phosphinate (TBP) used in this study were selected on
the basis of their low cost and to the best of our knowledge this is the first
report of employing these ILs for chitin pretreatment. IL pretreated
chitin is found to be highly porous and amorphous when compared to
untreated chitin, thereby facilitating chitinase accessibility during hy­
drolysis producing high COS yield when compared to untreated and
acid/based pretreated chitin. COS purification protocol has also been
developed using preparative cation exchange chromatography, and
quantified them using high performance liquid chromatography (HPLC).
2. Materials and methods
Two ILs EMB and TBP, N-Acetylglucosamine (GlcNAc), and dimeric
N-Acetylglucosamine (GlcNAc)2, and trimeric N-Acetylglucosamine
(GlcNAc)3 were acquired from Sigma-Aldrich, Germany. The α-chitin
processed from shrimp shells was obtained from HiMedia, India.
Colloidal chitin was prepared from the shrimp shell chitin flakes using
the reported protocol (Kumar et al., 2017). Briefly, 5 g of shrimp’s shells
chitin was mildly agitated with 100 ml of ice-cold concentrated hydro­
chloric acid in a glass bottle and maintained at 4℃ for 24 h. Then the
reaction mixture added to ice-cold ethanol (96%, 500 ml) and kept
under rapid stirring and maintained at 4℃ for 24 h. The precipitate was
separated by centrifugation (12,000g, 25 min, and 4℃) and washed
with distilled water until the pH reached neutral. The colloidal chitin
was further dried overnight, ground to fine powder using mortar and
pestle and stored at room temperature before further use. Thin layer
chromatography (TLC) silica gel sheets, chromatography cation ex­
change media such as SP-Sepharose were purchased from Sigma-
Aldrich, Germany. All other chemicals used in the study were analyt­
ical grade.
2.1. Fungal strain and chitinase production
The chitinase producing thermophilic (45 ◦ C) fungal strain used in
the present study, T. lanuginous ITCC 8895 (earlier known as Humicola
grisea) was isolated from the sandy loam soil of Jaisalmer, Rajasthan,
India. Chitinase was produced and purified at Microbial Catalysis and
Process Engineering Laboratory, Department of Microbiology, Central
University of Rajasthan, India using reported procedures (Kumar et al.,
2018; 2017). The chitinase production was carried out under submerged
fermentation employing the optimum levels of nutritional variables
(chitin, 7.49; colloidal chitin, 4.91; KH2PO4, 0.87; K2HPO4, 0.68; KCl,
0.19; NH4Cl, 1.0; MgSO4⋅7H2O, 0.20; yeast extract, 5.50 gl− 1) (Kumar
et al., 2017) and process conditions (pH, 6.50; Temperature, 47 ◦ C;
Inoculum size, 5 discs) (Kumar et al., 2018). Chitinase purification was
carried out by using cation and anion exchange chromatography (Kumar
et al., 2018).
M. Kumar et al.
2.2. Chitin pretreatment
The COS production was carried out by the employing a two-step
chemoenzymatic process. First, chitin was subjected to different pre­
treatment using chemicals such as H2SO4, NaOH and ILs (EMB and TBP)
followed by enzymatic hydrolysis. Chitin (10% w/v) was first combined
with different pretreatment chemicals at 10:1 ratio, followed by incu­
bation in a water bath (Tempo, India) at 100 ◦ C for 90 min using pre­
viously reported protocol (Usmani et al., 2020; Hou et al., 2017).
Further, the pretreated chitin was filtered, washed until pH reached 6.0,
and dried in an oven (50 ◦ C, overnight). The obtained pretreated dry
chitin was stored at room temperature for further use.
2.3. Physico-chemical analysis of chitin
The untreated as well as the pretreated chitin were subjected to
scanning electron microscopic (SEM) analysis to study the effect of
pretreatment on the structure and morphological properties. Briefly, the
dried chitin samples were taken in small amounts on double-sided car­
bon tape adhered to aluminum stubs. The stubs were then sputter coated
with gold and examined using FEI Nova NanoSEM 450 electron micro­
scope (Nova, Netherlands). The sample micrographs at different mag­
nifications were captured using a secondary electron detector at an
accelerating voltage of 15 kV.
Both untreated and pretreated chitin samples were subjected to
Fourier Transform Infrared (FTIR) spectroscopy (PerkinElmer, USA).
The spectra were generated by employing Attenuated Total Reflectance
(ATR) mode. All the chitin samples were scanned from 4000 to 400 cm− 1
to study the alternation in the functional groups, spectra were compared
and analysed through the stack graph prepared using OriginPro (Ori­
ginLab, USA). The DA (%) was determined by using the following
equation (Gbenebor et al., 2017):
DA(%) = A1655 / A3450 × 100 / 1.33 (1)
-1
Where, A1655 is the absorbance of amide at 1655 cm and A3450 is
the absorbance of the hydroxyl group at 3450 cm− 1.
The X-ray powder diffraction of untreated and pretreated chitin was
recorded using X-Ray Diffractometer, PANalytical X’Pert Pro (Malvern
Panalytical, Netherlands). The diffraction pattern was recorded at a scan
rate of 0.2◦ /s between 2θ angles of 5◦ and 30◦ under Cu radiation (40
mA, 40 kV). The X-ray diffractograms were analysed and plotted as a
stacked graph through OriginPro (OriginLab, USA). The crystallinity
index (CI %) was calculated using the following equation (Focher et al.,
1990):
CI (%) = [(I110 − − Iam )/I110 ] × 100 (2)
Where, I110 (arbitrary units) is the maximum intensity of the (1 1 0) peak
at 2θ = 19◦ , and Iam (arbitrary units) is the amorphous diffraction at 2θ
= 12◦ .
The thermogravimetric analysis (TGA) and differential thermal
analysis (DTA) of untreated and pretreated chitin were analysed with
the help of EXSTAR TG/DTA SII 6300 (Hitachi, Japan). The thermo­
grams were obtained by using chitin samples (5 mg) in an alumina pan
by purging nitrogen at 200 ml/min. Alumina powder was used as the
reference for TGA analysis. The DTA measurement was obtained at
35–700 ◦ C at a heating rate of 10 ◦ C/min. The graphical representation
of data was done employing OriginPro (OriginLab, USA).
2.4. Enzymatic hydrolysis of untreated and pretreated chitin
Chitin to COS conversion efficiency of untreated and pretreated
chitin samples was evaluated by using purified T. lanuginous chitinase.
The untreated and pretreated chitin samples (1%, w/v) were mixed with
citrate buffer (100 mM, pH 3.0). Enzyme hydrolysis was carried out for
48 h using chitinase at an enzyme loading of 2.5 U mg− 1 at 70 ◦ C.
3
Bioresource Technology 337 (2021) 125399
Chitinase assay was performed by incubating enzyme and 1% colloidal
chitin with citrate buffer (100 mM, pH 3.0) in 1:1 ratio (v/v) 70℃ for 60
min. The reaction mixture was centrifuged (10,000g, 5 min.) and the
supernatant was analysed for reducing sugar through 3,5-Dinitrosalicy­
clic acid method. Here, chitinase was used in the term of specific activity
and one unit (U) of chitinase activity was calculated as the amount of
enzyme required to release 1 µmol of GlcNAc per min under the standard
assay conditions (Kumar et al., 2018). Small aliquot of samples were
withdrawn using pipette at various time intervals (0, 0.25, 0.5, 1, 2, 4, 6,
8, 10, 12, 24 and 48 h) and the enzymatic reaction was stopped by
boiling (100 ◦ C, 10 min) using a heat block followed by cooling down on
ice and centrifugation (10,000g, 4 ◦ C, 5 min).
2.5. Thin-layer chromatography
The hydrolyzed products were analysed using TLC Silica gel 60
(Merck, Germany). Samples (1 µl) from untreated and pretreated enzy­
matic hydrolysate were spotted on the TLC. The spots were developed in
the solvent system containing n-butanol: methanol: ammonia solution
(25%): water (5:4:2:1, v/v/v/v) in closed jar. The developed spots were
analysed by spraying with aniline-diphenylamine reagent followed by
heating in a hot air oven set at 150 ◦ C (Tempo, India) for 10 min
(Purushotham et al., 2012). The quantitative analysis of spots present in
TLC plates were analysed using JustTLC software (Sweday, Sweden).
2.6. Design of experiments
The derivation of the optimum levels of T. lanuginous chitinase and
chitin (untreated and pretreated) could be achieved through statistical
optimization by two-factor design matrix. The range of TBP pretreated
chitin was kept between 1 and 10% (w/v), while purified T. lanuginosus
chitinase was taken in a range of 1–5 U mg− 1 (Table 1). A two-level 9 run
experiment was conducted and the response in terms of (GlcNAc)2 and
(GlcNAc)3 production was taken from the mean value of hydrolysis
experiment run in triplicates.
2.7. COS purification
The hydrolysis product obtained under optimized conditions con­
tains different DP’s. The hydrolysate was subjected to the extraction step
to remove GlcNAc and other impurities using cation exchange chro­
matography using SP-Sepharose media (Sigma-Aldrich, USA). The col­
umn was equilibrated with citrate buffer (100 mM, pH 3.0) and the
hydrolysis product was then loaded on the top of the column. Elution
buffer contains a varying concentration of sodium chloride (50–500
mM) and the collected fractions were subjected to TLC to determine the
relative concentration of GlcNAc and COS. The fraction containing
(GlcNAc)2 and (GlcNAc)3 were pooled and desalted using Sephadex G-
10 (Sigma-Aldrich, USA). Further, the active fractions were concen­
trated, forzen using deep freezer at − 80 ◦ C and lyophilized using a
FreeZone freeze dryer (Labconco, USA). The COS fractions were ana­
lysed using HPLC (Waters 1500 series, Ireland) using Asahipak NH2P-
50E column (4.6 × 250 mm) (Shodex, China) connected to a RI detector.
The HPLC separation was carried out at room temperature (25 ◦ C) using
acetonitrile water mixture (70:30, v/v) as the mobile phase at a flow rate
of 1.0 ml min− 1. The concentration of (GlcNAc)2 and (GlcNAc)3 was
quantified using a standard curve generated using commercial COS.
Table 1
Experimental variables at different levels in the two-factor design for COS
production.
Variable Component Levels
− 1 0 +1
X1 TBP pretreated chitin (%, w/v) 1.0 5.5 10.0
X2 T. lanuginosus chitinase (U mg− 1) 1.0 3.0 5.0
M. Kumar et al.
3. Results and discussion
3.1. Chitinase production, purification and characterization
Purified chitinase from T. lanuginosus ITCC 8895 was employed for
enzymatic hydrolysis of chitin following pretreatment. The chitinase
production, derivation of optimum nutritional and process conditions,
purification and characterization were performed in the earlier studies
(Kumar et al., 2017; 2018). Briefly, T. lanuginosus was isolated and pu­
rified from the soil sample of Jaisalmer, Rajasthan, India. The strain
showed the maximum level of chitinase activity (120 ± 0.90 U l− 1)
following 72 hr of submerged fermentation. Under derived nutritional
and process conditions the chitinase production was enhanced by 1.8
folds (213.57 ± 6.4 U l− 1).
Further, chitinase (50 kDa) was purified from the culture superna­
tants of T. lanuginosus to homogeneity using ion exchange chromatog­
raphy. The enzyme was purified to 3.8 fold with 17.1% recovery. The
optimal temperature and pH of the purified chitinase were 70℃ and 3.0
respectively. Chitinase showed stability over a wide range of pH (2.0 –
11.0) and temperature (30-90℃). Half life of the purified chitinase was
169.06 min at its optimal temperature. Moreover, detremination of ki­
netic parameters viz. Km, Vmax, kcat/Km and substrate specificity illus­
trated that the purified chitinase exhibited highest activity towards
colloidal chitin as depicted from its lower Km value (0.11 mg ml− 1). The
enzyme activity was notably enhanced by Mn2+ and Co2+ (173 and
144%).
3.2. Impact of pretreatment on the physicochemical properties of chitin
The pretreatment of chitin was performed by employing various
chemicals viz. H2SO4, NaOH and ILs (EMB and TBP) to reduce the
recalcitrance towards enzymatic action. Structural and physico-
chemical characterization (SEM, FTIR, XRD, TGA and DTA) was per­
formed following pretreatment to study the alterations occurred due to
the pretreatment to enhnace the substrate accessibility. The pretreated
chitin appeared more clear and amorphous as compared to the untreated
chitin. The effectiveness of the pretreatment process was illustrated by
decreased crystallinity, an opened structure and functional group
analysis.
3.2.1. SEM measurements
The alterations in the surface morphology of chitin following
chemical pretreatment could be observed by comparing the scanning
electron micrographs of untreated and pretreated chitin samples. The
micrographs were taken at 20,000x magnification and 5 µm resolution
illustrated significant variance in the morphology. The micrograph of
untreated chitin (see supplementary materials) showed an intact and
uniformly pitted crystalline structure with very random pores. The tight
and orderly fashion of the microfibril stacks could be seen on the surface
of the untreated chitin. However, in the case of H2SO4 and NaOH pre­
treated chitin (see supplementary materials), an improved surface
disintegration of the crystalline structure could be observed as compared
to the untreated chitin. Furthermore, the micrographs of EMB and TBP
pretreated chitin (see supplementary materials) revealed significant
disruption of the structure when compared to untreated chitin. The
microfibrils appeared to be arranged in bundles even after pretreatment.
Moreover, enhanced porosity of EMB and TBP pretreated chitin has
resulted into increased surface area to facilitate enzyme accessibility
during hydrolysis.
3.2.2. FTIR spectroscopy
The FTIR spectroscopy of untreated and pretreated chitin are shown
in supplementary materials with characteristic peaks for chitin as re­
ported before (Sivaramakrishna et al., 2020). A similar pattern of ab­
sorption bands was exhibited in both untreated and pretreated chitin
which confirm that chemical pretreatment process did not decompose
4
Bioresource Technology 337 (2021) 125399
chitin. The characteristic peaks of stretching vibrations of amide I, II,
and III were detected at 1626, 1552, and 1310 cm− 1. The bands at 3258
cm− 1 and 3100 cm− 1 in untreated and pretreated chitin indicated the
stretching and bending vibration of aliphatic –OH. The absorption peak
at 2878 cm− 1 represented the C–H stretching of the –CH3 group. The
absorption band at 1626 cm− 1 demonstrates the stretching vibration of
the carbonyl group. The bending vibration of amide II (N–H bending)
from acetamide groups was observed at 1552 cm− 1 and the stretching
vibration of amide III (C–N stretching) from acetamide group appeared
at 1310 cm− 1. Moreover, the peak at 1014 cm− 1 depicted the stretching
vibration for C–O–C of the glucosamine ring in the untreated as well as
pretreated chitin. The characteristic ring stretching band for β-1,4
glycosidic bonds was noticed in the range of 886 cm− 1 and 896 cm− 1
and in the case of pretreated chitin, a reduction in the peak intensity
could be observed. The decreased intensity of peak indicated the
disruption of chitin during pretreatment. (Sivaramakrishna et al., 2020;
Kumari et al., 2015; Dahmane et al., 2014). The DA of untreated and
pretreated chitin are presented in supplementary materials.
3.2.3. XRD
X-ray diffraction (XRD) analysis of untreated and pretreated chitin
was carried out to study the crystallinity and recalcitrance properties.
The crystallinity of chitin is a key attribute for their efficient enzymatic
hydrolysis (Mohan et al., 2018). A comparative x-ray diffractogram of
untreated and pretreated chitin samples are shown in supplementary
materials. The characteristic strong peaks at 2θ values of 9.5◦ , 19.2◦ ,
23.4◦ , and 26.4◦ can be observed in the untreated and the pretreated
chitin samples. The peak at 2θ value = 19.5◦ corresponds to the char­
acteristic plane (1 1 0) of crystalline chitin and a decrease in the in­
tensities of the peak was observed for pretreated chitin, indicating that
the crystallinity of the chitin was decreased following pretreatment,
which is further supported by the CI data obtained by using the equation
(1) (Ibitoye et al., 2018). The CI of untreated chitin was highest (81.0%),
followed by H2SO4, NaOH, TBP, and TBP pretreated chitin, i.e., 80.5%,
79.2%, 78.1%, and 76.6%, respectively (see supplementary materials).
The notable decrease in CI of EMB and TBP pretreated chitin suggested
that the IL pretreatment has made chitin amorphous and facilitate chi­
tinase accessibility and increase the production of COS.
3.2.4. TGA & DTA
The TGA and DTA analysis of untreated and pretreated chitin sam­
ples was performed to study thermal stability following pretreatment.
The degradation of untreated and pretreated chitin started in the range
of 50–100 ◦ C and is recognized by the evaporation of water molecules.
After the removal of water molecules, the decomposition occurred be­
tween 250 and 360 ◦ C that led to the degradation of chitin (see sup­
plementary materials). The temperature at which the sample began to
lose weight (Tonset) for untreated chitin was 285 ◦ C and the completion
of the degradation process occurred at 360 ◦ C. For H2SO4 and NaOH
pretreated chitin, the Tonset and the completion temperature were found
to be 275 and 355 ◦ C, respectively. While, the Tonset and the completion
temperature of EMB and TBP pretreated chitin was observed to be 265
and 350 ◦ C, respectively. The pretreated chitin experienced a slightly
earlier onset temperature (Tonset) when compared to the untreated chitin
due to the decrease in crystallinity after chemical pretreatment. Most of
the weight loss of chitin samples occurred before 360 ◦ C due to pyrolysis
and resulted in char residue of carbonaceous material. Similar pattern of
thermogram for chitin could be observed in the previous studies (Feás
et al., 2020).
Moreover, the DTA thermogram of untreated and pretreated chitin
show less difference (see supplementary materials) with one major sharp
peak for the core thermal decomposition at 360 ◦ C. Comparatively less
intense peaks were observed ~ 550 ◦ C due to the presence of recalcitrant
materials and traces amount of chemicals. A slight shift in the peak of
untreated chitin ~ 360 ◦ C indicated the requirement of high tempera­
ture for the decomposition of untreated chitin as compared to the
M. Kumar et al.
pretreated chitin. Hence, the pretreated chitin appeared to be more
amorphous, less thermostable and amenable for enzymatic hydrolysis.
The appearance of positive peaks in the TGA thermogram depicted the
evolution of heat, i.e., the reaction was exothermic suggesting the
change in the crystalline phase.
3.3. Enzymatic hydrolysis
Different pretreated chitin was subjected to purified T. lanuginosus
chitinase followed by TLC analysis to evaluate the COS conversion ef­
ficiency. TLC analysis revealed different hydrolyzed products GlcNAc,
chitobiose (GlcNAc)2, and chitotriose (GlcNAc)3 in the hydrolysate
(Fig. 1). Relatively high titers of COS (GlcNAc)2 and (GlcNAc)3 were
detected during the initial period of hydrolysis. However, a further in­
crease in hydrolysis time resulted in decrease of COS and increase in
GlcNAc. The production level of GlcNAc, (GlcNAc)2, and (GlcNAc)3 are
presented in Fig. 2.
The highest level of (GlcNAc)2 and (GlcNAc)3 produced after 1 hr of
enzymatic hydrolysis using untreated chitin was 0.35 and 0.33 mg ml− 1
respectively (Fig. 2). The H2SO4 pretreated chitin resulted in a
maximum of 0.76 and 0.43 mg ml− 1 of (GlcNAc)2 and (GlcNAc)3,
respectively (Fig. 2b). Further, NaOH pretreated chitin showed 0.72 mg
ml− 1 of (GlcNAc)2 after 0.5 h enzyme hydrolysis; while the highest level
of (GlcNAc)3, i.e., 0.69 mg ml− 1 was detected at the beginning of
enzymatic hydrolysis (Fig. 2c). The presence of (GlcNAc)2 and
(GlcNAc)3 at the beginning of hydrolysis for H2SO4 and NaOH pre­
treated chitin show the rapid disintegration of chitin. Moreover, EMB
and TBP pretreated chitin illustrated controlled conversion reaction
patterns (Fig. 2d & 2e). With EMB pretreated chitin, the highest level of
Fig. 1. Time-course TLC analysis of GlcNAc, (GlcNAc)2 and (GlcNAc)3 generation after hydrolysis. Here, substrates such as (a) untreated chitin; (b) H2SO4; (c) NaOH;
(d) EMB and (e) TBP pretreated chitin were combined with purified T. lanuginosus chitinase during hydrolysis. The COS formation were monitored at different
interval of time (given in hours).
5
Bioresource Technology 337 (2021) 125399
(GlcNAc)2 and (GlcNAc)3 were achieved after 2 h of the enzymatic hy­
drolysis, i.e., 0.88 and 0.78 mg ml− 1, respectively which is noticeably
higher when compared to the H2SO4 and NaOH pretreated chitin
(Fig. 2d). Similarly, TBP pretreated chitin resulted in 1.10 and 1.07 mg
ml− 1 of (GlcNAc)2 and (GlcNAc)3 (Fig. 2e). As the hydrolysis time pro­
gressed, prolonged COS hydrolysis has been resulted into accumulation
of GlcNAc. The GlcNAc concentration after 48 h of hydrolysis for un­
treated chitin, H2SO4, NaOH, EMB, and TBP pretreated chitin were 0.84,
0.57, 0.90, 0.51, and 1.65 mg ml− 1, respectively.
3.4. Derivation of optimum reaction conditions for enhanced COS
production
COS production could be augmented by deriving the optimum
enzyme and substrate concentration during hydrolysis employing two-
factor statistical optimization. The two-factor design and the corre­
sponding (GlcNAc)2 and (GlcNAc)3 yield are shown in Table 2. The two-
factor-9 run experiment showed significant variation in (GlcNAc)2 and
(GlcNAc)3 production levels i.e. 0.24–1.68 mg ml− 1 and 0.23–1.39 mg
ml− 1, respectively (Table 2). The TLC analysis of the hydrolysate from
the 9 run experiments has been shown in Fig. 3. The highest concen­
tration of (GlcNAc)2 and (GlcNAc)3 was obtained after 2 h of hydrolysis.
1.5 and 1.3 fold increase has been observed in (GlcNAc)2 and (GlcNAc)3
production following two-factor optimization. The observed variations
in the production levels of (GlcNAc)2 and (GlcNAc)3 depicted the sig­
nificance of optimizing enzyme and substrate ratio for the hydrolysis.
The highest level of (GlcNAc)2 and (GlcNAc)3 production (1.7 and 1.4
mg ml− 1) was achieved when the ratio of TBP pretreated chitin (%, w/v)
and chitinase (U mg− 1) from T. lanuginosus was 1:5 (Fig. 4 RUN1). The
M. Kumar et al. Bioresource Technology 337 (2021) 125399

Fig. 2. Time-course analysis of GlcNAc, (GlcNAc)2, and (GlcNAc)3 generation during hydrolysis of chitin. Here, different chitinase hydrolysis product produced using
(a) untreated chitin; (b) H2SO4; (c) NaOH; (d) EMB; and (e) TBP using chitinase from T. lanuginosus are shown.

COS production. However, the significant increment in (GlcNAc)2 and

Table 2
(GlcNAc)3 production could be observed with a comparatively low
Two-factor design matrix and (GlcNAc)2 and (GlcNAc)3 yield after chitinase
solids loading using TBP pretreated chitin. The decrease in levels of
hydrolysis when using TBP pretreated chitin. Here, X1 is TBP pretreated chitin
(GlcNAc)2 and (GlcNAc)3 production at high solids loading of pretreated
(%, w/v) and X2 is T. lanuginosus chitinase (U mg− 1). The (GlcNAc)2 and
(GlcNAc)3 concentrations in mg ml− 1 following hydrolysis (2 h) were taken as chitin suggested that substrate inhibition that could lower the rate of
response. hydrolysis.
Run X1 X2 Response
(GlcNAc)2 (mg ml− 1) (GlcNAc)3 (mg ml− 1) 3.5. COS purification
1 1 5 1.68 1.39
2 10 1 0.24 0.23 The removal of impurities from the hydrolysate was done by
3 1 1 0.38 0.24 empolying cation exchange chromatography. TLC analysis of the
4 10 5 0.40 0.31
different fractions showed that the major portion of impurities and
5 5.5 3 0.45 0.38
6 1 3 0.50 0.40 GlcNAc elute in the NaCl gradient of 50 to 200 mM. The elution of
7 5.5 5 0.41 0.30 (GlcNAc)2 and (GlcNAc)3 were mainly observed in the NaCl gradient of
8 5.5 1 0.31 0.30 250 and 300 mM respectively. Wu et al. (2017) also showed the appli­
9 10 3 0.35 0.29 cability of cation exchange chromatography fro the seperation of COS
from the mixture of various COS. The active fractions for (GlcNAc)2 and
effect of (GlcNAc)2 and (GlcNAc)3 production against the two variables, (GlcNAc)3 production were desalted and subjected to HPLC analysis.
i.e., TBP pretreated chitin (%, w/v) and chitinase (U mg− 1) from The quantification of (GlcNAc)2 and (GlcNAc)3 produced via coupled
T. lanuginosus could be studied through response surface plots (Fig. 5). chemoenzymatic disintegration was performed using HPLC. COS were
The increasing level of chitinase showed significant enhancement in separated using NH2P-50E (4.6 × 250 mm) cation exchange prepartive
column using 70% acetonitrile at a flow rate of 1 ml min− 1. The

6
M. Kumar et al.
Fig. 3. Time-course TLC analysis of GlcNAc, (GlcNAc)2 and (GlcNAc)3 generation from TBP pretreated chitin under different RUN conditions.
retention depends on the hydrophilic interaction between the stationary
phase and its hydroxyl amino group content in COS. The presence of
more hydroxyl and amino groups in COS enhances the hydrogen-
bonding interactions with the stationary phase, which influences the
retention time. The retention time for (GlcNAc)2 and (GlcNAc)3 in the
standard samples and TBP pretreated chitin hydrolysate after 2 h was
found 5.2 and 8.2 min. respectively (see supplementary materials). The
final concentration of (GlcNAc)2 and (GlcNAc)3 achieved was 1.05 and
1.02 mg ml− 1. About 0.1 g of both (GlcNAc)2 and (GlcNAc)3 could be
purified when 1 g of TBP pretreated chitin was hydrolyzed using chiti­
nase with a total COS yield of 20%. In another study, when chitin was
pretreated using 1-ethyl-3-methylimidazolium acetate IL followed by
enzymatic hydrolysis through chitinase from Streptomyces griseues that
resulted in the generation of 0.06 g of (GlcNAc)2 from 1 g of chitin
(Husson et al., 2017). Similarly, Significant production of (GlcNAc)2 has
been reported by Xu et al. (2018) from 1-butyl-3methylimidazolium
acetate pretreated crab shell chitin and double-chitinase (chip1 and
7
Bioresource Technology 337 (2021) 125399
chip2 from Paenibacilus pasadenesis) hydrolysis system. In another study
chitinase from S. albolongus ATCC 27,414 was employed on 1-ethyl-3-
methylimidazolium acetate pretreated chitin and resulted in the gen­
eration of (GlcNAc)2 following 48 hr of hydrolysis (Li et al., 2019).
Additionally, the present study could be considered significant in terms
of additional generation of (GlcNAc)3 besides (GlcNAc)2.
4. Conclusions
A new chemoenzymatic method for production of short chain COS
has been developed using ILs pretreated chitin followed by hydrolysis
using purified chitinase from T. lanuginosus. The highest levels of COS
((GlcNAc)2, 1.10; (GlcNAc)3, 1.07 mg ml− 1)) could be achieved from
TBP pretreated chitin. Further, two-factor optimization and chromato­
graphic seperation resulted in generation of 0.1 g of both (GlcNAc)2 and
(GlcNAc)3 from TBP pretreated chitin (1 g). The study will open up the
way for further studies to enhance the COS yield with simultaneous cost
M. Kumar et al. Bioresource Technology 337 (2021) 125399

Fig. 4. Time-course analysis of GlcNAc, (GlcNAc)2, and (GlcNAc)3 production from TBP pretreated chitin.

reduction to benefit industries that are dependent on these substrates for
development of several agricultural and health products along with
valorization of marine waste.
CRediT authorship contribution statement
Manish Kumar: Data curation, Methodology, Formal analysis,
Writing - original draft. Jogi Madhuprakash: Resources, Writing - re­
view & editing. Venkatesh Balan: Resources, Formal analysis, Writing -

8
M. Kumar et al.
Fig. 5. Response surface plots of (a) (GlcNAc)2 and (b) (GlcNAc)3 production as
a function of chitinase and TBP pretreated chitin. All the value of response are
expressed as the mean values of three independent assays ± stan­
dard deviations.
review & editing. Amit Kumar Singh: Formal analysis, Writing - review
& editing. V. Vivekanand: Formal analysis, Writing - review & editing.
Nidhi Pareek: Conceptulization, Funding acquisition, Supervision,
Project administration, Writing - review & editing.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgments
The authors would like to acknowledge the Department of Science
and Technology (Grant No. DST/INSPIRE/04/2014/002644) and Sci­
ence and Engineering Research Board (Grant No. ECR/2018/002633),
Government of India for financial support. Authors also acknowledged
Material Research Centre, MNIT, Jaipur, India and Institute Instru­
mentation Centre, IIT Roorkee, Roorkee, India for instrumentation
9
Bioresource Technology 337 (2021) 125399
facility. The authors also highly acknowledged the Central University of
Rajasthan, Rajasthan, India for providing the necessary facilities to
complete this work. Dr. Balan thank DOD (Grant No. W911NF2010281)
for partially funding this project and State of Texas for his startup funds.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.biortech.2021.125399.
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