AN INTRODUCTION TO ENZYME

ENGINEERING:DEFINITION,APPLICATIONS IN
INDUSTRY AND RECENT ADVANCEMENTS

A FINAL ESSAY SUBMITTED TO

ASSOC. PROF. DR 王亚军
OF THE SCHOOL OF BIOENGINEERING
AT
ZHEJIANG UNIVERSITY OF TECHNONOLGY

By
BUHLEBENKOSI JACKIE BHEBHE
(L201601020118)

In Partial Fulfillment of the Requirements for the

Degree of Bachelor of Science
in
Bioengineering
HANGZHOU, 2021

NEU
2019
 TABLE OF CONTENTS
ABSTRACT............................................................................................................................................... 1
CHAPTER ONE........................................................................................................................................ 2
INTRODUCTION..................................................................................................................................... 2
1.1. Background................................................................................................................................ 2
1.2 Improvement of enzymes for industrial use through protein engineering....................................3
1.2.1. Improvement of Enzyme Properties by Site-Directed Mutagenesis...............................4
1.2.2 Random mutagenesis to improve enzyme
properties…………………………………………………………………………...………………………………………… 5

1.3 Industrial applications of enzymes ....................................................................................................... 6

1.3.1 Enzymes in technical industries.......................................................................................................... 7
1.3.2 enzymes in food industries................................................................................................................ 8
1.3.3 enzymes in animal feed
industries………………………………………………………………………………………………….9
2.CHAPTERTWO………………………………………………………………………………………10
2.1 RECENT ADVANCEMENTS IN ENZYME ENGINEERING
2.1 Current technologies in enzyme engineering……………………………………………………….10
3CHAPTER THREE…………………………………………………………………………………… 15

1
 CONCLUSION…………………………………………………………………………………………15
REFERENCES…………………………………………………………………………………………16

ABSTRACT
Enzymes are intriguing biological catalysts that have made an immense contribution to
scientific progress. In industry, they play an important part and can be used in many
disciplines. Since society demands are becoming more amplified, the enzymes need to evolve
continuously. In techniques and machine tools, there has been an enormous growth that has
built the industries to meet the rising expectations. Techniques such as protein engineering
help to produce premium products by modifying amino acids in order to make products more
stable and effective. In addition, techniques such as enzyme immobilization provide the
ability to recycle the enzyme used with the same effectiveness, making it a budget
friendly industrial enzyme measure. In order to improve enzyme efficiency and its
characteristics, nanotechnology and CLEA formation are also integrated into enzyme
engineering. In designing efficient biocatalysts for biotechnology, biomedicine, and life
sciences, enzyme engineering features prominently. Data mining techniques have been
increasingly applied to detect patterns in data that better detect protein structures, enhance
enzyme stability, solubility, and function, predict substrate specificity, and guide rational
protein design, in addition to classical rational design and guided development
approaches.This short review will discuss how enzymes are applied in industry and how
enzyme or protein engineering plays a role in developing biocatalysts for usage in industrial
applications.
Key words:enzyme or protein engineering, nanotechnology, enzymes in industrial
application,immobilization.

2
 CHAPTER ONE
INTRODUCTION

1.1. Background

Enzyme engineering is the method of enhancing the efficacy of an enzyme that is already
accessible or of formulating a sophisticated enzyme function by adjusting its sequence of
amino acids. This technique has already produced positive performance in latest technologies
and refinement of enzymes for chemical and pharmaceutical biosynthesis, regenerative
medicine, food processing, biodegradation of waste and biosensing, considering the enormity
of potential transformations. Usually, but not specifically, enzymes are engineered for
catalytic activity, specificity of substrate, enantioselectivity, thermodynamic stability,
cosolvent stability, impressionability and solubility. Enzymes vary from living organisms to
factories that catalyze biochemical reactions in order to sustain the living system or to
synthesize a product or to degrade a material. (Shrivastava et al. 2012, 2013;Kumar et al.
2014, 2016). Enzymes have substituted factories with chemicals that are more
environmentally friendly and help to move towards a green world. Industries are now wholly
reliant on enzymes, and there is ongoing pressure for a variety of microbial enzymes with
new features and stability under severe industrial conditions. (Kumar and Shukla 2015). In
microbiology, biotechnology, and bioinformatics, there is ongoing advancement in providing
microbial enzymes with innovative features for the creation of better industrial processes
(Singh and Shukla 2012, 2015). Industrial applications consist of high-quantity procedures,
such as the transformation of biomass into fermentable sugars, to high-value, low-volume

3
processes, such as medical drug manufacturing and biosensors, for sustainable biofuel
processing. In such processes, the application efficiency of enzymes is greatly improved by
transforming the soluble enzymes into insoluble enzyme substances, which helps boost
stability and recyclability, while material structure, — in other words the reactor and the
process environment, need to be adapted to process application.This essay will provide an
overview of these industrial applications and technical advancements of enzyme engineering
throughout different sections of this essay.

1.2 Improvement of enzymes for industrial use through protein

engineering.

Protein engineering enables protein alteration itself to enhance the properties of industrial
process suitability. Mutation is the best way to enhance enzyme functions and investigating
protein expression in protein engineering. It is a method of altering a protein sequence in
order to achieve a desired result, such as a shift in the specificity of the substrate with
improved stability against pH and temperature extremes and in organic solvents. There are
two types of protein engineering: (1) site-directed mutagenesis or rational design and (2)
random mutagenesis.

1.2.1. Improvement of Enzyme Properties by Site-Directed Mutagenesis. 

As the title indicates, site-directed mutagenesis is a site-specific technique for enzyme

enhancement. It is also the protein method with total understanding of structure and
mechanism of action. The mutation type can differ as necessary including point mutation,
insertion, deletion, and substitution. Single site-directed mutagenesis and multiple site-
directed mutagenesis may be dependant on the number of mutations in a gene. The principal
objective of site-directed mutagenesis is the introduction to the biocatalyst of unique
properties such as improved specificity, stability, activity, solubility, expression, etc. There
are a variety of papers on Industrial Enzyme Improvement. Two examples of studies carried
out on two enzymes to improve their features are given here. A research in which alpha-
galactosidase characteristics were mutated at a specific site reported enhanced structural and
functional details (Xu et al. 2014). The thermostability of the immobilized protease was
improved in another study by adding residues of Cys to the surface of a cysteine-free
thermolysin-like B protease mutant. The site-directed immobilization of protease through a
single thiol group on thiol Sepharose was thus facilitated by stearothermophilus nd (Eijsink et
al. 1995). Rahimi et al.2016 reported that mutation in the nearby active site region promises
enhanced protein function.

4
1.2.2 Random mutagenesis to improve enzyme properties.

The second type of protein engineering is called random mutagenesis. Adopting the unbiased
approach, random mutagenesis imitates the variant creation mechanism of nature. It is
therefore a method of choice for those proteins whose structure has not been inferred because
it requires mutation in a randomized manner (Baweja et al. 2016). It is therefore quite a
simple method to use, but has a tedious screening process because there are often large
variations made. Different physical and chemical methods are used to produce random
mutations, such as chemical agents such as ethyl methane sulfonate (EMS),
methylnitronitrosoguanidine (MNNG) and ethylnitrosourea (ENU), using error-prone PCR
by using less FideliTaq polymerase instead of pfu polymerase; using base analogs, altering
nucleotide concentration; using PCR heavy water, mutator strain, and pfu polymerase
(ITCHY) (Sen et al. 2007; Rasila et al. 2009; Baweja et al. 2015). The error-prone PCR is the
most common technique in random mutagenesis with high success rates. The primary motto
behind random mutagenesis is to characterize the open reading frame (ORF) and to modify
the gene to obtain the desired product(Ramli et al. 2011). There are various computational
tools available that guide the library diversity and design, viz., ConSurf-HSSP GLUE,
PEDEL, DRIVeR, and SCHEMA (Labrou 2010). Different computer tools are available that
direct the diversity and design of the library, viz., ConSurf-HSSP GLUE, PEDEL, DRIVeR,
and SCHEMA (Labrou 2010). Various bioinformatics strategies are available that reduce the
tedious method of library screening. Techniques such as enzyme modeling and docking
prescreen the variants by providing the docking score that assesses their binding enzyme-
substrate relationship and efficacy. Different enzymes such as inulinase and xylanase have
been studied in modeling and docking studies. (Karthik and Shukla 2012; Singh et al. 2016).
The simulation of molecular dynamics helps determine the stability of various proteins in
specific conditions and thus filters out variants during library screening. (Singh et al. 2016).

1.3 Industrial applications of enzymes.

5
Natural enzymes have been used since ancient times in the manufacture of food and beverage
goods, including cheese, sourdough, beer, drinking spirits and vinegar, and in the production
of items such as leather and linen. The use of enzymes has grown over the past few years to
involve other industrial applications and systems spanning chemical-enzymatic synthesis to
the production of new biofuels from sustainable biomass. (Kim & Park, 2013; Wen et al.,
2009). In its present form, the enzyme industry is the product of the exponential growth of
modern biotechnology, namely recombinant DNA, high-throughput screening, and protein
engineering. Recent developments in recombinant gene technology have further optimized
manufacturing processes and allowed adequate amounts of enzymes that were not previously
suitable for production to be commercialized. In addition, recent breakthroughs in high-
performance and protein engineering technologies have revolutionized the development of
tailor-made enzymes that can operate under operational conditions, further broadening the
scope of their industrial application. (Kumar& Singh, 2013; Lutz, 2010).

1.3.1 Enzymes in technical industries.

In various industrial applications and processes, technological enzymes are usually used as
bulk enzymes, including in the detergent, textile and leather, pharmaceutical, cosmetic,
biofuel, and pulp and paper industries. Global technological enzyme sales in 2011 were
estimated at USD 1.2 billion (Prakash et al., 2013) and are forecast to cross USD 1.5 billion
by 2015. (Dewan, 2011). In terms of volume and value, the most relevant sector for applying
technological enzymes is the detergent industry. The use of proteases in detergents as
pancreatic extracts is old and dates back to 1913. However, only in the past five decades has
the use of enzymes in detergent formulations been introduced, particularly after the
availability of microbial enzymes in the 1960s (Maurer, 2004). Enzymes have been used for
removing dirt and challenging stains at washing temperatures in laundry detergents. The
major fraction of the technical enzymes used in detergents are proteases, but other hydrolases
(lipases, amylases and cellulases) are also used to achieve the different results needed. All of

6
the proteases used in the detergent industry are derived from Bacillus species with subtilisins.
A recent breakthrough in this area is the use of psychrophilic enzymes capable of functioning
efficiently at low temperatures, providing major opportunities for environmental and
economic benefits through energy saving (Cavicchioli et al., 2002; Mikhailova et al., 2014).
A increasing industry that has traditionally used a great deal of water, energy and harsh
chemicals is textile manufacturing. White biotechnology is a good example of enzyme use in
the textile industry, allowing environmentally-friendly innovations and low energy usage to
be applied in almost all phases of fiber production. Numerous enzymes, including a-
amylases, cellulases, laccases, peroxidases, lipases and pectate lyases, have been used in this
region since the middle of the last century (Araujo et al., 2008). Cellulases have widely been
used among these enzymes to enhance the look and feel of clothing made from a variety of
cellulosics such as cotton, linen and viscose. Pectate lyases have also been successfully
developed as replacements for traditional chemical processes used in cotton fabric
preparation (Kalantzi et al., 2008). Enzymes, especially proteases, have also been used to
remove collagen-associated proteins and replace toxic chemicals in many processes in the
leather industry, including the processing of skins and hides (Khan, 2013). A crucial factor in
regulating the consistency of final leather, including its longevity and softness, is the degree
of eliminating non-collagenous constituents. The substitution of chemicals with proteases has
also been often reported to offer the only way to limit the pollution caused by the leather
industry. The use of enzymes in the pulp and paper processes has grown rapidly since the mid
1980s (Demuner et al., 2011). Cellulases, xylanases, laccases and lipases are the most
important enzymes used in this industry. While xylanases and laccases have predominantly
been used in the production of pulp, cellulases and lipases have been widely used in the
production of paper. Laccases and xylanases were used in pulp for fiber delignification and
pulp bleach boosting. Manufacture. Similarly, cellulases and lipases, respectively, have found
extensive application for fiber modification and pitch control. The quest for enzymes that are
active at lower temperatures has created increasing interest in the pulp and paper industry for
quality, environmental and economic purposes. (van den Burg, 2003). With the growing
scarcity and cost of fossil fuels, the production of renewable energy sources and modern
approaches to their use are the future of energy supplies.Therefore, increasing attention has
recently been paid to the bioconversion of agricultural sources and abundant lignocellulosic

7
biomass as a feedstock for biofuels and biobased chemicals (Yamada et al., 2013). Because of
their powerful ability to minimize the high cost associated with lignocellulose conversion by
overcoming biomass recalcitrance, lignocellulose-degrading enzymes, including cellulases
and hemicellulases, have earned and increased interest in biofuel production (Himmel et al.,
2007; Wen et al., 2009). In the conversion of starchy materials to glucose that is subsequently
fermented by yeast to produce ethanol, amylases and glucoamylases have also gained
traction. (Kim et al., 2011)

1.3.2 Enzymes in food industries

Biocatalysts have a long history of use in food production. The earliest uses of beer brewing,
bread baking and cheese making go back to 6000 BC (Vasic-Racki,2006). A continuous
search for natural enzymes for application as replacements for traditional synthetic chemicals
used in food processing has recently been triggered by rising public awareness and market for
high and convenient food products. Enzymes are also widely used in a large variety of food
industries, including the bread baking, fruit juice, cheese, starch, wine, fat and oil industries.
The food texture, appearance, and nutritional value of food products can be enhanced by
enzymes and can produce desirable flavors and aromas. Pectinases were used in the 1930s to
clarify juices (Vasic-Racki, 2006). However, before the 1960s, with the use of amylases and
amyloglucosidases as replacements for conventional acid hydrolysis of starch, the widespread
application of food enzymes was not really known. (Fernandes, 2010). A few years later, the
use of glucose isomerase to generate high fructose syrup was also used in the starch
transformation approach. By 2009, the Association of Enzyme Products Manufacturers and
Formulators (AMFEP) had published a list of approximately 200 enzymes developed for use
in the food industry, of which at least 57 were derived from genetically modified
microorganisms. (2009 AMFEP). The toxigenic ability of the manufacturing microbe is the
primary criterion in determining food enzyme safety. In enzyme preparations for food
applications, only a few micro-organisms from a relatively limited number of bacterial and
fungal species, including Aspergillus oryzae, A.niger,Saccharomyces cerevisiae,
Streptomyces rubiginosus, Actinoplanes mussousriensis, Bacillus subtilis, and
B.licheniformis, classified as Commonly Recognized As Safe (GRAS), are used. The

8
nontoxic and nonpathogenic ability of the microorganisms used is one of the most significant
factors determining the safety of enzyme preparations. Characterized microbial strains with a
history of safe use in the production of food enzymes are often regarded as powerful
candidates for producing a safe lineage of strains from which improved strains can be
extracted through genetic modification (Pariza & Johnson, 2001). The authors of this
literature reported that the key elements required for the development of a safe strain lineage
include the systematic characterization of the host organism, the determination of the safety
of all new DNA introduced to it, and the confirmation that the procedure(s) used in the
modification of the host organism are appropriate for food use. Olempska-Beer et al.
(Olempska-Beer et al.2006) published on the engineering of microbial strains used for the
development of food enzymes by reducing or removing toxic secondary metabolites from
their possible production. Koushki et al. (Koushki et al., 2011) published on the genetic
modification of fungal strains to increase the yield of enzyme development, including
protease-deficient and sporulation-impaired fungi.

1.3.3 Enzymes in the animal feed industry

In the late 1980s, the use of enzymes as supplements in animal feed began and has since
developed significantly, particularly in the pig and poultry feed markets (Ravindran &Son,
2011). Global feed enzyme sales were valued at approximately USD 344 million in 2007 and
are forecast to hit USD 727 million in 2015. (Frost & Sullivian, 2011). Actually, for diet
formulations, exogenous enzymes provide various benefits. They enhance dietary component
digestion, which improves feed conversion and, hence, development. They also help to
overcome the adverse effects of anti-nutritional variables. (e.g. NSPs: non-starch
polysaccharides). This decreases the harmful environmental effects by reducing the excretion
of phosphorus and nitrogen and decreasing the discharge of manure. In several nations, cereal
grains (such as barley, rye and wheat) and their by-products are used in animal diets. They
have a high content of insoluble NSPs in these products. The creation of highly advanced

9
enzymes that target NSPs will make these polymers a major source of potential energy and a
promising class of prebiotics in a precise manner (Choct, 2006). The highest portion of
enzymes applied as feed additives is in poultry feed and is accompanied by swine feed.
Aquaculture and mammal feed have recently become common and rising markets for external
feed enzymes (Ravindran & Son, 2011). Those that have the ability to break down NSPs,
proteins, starch, and phytate are the enzymes usually used in animal feed. Thus, b-glucanases,
xylanases,pectinases, a-galactosidases, proteases, a-amylases, and phytases are the usually
commercialized feed enzymes. Phytases have drawn particular interest in animal feed
research and industry (Farhat-Khemakhem et al.2012; Selle & Ravindran, 2008).

They are especially utilised in order to supply phosphate and other phytate-bound nutrients
through the breakdown of phytic acid, a formidable antinutritional factor (Yi et al., 1996), and
their impressive ability to reduce phosphate pollution. Since high temperatures are involved
in the formulation of commercial animal feed, the use of feed enzymes in wide-scale
applications has often been conceived (Katrolia et al., 2013). Therefore, in this constantly
booming market, the search for and production of heat-stable enzymes has been imperative.

CHAPTER TWO
RECENT ADVANCEMENTS IN ENZYME ENGINEERING

2.1Current technologies of enzyme engineering.

Enzyme engineering is increasingly evolving from random (directed evolution) to semi-

rational or rational (data-driven) design approaches. Protein structures and molecular

10
modeling data are assessed in rational design biochemical data to suggest mutations that are
induced by site-specific mutagenesis discussed earlier in chapter one. An increased chance of
advantageous mutations and a substantial decrease in the size of the library is one of the
benefits of a rational design strategy and thus less effort and time has to be used for the
library screening. If no high-throughput assay method is available, this is especially
beneficial.

Semi-rational design incorporates the benefits of rational and random protein design to build
smaller, smarter libraries based on biochemical and/or structural data-based knowledge.
CASTing (combinatorial active site saturation test) is an example of a semi-rational
approach, which uses knowledge derived from, for example, structural data to classify amino
acids in interesting regions (e.g. active sites), which are then mutated randomly or one by one
or in combination by site-saturation mutagenesis. Synergistic effects that may have been
overlooked in single site-specific mutagenesis may result from a random combination of
mutations or associated mutations at targeted positions. These combinatorial approaches,
however, significantly increase the size of the library and different computational methods
have been developed in recent years, which help to reduce the size of the library by screening
virtual libraries and removing mutations that are anticipated to be unfavorable to the protein
fold. For the successful application of enzyme engineering, critical understanding of an
enzyme's structure and function is needed. In order to enhance the catalytic efficiency of
enzymes by altering their molecular composition and active structures, genetic engineering
techniques such as guided evolution, DNA shuffling, site-directed mutagenesis (previously
discussed in an earlier chapter), saturation mutagenesis, fusion and truncation have been used
to improve their functional attributes. Over the years, random screening of microorganisms
and mutagenesis has been commonly used, which significantly affects the properties of an
enzyme, such as thermostability, pH optimization, substrate specificity, inhibition of
feedback, Vmax, Km, and inhibition of carbon source (Adrio and Demain, 2010). Enzyme
immobilization, flow cytometry and peptidomimetics are other methods that assist in
enhancing enzymatic properties. For this review only direct evolution, dna shuffling,
saturation mutagenesis, fusion,enzyme immobilization and truncation will be discussed.

Directed evolution

This includes the repeated rounds of genetic diversification through the generation of a
mutant library through random mutagenesis, PCR vulnerable to error, DNA shuffling and
phased extension processes, and then high-throughput screening and library selection to
classify mutants with the desired characteristics. Driven evolution has become one of the
most effective strategies for any enzyme, metabolic pathway and even the whole organism to
evolve or modify. The key benefit of guided evolution is that no prior knowledge of protein

11
structure, function and various substitutions of amino acids is needed. In order to achieve
modified or completely new metabolic pathways for the creation of new whole-cell
biocatalysts for industrial applications, Guided Evolution has also been combined with
metabolic engineering.

Fusion

By merging the substrate domain and the catalytic domain, many chimeric enzymes were
created.Chimeric enzymes produced by fusion technique.Catalytic performance,
thermostability, product selectivity and specificity of the substrate have improved.
Oligopeptides and functional genes may also be used to generate innovative chimeric
enzymes. For example, the alkaline alpha-amylase enzyme containing the peptide
AEAEAKAKAEAEAKAK was updated by the fusion method and showed improved
operation and stability characteristics. By fusing the carbohydrate binding module (CBM) of
alpha-amylase with the C-terminus of CGTase, Han and colleagues developed a chimeric
cyclodextrin glycosyltransferase (CGTase) enzyme. This alteration led to a 5-fold
improvement of the soluble starch titer of 2-O-D-glucopyranosyl-Lascorbic acid relative to
wild strains.

Truncation
To extract the unwanted domains of proteins that inhibit enzyme function, random or guided
truncation strategies have been used. Enzymes or a truncated library with altered enzyme
properties, were built. For example, on 0.05 percent dextran after truncation, an endo-
dextranase mutant TM-NCG from Streptococcus mutant exhibited 1.4-2-fold increased
hydrolytic activity. A transglutaminase mutant E5D with improved activity and
thermostability was constructed by the use of a combination of truncation and site-directed
mutagenesis.Seven N-terminal residues of Streptomyces hygroscopicus transglutaminase
were deleted one by one to study their effect on enzyme activity. It was observed that the
specific activity of the mutant enzyme (with 4 residue long truncated N-terminus) increased
by 32.92%. The fifth residue in the N-terminus region (E5 or Glutamic acid residue) of the
wild-type enzyme was further substituted by 19 other amino acids. The mutant
transglutaminase having aspartic acid moiety at the fifth position exhibited a 1.85-fold higher
specific activity and 2.7-fold longer half-life at 50°C in comparison to the wild-type enzyme.
Along with this, the melting temperature of the mutant transglutaminase enzyme also
increased from 68.9ºC to 79.1ºC .

Rational design
Rational design is one of the most conventional methods of enzyme engineering in which
site-directed mutagenesis incorporates specific changes in the amino acid sequence. Rational
design is used where the comprehensive overview of the structure, function and mechanism
of proteins is already well understood. First, structural, biochemical and molecular modeling
data are precisely analyzed and then site-directed mutagenesis presents the proposed
mutations. Over the years, to improve desired enzyme properties for commercial applications,
rational design has been very successful. Rational design has also contributed to a deeper
understanding of protein structure, enzyme binding sites, and catalytic mechanisms, making a

12
significant contribution to the engineering of designer enzymes and opening up new views
for functional remodeling and prediction of new protein sequences and their properties.

Enzyme Immobilization
An immobilized enzyme is an enzyme attached to an inert, insoluble material—such as
calcium alginate (produced by reacting a mixture of sodium alginate solution and enzyme
solution with calcium chloride). This can provide increased resistance to changes in
conditions such as pH or temperature. It also lets enzymes be held in place throughout the
reaction, following which they are easily separated from the products and may be used again
- a far more efficient process and so is widely used in industry for enzyme catalysed
reactions. There are various ways by which an enzyme can be immobilized:

Affinity-tag binding: Enzymes may be immobilized to a surface, e.g. in a porous material,

using non-covalent or covalent Protein tags. This technology has been established for protein
purification purposes. This technique is the generally applicable, and can be performed
without prior enzyme purification with a pure preparation as the result. Porous glass and
derivatives thereof are used, where the porous surface can be adapted in terms of
hydrophobicity to suit the enzyme in question.
Adsorption on glass, alginate beads or matrix: Enzyme is attached to the outside of an inert
material. In general, this method is the slowest among those listed here. As adsorption is not a
chemical reaction, the active site of the immobilized enzyme may be blocked by the matrix or
bead, greatly reducing the activity of the enzyme.
Entrapment: The enzyme is trapped in insoluble beads or microspheres, such as calcium
alginate beads. However, these insoluble substances hinder the arrival of the substrate, and
the exit of products.
Cross-linkage: Enzyme molecules are covalently bonded to each other to create a matrix
consisting of almost only enzyme. The reaction ensures that the binding site does not cover
the enzyme's active site, the activity of the enzyme is only affected by immobility. However,
the inflexibility of the covalent bonds precludes the self-healing properties exhibited by
chemoadsorbed self-assembled monolayers. Use of a spacer molecule like poly(ethylene
glycol) helps reduce the steric hindrance by the substrate in this case.
Covalent bond: The enzyme is bound covalentely to an insoluble support (such as silica gel
or macroporous polymer beads with epoxide groups). This approach provides the strongest
enzyme/support interaction, and so the lowest protein leakage during catalysis.

13
 CHAPTER THREE
CONCLUSION

Enzymes for different industrial processes, along with the technical, food, beverage and
animal feed industries, are economic and ecologically desirable biocatalysts. World
consumption for enzymes is, therefore, projected to increase in the coming years. The use of
natural biocatalysts, however, has also been hindered in many cases by their low stability,
lack of specificity,and low catalytic efficiency under operational industrial conditions. Efforts
have been primarily devoted to engineering enzymes at gene and protein levels in order to

14
resolve these inadequacies and meet the increasing demands for strong, high turnover, and
inexpensive enzymes, with the goal of developing industrial biocatalysts with enhanced or
new catalytic functions. Due to major developments in structural biology, screening
protocols, and bioinformatic methods, substantial progress in enzyme engineering has been
achieved over the last few years. In designing effective strategies for the production of
modified or engineered enzymes, enzyme engineering has a huge effect. It has also assisted to
recognize new and improved metabolic pathways, opening up new perspectives for a range of
diverse products to be developed. Competent enzyme engineering will result  in the catalysis
of innovative non-physiological reactions, the creation of advanced and efficient pathways,
the optimization of ideal processes and the creation of new metabolites. This review has
aimed to discuss what enzyme engineering is, its applications in industry and current
advancements. Due to the dynamic, complex structure and wide occurrence of enzymes in
nature, new technologies are constantly being developed in order to learn the structure more
and enhance enzyme qualities for continued use. In coming years, enzyme engineering is
expected to rise to the occasion to fulfil the increasing demand.

15
 REFERENCES

Steiner, K., & Schwab, H. (2012). Recent advances in rational approaches for enzyme
engineering. Computational and structural biotechnology journal, 2(3), e201209010.
Liese, A., Seelbach, K., & Wandrey, C. (Eds.). (2006). Industrial biotransformations. John
Wiley & Sons.
Kumar, V., Baweja, M., Liu, H., & Shukla, P. (2017). Microbial enzyme engineering:
applications and perspectives. In Recent Advances in Applied Microbiology (pp. 259-273).
Springer, Singapore.
Bernal, C., Rodriguez, K., & Martinez, R. (2018). Integrating enzyme immobilization and
protein engineering: an alternative path for the development of novel and improved industrial
biocatalysts. Biotechnology advances, 36(5), 1470-1480.
Rehm, F. B., Chen, S., & Rehm, B. H. (2016). Enzyme engineering for in situ
immobilization. Molecules, 21(10), 1370.
Jemli, S., Ayadi-Zouari, D., Hlima, H. B., & Bejar, S. (2016). Biocatalysts: application and
engineering for industrial purposes. Critical Reviews in Biotechnology, 36(2), 246-258.
Sharma, A., Gupta, G., Ahmad, T., Mansoor, S., & Kaur, B. (2019). Enzyme Engineering:
Current trends and future perspectives. Food Reviews International, 1-34.
Gupta, G. K., & Shukla, P. (2020). Enzyme Engineering Techniques for Biotechnological
Applications. In Microbial Enzymes and Biotechniques (pp. 235-249). Springer, Singapore.
Kirk, O., Borchert, T. V., & Fuglsang, C. C. (2002). Industrial enzyme applications. Current
opinion in biotechnology, 13(4), 345-351.
Basheer, S. M., & Chellappan, S. (2017). Enzyme engineering. In Bioresources and
bioprocess in biotechnology (pp. 151-168). Springer, Singapore.
Wingard, L. B. (1972). Enzyme engineering. In Advances in Biochemical Engineering,
Volume 2 (pp. 1-48). Springer, Berlin, Heidelberg.
van Beilen, J. B., & Li, Z. (2002). Enzyme technology: an overview. Current Opinion in
biotechnology, 13(4), 338-344.
Toogood, H. S., & Scrutton, N. S. (2013). Enzyme engineering toolbox–a ‘catalyst’for
change. Catalysis Science & Technology, 3(9), 2182-2194.
Kragl, U., Kruse, W., Hummel, W., & Wandrey, C. (1996). Enzyme engineering aspects of
biocatalysis: cofactor regeneration as example. Biotechnology and bioengineering, 52(2),
309-319.
Chen, R. (2001). Enzyme engineering: rational redesign versus directed evolution. Trends in
biotechnology, 19(1), 13-14.
Won, Y., Pagar, A. D., Patil, M. D., Dawson, P. E., & Yun, H. (2019). Recent advances in
enzyme engineering through incorporation of unnatural amino acids. Biotechnology and
Bioprocess Engineering, 1-13.
Dubey, K. K., Pramanik, A., & Yadav, J. (2019). Enzyme Engineering. In Advances in
Enzyme Technology (pp. 325-347). Elsevier.
Deng, C., Huang, T., Jiang, Z., Lv, X., Liu, L., Chen, J., & Du, G. (2019). Enzyme
engineering and industrial bioprocess. In Current developments in biotechnology and
bioengineering (pp. 165-188). Elsevier.
Zhang, L. (2020). Protein Co-Assembly and Its Application in Enzyme Engineering.
Choi, J. M., Han, S. S., & Kim, H. S. (2015). Industrial applications of enzyme biocatalysis:

16
Current status and future aspects. Biotechnology advances, 33(7), 1443-1454.
Chowdhury, R., & Maranas, C. D. (2020). From directed evolution to computational enzyme
engineering—A review. AIChE Journal, 66(3), e16847.
Mazurenko, S., Prokop, Z., & Damborsky, J. (2019). Machine learning in enzyme
engineering. ACS Catalysis, 10(2), 1210-1223.
Agyei, D., Shanbhag, B. K., & He, L. (2015). Enzyme engineering (immobilization) for food
applications. In Improving and tailoring enzymes for food quality and functionality (pp. 213-
235). Woodhead Publishing.
Ali, M., Ishqi, H. M., & Husain, Q. (2020). Enzyme engineering: Reshaping the biocatalytic
functions. Biotechnology and Bioengineering, 117(6), 1877-1894.
Mandeep, G. K. G., & Shukla, P. (2020). Enzyme Engineering Techniques for
Biotechnological Applications. Microbial Enzymes and Biotechniques: Interdisciplinary
Perspectives, 235.
Rastetter, W. H. (1983). Enzyme engineering: applications and promise. Trends in
Biotechnology, 1(3), 80-84.
Davenport, A. (2019). Enzyme engineering for industrial applications at BASF.
Pickersgill, R. W., & Goodenough, P. W. (1991). Enzyme engineering. Trends in Food
Science & Technology, 2, 122-126.

17