CHAPTER ONE

1.0 INTRODUCTION

Enzymes are the set of biomolecules required for both syntheses as well as breakdown reactions

by living organisms (Choi et al., 2018). These are the active proteins (except RNAse) that can

catalyze the biochemical reaction. Like any other catalyst, enzyme brings the reaction catalyzed

to its equilibrium more quickly than would occur otherwise, which means they speed up the

reaction by lowering the activation energy (Choi et al., 2018). These are natural chemicals, made

and used by living organisms though they themselves are nonliving. The importance of enzymes

was recognized by the mankind thousands of years ago, clarification and filtration of wines and

beer being the earliest examples of application of industrial enzymes (Choi et al., 2018). Some

enzymes have been designed by the nature to form complex molecules from simpler ones while

others have been designed for breaking up the complex molecules into simpler ones; also few are

present for modifying the molecules. These reactions involve making and breaking of the

chemical bonds existing in the components (Madhavan et al., 2017). Owing to their

“specificity,” a property of enzyme that allows it to recognize a particular chemical compound or

substrate that they are designed to target, they are useful for industrial processes and are capable

of catalyzing the reaction between particular chemicals even though present in mixtures with

many chemicals (Madhavan et al., 2017). These enzymes are environmentally safe, natural and

are applied very safely in food and even pharmaceutical industries. Still, enzymes are proteins,

which like any protein can cause and have caused in the past allergic reactions, hence, protective

measures are necessary in their production and applications (Madhavan et al., 2017).

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1.1 BACKGROUND OF STUDY

By the late 17th and early 18th centuries, the digestion of meat by stomach secretions and the

conversion of starch to sugars by plant extracts and saliva were known but the mechanisms by

which these occurred had not been identified (Williams, 1904).

French chemist Anselme Payen was the first to discover an enzyme, diastase, in 1833 (Payen,

1833). A few decades later, when studying the fermentation of sugar to alcohol by yeast, Louis

Pasteur concluded that this fermentation was caused by a vital force contained within the yeast

cells called "ferments", which were thought to function only within living organisms. He wrote

that "alcoholic fermentation is an act correlated with the life and organization of the yeast cells,

not with the death or putrefaction of the cells" (Manchester, 1995).

Eduard Buchner submitted his first paper on the study of yeast extracts in 1897. In a series of

experiments at the University of Berlin, he found that sugar was fermented by yeast extracts even

when there were no living yeast cells in the mixture. He named the enzyme that brought about

the fermentation of sucrose "zymase". In 1907, he received the Nobel Prize in Chemistry for "his

discovery of cell-free fermentation". Following Buchner's example, enzymes are usually named

according to the reaction they carry out: the suffix -ase is combined with the name of

the substrate (e.g., lactase is the enzyme that cleaves lactose) or to the type of reaction

(e.g., DNA polymerase forms DNA polymers) (Manchester, 1995).

The biochemical identity of enzymes was still unknown in the early 1900s. Many scientists

observed that enzymatic activity was associated with proteins, but others (such as Nobel

laureate Richard Willstätter) argued that proteins were merely carriers for the true enzymes and

that proteins per se were incapable of catalysis. In 1926, James B. Sumner showed that the

enzyme urease was a pure protein and crystallized it; he did likewise for the enzyme catalase in

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1937. The conclusion that pure proteins can be enzymes was definitively demonstrated by John

Howard Northrop and Wendell Meredith Stanley, who worked on the digestive

enzymes pepsin (1930), trypsin and chymotrypsin. These three scientists were awarded the 1946

Nobel Prize in Chemistry.

The discovery that enzymes could be crystallized eventually allowed their structures to be solved

by x-ray crystallography. This was first done for lysozyme, an enzyme found in tears, saliva

and egg whites that digests the coating of some bacteria; the structure was solved by a group led

by David Chilton Phillips and published in 1965. This high-resolution structure of lysozyme

marked the beginning of the field of structural biology and the effort to understand how enzymes

work at an atomic level of detail (Johnson and Petsko, 1999).

1.3 Significance of Study

The enzyme industry is in continuous search for sustainable processes that enable higher yields

with improved efficiency and dynamic nature. From creating lactose-free dairy products to fast-

acting laundry detergents, innovation is the key in engineering improved, cost-effective end-

products for the textiles, foods, detergents, animals, biofuels, and more.

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 CHAPTER TWO

2.0 LITERATURE REVIEW

2.1 Enzymes

Enzymes are proteins that act as biological catalysts by accelerating chemical reactions. The

molecules upon which enzymes may act are called substrates, and the enzyme converts the

substrates into different molecules known as products. Almost all metabolic processes in

the cell need enzyme catalysis in order to occur at rates fast enough to sustain life (Murphy et

al., 2017). Metabolic pathways depend upon enzymes to catalyze individual steps. The study of

enzymes is called enzymology and the field of pseudoenzyme analysis recognizes that during

evolution, some enzymes have lost the ability to carry out biological catalysis, which is often

reflected in their amino acid sequences and unusual 'pseudocatalytic' properties (Murphy et al.,

2017).

Enzymes are known to catalyze more than 5,000 biochemical reaction types. Other biocatalysts

are catalytic RNA molecules, called ribozymes. Enzymes' specificity comes from their

unique three-dimensional structures (Murphy et al., 2017). .

Like all catalysts, enzymes increase the reaction rate by lowering its activation energy. Some

enzymes can make their conversion of substrate to product occur many millions of times faster.

An extreme example is orotidine 5'-phosphate decarboxylase, which allows a reaction that would

4
otherwise take millions of years to occur in milliseconds (Murphy et al., 2017).  Chemically,

enzymes are like any catalyst and are not consumed in chemical reactions, nor do they alter

the equilibrium of a reaction. Enzymes differ from most other catalysts by being much more

specific. Enzyme activity can be affected by other molecules: inhibitors are molecules that

decrease enzyme activity, and activators are molecules that increase activity. Many

therapeutic drugs and poisons are enzyme inhibitors. An enzyme's activity decreases markedly

outside its optimal temperature and pH, and many enzymes are (permanently) denatured when

exposed to excessive heat, losing their structure and catalytic properties. Some enzymes are used

commercially, for example, in the synthesis of antibiotics. Some household products use

enzymes to speed up chemical reactions: enzymes in biological washing powders break down

protein, starch or fat stains on clothes, and enzymes in meat tenderizer break down proteins into

smaller molecules, making the meat easier to chew (Murphy et al., 2017). .

2.2 Classification of Enzymes

2.2.1 Transferase

A transferase is any one of a class of enzymes that catalyse the transfer of specific functional

groups (e.g. a methyl or glycosyl group) from one molecule (called the donor) to another (called

the acceptor). They are involved in hundreds of different biochemical pathways throughout

biology, and are integral to some of life's most important processes (Herbst et al., 2020).

Transferases are involved in myriad reactions in the cell. Three examples of these reactions are

the activity of coenzyme A (CoA) transferase, which transfers thiol esters, the action of N-

acetyltransferase, which is part of the pathway that metabolizes tryptophan, and the regulation

of pyruvate dehydrogenase (PDH), which converts pyruvate to acetyl CoA (Herbst et al.,

5
2020). Transferases are also utilized during translation. In this case, an amino acid chain is the

functional group transferred by a peptidyl transferase. The transfer involves the removal of the

growing amino acid chain from the tRNA molecule in the A-site of the ribosome and its

subsequent addition to the amino acid attached to the tRNA in the P-site (Sun et al., 2017).

Transferase deficiencies are at the root of many common illnesses. The most common result of a

transferase deficiency is a buildup of a cellular product (Sun et al., 2017).

2.2.1.1 Uses of Transferase in biotechnology

2.2.1.1.1 Terminal transferases

Terminal transferases are transferases that can be used to label DNA or to produce plasmid

vectors. It accomplishes both of these tasks by adding deoxynucleotides in the form of a template

to the downstream end or 3' end of an existing DNA molecule. Terminal transferase is one of the

few DNA polymerases that can function without an RNA primer (Patel, 2017).

2.2.1.1.2 Glutathione transferases

The family of glutathione transferases (GST) is extremely diverse, and therefore can be used for

a number of biotechnological purposes. Plants use glutathione transferases as a means to

segregate toxic metals from the rest of the cell (Patel, 2017). These glutathione transferases can

be used to create biosensors to detect contaminants such as herbicides and insecticides.

 Glutathione transferases are also used in transgenic plants to increase resistance to both biotic
[103]

and abiotic stress.[103] Glutathione transferases are currently being explored as targets for anti-

cancer medications due to their role in drug resistance. Further, glutathione transferase genes

6
have been investigated due to their ability to prevent oxidative damage and have shown

improved resistance in transgenic cultigens (Patel, 2017).

2.2.1.1.3 Rubber transferases

Currently the only available commercial source of natural rubber is the Hevea plant (Hevea

brasiliensis). Natural rubber is superior to synthetic rubber in a number of commercial uses

(Patel, 2017). Efforts are being made to produce transgenic plants capable of synthesizing natural

rubber, including tobacco and sunflower. These efforts are focused on sequencing the subunits of

the rubber transferase enzyme complex in order to transfect these genes into other plants (Patel,

2017).

2.2.1.1.4 Membrane-associated transferases

Many transferases associate with biological membranes as peripheral membrane proteins or

anchored to membranes through a single transmembrane helix, for example

numerous glycosyltransferases in Golgi apparatus. Some others are multi-span transmembrane

proteins, for example certain oligosaccharyltransferases or microsomal glutathione S-

transferase from MAPEG family (Patel, 2017).

2.2.2 Oxidoreductase

Oxidoreductase is an enzyme that catalyzes the transfer of electrons from one molecule,

the reductant, also called the electron donor, to another, the oxidant, also called the electron

acceptor (Kapoor et al., 2017). This group of enzymes usually

utilizes NADP+ or NAD+ as cofactors (Kapoor et al., 2017). Transmembrane oxidoreductases

7
create electron transport chains in bacteria, chloroplasts and mitochondria, including respiratory

complexes I, II and III. Some others can associate with biological membranes as peripheral

membrane proteins or be anchored to the membranes through a single transmembrane helix

(Kapoor et al., 2017).

2.2.3 Hydrolase

Hydrolase is a class of enzyme that commonly perform as biochemical catalysts that use water to

break a chemical bond, which typically results in dividing a larger molecule into smaller

molecules. Some common examples of hydrolase enzymes

are esterases including lipases, phosphatases, glycosidases, peptidases, and nucleosidases

(Kapoor et al., 2017). Esterases cleave ester bonds in lipids and phosphatases cleave phosphate

groups off molecules. An example of crucial esterase is acetylcholine esterase, which assists in

transforming the neuron impulse into the acetate group after the hydrolase breaks

the acetylcholine into choline and acetic acid (Kapoor et al., 2017). Acetic acid is an important

metabolite in the body and a critical intermediate for other reactions such as glycolysis. Lipases

hydrolyze glycerides. Glycosidases cleave sugar molecules off carbohydrates and peptidases

hydrolyze peptide bonds. Nucleosidases hydrolyze the bonds of nucleotides (Pellis et al., 2018).

Hydrolase enzymes are important for the body because they have degradative properties. In

lipids, lipases contribute to the breakdown of fats and lipoproteins and other larger molecules

into smaller molecules like fatty acids and glycerol. Fatty acids and other small molecules are

used for synthesis and as a source of energy (Pellis et al., 2018).

2.2.4 Lyase

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Lyase is an enzyme that catalyzes the breaking (an elimination reaction) of various chemical

bonds by means other than hydrolysis (a substitution reaction) and oxidation, often forming a

new double bond or a new ring structure. Lyases differ from other enzymes in that they require

only one substrate for the reaction in one direction, but two substrates for the reverse reaction

(Pellis et al., 2018).

2.2.5 Isomerases

Isomerases are a general class of enzymes that convert a molecule from one isomer to another.

Isomerases facilitate intramolecular rearrangements in which bonds are broken and formed. The

general form of such a reaction is as follows: A–B → B–A (Pellis et al., 2018).

There is only one substrate yielding one product. This product has the same molecular formula as

the substrate but differs in bond connectivity or spatial arrangement. Isomerases catalyze

reactions across many biological processes, such as in glycolysis and carbohydrate metabolism

(Pellis et al., 2018).

2.2.6 Ligase

A ligase is an enzyme that can catalyze the joining (ligation) of two large molecules by forming

a new chemical bond. This is typically via hydrolysis of a small pendant chemical group on one

of the larger molecules or the enzyme catalyzing the linking together of two compounds, e.g.,

enzymes that catalyze joining of C-O, C-S, C-N, etc (Pellis et al., 2018). 

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2.3 Industrial Enzymes

Industrial enzymes are enzymes that are commercially used in a variety of industries such

as pharmaceuticals, chemical production, biofuels, food & beverage, and consumer products.

Due to advancements in recent years, biocatalysis through isolated enzymes is considered more

economical than use of whole cells (Fei et al., 2018). Enzymes may be used as a unit

operation within a process to generate a desired product, or may be the product of interest.

Industrial biological catalysis through enzymes has experienced rapid growth in recent years due

to their ability to operate at mild conditions, and exceptional chiral and positional specificity,

things that traditional chemical processes lack (Fei et al., 2018). Isolated enzymes are typically

used in hydrolytic and isomerization reactions. Whole cells are typically used when a reaction

requires a co-factor. Although co-factors may be generated in vitro, it is typically more cost-

effective to use metabolically active cells (Fei et al., 2018).

2.3.1 Amylase

An amylase is an enzyme that catalyses the hydrolysis of starch  into sugars. Amylase is present

in the saliva of humans and some other mammals, where it begins the chemical process

of digestion (Pajic et al., 2019). Foods that contain large amounts of starch but little sugar, such

as rice and potatoes, may acquire a slightly sweet taste as they are chewed because amylase

degrades some of their starch into sugar. The pancreas and salivary gland make amylase (alpha

amylase) to hydrolyse dietary starch into disaccharides and trisaccharides which are converted by

other enzymes to glucose to supply the body with energy. Plants and some bacteria also produce

amylase. Specific amylase proteins are designated by different Greek letters. All amylases

are glycoside hydrolases and act on α-1,4-glycosidic bonds (Pajic et al., 2019).

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2.3.1.1 α-Amylase

The α-amylases (EC 3.2.1.1 ) (CAS 9014-71-5) (alternative names: 1,4-α-D-glucan

glucanohydrolase; glycogenase) are calcium metalloenzymes. By acting at random locations

along the starch chain, α-amylase breaks down long-chain saccharides, ultimately yielding

either maltotriose and maltose from amylose, or maltose, glucose and "limit

dextrin" from amylopectin. They belong to glycoside hydrolase family 13 (Pajic et al., 2019).

Because it can act anywhere on the substrate, α-amylase tends to be faster-acting than β-amylase.

In animals, it is a major digestive enzyme, and its optimum pH is 6.7–7.0 (Pajic et al., 2019).

In human physiology, both the salivary and pancreatic amylases are α-amylases. The α-amylase

form is also found in plants, fungi (ascomycetes and basidiomycetes) and bacteria (Bacillus)

(Pajic et al., 2019).

2.3.1.2 β-Amylase

Another form of amylase, β-amylase (EC 3.2.1.2 ) (alternative names: 1,4-α-D-glucan

maltohydrolase; glycogenase; saccharogen amylase) is also synthesized by bacteria, fungi,

and plants. Working from the non-reducing end, β-amylase catalyzes the hydrolysis of the

second α-1,4 glycosidic bond, cleaving off two glucose units (maltose) at a time. During

the ripening of fruit, β-amylase breaks starch into maltose, resulting in the sweet flavor of ripe

fruit. They belong to glycoside hydrolase family 14 (Arendt et al., 2018).

Both α-amylase and β-amylase are present in seeds; β-amylase is present in an inactive form

prior to germination, whereas α-amylase and proteases appear once germination has begun.

Many microbes also produce amylase to degrade extracellular starches. Animal tissues do not

11
contain β-amylase, although it may be present in microorganisms contained within the digestive

tract. The optimum pH for β-amylase is 4.0–5.0 (Arendt et al., 2018).

2.3.1.3 γ-Amylase

γ-Amylase (EC 3.2.1.3 ) (alternative names: Glucan 1,4-a-glucosidase; amyloglucosidase; exo-

1,4-α-glucosidase; glucoamylase; lysosomal α-glucosidase; 1,4-α-D-glucan glucohydrolase) will

cleave α(1–6) glycosidic linkages, as well as the last α-1,4 glycosidic bond at the nonreducing

end of amylose and amylopectin, yielding glucose. The γ-amylase has the most acidic optimum

pH of all amylases because it is most active around pH 3 (Arendt et al., 2018). They belong to a

variety of different GH families, such as glycoside hydrolase family 15 in fungi, glycoside

hydrolase family 31 of human MGAM, and glycoside hydrolase family 97 of bacterial forms

(Arendt et al., 2018).

2.3.1.4 Uses of Amylases

2.3.1.4.1 Fermentation

α- and β-amylases are important in brewing beer and liquor made from sugars derived

from starch. In fermentation, yeast ingests sugars and excretes ethanol. In beer and some liquors,

the sugars present at the beginning of fermentation have been produced by "mashing" grains or

other starch sources (such as potatoes) (Arendt et al., 2018). In traditional beer brewing, malted

barley is mixed with hot water to create a "mash", which is held at a given temperature to allow

the amylases in the malted grain to convert the barley's starch into sugars. Different temperatures

optimize the activity of alpha or beta amylase, resulting in different mixtures of fermentable and

unfermentable sugars. In selecting mash temperature and grain-to-water ratio, a brewer can

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change the alcohol content, mouthfeel, aroma, and flavor of the finished beer (Arendt et al.,

2018).

2.3.1.4.2 Flour additive

Amylases are used in breadmaking and to break down complex sugars, such as starch (found

in flour), into simple sugars. Yeast then feeds on these simple sugars and converts it into the

waste products of ethanol and carbon dioxide (Arendt et al., 2018). This imparts flavour and

causes the bread to rise. While amylases are found naturally in yeast cells, it takes time for the

yeast to produce enough of these enzymes to break down significant quantities of starch in the

bread. This is the reason for long fermented doughs such as sourdough. Modern breadmaking

techniques have included amylases (often in the form of malted barley) into bread improver,

thereby making the process faster and more practical for commercial use (Arendt et al., 2018).

α-Amylase is often listed as an ingredient on commercially package-milled flour. Bakers with

long exposure to amylase-enriched flour are at risk of developing dermatitis or asthma (Arendt et

al., 2018).

2.3.2 Cellulase

Cellulases break down the cellulose molecule into monosaccharides such as β-glucose, or shorter

polysaccharides and oligosaccharides. Cellulose breakdown is of considerable economic

importance, because it makes a major constituent of plants available for consumption and use in

chemical reactions. The specific reaction involved is the hydrolysis of the 1,4-β-D-glycosidic

linkages in cellulose, hemicellulose, lichenin, and cereal β-D-glucans. Because cellulose

13
molecules bind strongly to each other, cellulolysis is relatively difficult compared to the

breakdown of other polysaccharides such as starch (Guerriero et al., 2018).

Most mammals have only very limited ability to digest dietary fibres like cellulose by

themselves. In many herbivorous animals such as ruminants like cattle and sheep and hindgut

fermenters like horses, cellulases are produced by symbiotic bacteria. Endogenous cellulases are

produced by a few types of metazoan animals, such as some termites, snails and earthworms

(Guerriero et al., 2018).

Recently, cellulases have also been found in green microalgae (Chlamydomonas

reinhardtii, Gonium pectorale and Volvox carteri) and their catalytic domains (CD) belonging

to GH9 Family show highest sequence homology to metazoan endogenous cellulases (Payne et

al., 2020). Algal cellulases are modular, consisting of putative novel cysteine-rich carbohydrate-

binding modules (CBMs), proline/serine-(PS) rich linkers in addition to putative Ig-like and

unknown domains in some members. Cellulase from Gonium pectorale consisted of two CDs

separated by linkers and with a C-terminal CBM (Payne et al., 2020).

2.3.3. Lipase 

Lipase are a family of enzymes that catalyzes the hydrolysis of fats. Some lipases display broad

substrate scope including esters of cholesterol, phospholipids, and of lipid-soluble vitamins [1]

 and sphingomyelinases; however, these are usually treated separately from "conventional"

[2]

lipases. Unlike esterases, which function in water, lipases "are activated only when adsorbed to

an oil–water interface" (Diaz and Arm, 2018). Lipases perform essential roles in digestion,

transport and processing of dietary lipids in most, if not all, organisms (Diaz and Arm, 2018).

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Lipases are serine hydrolases, i.e. they function by transesterification generating an acyl serine

intermediate. Most lipases act at a specific position on the glycerol backbone of a

lipid substrate (A1, A2 or A3). For example, human pancreatic

lipase (HPL), converts triglyceride substrates found in ingested oils to monoglycerides and

two fatty acids (Diaz and Arm, 2018).

A diverse array of genetically distinct lipase enzymes are found in nature, and they represent

several types of protein folds and catalytic mechanisms. However, most are built on

an alpha/beta hydrolase fold and employ a chymotrypsin-like hydrolysis mechanism using

a catalytic triad consisting of a serine nucleophile, a histidine base, and an acid residue,

usually aspartic acid (Diaz and Arm, 2018).

2.3.3.1 Uses of Lipases

In the commercial sphere, lipases are widely used in laundry detergents. Several thousand tons

per year a produced for this role (Diaz and Arm, 2018).

Lipases are catalysts for hydrolysis of esters and are useful outside of the cell, a testament to

their wide substrate scope and ruggedness. The ester hydrolysis activity of lipases has been well

evaluated for the conversion of triglycerides into biofuels or their precursors (Cao et al., 2018).

Lipases are of course chiral, which means that they can be used for the enantioselective

hydrolysis prochiral diesters. Several procedures have been reported for applications in the

synthesis of fine chemicals (Cao et al., 2018). Lipases are generally animal sourced, but can also

be sourced microbially (Cao et al., 2018).

2.3.4 Xylose isomerase

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Xylose isomerase (EC 5.3.1.5) is an enzyme that catalyzes the interconversion of D-

xylose and D-xylulose (Mehta et al., 2018). This enzyme belongs to the family of isomerases,

specifically those intramolecular oxidoreductases interconverting aldoses and ketoses. The

isomerase has now been observed in nearly a hundred species of bacteria. Xylose-isomerases are

also commonly called fructose-isomerases due to their ability to interconvert glucose and

fructose. The systematic name of this enzyme class is D-xylose aldose-ketose-isomerase. Other

names in common use include D-xylose isomerase, D-xylose ketoisomerase, and D-xylose ketol-

isomerase (Mehta et al., 2018). This enzyme participates in pentose and glucuronate

interconversions and fructose and mannose metabolism. The most bio-available sugars according

to the International Society of Rare Sugars are: glucose, galactose, mannose, fructose, xylose,

ribose, and L-arabinose. Twenty hexoses and nine pentoses, including xylulose, were considered

to be "rare sugars". Hence D-xylose isomerase is used to produce these rare sugars which have

very important applications in biology despite their low abundance (Cipolatti et al., 2019).

2.3.5 Pectinase

Pectinases are a heterogeneous group of related enzymes that hydrolyze the pectin substances,

present mostly in plants. Pectic enzymes are widely distributed in nature and are produced by

bacteria, yeast, fungi and plants (Babu and Bayer 2017). In plants, pectic enzymes are very

important since they play a role in elongation and cellular growth as well as in fruit ripening.

Pectolytic activity of microorganisms plays a significant role, firstly, in the pathogenesis of

plants since these enzymes are the first to attack the tissue. In addition, they are also involved in

the process of symbiosis and the decay of vegetable residues. Thus by breaking down pectin

polymer for nutritional purposes, microbial pectolytic enzymes play an important role in nature

16
(Yadav et al., 2019). These enzymes are inducible, produced only when needed and they

contribute to the natural carbon cycle.

2.4 Industrial Enzyme Production technology

2.4.1 Submerged Fermentation

Fermentation done in the presence of excess of free water is termed as submerged fermentation

(SmF) (Chao et al., 2019). It is the preferred technology for industrial enzyme production due to

ease of handling at large-scale when compared to SSF (Chao et al., 2019). Large-scale

fermenters, varying in volumes of thousands of liters for SmF are well developed and offer

online control over several parameters such as pH, temperature, dissolved oxygen (DO), and

froth formation. Moreover, there is no problem of mass transfer and heat removal. These benefits

make SmF production technology superior than SSF and widely accepted for industrial

metabolites production (Li et al., 2020). The medium in SmF is liquid, which remains in contact

with the microorganisms. A supply of the oxygen is essential in the SmF, which is done through

sparger. Stirrer and impellers and their design play an important role in these fermenters for

mixing the gas (air), biomass, and suspended particles. There are four main ways of growing the

microorganisms in the SmF (Li et al., 2020). They are batch culture, fed-batch culture, perfusion

batch culture, and continuous culture. In the batch culture, the microorganisms are inoculated in

fixed volume of the medium. In the case of fed-batch culture, the concentrated components of

the nutrient are gradually added to the batch culture. In the perfusion batch culture, the addition

of the culture and withdrawal of an equal volume of used cell-free medium is performed. In the

continuous culture, fresh medium is added into the batch system at the exponential phase of the

microbial growth with a corresponding withdrawal of the medium containing the product (Desai

17
et al., 2019). The continuous cultivation gives a near-balanced growth, with little fluctuation of

the nutrients, metabolites, cell numbers, or biomass. Some enzymes are produced more as a

secondary metabolite, and specific productivity may then be an inverse function of the growth

rate, i.e., nongrowth-associated production (Desai et al., 2019). Here, a recycling reactor may be

the most suitable. A recycling reactor is similar to the continuous culture, but a device is added

to return a significant fraction of the cells to the reactor. Low growth rates with high cell

concentration can often be achieved in such systems. In practice, scale-up effects are more

pronounced for aerobic process than anaerobic process. Hence, aeration and agitation are

maintained during the scale-up in the fermenter to have constant oxygen supply (Desai et al.,

2019). Scale-up complications arise from cell response to distributed values of DO, temperature,

pH, and nutrients. For enzyme production, economy of scale leads to the use of fermenters with a

volume of 20–200m3. The concomitant problems of mass and heat transfer are usually neglected

in small fermenters and at low cell densities (Kjellin et al., 2018). However, in industrial

microbiology, with the above mentioned fermenter volumes and the economic necessity of using

the highest possible cell densities, transport processes must be considered. These can limit the

metabolic rates such as oxygen limitation, which leads the microorganisms to respond with

changes in physiological pattern. In these conditions, the desired control of microbial metabolism

is lost. In controlled operation of an industrial process, metabolic rates must be limited to a level

just below the transport capacity of the fermenter. Therefore, the highest possible productivity in

a fermenter is obtained at maximal transport capacity (Kjellin et al., 2018).

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2.4.2 Solid State Fermentation

SSF is defined as the fermentation process occurring in the absence or near-absence of free

water. SSF is known since antiquity but has gained current scientific attention due to several

environmental and apparent economic benefits it offers (Marouf et al., 2018). It presents

potential opportunities for developing “green processes” employing agro-industrial residues. The

cost of production of several enzymes when produced in SSF is lower in comparison to that

produced in SmF. At industrial scale, SSF is considered for the production of lowvalue enzymes

such as cellulase, xylanase, amylase, etc In addition to above, SSF appears to possess several

other biotechnological advantages such as higher fermentation productivity, higher end-

concentration of products, higher product stability, lower catabolic repression, cultivation of

microorganisms specialized for the water-insoluble substrates or mixed cultivation of various

fungi, and last but not least, lower demand on sterility due to the low water activity used in

SSF,8 though at present mostly on a laboratory-scale (Marouf et al., 2018).

2.4.3 Strain Improvement

2.4.3.1 Mutation

Most of the strains used for industrial enzyme production have been improved by classical

selection (Elavarasan et al., 2018). There are four classes of mutations: (1) spontaneous

mutations (molecular decay), (2) mutations due to error-prone replication bypass of naturally

occurring DNA damage (also called error-prone translation synthesis), (3) errors introduced

during DNA repair, and (4) induced mutations caused by mutagens. Scientists may also

deliberately introduce mutant sequences through DNA manipulation for the sake of scientific

19
experimentation. Mutagenesis by UV radiation or chemical mutagens has been applied to

quickly find the useful variants. Many cells are subjected to mutation and the resulting mutants

are selected for the desired combination of traits (Elavarasan et al., 2018). Usually, mutation

causes changes of protein structure, which results in deterioration of function. Rarely, changes in

the structural components by mutation result in improvements unless the specific loss of function

is required for the production purposes, e.g., when a loss of regulatory function results in

enhanced enzyme production. Mutation and selection are directed primarily toward higher

overall productivity rather than mutation of a specific function, but a loss of regulatory function

is highly probable. Literature says that about 8% of overall mutation results have been useful.

There are several examples of mutant strains, which are known as hyper producers such as

Trichoderma reesei RUT C-30, which has been described as one of the best cellulase producers

since decades (Liu et al., 2017).

2.4.3.2 Recombinant DNA Technology

Microorganisms isolated from diverse environments represent a source of enzymes that can be

used for industrial process chemistry. Though the use of high-throughput screening methods

have enabled to find novel and potent enzymes from the microorganisms, many of those

microorganisms are not easily cultivable in laboratory conditions or their enzyme yield is too low

for economical use (SA et al., 2017). Using recombinant DNA (rDNA) technology, cloning the

genes encoding these enzymes and heterologously expressing them in commonly used industrial

strains has become a common practice. The novel enzymes suitable for the specific conditions

may be obtained by genetically modifying the microorganism. Industrial production of insulin is

produced by genetically modified Escherichia coli. rDNA technology enables the production of

20
enzymes at levels 100-fold higher than the native expression, making them available at low cost

and in large quantities (Badgujar et al., 2018). As a result, several important food processing

enzymes such as amylases and lipases with the properties tailored to particular food applications

have become available. Several microbial strains have been engineered to increase the enzyme

yield by deleting native genes encoding the extracellular proteases. Moreover, certain fungal

production strains have been modified to reduce or eliminate their potential for the production of

toxic secondary metabolites (Kumar et al., 2018). Although the use of rDNA technology

significantly lowers the cost of enzyme production, the applications of enzymes are still limited.

Most chemicals with industrial interest are not natural substrates for these enzymes. If a desired

enzyme activity is found, the yield is often low. Moreover, enzymes are not usually stable in

harsh reaction conditions such as pH higher or lower than the physiological pH 7.0, high

temperature, or the presence of organic solvents required to solubilize many substrates. This

approach precludes the transfer of any extraneous or unidentified DNA from the donor

organisms to the production strain (Kumar et al., 2018).

2.4.3.2 Protein Engineering

With recent advances in polymerase chain reaction technology, site-specific and random

mutagenesis is readily available to improve the stability of the enzyme in a wider range of pH

and temperature and tolerance to a variety of organic solvents (Singh et al., 2020). Since a large

quantity of enzymes can be obtained by recombinant expression, X-ray crystallography can

facilitate the understanding of the tertiary structure of an enzyme and its substrate

binding/recognition sites. This information may assist a rational design of the enzyme, predicting

amino acid changes for altering the substrate specificity, catalytic, rate and enantioselectivity (in

21
the case of chiral compound synthesis) (Singh et al., 2020). To engineer a commercially

available enzyme to be a better industrial catalyst, two different approaches are presently

available: a random method, called directed evolution and a protein engineering method, called

rational design. The protein engineering is a method of changing a protein sequence to achieve a

desired result, such as a change in the substrate specificity or increased stability to the

temperature, organic solvents, and/or extremes of pH (Zhang et al., 2019). Many specific

methods for the protein engineering exist, but they can be grouped into two major categories:

those involving the rational design of the protein changes, and the combinatorial methods, which

make changes in a more random fashion. Protein engineering or rational methods such as the

site-directed mutagenesis, require targeted amino acid substitutions, and therefore, require a large

body of the knowledge about the biocatalyst being improved, including the three-dimensional

structure and the chemical mechanism of the reaction. The main advantage of the rational design

is that a very small number of protein variants are created, meaning that very little effort is

necessary to screen for the improved properties. The combinatorial methods, on the other hand,

create a large number of variants that must be assayed; however, they have the advantage of not

requiring such extensive knowledge about the protein. In addition, often nonobvious changes in

the protein sequence lead to large improvements in their properties, which are extremely hard to

predict rationally, and thus, can only be identified by the combinatorial methods (Zhang et al.,

2019). Several enzymes have already been engineered to function better in the industrial

processes. These include the proteinases, lipases, cellulases, α-amylases, and glucoamylases.

Xylanase is a good example of an industrial enzyme, which needs to be stable at high

temperature and active at the physiological temperature and pH when used as the feed additive

and in the alkaline conditions when used in the bleaching in the pulp and paper industry. One of

22
the industrial production organisms of the xylanases is Trichoderma sp. Its xylanase has been

purified and crystallized. By the designed mutagenesis, its thermal stability increased by about

15 °C (Olofsson et al., 2017). The mutational changes increased the half-life in the thermal

inactivation of this enzyme from approximately 40 s to approximately 20min at 65 °C, and from

less than 10 s to approximately 6min at 70 °C.11 Thus, the designed mutagenesis increased its

thermal stability about 2000 times and its pH optimum shifted toward alkaline region by one pH-

unit. The most successful strategies to improve the stability of the Trichoderma xylanase include

the stabilization of the α-helix region and the N-terminus (Olofsson et al., 2017).

2.4.4 Downstream Processing

The goal of the fermentation processes is to produce a final formulated enzyme product and it

also includes many postfermentation unit operations. Still the maximum production rate could be

the most important factor (Khorshid et al., 2018). However, lowest unit production cost could

also be an important driving force. Optimization of each individual unit operation does not

always lead to an optimal overall process performance, especially when there are strong

interactions between unit operations (Khorshid et al., 2018). Understanding of these interactions

is crucial to overall process optimization. For instance, product concentration or purity in the

fermentation broth can significantly impact the downstream purification unit operation. If the

fermentation is optimized for the productivity, without taking into account its effect on the

purification step, the overall process productivity can be negatively affected. The use of

antifoaming agents in the fermentation process is another example of such a trade-off. By

reducing foaming in the fermentation, a higher working volume can be used to optimize the

fermentation unit operations. Care has to be taken as antifoams could have adverse effect on

23
filtration unit membranes. Thus, it is important to have knowledge of how the fermentation

process is going to affect the other unit operations of downstream processing (Lima et al., 2018).

2.4.5 Product Formulation

The primary task of formulation is to minimize the losses in enzymatic activity during the

transport, storage, and usage. Hence, enzymes are sold as stabilized liquid concentrates or as

particulate solids (Tan et al., 2020). Enzymes are often exposed to humid, hot, or oxidative

environments in industrial applications such as detergents, textile formulations as well as food

and beverage processing. Chemical stabilizers are available to protect the thermolabile enzymes

thermally and chemically to an extent. Hence, it is preferred to select the enzymes that are

structurally more stable or resistant to oxidation during screening itself. Formulations enhance

the stability by counteracting the primary forces of deactivation: denaturation, catalytic-site

deactivation, and proteolysis (Tan et al., 2020). Denaturation occurs by physical unfolding of an

enzyme’s tertiary protein structure under thermal or chemical stress. Once an enzyme begins to

unfold, it becomes dramatically more vulnerable to deactivation and proteolysis. In order to

minimize the unfolding, the formulator can alter the protein’s environment so as to induce a

compact protein structure. This is done most effectively by adding water-associating compounds

such as sugars, polyhydric alcohols, and lyotropic salts, which via “preferential exclusion”

detach water molecules from the protein surface. The best ways to combat the active site

inactivation are to ensure sufficient levels of any required cofactors, to add reversible inhibitors,

and to exclude reactive or oxidizing species from the formulation (Tan et al., 2020).

24
2.5 Application of Industrial Enzymes

2.5.1 Food Industry

2.5.1.1 Starch Industry

The use of starch degrading enzymes was the first large-scale application of microbial enzymes

in the food industry. Mainly two enzymes carry out the conversion of starch to glucose: α-

amylase and glucoamylase. Sometimes additional debranching enzymes, e.g., pullulanase are

added to improve the glucose yield. β-amylase is commercially produced from barley grains and

used for the production of the disaccharide maltose (Jesionowski et al., 2020). Recently, much

work has been carried out on the application of transglutaminase as a texturing agent in the

processing of sausages, noodles, and yoghurt, where cross-linking of proteins provides improved

viscoelastic properties of the products. (Jesionowski et al., 2020). Industrial production of

fructose syrups is carried out employing glucose isomerase after Ca2+ removal (α-amylase needs

Ca2+ for activity but it inhibits glucose isomerase). This is done by bacterial enzymes, which

need Mg2+ ions for activity. Fructose is separated from glucose by chromatographic separation

and crystallized. Alternatively, fructose is concentrated to 55% and used as high fructose syrup

in soft drink or other food industries (Jesionowski et al., 2020).

2.5.1.2 Baking Industry

25
α-amylases have been most widely studied in connection with improved bread quality and

increased shelf life. Both fungal and bacterial amylases are used. The added amount needs to be

carefully controlled as overdosage may lead to sticky dough (Feng et al., 2019). One of the goals

to study the effect of enzymes on dough and bread qualities is to reduce other additives. In

addition to starch, flour typically contains minor amounts of cellulose, glucans, and

hemicelluloses like arabinoxylan and arabinogalactan. There is evidence that the use of

xylanases decreases the water absorption, and thus reduces the amount of water needed in

baking. This leads to more stable dough. Xylanases are used especially in wholemeal rye baking

and dry crisps common in Scandinavia. Proteinases can be added to improve dough-handling

properties; glucose oxidase has been used to replace the chemical oxidants and lipases to

strengthen gluten, which leads to more stable dough and better bread quality (Feng et al., 2019).

2.5.1.3 Dairy Industry

Enzymes have many applications in dairy industry. Chymosin is used in cheese making to

coagulate milk protein. Another enzyme used in milk industry is β-galactosidase or lactase,

which splits milk-sugar lactose into glucose and galactose. This process is used for milk products

that are consumed by lactose intolerant-consumers (Feng et al., 2019).

2.5.1.4 Fruit Juice Industry

Enzymes are used also in fruit juice manufacturing. Addition of pectinase, xylanase, and

cellulase improve the liberation of the juice from the pulp (Kumar et al., 2018). Pectinases and

amylases are used in juice clarification. Similarly enzymes are widely used in wine production to

26
obtain a better extraction of the necessary components, and thus improving the yield. Enzymes

hydrolyze the high molecular weight substances like pectin. Enzymes can be used to help the

starch hydrolysis (typically α-amylases), solve filtration problems caused by β-glucans present in

malt (β-glucanases), hydrolyze proteins (neutral proteinase), and control haze during maturation,

filtration, and storage (papain, α-amylase, and β-glucanase) (Kumar et al., 2018).

2.5.2 Textile Industry

The use of enzymes in the textile industry is one of the most rapidly growing fields in industrial

enzymology. Amylases are used for desizing of textile fibers. Another important enzyme used in

the textile industry is the cellulase (Goncalves et al., 2018). Due to their ability in modifying the

cellulosic fibers in a controlled and desired fashion so as to improve the quality of fabrics, these

(neutral or acidic) offer an excellent replacement for stonewashing of the blue denim garments as

it eliminates the disadvantages caused due to use of the stones, such as damage to the washers

and garments, handling and environment problems. The enzymatic stonewashing allows up to

50% higher jean load and yields the desired look and softer finish. The neutral cellulase is the

enzyme of choice for stonewashing because of the reduction in back-staining and its broader pH

profile. This latter property reduces the need for the rigid pH control of the wash, resulting in a

more reproducible finish from wash to wash (Goncalves et al., 2018). Fuzz formation and pilling

are the common problems associated with the fabric using the cotton or other natural fibers;

cellulases are also utilized for digesting off the small fiber ends protruding from the fabric,

resulting in a better finish (Goncalves et al., 2018). Catalase is used to degrade the excess

peroxide, as hydrogen peroxides are used as bleaching agents to replace chlorine-based

chemicals. Another recent approach is to use oxidative enzymes directly to bleach the textiles.

27
Laccase—a polyphenol oxidase from fungi—is a new candidate in this field. It is a copper-

containing enzyme, which is oxidized by oxygen, and which in an oxidized state can oxidatively

degrade many different types of molecules like dye pigments (Krell et al., 2018).

2.5.3 Detergent Industry

Detergent industry is the largest single industry for the use of enzymes using about 25–30% of

total industrial enzyme. About half of the detergents available in the market contain enzymes in

their formulations; however, rarely the information is published about the formulations Dirt in

the clothes could be proteins, starches, or lipids in nature (Hayes et al., 2020). It is possible to

remove most types of the dirt using detergents in water at high temperatures with vigorous

mixing but the cost of heating the water is high and lengthy (which also requires mixing or

beating). This shortens the life of cloths. The use of enzymes allows lower temperatures to be

employed with shorter periods of agitation, often after a preliminary period of soaking. In

general, enzyme detergents remove the proteins from clothes soiled with blood, milk, sweat,

grass, etc. far more effectively than nonenzyme detergents (Hayes et al., 2020). Cellulases are

employed for loosening the fibers so as to remove the dirt easily and also it gives a finishing

touch by digesting fine fibers during washing. At present, only proteases, amylases, cellulases,

and lipases are commonly used in detergent industry. Enzymes are used in surprisingly small

amounts in most detergent preparations (only 0.4–0.8% crude enzyme by weight, which is about

1% by cost). The ability of enzymes to withstand the conditions of use is a more important

criterion than its cost. With the current availability of second generation detergent enzymes,

detergents are quite efficient at low temperature and alkaline pH (Hayes et al., 2020).

2.5.3 Pulp and Paper Industry

28
Intensive studies have been carried out during the last 20 years to apply many different enzymes

in the pulp and paper industry (Hayes et al., 2020). Xylanases are applied in pulp bleaching,

which liberate lignin fragments by hydrolyzing the residual xylan. This reduces considerably the

need for chlorine-based bleaching chemicals. Cellulases are used for deinking the cellulose fibers

during recycling. In papermaking, amylases are used especially in modification of starch, which

improves the strength, stiffness, and erasability of the paper. The starch suspension must have a

certain viscosity, which is achieved by adding amylases in a controlled process. Pitch is a sticky

substance, composed of lipids present mainly in softwoods. It causes problem for the paper

machine when mechanical pulps of red pine are used as a raw material, which can be removed by

lipases (Hayes et al., 2020).

2.5.5 Animal Feed Industry

Enzyme addition in the animal feed has been intensively started in 1980s, which reduces

viscosity, increases absorption of nutrients, liberates nutrients either by hydrolysis of

nondegradable fibers or by liberating nutrients blocked by these fibers, and reduces the amount

of feces (Vosmann et al., 2018). They are added as enzyme premixes (enzyme–flour mixture)

during the feed manufacturing process, which involves extrusion of wet feed mass in high

temperature (80–90 °C). Therefore, the feed enzymes need to be thermotolerant during the feed

manufacturing and operative in the animal body temperature. The first commercial success was

the addition of β-glucanase into barley-based feed diets. Barley contains β-glucan, which causes

high viscosity in the chicken gut. The net effect of enzyme usage in the feed has been increased

animal weight gain with the same amount of barley, resulting in increased feed conversion ratio.

Addition of xylanase to wheatbased broiler feed is capable of increasing the available

29
metabolizable energy to 7–10%. Another important feed enzyme is the phytase, which is a

phosphoesterase and liberates the phosphate from the phytic acid. Phytic acid is commonly

present in the plantbased feed materials. The supplementation of the phytase results in reduced

amount of the phosphorous in the feces resulting in reduced environmental pollution. It also

minimizes the need to add the phosphorus in the feed. Currently, phytases from the fungal

sources are potent feed enzymes (Vosmann et al., 2018). Usually, a feed-enzyme preparation is a

multienzyme cocktail containing glucanases, xylanases, proteinases, phytases, and amylases

(Vosmann et al., 2018).

2.5.6 Leather Industry

Leather industry uses proteolytic and lipolytic enzymes in leather processing. The use of these

enzymes is associated with the structure of animal skin as a raw material. Enzymes are used to

remove unwanted parts (Martinez et al., 2018). Alkaline proteases are added in the soaking

phase. This improves water uptake by the dry skins, removal and degradation of protein, dirt, and

fats and reduces the processing time. In some cases, pancreatic trypsin is also used in this phase.

Proteases are used in dehairing and dewooling of leather, and improve its quality (cleaner and

stronger surface, softer leather, less spots). Lipases are used in this phase or in bating phase to

specifically remove grease. The use of lipases is a fairly new development in leather industry

(Martinez et al., 2018).

2.5.7 Biofuel from Biomass

Perhaps the most important emerging application of the enzymes currently being investigated

actively is in the utilization of the lignocellulosic biomass for the production of biofuels

30
(Martinez et al., 2018). Lignocellulosic biomass represents the most abundant renewable

resource available to the mankind for the effective utilization. However, lack of cost-effective

enzyme conversion technologies has made the biofuels program currently economically not

feasible, basically due to high cost of the cellulases and also lack of specificities for various

lignocellulosic substrates. The strategy employed currently in the bioethanol production from the

biomass is a multistep process in which enzymatic hydrolysis is a crucial step. In the effort to

develop the efficient technologies for biofuel production, significant studies have been carried

out on the identification of efficient cellulase systems and process conditions, besides on the

biochemical and genetic improvement of the existing organisms utilized in the process Several

industries such as Genencor and Novozymes have been actively involved in cellulases research

and have claimed attaining significantly reduced cost of the enzyme with improved efficiency

(Martinez et al., 2018).

31
 CHAPTER THREE

3.0 CONCLUSION

Though enzyme technology is a well-established branch of science, it is passing through a

continuous phase of maturation as well as evolution. Search for novel enzymes based on

particular application as well as a potential application for a known enzyme are moving ahead

simultaneously. Our society is moving toward eco-friendly technologies, which can help saving

the planet earth for the future generations. Enzymes, the biocatalysts have shown tremendous

capacity to guide us toward bio-based processes. Several chemical processes have been replaced

by the biological processes with several benefits such as mild operating conditions, specificity,

and environmental feasibility. Enzymes could be used in almost all the major commercial sectors

with domination over pharmaceutical and food industry. Application of enzymes in few sectors,

such as in biofuels, has emerged with potential offerings. . Enzymes role in all living beings life

and the society is inevitable.

3.1 RECOMMENDATION

32
To reduce enzyme cost, significant and tremendous contributions have to be made by enzyme

producing companies so as to have economically feasible enzymatic technologies

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