Content uploaded by Michele Vitolo
Author content
All content in this area was uploaded by Michele Vitolo on Feb 03, 2020
Content may be subject to copyright.
www.wjpr.net Vol 9, Issue 2, 2020.
Vitolo. World Journal of Pharmaceutical Research
199
MISCELLANEOUS USE OF ENZYMES
*Michele Vitolo
School of Pharmaceutical Sciences, University of São Paulo, Brazil.
ABSTRACT
Enzymes are catalysts that act under gentle reaction conditions (room
temperature and 4.0 < pH < 9.0). They present high specificity and
enantiomer selectivity, enabling them to be largely used in industry
(food, animal feeding, pharmaceutical, biotechnological, chemical,
textile, laundry, waste treatments, leather, pulp and paper), clinical and
chemical analyses, and therapy. Due to their specificity (hydrolysis of
prostaglandin ethyl ester by esterase, for instance) and enantiomer
selectivity (conversion of fumaric acid into malic acid or L-aspartic
acid by fumarase), they have been used in the modification of complex
and labile substances – largely used in immunology, endocrinology,
among others – by introducing in the molecule structure one or two chiral carbon and/or a
specific chemical group at a pre-defined position (conversion of progesterone into 11-
hydroxyprogesterone, for example).
KEYWORDS: Enzymes, industrial enzymes.
INTRODUCTION
Enzymes are specialized high-molecular weight proteins composed of amino acid building
blocks and are in general natural substances produced by all living organisms. There is a
special group of intracellular enzymes – called ribozymes –, whose building blocks are
ribonucleotides.[1] Enzymes act as catalysts and conduct about 95% of all physiological
processes pivotal for the growth and life of all living matter. They can accelerate the
processes of synthesis or decomposition of organic substances (prostaglandins, hormones,
antibiotics, fats, carbohydrates, among others) under moderate conditions of pH, pressure and
temperature.
World Journal of Pharmaceutical Research
SJIF Impact Factor 8.084
Volume 9, Issue 2, 199-224. Review Article ISSN 2277– 7105
Article Received on
15 Dec. 2019,
Revised on 05 Jan. 2020,
Accepted on 25 Jan. 2020,
DOI: 10.20959/wjpr20202-16783
*Corresponding Author
Michele Vitolo
School of Pharmaceutical
Sciences, University of São
Paulo, Brazil.
www.wjpr.net Vol 9, Issue 2, 2020.
Vitolo. World Journal of Pharmaceutical Research
200
When compared with another catalysts, organic or inorganic, the enzymes have uncommon
characteristics regarding specificity (they act on a particular substance or insert a chemical
group – such as the hydroxyl group – at a pre-selected position inside the substrate molecule,
as in the conversion of progesterone into 11-hydroxyprogesterone) and enantiomer selectivity
(they sort out only one of the two isomers present in a racemic mixture).
The initial use of enzymes was set when -amylase from Aspergillus oryzae and invertase
from Saccharomyces cerevisiae were produced at an industrial scale in the beginning of the
twentieth century.[2] Since then, more and more enzymes were identified and produced in
significant amounts that led to applications in industry (food, animal feeding, pharmaceutical,
cosmetic, fragrance, chemical etc.), in medicine (as drugs), and in clinical and chemical
analytical procedures (as reagents).
The aim of this review is to analyze the use of enzymes in detergents, effluent and waste
treatments, flavor production, leather, textiles, pulp and paper, edible oils, animal feeding,
analytical procedures, medicine, and organic synthesis (biotransformations).
ENZYMES IN DETERGENTS
Detergent is a generic term that encompasses all cleaning products available in the market.
However, those containing enzymes (proteases, lipases, amylases, and cellulase) are used in
households, industrial and hospital laundry, and household dishwashing since 1960.
Approximately 30% of the overall worldwide enzyme production is funneled to the detergent
industry. Enzymes line with surfactants, bleaching compounds and builders as the main
formulation ingredients of cleaning products.[3]
The most used are proteases due to their broad substrate specificities and capability of
functioning to some extent some under extreme conditions found in domestic washing
(temperatures of 20 to 70oC, at a pH up to 11, and high concentrations of surfactants,
polyphosphates and chelating agents).
Current trends on energy conservation promote low temperature fabric washing, requiring
enzymes with a high catalytic activity at room temperature.
www.wjpr.net Vol 9, Issue 2, 2020.
Vitolo. World Journal of Pharmaceutical Research
201
In laundry, proteases touch their substrates on stains glued on a fabric surface. Therefore,
they must have the ability to be adsorbed by insoluble fragments, which occurs optimally
when the pH value of the detergent solution is close to that of the enzyme pI (isolectric pH).
Lipases have been used in detergent formulations since 1988, aiming to wash out fatty stains
deposited on the fabric surface. The lipase hydrolyzes the triglycerides present in fats, freeing
hydrophilic compounds such as fatty acids, diglycerides, monoglycerides and glycerol.[4] All
of them are removed by washing the fabric in an alkaline condition (pH: 8.0-11.0). In alkaline
pH, the lipase maintains at least 60% of its overall activity as the temperature ranges from
20oC to 55oC.[3] Under these conditions, a complete wash out of a fatty stain from the fabric
occurs after two or three washing cycles, shorter than the seven/eight washing cycles required
for non-lipolytic washing.
Amylases are enzymes that catalyze the hydrolysis of starch in low MW sugars and
oligosaccharides. The starch adheres to the fabric surface aggregating other dirty on stains.
The most used are heat stable microbial α-amylases. Depending on the origin of the -
amylase, its optimal activity pH can vary from 6.5 to 9.0 (Bacillus licheniformis α-amylase)
or from 4.5 to 7.0 (Bacillus amyloliquefaciens α-amylase).
Cellulases have been used in detergent formulations since the 1980s. They catalyze the
hydrolysis of -1,4 glycosidic bonds in cellulose, freeing short chain oligosaccharides
consisting of glucose units. Unlike other detergent enzymes, cellulases do not act on stains,
but on cellulose, reconstituting the microfibers and damaged fibers which appear on the tissue
during wash and wear of a garment. Till now, no damage was observed to the strength of
textiles despite the cellulose being the main constituent of fabrics. The macroscopic effects
resulting from the cellulase action on the garment are color intensification, enhancing the
softness of the fabric surface, and improving the removal of particles of grime entrapped in
the twill of the fabric. The intensity of the effects on garments depends on the dose, the
washing conditions, and the detergent formulation.
Detergents containing enzymes are commercialized as either powder or liquid. Currently, the
enzyme itself is presented as granules as a preventive measure against allergenic problems
which can affect either domestic users or workers at detergent factories.[5]
www.wjpr.net Vol 9, Issue 2, 2020.
Vitolo. World Journal of Pharmaceutical Research
202
Enzyme compatibility with detergents involves stability during storage and washing. During
the use, enzymes are exposed to denaturation (unfolding of the molecule structure due to
high temperature and/or harsh environment; the addition of Ca2+ at 500ppm can stabilize
alkaline and super-alkaline proteases), undesired chemical reactions (an oxidizing agent
modifying a critical amino acid residue located at the active site, for example), and
proteolysis (which can be minimized by adding to the formulation propylene glycol and/or
reversible protease inhibitors such as glycine and borates). Storage stability depends on
whether the product is a liquid or a powder detergent.
In a liquid detergent, all ingredients can have a direct impact on the enzyme. Thereby, the
addition of an adequate surfactant, mainly non-ionic, can reduce the rate of denaturation. This
is not the case for a granulated enzyme in powder detergents. The formulation and the type of
granulation have a major influence on the stability. Granulation introduces a barrier between
the harmful surrounding and the enzyme inside granules. The major parameters for
stabilizing enzymes in powder detergents are storage at low temperature and humidity.
However, if the formulation has a bleaching system (perborate plus tetra acetyl ethylene
diamine, for instance), the enzyme activity can decrease due to the oxidative action of oxygen
free radicals – generated during the reaction promoted by bleaches – on amino acids that are
constituent of the enzyme. In this case, an oxygen free radical sequester, such as ascorbic
acid, must be added.[6]
During washing, enzyme stability depends on factors such as detergent composition and
dosage, pH of the detergent solution, ionic strength of the detergent solution, washing
temperature, washing time, mechanical handling, water hardness, level of soiling, and type of
textile.
Finally, enzyme detergents can also be used in cleaning machinery parts, ultrafiltration and
reverse osmosis membranes and gadgets such as lens, endoscopes, dentures and electrodes.[7]
EFFLUENT AND WASTE TREATMENTS
Currently, one of the greater aims is to minimize damages inflicted to the environment by any
form of residue (gaseous, liquid or solid) generated by the industrial activity.
The best situation would be that where no waste is discharged into the environment, i.e., the
plant is structured for recovering and reworking all residues generated. Unfortunately, such
www.wjpr.net Vol 9, Issue 2, 2020.
Vitolo. World Journal of Pharmaceutical Research
203
situation is almost never achieved. Thereby, the only way to avoid damages to the
environment is treating the waste.
Waste treatment can result in a marketable byproduct – a situation in which financial benefit
can be reverted to the company – or in an effluent inoffensive to the environment, resulting in
expenditure to the company. However, this expense can be reverted into benefits to the
company since the plant is operated using good environmental practices. Thereby, the
company could ask for an environment certification (based on the ISO 14001), which could
aggregate value to the company assets.
The ways of treating wastes and pollution clean-up can be carried out by chemical and/or
physical processes or biological agents (microorganisms and/or enzymes).
The use of microorganisms in industrial waste streams can be by activated sludge with or
without anaerobic digestion. Moreover, the application of microorganisms to waste treatment
and pollution clean-up can involve the disposal of municipal waste into methanogenic
landfills and the treatment of land which has already been polluted with undesirable and
noxious compounds.
Enzymes acting on wastes generated by food industry – still rich in sugars, proteins and fats –
can result in valuable byproducts. However, if the waste effluent comes from another type of
industry – normally rich in noxious chemicals –, the direct treatment of enzymes aims to
reduce its pollution power before throwing it to the environment.
Evaluation of biological waste upgrade viability is based on a) the knowledge about the
chemical and physical characteristics of the waste, b) the waste nature – if it is a genuine one
or an underutilized byproduct, c) the commercial novelty of the byproduct obtained, d) the
waste abundance, e) the economic viability of waste treatment considering legal
requirements, cost and logistic for collecting, and long-term policy on waste source
processing.
Some aspects regarding the enzyme-assisted processing of waste and byproducts are a) the
economic recovery and reuse (sugars recycling in confectionary after -amylase hydrolysis
of starch present in the waste), b) the energy conservation and material economy (cellulases
in the extraction of flavors and colors, byproducts with high market values), c) process
www.wjpr.net Vol 9, Issue 2, 2020.
Vitolo. World Journal of Pharmaceutical Research
204
economy and upgraded byproducts (for example, use of proteases in the reduction of
viscosity and ―stickiness‖ of concentrated wash water from fish meal industry used as
supplement of culture media for fermentative processes), d) alternative higher value
derivatives (when the raw waste is a complex mixture of substances, polymers included, and
the separation of one of them is not economically viable, a pool of enzymes – proteases,
amylases and pectinases – can be used to hydrolyze the polymers resulting in a medium
valuable as a fermentation feedstock, and e) the attainment of new source materials (for
example, using low active proteases in cheese whey concentrate leads to obtaining a mixture
of proteins with changed foaming and gelling capabilities, enabling whey to be used in food
formulations).[8]
The direct use of enzymes in effluent treatment originated from industries other than the food
industry is still incipient. This is due to the harshness of the chemical composition of wastes
against the delicate enzyme structure. The peroxidase/hydrogen peroxide combination
(PHPC) is the best studied system for treating wastes rich in phenols and aromatic amines.
These compounds, in presence of free hydroxyl radicals (generated by PHPC), are converted
into insoluble polymers, which are separated by filtration or decantation. After drying, they
are incinerated.[9]
Growing public awareness about environmental problems in recent years will push the
academic and industrial researches to developing new enzyme means for waste disposal.
FLAVOR PRODUCTION WITH ENZYMES
Practically all types of natural and industrialized foods have characteristic flavors and
aromas. For example, citrus juices have a little bitter taste due to the presence of naringin and
limonin, two compounds located at the white layer of citrus fruits.
Thousands of volatile chemicals belonging to different classes of organic compounds (esters,
amines, alcohols, alkenes, terpenes, aldehydes and ketones) have been identified in food. The
―food aroma‖ is a mixture of hundreds of compounds. For example, the apple aroma/flavor is
composed by at least fifteen different chemicals (1-butanol, 2-methyl-1-butanol, hexanal, 1-
hexanol, furfural, among others).
Market surveys on flavoring compounds have demonstrated that consumers prefer foodstuffs
that can be labeled ―natural.‖ The perception of ―natural‖ as better has led to an increased
www.wjpr.net Vol 9, Issue 2, 2020.
Vitolo. World Journal of Pharmaceutical Research
205
demand for flavor and fragrance chemicals that may be considered ―natural.‖ Plants are the
best natural source of flavor chemicals. However, there are setbacks regarding plant source,
such as few suppliers, expense of isolation, variability in the amount, and quality of final
product from different geographical sources. These concerns have resulted in a high price for
natural source chemicals and a search for alternate supplies of the desired compounds. One
way of alleviating this problem has been the development of biotechnological processes –
either via microbial fermentation or enzymatic reaction – for the production of specific flavor
and fragrance chemicals and complex mixtures with a cheese, fish and meat aroma, among
others.
Food aroma/flavor can be produced by a variety of processes including enzymatic and
microbial action, food processing, cooking, and chemical interactions.[10] A convenient way
to consider these chemicals is to divide them into three broad groups: a) the heat-derived or
Maillard browning aroma chemicals, which are formed when food is cooked or heat-
processed, such as the aroma of meat and coffee, b) chemicals formed during heat-processing
(via Maillard reaction) from a nonvolatile chemical precursor formed in the course of a
fermentation step, such as observed in cocoa and bread, c) biologically-derived aroma
chemicals, often referred to as secondary metabolites, arise by microbial fermentation, action
of endogenous enzymes, end-products of plant metabolism or enzymes added during
processing.
Regarding aroma/flavor production by enzymatic ways, there are three main approaches, i.e.,
in situ – for example, the taste of beer is formed by cooking the mashed cereal must with
hops before the fermentation of the broth by the beer-yeast –, enzyme catalyzed reaction for
the production of specific flavor chemicals (for instance, the sweetener aspartame is
synthesized by thermolysin from aspartic acid and phenylalanine methyl ester;), and enzyme-
modified foods for the production of savory flavors such as meat, cheese, and fish.[11]
The cheese manufacture is an example of in situ and enzyme-modified-food approaches.
The flavor generation in situ is promoted by leaving the whey-free p-k-casein coagulum in a
room for a long time, during which the characteristic flavor and aroma appear. This is mainly
due to the action of selected or wild strains of microorganisms (Lactobacillus sp.) associated
with the proteolytic and lipolytic activities of residual protease and lipase added in the first
step of cheese making (enzymatic p-k-casein coagulation). The enzyme-modified cheese
www.wjpr.net Vol 9, Issue 2, 2020.
Vitolo. World Journal of Pharmaceutical Research
206
production can be summarized as follows: off-cuts of cheese (with about 63% of dry solids)
are mixed with water and emulsifiers; the cheese slurry (with about 45% of dry solids) is
pasteurized (72oC for ten minutes) and cooled at 50oC. Enzymes (porcine pancreatic lipase –
PPL – or microbial lipase – ML – and fungal protease) are added to the paste, which is left
for at least 8 h at 50oC. Then, the paste is pasteurized (72oC for 25-35 min) and, finally,
spray-dried. Normally, ML is preferred over PPL because a) the PPL is always contaminated
by trypsin, whose proteolytic activity causes the appearance of bitter peptides in the final
product, b) the PPL cannot be used for vegetarian and kosher products, and c) the fear of
virus or prion presence in products of animal origin. Fungal proteases are the enzymes of
choice because they do not produce a high level of bitter peptides and some of them contain
very high levels of both carboxy- and amino-peptidases, which hydrolyze bitter peptides, a
well prized side effect.[11]
The attainment of meat flavor is an example of enzyme-catalyzed reaction, although it can
be obtained by acid hydrolysis (6M HCl) of soya protein at 180oC and pressure of 6 atm. The
meaty flavor of soya hydrolyzed protein is due to the Maillard reaction involving sugars
(from hydrolyzed carbohydrates) and amino acids (from hydrolyzed protein). The acidic soya
hydrolysate is used in formulations of soups, sauces, snacks, pies etc., in spite of presenting
the following as disadvantages: a) many countries consider it an artificial instead of natural
flavoring, b) containing sodium chloride over 40% (residue of hydrochloric acid
neutralization), c) high glutamate content, which is reproved by several countries (USA,
included), and d) containing a low amount of mono- and dichloropropanol, potential
carcinogens. The resilience on HCl substitution for proteases is due to its low cost. However,
the substitution of acid hydrolysis for enzyme hydrolysis is possible when high-cost foods are
produced. Moreover, as the ecological conscience of the community increases, entrepreneurs
of this kind of industry will be pressured to take measures to reduce the probability of
environment damages, leading necessarily to a full substitution of the acid for enzymatic
hydrolysis. In this point of view, an alternative to the enzyme soya hydrolysate could be the
use of yeast extract – previously treated with ribonuclease and deaminase – as a meat savory
enhancer.[11]
Finally, flavor enhancer compounds of microbial origin such as monosodium glutamate,
inosine monophosphate and guanosine monophosphate deserve to be remembered because
www.wjpr.net Vol 9, Issue 2, 2020.
Vitolo. World Journal of Pharmaceutical Research
207
they help to obtain a certain type of flavor (meat, for instance) even in the presence of low
concentrations of natural flavoring substances.[11]
LEATHER
Leather processing consists of six clearly defined steps, i.e., curing (the fresh skin is placed
into a salt concentrated solution, then draining, addition of antimicrobials – non-ionic or
anionic surfactant – and drying the flesh under the sun), soaking (consists on rehydration,
washing away fat and dirt, swelling and cleansing the cured skin), dehairing (consists on
removal of hairs from cured and soaked skin by using an alkaline mixture of hydrated lime,
inorganic sulfides and amines (pH=11.0). Sulfides break the bonding protein fibrils within the
hair and dissolve the proteins of the hair root. The skin is left in contact with this solution
under agitation for several days), dewooling (consists on painting the skin with an aqueous
solution constituted by hydrated lime, sodium chlorite and protease. The painted skin is left
overnight at 30oC, resulting a bright and clean skin), bating (consists on deliming, deswelling
the collagen of the skin, degrading the protein fibers partially so that they become soft and
able to accept an even dye), and tanning (consists on treating the bated skin with acid
solutions in order to produce a further deliming without reswelling the collagen fibers.
Staining chemicals are used obtaining the final leather with chromatic nuances).
Enzymes are useful catalysts in some stages of leathering (soaking, dehairing, dewooling and
bating). The most valuable in leather industry are proteases – mainly the neutral and alkaline
– and a raw extract of bovine and porcine pancreas called ―pancreatin.‖ Indeed, pancreatin
has amylolytic, lipolytic and proteolytic activities. Its use becomes more advantageous than
the protease alone.
The use of pancreatin at the soaking step enables the tanner to handle fat skins. Furthermore,
protease promotes subtle modifications on the proteins, favoring water absorption by the skin.
A mixture of alkaline protease and lime in dehairing the skin promotes hair detachment from
the roots (a smooth leather is thus obtained). Dehairing by enzymes leads to a decrease in
sulfured chemicals needed, contributing to diminish the environmental pollution near the
tannery because less polluted effluents and unpleasant odors are emitted, respectively, into
water streams (or urban sewage system) and in the air. Dewooling involves two steps: 1st)
dehaired skin is embedded in a water solution containing hydrated lime, sodium chloride and
alkaline bacterial protease; and 2nd) the flesh side of the skin is evenly sprinkled with a
powder constituted by sodium sulphate, sodium sulfite, ammonium chloride, ammonium
www.wjpr.net Vol 9, Issue 2, 2020.
Vitolo. World Journal of Pharmaceutical Research
208
sulphate and alkaline or neutral protease. Next, the skin is hung in a conditioning room at 25-
30oC for 24 h before the wool is pulled out. Since 1908, pancreatic trypsin substitutes animal
excrements (used by millenniums) in bating. The action of trypsin, in combination with the
added chemicals, is to remove any hair residues, allow water penetration deswelling the
collagen fibers, and have a minimal influence on collagen. The proteases used for bating are
selected for their pattern of specificity to the various proteins of the skin. Thereby, trypsin,
fungal acid protease, bacterial neutral and alkaline proteases are active on muscles; bacterial
super-alkaline protease acts on muscles and keratin; papain acts on muscles, collagen and
elastin; ficin acts on muscles, collagen, elastin and keratin; and bromelain acts on collagen
and elastin. By using adequate activity combinations of pancreatin, plant proteases (papain,
bromelain and ficin) and microbial proteases (acid, neutral, alkaline or super-alkaline), the
tanner can perform a weak, medium or strong bating.[12]
In fact, the incorporation of enzymes into some steps of leathering has proved very successful
both in improving leather quality and reducing environment pollution.
TEXTILES
During the production of fabrics, sizing the treads with starch combined or not with polyvinyl
alcohol, gelatin, gums or carboxymethyl cellulose is fundamental to obtain a good weaving.
In weaving, starch paste is applied for warping aiming to provide strength to the textile and
prevent the loss of string by friction/cutting and generation of static electricity on the string.
The desizing of the treads – to allow an efficient adsorption of dyes, bleaches and texture
enhancer on the cloth – is accomplished by using a thermal (85-110oC) and a chemical
resistant microbial -amylase. The operating pH ranges from 5.0 to 7.5, and the addition of
calcium ions (about 0.5 g/L) for stabilizing the enzyme is required when very soft water
(hardness lower than 50 ppm) is used as solvent. The starch hydrolysis during desizing must
occur in the shortest time possible.
The desizing process can be divided as follows: a) prewashing (removing of waxes or other
additives by passing the fabric through boiling water and surfactant at 0.5 g/L; moreover, the
starch glued on the treads swallows in, facilitating the -amylase hydrolysis in the next
stage); b) impregnation (starched-treads are treated with the enzyme solution (0.05% w/v)
so that the hydrolysis begins slowly, but consistently); c) starch hydrolysis (the polymer
breakdown can be programmed to occur for 12-16 h at 35-40oC or for 1-4 h at 70-75oC
www.wjpr.net Vol 9, Issue 2, 2020.
Vitolo. World Journal of Pharmaceutical Research
209
depending on the fabric desired. Long reaction times at low enzyme levels can be economic if
stability is ensured, otherwise it is better to use high enzyme levels at high temperatures,
reducing the duration of the hydrolysis); and d) after-wash (the complete removal of the
hydrolysis products (low MW oligosaccharides) is pivotal for the success of bleaching, the
step following desizing. In this stage, hot water containing synthetic detergent (5 g/L) and
sodium hydroxide (10 g/L) is added aiming the complete removal of oligosaccharides
following the neutralization of alkalis with acid and the thorough rinsing with water).[11]
In denim processing, a mixture of -amylase and cellulase is used aiming the substitution of
pumice stones. The stones are used for removing the excess of indigo blue adsorbed by twill
cotton in the production of the fabric directed to jeans confection. This procedure is carried
out by suspending the tinted-blue cotton with stones in a water-loaded tumbling machine.
After an intense agitation, the excess of indigo dissolves into the water, leading to a color-
faded denim jean. This procedure creates problems such as the disposal of the sand –
resulting from stone eroding –, which, if not adequately treated, affects the environment
surrounding the facility; the decrease of the operational half-life of the tumbler; the
production of a fabric with a low tensile strength, and difficulty for reproducing a particular
combination of fade and abrasion to create very large consignments of identical
products.[11][13]
Cellulase was introduced into the denim processing in order to fade homogeneously the blue
color of the fabric with a wider range of color tones. The enzyme is directly added to the
fabric as soon as it is completely desized and fully water rinsed.[13]
During the last years, the textile industry has been pressured to move away from chemical
bleaches to meet ecological demands of the society. The response resulted on the substitution
of chlorine-based chemicals for hydrogen peroxide in bleaching (a less environment
damaging chemical; the excess is removed by catalase).[14]
The textile industry is intensely pressured to minimize the consumption of treated water, the
pollution of water streams surrounding the facility, and the overall volume of dischargeable
effluents resulting from processing operations. The use of enzymes may be expected to play a
relevant role in helping the textile industry with its effort to meet all such demands.
www.wjpr.net Vol 9, Issue 2, 2020.
Vitolo. World Journal of Pharmaceutical Research
210
PULP AND PAPER
The invention of writing (about 3,300 BC) was fundamental for the humankind, insofar as the
registration of administrative acts and the knowledge accumulated along the centuries were
made possible.
However, with the advent of writing, it was peremptory to find a suitable material on which
the words could be written. The first writings were recorded on solid surfaces (wood, animal
bones and stones), followed by papyrus leaves (prepared with the stalk of leaves of the plant
Cyperus papyrus), parchment (prepared with animal skin), and, finally, paper (made of
cellulose fibers prepared from trees of hardwood and softwood). The development of printing
led to the consolidation of the paper industry worldwide.
The paper production involves the feedstock, handling, pulping, refining, dewatering,
bleaching, and papermaking. Regardless of the fiber source, physical handling is carried out
to obtain cleanliness and a particle size suitable for pulping. This processing takes the form of
debarking, washing and screening to remove the foreign matter, and mechanical chipping for
most pulping processes.[11]
The use of enzymes in the paper industry – more precisely, in pulp refining/dewatering,
bleaching and papermaking – has grown rapidly since the mid-1980s.
Pulping is carried out by either mechanical (for newsprint and other bulk papers) or chemical
(writing and wrapping papers) processes.
In the mechanical process, the fiber feedstock is cleaned and chipped to a uniform size. The
raw fiber is torn apart by mechanical means during pulping (the process by which the
macroscopic structure of raw wood fiber is broken apart, rendering a pliable fiber). Closely
associated with the pulping is refining, which improves the strength of the pulp. Then, the
pulp dewatering (removing the water retained within the wooden lattice) is made in presence
of enzymes (cellulases and hemicellulases).[15] A careful control of the enzyme dose is
important; excessive enzyme treatment can weaken the pulp because of cellulose hydrolysis.
In addition, the excessive creation or destruction of fines (particles constituted by cellulose
and hemicellulose that pass through a 200-mesh screen) can affect the opacity of the sheet.
Following dewatering/refining, the pulp is treated with peroxide for bleaching, from which a
pulp ready for papermaking is obtained.
www.wjpr.net Vol 9, Issue 2, 2020.
Vitolo. World Journal of Pharmaceutical Research
211
In chemical pulping (designed for dissolving lignin), the wood chips are cooked between
160oC and 190oC for 3 h in either a concentrated solution of sodium hydroxide and sodium
sulfide (Kraft pulp) or sulfurous acid (sulfite pulp).[11]
Today, recycled paper is becoming an important feedstock for paper industry, being known
as secondary fiber processing. In this case, the feedstock is shredded and pulped again. This
raw material comes from discharged printed paper. Therefore, the removal of ink (deinking
process) precedes bleaching. Deinking is carried out by pulping again at a 3-4% solids
consistency. The slurry is diluted into a 1% solid consistency followed by the addition of
flocculating surfactants, ink solvents and cellulase (used for releasing ink particles from fiber
fines). The ink particles float in the surface and are collected and removed.[11]
Bleaching – the process by which the pulp is brightened or made completely white by
oxidizing chemicals. It involves the complete removal of lignin without any alteration on the
amount and structure of cellulose. Xylanase is added into Kraft pulp slurry to change the pulp
structure and facilitate the action of oxidizing bleaching chemicals (chlorine gas, alkali
extraction and chlorine dioxide), and diminish the amount needed by 20%.[11] Xylanase
preparation – which must be devoid of cellulase activity, otherwise cellulose would be
hydrolyzed and papermaking would be hindered – is characterized by a range of pH and
temperatures from 3.0 to 8.0 and from 30oC to 60oC, respectively.[16]
The finished pulp directed to papermaking is combined with chemicals (clay or starch) to
improve paper properties such as strength, stiffness and erasability (important in good typing
paper), as well as the performance of paper machines. In this phase, -amylase (removes
excess of starch), lipase (removes pitch, constituted of highly water insoluble lipids, which
adheres to the paper machine) and levan-hydrolase (eliminates slime – such as levan, a
bacterial -2,6-linked fructose polymer – that accumulates in the paper machine) are used.
In short, enzymes are used in pulp and paper industry to modify substrates such as fiber
constituents (cellulose, lipids and hemicellulose) and paper contaminants and additives
(starch, pitch and slime).
EDIBLE OILS
Pulp and seeds of oleaginous plants (palm, olive, soybean, sunflower, cottonseed, canola,
rapeseed etc.) are raw materials for the extraction of edible oils. The oils from the pulp of
www.wjpr.net Vol 9, Issue 2, 2020.
Vitolo. World Journal of Pharmaceutical Research
212
oleaginous fruits (olive and palm oils) and from seeds (rapeseed and cottonseed oils) are
extracted by water and organic solvent, respectively.
As edible oils are sold at low prices, the use of enzymes tends to increase their market price.
Thereby, the use of enzymes is justified if the oil is sold at a high price (olive oil) or if a
production increase is achieved (palm oil).
The production of olive oil consists on olive milling in presence of water to make a paste
with an adequate texture for pressing. The pressed slurry has a solid phase (composed by 3-
5% of residual oil, 50% of water and 43-45% of solids), an aqueous phase (composed by less
than 1% of residual oil, 90-95% of water and 5-10% of solids), and an oil phase (composed
by 98-99% of oil and water and solids less than 1%).[11] By carrying out pressing in presence
of cellulase and pectinase, less water will appear in the oil phase. Consequently, less water
separates from the oil during the storage period (obligatory step for oil maturation). Such
procedure leads to an increase of oil yield by 1%.[17] Considering that thousands of cubic
meters of olive oil are processed, any volume close to 1% of water over the whole oil volume
will certainly represent some cubic meters more of oil bottled and sold.
In oleaginous seeds and fishes, the oil is often bound to proteins. Therefore, the use of
proteases increases the extraction yield.
When processing oleaginous seeds, it is common to obtain oil contaminated by phospholipids
(e.g., lecithin) and/or phosphatides, which confer gumminess to the final product. Although
degumming could be made with enzymes (phospholipases A1, A2, C and D), the industry
prefers removing them with organic compounds or hot water for economic reasons.
Microbial lipases can be used in inter-esterification processes – such as the conversion of
palm oil (rich in lauric acid but poor in stearic and palmitic acids) into cocoa butter (rich in
stearic and palmitic acids) – and in the hydrolysis of oily triglycerides.[4][11]
The trends for using enzymes in edible oil technology are promising despite their high cost.
Environmental concerns, pushed over by humans each day more conscious on better
ecological environments, will arise due to the huge wasting of water during oil processing as
well as the high volume of pollutant effluents generated.
www.wjpr.net Vol 9, Issue 2, 2020.
Vitolo. World Journal of Pharmaceutical Research
213
ENZYMES IN ANIMAL FEEDING
Enzymes can be added in animal food aiming to improve the digestibility of raw materials
(starches, proteins, fats, fibers etc.) and to reduce the excretion of nitrogenous and
phosphorous substances to the environment.
The main feed raw materials in terms of fiber composition can be divided as follows: group I
(materials rich in β-glucan, e.g., barley and oats), group II (materials rich in pentosans –
arabino-xylans –, e.g., wheat, rye and triticale), group III (materials not susceptible to the
enzyme decomposition; e.g., white sorghum and maize), and group IV (vegetable protein
sources with pectic and galactosaccharide substances in their fiber structure).[11] The raw
materials belonging to the groups I, II and IV are susceptible to enzymatic attacks.
The rationale for using enzymes in animal food assumes that catalysts, when added, must
degrade soluble fibers – normally with an anti-nutritional effect – and/or to supplement the
animals’ own digestive enzymes. In the latter case, the pig pancreas at weaning is incapable
of producing a pancreatic juice with amylase and protease activities enough to carry out an
efficient digestion of the meal. This physiological condition lasts four weeks. After the full
pancreas functioning is reached. Thereby, during the enzyme insufficiency period, the
supplementation with exogenous amylases and protease is beneficial to the development of
the animal. Besides the supplementation of diets for young animals, enzymes are also
important in the degradation of non-starch polysaccharides found in cereals and vegetable
proteins.
Diets for poultry can be based on barley, maize, wheat and/or vegetable protein sources.
Soybean meal is the most largely used. Broilers are negatively affected by diets containing
more than 10% (w/w) of barley due to their susceptibility to the high β-glucan content of
barley. Therefore, the feed for them must be added with β-glucanases. In the case of wheat-
based diets – rich in water-soluble pentosans –, the addition of pentosanases or endo-
xylanases will improve meal use by broilers. Laying hens and turkeys are also positively
affected by the cited enzymes. For laying hens, egg cleanliness is lost when diets with high
non-starch polysaccharides levels (barley and wheat) are used because these cereals promote
egg dirtiness with fecal material. This problem can be minimized by using β-glucanases and
xylanases in the feed formulation. In addition, when the overall diet composition is
considered in terms of nutrition evaluation, the metabolized sugars resulting from the enzyme
www.wjpr.net Vol 9, Issue 2, 2020.
Vitolo. World Journal of Pharmaceutical Research
214
action generate an extra metabolic energy, which must be considered in formulating the
diet.[18]
Diets for pigs formulated with barley, wheat and agricultural and milling byproducts require
essentially the same enzymes as poultry diets. Poultry and pigs present physiological
differences (such as in secretion of endogenous enzymes) regarding the response to their
needs for exogenous enzymes. The activity peaks of amylase, lipase, pepsin and trypsin in the
small intestine of pigs are, respectively, reached after 33, from birth, 49 and 25 days after
birth, whereas for broilers, the peaks are reached after 5, 7, 10 and 15 days after birth,
respectively.[19]
Enzymes for animal feeding are additives whose assessment must match that of substrates –
whose contents in raw materials vary regarding the crop site, time of harvesting, climatic
conditions and species of cereal – in order to present a high efficiency. Consequently, a large
variety of enzyme preparations are available in the market. Thereby, the user can only
compare different enzyme preparations based on feeding trial, i.e., under practical conditions.
The enzyme producer, in turn, must guarantee that the enzyme is resistant to thermal
treatment (during the feed processing: 90oC/30 min) and transiting the digestive system intact
to the point of action (during animal feeding).
Cattle rearing generate huge amounts of manure rich in nitrogen and phosphorous
compounds, which ends up reaching subsoil and water supplies, rivers and the ocean coast
after being dissolved by rainwater. To minimize the environmental damage, the best way is to
optimize the metabolism of nitrogen and phosphorous by the animal, assuring that only a
small portion of these compounds is present in the manure. This can be achieved by
improving feed digestibility, adding amylases, proteases, xylanases, β-glucanase, β-
galactosidase and phytase to the animal feed. Phytase is an enzyme that decomposes phytate
salts in which the phosphorous is retained. By decomposing such salts, the phosphorous
element is freed and fully metabolized by the animal. A huge amount of phytase is needed to
meet the increase in animal rearing worldwide. Undoubtedly, the main trend on developing
enzymes for animal feeding is the optimization of the phytase production in large scale.[20]
ENZYMES AS ANALYTICAL TOOLS
Enzymes have been important reagents in analytical techniques used in clinical chemistry,
food and chemical analysis since 1960.
www.wjpr.net Vol 9, Issue 2, 2020.
Vitolo. World Journal of Pharmaceutical Research
215
Enzyme assays are often the method of choice in analysis because of their high specificity
and sensitivity. Because of their high specificity, samples often require little or no
purification prior to analyses. Enzymatic reactions can be run rapidly at or below room
temperature, often close to pH neutrality and in a few minutes. Under these conditions,
instability of compounds and/or enzyme is not a problem. Side reactions do not occur when
purified enzymes are used.
Enzymes can be used in soluble or insoluble forms.[21]
The soluble enzymes can be used either as catalysts for the determination of a compound
concentration (any inorganic or organic substance that serves as substrate, activator or
inhibitor) even at the order of ng/mL or pg/mL, or as a target for the substrate in order to
measure its catalytic activity. The latter approach leads to measuring the enzyme activity
present in a biological tissue, becoming a diagnostic tool to determine the state of health or
illness of humans. Moreover, enzyme activities are fast indicators of the quality of foods (for
example, zero activity of peroxidase and alkaline phosphatase, respectively, in blanched
vegetables and pasteurized milk is an indication that the heat treatment was properly
executed). Another use of enzymes is for the determination of absolute stereochemistry
and/or the primary, secondary or higher order structures of complex chemicals.
Substrate concentrations are determined enzymatically in two ways, i.e., end-point method
(in which the substrate is converted completely) and measurement of reaction rate (useful
when the reaction reach rapidly the equilibrium and/or the product is insoluble or volatile).
Some examples of reactions are:
1. Determination of glucose with glucose oxidase (GO), peroxidase (PER) and a chromogen
(pyrogalol, o-dianisidine; CH):
GLUCOSE + O2 + H2O (GO) → GLUCONATE + H2O2
H2O2 + CHREDUCED (PER) → H2O + CHOXIDIZED
2. Determination of cholesterol with cholesterol esterase (CE), cholesterol oxidase (CO),
peroxidase (PER) and a chromogen (CH):
CHOLESTEROLesterified + H2O (CE) → CHOLESTEROL + FATTY ACID
CHOLESTEROL + O2 (CO) → CHOLESTENONE + H2O2
H2O2 + CHREDUCED (PER) → H2O + CHOXIDIZED
www.wjpr.net Vol 9, Issue 2, 2020.
Vitolo. World Journal of Pharmaceutical Research
216
3. Determination of glucose with hexokinase (HK), glucose 6-phosphate dehydrogenase
(G6PDH) and NADP:
GLUCOSE + ATP — (HK) → GLUCOSE 6-PHOSPHATE + ADP
GLUCOSE-6-PHOSPHATE + NADP — (G6PDH) → GLUCONO--LACTONE + NADPH
4. Determination of triglycerides with lipase (LIP), glycerol kinase (GK), pyruvate kinase
(PK), lactate-dehydrogenase (LAD), ATP and phosphoenolpyruvate (PPP):
TRIGLYCERIDE + 3 H2O — (LIP )→ GLYCEROL + 3 FATTY ACID
GLYCEROL + ATP — (GK) → GLYCEROL 3-PHOSPHATE + ADP
ADP + PPP — (PK) → ATP + PYRUVATE
PYRUVATE + NADH+ H+ — (LAD) → L-LACTATE + NAD
5. Determination of the activity of alkaline phosphatase (AP) using the 4-
nitrophenylphosphate (4-NPP):
4-NPP + H2O — (AP) → PHOSPHATE + 4-NITROPHENOLATE
Enzymes in the immobilized form[21] can be used as analytical tools, presenting advantages
over the soluble form such as the repeated use for many assays and increased sensitivity and
stability. Moreover, immobilized enzymes are pivotal in diagnostic procedures because they
are components of auto-analyzers, test strips and biosensors.
Auto-analyzers incorporate in their configuration devices such as electronic circuit and
column filled with an immobilized enzyme. There are auto-analyzers for measuring ethanol
(alcohol dehydrogenase: ethanol + NAD → acetaldehyde + NADH), ammonia (L-
glutamate dehydrogenase: 2-oxoglutarate + ammonia + NADPH → glutamate + NADP),
glucose (glucose oxidase/peroxidase: glucose + O2 + 2H2O → gluconic acid + H2O + O2),
and uric acid (uricase/peroxidase: uric acid + 2H2O + O2 → allantoin + CO2 + H2O + O2).
The test strip consists on the immobilization of one or more enzymes in a flexible and porous
material, such as filter paper, which is also impregnated with a chromogen. The glucose test
strip for diabetics is by far the most sold device, in which glucose oxidase, peroxidase and o-
dianisidine are adsorbed on the dried surface of filter paper or plastic strip, which becomes
green when immersed in body fluids (blood or urine). High green intensity indicates high
glucose concentration, alerting the person to take a shot of insulin.[22]
www.wjpr.net Vol 9, Issue 2, 2020.
Vitolo. World Journal of Pharmaceutical Research
217
Biosensor or enzymatic electrode is a device whose sensing element is usually an enzyme
which is immobilized in close proximity to an electrode capable of collecting electrons from
or donating electrons to the enzyme reaction. The sensing element is reusable and does not
require any reagent to make the measurement, except the buffer for flushing the sample
chamber. The electrode allows carrying out dozens of determinations in a short period,
reducing significantly the cost of the clinical/chemical analysis. Moreover, this device does
not need sample preparation to remove colored compounds (e.g., whole blood) or solids (cell
debris) from the sample.[23]
Enzymes such as horseradish peroxidase, alkaline phosphatase, glucose 6-phosphate
dehydrogenase and lysozyme can be used as antibody markers in the enzyme-linked
immunochemical assay (ELISA). Enzymes can be bound to antibodies using such
bifunctional coupling reagents such as glutaraldehyde and 3-maleinimidobenzoyl-N-
hydroxysuccinimide depending on the nature of the reactive groups of the enzyme. One of
them consists of rabbit antibodies adsorption to the walls of a plastic micro-titer tray.
Antigen, at a concentration lower than the concentration needed to bind to all antibody sites,
is added and binds specifically to antibodies. Following washing, horse antibodies against the
antigen are added to form the rabbit antibody-antigen-horse antibody complex. Then, the
complex is treated with an enzyme coupled to rabbit anti-horse antibodies. The complex is
washed and evaluated as to bound enzyme by adding substrate and buffer at the desired pH.
Thereby, the components immobilized are rabbit antibodies, whereas the enzyme is the
marker of the rabbit anti-horse antibody, but this complex is soluble. ELISA can be
considered as a type of immobilization technique, in which the enzyme does not remain
immobilized all the time.[24]
ENZYMES AS DRUGS
The use of enzymes in therapeutics is not a typical industrial use. However, it deserves a few
words due to the significant commercial role enzymes play in healthcare, whose selling
revenues surpass US$ 1 billion per year. Only the mucopolysaccharosidases – traded as
Cerezyme® and Fabrazyme® and used to treat, respectively, Gaucher’s and Fabry’s
syndromes, both of genetic origin – has sales (in a conservative estimative) of about US$ 400
million per year.
The production of therapeutic enzymes involve a large diversity of industrial unit operations
to obtain them from cell culture, microbial fermentation or extraction from plants and
www.wjpr.net Vol 9, Issue 2, 2020.
Vitolo. World Journal of Pharmaceutical Research
218
animals, and to purify them using a variety of downstream protocols (salt/solvent
precipitation, filtration through ultra- or nanofiltration membranes, reverse osmosis,
chromatographic techniques etc.). Moreover, therapeutic enzymes are needed at low amounts
but at a high purity, contrarily to industrial enzymes, which are less purified preparations.
Enzymes in therapy resulted from attempts to take advantage of the specificity and efficiency
characteristics of enzymatic reactions. As any drug, they must meet requirements regarding
issues such as dosage, route of administration (intravenous or subcutaneous injection,
pulmonary or gastrointestinal tract delivery. In any case, the enzyme must be protected
against natural occurring proteases), bioavailability, mode of action (in the right tissue
compartment and under physiological conditions with respect to ionic milieu, substrate and
cofactor supply, and presence of endogenous inhibitors), and therapeutic value (outweighing
the adverse reactions as immunogenicity and cross-reactivity).[25]
Perhaps the most significant approach regarding the therapeutic use of enzymes (powerful but
delicate macromolecules) is the establishment of the adequate route for an effective
administration.[26]
The main pathways for administering enzymes to the body are a) nasal epithelium and
lungs: the challenge is to direct the enzyme to the alveoli and through them to enter the blood
stream without being destroyed by macrophages. The use of spray containing enzymes
molecules confined inside nanometer droplets seems a viable solution; b) skin: the enzyme
molecules must cross the epidermis and the endothelium of blood vessels. Such barriers can
be circumvented by using Iontophoresis (harmless electrical pulses) or ultra-sound vibrations
(sonication);[27] c) intestine: the enzyme must cross the epithelium and avoid being
hydrolyzed by proteases (abundant in the juice of intestinal lumen). To use this path, delivery
technology methods have been used: c1) the particles containing enzyme molecules are
covered with bio-adhesives, which adhere to the epithelium facilitating the crossing of the
intestine wall; c2) increasing the enzyme affinity with surface cell receptors by inserting
enzyme molecules within liposomes (encapsulated particles containing enzyme molecules).
They are absorbed by lipoprotein-coated cells of the reticulum-endothelial system, making
them reach the blood stream.[28] c3) linking the enzyme to a carrier molecule (salycilate, for
example), which normally cross the intestinal wall.
www.wjpr.net Vol 9, Issue 2, 2020.
Vitolo. World Journal of Pharmaceutical Research
219
A different form of delivering enzymes is the controlled liberation approach. It aims to
maintain the enzyme at a desirable level in the blood without repeated administration. This
can be achieved through implantable microchips containing reservoirs filled with enzyme,
which are covered by a thin gold sheet. This sheet can be dissolved by applying an electric
field, freeing the catalyst from time to time according to a delivering schedule.[27]
The enzyme therapy comprises controlling the activity of a metabolic enzyme – a specific
inhibitor is administered to the patient (Table 1) – or the enzyme is a drug per se, i.e., it must
be administered to the patient by pharmaceutical delivery form such as tablets, capsules,
injections, inhalation sprays, creams, ointments etc. (Table 2).
Table 1: Drugs used as inhibitors of human metabolic enzymes.
INHIBITOR
TARGET ENZYME
EFFECT
Zileutine
5-lipoxygenase
Anti-asthma, anti-inflammatory
Omeprazol
H+/K+-ATPase
Inhibition of gastric juice secretion
Aspirine
Ciclooxigenase
Anti-inflammatory
Alopurinol
Xantine oxidase
Inhibition of uric acid synthesis
Acetazolamide
Carbonic anhydrase
Interfere on the [(H2O + CO2)/H2CO3]
equilibrium
Zidovudine (AZT)
Reverse transcriptase
Anti-HIV
Sildenafil citrate
phosphodiesterase
GMPc accumulation
Non steroidal anti-
inflammatory
IKB-cinase
Decreases the action of the promoter NFKB
on RNA polymerase II of immune cells
Table 2: Examples of enzymes used as drugs.
ENZYME
ILNESS
Cerezyme®
Gaucher’s syndrome (type 1): congenital deficiency of -
glucocerebrosidase. Chronic, progressive and multi-systemic infirmity
Fabrazyme®
Fabry syndrome: congenital deficiency of -galactosidase, leading to
the accumulation of glycosphingolipids into the endothelium of blood
vessels, damaging several tissues and organs.
Aldurazyme®
Mucopolysaccharidose I resulting from the congenital deficiency of -
L-iduronidase
Streptokinase
Acute myocardial infarction
Hyaluronidase
Diffusion of local anesthetics; dissolution of hyaluronic acid deposited
inside the derma
Lysozyme
Anti-microbial
Pancreatin (trypsin, chymotrypsin,
-amylase and lipase),
Digestive disorder
Papain, trypsin, collagenase
Debridement of wounds
Snake venom protease
Blood rheology disorders
Urokinase
Thrombolysis
Thrombin
Blood clotting
www.wjpr.net Vol 9, Issue 2, 2020.
Vitolo. World Journal of Pharmaceutical Research
220
Asparaginase
Malignancies
Chymopapain
Intervertebral disk herniation
Superoxide dismutase
Inflammation and reperfusion injury
ENZYMATIC BIOTRANSFORMATIONS
The potential use of enzymes in organic synthesis was recognized since the beginning of the
last century. Probably the first reaction catalyzed by an enzyme was the hydrolysis of sucrose
by invertase to produce inverted syrup (a mixture of glucose and fructose at a 1:1 ratio).[29]
The broadening of enzyme use in organic synthesis depends on circumventing technical
handicaps such as cofactor regeneration, enzyme immobilization and enzyme stabilization.
Nevertheless, enzyme-based synthetic chemistry has grown because enzymes operate under
mild conditions of temperature and pH – so that sensitive substances can be handled. They
can start reactions which are difficult to emulate using more conventional chemical methods,
and they are able to distinguish an enantiomer in racemic mixture, and/or identify a
functional chemical group in a prochiral molecule to generate an optically active compound
(generally a pivotal intermediary in the synthesis of new pharmaceuticals, agrochemicals,
fragrances, flavors etc.).
Enzymes used in synthetic chemistry do not need to be extensively purified. Sometimes,
intact cells such as Saccharomyces cerevisiae, Pseudomonas putrida and Aspergillus niger
are used in the sucrose hydrolysis, conversion of methylbenzene into 1-methyl-2,3 dihydroxi-
5,6 cycle hexane and progesterone into 11-hydroxi-progesterone, respectively. Moreover,
enzymes may be used in immobilized forms (recovery and enhancing stability), and the
catalysis can occur in aqueous (for most enzymes) or organic solvent-water (for few
enzymes) medium.
The enzymes can be divided as follows: a) enzymes not requiring coenzymes: esterases,
lipases, amidases (e.g., penicillinamidase), aldolases (e.g., fructose 1,6-diphosphate aldolase),
lyases, hydrolases (e.g., -amylase and thermolisin) and isomerases (e.g., glucose isomerase);
b) enzymes requiring coenzymes, but not cofactor regeneration systems: the cofactor
(flavins, pyridoxal phosphate, thiamine pyrophosphate, lipoamide, and metal ions) is bound
tightly to enzyme molecule (e.g., glucose oxidase and peroxidase), often in a domain
different of the active site, and regenerates during the course of the catalysis; c) enzymes
requiring added coenzymes: approximately seventy percent of all enzymes require one
coenzyme (nucleoside triphosphate, nicotinamide derivatives, or coenzyme A). As
www.wjpr.net Vol 9, Issue 2, 2020.
Vitolo. World Journal of Pharmaceutical Research
221
coenzymes are expensive compounds, they must be regenerated in situ. For example, the
xylose conversion into xylitol by xylose reductase (NADPH-dependent enzyme) coupled
with the glucose 6-phosphate conversion into 6-phosphate gluconic acid by glucose 6-
phosphate dehydrogenase (NADP-dependent enzyme) was recently described.[30] There are
several types of organic synthesis reactions (Table 3).
Table 3: Examples of enzymes used in organic synthesis.
REACTION
ENZYME
EXAMPLE
Hydrolysis
Lipase
Ester Alcohol + Acid
Penicillinamidase
Penicillin G 6-aminopenicillanic acid
Nitrile hydratase
Acrylonitrile Acrylamide
Esterification
Lipase
2-chloropropanoic acid + n-butanol Butyl 2-
chloropropanoate + 2-chloropropanoic acid
Transesterification
Lipase
2-methyl-6-hydroxy-2-heptene + trichloroethyl
butanoate 2-methyl-2-heptenoyl butanoate +
2-methyl-6-hydroxy-2-heptene
Synthesis
Thermolisin
L-methyl-phenylalanine + N-carbobenzoxy-
(L)-aspartic acid N-carboxy aspartame
Reduction
Xylose reductase
Xylose Xylitol
Oxidation
Glucose oxidase
Glucose Gluconic acid + H2O2
Condensation
Fructose-1,6-
diphospho aldolase
D-glyceraldehyde-3-phosphate + ketone
dihydroxy-phosphate Fructose-1,6-
diphosphate
Isomerization
Glucose isomerase
Glucose Fructose
Addition
Fumarase
Fumaric acid + H2O Malic acid
Fumaric acid + NH4+ L-aspartic acid
Complex and labile molecules of large use in immunology, endocrinology, intermediary
metabolism, molecular genetics, plant and insect biology (pheromones) can be handled only
by enzymes, which are enantiomer and regioselective catalysts. Undoubtedly, the classical
synthetic organic chemistry techniques are well complemented by enzymology. Today 500
out of 3,000 known enzymes are used in industry, analysis, waste treatment, and therapy.
However, the portfolio of enzymes marketed has been growing as recombinant DNA and cell
fusion techniques develop. Moreover, progress in protein engineering has been remarkable in
the production of synthetic catalysts.
CONCLUSION
Enzymes constitute a versatile group of catalysts as can be seen through the diversity of uses
analyzed. Undoubtedly, this is a consequence of the incomparable qualities presented by
these catalysts, i.e., specificity, enantiomer selectivity and plentiful activity under mild
reaction conditions. Although only 500 out of 3,000 enzymes known are used, they share
www.wjpr.net Vol 9, Issue 2, 2020.
Vitolo. World Journal of Pharmaceutical Research
222
about 20% (a conservative estimation) of the worldwide biotechnological market.
Furthermore, the number of enzymes marketed has been growing because of the
developments in genetic, protein engineering and immobilization techniques.
FUNDING: This work was supported by the National Council for Scientific and
Technological Development CNPq (grant no. 303082/2015-1).
REFERENCES
1. Müller S, Appel B, Balke D, Hieronymus R, Nübel C. Thirty-five years of research into
ribozymes and nucleic acid catalysis: where do we stand today? F1000 research, 2016; 5:
doi 10.12688/f1000 research.8601.1.
2. Vitolo M. Extraction of invertase from Saccharomyces cerevisiae: effect of rheological
parameters. World Journal of Pharmaceutical Research, 2019; 8(6): 12-19.
3. Eriksen N. Detergents. In: Godfrey T, West S (eds). Industrial Enzymology, London;
MacMillan Press, 1996; 187- 200.
4. Hares-Jr SJ, Ract JRN, Gioielli LA, Vitolo M. Medium oleic sunflower oil hydrolysis by
microbial lipase in a fed-batch bioreactor. World Journal of Pharmacy and
Pharmaceutical Sciences, 2019; 8(12): 94-108.
5. Basketter DA, Kruszenski FH, Mathieu S, Kirchner DB, Panepinto A, Fieldsend M.
Managing the risk of occupational allergy in the enzyme detergent industry. Journal of
Occupational and Environmental Hygiene, 2015; 12(7): 431-437.
6. Vitolo M. Overview on the biological role of free radicals. World Journal of
Pharmaceutical Research, 2019; 8(13): 23-46.
7. Moses V, Cape RE. Biotechnology: the science and the business, Chur; Harwood
Academic Publishers, 1994.
8. Wigley RC. Cheese and Whey. In: Godfrey T, West S (eds). Industrial Enzymology,
London; MacMillan Press, 1996; 133-154.
9. Na SY, Lee Y. Elimination of trace organic contaminants during enhanced wastewater
treatment with horseradish peroxidase/hydrogen peroxide catalytic process. Catalysis
Today, 2017; 282(1): 86-94.
10. Schwendenwein D, Fuime G, Weber H, Rudroff F, Winkler M. Selective enzymatic
transformation to aldehydes in vivo by fungal carboxylate reductase from Neurospora
crassa. Advanced Synthesis and Catalysis, 2016; 358(21): 3414-3421.
11. Godfrey T, West S. Industrial Enzymology. New York; Stockton Press, 1996.
www.wjpr.net Vol 9, Issue 2, 2020.
Vitolo. World Journal of Pharmaceutical Research
223
12. Souza FR, Guterres M. Application of enzymes in leather processing: a comparison
between chemical and coenzymatic processes. Brazilian Journal of Chemical
Engineering, 2012; 29(3): 473-482.
13. Araújo R, Casal M, Cavaco-Paulo A. Application of enzymes for textile fiber processing.
Biocatalysis and Biotransformations, 2008; 26(5): 332-349.
14. Milek J, Wójcik M, Verschelde W. Thermal stability for the effective use of commercial
catalase. Polish Journal of Chemical Technology, 2014; 16(4): 75-79.
15. Singh S, Singh VK, Aamir M, Dubey MK, Patel JS, Upadhyay RS, Gupta VK. Cellulase
in pulp and paper industry. In: Elsevier (ed). New and Future Developments in Microbial
Biotechnology and Bioengineering, New York, Elsevier, 2016; 152-162.
16. Buzala KP, Kalinowska H, Borkowski J, Przybysz P. Effect of xylanases on refining
process and Kraft pulp properties. Cellulose, 2017; DOI:10.1007/s 10570-017-1609-y.
17. Puri M, Sharma D, Barrow CJ. Enzyme-assisted extraction of bioactives from plants.
Trends in Biotechnology, 2012; 30(1): 1-8.
18. Dalolio FS, Moreira J, Vaz DP, Albino LFT, Valadares LR, Pires AV, Pinheiro SRF.
Exogenous enzymes in diets for broilers. Revista Brasileira de Saúde e Produção Animal,
2016; 17(2): 149-161.
19. Ojha BK, Singh PK, Shrivastava N. Enzymes in the animal feed industry. In: Enzymes in
Food Biotechnology, Boston, Academic Press, 2019; 93-109.
20. Romano N, Kumar V. Phytase in animal feed. In: Enzymes in Human and Animal
Nutrition, Boston, Academic Press, 2018; 73-88.
21. Vitolo M. Calcium-alginate beads as carriers for biocatalyst encapsulation. World Journal
of Pharmaceutical Research, 2019; 8(10): 1-17.
22. Wilson KG, Ovington PBS, Dean D. A low-cost inkjet-printed glucose test strip system
for resource-poor settings. Journal of Diabetes Science and Technology, 2015; 9(6):
1275-1281.
23. Pereira AR, Sedenho GC, Souza JCP, Crespilho FN. Advances in enzyme
bioelectrochemistry. Annals of the Brazilian Academy of Sciences, 2018; 90(1): 825-857.
24. Tian W, Wang L, Lei H, Sun Y, Xiao Z. Antibody production and application for
immunoassay development of environmental hormones: a review. Chemical and
Biological Technologies in Agriculture, 2018; DOI: 10.1186/s 40538-018-0117-0.
25. Vellard M. The enzymes as drug: application of enzymes as pharmaceuticals. Current
Opinion in Biotechnology, 2003; 14(4): 444-450.
26. Lauwers, A.; Scharpè, S. Pharmaceutical Enzymes. New York; Marcel Dekker, 1997.
www.wjpr.net Vol 9, Issue 2, 2020.
Vitolo. World Journal of Pharmaceutical Research
224
27. Langer R. Drug delivery: drugs on target. Science, 2001; 293: 58-59.
28. Daraee H, Etemadi A, Kouhi M, Alimirzolu S, Akbarzadeh A. Application of liposomes
in medicine and drug delivery. Artificial Cells, Nanomedicine and Biotechnology, 2016;
44: 381-391.
29. Tomotani, E.J.; Vitolo, M. Production of high fructose syrup using immobilized invertase
in a membrane reactor. Journal of Food Engineering, 2007; 80: 662-667.
30. Tomotani EJ, Arruda PV, Vitolo M, Felipe MGA. Obtainment of partial purified xylose
reductase from Candida guilliermondii. Brazilian Journal of Microbiology, 2009; 40:
631-635.
