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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5864_Библиотеки_им_академика_М_И_Перельмана.pdf
Introduction to Pharmaceutical Biotechnology, Volume 2 (Second Edition)
companies have emerged, such as Novozymes dominating with 45% sales, followed
by Danisco which holds a 20% share of the market. Bulk enzymes, such as proteases,
amylases, lipases, etc, which are required in large quantities have, however, an
intrinsically low unit value, so they demand significantly lower manufacturing costs.
In contrast, the therapeutics sector includes enzymes such as urokinase, which are
produced in lower quantities and at greater manufacturing cost. The technologies
involved in the production of enzymes unite the disciplines of microbiology,
genetics, biochemistry and engineering. Requirements for new enzymes can be
achieved by the development of new procedures or the development of new
procedures to address the unsatisfactory performance of known enzymes. Recent
developments in gene technology have had a large impact on the enzyme industry.
Genetic engineering tools have allowed enzyme manufacturers to synthesize
adequate amounts of any enzyme irrespective of source, whereas protein engineering
facilitates alteration of the enzymes before production. This chapter offers an outline
of enzyme production procedures beginning from raw materials to the finished
product, and also offers an understanding of the different alternative technologies
existing for different phases of production.
2.1.1 Sources of enzymes
These enzymes are used for applications in industries on commercial scales [4]. There
are various applications of these enzymes, for example, softening hides by using the
feces of dogs and pigeons before tanning. A German researcher named Otto Rohm
reported in 1905 that extracts from animal organs (pig and cow pancrease) could be
used as the source of the enzyme protase, for leather softening [4]. The utilization of
enzymes (mainly proteases) for cleaning laundry began in 1915. However, this was
not initially successful due to allergic reactions to impurities in the enzymes.
Currently, advance techniques are available for purification and the use of enzymes
in washing powders (without allergic reactions) is common. A real boom in the
large-scale industrial production of enzymes from microorganisms occurred after the
1950s. From the industrial point of view, enzymes can be derived from different
sources, however, microbes act as an active source for unique biological enzymes
and thus most enzymes are derived from microbial sources [4]. A wide variety of
living organisms can serve as sources of enzymes, as depicted in figure 2.1.
2.1.1.1 Enzymes from animal and plant sources
A few decades ago, plants and animals were considered the major sources for
enzymes. Even today, they are still major sources of certain enzymes [4], such as
lipases, esterases and protease. Hen’s eggs and human milk contain high amounts of
lysozyme. This lysozyme with bovine lactoferrin offers innate immunity to an infant
by killing gram-negative bacteria. Certain plants such as papaya (papain) and
pineapple (bromelain) are excellent sources of enzymes. The following lists a few
recent breakthroughs and discoveries:
• Enzymes present in herbivore gut fungi and their potential applications in
fuels and chemical industry [5].
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Introduction to Pharmaceutical Biotechnology, Volume 2 (Second Edition)
70
60
50
40
30
20
10
0
Enzyme sources
Overall %age
Figure 2.1. Enzyme sources.
• Bacterial enzymes responsible for human infection [6].
• Gro3P phosphatase responsible for the conversion of glucose and fats into
other compounds (i.e., regulate how your body converts sugar and fats) [7].
• A class of DNA repair enzymes (DNA glycosylase) [8].
• An enzyme (reductive aminase) that will make a drug used to treat
Parkinson’s disease [9].
Enzyme production involves many critical steps which present major challenges in
the production of enzymes from different sources. Lack of suitable isolation and
purification procedures, low yield and high cost of overall process are the major
challenges facing enzyme-based industries. Certain enzymes are more closely associated with cell walls and face certain problems such as low concentration, insolubility
and maintaining biological activity. Offering an optimal pH and isoelectric point
during extraction is an important factor in determining the biological activity of these
enzymes. Thus selection of the purification procedure and the correct physical and
chemical conditions also helps to retain the biological activity of an enzyme. Certain
enzymes are very sensitive to variation in environmental factors, and can immediately
lose activity, so their catalytic activity needs to be maintained by using a continuous
process to accelerate the purification process. One of the recent developments for
purification is a miniaturization automated approach that not only accelerates but also
optimizes and statistically analyzes the enzymes during purification. The utilization of
chromatographic techniques (aided by the development of new resins) and ultrafiltration are the main approaches to enzyme purification. The contamination and
toxicity of enzymes are again major challenges that can be minimized during the
production procedure, e.g. enzymes derived from bovine sources involve a high risk of
contamination with bovine spongiform encephalopathy (an illness caused by the
ingestion of abnormal proteins called prions). These prions can be carefully heatinactivated at a certain temperature which will not affect the enzyme properties. Due
to these serious concerns, microbial sources are generally preferred over plant- and
animal-based enzymes [10, 11].
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Introduction to Pharmaceutical Biotechnology, Volume 2 (Second Edition)
2.1.1.2 Enzymes derived from mammalian cell cultures
Mammalian cell cultures offer a continuous supply of enzymes that are commercially important (table 2.1), however, a major constraint is the cost, which can be
extremely high [12]. With more advancements in tissue culture, in particular in the
mass cultivation of cells and the availability of cheaper culture media, it is now
possible to affordably produce many therapeutic enzymes such as urokinase.
Further advancements in mammalian cell culture are exploring the application of
cell immobilization, capillary culture, large-scale suspension culture and perfusion
techniques. By using such approaches it is now possible to produce therapeutic
enzymes, such as tissue plasminogen activator, at a large scale.
2.1.1.3 Microbial enzymes
Enzymes derived from microbes are referred to as microbial enzymes. Microbial
enzymes were exploited for many decades without a complete understanding of how
they work. Microbes are considered as the most reliable source for the production of
commercial enzymes. In fact, many such enzymes are already being used on a
commercial scale (table 2.2).
Since the rate of cell growth and proliferation in microbes is very high, a large
amount of enzymes can be produced by culturing microorganisms under controlled
in vitro environments. The media and other facilities involved in culturing microbes
are economical in comparison to using plants and animals [4]. Moreover, plant and
animal cells are more susceptible to contamination than microorganisms.
Recombinant DNA technology can be utilized to increase the production of a
desired protein in microorganisms, which is more challenging in plant and animal
cells because of their high genetic complexity as eukaryotes compared to prokaryotes. This notion is based on the sexual mode of reproduction in eukaryotes which
offers more genetic variation then prokaryotes (asexual reproduction).
To satisfy industrial demands, microbial sources must allow the proper recovery,
isolation and purification of enzymes. The production procedure for enzymes is
described in figure 2.2. The initial step in isolation is to select the best source that
allows easy production of the enzyme within the shortest amount of time. Since
microbial strains can grow rapidly and can be manipulated with the help of genetic
Table 2.1. Commercially produced enzymes from animal sources and their applications.
Enzyme(s) Source(s) Application(s)
Amylase, esterase Lamb,
calf
Pepsin, trypsin Bovine
Lipase, rennin (chymosin) phospholipase,
phytase
Lysozyme Hen eggs Cell wall breakage in bacteria
Human urine Urokinase For dissolution of blood clots
Porcine
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Digestive aids, preparation of
cheese
Introduction to Pharmaceutical Biotechnology, Volume 2 (Second Edition)
Table 2.2. Selected list of industrially (microbially) produced enzymes, their sources and applications.
Enzyme Source(s) Application(s)
α-amylase Aspergillus oryzae, Aspergillus
niger, Bacillus subtilis,
Bacillus licheniforms
Amyloglucosidase A. niger, Rhizopus niveus Starch hydrolysis
Cellulase A. niger, Tricoderma koningi Alcohol and glucose production
Glucoamylase A. niger, Bacillus
amyloliquefaciens
Glucose
isomerase
Glucose oxidase A. niger Antioxidant in prepared foods
Invertase Saccharomyces cerevisiae Sucrose inversion; preparation of artificial
Keratinase Streptomyces fradiae Removal of hair from hides
Lactase Kluyveromyus spp.,
Lipase Candida lipolytica, A. niger Preparation of cheese; flavor production
Pectinase Aspergillus spp., Sclerotina
Penicillin acylase Escherichia coli Production of 6-aminopenicillanic acid
Penicillanase B. subtilis Removal of penicillin
Protease, acid A. niger Digestive aid; substitute for calf rennet
Protease, neutral B. amyloliquefaciens Fish and meat tenderizer
Protease, alkaline A. oryzae, Streptomyces griseus,
Pollulanase Klebsiella aerogens Hydrolysis of starch
Takadiastase A. oryzae Bread supplement; digestive aid
Arthrobacter spp., Bacillus spp. Manufacture of high fructose syrups
Saccharomyces fragilis
medina
Bacillus spp.
Production of beer and alcohol;
preparation of glucose syrups; as a
digestive aid; removal of starch sizes
Production of beer and alcohol; starch
hydrolysis
honey; confectionaries
Lactose hydrolysis; removal of lactose
from whey
Clarification of fruit juices and wines;
alcohol production; coffee
concentration
Meat tenderizer; detergent additive; beer
stabilizer
engineering, i.e., rDNA and gene editing tools, they are considered as the best
sources for the production of a large variety of enzymes. Several therapeutic
enzymes have been successfully produced from different microbial sources, as shown
in table 2.2.
2.1.1.3.1 Aspergillus niger for the production of bulk enzymes
Enzymes derived from fungal sources such as Aspergillus, Rhizopus and Penicillium
are considered as safe. These sources all produce extracellular enzymes which can be
conveniently recovered. Although various microorganisms produce extracellular
enzymes, the most frequently utilized in industrial applications is Aspergillus niger.
A. niger,afilamentous fungi, produces significant amounts of extracellular enzymes
and citric acid, which are widely used for industrial purposes. So far, more than 50
enzymes have been isolated from this source, including various commercial enzymes
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Introduction to Pharmaceutical Biotechnology, Volume 2 (Second Edition)
Figure 2.2. Schematic representation of the production of enzymes by microorganisms.
that are conveniently produced by A. niger. Some of the key enzymes are protease,
insulinase phytase, catalase α-amylase, lipase, pectinase and cellulase [4]. This acid
tolerant microorganism has significant hydrolytic potential in the production of αamylase, and it also prevents bacteria-mediated contamination [13].
2.2 Enzyme production technology
Generally, the procedures used for microbial production of enzymes are equivalent
to the methods used for the production of other industrial products. The significant
features are, briefly[4]:
• Selection of organisms.
• Formulation of medium.
• Production process.
• Recovery and purification of enzymes.
A flow chart for enzyme production by microorganisms is depicted in figure 2.3.
2.2.1 Selection of microorganisms
Appropriate selection of microorganisms allows a high rate of production of
enzymes. Selection can be achieved using different approaches, such as mutagens
and radiation, in particular UV rays. The selection procedure for microorganisms is
described in figure 2.4. The most significant factor in selecting microorganisms is
that the organism should produce a high amount of the desired enzyme in a very
short time, while the quantities of other metabolites produced should be low. After
selecting the organism, strain improvement to optimize enzyme production can be
achieved using suitable methods (as above, using mutagens or UV rays). After
selection of the microorganisms, inoculum can be prepared in a liquid medium.
In enzyme research, the identification of enzymes is an important goal. The
classical selective microbial screening method helps in discovering enzymes in
populations of microorganisms. During this procedure organisms are selected and
then individually isolated to develop pure cultures. A pure culture can be propagated
to a higher level, however, only few colonies are obtained from this pure culture.
Due to syntrophic benefits (interspecies interaction) it is again challenging to isolate
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Introduction to Pharmaceutical Biotechnology, Volume 2 (Second Edition)
Figure 2.3. Enzyme production at an industrial scale.
pure colonies under standard conditions. Thus only 1% of pure culture can be
derived from this classical selection method [14].
However, if pure cultures are only considered for enzyme isolation, then the
maximum potential of the microorganisms, which are capable of producing other
novel enzymes, will be ignored. There are vast numbers of microorganisms present in
nature and it is challenging to cultivate all of them using classical microbiological
techniques. The arrival of metagenomics allowed the retrieval of genetic material from
the microbial environment without using any cultivation methods. At present,
metagenomics are extensively used as a technique to isolate and identify enzymes
with unique properties from the uncultivable part of microbial populations. These
techniques allow researchers to establish the relationship between the genetic material
(the genes responsible for enzyme production), the enzyme utilized (or whether an
enzyme is utilized or not) and their properties. Recent developments in the field of
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Introduction to Pharmaceutical Biotechnology, Volume 2 (Second Edition)
Figure 2.4. Microorganism selection for enzyme production by the classical method.
enzyme technology allow the exploration of new approaches, such as metaproteomics.
Most importantly, using metagenomics it is possible to discover the genetic potential
required by a cell to produce enzymes, i.e., identification of those particular genes that
are responsible for the synthesis of the desired enzyme. After identification of the gene,
there remains the challenge to discover whether the enzyme is synthesized under a
particular environment and also whether the enzyme produced will carry all the
desired properties. To further improve the selection process, the study of symbiotic
environments (consortiums) was suggested, by integrating various omics-techniques,
such as metagenomics (shotgun sequencing methods), metatranscriptomics and
metaproteomics (extensive characterization of the whole protein complement of one
microbiota condition at a particular time) [15, 16]. ‘At a particular time’ is significant,
as the protein expression at different times helps in understanding protein dynamics.
Consequently, metaproteomics allows temporal interpretation studies of protein
expression, in comparison to the observational studies of the static metagenome.
2.2.2 Medium selection
In the classical approach, a suitable culture medium with defined composition is
selected that contains all the vital components to encourage the growth of only those
microorganisms that are capable of producing relatively large amounts of enzymes.
The nutritional component of the culture medium should be available at a low price
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Introduction to Pharmaceutical Biotechnology, Volume 2 (Second Edition)
and should be safe. For the growth of anchorage-dependent microorganisms, a
number of substrates in the medium have been explored, such as yeast extract, whey,
starch hydrolysate, corn steep liquor, molasses and soybean meal. In traditional
approaches of fermentation, cereals (wheat) and pulses (peanut) have been utilized
as substrates. Optimal pH is required for significant microbial growth and also plays
an important role in enzyme production.
2.2.3 Production process
The production of enzymes is often performed at larger scales using fermentation
techniques, in particular submerged fermentation (the development of microorganisms
in a liquid broth) and solid-substrate fermentation (the development of microorganisms on a solid substrate, e.g. rice bran or wheat bran). In submerged fermentation a
liquid broth is utilized to offer nutrients that result in the production of industrial
enzymes, antibiotics or other products. At the industrial scale, submerged liquid
fermentation procedures are classically employed for the production of enzymes from
microbial sources, whereas solid-substrate fermentation is rarely used for this purpose.
This is because submerged liquid fermentation offers a number of advantages, such as
ease of handling and greater control of environmental factors (such as temperature
and pH). However, the solid-substrate fermentation method can also be utilized to
improve the yield and decrease the cost of enzyme production [17, 18].
Solid-substrate fermentation is an alternative approach to submerged fermentation which is often employed for the production of fungal enzymes such as cellulases,
amylases, proteases and pectioases [19]. During this process microorganisms are
developed under a controlled environment to produce enzymes, fuel and nutrients.
Sterilization is not essentially required for solid-substrate fermentation, as the
fermentation substrate starts sterilization and the microbes inhibit the growth of
micro-flora. It has been reported that both approaches (submerged and solidsubstrate fermentation) are considered as effective techniques to produce several
valuable products. Due to advancements in fermentation technology, particularly in
solid-substrate fermentation, bioreactors of different sizes have now been designed.
The design of bioreactors is based on the principles of biochemical engineering,
including mathematical modeling, which is important in defining the cellular
development of microorganisms. Several problems, such as heat and mass transfer,
can be overcome using such bioreactors.
For fermentation (if required), batch and continuous sterilization techniques can
be employed to sterilize a suitable medium. Afterwards the medium is inoculated
with a suitable microorganism to initiate the fermentation process. The culture’s
environmental conditions, such as pH, temperature, O
and nutrient supplementa-
2
tion are initially optimized and later maintained to achieve the desired growth of
microorganisms. The addition of an antifoaming agent is required to inhibit surface
tension. Production also involves fermentation kinetics, the mathematical analysis
of certain factors that helps in designing or improving the batch and continuous
process. It has been observed that continuous fermentation processes give a lower
enzyme yield, thus enzyme production is often conducted using batch fermentation,
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Introduction to Pharmaceutical Biotechnology, Volume 2 (Second Edition)
and only rarely using the continuous process. The vessel should be sterile during the
fermentation process. Aseptic conditions can be maintained by effective management of vessel design, operating procedures, continuous monitoring and maintenance, and skilled technicians. Usually, the duration of the fermentation process
varies from 2 to 7 days. Certain additional metabolites are also produced in the
medium which can interfere with recovery of the desired enzyme, however, only
desired enzyme(s) should be recovered and purified.
2.2.4 Recovery and purification of enzymes
After developing the selected microorganisms or establishing a pure culture, the
recovery of suitable extracellular enzymes is again a challenge, since the culture
medium can be full of unwanted end-products released by the stationary phase of the
microorganisms. These unwanted substances can interfere with the recovery of
suitable enzymes. Since the recovery of extracellular enzymes is easier than that of
intracellular enzymes, based on the recovery process used, commercial enzyme
preparations are available in crude or highly purified form. However, other factors
such as the form of medium (solid or liquid), viscosity, the nature of the enzyme and
the degree of purity desired, etc, affect the recovery of enzymes. As discussed,
downstream processing, which involves recovery and purification steps for extracellular
enzymes, is easier compared to intracellular enzymes which require more advance
techniques for cell lysis. The selection of a suitable hydrolyzing agent for intracellular
enzyme recovery is a crucial step. It requires a basic understanding of cell membrane
physiology and should be carried out such that the enzyme properties are not affected.
Different physical, chemical and enzymatic hydrolyzing agents have been reported to
break the cell wall and release their intracellular material [20]. However, the recovery of
enzymes closely associated with the membrane is challenging. Cell lysis is always
followed by differential centrifugation which allows the separation of cytoplasmic
particles on the basis of size and specific gravity. For recovery from microbial sources,
the cell membrane can be lysed by physical means, such as sonication, high pressure
and glass beads. Lysozyme is often used as a hydrolyzing agent for the lysis of the
bacterial cell wall. For cell wall breakdown in yeasts, the carbohydrate enzyme β-
glucanase is used to break down the glycosidic bonds within beta-glucans, which allows
the release and solubilization of these polymers from the cell walls. However,
enzymatic procedures have the disadvantage of their high cost. Cellulose-degrading
enzyme methods are often employed for intracellular enzyme recovery. The methodologies utilized for both intracellular and extracellular enzyme extraction always
remain same, however, the hydrolyzing agents often differ. The objective of both
procedures is to decrease the loss of the anticipated enzyme and its activity [20].
2.2.5 Cell debris removal
Procedures such as cross-flow microfiltration with rapid back pulsing and differential
centrifugation can be employed to remove cell debris. For thermostable enzymes, a
more convenient method is to denature the unwanted proteins by heating, then cooling
the solution, and finally unwanted proteins can be removed by centrifugation.
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Introduction to Pharmaceutical Biotechnology, Volume 2 (Second Edition)
2.2.6 Nucleic acid removal
Non-proteinaceous contamination, such as nucleic acids, restricts recovery and
purification of enzymes. These contaminants can be removed by precipitation
processes using polyamines, streptomycin and polyethyleneimine [21]. The presence
of DNA/RNA contaminants can be examined by blotting techniques and simple
wavelength scan assessment of DNA/RNA.
2.2.7 Precipitation of enzymes
In a solution, salt concentration generally determines the protein solubility. During
the process called salting-in, the concentration of salts is low, which can stabilize
several charged groups over the protein molecule, and which can ultimately increase
the solubility of the protein. In contrast, an increase in salt concentration increases the
solubility to a particular point, afterwards a further increase in the salt concentration
increases precipitation of proteins as water molecules are now replaced with salt
concentration, so insufficient water molecules for solubilization of the protein are
available. Precipitation of protein in the presence of a surplus amount of salt is called
salting-out. Various salts can be utilized for this phenomenon. Ammonium sulfate is
often used for this purpose as it has high solubility and is comparatively inexpensive.
Ammonium sulfate reduces the solubility of proteins and enhances the precipitation,
which may further increase the stability of the native conformation. Precipitation is
very beneficial in downstream processing, as the precipitated enzyme can be
solubilized in a minimal volume to concentrate the enzyme [22].
2.2.8 Liquid–liquid partition
The desired enzymes can be obtained by liquid–liquid chromatography using poly-
ethylene glycol or polyamines. Liquid–liquid partition chromatography can also be
utilized to identify conformational changesin well-characterized enzymes [23]. Various
advanced liquid–liquid fractionation techniques have emerged, such as the aqueous
two-phase system, which was initially accidently utilized by Martinus Willem
Beijerinck (1896) by mixing an aqueous solution of starch and gelatin [24, 25]. The
influence of organic solvents on the partitioning of enzymes in aqueous twophase systems was studied in 1987 [24]. As per this report, partition coefficients for
alkaline phosphatase decrease with ethylene glycol, glycerol, sucrose and urea. In
1996, the effects of low temperature (−18 °C) on glycolytic enzymes (e.g. phospho-
fructokinase) were investigated in terms of the stability and partitioning within an
aqueous two-phase system [26]. It was found that the presence of ethylene glycol,
phase polymers and low temperature stabilized enzyme activities.
This technique has now gained much attention because of its great potential for
the extraction, separation, purification and enrichment of proteins, in particular
enzymes, nucleic acids and other biomolecules. This complex partition behavior is
formed by mixing a variety of components in water. As water is the main component
of both phases in aqueous two-phase systems, it offers a protected environment to
biomolecules for their separation and polymers to stabilize their structure and
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