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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5586_Библиотеки_им_академика_М_И_Перельмана.pdf
Introduction to Pharmaceutical Biotechnology, Volume 2 (Second Edition)
effectiveness. This section will look at the various techniques used in enzymatic
engineering [105].
2.6.1 Protein engineering strategies
The study of protein engineering is a multidisciplinary field that aims to increase the
functioning of proteins and enzymes by improving their balance, procedure, and
substrate selection via a holistic strategy. Protein engineering uses a broad number of
strategies to accomplish these objectives. This study will examine various methodologies frequently employed in protein engineering [106]. In rational protein design,
one of the most critical steps is the alteration of the amino acid sequence of an
enzyme on purpose. This modification must be based on previous knowledge of the
connection between the structure of the enzyme and its function. The combined
application of computational analysis and molecular modelling approaches enables
the identification of specific amino acids that might be modified to enhance the
stability, attachment of substrates, and catalytic effectiveness of an enzyme [107].
Enzymes with thermophilic properties may be produced in a controlled laboratory
setting. Scientists have effectively created thermophilic enzymes, which are characterized by high levels of catalytic effectiveness and stability. This phenomenon may
be linked to their capacity to operate at heightened temperatures with optimum
efficiency. Thermophilic DNA polymerases have shown benefits for polymerase
chain reaction (PCR) and other molecular biology techniques, owing to their
increased resilience against denaturation at high temperatures [108] (table 2.14).
2.6.1.1 Directed evolution
The method of continually changing the enzyme via regulated mutagenesis and
choosing mutants with desired features is directed evolution. Mutants with improved
stability, substrate selectivity, or activity may be generated by subjecting the enzyme
to different selection pressures. Temperature stability of evolved enzymes, enzyme
thermostability, has been successfully enhanced by researchers using directed
Table 2.14. Examples of rational protein design for enhanced stability and activity.
Enzyme Modification Impact on stability/activity
DNA
polymerase
α-Amylase Creating mutations at specific sites to
Proteases Designed protection from proteolytic
Lipases Durability in organic solvents is much
Chimeric
enzymes
Mutations that are heat-resistant are
introduced
enhance the binding of substrates
breakdown
improved
Combining the work of multiple enzymes
into one
A more stable and active state at
elevated temperatures
Increased catalytic activity
Sustained steadiness under
pressure
Enhanced behaviour in non-
aqueous settings
Increased substrate and catalytic
diversity
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Introduction to Pharmaceutical Biotechnology, Volume 2 (Second Edition)
Table 2.15. Examples of directed evolution for enhanced stability and activity.
Enzyme Mutation technique Impact on stability/activity
Lipases PCR errors, DNA jiggery-pokery, and site-
specific mutagenesis
Cellulases Development on a cellulose-based substrate
via guided development
Proteases Saturation mutagenesis iteratively Modified stability and substrate
Peroxidases Hydrogen peroxide-mediated laboratory
evolution
Table 2.16. Examples of immobilization-assisted enzyme engineering.
Enzyme Immobilization strategy Impact on stability/activity
Lipases High-throughput screening by
immobilization on solid frameworks
Glucose
oxidase
Proteases Capture on chromatographic resins Proteolytic processes with enhanced
Cytochrome
P450s
Adherence to biosensor surfaces Improved glucose detection stability
Investigations of drug metabolism via
immobilization in microreactors
Heat regulation and reactivity are
both improved
Enhancement of substrate binding
and catalytic activity
specificity
Peroxide reduction catalysis is
accelerated
Finding more effective variations for
catalysis in organic solvents
and sensitivity
substrate selectivity and stability
Streamlined variant screening for
medical uses
evolution. Guided evolution has resulted in more robust lipases to higher temperatures, allowing them to function in high-temperature industrial settings [109]
(table 2.15).
2.6.1.2 Immobilization-assisted enzyme engineering
Enzyme immobilization has the potential to serve as a convenient platform for
testing mutant libraries for enhanced stability and activity in enzyme engineering.
Screening immobilized enzymes using high-throughput technologies might expedite
the development of superior variants. For enzyme immobilization at high throughput in a lipase engineering study, immobilization facilitated a rapid screening of
mutant libraries for improved catalytic efficiency and substrate selectivity. This
technique was used to identify variations of lipase with enhanced activity in organic
solvents. These are helpful for commercial uses that do not include water [110]
(table 2.16).
2.6.1.3 Site-directed mutagenesis
The protein-coding gene is targeted for precise modification in site-directed mutagenesis. Amino acid substitutions brought on by these mutations alter the protein’s
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Introduction to Pharmaceutical Biotechnology, Volume 2 (Second Edition)
Table 2.17. Examples of site-directed mutagenesis for enhanced protein properties.
Protein Mutation(s) Impact on protein properties
HIV protease Sub-stratum-binding residues improved catalytic activity and
substrate selectivity
Green fluorescent
protein (GFP)
Serine protease Mutations that are thermostable are
α-Amylase Substrate-specific amino acid
Peroxidase Substrate specificity modification
Fluorescence-altering amino acid
substitutions
introduced
substitutions
by site-directed mutagenesis
Fluorescence emission tuning for
various uses
Enhanced stability and activity in the
presence of heat
Increased catalytic activity on some
substrates
Peroxide reduction now works in a
broader variety of substrates.
amino acid sequence and, by extension, its structure and function. Researchers may
study an enzyme’s stability and activity after introducing different amino acids to
tailor an enzyme to a particular task. Enhanced thermostability of DNA polymerase
site-directed mutagenesis has been used to introduce thermostable mutations into
DNA polymerases for use in PCR. These modifications have increased the enzyme’s
stability against denaturation in PCR processes at high temperatures [111]
(table 2.17).
2.6.1.4 Circular permutation
Protein engineering techniques like circular permutation allow for modifying a
protein’s amino acid sequence. In this technique, the peptide backbone is cleaved in
a specific spot and then rejoined to create a circularized variant. This procedure
results in the creation of novel N- and C-termini for the protein, which may
drastically change its structure and function [112]. Circular permutation may be
used to study unexplored protein conformations, tune protein stability, and alter
ligand-binding properties. The presence of a circular mutation in the green
fluorescent protein (cpGFP) One prominent illustration of this phenomenon is the
circular permutation of green fluorescent protein (cpGFP). In the cpGFP variant,
the N- and C-termini of the native GFP molecule are connected, resulting in the
formation of novel termini. GFP’s potential reorganization could impact its folding
and fluorescence characteristics, diminishing its utility as a research tool for
investigating protein folding and biological processes [113] (table 2.18).
2.6.1.5 Domain swapping
The approach of domain swapping in protein engineering enables the assembly of
oligomeric structures by the exchange of domains among protein subunits, irrespective of their structural resemblance [114]. Domain flipping can lead to the
formation of novel, more complicated protein structures in certain circumstances
[115]. Antibodies are Y-shaped proteins with two heavy chains and two light chains
that are similar to one another. Light and heavy chain variable domains combine to
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Introduction to Pharmaceutical Biotechnology, Volume 2 (Second Edition)
Table 2.18. In protein properties caused by circular permutations.
Strategy based on iterative
Protein
permutations Effects on protein features
Green fluorescent
protein (GFP)
β-Lactamase Rearrangement of the active site’s
Lipases Rotation in a circle of the eyelid Activation at the interface and
Protein kinases The kinase domain undergoes a
Eukaryotic
initiation factors
Table 2.19. This study examines instances of domain swapping in proteins and investigates the effects on
protein properties.
Protein Domain name swap technique Effects on protein features
Immunoglobulins
(antibodies)
α-Amylase Interchange of domains
Serpins Inter-serpin family reactive
Tissue plasminogen
activator (tPA)
Hemagglutinin
protein (influenza
virus)
Changing the order of the ends to
make a circle
loop in a circle
circular permutation
The RNA-binding domain
undergoes a circular permutation
Antibody fragments swapping
variable domains
between monomeric
components
centre loop exchange
Kringle domains allow for
domain shifting
Flu hemagglutinin domain
swapping
Changed fluorescence
characteristics and protein
folding
Elevated beta-lactam antibiotic
resistance
altered substrate specificity
Substrate recognition and kinase
activity are altered
Shifts in the control of RNA
binding and translation
Adaptations in antigen binding and
immunological responses
The stability was improved, and the
substrate specificity was changed
Changes in substrate specificity and
protease inhibition
Modified plasminogen ligand binding
and interactions
Variations in viral entry characteristics
and receptor binding
form the antigen-binding site. Domain swapping occurs in immunoglobulins,
wherein dimeric or higher-order complexes are formed from the interchangeable
domains of different antibody subunits. Immune reactions and antigen-binding
properties may be affected by this technique [116] (table 2.19).
2.6.2 Improving enzyme thermostability
Enzymes’ capacity to withstand heat is crucial for their use in various industrial
procedures. Enzymes may be more robust and efficient catalysts if protein engineering techniques are used to increase their stability at high temperatures. Enzyme
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Introduction to Pharmaceutical Biotechnology, Volume 2 (Second Edition)
Table 2.20. Modifications that increase the thermal stability of enzymes.
Enzyme Method for increasing thermal stability Thermodynamic effect
DNA polymerases Extremophiles’
thermostable mutations are
incorporated into the population.
Enhanced
high-
Lipases Improved thermal stability through
Amylases Disulfide linkages and salt bridges that
Cellulases The addition of disulfide bonds and
Proteases Substitution of residues in deformable
temperature stability and activity
guided evolution
make sense
tweaks to the loop structure
domains
High-temperature organic synthesis
with enhanced performance
Increased resistance to heat and
denaturation
Increased resistance to deterioration
from high temperatures in biomass
Increased catalytic activity and
thermal stability
thermostability may be improved in several ways, including rational protein design,
controlled evolution, and the incorporation of thermostable patterns seen in
extremophilic species [117]. The development of thermostable DNA polymerases
like Taq polymerase has dramatically impacted the field of molecular biology by
making PCR possible at higher temperatures. The thermostability of DNA polymerases has been significantly enhanced by protein engineering. PCR amplification has
become more efficient and reliable by extending the half-life of DNA polymerases,
as shown in thermostable mutations in extremophilic bacteria [118] (table 2.20).
2.6.3 Enhancing enzyme substrate specificity
Substrate specificity is a crucial characteristic of an enzyme that determines its
ability to recognize and bind specific substrates, leading to efficient catalysis. Protein
engineering to improve enzyme substrate specificity might expand the range of
substrates that enzymes can process [119]. To do so, we must use rational design or
controlled evolution to alter the enzyme’s active site or binding pockets. The
substrate specificity of chymotrypsin was altered. Chymotrypsin is a protease that
cleaves peptide bonds in specific spots within proteins. Using directed evolution,
scientists have developed variations of chymotrypsin with altered substrate specificities. Since these improved forms can now break peptide bonds at a wider variety of
amino acid residues, they are of greater use in proteolytic processes and peptide
synthesis [120] (table 2.21).
2.7 Upstream process intensification
In bioprocessing, upstream process intensification refers to improving the efficiency
and output of the first steps in producing industrial and biopharmaceutical enzymes.
Increased yields, shorter production cycles, and reduced production costs may be
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Introduction to Pharmaceutical Biotechnology, Volume 2 (Second Edition)
Table 2.21. Improving the substrate’s specificity of enzymes: a few case studies.
Method for improving
Enzyme
substrate selectivity Substrate specificity effects
Chymotrypsin Substrate recognition evolution
with a purpose
Lipases Designing residues in the active
site
Cytochrome
P450s
β-lactamase Mutations introduced into the
Glycosidases Evolutionary manipulation of
Table 2.22. The benefits of high cell-density fermentation are numerous.
Advantages Explanation
Increased productivity Increased cell densities lead to elevated product concentration levels
Reduced production
time
Lower production costs Increased crop productivity and decreased fermentation duration lead
Decreased risk of
contamination
Simplified downstream
processing
Changes to the substrate-
binding residues
active site
substrate-binding sites
and enhanced overall production efficiency.
Reduced fermentation cycles result in accelerated manufacturing,
facilitating increased frequency of product batches.
to decreased utilization of resources and reduced expenses.
Increasing cell densities can mitigate the potential for contamination,
enhancing the output’s overall quality.
Increased concentrations of the product result in enhanced efficiency
and cost-effectiveness of downstream purifying procedures.
Enhancements to peptide synthesis and the
number of sites for cleavage
Increased generality in enantioselective
reactions
Accelerated reaction on selected substrates
The hydrolysis substrate range of beta-lactam
antibiotics has been expanded.
A shift in the glycosylation reaction’s
specificity
achieved by enhancing and optimizing the fermentation, cell culture, and enzyme
manufacturing processes. Process intensification aims to enhance efficiency by
maximizing output while decreasing inputs [121].
2.7.1 High cell density fermentation
High cell density fermentation is a method of upstream process intensification that
includes cultivating microorganisms at densities far higher than those employed in
conventional fermentation. This is achieved via the use of upgraded bioreactor
designs, medium formulation improvement, and cutting-edge control methods. High
cell-density fermentation can boost yields, decrease fermentation periods, and cut
production costs [122] (table 2.22).
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Introduction to Pharmaceutical Biotechnology, Volume 2 (Second Edition)
2.7.2 Solid-state fermentation
Solid-state fermentation (SSF) is a technique used in bioprocessing in which
microorganisms are cultured in the absence or minimal presence of free-flowing
water on solid substrates. It is an alternative to conventional submerged fermentation that is just as efficient at making bioproducts, including enzymes, organic acids,
bioactive compounds, and more [123]. The solid substrate provides food and
structural support for the microbes in solid-state fermentation. Solid-state fermentation has been widely used in the production of several enzymes. One such instance
is the evolution of amylases, enzymes that catalyze the breakdown of starch into
more straightforward carbohydrates. Aspergillus oryzae and Rhizopus species are
two common filamentous fungi used in SSF-based amylase production. SSF is a
valuable and cost-effective method of enzyme production since the fungus can grow
and release amylases directly onto solid substrates like wheat bran or rice husk [124]
(table 2.23).
2.7.3 Continuous fermentation
The fermented broth is constantly drained from the bioreactor and replaced with
fresh nutrient-rich medium in the bioprocessing method known as continuous
fermentation [125]. In contrast to batch fermentation, which occurs in a sealed
vessel with a finite capacity, continuous fermentation permits continuous growth
and is, therefore, well-suited for long-term and enormous bioproduct synthesis. The
production of biofuels like ethanol relies heavily on continuous fermentation. In a
bioreactor, sugar-containing feedstock such as sugarcane juice or molasses is
continuously added, and the fermented broth containing the ethanol is continuously
removed to produce ethanol in a continuous process. This setup ensures a steady
substrate supply and a consistent condition, boosting productivity and efficiency in
the process [126] (table 2.24).
Table 2.23. The benefits of solid-state fermentation.
Advantages Explanation
Lower water
requirements
Cost-effectiveness The utilization of solid substrates is typically more cost-effective than
Enhanced enzyme
stability
Higher product
concentrations
Simplicity of scale-up The method of scaling up SSF is very uncomplicated due to its little
The utilization of SSF results in reduced water consumption and
subsequent expenditures due to its minimal water requirements.
liquid media, leading to cost reduction.
Enzymes generated using SSF frequently demonstrate enhanced
stability, rendering them well-suited for various applications.
The absence of diluting effects in SSF can increase product
concentrations.
requirement for liquid handling procedures.
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Introduction to Pharmaceutical Biotechnology, Volume 2 (Second Edition)
Table 2.24. Benefits of constant fermentation.
Advantages Explanation
Steady-state operation Constant substrate addition and product removal are possible during
continuous fermentation.
Increased process
productivity
Reduced labor and
downtime
Enhanced process
control
Scalability Manufacturing bioproducts on a massive scale is well suited to
Table 2.25. Benefits of enzyme production by microbial consortia.
Advantages Explanation
A more extended fermentation period is achieved through continuous
operation, increasing product yields.
There is less downtime due to interruptions and less need for manual
intervention when fermentation is continuous.
Continuous fermentation allows for fine-tuned regulation of critical
process parameters thanks to its steady-state character.
continuous fermentation because of its scalability.
Enhanced enzyme
production
Efficient substrate
utilization
Process robustness and
stability
Expanded substrate
range
Lower production costs Increases in efficiency and output lower the price per unit of enzyme
Microbial consortiums exploit synergistic interactions to increase
enzyme production and productivity.
The various microorganisms in the consortium each have their area of
expertise, allowing for more efficient substrate use.
When a wide variety of microorganisms are present, the process
becomes more stable and resistant to external changes.
Multi-enzyme synthesis is made possible by microbial consortia’
increased substrate versatility.
produced.
2.7.4 Microbial consortia for enzyme production
Groups of microorganisms called consortia work together to accomplish complex
tasks like enzyme synthesis. Microbial consortia have gained popularity in the
bioprocessing industry due to their increased efficiency in enzyme synthesis,
substrate use, and product diversity. These consortia use the synergistic interactions
between several microbial species to boost enzyme output and enhance bioprocess
conditions [127]. Plant biomass contains cellulose, a complex polymer that is
difficult to break down. However, bacterial and fungal cellulolytic microbial
consortia can now break cellulose into simpler sugars, making it more digestible.
The fungi in these groups secrete enzymes called cellulases that degrade cellulose
into soluble sugars that the bacteria may then consume. The microorganisms’
collaborative activity results in higher cellulase yields and more efficient biomass
conversion than with individual cultures alone [128] (table 2.25).
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Introduction to Pharmaceutical Biotechnology, Volume 2 (Second Edition)
Table 2.26. There are benefits to using in situ product removal methods.
Advantages Explanation
Shift in reaction
equilibrium
Increased enzyme
productivity
Enhanced reaction
yield
Prolonged enzyme
lifespan
Efficient scale-up ISPR methods are easily reproducible, opening the door to industrial-
Constant removal of the product prevents the buildup of the product and
drives the reaction to completion.
Enzyme output and overall reaction efficiency are improved due to less
product inhibition.
Keeping a positive concentration gradient via constant product removal
allows for increased yields.
Enzyme inactivation is a possible side effect of product inhibition.
Enzyme stability is improved through ISPR methods.
scale bioprocessing and enzyme manufacturing.
2.7.5 In situ product removal strategies
In situ, product removal (ISPR) removes product molecules from the reaction mixture
in real time during bioprocessing. ISPR methods boost enzyme output by rebalancing
the reaction in favour of product formation rather than product inhibition, hence
shifting the equilibrium of the process [129]. Physical separation techniques such as
adsorption, extraction, or membrane filtering may be used regularly or continuously to
remove the product from the reaction fluid. The process of lactose hydrolysis is
essential in producing lactose-free dairy products. Glucose and galactose are produced
when lactase digests lactose. Keeping the reaction mixture free of by-products and
maximizing lactose hydrolysis efficiency calls for a membrane filtration system that
continuously removes glucose and galactose [130](table2.26).
2.8 Enzyme production from extreme environments
Enzymes produced by organisms that can survive in extreme conditions are of great
interest in biotechnology and industry. Heat (thermophiles), cold (psychrophiles),
acid, alkali, pressure, and salt (halophiles) are all examples of extreme environments
in which certain organisms may thrive. High stability, activity, and selectivity under
extreme conditions characterize extremophile enzymes, making them promising
biocatalysts for various industrial applications [131]. Extremophiles that thrive in
warm environments include thermophilic bacteria like Thermus aquaticus. Many
biotechnological procedures, such as PCR, need thermostable enzymes produced by
these microbes (PCR). Thermus aquaticus (Taq) polymerase is one of the most wellknown thermostable enzymes in PCRs. Due to its stability up to 95 °C, it is ideal for
PCR applications (figure 2.8) (table 2.27)[132].
2.8.1 Psychrophiles (cold-loving)
Psychrophiles are a subset of extremophiles that may thrive at temperatures as low
as 20 °C. To overcome the challenges given by the cold, these bacteria have evolved
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Introduction to Pharmaceutical Biotechnology, Volume 2 (Second Edition)
Figure 2.8. Classification of extremophiles based on environmental conditions, including pH, temperature,
salinity, pressure, and other extreme factors.
Table 2.27. Extremophile enzymes are used as examples.
Use of polymerase chain reaction
Source
Enzyme
Taq polymerase Aquatic
Cold-adapted
lipases
psychrophile)
Acid proteases Fungi with a low pH The food and textile industries Low pH (Acid
Alkaline proteases Alkali-loving
Halophilic
α-amylase
(extremophile)
thermophilus
Psychotic-loving
microbes
microorganisms
Archaea that thrive
in salty
environments
in organic synthesis at low
temperature
Manufacturing of cleaning
products and food
Fabric softeners and leather
cleaners
Use of polymerase chain reaction
in organic synthesis at low
temperature
Manufacturing of cleaning
products and food
Dangerous
situation
Thermophilic
(loving heat)
Cold-loving
(or
lover).
Low acidity
(Basophile)
Halophilic
(high salt)
enzymes with specific properties adapted for functioning well in such environments.
Because of their high catalytic activity and versatility at low temperatures,
psychrophilic enzymes play a significant role in various applications as biocatalysts.
Lipases are enzymes that catalyze the breakdown of lipids, and it is known that
psychrophilic bacteria may create cold-adapted lipases. These lipases are essential
for the synthesis of organic molecules at mild temperatures. In producing fine
chemicals and medications, for instance, cold-adapted lipases have been shown to
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