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Introduction to Pharmaceutical Biotechnology, Volume 2 (Second Edition)
Table 2.28. Psychrophilic enzymes have these distinctive features.
Source
Enzyme
(psychrophile) Applications
Cold
temperature
range
Cold-adapted
lipases
Psychrophilic
proteases
Cold-active
amylases
Table 2.29. Characteristics of thermophilic enzymes.
Enzyme
Thermostable DNA
polymerases
Thermophilic
proteases
Heat-resistant
amylases
Psychrophilic
bacteria
Psychrophilic
fungi
Psychrophilic
archaea
Source
(thermophile) Applications
Thermus
aquatics
Thermophilic
bacteria
Thermophilic
fungi
Organic synthesis, speciality compounds,
and medicinal drugs
Industrial kitchens, detergent creation, and
frigid cleaning agents
Baking resistant to low temperatures and
starch hydrolysis
Below 20 °C
Below 15 °C
Below ten °C
Temperature
range
Industrial applications of PCR in the
food and pharmaceutical sectors
Baking and hydrolysis of starch 60 °C–80 °C
Industrial applications of PCR in the
food and pharmaceutical sectors
50 °C–95 °C
70 °C–90 °C
catalyze esterification and transesterification processes well, even at refrigerator
temperatures [133] (table 2.28).
2.8.2 Thermophiles (heat-loving)
Thermophiles are a kind of extremophile that can survive at hot temperatures (over
50 °C). These creatures have adapted in unique ways to live in such extreme heat.
Because of their remarkable durability and activity at high temperatures, thermophilic enzymes are an excellent choice for industrial applications that call for high
heat. PCR requires thermostable DNA polymerases, which are generated by
thermophilic bacteria like Thermus aquaticus (PCR). PCR is a vital tool in molecular
biology since it allows for the amplification of DNA sequences. DNA polymerases
from thermophiles may continue to operate and keep their catalytic efficiency even
at the high temperatures required for DNA denaturation during the PCR process
[134] (table 2.29).
2.8.3 Acidophiles (acid-loving)
Acidophiles are extremophiles that thrive in environments with a pH level of 3 or
below. To thrive in these harsh conditions, these bacteria have evolved remarkably
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Introduction to Pharmaceutical Biotechnology, Volume 2 (Second Edition)
Table 2.30. Characteristics of acidophilic enzymes.
Source
Enzyme
Acid proteases Aspergillus niger Manufacturing of cleaning products and
Acid cellulases Acidophilic
Acidophilic
amylases
(acidophile) Applications
food
Hydrolysis of cellulose for use in making
bacteria
Acidophilic
archaea
biofuels
Low-pH starch hydrolysis pH < 4
pH
range
pH < 3
pH < 4
stable enzymes and are active at low pH levels. Food processing and detergent
manufacturing are only two examples of industries that benefit from using acidophilic enzymes. Fungi that thrive in acidic environments, such as Aspergillus niger,
produce acid proteases, which are most active and stable at acidic pH values. In the
food industry, these acid proteases play a vital role in cheesemaking, meat tenderization, and protein breakdown. Their high acidic activity facilitates protein
hydrolysis and modification in several food processing contexts [135] (table 2.30).
2.8.4 Alkaliphiles (alkaline-loving)
Alkaliphiles are extremophiles that thrive in environments with a pH greater than 9.
Because of the challenges of high alkalinity levels, these microorganisms have
evolved to produce alkaliphilic enzymes. The capacity of alkaliphilic enzymes to
function normally and remain stable in an alkaline environment sets them apart.
Since many manufacturing procedures need an environment with a high pH, they
have attracted much attention in biotechnology [136]. Bacillus species, which are
alkaliphilic bacteria, produce alkaline proteases, an enzyme. Due to their stability
and activity at high pH levels, these enzymes find widespread application in laundry
detergents. Stains on clothes, such as those caused by food and blood, maybe more
easily removed with the help of alkaline proteases, which are enzymes that break
down proteins. Alkaline protease detergents allow for more eco-friendly, lowertemperature washing without sacrificing fabric quality [137 ] (table 2.31).
2.8.5 Halophiles (salt-loving)
Some organisms, called halophiles, can tolerate far higher salt concentrations than is
typical, and hence, they thrive in environments like salt flats and salty lakes. These
microorganisms produce halophilic enzymes to help them survive in salty conditions. Because of their stability and activity in high salt concentrations, halophilic
enzymes play a significant role in various industrial applications. Halophilic archaea
generate halophilic-amylases, enzymes that hydrolyze starch efficiently in high-salt
environments. Enzymes like this are helpful in both the food and textile industries.
In the textile industry, halophilic-amylases are used to break down starch-based
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Introduction to Pharmaceutical Biotechnology, Volume 2 (Second Edition)
Table 2.31. Characteristics of alkaliphilic enzymes.
Source
Enzyme
Alkaline
proteases
Alkaliphilic
lipases
Alkaline
amylases
Table 2.32. Characteristics of halophilic enzymes.
Enzyme
Halophilic α-
amylases
Halophilic
proteases
Halophilic
lipases
(alkaliphile) Applications
Bacillus species Cleaning agents, leather tanning pH > 9
Alkaliphilic
bacteria
Alkaliphilic fungi Detergent production and the hydrolysis of
Source
(halophile) Applications
Halophilic
archaea
Halophilic
bacteria
Halophilic
fungi
Cleaning product creation and organic
synthesis
starch
Food processing and the de-sizing of
textiles
Protein breakdown and bioprocessing High-salt
Synthetic organic compounds and the
creation of detergents
pH
Range
pH > 9
pH > 9
Salt
concentration
High-salt
environments
environments
High-salt
environments
sizing agents during the designing process. These enzymes aid in the starch
hydrolysis process, improving processed foods’ texture and quality [138] (table 2.32).
2.8.6 Applications of extremozymes in biotechnology
Extremozymes, enzymes produced by extremophiles, have several uses in biotechnological processes because of their unique properties and adaptability to extreme
conditions. Several industries, including biofuels, food processing, detergents, and
medications, have been revolutionized by these enzymes’ increased efficiency and
selectivity [139] (table 2.33).
2.9 Downstream process intensification
The enzyme synthesis processing steps in the bioprocessing pipeline may be made
more efficient and productive using a novel approach known as downstream process
intensification. The purification, separation, and recovery of enzymes must be
accelerated and improved by incorporating and using state-of-the-art procedures
as part of this process optimization. Researchers in biotechnology may be able to
increase production output, lower costs, and develop greener production processes
by concentrating on the process that follows [140].
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Introduction to Pharmaceutical Biotechnology, Volume 2 (Second Edition)
Table 2.33. Applications of extremozymes in biotechnology.
Enzyme Application
Cold-adapted lipases Synthetic organic chemistry and detergent development
Thermostable DNA polymerases Reverse transcription-PCR
Acid proteases Manufacturing of cleaning agents and foods
Alkaline proteases Cleaning agents, leather tanning
Alkaliphilic lipases Cleaning product creation and organic synthesis
Alkaline amylases Detergent production and the hydrolysis of starch
Halophilic α-amylases De-sizing in the textile industry, industrial cooking
Halophilic proteases Protein breakdown in bioprocessing
Halophilic lipases Cleaning product creation and organic synthesis
2.9.1 Continuous chromatography
Ongoing chromatography has some benefits over batch chromatography, and is a
cutting-edge method for improving downstream processes. Continuous chromatography was developed in the 1970s. It is a technique that facilitates the uninterrupted
purification and segregation of enzymes in a way that allows for a continuous flow,
resulting in increased efficiency and decreased processing duration [141]. Continuous
chromatography, also sometimes called Simulated Moving Bed (SMB) chromatography, can be used to clean enzymes. SMB chromatography is a constant procedure
whereby feed, adsorption, and eluent are continuously transported through a series
of static columns. As a result, a counter-current flow pattern is noticed, leading to an
improvement in the yield of the chromatographic splitting phase. SMB chromatography in industrial-scale enzyme manufacturing is beneficial since it needs a much
lower amount of solvent, which lowers costs and enhances output rates [142]. Some
advantages of continuous chromatography are:
• method of purification and isolation that occurs continuously;
• continuous tracking and regulation;
• decreased use of reducing agents;
• improvements in output and productivity;
• lower infrastructure requirements.
2.9.2 Process integration and optimization
Process integration and optimization include the systematic examination and
improvement of various bioprocessing procedures to create a workflow that is
both more efficient in terms of productivity and more economical in terms of cost.
This technique utilizes several unit operations, state-of-the-art analytical techniques,
and process modelling to improve enzyme synthesis and subsequent processing. The
production of enzymes may be optimized with the use of computer-aided process
design and cutting-edge software tools and algorithms. Biotechnologists may
enhance process yields and decrease production costs by simulating different process
settings and parameters to find the optimal operating conditions. Computer-aided
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Introduction to Pharmaceutical Biotechnology, Volume 2 (Second Edition)
process design (CAPD) is advantageous when dealing with complex multistage
processes since it allows the exploration of various scenarios without extensive
testing [143]. Benefits of process integration and optimization are:
• enhanced performance and output;
• reduced enzyme production costs Rapid prototyping and scaling;
• improved steady operation and stability;
• methods that do not harm the environment.
2.9.3 Advanced filtration techniques
One of the most critical ways cutting-edge filtering techniques contribute to ramping
up downstream processes is by improving enzyme separation and purification. These
strategies boost filtration output and efficiency using modern membrane materials,
cutting-edge mediums, and specialized equipment.
2.9.3.1 Tangential flow filtration (TFF)
TFF is a more efficient filtering technology often used in the enzyme purification). It
employs a semi-permeable membrane to size-separate enzymes from the other
components of the feed solution. Compared to traditional dead-end filtration,
TFF’s cross-flow mode reduces the likelihood of filter clogging while increasing
filtration efficiency. TFF may be used to concentrate and purify large volumes of
fermentation broth, hence boosting the efficiency of the process [144]. Advantages of
advanced filtration techniques are:
• enzyme purification and isolation at high yields;
• filter fouling is reduced, increasing filter life and decreasing processing times;
• production of enzymes on a massive scale is feasible;
• reduced loss of enzyme activity during filtering;
• enzyme purification and isolation at high yields.
2.9.3.2 Depth filtration
Depth filtration is often used in the enzyme industry for purification and downstream process intensification. This technique involves filtering the enzyme solution
through a porous matrix, such as a fibre depth filter, which traps particles and
impurities while letting the enzymes pass through. Depth filtering is a straightforward
and cheap method for clarifying and partially purifying enzyme solutions. Clarifying
the fermentation broth often involves depth
filtration as part of the enzyme production
process. Most of the time, the fermentation broth has foreign substances like microbial
cells or cell debris. During a depth filtering step, the bulk of the solid particles in the
broth are removed, leaving a somewhat clarified enzyme solution. This partial
purification facilitates further downstream processing steps, including chromatography and ultrafiltration [145]. Advantages of depth filtration are:
• method of inexpensive purification;
• strong ability to trap particles;
• enzyme breakdown is kept to a minimum during filtration, making it feasible
for industrial enzyme manufacturing;
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Introduction to Pharmaceutical Biotechnology, Volume 2 (Second Edition)
• easy to understand and implement procedure;
• method of inexpensive purification.
2.9.3.3 Crossflow filtration
Crossflow filtration is a sophisticated downstream process intensification strategy for
the concentration and purification of enzymes. In contrast to traditional dead-end
filtration, in which the feed is constantly passed through the filter, cross-flow
filtration employs a tangential flow that decreases filter clogging. This technique
isolates and concentrates enzymes by permitting continuous feed circulation across
the filter surface. Enzyme production using ultrafiltration often employs cross-flow
filtration. Ultrafiltration concentrates enzymes by rejecting molecules too small to
pass through the ultrafiltration membrane. This approach is ideal when dealing with
enzymes that are easily damaged by heat since it may be used at milder temperatures. Ultrafiltration’s ability to recover concentrated enzyme solutions aids in
subsequent purification steps [146]. Advantages of cross-flow filtration are:
• effective and continuous separation method;
• reduced filter clogging and increased filter longevity;
• heat-sensitive enzymes can function at a low temperature;
• effective factor concentration at a high level;
• production of enzymes in large quantities.
2.9.4 Automation and robotics in downstream processing
Automation and robotics have made downstream processing more efficient, precise,
and repeatable. Complex tasks, including sample management, liquid handling,
chromatography, and filtration, may be performed by automated systems with little
or no human intervention [147]. Adopting this technology allows scientists to increase
enzyme output while decreasing processing times and improving efficiency.
Automation and robotics have enabled high-throughput screening of enzyme variants
to discover mutants with enhanced stability, activity, or substrate selectivity. Scientists
can rapidly screen hundreds of mutant enzymes thanks to automated liquid handling
systems’ ability to produce and analyze various enzyme variants. These speed up
enzyme engineering, allowing for the development of more valuable enzymes [148].
Advantages of automation and robotics in downstream processing are:
• increased effectiveness and output in all processes;
• prevention of mistakes made by humans;
• the engineering of enzymes sped up;
• improved ability to replicate studies;
• ability to expand manufacturing capacity.
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