Добавил:
Sekretar
kiopkiopkiop18@yandex.ru
t.me/Prokururor I Вовсе не секретарь, но почту проверяю
Опубликованный материал нарушает ваши авторские права? Сообщите нам.
Вуз:
Предмет:
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)
In the context of biotransformation, enzymes act as biocatalysts, speeding up
biochemical reactions without being consumed. They can be extracted from cells
and catalyze a wide range of commercially important processes. Enzymes are known
for their ability to direct groups controlling selectivity alongside acting as catalysts,
making them invaluable in biotransformation processes.
In some biotransformation scenarios, enzymes exhibit synergistic catalytic
activity. For instance, the combined action of different enzymes can lead to efficient
biotransformation in a one-pot reaction, as demonstrated in a study where a
hypothetical synergistic mechanism of two enzymes (CDase and MTSase) was
proposed through analyzing enzyme reaction pathways [30].
6.6.2 Cofactors and coenzymes in biotransformation
Cofactors and coenzymes play a crucial role in facilitating enzymatic processes.
Enzymes that rely on cofactors facilitate a diverse array of chemically valuable
reactions. However, the need for cofactors presents significant economic and
practical obstacles to effectively using these biocatalysts. Enzymes bind cofactors,
either tiny molecules or metal ions, to aid in catalysis and activity. Approximately
50% of all enzyme activities need a cofactor, either a firmly attached prosthetic
group or a loosely linked coenzyme [31]. Acetyl coenzyme A, nicotinamide adenine
dinucleotide phosphate (NAD(P)+), and adenosine triphosphate (ADP) all play
important roles in microbial metabolism3. These cofactors and coenzymes are
essential in assisting enzymes in catalyzing reactions, highlighting their importance
in biotransformation [32 ]. The general mechanism of the enzyme activation is shown
in figure 6.6.
6.6.3 Enzyme inhibition and activation
Enzyme activity modulation through inhibition and activation plays a significant
role in regulating cellular metabolism. Enzyme inhibition is often used in drug
Figure 6.6. Enzyme activation mechanism: transition from apoenzyme to holoenzyme upon cofactor binding.
6-12
Introduction to Pharmaceutical Biotechnology, Volume 2 (Second Edition)
design to treat various conditions or elucidate mechanisms involved, with many
drugs functioning through enzyme inhibition. On the other hand, enzyme activation
aims to intensify reaction processes for higher yield quickly and can be utilized for
various applications in biotransformation. Factors such as proinflammatory cytokines can modulate enzymatic activity, affecting an organism’s response to medications. The balance between enzyme inhibition and activation is crucial for
effective biotransformation processes [33].
6.6.4 Role of enzyme specificity and selectivity
Enzyme specificity and selectivity are crucial for achieving desired reactions in
biotransformation. Specific enzymes highly catalyze reaction-specific and stereospecific reactions to synthesize compounds that cannot be produced by chemical
means. Specificity is often assessed using the value of k
, which is more manifest
cat/Km
in the rate at which a substrate reacts rather than the affinity of substrate binding
[34]. Enzyme engineering can alter substrate specificity and product selectivity to
enhance enzyme activity, illustrating the potential for optimizing biotransformation
processes through enzyme engineering. Furthermore, the substrate specificity of
enzymes has unavoidable physicochemical limits, underlining the importance of
understanding enzyme specificity and selectivity in biotransformation [35]. An
overview of the enzyme inhibition is given in figure 6.7.
6.7 Biotransformation in environmental applications
Biotransformation in environmental contexts primarily involves using microorganisms or enzymes to detoxify or mineralize chemical contaminants. This process is
Figure 6.7. Comparative diagram of enzyme inhibition: normal reaction versus competitive and noncompetitive inhibition.
6-13
Introduction to Pharmaceutical Biotechnology, Volume 2 (Second Edition)
Figure 6.8. Diverse applications of biotransformation in producing food, feed supplements, pharmaceuticals,
and environmental waste treatment.
instrumental in transforming harmful compounds into less active metabolites or
complete mineralization, which is crucial for environmental remediation [36]. Over
the past few decades, substantial knowledge has accrued regarding the biotransformation of major legacy contaminants like chlorinated solvents, BTEX (Benzene,
Toluene, Ethylbenzene, and Xylenes), and explosives/munitions chemicals, primarily in a bioremediation context where these contaminants are metabolically
degraded [37]. Several applications of the biotransformations are reported in
figure 6.8.
6.7.1 Degradation of pollutants
The degradation of pollutants through biotransformation is a significant aspect of
environmental remediation. Various strategies and methodologies have been developed to enhance the efficiency of this process. For instance: PAHs are a cluster of
organic contaminants that pose environmental and human health risks.
Biotransformation is an eco-friendly biological treatment solution for PAH remediation, with microbial remediation approaches being prominently utilized [38].
Biotransformation helps remove contamination from soil and water systems by
transforming unwanted complexes into desired products. This process is achieved
through microorganisms or enzymes, with recent research focusing on implementing
biocatalysts to enhance the stability and efficiency of enzymes involved in this
process [39]. Catalases play a crucial role in breaking down various contaminants,
such as phenolic chemicals, insecticides, dyes, and poly-aromatic hydrocarbons.
Furthermore, the utilization of catalase-based biosensors has been implemented to
monitor hazardous waste, hence demonstrating the adaptability and potential of
enzyme-mediated biotransformation in the realm of environmental applications [40].
Biotransformation of plastics and microplastics involves breaking these materials
into simpler substances by biological agents. The entire procedure starts with plastic
6-14
Introduction to Pharmaceutical Biotechnology, Volume 2 (Second Edition)
waste in an aqueous medium split into micro- and nano-plastics by ultraviolet (UV)
radiation and the action of microbes. This is how the process starts. Over time,
microplastics in the soil undergo transformations influenced by biotic and abiotic
factors, which alter their morphology and particle size [41]. Biotechnological
advancements such as biostimulation, bioaugmentation, and enzymatic biodegradation have been proposed for removing microplastics (MPs) by biodegradation and
bioaccumulation processes [42].
Moreover, algae, fungi, and bacteria play significant roles in the biodegradation
of microplastics, with biotechnological methods like gene editing tools and bioinformatics being utilized to enhance degradation processes. The review articles
further discuss various microorganisms and enzymes capable of degrading synthetic
plastics like polyethene, polypropylene, and polyvinyl chloride, among others. To
provide an all-encompassing comprehension of the biotransformation procedure, a
summary of the various microbial species, genes, biochemical reaction pathways,
and enzymes involved in plastic decomposition has also been included [43].
Bioremediation uses biological agents, such as fungi and bacteria, to remove or
reduce environmental pollutants. The metabolic and ecological features of fungi
make them suitable for bioremediation and waste treatment processes. The synthesis
of practical microbial taxa, including bacteria, fungi, and microalgae for hydrocarbon removal in marine systems has been reviewed, showcasing the importance of
these organisms in bioremediation processes [44]. Additionally, fungi are deemed
sustainable and green agents for the clean-up of contaminated sites due to their
diverse metabolic capacities, which enable them to detoxify various toxic and
recalcitrant compounds
8
. Bacteria and fungi have shown potential in bioremediation
strategies, particularly in the environmental degradation of xenobiotic compounds
The potential of bacteria and fungi, individually and in association with plants, has
been critically examined, showing high metal tolerance and bioremediation potential
up to 98% when associated with plants [45].
9
.
6.7.2 Enzymatic breakdown of pesticides
Enzymes play a crucial part in the process of pesticide biodegradation. The
compounds in question play a crucial role in the biological aspects of numerous
pesticides, impacting their methods of operation, environmental outcomes, and the
development of resistance in target species. Specific enzymes are crucial in the
degradation of certain pesticides, such as the enzymatic degradation of the organophosphate pesticide malathion [46]. Microbe-derived enzymes are considered
excellent candidates for pesticide remediation, and screening elite microbial strains
is crucial during biodegradation [47]. The significance of enzymes in mitigating
environmental contamination resulting from pesticides has been the subject of
numerous studies, highlighting their pivotal role in the biodegradation process.
Furthermore, the focus has been placed on the remediation of chemical pesticides
from polluted areas using possible microorganisms and their functional enzymes.
This emphasizes this study area’s potential opportunities and obstacles [48].
6-15
Introduction to Pharmaceutical Biotechnology, Volume 2 (Second Edition)
6.8 Emerging technologies in biotransformation
Emerging technologies in biotransformation involve novel methods and advancements that enable the conversion of substances through biological agents or systems.
These advancements encompass various fields, including enzyme engineering,
biocatalyst design, and precision biotransformation. Developing new and successful
biotransformation processes in medicinal and pharmaceutical chemistry often
entails creating new biocatalysts with improved or even new activities and selectivity
[49]. Moreover, the emergence of precision biotransformation strategies has
provided detailed insights into biotransformation pathways in natural environmental settings using high-accuracy quantum chemistry, particularly for emerging
pollutants whose metabolites are easily overlooked but may cause idiosyncratic
toxicity [50].
6.8.1 Enzyme engineering and directed evolution
Enzyme engineering and directed evolution are pivotal in modifying enzymes to
meet industrial applications and research requirements. Enzyme engineering focuses
on modifying the properties of enzymes to improve their performance for specific
tasks. Engineering enzyme properties has facilitated the biocatalysis industry in
performing highly selective chemical syntheses, which was achievable through the
development of effective methods in enzyme engineering to overcome the limitations
of naturally occurring enzymes. The field of enzyme engineering has seen a
significant transformation, resulting in a remarkable increase in the range and
potential uses of enzymes that have been modified or enhanced to possess specific
physical and catalytic characteristics [51]. Directed evolution accelerates the organic
process of evolution in biological molecules and systems inside a controlled
laboratory environment. This is achieved through repeated rounds involving gene
divergence and library screening/selection. In recent years, there has been a
significant advancement in the utilization of this instrument for enhancing or
creating new functionalities in enzymes and biocatalysts based on entire cells.
Notable progress has been made in generating novel enzyme activities and establishing many practical strategies for directed evolution. The approach described herein
entails emulating the inherent evolutionary mechanisms to attain specific enzyme
characteristics, rendering it an indispensable technique in advancing enzymes for
applications in industrial biocatalysis [52]. The integration of nanotechnology and
biotechnology occurs through the manipulation of nanostructures, resulting in the
modification of their inherent properties. This interaction influences the dispersion
of particles and the biotransformation processes they induce. The biotransformation
of nanomaterials like silver nanoparticles (AgNPs) is noted, where sulfidation
dominates as a cellular detoxification pathway [53]. Though direct information on
the integration with bioinformatics and computational biology was not obtained, it
is reasonable to infer that nanotechnology, when coupled with these fields, can
enhance data analysis and modeling, thus facilitating a better understanding and
manipulation of biological systems at the nanoscale. Nanotechnology exhibits the
potential to address a diverse range of issues within the domains of medical
6-16
Introduction to Pharmaceutical Biotechnology, Volume 2 (Second Edition)
technology, biosensors, cancer therapy, bioimaging, pharmaceutical administration,
precision agriculture, water treatment, and environmental remediation. Through a
systems biology and metabolic engineering lens, nanotechnology can be utilized to
develop systemic approaches for addressing environmental contaminants and health
issues. For instance, bioremediation combined with nanotechnology has been
explored to develop more systemic approaches for removing contaminants from
environmental matrices [54]. This process within the food and beverage sector
pertains to converting a given substance, denoted as A (substrate), into another
substance, referred to as B (product), through a biocatalyst. This process can be
carried out using purified and immobilized enzymes, growing cultures, resting or
washed microbial cells, immobilized cells, or a combination of two sequential
biotransformation steps catalyzed by different microorganisms. Here is a deeper
dive into the sub-topic.
6.8.2 Production of flavors and fragrances
Enzymes play an imperative starring role in the biotransformation processes within
the food and beverage industry. For instance, the beverage industry extensively uses
amylases, pectinases, and cellulases to derive flavors and fragrances from natural
substrates like grapes and other fruit [55]. Microorganisms are pivotal in biotransformation to produce enzymes and metabolites of industrial interest. For example,
the role of microorganisms in brewers’ spent grain (BSG) exploitation to produce
enzymes and metabolites of industrial interest is well documented [56].
Biotransformation categories such as glycosidic cleavage, terpenol transformation,
organic acid conversion, and thiol release are key to flavor and fragrance development in brewing. Glycosidic cleavage, for instance, can release aglycones, often
aromatic compounds contributing to the flavor and aroma of the beverage [57]. The
end goal of these biotransformations is often to enhance the consumer experience by
improving the nutritional profiles and bioavailability of nutrients and creating
appealing flavors and fragrances that connect with the consumers on an emotional
level, enriching the overall sensory experience of the food or beverage product [58].
6.8.3 Biotransformation of lipids for healthy oils
The biotransformation of lipids to produce healthy oils involves modifying the
structure and composition of fats and oils to enhance their nutritional profiles and
meet consumer preferences for healthier fat options. Biochemical processing
techniques alter the physicochemical and nutritional properties of natural fats and
oils, enabling the production of health-promoting lipid products [59]. SLs are
designed through the rearrangement or modification of fatty acids on a glycerol
backbone, often targeting the incorporation of n−3 long-chain polyunsaturated
fatty acids (LCPUFAs) which are known for their health benefits [60]. Enzymatic
processes produce bioactive, SLs with improved nutritional characteristics. These
processes utilize enzymes to modify lipids in fi sh oil, microalgal lipids, and new seed
oils high in omega-3 fatty acids, enhancing their health-promoting properties [61].
Naturally occurring and synthetic ether lipids can be biotransformed in
6-17
Introduction to Pharmaceutical Biotechnology, Volume 2 (Second Edition)
microorganisms, plants, or animal tissues, which can enhance or inhibit the effects of
these lipids depending on the compounds involved in the biotransformation process.
6.8.4 Fermentation and enzymatic modification of food products
Fermentation and enzymatic modification are crucial processes in the food industry
that enhance food products’ nutritional, biochemical, and sensory qualities.
Fermentation, driven by microorganisms and enzymes from raw ingredients, alters
foods’ nutritional and biochemical quality, creating complex ecosystems within
fermented products. This process also contributes to food stability through physical
and biochemical changes [62]. Enzymatic processes are suitable for converting food
waste into valuable products, with potential biotechnological applications such as
creating functional foods with probiotic properties. Microbial enzymes, including
lipases produced by bacteria, fungi, and yeast, find extensive application in the food
industry, contributing significantly to the global market of lipases. They are used in
various industries, including food, biofuel, and others [63]. Enzymatic treatment,
particularly employing non-Saccharomyces yeast with enzymatic activities, produces
complex flavor profiles in fermented fruit-based products, aligning with new market
trends and consumer preferences. The fermentation process also produces enzymes,
organic acids, Baker’s yeast, ethanol, vitamins, and other products, which are
crucial for various industrial and food applications.
6.9 Biotransformation challenges and future perspectives
The challenges and prospects in biotransformation span across analytical, biotechnological, and industrial sectors. The advancement in biotransformation technologies is seen as a potent tool for addressing various environmental and industrial
challenges. Current challenges in biotransformation are primarily analytical and
biotechnological, with a specific mention of challenges associated with the bioconversion of tetracyclines by fungi, as an example, illustrating the need for research
to advance technology for applications like wastewater and manure treatment [64].
Scalability issues are particularly significant when considering the industrial applications of biotransformation. An integrative approach involving the design of
biocatalysts using enzyme engineering and metabolic engineering tools is suggested
to enhance biocatalytic efficiency, making it a feasible tool for industrial and
pharmaceutical applications. In another instance, the scalability of bioprocesses for
the purification of stem cells was discussed, indicating the breadth of scalability
challenges across different domains of biotransformation [65]. The necessity of
overcoming technical challenges before large-scale implementation can be achieved
in the biotransformation of carbon dioxide in bioelectrochemical systems, with
recommendations on technical perspectives for successful implementation and
future research directions being provided [66].
6.9.1 Scalability issues in industrial applications
Scalability issues in industrial applications of biotransformation technologies are a
significant challenge. The transition from lab-scale or pilot-scale to industrial-scale
6-18
Introduction to Pharmaceutical Biotechnology, Volume 2 (Second Edition)
operations often entails numerous challenges. Ensuring the process is optimized for
large-scale operations, including the efficient design of biocatalysts and the utilization
of suitable engineering tools. Ensuring that the scaled-up process is cost-effective and
economically viable is critical for industrial adoption. Adequate infrastructure and
equipment are necessary to handle large-scale biotransformation processes. Adhering
to industrial standards and regulatory compliance is crucial, especially in pharmaceutical and food industry applications. Continuous technological advancements are
needed to address the evolving challenges associated with scalability.
6.9.2 Regulatory and safety concerns
The regulatory and safety concerns surrounding enzymes, particularly food
enzymes, primarily revolve around allergies, irritation, and potential toxic properties. There is a lack of consistency in the global regulatory landscape for enzymes
despite authorities’ familiarity with enzyme technology and its applications. The
safety assessment of enzyme production strains is crucial and involves repeated
toxicological testing to establish a safe strain lineage (SSL) when no adverse effects
are identified from enzymes derived from the same strain lineage. Enzyme
production and use regulation is overseen by three central bodies: the Joint Food
and Agriculture Organization of the United Nations/World Health Organization
Expert Committee on Food Additives, the European Food Safety Authority, and
the US Food and Drug Administration [ 67].
6.9.3 Challenges in enzyme storage and stability
Enzyme stability during storage and operational usage is a critical aspect that
impacts the efficacy and shelf-life of enzymes. The challenges include maintaining
the enzyme’s catalytic abilities from the point of manufacture to eventual use,
known as storage stability, and ensuring the persistence of enzyme activity during
operational stability. Various strategies for enzyme stabilization have been reviewed,
although a practical approach towards this is often seen as lacking. This includes
using certain chemicals and conditions to maintain enzyme activity over time. The
stability of enzymes is a crucial issue, especially when implementing them as
catalysts in industrial processes, which may involve extreme environmental conditions that can induce enzyme instability [68].
6.9.4 Future trends and emerging areas of research
Various emerging trends and prospects in enzyme technology have been identified.
For instance, the application of enzyme technologies in developing ‘future foods’ is
seen as a growing research area, focusing on creating healthier, more nutritious, and
sustainable food options, although challenges with texture, nutrition, and flavor
remain. Enzyme engineering is advancing to address limitations such as lower
catalytic efficiency at ambient conditions and poor productivity in native microbial
cultures. Additionally, the utilization of enzymes for environmental applications is
expanding with the advent of recombinant DNA technology, protein engineering,
and rational enzyme design. These emerging research areas aim to leverage various
6-19
Introduction to Pharmaceutical Biotechnology, Volume 2 (Second Edition)
technologies, including gene shuffling, high throughput screening, and nanotechnology, to further the environmental applications of enzymes [69].
6.9.5 Biotransformation in biofuel production
The process of biotransformation plays a crucial role in the production of biofuels,
namely in the conversion of lignocellulosic biomass into bioethanol, biodiesel, and
biohydrogen through biological deterioration. A principal part of this process entails
engineering microbial strains to optimize substrate utilization, sugar transportation,
tolerance to inhibitory compounds, and increased metabolic fluxes for improved
fermentation outcomes. Such engineered microbes help economically exploit metabolic pathways for biofuel production from lignocellulosic biomass. Metabolic
engineering, a significant aspect of biotransformation, has been extensively utilized
to alter biosynthetic pathways for desired product formation in native or engineered
hosts. The challenges in this domain include the cost of enzymes required for
biomass conversion and the optimization of diverse practices for most harvests. The
integration of process engineering, fermenting technology, enzyme engineering, and
metabolic engineering has significantly propelled the progress of the biofuel sector.
The emergence of metabolic engineering and the proliferation of whole-genome
sequenced organisms have facilitated the enhancement of microbial metabolic
pathways and the synthesis of crucial molecules for biofuel production [70].
6.9.6 Biotransformation in the cosmetic industry
Biotransformation has found significant application in the cosmetic industry,
contributing to developing more environmentally friendly products. There is a
growing trend towards leveraging marine species to produce green and sustainable
cosmetic products via biotransformation. Marine microorganisms, for instance,
have been reviewed for their potential in various cosmetic applications owing to
their natural products [71]. Moreover, advancements in recombinant DNA technology and genetic engineering have facilitated the large-scale production of
bioactive molecules essential for cosmetics, addressing various ethical concerns
traditionally associated with production. Dedicated symposia and discussions in
academic and industrial circles emphasize biotransformation’s widening scope in the
pharmaceutical and cosmetic industries [72].
6.9.7 Specialized enzyme systems: lignin-modifying enzymes in biotransformation
Lignin-modifying enzymes (LMEs) are critical in acting upon lignin polymers
through catalytic cleavage, depolymerization, and valorization processes. These
enzymes are emerging as versatile biocatalysts, with applications extending to
developing environmentally friendly products like lignin biopolymers, binders,
and bio-vanillin, among others. Particularly, LMEs produced by white rot fungi
are noted for their high specificity, which enhances the yield of biotechnological
processes, ultimately aiding in the generation of biodegradable products and
reducing waste. Genome-based engineering is being employed to modify and
enhance the capabilities of ligninolytic enzymes, facilitating the breakdown of
6-20
Introduction to Pharmaceutical Biotechnology, Volume 2 (Second Edition)
lignin into less complex molecules, which holds promise for various industrial
applications [73]. Biotransformation is continuously evolving with advancements in
molecular structures and metabolic pathways. The exploration of enzyme technology, especially in the context of lignocellulosic biomass, is considered a viable
alternative for bioethanol production, although challenges related to pre-treatment
requirements and economic considerations remain. Moreover, as drug molecules
grow in complexity, the role of drug-metabolizing enzymes, a facet of biotransformation, is expanding, signifying the potential for further research and development
in this area. Furthermore, the prospective trajectory of biotransformation research
within the pharmaceutical sector suggests a significant need for specialist expertise,
notably in drug metabolism and pharmacokinetics departments [74].
References
[1] Leresche J E and Meyer H-P 2006 Chemocatalysis and biocatalysis (biotransformation):
some thoughts of a chemist and of a biotechnologist Org. Process Res. Dev.
[2] Collins A M and Kennedy M J 1999 Biotransformations and bioconversions in New
Zealand: past endeavours and future potential Aus. Biotech. 9 86–94
[3] Woodley J M and Lilly M D 1994 Biotransformation reactor selection and operation Applied
Biocatalysis ed J M S Cabral, D Best, L Boross and J Tramper (Chur: Harwood Academic)
371–93
[4] Mustacchi R et al 2005 Enhanced biotransformations and product recovery in a membrane
bioreactor through application of a direct electric current Biotechnol. Bioeng.
[5] Rojas A et al 2004 Biotransformation in double-phase systems: physiological responses of
Pseudomonas putida DOT-T1E to a double phase made of aliphatic alcohols and biosynthesis of substituted catechols Appl. Environ. Microbiol.
[6] Gao P et al 2017 Enhancing productivity for cascade biotransformation of styrene to (S)-
vicinal diol with biphasic system in hollow fiber membrane bioreactor Appl. Microbiol.
Biotechnol.
[7] Takamatsu S and Ryu D D 1988 Recirculating bioreactor–separator system for simulta-
neous biotransformation and recovery of product: immobilized l-aspartate β-decarboxylase
reactor system Biotechnol. Bioeng.
[8] Weber J M et al 2013 Biotransformation and recovery of the isoflavones genistein and
daidzein from industrial antibiotic fermentations Appl. Microbiol. Biotechnol.
[9] Siddiqui M et al 2017 Biotransformation of a potent anabolic steroid, mibolerone, with
Cunninghamella blakesleeana, C. echinulata, and macrophomina phaseolina, and biological
activity evaluation of its metabolites PLoS One
[10] Baydoun E et al 2017 Biotransformation of drospirenone, a contraceptive drug, with
Cunninghamella elegans Steroids
[11] Wang H H et al 2018 Cholesterol and lipoprotein metabolism and atherosclerosis: recent
advances in reverse cholesterol transport Ann. Hepatol.
[12] Bauer A and Brönstrup M 2014 Industrial natural product chemistry for drug discovery and
development Nat. Prod. Rep.
[13] Lavrnja I et al 2017 Expression profiles of cholesterol metabolism-related genes are altered
during development of experimental autoimmune encephalomyelitis in the rat spinal cord
Sci. Rep.
101 1857–68
32 184–91
12 e0171476
126 30–4
31 35–60
7 2702
70 3637–43
16 27–42
10 572–80
89 18–23
97 6427–37
6-21