Polymeric Supports for Enzyme Immobilization: Opportunities and Applications

By Alka Dwevedi

Enzyme immobilization has been approached for finding solutions for various critical problems associated with industries, medicine, environment, agriculture, etc. Especially since last decades, several innovative researches have come up to look for enhancing catalytic efficiency, reusability of immobilized enzyme and longer stability by introducing range of immobilizing supports, supports modifiers through introduction of several chemical agents (non-toxic) and adopting innovative enzyme immobilization methods. In the present book, polymeric supports have been focussed for enzyme immobilization, especially due to their versatility in immobilizing different enzymes for different large scale enzyme reactors to be used for several applications. Especially, polymers can be modified according to applications and enzyme properties which have made it supports of choice for all several enzyme based applications. Polymeric Supports for Enzyme Immobilization: Opportunities and Applications offers in-depth discussions of known polymeric enzyme support materials, reaction processes, and optimized methods to enhance enzyme immobilization. Case-based chapters examine methods of enzyme immobilization onto various polymeric supports, their surface chemistry and physical morphology followed by implementation of polymers based immobilized enzymes in various applications, viz. medicine, environment, industries, clean energy, disease diagnosis, drug delivery etc. This book has prime focus to allow several researchers across the world to provide updated technological details and incite to contribute more innovative work in coming years to find solutions to several critical problems.

• Offers an in-depth, case-driven discussion of known polymeric enzyme support materials, associated reaction processes, and methods to enhance enzyme immobilization
• Provides optimal strategies for various enzymes, processes, and applications, considering the enzyme itself, substrate, and available support properties
• Provides complete details on applications of polymeric based immobilized enzymes in various applications ranging from chemical; or pharmaceutical synthesis, food processing, bioremediation, industrial catalysis, etc.

• Language: English
• Publisher: Academic Press
• Release date: Jun 22, 2021
• ISBN: 9780128192078

Alka Dwevedi

She has joined UNESCO-Regional Centre for Biotechnology, India as young investigator in 2011. She is presently a guest faculty at University of Delhi. She has published 20 articles in peer review International and National Journals in the fields of Biochemistry and Molecular Biology, Microbiology, Biophysics, Proteomics, Food Chemistry, Nanotechnology, Biotechnology, Medicinal Chemistry and Pharmacology. She has published 6 book chapters, 2 monographs. She is the first author in almost all published articles, reviews and book chapters. Dr Dwevedi is on Authorial board in various international journals including Developmental Microbiology and Molecular Biology, International Journal of Biotechnology and Bioengineering Research, International Journal of Applied Biotechnology and Biochemistry, Wyno Journal of Biological Sciences, International Journal of Genetic Engineering and Biotechnology, Global Journal of Microbiology and Biotechnology, International Journal of Molecular Biology and Biochemistry, World Journal of Biotechnology and reviewer in several journals. Further, she is a life member of Indian Biophysical Society and also a member of American Chemical Society.

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Preface

Alka Dwevedi

Science has become an integral part of our lives, it has become essentially important to know important facts to never get into loop of confusion. Corona pandemic has proved it correctly. It has become a moral responsibility of the people in science to present their facts in legitimate manner (rather glamourizing) to be easily approachable by common man. It would help them to make correct decisions in adapting any scientific technology in their life.

This book is an initiative to present all the facts in an easiest and most apprehensible way to provide complete details on enzyme technology based on polymers and their applications in various streams.

In Chapter 1, Current and Future Trends on Polymer-based Enzyme Immobilization, enzymes (nature’s catalysts) have been the crucial partner in various human activities, from daily chores to commercial market. It has been well validated now that enzymes have the solutions for every problem ranging from clinical, industrial, environmental, physiological, and so on. The best alluring aspect of enzymes is that they can perform actions under mild conditions with a very high degree of substrate specificity with least generation of by-products. However, their utilization for various applications has been limited due to their relative instability and their high costs of isolation. Enzyme immobilization has found solution to this problem as it allows reusability of enzymes in addition to its stability, activity, inhibition by reaction products, and selectivity towards nonnatural substrates. Thorough studies have been going on across the world to look for excellent matrices for enzyme immobilization as well regular improvization of immobilization techniques to obtain immobilized enzyme with excellent physicochemical properties. The parameters like matrix properties including mean particle diameter, swelling behavior, mechanical strength, compression behavior, and surface area have been most critical in determining the performance of immobilized enzymes. These parameters are useful in designing correct bioreactor for any given large-scale industrial processes. This chapter has provided a summary on a wide range of polymeric matrices and their utilization for enzyme immobilization for various industrial applications.

In Chapter 2, Implication of Polymer-based Immobilized Enzymes in Medicine, enzymatic catalysis has been extensively used for wide applications ranging from chemical to pharmaceutical synthesis, food processing, bioremediation, industrial catalysis, and so on under mild conditions with utmost efficacy with respect to chemical processes without any usage of hazardous solvents under mild conditions (temperature: 10°C–50°C, pH: 4.0–8.0) in an inexpensive and environmentally friendly manner. Enzymes are nontoxic, readily degradable, and can be produced in unlimited quantities; therefore their derived processes are several folds higher economical than chemical based processes. Enzymes cannot be reused, relatively fragile, much susceptible in the presence of extreme physicochemical environment, and is almost impossible to separate them from reaction mixtures. Immobilization of enzymes onto suitable matrices has been very useful to overcome various shortcomings as mentioned for soluble enzymes. It helps in protecting enzyme from inactivation due to any physicochemical challenges and optimizing in-service performance of enzyme in various industrial processes in addition to drastic decrease in overall cost of industrial productions. The immobilizing matrix (especially its chemical composition and surface morphology) and immobilization methods are the crucial determinants for catalytic efficiency of immobilized enzymes during various industrial processes.

Polymeric materials as immobilization matrix for enzyme have provided versatility in immobilizing vast variety of enzymes involve in various types of applications such as oxidation, reduction, inter- and intra-molecular transfer of groups, hydrolysis, cleavage of covalent bonds by elimination, addition of groups to double bonds, and isomerization. It is so because polymeric materials can be modulated into various forms such as particles, membranes, and nanofibers with adjustable physicochemical properties making it more desirous for various enzyme-based industrial applications. Most importantly, with the presence of such versatility in polymeric materials, it is possible to improvize catalytic efficiency, stability, reusability cycles, as well as minimizing nonspecific interactions. It is extremely important to scrutinize the type of polymer and its chemical nature before using it for any specific enzyme immobilization. The chosen polymeric immobilizing support should protect the enzyme structure by providing stable enzyme–matrix interactions by thoroughly understanding the type of functional groups (important in enzyme attachment as well as its microenvironment) present on the matrix. This chapter is based on applications of polymer-based immobilized enzyme in medicine (viz. disease diagnostics, therapeutics, strengthening immune system, disease marker, etc.).

In Chapter 3, Modulation of Polymer-based Immobilized Enzymes for Industrial Scale Applications, enzymes have become a crucial part of various industries like development and production of nutritious food and beverage products, processing of fruit and vegetable, cheese, protein, grains, and fats and oils, as well as in several other industries like dairy, baking, brewing, and cereal extraction. It has been seen since last decade that enzymes have been sharing a major part in global market with its increasing usage in various sectors. R&D centers of several companies have been now focusing on evaluation, development, upscaling (usually by expression and cloning of enzymes), validation of technologies, and innovative enzyme formulation for various commercial processes. Enzyme technology has been known for several hundreds of years; however, 21st century has revolutionized it due to its increased applications in almost every sector. According to Markets and Markets, the enzymes have market of USD 5.9 billion (estimated for 2020) while it has been expected to reach USD 8.7 billion by 2026 according to CAGR. The industrial applications of enzymes have a prerequisite of enzyme to be in its insoluble state as soluble state is fragile requiring extensive protocols for its stability during long storage as well as cost-expensive due to its single use. The enzymes in its immobilized state can be used for several times with easier recovery from reaction mixture and maintained for long time periods. The enzyme immobilization is actually a platform which involves a combination of enzyme selectivity and kinetics with the physical and chemical properties of the immobilizing matrix in a specialized formulation to maximize both its stability and catalysis and make cost-effective industrial process. Most importantly, scaling up of immobilized process from laboratories to industries requires lesser optimization in a cost-effective manner than that based on soluble enzymes. Furthermore, immobilized enzyme-based industrial processes have been enticing due to least time consumption and cost-efficacious downstream processing. This chapter is based on modulating enzyme immobilization so that lab-scale experimentation can be easily scaled up to industrial scale processes with maximum product yield and minimal downstream processing.

In Chapter 4, Polymer-based Immobilized Enzymes in Environmental Remediation, environmental pollution leads to the release of harmful substances into air, water, and soil and get accumulated due to their recalcitrance. It has become extremely important now to develop new degradative low-cost and eco-friendly technologies which would be helpful in the removal of recalcitrant pollutants from the environment. The pollutants have severe effects on the entire available flora and fauna (affect crucial processes like respiration, photosynthesis, reproduction as well as birth defects like in humans, Down’s syndrome, anencephaly, spina bifida, etc.). Environmental pollution has affected all the countries of the world, however with a varied rate of pollutants accumulation. It has been seen that there is almost direct relationship with the development and extent of pollution. Developed countries have evolved several technologies to control environmental pollution; however, it has become more problematic for developing countries due to expensive available technologies for environmental remediation. It has become extremely important now to develop efficient and cost-effective technologies for environmental remediation. Major goals are recovery of soil health and fertility, reutilization of wastewater (removal of recalcitrant compounds), detoxification of groundwater and production of healthy air. We have several methodologies for the remediation of polluted systems with successful results, particularly categorized into engineering (physical and chemical methods) and biological (composting, land fanning, bioreactors, bioremediation, phytoremediation and enzyme-based remediation) methods. It has been observed that biological methods are commonly used for remediation of soil and groundwater, while physical and chemical methods are being used for the purification of polluted air. Enzyme-based environmental remediation has been very successful in the removal of recalcitrant compounds in a very short duration under mild conditions. It has been recommended to use immobilized enzyme for environmental remediation as degrading enzymes are rare (found only in specific species) leading to cost-expensive isolation and purification protocols. Furthermore, enzyme immobilization allows enzyme reusability and stability, thus decontamination of polluted sites can be carried out in both in situ (directly on contaminated site) as well as ex situ (away from contaminated site). There is a range of immobilizing matrices for enzyme immobilization, it is often recommended to use eco-friendly matrices especially when immobilized enzyme is to be used for environmental remediation. It has been argued that environmental remediation should be such that purified air, water, and soil could be directly used by the living organisms without producing any innocuous affects. This chapter is based on providing an overview of enzymes important in environmental remediation, commonly used eco-friendly polymers for their immobilization, as well as patented enzyme-based technology for environmental remediation.

Chapter 1

Current and future trends on polymer-based enzyme immobilization

Ranjana Das¹, Alka Dwevedi² and Arvind M. Kayastha¹,    ¹School of Biotechnology, Institute of Science, Banaras Hindu University, Varanasi, India,    ²Department of Biotechnology, Delhi Technological University, New Delhi, India

Abstract

Enzymes (nature’s catalysts) have been the crucial partner in various human activities, from daily chores to commercial market. It has been well validated now that enzymes have the solutions for every problem ranging from clinical, industrial, environmental, physiological, etc. The best alluring aspect of enzymes is that they can perform actions under mild conditions with a very high degree of substrate specificity with least generation of by-products. However, their utilization for various applications has been limited due to their relative instability and their high costs of isolation. Enzyme immobilization has found solution to this problem as it allows reusability of enzymes in addition to its stability, activity, inhibition by reaction products, and selectivity toward nonnatural substrates. Thorough studies have been going on across the world to look for excellent matrices for enzyme immobilization as well regular improvisation of immobilization techniques to obtain immobilized enzyme with excellent physicochemical properties. The parameters like matrix properties, including mean particle diameter, swelling behavior, mechanical strength, compression behavior, and surface area, have been most critical in determining the performance of immobilized enzymes. These parameters are useful in designing correct bioreactor for any given large-scale industrial processes. The present chapter has provided a summary on the wide range of polymeric matrices and their utilization for enzyme immobilization for various industrial applications.

Keywords

Enzymes; polymers; immobilization; applications; matrices; stability; reusability

1.1 Introduction

The enzymes are the nature’s catalysts that facilitate the conversion of chemical species present in living system and, thus, play a key role in the normal functioning of biological cell. Enzymes have been the crucial partner in various human activities, from daily chores to commercial market. It has been well validated now that enzymes have the solutions for every problem ranging from clinical, industrial, environmental, physiological, etc. The best alluring aspect of enzymes is that they can perform actions under mild conditions with a very high degree of substrate specificity with least generation of by-products. However, their utilization for various applications has been limited due to their relative instability and high costs of isolation. The solution to this has come from immobilization, involving enzyme attachment or encapsulation on/in to suitable matrices that allow reusability of enzymes as well as stability, activity, inhibition by reaction products, and selectivity toward nonnatural substrates.

Enzyme immobilization has been useful as it has led to substantial reduction in the operational expenses for various potentially commercial applications. Thorough research has been going on across the world to look for excellent matrices for enzyme immobilization as well regular improvisation of immobilization techniques to obtain immobilized enzyme with excellent physicochemical properties. The parameters like matrix properties, including mean particle diameter, swelling behavior, mechanical strength, and compression behavior, have been most critical in determining the performance of immobilized enzymes. Further, it is the determinant factor in the type of reactor to be used under technical conditions (i.e., stirred tank, fluidized, and fixed beds). Further, pore parameters and particle size of the matrices determine the total surface area and, thus, specifically affect the capacity for binding of enzymes. For example, nonporous supports have been found to have lesser diffusional constrains with lower enzyme loading capacity with respect to porous supports. Further, porous supports have precise pore distribution that is helpful in optimizing flow properties. On the basis of chemical properties of matrices, they are categorized as organic and inorganic, with former having wide industrial applications, while later having high stability against physical, chemical, and microbial degradation [1]. The present chapter is based on overview on the wide range of polymeric matrices and their utilization for enzyme immobilization for various applications. Further, it has also discussed various immobilization methods like surface attachment and encapsulation, including some modification strategies like spacer arm introduction in covalent bonding.

1.2 Immobilization techniques

The criteria deciding best immobilization strategy depend on both enzymes and matrix types. In general, immobilization techniques (Fig. 1.1) can be divided into four types with each one having their characteristic advantages and disadvantages as discussed in following sections.

Figure 1.1 Approaches to enzyme immobilization: (A) adsorption, (B) covalent, (C) entrapment in beads, (D) entrapment in fibers, (E) microencapsulation, and (F) cross-linking.

1.2.1 Noncovalent adsorption, deposition, and hydrogen bonds

This method relies upon physical interactions between enzyme and carrier matrix involving weak forces like Van der Waals forces, hydrogen bond, entropy changes, or ionic interactions holding both the components together. Whenever an enzyme with lipophilic surface area comes in contact with a hydrophobic support matrix, entropy changes or Van der Waal forces play actively in the process of immobilization. A hydrophilic enzyme using a hydrophilic carrier guarantees hydrogen bonding between the enzyme and carrier matrix. Regardless of weak bonding strength, the greatest advantage of this method is that it does not alter the enzyme conformation, maintaining the enzyme active site, withholding its activity. This type of immobilization technique makes a use of functional groups present on the carrier material. Enzyme and carrier, both have affinity for each other and in the absence of groups, an intermediate agent can be used as a modifier. Major drawback of this method is the leaching of enzyme from the support as the reaction proceeds in the absence of any strong covalent bond. Thus this technique is ineffective for long-term procedures.

1.2.2 Cross-linking

It is an irreversible form of enzyme immobilization. It is a matrix-free technique where every enzyme molecule acts as a carrier for each other. This involves intermolecular cross-linking of enzyme molecule via surface functional groups of insoluble support matrices [2]. Cross-linked enzyme aggregates (CLEAs) are usually prepared by using precipitating agents like organic solvents (acetone, ethanol, propanol, and tert-butanol), salts (ammonium sulfate), or nonionic polymers (polyethylene glycol) and subsequent binding to each other using glutaraldehyde as bifunctional cross-linking agent [3–5]. This process imparts comparable stability, preventing enzyme leakage with lower desorption. Hence, CLEAs owing higher enzymatic activity and stability eliminate the use of additional carrier matrix with the ease of separation, which enables simple and cost-effective production technique [6,7]. However, in some cases enzyme may be rendered ineffective due to cross-linking. This could be attributed to low amine content of the enzyme, since efficiency of cross-linking depends on amine content of enzyme. Fig. 1.1F shows how free enzymes are transformed into aggregates with the help of glutaraldehyde.

1.2.3 Encapsulation/microentrapment

The entrapment/encapsulation method is based on the confinement of an enzyme within a polymeric network that allows the substrate and products to pass through but retains the enzyme. It is a unique method in that it cages the enzyme rather than binding the enzyme to a matrix or membrane. There are various methods of entrapping enzymes such as gel or fiber entrapping and microencapsulation [8]. Application in practice of these methods is limited by mass transfer limitations through membranes or gels and enzyme outflow. Probability of enzyme damage due to chemical and heat used during the formation of gel [9] is quite frequent. Nevertheless, problem of enzyme outflow can be overcome by controlling the polymerization conditions. This could be attained by altering the pore size, surface functional group, and network structure of polymeric material [10]. Recently, entrapment of enzyme on nanostructured support materials, namely, electrospun, nanofibers, and pristine materials, has attracted wider interest for various industrial applications