Enzyme Immobilization: Advances in Industry, Agriculture, Medicine, and the Environment

141 Pages·2016·1.92 MB·English

by  Alka Dwevedi (auth.)

Alka Dwevedi
Enzyme
Immobilization
Advances in Industry, Agriculture,
Medicine, and the Environment
Enzyme Immobilization
Alka Dwevedi
Enzyme Immobilization
Advances in Industry, Agriculture,
Medicine, and the Environment
1 3
Alka Dwevedi
Sri Aurobindo College
University of Delhi
New Delhi
India
ISBN 978-3-319-41416-4 ISBN 978-3-319-41418-8 (eBook)
DOI 10.1007/978-3-319-41418-8
Library of Congress Control Number: 2016943793
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Preface
Immobilizing body at a particular position is a mechanism of meditation that concentrates
complete energy and allows its linear alignment from head to toe.
Chapter 1: The concept of enzyme immobilization first arose in 1916 when it was
found that enzymes could perform catalysis even in a fixed state with movement
only at the active site. Technology has evolved tremendously since then, particu-
larly with respect to immobilizing matrices. Enzyme immobilization has solutions
to various problems related to the industrial, agricultural and environmental sec-
tors, and it has a higher number of patents across the world than any other technol-
ogy known to date. However, the connectivity to their arrival in the market and
patents filed is not very appreciative. An immobilized enzyme has various height-
ened and additional properties with respect to its soluble state, such as higher
stability, more reusability, longer storage time, a broad range of activities in the
presence of various physical and chemical factors, as well as easier product recov-
ery. Chapter 1 is the compiled summary of the innovative research carried out in
the field over the last 100 years.
Chapter 2: Immobilized enzymes have a broad range of applications, including
synthesis of complex drug intermediates; chemical synthesis without toxic by-
products under mild conditions; remediation of polluted water, air and soil; and
disease diagnosis and its treatment. Enzyme immobilization is preferable to other
known technologies due to the convenience of enzyme handling, the ease of prod-
uct recovery, the possibility of enzyme reusability, the stability under extreme
physical and chemical conditions, the ease of shipment and the fact that it is appli-
cable for all reactor types and enables easier process control. There are numerous
protocols for enzyme immobilization and several modifications are emerging to
make it adaptable for any process. However, it is still a technique of trial and error;
this is supported by the fact that enzyme behavior could not be predicted with the
given matrix being implemented when a number of enzymes with similar struc-
tures that were from the same family were investigated. It has been found from a
number of studies that the immobilizing matrix plays a key role in the properties
v
vi Preface
of the immobilized enzyme. Chapter 2 is the compiled summary of recent devel-
opments in immobilization techniques with respect to the types of matrices and the
immobilization methods. Furthermore, it also discusses immobilizing enzymes in
the absence of a matrix, and the associated properties.
Chapter 3: There are a number of enzyme sources, among the most important
of which are bacteria, fungi, plants and animals. Structural studies of enzymes
from any of these sources have found a similarity of more than 70 % with an
almost negligible difference at their active sites. Bacterial and fungal sources are
widely used for various industrial processes such as the production of organic
acids, amino acids, antibiotics, food and beverages, including alcohol, cleaning
supplies, clothing, paper products, transportation fuels, agrochemicals, monitor-
ing devices, and pharmaceuticals, as well as in disease diagnosis and chemical
feedstock. This is a result of the ease with which they can be cultured and the
associated high enzyme recovery. With the evolution in purification protocols
and improvement of novel chromatographic techniques as well as instrumenta-
tion, the concept has moved for enzymes usage from microbial to plant sources.
Furthermore, plant enzymes have good catalytic properties and are high in abun-
dance. Plant carbohydrases and proteases are the main enzyme classes which
are found to have extensive industrial applications. Financial statistics of the
global market have revealed that there is tremendous requirement for enzymes.
Therefore, enzymes obtained from the best source for a particular process would
accelerate it by several fold. In addition, enzymes from plant sources have addi-
tional benefits due to the generation of huge biomass, which is of economic sig-
nificance. Chapter 3 is the compiled summary of the physiological and industrial
significance of enzymes from plants. Furthermore, it also discusses the usage of
leftover biomass for various applications.
Chapter 4: Enzymes are now known to be the important constituents of our
daily diet due to their capabilities for treating several diseases. It has been found
that eating highly processed food laden with preservatives is not good for health
as it hampers the body’s immune mechanisms and diverts energy to the produc-
tion of only digestive enzymes. This can lead to the development of several seri-
ous diseases such as cancer, cardiac arrest, and autoimmune diseases (allergies and
arthritis). There are several medications known that use enzymes for the treatment
of various diseases. However, the popularity of enzyme therapy has been limited
for various reasons, the most important of which are the state and route of admin-
istration. Soluble enzymes are prone to degradation inside the body due to various
factors, as well as to immunogenicity. Enzyme immobilization has the solution by
employing biocompatible matrices. Immobilized enzymes have enormous applica-
tions in bioanalytical and biomedical fields including in biosensors, disease diag-
nosis, bioreactors in the removal of waste metabolites and the correction of inborn
metabolic deficiencies, development of controlled release drug delivery systems,
prediction of species-dependent metabolic pathways, large-scale synthesis of
drug and xenobiotic metabolites. Furthermore, immobilized enzymes are helpful
in making drugs pharmacologically more active by conjugating with various side
groups. Chapter 4 is the compiled summary of the biomedical and bioanalytical
Preface vii
applications of immobilized enzymes as well as a discussion of their efficacy
compared with other techniques known to date.
Chapter 5: Environmental pollution is responsible for about 12.6 million deaths
across the world, annually (WHO), which is much higher than any other cause.
Developing countries (which constitute one fourth of the world’s population) are
at major risk from environmental pollution due to their unstable economy, political
instability, low productivity, high rate of damage to ecosystems and almost neg-
ligible budget towards public health. Children are at a highter risk than adults as
they have a higher metabolic rate, due to which they can ingest larger concentra-
tions of pollutants and retain them for longer durations. Furthermore, pollutants
such as lead and mercury can have toxic effects even before child is born, when it
is living inside the mother’s womb, since pollutants can easily cross the placenta.
It has been found that poor sewage systems have worsened the situation by several
fold. It has been estimated that about 30–50 % of solid wastes are not collected
and disposed of properly, which creates ideal breeding conditions for mosquitoes,
rodents and other disease-bearing organisms. Furthermore, the leaching of waste
materials from unsealed dumpsites into soil and drinking water supplies as a result
of poor landfill management has increased the severity. There is an urgent need for
effective waste management via cheaper technology. Immobilized enzymes have
solutions for the remediation of various pollutants, especially from water and air.
Chapter 5 is the compiled summary of various immobilized enzymes being used in
the treatment of waste water and polluted air.
This book is the outcome of long hours of immobilizing myself onto the chair.
It has an emphasis on simplicity so as to reach all classes, whether from the field
or not. I am very thankful to my Ph.D. supervisor, Prof. Arvind M. Kayastha
(School of Biotechnology, Banaras Hindu University), who introduced me to the
field and for his continuous motivation, which has always made me move ahead
with keen interest and enthusiasm. This book would not have been completed
without the support from my family members (mummy, papa, raman, muniya,
anshu and yogesh) who have spared their time to allow me to focus on book writ-
ing. Furthermore, I make a special thanks to the various living creatures around
me; they have motivated me in various respects and for this I find no words to
express my feelings.
New Delhi, India Dr. Alka Dwevedi
Contents
1 100 Years of Enzyme Immobilization ........................... 1
1.1 Introduction ............................................ 1
1.2 100 Years of Enzyme Immobilization ........................ 2
1.2.1 Before 1970s. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
1.2.2 Between 1970–1990 ................................ 3
1.2.3 After the 1990s .................................... 7
1.3 Important Patent Applications for the Last 25 Years ............. 13
1.4 Commercialization of Immobilized Enzymes .................. 16
2 Basics of Enzyme Immobilization .............................. 21
2.1 Introduction ............................................ 21
2.2 Methods of Enzyme Immobilization ......................... 23
2.2.1 Physical Method ................................... 23
2.2.2 Chemical Method .................................. 24
2.3 Properties of Immobilized Enzymes ......................... 26
2.4 Matrices for Enzyme Immobilization. . . . . . . . . . . . . . . . . . . . . . . . . 30
2.5 Structure Based Immobilization ............................. 33
2.6 Cross-Linked Enzyme Aggregates and Crystals ................ 40
2.7 Summary .............................................. 43
3 Enzyme Immobilization: An Important Link Between
Agriculture and Industries .................................... 45
3.1 Introduction ............................................ 45
3.2 Plants as Source of Various Industrial Enzymes ................ 46
3.3 Correlation Between Agricultural Waste and Industries .......... 54
3.3.1 Applications of Agro-Wastes in the Production
of Various Industrially Valuable Compounds
Using SSF Processes ............................... 55
3.4 Effective Ways of Immobilization of Plant Enzymes
for Commercial Applications ............................... 57
3.5 Connectivity of Economic Growth with Enzyme
Immobilization .......................................... 62
ix
x Contents
4 Implication of Enzyme Immobilization in Therapeutics
as Well as Diagnostics of Various Diseases ....................... 65
4.1 Introduction ............................................ 65
4.2 An Overview: Biomedical Applications of Enzymes ............. 67
4.2.1 Various Biological Enzyme Applications ................ 67
4.2.2 Commercialized Soluble Biomedical Enzymes ........... 70
4.3 Significance of Enzyme Immobilization for Their Biomedical
Applications ............................................ 72
4.4 Implementation of Enzymes in the Treatment of Human
Diseases ............................................... 75
4.4.1 Intra-corporeal Enzyme Therapy ...................... 75
4.4.2 Extra-Corporeal Enzyme Therapy ..................... 76
4.5 Role of Immobilized Enzymes in Pharmaceuticals .............. 78
4.5.1 Biosensors ....................................... 78
4.6 Immobilized Enzymes Used in Bioreactors .................... 81
4.6.1 Red Blood Cell as Enzymes Carrier .................... 81
4.6.2 Liposome as a Carrier for Enzymes .................... 81
4.6.3 Enzymes Encapsulation in Sol-Gel .................... 82
4.7 Immobilized Enzymes as Effective Tool for Disease
Diagnostics ............................................. 82
4.8 Future Prospects ......................................... 85
5 Enzyme Immobilization: Solution Towards Various
Environmental Issues ........................................ 87
5.1 Introduction ............................................ 87
5.2 Immobilized Enzymes in Waste Water Treatment ............... 89
5.3 Immobilized Enzymes in the Synthesis of Biodiesel ............. 97
5.4 Air Pollution and Enzyme Immobilization .................... 101
5.5 Effective Pollution Control Across the World
Using Immobilized Enzymes ............................... 104
References .................................................... 107
Chapter 1
100 Years of Enzyme Immobilization
1.1 Introduction
The concept of changing freely moving enzymes (in the solvent) into a fixed state
with movement possible only at the active site came at the beginning of 1916. It
was provided by various reports emphasizing the role of the enzymatic portion
beyond the active site during catalysis. Those portions are required for the mainte-
nance of the actual shape and orientation of the active site to allow the enzyme to
accurately fit with the substrate and carry out catalysis [1, 2].
The invertase was the first enzyme to be immobilized onto matrices such as
charcoal and aluminium hydroxide in late 1916. Immobilized invertase had exhib-
ited a similar activity to that of its soluble state [3]. This discovery provided the
basis for the currently available enzyme immobilization techniques. During the
early days (before the 1940s), available immobilization techniques were based on
using a very high amount of enzyme as compared with matrix. This constricted
the use of immobilization for all enzymes, especially those not available in large
quantities.
There are more than 10,000 publications (Fig. 1.1) and a number of patents
that have been published based on enzyme immobilization, including immobili-
zation techniques, matrices and applications, and more will be published in the
future. There are various immobilized enzymes that have been commercialized for
various applications. Enzymes such as penicillin G acylase, invertase, lipases and
proteases are being implicated in various large-scale processes [4]. Even though
enzyme immobilization has come a long way, it is still a subject for research as
shown by the number of published research and review articles working on its
various aspects. The year 2010 is thought of as wondrous in the field of enzyme
immobilization since there were about 200 articles that were published in the first
6 months, as per PubMed database. Enzyme immobilization has made enzymes a
commercially valuable biomolecule due to various additional properties such as
© Springer International Publishing Switzerland 2016 1
A. Dwevedi, Enzyme Immobilization, DOI 10.1007/978-3-319-41418-8_1