Current Developments in Biotechnology and Bioengineering. Production, Isolation and Purification of Industrial Products 1st Edition Ashok Pandey

Current Developments in
Biotechnology and Bioengineering.

Production, Isolation and Purification of
Industrial Products 1st Edition Ashok
Pandey
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Current Developments
in Biotechnology and
Bioengineering
Production, Isolation and Purification
of Industrial Products
Edited by
Ashok Pandey, Sangeeta Negi,
Carlos Ricardo Soccol
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List of Contributors
M. Adsul DBT-IOC Centre for Advanced Bioenergy Research, IndianOil Corporation

Limited
Cristóbal N. Aguilar Food Research Department, School of Chemistry,

Autonomous University of Coahuila, Saltillo, Coahuila, México
A. Angel-Cuapio Universidad Autónoma Metropolitana-Iztapalapa, Mexico City,

DF, Mexico
G.S. Anisha Government College for Women, Trivandrum, Kerala, India
P. Binod CSIR-National Institute for Interdisciplinary Science and Technology (NIIST),

Trivandrum, India
J. Buenrostro-Figueroa Department of Biotechnology, Division of Health and

Biological Sciences, Metropolitan Autonomous University, Iztapalapa, México
S. Chakraborty Indian Institute of Technology Guwahati, Guwahati, Assam, India
M.L. Chávez González Food Research Department, School of Chemistry,

G.-Q. Chen Tsinghua University, Beijing, China
S. Chen Hubei University, Wuhan, PR China
Juan C. Contreras-Esquivel Food Research Department, School of Chemistry,

J.D. Coral Bioprocess Engineering and Biotechnology Department, Federal

University of Paraná (UFPR), Curitiba, PR, Brazil
J.C. de Carvalho Bioprocess Engineering and Biotechnology Department, Federal

xxi
xxii List of Contributors
J. de Oliveira Bioprocess Engineering and Biotechnology Department, Federal

A. Dhillon Indian Institute of Technology Guwahati, Guwahati, Assam, India
M.J. Fernandes Bioprocess Engineering and Biotechnology Department, Federal

R. Gaur Indian Oil Corporation Limited, R&D Centre, Faridabad, India
A. Goyal Indian Institute of Technology Guwahati, Guwahati, Assam, India
L.R.C. Guimarães Bioprocess Engineering and Biotechnology Department, Federal

M. Haridas Kannur University, Kannur, India
R. Hemamalini Indian Institute of Technology Delhi, New Delhi, India
Ayerim Hernandez-Almanza Food Research Department, School of Chemistry,

A. Illanes Pontificia Universidad Católica de Valparaíso, Valparaíso, Chile
J. Isar University of Delhi South Campus, New Delhi, India
A. Joseph Kannur University, Kannur, India
S.G. Karp Bioprocess Engineering and Biotechnology Department, Federal

N. Karthik CSIR-National Institute for Interdisciplinary Science and Technology

(NIIST), Trivandrum, India
R. Kaushik University of Delhi South Campus, New Delhi, India
S.K. Khare Indian Institute of Technology Delhi, New Delhi, India
P.C.S. Kirnev Bioprocess Engineering and Biotechnology Department, Federal

D. Kothari Indian Institute of Technology Guwahati, Guwahati, Assam, India

List of Contributors xxiii
C. Larroche Blaise Pascal University, Aubière Cedex, France
L.A.J. Letti Bioprocess Engineering and Biotechnology Department, Federal

O. Loera-Corral Universidad Autónoma Metropolitana-Iztapalapa, Mexico City, DF,

Mexico
A.I. Magalhães, Jr. Bioprocess Engineering and Biotechnology Department,

Federal University of Paraná (UFPR), Curitiba, PR, Brazil
A.B.P. Medeiros Bioprocess Engineering and Biotechnology Department, Federal

J.D.C. Medina Bioprocess Engineering and Biotechnology Department, Federal

F. Miranda-Hernández Universidad Autónoma Metropolitana-Iztapalapa, Mexico

City, DF, Mexico
N.R. Nair CSIR-National Institute for Interdisciplinary Science and Technology

S. Nair Dow Chemicals GmBH, Dubai, UAE
K.M. Nampoothiri CSIR-National Institute for Interdisciplinary Science and

Technology (NIIST), Trivandrum, India
A. Nandan CSIR-National Institute for Interdisciplinary Science and Technology

S. Negi Motilal Nehru National Institute of Technology, Allahabad, India
M.G.B. Pagnoncelli Bioprocess Engineering and Biotechnology Department,

Federal University of Paraná (UFPR), Curitiba, PR, Brazil; Federal Technological
University of Parana, Dois Vizinhos, Brazil
A. Pandey Center of Innovative and Applied Bioprocessing, (a national institute

under Dept of Biotechnology, Ministry of S&T, Govt of India), Mohali, Punjab, India
A.K. Patel DBT-IOC Centre for Advanced Bioenergy Research, IndianOil Corporation
Limited
xxiv List of Contributors
V. Rajulapati Indian Institute of Technology Guwahati, Guwahati, Assam, India
S. Ramachandran Insight Professional Institute, Dubai, UAE
A. Rani Indian Institute of Technology Guwahati, Guwahati, Assam, India
C. Rodrigues Bioprocess Engineering and Biotechnology Department, Federal

Rosa M. Rodríguez-Jasso Food Research Department, School of Chemistry,

R. Rodríguez Food Research Department, School of Chemistry, Autonomous

University of Coahuila, Saltillo, Coahuila, México
L.V. Rodríguez Durán Department of Biotechnology, Division of Health and

Biological Sciences, Metropolitan Autonomous University, Iztapalapa, México
Héctor A. Ruiz Food Research Department, School of Chemistry, Autonomous

A. Sabu Kannur University, Kannur, India
R. Saini DBT-IOC Centre for Advanced Bioenergy Research, IndianOil Corporation

Limited
S. Sajitha CSIR-National Institute for Interdisciplinary Science and Technology

S. Saran University of Delhi South Campus, New Delhi, India
R.K. Saxena University of Delhi South Campus, New Delhi, India
V.C. Sekhar CSIR-National Institute for Interdisciplinary Science and Technology

K. Sharma Indian Institute of Technology Guwahati, Guwahati, Assam, India
R. Sindhu CSIR-National Institute for Interdisciplinary Science and Technology

R.P. Singh Punjabi University, Patiala, Punjab, India

List of Contributors xxv
R.S. Singh Punjabi University, Patiala, Punjab, India
Reeta R. Singhania DBT-IOC Centre for Advanced Bioenergy Research, IndianOil

Corporation Limited
C.R. Soccol Bioprocess Engineering and Biotechnology Department, Federal

T.S. Swapna Government Victoria College, Palakkad, India
D. Tan Xían Jiaotong University, Xían, China
L. Thomas CSIR-National Institute for Interdisciplinary Science and Technology

M.V. Ushasree CSIR-National Institute for Interdisciplinary Science and Technology

P. Valencia Universidad Técnica Federico Santa María, Valparaíso, Chile
L.P.S. Vandenberghe Bioprocess Engineering and Biotechnology Department,

K. Vibha Motilal Nehru National Institute of Technology, Allahabad, India
J. Vidya CSIR-National Institute for Interdisciplinary Science and Technology (NIIST),

Trivandrum, India
N. Vijayan Kannur University, Kannur, India
N. Vivek CSIR-National Institute for Interdisciplinary Science and Technology (NIIST),

Trivandrum, India
Q. Wang Hubei University, Wuhan, PR China
X. Wei Hubei University, Wuhan, PR China
A.L. Woiciechowski Bioprocess Engineering and Biotechnology Department,

J. Yin Tsinghua University, Beijing, China

xxvi List of Contributors
A. Zandoná Filho Bioprocess Engineering and Biotechnology Department, Federal

P.A. Zárate Food Research Department, School of Chemistry, Autonomous

S.F. Zawadzki Bioprocess Engineering and Biotechnology Department, Federal

About the Editors
Ashok Pandey
Professor Ashok Pandey is Eminent Scientist at the Center of
Innovative and Applied Bioprocessing, Mohali (a national
institute under the Department of Biotechnology, Ministry
of Science and Technology, Government of India), and
former chief scientist and head of the Biotechnology
Division at the CSIR’s National Institute for Interdisciplinary
Science and Technology at Trivandrum. He is an adjunct
professor at Mar Athanasios College for Advanced Studies
Thiruvalla, Kerala, and at Kalasalingam University, Krishnan
Koil, Tamil Nadu. His major research interests are in the
areas of microbial, enzyme, and bioprocess technology,
which span various programs, including biomass to fuels
and chemicals, probiotics and nutraceuticals, industrial
enzymes, solid-state fermentation, etc. He has more than
1100 publications and communications, which include 16
patents, 50+ books, 125 book chapters, and 425 original and review papers, with an h index
of 75 and more than 23,500 citations (Google Scholar). He has transferred several tech-
nologies to industries and has been an industrial consultant for about a dozen projects for
Indian and international industries.
Professor Pandey is the recipient of many national and international awards
and fellowships, which include Elected Member of the European Academy of Sciences
and Arts, Germany; Fellow of the International Society for Energy, Environment and
Sustainability; Fellow of the National Academy of Science (India); Fellow of the Biotech
Research Society, India; Fellow of the International Organization of Biotechnology and
Bioengineering; Fellow of the Association of Microbiologists of India; honorary doctorate
degree from the Université Blaise Pascal, France; Thomson Scientific India Citation
Laureate Award, United States; Lupin Visiting Fellowship; Visiting Professor at the
Université Blaise Pascal, France, the Federal University of Parana, Brazil, and the École
Polytechnique Fédérale de Lausanne, Switzerland; Best Scientific Work Achievement
Award, Government of Cuba; UNESCO Professor; Raman Research Fellowship Award,
CSIR; GBF, Germany, and CNRS, France fellowships; Young Scientist Award; and others.
He was chairman of the International Society of Food, Agriculture and Environment,
Finland (Food & Health) during 2003e04. He is the Founder President of the Biotech
xxvii
xxviii About the Editors
Research Society, India (www.brsi.in); International Coordinator of the International

Forum on Industrial Bioprocesses, France (www.ifibiop.org); chairman of the
International Society for Energy, Environment & Sustainability (www.isees.org); and vice
president of the All India Biotech Association (www.aibaonline.com). Professor Pandey
is editor-in-chief of Bioresource Technology, Honorary Executive Advisor of the Journal of
Water Sustainability and Journal of Energy and Environmental Sustainability, subject
editor of the Proceedings of the National Academy of Sciences (India), and editorial board
member of several international and Indian journals, and also a member of several
national and international committees.
Sangeeta Negi
Dr. Sangeeta Negi is an assistant professor in the Department
of Biotechnology at the Motilal Nehru National Institute of
Technology, India. She has a First Class Master’s degree in
biochemistry and a PhD in biotechnology from the Indian
Institute of Technology, Kharagpur. She has also worked as
an academic guest at the Biological Engineering Department,
Polytech Clermont-Ferrand; the Université Blaise Pascal,
France; and the Bioenergy and Energy Planning Research
Group, Swiss Federal Institute of Technology, Lausanne,
Switzerland. Dr. Negi’s current research interests are in the
areas of biofuels, industrial enzymes, and bioremediation. She is an editorial board
member of the Journal of Waste Conversion, Bioproducts and Biotechnology and the Journal
of Environmental Science and Sustainability. She has been awarded as Outstanding
Reviewer by Elsevier and has won the Young Scientist Award by DST at the National
Seminar on Biological and Alternative Energies Present and Future, organized by Andhra
University, Visakhapatnam, in 2009. She has also won the Best Poster Award at the
International Congress on Bioprocesses in Food Industries (2008) at Hyderabad. Dr. Negi
has contributed to nearly 70 publications, including review articles, original papers, and
conference communications.
About the Editors xxix

Professor Carlos Ricardo Soccol is the research group leader
of the Department of Bioprocesses Engineering and
Biotechnology at the Federal University of Paraná (UFPR),
Brazil, with 20 years of experience in biotechnological
research and development of bioprocesses with industrial
application. He graduated with a BSc in chemical engi-
neering (UFPR, 1979), Master’s in food technology (UFPR,
1986), and PhD in Génie Enzymatique, Microbiologie et
Bioconversion (Université de Technologie de Compiègne,
France, 1992). He did his postdoctoral work at the Institut
ORSTOM/IRD (Montpellier, 1994 and 1997) and at the
Université de Provence et de la Méditerranée (Marseille,
2000). He is an HDR Professor at the École d’Ingénieurs Supériure of Luminy,
MarseilleeFrance. He has experience in the areas of science and food technology, with
emphasis on agro-industrial and agro-alimentary biotechnology, acting in the following
areas: bioprocess engineering and solid-state fermentation, submerged fermentation,
bioseparations, industrial bioprocesses, enzyme technology, tissue culture, bio-
industrial projects, and bio-production. He is currently the Coordinator of Master
BIODEV-UNESCO, associate editor of five international journals, and editor-in-chief of
the journal Brazilian Archives of Biology and Technology. Professor Soccol has received
several national and international awards, which include the Science and Technology
Award of the Government of Paraná (1996); Scopus/Elsevier Award (2009); Dr. Honoris
Causa, Université Blaise Pascal, France (2010); Outstanding Scientist at the 5th
International Conference on Industrial Bioprocesses, Taipei, Taiwan (2012); and Elected
Titular Member of the Brazilian Academy of Sciences (2014). He is a technical and sci-
entific consultant for several companies, agencies, and scientific journals in Brazil and
abroad. He has supervised and mentored 96 Master of Science students, 48 PhD stu-
dents, and 14 postdoctoral students. He has 995 publications and communications,
which include 17 books, 107 book chapters, 270 original research papers, and 557
research communications in international and national conferences and has registered
44 patents. His research articles as of this writing have been cited (Scopus database) 5600
times with an h index of 36.
Preface
This book is a part of the comprehensive series Current Developments in Biotechnology and
Bioengineering, comprising nine volumes (Editor-in-chief: Ashok Pandey), and deals with the
production, isolation, and purification of industrial products produced by biotechnological
processes. This book covers recent technological advances of a great number of biotechno-
logical products and is divided into four different parts: Production of Industrial and
Therapeutic Enzymes, Organic Acids, Biopolymers and Other Products, and Products
Isolation and Purification.
Part 1 is devoted to the production of industrial and therapeutic enzymes. The first
chapter describes the current and future trends of production, application, and strain
improvement of a-amylases, one of the most important enzymes used in industry.
a-Amylases find application in several industrial processes, such as starch liquefaction,
desizing of textiles, detergents, baking, bioethanol production, etc. Glucoamylase is another
enzyme extensively used in the food and fermentation industries, mainly for the saccharifi-
cation of starch, brewing, and production of high-fructose syrup, which are discussed in
Chapter 2. Cellulases, b-glucosidases, and xylanases are the second most used enzymes in
industry by sales volume, with an increasing demand since 1995 in several industrial appli-
cations, comprising detergents and textiles, animal feed, food, paper, and biofuels. These
enzymes are discussed in Chapters 4, 5, and 6 of this book. Chapter 7 discusses proteolytic
enzymes, also known as “proteases,” which are used to cleave the peptide bonds connecting
two amino acids. They are produced mainly by microorganisms and have great commercial
value, being used in food, dairy, detergents, and leather processing. Lipolytic enzymes are
hydrolases comprising 15 families of lipases, as shown in Chapter 8 of this book through a
study of the industrial applications and other important aspects of these enzymes. The
purpose of Chapters 9 and 10 is to present an overview of laccases and peroxidases, covering
their production and use in the pretreatment of lignocellulosic biomass and biopulping, and
also projecting new perspectives on improving such processes and products using these
enzymes. Sources of production, strategies, characteristics, applications, and industrial
importance of therapeutic enzymes, such as L-glutaminase, L-asparaginase, and penicillin
acylase, are presented and discussed in Chapters 11, 12, and 13. Other enzymes, such as
phytases, chitinases, keratinases, tannases, aminopeptidases, nattokinases, and poly-
saccharide lyases, are reviewed in Chapters 14 to 23, covering recent advances, production
methods, potential applications, and the global market.
The second part of the book is dedicated to organic acids. In Chapters 24 and 25, lactic
acid and citric acid production, synthesis (covering factors that affect biochemical pathways),
and recovery are addressed. Chapter 26 reviews the microbial production of gluconic acid,
properties of glucose oxidase, production, recovery, and applications. Succinic acid is an
important platform molecule, used as an intermediate in the production of numerous
everyday products, among which are pharmaceuticals and adhesives, representing a total
immediate addressable market of more than $7.2 billion. Chapter 27 presents an analysis of
the current market, biological-based production processes, enzymatic regulation, and
recovery systems of succinic acid.
xxxi
xxxii Preface
Part 3 discusses polymer production and other products. Polylactide (PLA), derived
from lactic acid, a biodegradable polyester, has applications in packaging, textiles, and the
biomedical and pharmaceutical industries. Chapter 28 reviews the properties and applica-
tions of PLA, focusing on recent technologies and improvement of production techniques.
Polyhydroxyalkanoates (PHAs), a family of environmentally friendly polyesters that can be
synthesized by a wide range of microorganisms as carbon and energy reserves, have been
considered an alternative to petroleum-based chemicals. The composition and structural
diversity of PHAs have led to various properties and endless applications to form a PHA value
chain. Chapter 29 briefly introduces their production and application, highlighting the lab-
oratory production by the microbial strains developed using genetic and/or metabolic en-
gineering or synthetic biology techniques. Industrial production, recent technologies, and
improvement of PHA production are also discussed. Poly-g-glutamic acid (g-PGA) is a natural
polymer, synthesized by various strains of Bacillus spp., that is used in food, cosmetics,
agriculture, and the wastewater industry. Chapter 30 provides updated information on the
biosynthesis, fermentation, purification, and application of g-PGA. In Chapter 31, recent
developments in the biological production of 1,3-propanediol by various natural and
genetically engineered microorganisms, nonnative 1,3-propanediol producers, as well as
mixed cultures, are discussed. Important aspects of downstream processing and various
methods and steps involved in the extraction and purification of 1,3-propanediol from the
fermentation broth are also covered in this chapter. The production of petroleum-based
plastics is a challenging environmental problem, causing the production and consumption
of biodegradable plastics to receive considerable attention nowadays. Chapter 32 provides an
overview of the degradation mechanisms of biodegradable polymers, with particular
emphasis on the main parameters affecting the degradation of these polymeric biomaterials.
In Chapter 33 the potential of biological control is presented and discussed as a promising
alternative to chemical pesticides. The final two chapters of this book, Chapters 34 and 35,
present the most relevant downstream processes to extract, isolate, purify, and refine
fermentation products.
We are confident that this book will be profitable to students, professors, researchers,
and professionals interested in studying biotechnology and bioengineering. We thank
Dr. Kostas Marinakis, Book Acquisition Editor; Ms. Anneka Hess; and entire production team
at Elsevier for their help and support in bringing out this volume.
Editors
Ashok Pandey
Sangeeta Negi
1
a-Amylases
R. Sindhu1, *, P. Binod1, A. Pandey2

1
CSIR-NATIONAL INSTITUTE F OR INTERDISCIPLINARY SCIENCE AND TECHNOLOGY (NIIST),
TRIVANDRUM, INDIA; 2 CE NTER OF INNOVATIVE AND APPLIED BIOPROCESSING,
(A NATIONAL INSTITUTE UNDER DEPT OF BIOTECHNOL OGY, MINISTRY OF S&T, GOVT OF
INDIA), MOHALI, PUNJAB , INDIA
1.1 Introduction
1.1.1 Starch
Starch is the major polysaccharide food reserve in nature after cellulose. It serves as an
important source of nutrition for other living organisms [1]. It is synthesized in the
plastids present in leaves and accumulates as insoluble granules in higher and lower
plants. Starch is composed of a large number of glucose units joined by glycosidic bonds.
It consists of two types of molecules: amylose and amylopectin. Amylose is a linear,
water-insoluble polymer of glucose joined by a-1,4 bonds, whereas amylopectin is a
branched, water-soluble polysaccharide with short a-1,4-linked linear chains of 10e60
glucose units and a-1,6-linked side chains with 15e45 glucose units. The levels of
amylase and amylopectin vary among different starches. Generally, starch is composed
of amylose and amylopectin in the range 25e28% and 72e75%, respectively.
1.1.2 Amylases
Amylases are the enzymes that break down starch, or glycogen. These enzymes are
produced by a variety of living organisms, ranging from bacteria to plants to humans.
Though amylases are produced by several microorganisms, those produced by fungi and
bacteria have dominated applications in the industrial sector [2]. Bacteria and fungi
secrete amylases to the outside of their cells to carry out extracellular digestion, which
breaks down the insoluble starch, and then the soluble end products (such as glucose or
maltose) are absorbed into the cells.
Amylases constitute a class of industrial enzymes occupying about 25% of the enzyme
market. Because of the increasing demand for these enzymes in various industries, there
is enormous interest in developing them with better properties, such as raw starch-
degrading amylases suitable for industrial applications, and cost-effective production
*
Corresponding Author.
Current Developments in Biotechnology and Bioengineering: Production, Isolation and Purification of Industrial Products
http://dx.doi.org/10.1016/B978-0-444-63662-1.00001-4 3
Copyright © 2017 Elsevier B.V. All rights reserved.
4 CURRENT DEVELOPMENTS IN BIOTECHNOLOGY AND BIOENGINEERING
techniques. Although amylases can be derived from several sources, including plants,
animals, and microorganisms, microbial enzymes generally meet industrial demands.
A large number of microbial amylases are available commercially and they have almost
completely replaced the chemical hydrolysis of starch in the starch processing industry
[3]. One of the most important advantages of using microbes for the production of
amylases is the bulk production capacity and the fact that microbes can be genetically
modified to produce enzymes with desired characteristics [4]. These enzymes are of great
significance in biotechnology, with various applications ranging from food, fermentation,
and textiles to the paper industry. Each application of a-amylase requires unique
properties with respect to specificity, stability, and temperature and pH dependence.
Modern technologies such as computational packages and online servers are the
current tools used in protein sequence analysis and characterization. The physico-
chemical and structural properties of these proteins are well understood with the use of
computational tools. The protein sequence profile, such as number of amino acids and
sequence length, and the physicochemical properties of the protein, such as molecular
weight, atomic composition, extinction coefficient, aliphatic index, instability index, etc.,
can be computed by ProtParam, and the secondary structure prediction, sequence
similarity, evolutionary relationships, and 3-D structure of various proteins can be
computed using the ESyPred3D server [5].
1.1.3 Classification of Amylases

Based on the mechanism of breakdown of starch, the molecules are classified into three
types: a-amylase, b-amylase, and amyloglucosidase. a-Amylase reduces the viscosity of
starch by breaking down the bonds at random, thereby producing variably sized chains
of glucose. b-Amylase enzyme breaks the glucoseeglucose bonds by removing two
glucose units at a time, thereby producing maltose. Amyloglucosidase is the enzyme that
breaks successive bonds from the nonreducing end of the straight chain, producing
glucose. Many microbial amylases usually contain a mixture of these amylases. This
chapter focuses only on a-amylases.
a-Amylases (EC 3.2.1.1) are starch-degrading enzymes that catalyze the hydrolysis of
internal a-1,4-O-glycosidic bonds in the polysaccharides with the retention of the
a-anomeric configuration in the products. Most of the a-amylases are metalloenzymes,
which require calcium ions (Ca2þ) for their activity, structural integrity, and stability.
They belong to family 13 (GH-13) of the glycoside hydrolase group of enzymes [6,7].
Based on the end-product formation a-amylases are classified as saccharifying and
liquefying amylases. The saccharifying a-amylases are further classified as maltose
forming, maltotetraose forming, maltopentaose forming and maltohexaose forming
based on the end products formed [1].
The a-amylase family is the largest family of glycoside hydrolases, transferases, and
isomerases, comprising 30 different enzyme specificities. These enzymes are classified
into four groups: endoamylases, exoamylases, debranching enzymes, and transferases.
Endoamylases are enzymes that cleave internal a-1,4 bonds resulting in a-anomeric
Chapter 1 a-Amylases 5
products. Exoamylases are enzymes that cleave a-1,4, or a-1,6 bonds of the external
glucose residues resulting in a- or b-anomeric products. Debranching enzymes are
enzymes that hydrolyze a-1,6 bonds leaving linear polysaccharides. Transferases are
enzymes that cleave a-1,4 glycosidic bonds of the donor molecule and transfer part of
the donor molecule to a glycosidic acceptor, forming a new glycosidic bond [7].
1.2 Sources of a-Amylase

a-Amylases are universally distributed throughout the plant, animal, and microbial
kingdoms. The enzymes from microbial sources have dominated applications in in-
dustrial processes [2]. Though a-amylases have been derived from several microbial
sources, including bacteria, fungi, yeast, and actinomycetes, the enzymes produced from
bacterial and fungal sources have dominated applications in industrial sectors. Because
of their short growth period, their biochemical diversity, and the ease with which
enzyme concentrations might be increased by environmental and genetic manipulation,
the enzymes from microbial sources generally meet industrial demands.
1.2.1 Plant a-Amylases

Plants store carbon predominantly as starch and the metabolism of starch is essential to
all life. Family 1 a-amylases are characterized by having a secretary signal peptide.
This plays an important role in the degradation of extracellular starch in cereal grain
endosperms. Family 2 a-amylases are characterized by having no predicted targeted
peptide and are localized in the cytoplasm. These amylases have been identified from
monocotyledons, dicotyledons, and gymnosperms. They become most active when the
plastidial starch reserves of leaves are more depleted. They are involved in general stress
responses. Family 3 a-amylases are characterized by having a large N-terminal domain,
which contains a large predicted chloroplast transit peptide. These enzymes are
responsible for degrading plastid-bound starch in storage tissues and leaves [8].
1.2.2 Bacterial a-Amylases

a-Amylases are produced from various bacterial sources, including Bacillus, Brevi-
bacterium, Clostridium, Halomonas, Naxibacter, Nesterenkonia, Paenibacillus,
Pseudomonas, Streptomyces sp., etc. Among the bacterial sources, Bacillus sp. is widely
used, especially for the production of thermostable a-amylases. Bacillus subtilis, Bacillus
stearothermophilus, Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus acid-
ocaldarius, Bifidobacterium bifidum, and Bifidobacterium acerans are important sources
used for a-amylase production [9]. Alkaline and thermotolerant amylases have been
reported from Bacillus sp., B. licheniformis, and Bacillus halodurans [10]. Other bacteria
producing a-amylase include Anoxybacillus beppuensis [11], Bacillus laterosporus [12],
Bacillus acidicola [13], Chryseobacterium taeanense [14], Clostridium sp. [15],
Microbacterium foliorum [16], Nesterenkonia sp. [17], Thermococcus sp. [18], Anoxybacillus
flavithermus [19] etc.
1.2.3 Fungal a-Amylases

Several fungal species also produce a-amylases, including Acremonium, Aspergillus,
Penicillium, Mucor, Neurospora, and Thermomyces sp. Among the fungal sources, the
genus Aspergillus has been widely used for the production of a-amylases. Aspergillus
niger, Aspergillus flavus, and Aspergillus oryzae are important sources used among the
fungal sources [20,21]. Other fungal strains producing a-amylase include Thermomyces
lanuginosus [22].
1.3 Production of a-Amylase

1.3.1 Production Methods
To meet the industrial demand, it is essential to develop a low-cost medium for the
production of a-amylase. It can be produced by submerged fermentation (SmF) and
solid-state fermentation (SSF). The production is affected by a variety of physiological
factors, which include pH, temperature, aeration, inoculum concentration, inoculum
age, composition of the growth medium, surfactants, carbon source, nitrogen source,
etc. [23]. Interactions of these parameters have a significant influence on the production
of the enzyme. Generally, SmF is carried out using synthetic media, incorporating me-
dium constituents such as nutrient broth and soluble starch, as well as other compo-
nents, which are very expensive. Replacement of such constituents by cheaper carbon
and nitrogen sources as well as nutrients would benefit the process in cost reduction.
Agricultural by-products offer potential benefits in this regard [7].
SSF is defined as the process in which the growth of microorganisms is carried out on
solid substrates with negligible free water, or free-flowing water [24]. SSF plays an
important role in the production of enzymes. Agro-industrial substrates are considered
the best substrates for SSF processes. It is of special interest in those processes in which a
crude fermented product may be used directly as an enzyme source. The common
substrates used for SSF processes are wheat bran, rice bran, cassava waste, palm oil
waste, banana waste, tea waste, coconut oil cake, coir pith, corn cobs, etc. In SSF, it is
important to provide optimized water content and to control the water activity of the
fermenting substrate. At times, SSF is preferred to SmF because of its simple technique,
low capital investment, lower levels of catabolite repression and end-product inhibition,
low wastewater output, better product recovery, and high-quality production [25].
Continuous and fed-batch studies are more effective for the production of a-amylase.
The study conducted by Lee and Parulekar [26] revealed that the a-amylase production
by B. subtilis TN 106 was enhanced when batch cultivation was extended with fed-batch
cultivation, and the enzyme activity was 54% higher in a two-stage fed-batch operation
compared to a single-stage batch culture. Mishra and Maheswari [27] reported
a-amylase from a thermophilic fungus, T. lanuginosus; the enzyme was a dimeric protein
with a molecular mass of 42 kDa with optimum pH and temperature of 5.6 and 65 C,
respectively. The enzyme produced high levels of maltose from potato starch, suggesting
its usefulness in the commercial production of maltose and glucose syrups. The study
conducted by Krishna and Chandrasekharan [28] revealed that banana peel could be
utilized as a potential substrate for a-amylase production by A. niger. Saxena and Singh
[29] screened various agro-industrial residues for amylase production from Bacillus sp.
and found mustard oil cake to be the best substrate. The strain produced 5400 U/g of
amylase at 1:3 moisture content, 20% inoculum, and an incubation period of 72 h. Yang
and Wang [30] reported a-amylase production by Streptomyces rimosus TM 55 using
sweet potato residue and peanut meal residue as a substrate. The strain produced
1903 U of a-amylase after 96 h of incubation.
Ramachandran et al. [20] used coconut oil cake (COC), a by-product of oil extraction
from dried copra, as a substrate for the production of a-amylase from fungi. COC sup-
plemented with 0.5% starch and 1% peptone enhanced a-amylase production by
A. oryzae. COC serves as a source of soluble proteins and lipids thus providing essential
nutrients for the growth of and enzyme synthesis by the organism. Production of
a-amylase by B. amyloliquefaciens under SSF using corn gluten meal (CGM) was re-
ported by Saban et al. [31]. The study revealed that a-amylase production in a medium
with CGM was five times higher than that in a medium containing starch and other
components. Utilization of CGM as a substrate makes the process economically viable
because CGM is a by-product of starch-based industries.
Production and optimization of a-amylase from A. oryzae CBS 819 using a by-product
of wheat grinding (gruel) as the sole carbon source was done by Kammoun et al. [32].
Various process parameters affecting the production were optimized by adopting a
BoxeBehnken design, which increased the enzyme production from 40.1 to 151.1 U/mL.
Murthy et al. [33] reported coffee by-products as suitable substrate for the production of
a-amylase under SSF. Coffee waste was converted into value-added products by
fermentation using Neurospora crassa CFR 308. The optimum conditions for a-amylase
production were moisture content of 60%, pH 4.5, incubation temperature of 27 C,
particle size of 1 mm, and incubation time of 5 days. Under optimized conditions the
strain produced 7084 U/gds of a-amylase.
Syed et al. [34] reported extracellular amylase production by Streptomyces gulbar-
gensis DAS 131 by SmF. The highest amylase production was observed when the medium
was supplemented with 1% starch. The enzyme was thermotolerant and stable at pH 9.0.
Starch and peptone were good sources of carbon and nitrogen. Sharma and
Satyanarayana [13] reported enhanced production of acidic high-maltose-forming and
Ca2þ-independent a-amylase by B. acidicola; a maximum enzyme titer of 366 IU/L was
attained after 36 h of fermentation at pH 4.5, 33 C, with 0.5 vvm aeration. The enzyme
titer was 10,100 IU/L in fed-batch fermentation. One of the main advantages of fed-
batch fermentation over the batch fermentation is that the concentration of limiting
substrate is maintained at low levels, thus avoiding the repressing effect of high substrate
concentration and thereby minimizing the accumulation of inhibitory metabolites.
A highly thermostable and calcium-independent a-amylase from A. beppuensis

TSSC-1 was reported by Kikani and Singh [11]. This organism produced a monomeric
a-amylase with optimal pH and temperature of 7.0 and 55 C, respectively. The key
findings of this study were cost-effective purification, high thermostability, and broad
pH stability. The enzyme exhibited Ca2þ independence and resistance to chemical
denaturation, which could make it suitable for many industrial applications. Another
agro-industrial residue, date waste, has also been used as the substrate for the pro-
duction of a-amylase using yeast, Candida guilliermondii CGL-A10 [35]. Maximum
enzyme production was attained in SmF (2056 mmol/L/min). Rajagopalan et al. [15]
used sugarcane bagasse hydrolyzate for the production of a-amylase produced by a
solventogenic Clostridium sp. BOH3. The strain used starch directly without any pre-
treatment and produced extracellular amylase (7.15 U/mg protein) and butanol almost
equivalent to 90% of the yield equivalent to glucose. Sugarcane bagasse was used by
Roohi and Kuddus [16] to produce a cold-active a-amylase from M. foliorum GA2.
Maximum enzyme production (6610 U) was observed when fermentation was carried
out in a medium containing 40% bagasse, 0.0003 M lactose, at pH 8.0, with incubation
temperature of 20 C for 5 day at static conditions. This was the first report on cold-
active a-amylase production from M. foliorum GA2. Table 1.1 shows various microor-
ganisms used for the production of a-amylase.
1.3.2 Factors Influencing the Production of a-Amylase

Production of a-amylase by SSF and SmF is affected by a variety of physicochemical
factors [3]. These include media composition, incubation temperature, inoculum age,
carbon source, nitrogen source, pH, phosphate concentration, aeration, and others.
Table 1.1 Strains and Strategies Adopted for a-Amylase Production

Method of
Microorganism Production Substrate Enzyme Yield References
Bacillus subtilis TN 106 Fed batch [26]
Streptomyces rimosus TM55 SSF Sweet potato residue/ 2642.7 U/gds [30]
peanut residue
Aspergillus oryzae SSF Oil cake 9196 U/gds [20]
Aspergillus oryzae OBS819 SmF 151.1 U/mL [32]
Neurospora crassa CFR 308 SSF Coffee waste 7084 U/gds [33]
Streptomyces gulbargensis DAS 131 SmF Starch [34]
Bacillus acidicola SmF 366 IU/L [13]
Anoxybacillus beppuensis TSSC-1 [11]
Candida guilliermondii CGL-A10 SmF 2056 mmol/L/min [35]
Clostridium sp. BOH3 SmF Sugarcane bagasse 7.15 U/mg protein [15]
hydrolyzate
Microbacterium foliorum GA2 SmF Bagasse 6610 U/mL [16]
SmF, submerged fermentation; SSF, solid-state fermentation.

1.3.2.1 Incubation Temperature

The effect of temperature on a-amylase production is related to the growth of the or-
ganism. Temperature control is very important in fermentation processes because
growth and production of enzymes are sensitive to temperature. Hence, the optimum
temperature varies with the culture. a-Amylases have been produced by various mi-
crobes over a wide range of temperature. Productions in SSF as well as in SmF are
usually carried out in the range 25e37 C. However, psychrophilic and thermophilic
temperatures have also been reported for the production. For example, a-amylase
production was attained at 55 C by the thermophilic fungi Thermomonospora fusca [36]
and T. lanuginosus [27] and at 80 C by a hyperthermophilic bacterium, Thermococcus
profundus [36]. A psychrophilic bacterium, Alteromonas haloplanktis, produced
a-amylase at 4 C [37].
1.3.2.2 pH
The pH of the fermentation medium plays an important role in enzyme production. It
induces morphological changes in the organisms as they are sensitive to the concen-
tration of hydrogen ions present in the medium. A pH change in the medium affects the
growth as well as the product stability. Unlike SmF, in which pH control is almost
mandatory for a-amylase production, in SSF processes, generally there is no need to set,
or control, the pH, as the substrates (agro-industrial residues) mostly possess excellent
buffering capacity and keep the pH favorable for the growth and activity of the culture.
Most of the Bacillus strains used commercially for the production of a-amylases have an
optimum pH of 6.0 or 7.0. Some of the medium components eliminate the need for pH
control. Yabuki et al. [38] reported that A. oryzae 557 accumulated a-amylase in the
mycelia when grown in phosphate, or sulfate-deficient, medium and it was released
when the mycelia were placed in a medium with pH above 7.2. Based on the optimal pH
for activity, a-amylases are classified as acidic, neutral, and alkaline [1].
1.3.2.3 Carbon Sources

a-Amylase production could be either constitutive or inducible. Galactose, inulin, and
glycogen are suitable substrates for a-amylase production in SmF. Supplementation with
lactose; an analog of maltose, a-methyl-D-glucoside; and yeast extract induces the
production [7]. Several agro-residues such as wheat bran, rice bran, vegetable peels, fruit
peels, cassava bagasse, and vegetable-oil-extracted residues are used as substrates for
a-amylase production in SSF. Most studies on a-amylase production by A. oryzae suggest
that the general inducer molecule is maltose. Eratt et al. [39] observed a 20-fold increase
in enzyme activity when maltose and starch were used as inducers in A. oryzae NRC
401013. Xylose and fructose support good growth, but they are strongly repressive [40].
1.3.2.4 Nitrogen Sources

Organic as well inorganic nitrogen sources are used for the production of a-amylases,
although organic sources have been preferred over inorganic nitrogen sources. Commonly
used nitrogen sources include bactopeptone, ammonium sulfate, ammonium nitrate, Vogel
salts, casein, meat extract, beef extract, yeast extract, corn steep liquor, and soybean flour.
There are reports on the use of several other nitrogenous sources for a-amylase production.
For example, L-asparagine was reported as the better nitrogen source for enzyme produc-
tion by T. lanuginosus; casein hydrolyzate and yeast extract improved a-amylase
production several fold and by 110e156%, respectively, by A. oryzae [41]. Complex nitrogen
sources in the medium influence the production of a-amylases. Studies carried out by
Dettori et al. [42] revealed that the supplementation of two organic nitrogen sources
enhanced amylase production and this was better than a single organic nitrogen source.
1.3.2.5 Metal Ions

Supplementation of metal ions in the fermentation medium promotes microbial growth,
which in turn accelerates the enzyme production. Most a-amylases are known to be
metal dependent for divalent ions, e.g., Ca2þ, Mg2þ, Mn2þ, and Zn2þ [2].
Supplementation with Ca2þ is generally required for an increased in a-amylase pro-
duction by several bacteria. Ca2þ imparts thermostability of the enzyme due to salting
out of hydrophobic residues by Ca2þ in the protein. The production was reduced to 50%
when Mg2þ was omitted from the medium; Naþ and Mg2þ showed coordinated stimu-
lation of enzyme production by Bacillus sp. CRP strain [43]. However, some metal ions
could have a negative impact on the microbes for a-amylase production, e.g., Li2þ and
Hg2þ have negative effects on a-amylase production. Mg2þ also plays an important role
in a-amylase production.
1.3.2.6 Surfactants
Addition of surfactants to the fermentation medium is generally known to increase the
secretion of proteins by increasing cell membrane permeability. The commonly used
surfactants are Tween 80, Tween 40, Triton X-100, sodium dodecyl sulfate (SDS), poly-
ethylene glycol, and glycerol. These surfactants are reported to increase cell perme-
ability, thereby enhancing enzyme yield. Arneson et al. [44] reported a twofold increase
in a-amylase production by T. lanuginosus. Goes and Sheppard [45] reported a signifi-
cant advantage in using the bio-surfactant surfactin to enhance the production of
a-amylase by B. subtilis in SSF. In addition to increasing the enzyme activity, surfactin
offers other advantages, including eco-friendliness, less sensitivity to extremes of tem-
perature and pH, and being a potential fungicide, thereby eliminating contamination of
the exposed substrate, compared to synthetic surfactants.
1.3.2.7 Agitation
Agitation influences the mixing as well as the oxygen transfer rate in most fermentations
and thus influences cell morphology and product formation [46,47]. It is generally
believed that higher agitation is detrimental to cell growth, which in turn could decrease
enzyme production. Agitation intensities up to 300 rpm are normally employed for the
production of a-amylase in SmF from various microorganisms.
1.4 Assay of a-Amylases

Activity of a-amylases is quantified by measuring either the end products, like glucose or
maltose, or the amount of substrate that remains after enzymatic hydrolysis. a-Amylases
are assayed using soluble starch or modified starch as the substrate. They catalyze the
hydrolysis of a-1,4 glycosidic linkages in starch to produce glucose, dextrins, and limit
dextrins. The reaction is monitored by an increase in the reducing sugar levels or a
decrease in the iodine color of the treated substrate. Various methods are available for the
determination of a-amylase activity [48]. These are based on a decrease in starcheiodine
color intensity, increase in reducing sugars, degradation of color-complexed substrate,
and decrease in viscosity of the starch suspension [3]. The common methods employed
for the determination of a-amylase activity are the iodine method [49]; dextrinizing ac-
tivity [50]; Sandstedt, Kneen, and Blish method [51]; dinitrosalicylic acid method [52];
and degradation of color-complexed substrate [53,54].
The dinitrosalicylic acid (DNS) method [52] is among the most commonly used
methods for estimating the reducing sugars. The DNS reacts with reducing sugar under
boiling and turns to red from yellow. In the method of Fuwa et al. [50], the starch reacts
with iodine and forms a blue solution and the intensity of the color is directly propor-
tional to the starch concentration. Boron dipyrromethene-labeled substrate releases a
fluorescent fragment upon digestion with the enzyme and has been developed for
determining a-amylase activity in foods [55].
1.5 a-Amylase Inhibitors

Proteinaceous a-amylase inhibitors have been isolated from plants and microorganisms
[56]. These inhibitors control endogenous a-amylase activity or work in defense against
pests and pathogens; some inhibitors are antinutritional factors. a-Amylase inhibitors
belong to seven different protein structural families. Six types are from higher plants and
one is from Streptomyces sp. High-resolution structures are available for target
a-amylase and these structures indicate major diversity and some similarities in the
structural basis of a-amylase inhibition. Various types of inhibitors include Streptomyces
inhibitors, knottins, g-thionins, CM proteins, and kunitz-type, thaumatin-like, and
lectin-like inhibitors. Some a-amylase inhibitors have adverse effects on nutrition due to
their inhibition of digestive enzymes in humans and animals. a-Amylase inhibitors find
application in obesity and diabetic therapy.
1.6 Strain Improvement

Strain improvement is usually carried out to increase production as well as to improve
the properties of the enzyme. The catalytic properties of enzymes are determined by
their 3-D structure. Hence, enzyme properties can be altered by site-directed muta-
genesis. Using this method, the properties of an enzyme can be improved, by making
Table 1.2 Some Strategies Adopted for Strain Improvement/Properties of a-Amylase

Microorganism Improved Property References
Bacillus subtilis BR151 Thermostability [60]
Alternaria tenuissima FCBP 252 2.39-fold increased production [62]
Thermobifida fusca NTU22 Increased production [63]
Bacillus amyloliquefaciens Increased production (1.4-fold) [71]
Anoxybacillus sp. High stability in absence of Ca2þ ions at 60 C [66]
and high levels of maltose production
Aspergillus oryzae IIB 30 Increased production (2.1-fold) [70]
Paenibacillus sp. High rate of maltose production [67]
Bacillus licheniformis MSG Self-inducible, catabolite repression free, and [68]
glucose-activated expression system
B. subtilis ASO1a Increased production (7-fold) and high stability [69]
in absence of Ca2þ
Thermotoga maritima Oxidative stability [72]
B. subtilis Improved protein stability and catalytic efficiency [73]
Bacillus sp. AAH-31 Increased production [74]
it thermostable, reducing its dependence on cofactors, or increasing its activity at low

temperature. Studies on the cloning of the a-amylase gene have been extensively carried
out for hyperproduction [7]. Table 1.2 presents some strategies that have been adopted
for strain improvement of a-amylase.
a-Amylases have been engineered for the improvement of properties such as pH
tolerance, thermotolerance, etc. [57e59]. Barnett et al. [58] found that the introduction
of disulfide bonds in the enzymes and alteration of amino acids prone to oxidation by
an amino acid resistant to oxidizing agents improved the stability of the enzyme.
Suzuki et al. [57] constructed hybrids of homologous strains of the B. licheniformis and
B. amyloliquefaciens with improved thermostability. Ozcan and Ozcan [60] introduced
the thermostable plasmid pC194Amy, harboring a 5.2-kb DNA fragment encoding a
gene of B. stearothermophilus, into B. subtilis BR151 by electroporation. The recom-
binant strains produced more thermostable a-amylase compared to the wild-type
strain. A new strain of B. licheniformis CBBD302, carrying a recombinant plasmid,
pHY-amyL, for B. licheniformis a-amylase (BLA) production, was constructed by Niu
et al. [61]. The combination of target-directed screening and genetic recombination led
to an approximately 26-fold improvement in BLA production and export in
B. licheniformis. Shafique et al. [62] reported the production of an extracellular amylase
from Alternaria tenuissima FCBP 252 in SSF. Chemical mutagenesis using ethyl
methanesulfonate (EMS) produced mutants with a high level of a-amylase activity
(2.39-fold) compared to the parental strain. Genetic characterization of the mutants
using random amplified polymorphic DNA PCR revealed that the expression patterns
of the mutants were isogenic variants of the parent strain. Yang et al. [63] expressed
an a-amylase gene from Thermobifida fusca NTU22 in Pichia pastoris X33 because of
its potential application as a food supplement. Recombinant expression resulted in
higher levels of extracellular enzyme production (510 U/L), indicating constitutive
expression and secretion of the protein. The amount of extracellular protein in the
culture of P. pastoris transformants was less than that in the cell-free extract of
Escherichia coli transformants, hence facilitating the application of crude amylase in
industry without purification.
The gene encoding the a-amylase enzyme in B. subtilis PY22 was amplified by PCR,
sequenced, and cloned into P. pastoris KM71H strain using the vector Ppicz A, allowing
methanol-induced expression and secretion of the protein [64]. Recombinant expres-
sion resulted in high levels of extracellular amylase production (22 mg/L). The presence
of Ca2þ ions in the medium resulted in a 41% increase in a-amylase activity. Expression
in P. pastoris not only increased the yield of production but also potentially helped
facilitate purification. Gene cloning and heterologous expression of the high-maltose-
producing a-amylase of Rhizopus oryzae showed successful expression of R. oryzae
a-amylase in P. pastoris at a high level (382 mg/L) [65]. The enzyme had an extremely
high affinity for maltotriose and no maltotriose remained after hydrolysis. Chai et al.
[66] cloned two genes that encoded a-amylases from Anoxybacillus sp. and expressed
them in E. coli. The enzymes produced by the recombinant strains were highly stable
even in the absence of calcium at 60 C for 48 h and they produced high levels of
maltose. Protein sequencing revealed that the recombinant a-amylase differed in 17
amino acids compared to the amylase produced by the wild-type strain. A gene
encoding a-amylase from the genomic DNA of Paenibacillus sp. and the heterologous
expression of recombinant Amy1 in E. coli BL21 (DE3) facilitated the recovery of this
protein in soluble form. The high rate of maltose production due to the action of Amy1
could be exploited for the production of simple sugars as a by-product in food waste
processing [67].
The use of an expression system to overcome catabolite repression opens up an
avenue for exploiting cheap carbon sources for the production of recombinant enzyme.
Nathan and Nair [68] developed a repression-free catabolite-enhanced expression sys-
tem for a thermophilic a-amylase from B. licheniformis MSG. A self-inducible, catabolite
repression-free, and glucose-activated expression system was developed using a ther-
mophilic a-amylase as a model. The a-amylase gene from B. licheniformis MSG without
any 50 cre operator produced unimpeded glucose-enhanced expression when fused to
the phosphate starvation-inducible strong pst promoter with optimum translation sig-
nals in a protease-deficient B. subtilis. The yield was 18.5-fold higher than that of native
promoter. Roy et al. [69] cloned and overexpressed a raw-starch-digesting a-amylase
gene (AmyBS-I) from B. subtilis strain ASO1a in E. coli BL21. The gene also included its
signal peptide sequence for the efficient extracellular expression of recombinant
a-amylase in correctly folded form. The extracellular secretion of AmyBS-I was sevenfold
higher and it did not require Ca2þ ions for its a-amylase activity/thermostability, which
was an added advantage for its use in the starch industry.
Random mutagenesis has also been used for enhanced production of a-amylase. A
strain of A. oryzae IIB 30 was subjected to physical (using UV light) and chemical
mutagenesis (using nitrous acid and EMS). Mutation using EMS-20 showed a 2.1-fold
increased amylase activity compared to the wild-type strain [70]. An identical observa-
tion was earlier reported for a B. amyloliquefaciens strain in which mutation using EMS
improved enzyme activity by 1.4-fold higher than that of the parental strain [71]. Ozturk
et al. [72] reported site-directed mutagenesis of methionine residues for improving the
oxidative stability of a-amylase from Thermotoga maritima. The oxidative stability of
a-amylase (AmyC) was improved by mutating the methionine residues at positions 43
and 44, and 55 and 62, to oxidative-resistant alanine residues. The mutant exhibited
improved oxidative properties. The engineered AmyC could be a potential candidate for
industrial applications, especially in the presence of oxidizing agents. This is the first
protein engineering attempt for AmyC from T. maritima. Yang et al. [73] carried out
structural engineering of histidine residues in the catalytic domain of a-amylase from
B. subtilis for improved protein stability and catalytic efficiency under acidic conditions
by site-directed mutagenesis. The four histidine residues His222, His275, His293, and
His310 in the catalytic domain were selected as the mutation sites and were further
replaced with acidic aspartic acid, respectively yielding four mutants H222D, H275D,
H293D, and H310D. The acidic stability of the enzyme was significantly enhanced after
mutation, and 45e92% of the initial activity of the mutants was retained after incubation
at pH 4.5 and 25 C for 24 h, whereas that for the wild type was only 39.5%. As revealed by
the structure models of the wild-type and mutant enzymes, the hydrogen bonds and salt
bridges were increased after mutation, and an obvious shift of the basic limb toward
acidity was observed for the mutants. These changes around the catalytic domain
contributed to the significantly improved protein stability and catalytic efficiency at low
pH. This work provided an effective strategy to improve the catalytic activity and stability
of a-amylase under acidic conditions, and the results indicated potential application for
the improvement of acid resistance of other enzymes.
The hydrolytic activity of thermophilic, alkalophilic a-amylase could also be
enhanced through the optimization of amino acid residues surrounding the substrate
binding site [74]. Twenty-four selected amino acid residues were replaced with Ala, and
Gly429 and Gly550 were altered to Lys and Glu, respectively, based on comparison of
AmyL’s amino acid sequence with related enzymes. Y426A, H431A, I509A, and K549A
showed higher activity than the wild type at 162e254% of wild-type activity. Tyr426,
His431, and Ile509 were predicted to be located near subsite 2, and Lys549 was near
subsite þ2. Ser, Ala, Ala, and Met were the best amino acid residues for the positions of
Tyr426, His431, Ile509, and Lys549, respectively. Combinations of the optimized single
mutations at distant positions were effective in enhancing catalytic activity. The double-
mutant enzymes Y426S/K549M, H431A/K549M, and I509A/K549M, combining two of
the selected single mutations, showed 340%, 252%, and 271% of wild-type activity,
respectively. Triple- and quadruple-mutant enzymes of the selected mutations did not
show higher activity than the best double mutant, Y426S/K549M.
1.7 Purification and Characterization of a-Amylases

a-Amylases produced by fermentation are relatively crude preparations. Most of the
commercial use of a-amylase does not require 100% purification of the enzyme. But,
high-purity enzymes are required when they are used in clinical and pharmaceutical
sectors. The first steps in the purification involve the isolation of crude enzyme after the
fermentation. In SmF, this is usually done by centrifuging the fermented medium and
taking the supernatant as the source of crude enzyme; in the case of SSF, the fermented
matter is usually mixed with water or buffers, and after suitable mixing the contents are
filtered, whereby the filtrate contains the crude enzyme. Then, the enzyme is concen-
trated (in the supernatant/filtrate), precipitated (using salts/solvents), and purified using
various chromatographic techniques such as ion-exchange chromatography, gel filtra-
tion, isoelectric focusing, etc. Table 1.3 presents strategies adopted for the purification of
a-amylase from various microorganisms.
There are a large number of reports on the purification and characterization of
a-amylases produced by bacterial or fungal sources in SmF and SSF [75e87]. An enzyme
produced in SSF was partially purified by ammonium sulfate fractionation. The enzyme
was optimally active at pH 5.0 and 50 C with a molecular mass of 66 kDa. The presence of
Mn2þ and Fe2þ enhanced the enzyme activity, whereas in the presence of Hg2þ and Cu2þ
the activity was reduced [76]. A partially purified a-amylase from Streptomyces erumpens
MTCC 7317 showed a molecular mass of 54,500 Da [77]. a-Amylase from B. subtilis
KIBGE-HAS was purified by ultrafiltration and ammonium sulfate precipitation with 19.2-
fold purification and specific activity of 4195 U/mg. The enzyme was highly stable in the
presence of various surfactants and detergents. Metal ions such as Mn2þ, Ca2þ, Mg2þ, Kþ,
Co2þ, and Fe3þ activated the enzyme, whereas Ba2þ, Cu2þ, Naþ, and Al3þ strongly
inhibited the activity. A highly efficient raw-starch-digesting a-amylase from B. lichen-
iformis ATCC 9945a was purified by gel-filtration chromatography with a sixfold increase
in specific activity and recovery of 38% with a molecular mass of 31 kDa [82]. The purified
enzyme showed an optimum pH and temperature of 6.5 and 90 C, respectively. Co2þ,
Ni2þ, and Ca2þ slightly stimulated, whereas Hg2þ completely inhibited, a-amylase ac-
tivity. An a-amylase from Brevibacterium linens DSM 20158, purified by ion-exchange
chromatography on a DEAEeSephadex column, showed a 7.88-fold increase in purity
with a 16.80% yield, and it appeared homogeneous on SDSepolyacrylamide gel elec-
trophoresis with a molecular mass of 58 kDa. EDTA and Hg2þ inhibited the enzyme
activity, whereas Mn2þ and Ca2þ enhanced the enzyme activity [83].
A novel SDS- and surfactant-stable, raw-starch-digesting, and halophilic a-amylase
was purified from Nesterenkonia sp. [17]. The extracellular a-amylase was purified to
homogeneity by 80% ethanol precipitation, Q-Sepharose anion-exchange chromatog-
raphy, and Sephacryl S-200 gel-filtration chromatography. The optimum temperature
and pH were 45.8 C and 7.5, respectively. The molecular mass was estimated as 100 kDa.
The enzyme was inhibited by EDTA, but was not inhibited by phenylmethanesulfonyl
fluoride and b-mercaptoethanol. Ca2þ stimulated enzyme activity, whereas the enzyme
Table 1.3 Strategies Adopted for Purification of a-Amylase from Various Microorganisms

Optimum Optimum Molecular
Microorganism Purification Strategy Temperature pH Activators Inhibitors Mass References
Bacillus subtilis ATCC [75]
465
Aspergillus oryzae Ammonium sulfate fractionation 50 C 5.0 Mn2þ and Fe2þ Hg2þ and Cu2þ 66 kDa [76]
Streptomyces erumpens 54.5 kDa [77]
MTCC 7317
B. subtilis KIBGE-HAS Ultrafiltration and ammonium Mn2þ, Ca2þ, Ba2þ, Cu2þ, Naþ, [78]
sulfate precipitation Mg2þ, Kþ, Co2þ, and Al3þ
and Fe3þ
B. subtilis C10 Polyethylene glycol/potassium [79]
phosphate aqueous two-phase
system
Penicillium janthinellum Ammonium sulfate fractionation/ 50 C 5.0 EDTA 42.7 kDa [80]
NCIM 4960 anion-exchange chromatography
(DEAE)
Nesterenkonia sp. 80% ethanol precipitation, Q- 45.8 C 7.5 Ca2þ Fe3þ, Cu2þ, 100 kDa [17]
Sepharose anion-exchange Zn2þ, and Al3þ
chromatography, and Sephacryl S-
200 gel-filtration chromatography
Aspergillus flavus 55 C 5.0 55 kDa [81]
Bacillus licheniformis Gel-filtration chromatography 90 C 6.5 Co2þ, Ni2þ, and Hg2þ 31 kDa [82]
ATCC 9945a Ca2þ
Brevibacterium linens Ion-exchange chromatography on Mn2þ and Ca2þ EDTA and Hg2þ 58 kDa [83]
DSM 20158 a DEAEeSephadex column
Rhizopus microsporus Ammonium sulfate precipitation, 70 C 5.0 75 kDa [84]
Sephadex G25 desalination, and
DEAE-52 cellulose chromatography
Aspergillus oryzae strain Acetone precipitation, size- 50 C 5.6 50 and [85]
S2 exclusion and anion-exchange 42 kDa
chromatography
Penicillium chrysogenum Ammonium sulfate precipitation 60 C 6.0 [86]
and Sephadex G50 filtration
Bacillus 80% ammonium sulfate 70 C 7.0 Mg2þ, Ba2þ, 44 kDa [87]
methylotrophicus strain precipitation, DEAE FF anion Al3þ, and
P11-2 exchange, and Superdex 75 10/ dithiothreitol
300 gel-filtration chromatography
was strongly inhibited by Fe3þ, Cu2þ, Zn2þ, and Al3þ. An aqueous two-phase system
comprising polyethylene glycol/potassium phosphate was used for the partition and
purification of a-amylase from the culture supernatant of B. subtilis C10 [79], which
resulted in a 3.56-fold purification of enzyme with a recovery of 59.37%.
An a-amylase from Penicillium janthinellum NCIM 4960, purified by ammonium
sulfate, showed an almost 20-fold increase in specific activity with a 30.73% yield after
anion-exchange chromatography on DEAE cellulose. The purified enzyme had a mo-
lecular mass of 42.7 kDa. The optimum pH and temperature were 5.0 and 50 C,
respectively. The enzyme showed substrate specificity toward amylose and amylopectin.
The chelating agent EDTA inhibited enzyme activity. The enzyme was stable in the
presence of commercial detergents and stability increased in the presence of CaCl2 [80].
An a-amylase produced by A. flavus isolated from mangrove soil was partially purified
using ammonium sulfate, which resulted in a fivefold increase in enzyme activity. The
partially purified enzyme was optimally active at pH 5.0, temperature of 55 C, with a
molecular mass of 55 kDa. The extracellular amylase was purified by anionic- and
cationic-exchange chromatography and preparative electrophoresis, which resulted in
38-fold purity [81]. Shen et al. [84] purified an acid-stable and thermostable a-amylase
from Rhizopus microsporus isolated from distilled liquor. The crude extract was purified
using ammonium sulfate precipitation, Sephadex G25 desalination, and DEAE-52 cel-
lulose chromatography. The optimum pH and temperature were 5.0 and 70 C, respec-
tively, with a molecular mass of 75 kDa.
1.8 Applications of a-Amylase

a-Amylases find a wide range of biotechnological applications in the textile, food,
pharmaceutical, and detergent industries. With the current developments in biotech-
nology, the applications of a-amylases have been widened to other fields like clinical,
medicinal, and analytical chemistry.
1.8.1 Detergent Applications

a-Amylases comprise one of the ingredients of modern compact detergents. One of the
main advantages of using enzymes in detergents is that much milder conditions can be
used than with enzyme-free detergents [3]. Enzymes help in lowering of the washing
temperature. a-Amylases have been used as powerful laundry detergents since 1975.
Currently, 90% of all commercially available detergents contain a-amylase and the de-
mand for automatic dishwasher detergents is growing. One of the main limitations of
a-amylases in detergents is that it is highly sensitive to calcium and oxidants, which are
components of detergents. However, the development of genetically modified strains for
the production of a-amylases to improve their bleach stability has been achieved by
replacing the oxidation-sensitive amino acids with other amino acids. Replacement
of methionine at position 197 by leucine in B. amyloliquefaciens amylase has resulted in
improved resistance against oxidative compounds [88e91].
1.8.2 Textile Desizing

a-Amylases find application in the textile industry for textile desizing. To protect yarn
from breaking, a removable protective layer is applied to the threads. Starch is an ideal
desizing agent because it is cheap, is readily available, and can be removed very easily
[3]. An effective desizing of starch-sized textiles is achieved by the application of
a-amylases, which selectively remove the sizing and do not attack the fibers. It randomly
cleaves the starch into dextrins, which are water soluble and can be removed by washing.
Amylase from Bacillus sp. has been widely used in textile industries for a long time.
Sarvanan et al. [92] studied the desizing of cotton fabrics using a-amylase from
B. licheniformis. The study revealed that pH and amylase concentration exhibited the
dominant effect, followed by treatment time, on desizing efficiency.
1.8.3 Medicinal and Clinical Chemistry

There are several processes in the medicinal and clinical areas that require the appli-
cation of a-amylases [3]. The first enzyme produced industrially was a fungal amylase in
1894 and was used as a pharmaceutical aid for the treatment of digestive disorders.
Dumoulin et al. [93] observed that the addition of a-amylase to cross-linked amylose
tablets could modulate the release kinetics of the drug.
1.8.4 Paper Industry

a-Amylases find application in the paper and pulp industry for the modification of
starches for coating paper. The coating treatment serves to make the surface of the paper
smooth and strong to improve the writing quality of the paper. Starch acts as a good
sizing agent for the finishing of paper, improving the quality and erasability, in addition
to being a good coating for the paper. The sizing enhances the stiffness and strength of
the paper [94]. Cold-active a-amylase is used in the paper industry because it reduces the
viscosity of starch for the appropriate coating of paper [95].
1.8.5 Starch Liquefaction and Saccharification

One of the most important applications of a-amylase is in starch liquefaction for the
production of glucose and fructose syrups. Starch is converted to high-fructose corn
syrup and is widely used in the beverage industry as a sweetener for soft drinks
because of its high sweetening property. The process requires the usage of highly
thermostable a-amylase for starch liquefaction [3]. The enzymatic conversion involves
three processes, which include gelatinization, liquefaction, and saccharification.
Gelatinization involves dissolution of starch granules, forming a viscous solution;
liquefaction involves partial hydrolysis and leads to a loss in viscosity, followed by
saccharification involving the production of maltose and fructose [96]. The enzymes
from B. licheniformis and B. stearothermophilus are widely used because of their
remarkable thermostability.
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TARTAR HORSES AND HORSEMEN.
The people of Toorkistan, or Independent Tartary, are splendid

riders. They have fine horses, of which they take the greatest care.
But their way of taking care of horses is very different from ours.
The saddles are never taken off, night or day; and many Tartars
will not allow their horses to lie down at all because they say the corn
settles in their legs, and makes them lame! They are walked about a
great part of the time they are not on the road, sometimes for four or
five hours after coming in. At the beginning of the day’s march they
are allowed a full drink of water, but none during the day, while the
sun is hot. On first coming in from a journey, they are walked up and
down a long time, after which without being unsaddled, the bit is
taken out, and they are tied up, and covered from head to tail with
thick horse cloths, even in hot weather. Then they are fed with
barley, and Indian corn, and a very little grass.
We would think it cruel to keep a horse saddled and tied up in this
way, but the Tartar horses seem to thrive on this treatment. Their
saddles are more comfortable for the horse than ours, being well
raised above the back bone. These saddles are of wood, with a high
peak in front; and the rich Tartars cover them with embroidered
cloths, and silver mountings.
The horses are kept beautifully clean, and their coats are as
smooth and glossy as satin. In order to test whether his horse has
been properly groomed, the owner will wet the white sleeve of his
shirt, and rub it upon the horse’s coat. If there is not the least mark
on the sleeve he is satisfied.
The Tartars hunt birds on horseback, with great success. In the
case of partridges they gallop after the birds until they run them
down, and tire them out, when they can catch them alive.
TARTAR HORSES AND HORSEMEN.
They have several games which they play on horseback. In one of
them the riders all try to get possession of each other’s turbans! This
seems rather childish, but it is no child’s sport to accomplish this,
and the players perform most surprising feats of horsemanship.
They also have wrestling matches on horseback, trying to dislodge
one another from the saddle, while the horses are galloping furiously
and jumping ditches.
I suppose it would be almost an impossibility for a horse to throw a
Tartar rider.
TWO HAPPY MEN.
When we have our minds set upon some pursuit in which we are
resolved to excel, we are likely to forget any little disagreeable thing
that troubles us at other times, and we are happy in our work. What
pleasure a boy takes in fashioning his kite! What delight is it to a girl
to put together ends of silks, ribbons and laces into a pretty bonnet
for her doll! There is even pleasure in learning a Latin lesson, or in
working out a difficult problem when we are interested, and are
determined to do it well. The reason why so many grown persons
are unhappy is because they have no occupation at all, or because
they are engaged in some business which they do not like.
The best cure for this is to take up some business, and make up
your mind you will like it, and try to do your very best.
When a man’s business is in any branch of what we call Art he is,
perhaps, happier than he could be at anything else; for, besides the
satisfaction of doing the work, it is a pleasure to see beautiful things
grow under our hands.
I am going to tell you about two very happy men, who both lived in
the same place—a small city in Peru. One was an artist, who spent
all his time painting pictures. Let me introduce you into his home,
that you may see in what kind of place this happy mortal passed his
days.
THE ARTIST AT WORK.
The room in which he painted—his studio—was below the level of
the ground. To reach it from the street you went down three broken
stone steps. Pretty much all the light the artist had came from the
ever-open doorway. The floor was covered with straw, and scraps of
vegetables, among which chickens and guinea-pigs picked up a
living. His two best friends, a dog and a cat, usually shared the room
with him. The cat had lost its ears and its tail, but was not the less
liked by her master on that account. She was very fond of getting on
his shoulder as he bent over his work, and sometimes would take a
quite comfortable nap there.
Certainly it was not a beautiful home that made the artist happy.
He had the misfortune to be married to a woman who would have
made most men miserable. She scolded from morning to night. The
artist never could please her. No matter what he did, it was sure to
be wrong in her eyes. She would stop while stirring the pot, and rail
at him, shaking her greasy spoon to give emphasis to what she was
saying. But the artist answered her never a word. He was so
absorbed in his work that it is probable he did not hear her, half the
time.
And so it was not pleasant companionship, and loving words that
made him happy.
He could not even procure the proper materials for the work he
loved so much. There were no shops in all that region where such
things were sold. In our cities there are shops in which an artist can
buy everything he needs. But our happy man could only pick up a
few colors from the apothecary—the others he got himself from
earths and stones he found among the mountains. From the grocer
he obtained oil. The smoke of his candle furnished him with black,
and his brushes he manufactured himself from the hair of the dogs
killed in the city. Instead of canvas he used white cotton cloth, which
he prepared in some sort of fashion; and then stretched, and tacked
to a board.
With these materials, and under such disadvantages did our artist
work. And he painted very good pictures too. Some of them were
taken to Europe, and to the United States, and sold for twenty times
more than was paid to our artist for them. But he did not know this;
and the small sums he received sufficed for his simple wants.
He was always happy because his painting was to him a perpetual
delight. His business was his pleasure.
THE SCULPTOR AT WORK.
The other happy man was also an artist. He was a sculptor. His
statues were very singular-looking; and to our eyes, very ugly. But
the people in that Peruvian town admired them greatly, and the
sculptor himself thought them beautiful, and so it was all the same,
as far as he was concerned, as if they really had been beautiful.
Clothed in rags and tatters, he worked faithfully in his studio,
piecing together legs, and arms, and bodies, and heads, until he had
an image of a man, woman, or child, that satisfied him. His room was
a little better than the painter’s, but the walls were of rough stone;
and, as for furniture, he would have laughed at the idea of having
any.
He had such strongly marked Indian features that his face was not
pleasant at first sight, but he was always in such a good humor that
one soon forgot he was not handsome.
This sculptor worked in plaster. He moulded different parts of the
body, and hung them up on his walls. The legs, arms, &c. were
provided with wooden pegs, so that they could be properly fastened
together. When he wanted to make an image he would take down
the different members he required, and put them together. If they did
not fit properly he would cut out blocks of plaster, and patch them up.
These statues were all colored, and the sculptor had as much
difficulty in getting his colors as the painter, only he did not require so
many.
One of the queer things about his statues was that they all had
glass eyes! And this is the way he made them. He put fragments of
window glass, cut in the shape of eyes, into a frying pan pierced with
holes about an inch in diameter. As soon as the heat softened the
glass sufficiently he would press the pieces down into the holes with
a metal stick, and thus they would be rounded like eyes.
He procured his tools how and where he could. Old nails, old
brushes, worn-out knife blades, and even sheep bones, furnished
him materials.
But he took great pleasure in making these images that he thought
so lovely, and which charmed his neighbors. And, occupied in this
fascinating business, he had no time to think of his poverty, and
troubles. He was as happy as the day is long.
THE WONDERFUL ASH TREE.
The people who used to live in the northern parts of Europe were
not very pleasant people, if we are to believe all the blood-thirsty
stories we have heard about them, but they had a religion, although
it was rather a queer one. There is one thing, however, to be said of
their gods and goddesses, which is very much in their favor. They
were generally honest, and tolerably strong-minded, which is much
more than we can say about some of the gods of ancient Greece
and Rome. Mercury, you know, was a great thief, and even Jupiter
was none too good.
The Scandinavians believed
that Ymer was the very first god
of all, and he made his
appearance in the following
manner. Before the world was
created heavy mists filled all the
dark space. This space must
have been have been very cold,
for the frosty air condensed the
mists, and out of this
compressed fog, the god Ymer
came into existence.
But his brain does not seem to
have been at all foggy; for, after a
short time, becoming tired of
being alone, he set his wits to
work to find out how he could
have the company of other
beings like himself. He made a
very good guess as to how he
had taken shape; and, gathering
THE GOD YMER. the mists around him into foggy
masses, he shaped them into forms like his own; and then waited to
see what would happen. Soon the cold winds came and congealed
the mists, and behold! a number of gigantic companions for the
lonely god! He took good care, however, to make them smaller than
himself; for, although they were twice the height of the tallest
mountains on our earth, yet Ymer himself, when he laid down (if he
ever did lie down) required about half the world for his bed.
Ymer was so much pleased with his success that he concluded he
would make some more things out of the mists. He spread some of it
out in great smooth surfaces, some he collected in small piles, and
some he heaped up in great masses of many curious shapes. And
that is the way the valleys, mountains, and hills were created.
The foggy material that was left fell down to an immense depth,
and became the ocean.
Ymer made nothing more, for he did not know how to work in
anything but mists, and they were all gone.
What he and his companions did in the way of employment or
amusement I cannot say. Let us hope they took comfort in striding
around the world—a walk of an hour or so—and in talking with each
other. They could not see anything except by occasional gleams of
lightning, for there was no light anywhere.
Monstrous creatures, such as dragons, hydras, griffins, and the
like, now made their appearance in the world, but there is no account
of their creation, and they must have come of their own accord.
One day a marvelous thing happened. Ymer and his giants saw a
pink flush spreading over the black sky. This grew brighter, and
brighter, until the whole firmament was a brilliant flame color. And,
while they were wondering what this could mean, whizz! came in
sight a great ball of fire! This was nothing less than a new god,
named Odin. Where he came from nobody knew, but there he was.
He descended upon the mountains, and took possession of Ymer’s
world.
He brought with him the Sun, the Moon, and the Stars. He told the
Sun to light up the world, and to warm up things generally, and to be
sure to melt the ice that covered a great part of the earth. The Moon
and the Stars were to take care of the earth during the night.
Odin brought with him also, a large number of followers; and,
according to the invariable rule of all discoverers of new countries,
he proceeded to kill all the original inhabitants; beginning with great
Ymer himself, and ending with the land and sea monsters. That is,
he intended to kill them all, and he thought he had. But one of the
giants escaped, and also a wolf, named Fenris (a terrible creature
that made nothing of crushing a mountain with his teeth). And the
great sea-serpent Iormungandur was not slain.
The warmth of the sun soon called into life the grasses, the
flowers, and the trees; springs welled up in the woods; and brooks
and rivers flowed through the plains to the sea; and a great variety of
animals took possession of the world, now so beautiful.
Odin was charmed with all this, but not quite satisfied. He wanted
some beings on the earth that should be less than gods, and yet of a
finer intelligence than the beasts. Thinking about this one day, as he
walked by the sea-shore, his eyes chanced to fall upon a large
branch that had blown off a tree into the water. This put a bright idea
into his mind. He drew the wood towards him; and, splitting it in two,
made a man and a woman out of the two parts. From this couple,
according to the Scandinavian legends, all the people in the world
are descended.
People increased so fast, and were so rude and savage, and
quarrelled and fought so much, that Odin found he had his hands too
full of business, and he thought it was about time for his lazy
followers to help him. So he set them all to work.
Forseti was to make peace among men. Vali was to teach them
the use of the bow, not for the purpose of killing each other, but for
slaughtering game for food. Uller was to teach skating. The goddess
Gefione taught men to labor, and how to break up the earth for seed,
and to raise crops. I think you will agree with me that she was one of
the very best of all the Scandinavian gods, and goddesses.
THE GOD EGIR.
Egir was a very important god. He showed men how to build ships,
and how to manage the sails, and the rudder. And not only did he do
this, but, he very obligingly, blew the vessels along with his powerful
breath, so that men were not afraid to trust themselves on the rivers
in these frail-looking crafts, but even boldly launched out upon the
ocean.
Widar taught people a most excellent thing—when to hold their
tongues. This he did by his example, for he was dumb, and could not
talk at all.
Balder was called the Bright God. He was the most beloved of
them all. He put good thoughts into the hearts of men, and
encouraged them to be loving and patient with each other. A
beautiful silvery light always shone around him.
Now, where do you suppose all these gods lived? You would
probably answer that they dwelt up in the sky, or on the tops of high
mountains. No. They lived in an ash tree!
This wonderful tree bore the name of Ygdrasil. Its branches
overshadowed the whole world; its top supported the sky, and its
roots went so far down that no one could find the end. This tree was
the home of Odin and his gods, and there they stayed, except when
business called them elsewhere.
This is the way the gods found out what was going on in the world,
while they were having a good time in Ygdrasil. Two ravens were
always flying to and fro through the Universe, and, once a day, they
would perch on Odin’s shoulders and tell him the news. A little
squirrel darted swiftly up and down the tree, and picked up all the
scraps of gossip it could. Near the top of the tree a great eagle kept
perpetual watch, and on the very topmost branch perched a vulture;
and these birds, which could see to the horizon on every side cried
out, and flapped their wings when any strange thing happened.
Besides all these there was the watch-god, Heimdall. His sight and
hearing were marvelous. He could hear the grass grow in the fields,
and hear the wool grow on the backs of the sheep. He could not only
watch a fly from the one end of the world to the other, but could
count the spots on its wings, and the joints in its little legs, if it was at
the opposite side of the universe from himself. He could see the
smallest atom that moved at the bottom of the ocean. And, what was
the most astonishing of all, he could see in the darkest night as well
as in the brightest day.
It is a pity this god is not living now, for he could describe to us the
bottom of the ocean, and tell us if there is an open sea at the North
Pole, and an icy continent at the South Pole, and a great many
things we want very much to know, and have not been able to find
out.
THE YOUNG GOD JARL.
This Heimdall had golden teeth. He had also a son, named Jarl,
who was a very famous god. When he was only a child he could give
heavy blows with a great club, and swim like a fish, and ride on
horseback as swiftly as the wind. And he understood the language of
birds and beasts, and could converse with them.
There were some very queer things about these gods. We might
suppose these powerful beings would be perfectly formed, but they
were not. Heimdall, as we have seen, had false teeth; Tyr had but
one hand; Widar was dumb; Hoder was blind; and the great Odin
himself had but one eye. And it seems, too, that they did not know
everything there was to be known, as the following story will show
you.
There lived in the world, in those days, a very wise man, named
Kvasir. He noticed how much trouble men had in expressing their
thoughts in any way but speech. If one wanted to send a message to
another he could only make a rude drawing on a piece of stone to
represent what he wanted to say, or paint it in certain colors that
stood for certain things. There was not much of this done, for not
only was this process troublesome, but it was easy to misunderstand
these messages; and they caused a great deal of confusion, and
many quarrels, and much fighting. Kvasir wanted to remedy this;
and, after a great deal of hard study, and many experiments he
invented the art of writing. He also invented poetry. He called his
verses runes, and he wrote them on beech bark, which he made into
tablets.
The gods had never thought of doing anything like this.
There is no knowing how much Kvasir would have done if he had
lived longer. Perhaps he would have invented printing and paper;
which, as matters turned out, nobody thought of doing until many
hundred years later.
But this wise and good man was killed by two wicked dwarfs. They
did this in order to steal from him this treasure of poetry, and the art
of writing. You may wonder how they were going to get at the
treasure, for, after they had killed him, there could be no more
poetry; and they could not pick it out of his brain as a thief takes a
pocket-book out of the pocket. But these dwarfs were magicians,
and such people, you know, have a pretty good idea what they are
about. They collected his blood, and mixed it with honey in three
separate proportions. These they put into three jars which they
closely sealed, and buried in a cave which had never been seen
either by gods or men.
These three compounds were Logic, Eloquence, and Poetry. We
shall never know what the dwarfs were going to do with them, for I
am happy to say that they were not allowed to keep them.
THE THREE PRECIOUS JARS.
Odin’s two ravens had witnessed the whole performance of the
dwarfs, and the sensible birds concluded this must be a great
treasure, or it would not be worth so much trouble. So they flew
straight to Odin, and told him all about it. Odin sent the squirrel up
the tree Ygdrasil with an order for the eagle to leave his post, to fly to
the cave, and to bring the jars to him.
This the eagle accomplished in a very short space of time, and
Odin immediately opened each jar, and tasted the contents. He at
once commenced reasoning eloquently in the most ravishing strains
of poetry. His daughter Saga, and his son Bragi, were with their
father; and, seeing how he enjoyed these new dishes, they wanted
some too. Odin politely offered the first jar to Saga, but it probably
did not taste pleasantly, as she declined to do more than just touch
its contents to her lips. But Bragi drank up all his father had left, and
immediately began to sing a magnificent chant. From that time he
was called the god of poetry.
Bragi was not stingy with his treasure, but gave some of it to men,
and thus the invention of the good Kvasir was used as he would
have used it had he lived; and men learned to write, and to sing.
The greatest of the gods, next to Odin, was his son Thor. He was
the god of tempests. He held thunderbolts shut up in his fists, and
flung lightning from his fingers’ ends. He had a mighty hammer with
which he reconstructed the world after Ymer had been killed. He
splintered up the mountains, and made them all over again, and he
knocked away at the crust of the earth, and made valleys and caves,
and sometimes he amused himself by splitting open the earth, and
tumbling a mountain or two into the abyss. And that was the way
earthquakes came about. He made holes in some of the mountains,
and let the imprisoned fire out of them.
Odin gave Thor three wonderful gifts. The first was his great
hammer. It would go out of his hand to do his bidding, and then
return of its own accord. The second was a pair of iron gloves. He
had only to put these on, and his spear would come back into his
hand after having destroyed his victim. The third was a war belt,
which made him stronger than any other being while he wore it.
It is no great wonder that with all these things to help him Thor
succeeded in killing off Ymer, and his race of giants, for he did most
of this work.
THE GOD THOR.
But, you remember, in the account given of the destruction of the
giants, and the land and sea monsters, that one giant escaped, and
the wolf Fenris, and the great sea-serpent, Iormungandur. And, by
these three, after a great number of years, Odin and his gods came
to grief.
The gods all understood that their fates depended upon the god of
love, the bright and beautiful Balder. If he died they must die. Think
then how troubled they must have been, when, one day, they heard
a great cry ringing through the earth, and up to the very top of the
ash tree, where was placed their highest heaven, called Walhalla.
This piercing cry was: “Balder, fair Balder is going to die!” They had
never thought before that their beloved Balder could die, but now
they were sore afraid, not only for him, but for themselves. They
were told by some wise woman that Balder would surely die unless
all substances that could inflict death were made powerless. Upon
hearing this his mother, Frigg, travelled over the whole world, and
asked the rocks, and the pebbles, frost and rain, and wood and iron;
everything, in short, to spare her son. And they all promised not to
hurt him.
There was great joy among the gods when Frigg returned with this
good news. So Balder was not to die, after all. And there was a great
feast held in Walhalla to celebrate the glad tiding. In the midst of the
merriment it was proposed to try some of these things that had
promised not to hurt Balder, to see how they would avoid injuring
him. One of the gods threw a clod of earth at Balder, and it broke into
a cloud of dust before it reached him. Another poured a pitcher of
water over him, and the water formed a cascade over him without
wetting his clothes. Then they tried more dangerous weapons; a
rock; a club; a sword; and Vali shot an arrow at him. All passed by
him, or fell harmless. Even Thor’s mighty hammer refused to hit
Balder.
At last a brother of Balder’s approached, holding in his hand a
small bunch of leaves. All laughed at the sight of this harmless
weapon. But alas! when the leaves struck Balder’s breast he fell,
and died instantly. They were mistletoe leaves, and when Frigg had
asked the oak tree to spare her son, she forgot to ask the mistletoe,
which grows upon the oak. So the mistletoe had given no promise;
and now Balder was dead. The brother who had thrown the leaves
was greatly distressed, and all Walhalla was filled with mourning.
Balder being dead, the other gods must die. The giant, who had
escaped Thor’s hammer, killed some of them, and others died in
various ways. Finally Thor was killed by the sea-serpent; and the
great Odin was torn in pieces by the wolf Fenris.
And that was the end of the Scandinavian gods.
Then the Druid priests brought their religion into the country; and,
after many years, the Romans came, and taught the Scandinavians
the gospel of Jesus Christ.
WORK AND WATER.
It is so easy for most of us to get a drink of water when we feel

thirsty, that we are not apt to even think of the vast amount of
thought and labor and money that is necessary in many parts of
every country in the world in order to give people a glass or a cup of
water when they want it.
And yet water is often a very costly thing, so much so indeed that
there are lands where people, and civilized people too, cannot afford
a drink of it every time they feel thirsty.
If we live in the country we go to our well, or our spring, or our
pump from the cistern, and we get all the water we want. If we live in
the city we have our hydrants, and perhaps have the water carried to
every floor of the house. This is because we are Americans, and, as
a nation, we believe that we cannot spend too much money in
making ourselves comfortable, and having thing’s convenient around
us.
We build great reservoirs and conduct into them the pure water
from the streams, often far distant from our cities, and we have pipes
running through every street, and into every house, so that even the
poorest people can always have plenty of water, no matter what else
they may have to go without.
But in many countries that were civilized and enlightened long
before America was ever thought of, there are to-day no such
conveniences for obtaining a drink of water.
In some places in Europe water is carried about from house to
house, as the milk-man brings us milk, and some of the plans of
carrying it are very curious.
In parts of Holland where the canals serve as roads, there are
water-boats, that go up and down the canals serving water to
everyone who wishes to buy it, and has money to pay for it. And
sometimes it is pretty stale water when the last families get their

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AMSTERDAM l BOSTON l HEIDELBERG l LONDON l NEW YORK l OXFORD

Copyright © 2017 Elsevier B.V. All rights reserved.

British Library Cataloguing-in-Publication Data

For information on all Elsevier publications

Publisher: John Fedor

Typeset by TNQ Books and Journals

M. Adsul DBT-IOC Centre for Advanced Bioenergy Research, IndianOil Corporation

Cristóbal N. Aguilar Food Research Department, School of Chemistry,

A. Angel-Cuapio Universidad Autónoma Metropolitana-Iztapalapa, Mexico City,

G.S. Anisha Government College for Women, Trivandrum, Kerala, India

P. Binod CSIR-National Institute for Interdisciplinary Science and Technology (NIIST),

J. Buenrostro-Figueroa Department of Biotechnology, Division of Health and

S. Chakraborty Indian Institute of Technology Guwahati, Guwahati, Assam, India

M.L. Chávez González Food Research Department, School of Chemistry,

G.-Q. Chen Tsinghua University, Beijing, China

S. Chen Hubei University, Wuhan, PR China

Juan C. Contreras-Esquivel Food Research Department, School of Chemistry,

J.D. Coral Bioprocess Engineering and Biotechnology Department, Federal

J.C. de Carvalho Bioprocess Engineering and Biotechnology Department, Federal

J. de Oliveira Bioprocess Engineering and Biotechnology Department, Federal

A. Dhillon Indian Institute of Technology Guwahati, Guwahati, Assam, India

M.J. Fernandes Bioprocess Engineering and Biotechnology Department, Federal

R. Gaur Indian Oil Corporation Limited, R&D Centre, Faridabad, India

A. Goyal Indian Institute of Technology Guwahati, Guwahati, Assam, India

L.R.C. Guimarães Bioprocess Engineering and Biotechnology Department, Federal

M. Haridas Kannur University, Kannur, India

R. Hemamalini Indian Institute of Technology Delhi, New Delhi, India

Ayerim Hernandez-Almanza Food Research Department, School of Chemistry,

A. Illanes Pontiﬁcia Universidad Católica de Valparaíso, Valparaíso, Chile

J. Isar University of Delhi South Campus, New Delhi, India

A. Joseph Kannur University, Kannur, India

S.G. Karp Bioprocess Engineering and Biotechnology Department, Federal

N. Karthik CSIR-National Institute for Interdisciplinary Science and Technology

R. Kaushik University of Delhi South Campus, New Delhi, India

S.K. Khare Indian Institute of Technology Delhi, New Delhi, India

P.C.S. Kirnev Bioprocess Engineering and Biotechnology Department, Federal

D. Kothari Indian Institute of Technology Guwahati, Guwahati, Assam, India

C. Larroche Blaise Pascal University, Aubière Cedex, France

L.A.J. Letti Bioprocess Engineering and Biotechnology Department, Federal

O. Loera-Corral Universidad Autónoma Metropolitana-Iztapalapa, Mexico City, DF,

A.I. Magalhães, Jr. Bioprocess Engineering and Biotechnology Department,

A.B.P. Medeiros Bioprocess Engineering and Biotechnology Department, Federal

J.D.C. Medina Bioprocess Engineering and Biotechnology Department, Federal

F. Miranda-Hernández Universidad Autónoma Metropolitana-Iztapalapa, Mexico

N.R. Nair CSIR-National Institute for Interdisciplinary Science and Technology

S. Nair Dow Chemicals GmBH, Dubai, UAE

K.M. Nampoothiri CSIR-National Institute for Interdisciplinary Science and

A. Nandan CSIR-National Institute for Interdisciplinary Science and Technology

S. Negi Motilal Nehru National Institute of Technology, Allahabad, India

M.G.B. Pagnoncelli Bioprocess Engineering and Biotechnology Department,

A. Pandey Center of Innovative and Applied Bioprocessing, (a national institute

V. Rajulapati Indian Institute of Technology Guwahati, Guwahati, Assam, India

S. Ramachandran Insight Professional Institute, Dubai, UAE

A. Rani Indian Institute of Technology Guwahati, Guwahati, Assam, India

C. Rodrigues Bioprocess Engineering and Biotechnology Department, Federal

Rosa M. Rodríguez-Jasso Food Research Department, School of Chemistry,