Recent trends in applied sciences: 1

Recent Trends in Life Sciences
1
: I

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Title of the Book: Recent Trends in Life Sciences
Edited:
Dr. Rohit Shankar Mane
Mukul Barwant
Divya Makwana
Publisher: Scientist R Academy, Bangalore, India
Publishers Address:
Scientist R Academy,
C.V. Raman Road, 4th
Cross,
Yashwantpur, Bangalore, India 560012
www.scientistracademy.com
Edition: First
ISBN: 978-93-5593-624-3
©2022, Scientist R Academy, Bangalore, India
Book Price: 320
Disclaimer: The information compiled in this book is sourced from
public domain like Wikipedia, Google search, etc., and the writer is
responsible for complete authenticity of it. Full author is responsible
for plagiarism not publisher.

3
About Editors
Dr. Rohit Shankar Mane M.Sc. NET. DPM. PGDFSQM. Ph.D.,
Dr. Rohit Shankar Mane is Microbiology Scientist. His
educational qualification is B.Sc. M.Sc. NET. DPM. PGDFSQM.
Ph.D., He is Principal Investigator of DST, DBT and National
geography research projects. He is inventor of ROVE
sterilization method. He is fellow of Dr. Babasaheb Ambedkar
National Research Fellowship, BARTI, India. He has given
International Young scientist award by AEI, India. He has given
National Young Scientist award by American Microbiology
Society of America. He is the 4th
runner up of Citrinin against
MCF-7 cell line at Dr. Raghunath Mashelkar Award, India. His
project entitled “Chitinase and their antifungal effects” got
nominated for GYTI-2018 award under Indian government. He
has published 36 Books and 56 research papers at International
and National level. He is reviewer of 26 International Journals
and associate editor of 12 International Journals. His interested
research area is Microbiology and Agriculture. He is member of
American Microbiology Society of America and Canadian
Microbiology Society of Canada. He is founder and Director of
“Scientist R academy” Research and Publication Institute of
India. He is poet, activist and always engaged in microbiology
awareness programs.
Contact Details:
Dr. Rohit Shankar Mane
rohitmane2025@gmail.com
+91 7972440873

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Mr.Mukul Barwant M.Sc. (Ph.D)
Assistant Professor, Department of Botany, Sanjivani Rural
Education Society, Sanjivani Arts Commerce and Science College
Kopargaon Ahmednagar, Maharashtra, India, Ph.D. Pursuing Shivaji
University Kolhapur Maharashtra India, Master of Science
Completed From Savitribai Phule Pune University Pune India. his
field of study and expertise in algal research, ecology physiology,
and another stream of plant science. He has a different award in
research and academics like best presenter award-2021, best
young speaker award-2021, -vicaash, young researcher award
2021 -(ijimer) elsevier, dr. Sarvepalli radhakrishnan best teacher
award in dsrbta meet 2021, rajyastariya gunwant shikshak
gurugaurav shikshanratn puraskar 2021 by mvla trust (
manushybal vikas lokseva akademi ), young scholar - award –iardo.
Best book chapter He has 02 patents to his credit so far. He has
also various research publications, and book chapters both
internationally and nationally to his credit. He has bee published 02
books He has published 28 research papers, in reputed national,
International Journal, like the UGC Care list, Springer, and 8 book
chapters has published. He is also a reviewer of 19 Journal and 6
Editorial board members of journals and publisher.
Contact Details:
Mr.Mukul Barwant
mukulbarwant97@gmail.com

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Ms. Divya Makwana
Divya Makwana is Assistant Professor at Mahi Girls College
Pratapgarh, MP, India. Her educational qualification is B.Sc. M.Sc.
She is Principal Investigator of DST, and DBT research projects. She
is a hardworking, motivated biotechnologist is seeking for entry
level opportunity to utilize my skills and knowledge while making a
significant contribution to achieve organization’s goal. Diploma in
Computer Application, Makhan Lal Chaturvedi University, Bhopal.
She worked on Studies of Chitinolytic Actinobacteria. Participated
in CSIR-Online Summer Research Training Programmed 2020. She
has participated in two-days international conference on “Microbial
biotech interactions for sustainable agriculture and societal
development”. She has participated in two-day work shop on
“Identification of microorganism using PCR based methods and
Bioinformatics analysis”. She has participated in four-days National
Webinar on “Recent Trends in Life Science Research”.She is
participated in Rangoli Competition Based on Immunology at
National Level. She is also associtaed with “Scientist R academy”
Research and Publication Institute of India. She is always engaged
in microbiology awareness programs.
Contact Details:
Ms. Divya Makwana
divyamakwana628@gmail.com

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Preface
The recent trends in Life Sciences have been experiencing rapid
transformation in recent years due to development of technology by
considering available ancient techniques. For this change, most
importunately different scientist was discovered scientific
technologies, methods, concepts, and microorganisms. All this
research helped to develop society in all aspects including medicinal
plants research. Different plants are widely known for their medicinal
properties, food properties, industrial important products formation
properties etc., Due to our improved understanding and different
methodology, even our meanings of familiar words, such as antibiotic
and species appear to be shifting. This book is coordinated towards
students, researchers, scientists and starting alumni understudies in
medicinal plants and Botany. However, the book is fully focused on
different plants and their applications in different fields. We would like
to offer our thanks to all authors, parents, teachers, and friends.
2022 Editors

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Acknowledgments
How does a person say “Thanks a lot” when there are so many people
around to say “Thanks a lot”? Many people helped and submitted their
chapters to enhance the research ideas, research skills, writing skills
and study area. I would like to thanks to all authors. I would like to
thanks to all editors. I would like to thanks to the First Professor whose
guest lecture I attended and after that many people from my society,
college, university, who helped me too etc.
I would like to thank my Mother, Father, Sister, and Brother for
supporting and encouraging me.
They make my life complete.
Editors

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Index
Sr. No. Chapter Title Page
No.
1. Application of crispr-cas9 in breeding of crop
plants
10-17
2. Enzymes and their applications 18-42
3. Molecular characterization of unicellular
cyanobacteria isolated from mangrove
biotope
43-52
4. Pollution: password to doomsday 53-58
5. In silico validation of protein models and
evolutionary analysis of trichophyton fungi
(ringworm disease)
59-67
6. Plant based microbial fuel cells-recent
trends
68-73
7. Biodiversity and its conservation 74-85
8. Salicylic acid-mediated defense signaling
networks in plant cicer arietinum (l.)
86-97
9. Ecological alteration and its impact on health
and well-being of children
98-109
10. Air purifier and human health 110-117
11. Biological activities of medicinal plants
12. Medicinal properties of garlic (Allium
sativum) and ginger (Zingiber officinale)
118-132
13. Basic laboratory equipment and their uses in
fungal dna extraction and molecular studies
133-155
14. Effect of vanadium metal complexes on
reducing serum glucose levels in wistar rats
156-174
15. Review on effect of beneficial elements in the
growth, development and quality yield of
medicinal plants
175-233

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Recent Trends in Life sciences-I
is
dedicated to
all authors of the book

10
ABSTRACT ARTICLE HISTORY
Received: 19.01.2022
Revised: 26.01.2022
Accepted: 30.01.2022
KEYWORDS:
1, Genome editing, ,
2, CRISPR/Cas9,
3. GMO,
4. SDNs
Ensuring food security is the most urgent and critical problem
facing in the present scenario with the expanding world
population. It is said that by 2050, the population of the world
would be increased to ten billion people with the limited land
availability which necessitates an annual increase in the global
food demand by 60-100 %. Besides the limited agricultural land
supply, extreme weather condition, biotic and abiotic pressure also
acts as important constraints to food production. So, to increase
the productivity of a crop several conventional breeding methods
were used before, which is modified by using with molecular
breeding technique, but these tools has some defects like those of
time consuming, expensive process, laborious to obtain the
desired outcome. Transgenic methods has come into existence two
decades back with the production of GMO but its adoption is
limited due to laborious, societal distrust, time-consuming
method. Although these techniques have been successfully use
over the ages to augment the crop production, modern approaches
like genome editing further offer a rapid and precise development
of improved plants than those techniques. Application of genome
editing in plants by using site directed nucleases (SDNs) boost in
the research work of several plant breeders. So, the evolution of
genome editing tool, particularly the CRISPR/Cas9 system are
nowadays widely used in the improvement of plant species.
CRISPR/Cas9 system of genome editing method is widely used in
a variety of crop of about 40 different plants with different
purposes.
Book Chapter 1
ISBN: 978-93-5593-624-3
APPLICATION OF CRISPR-CAS9 IN BREEDING OF CROP PLANTS
Yengkhom Linthoingambi1*
, Rajeev Shrivastava2
Department of Genetics and Plant Breeding,
College of Agriculture, IGKV, Raipur, Chhattisgarh
linthoiyengkhom123@gmail.com

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INTRODUCTION
Maintaining a sustainable environment in the increasing population with global climate change, food
scarcity and also with the evolution of new pathogens and pest, requires a more advance form of novel
technology for crop improvement. Conventional or molecular breeding was used before to achieve
the goal but because of its limitation due to more labour intensive, expensive and more time
consuming to achieve the desirable result, it is being replaced by more advance methods. Later,
genetic modification (GM) technologies have been used to produce value-added crops by transferring
of desirable genes to the target plant. But this method is associated either with unsubstantiated health
effect or environmental safety concerns. And also for the approval of a particular GM crops for
commercial cultivation, it requires a significant cost and time for regulation for approval of the plant.
Besides this, the integration of the gene in another genome is random and sometimes it may not come
out as the expected result. Therefore, more precise advanced form of genome editing tools came to
use (1). Genome editing tools using site-directed nucleases (SDNs) such as transcription activator-
like effector nucleases (TALENs), zinc-finger nucleases (ZFNs), and clustered regularly interspaced
short palindrome repeats (CRISPR)-associated (Cas) nucleases. Among the genome editing tools, the
most popularly used one being the CRISPR/Cas9 system which contain the Cas9 protein. The
CRISPR/cas system was discovered by identifying the unusual structure which is present in the
downstream of the iap gene of Escherichia coli. The structure is consisting of direct repeats of five
homologous sequences of 29 nucleotides separated by 32 nucleotides as spacers [2]. These repeats
interspaced structure later came to known as CRISPR, which is associated with cas genes. Thus, the
CRISPR-Cas system is represented by a genomic locus which consisting of a series of direct repeat
sequence which is separated by CRISPR array, also known as variable spacer and also has a
diversified flanking cas genes[3,4]. CRISPR-Cas system show exceptional diversity and they can be
classified into class1 and class 2. The classification mode is based on the phylogeny and the signature
of the cas gene in the CRISPR/ cas system, so the two classes are mainly differ in their effectors
modules [5, 6]. Class 1 CRISPR-Cas system has effector complexes of multiple subunits of Cas
protein. It again consists of type I, III and IV CRISPR-Cas system. While class 2 CRISPR-Cas system
have a simple architecture of single protein with multiple domains and function [5, 7, and 8]. It again
consists of type II, V and VI CRISPR-Cas system. Class 2 system becomes attractive for genome
editing platform owing to its simple organisation of effector complexes. Particularly, the type II
CRISPR-Cas system having the Cas 9 endonuclease system have been exploited mostly in the present
day genome editing of several plant species[10]. The invading foreign DNA is inserted in the CRISPR
array of Type II CRISPR-Cas system in the host genome. The CRISPR array transcribed and form a
single pre-crRNA transcript which will be matured to form mature short crRNAs which can pair up
with the protospacers of invading foreign DNA whenever complementary sequence is present. Trans-
activating crRNA (tracrRNA) will hybridized the pre-crRNA and help in its maturation process and
this activity is facilitated by RNase III and Cas9 protein [11]. Thus, the base pair structure of crRNA
and tracrRNA structure form a complex which activate the Cas9 and help in site-directed cleavage of
DNA. The target DNA was cleaved by Cas9 nuclease at sites discovered by the crRNA with
complementary sequence to the target protospacer which is present near to the protospacer adjacent

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motifs (PAMs). PAM is considered important for recognition sequence which will determine the
target strand for cleavage. Different CRISPR-Cas systems need different PAM sequence [12, 13]. The
type II CRISPR-Cas system generally requires three different components viz. crRNA, tracrRNA,
and Cas9 nuclease to target and induce the cleavage of DNA. Nowadays, the characterized Type II
CRISPR-Cas system of Streptococcus pyogenes is mostly used platform for genome editing. The dual
structure crRNA-tracrRNA in S. pyogenes has been fused as a single RNA chimera known as the
single guide RNA (sgRNA). This dual structure has simplified the system from the typical three
component system to two component system i.e. sgRNA and Cas9 nuclease which is used in genome
editing. The sgRNA helps in directing of Cas9 nuclease to the target DNA site through RNA-DNA
base-pairing rules and it usually has 20 nucleotides corresponding to the protospacer sequence. The
PAM sequence which help in cleavage to occur present at the 30 end of the protospacer sequence.
The Cas nuclease will be directed by sgRNA and helps in recognition of the target DNA sequence
flanked by PAM and generate the site-specific double strand breaks (DSBs). The resulting DSBs can
be repaired by non-homologous end joining (NHEJ) pathway, and homology-directed repair (HDR)
pathway. The HDR system is used when there is the presence of homologous donor templates at the
time of DSB formation and NHEJ pathway mostly result in the formation of nucleotide insertions/
deletions (indels) and/or substitutions. Diverse methodology is present to deliver GE tools into plants,
which include those of polyethylene glycol-mediated protoplast transformation of DNA,
Agrobacterium-mediated transformation, viral vector-based delivery, ribo-nucleoprotein (RNP)
complexes, and particle bombardment. Recently haploid inducer-mediated GE (HI-Edit) have
recently used for GE of recalcitrant plant.
APPLICATION OF CRISPR-CAS9 IN BREEDING OF CROP PLANTS
After the introduction of CRISPR-Cas9 system as a programmable RNA guided editing system,
several studies have been conducted on the various organisms and proven the efficiency of this
system. The application of the system influenced greatly in advancement of genome manipulations in
plant systems. Utilizing the platform of genome editing tools which can induce the DSBs helps in
gene knockout and knockin of the plants either through NHEJ and HR endogenous DSB repair
pathway. NHEJ associated system of gene knockout is used either to silence or eliminate the genes
that has negative impact on the growth and productivity of plant. HR otherwise is useful for integration
of transgene with desirable traits in plants [14]. Thus, CRISPR-Cas system has been rapidly used for
genome modifications and is used for the improvement of plant in terms of quality and yield, tolerant
against stress, characterisation of gene functions and alter metabolic pathway.
ROLE OF CRISPR/CAS9 IN BREEDING FOR IMPROVEMENT OF YIELD AND
NUTRITIONAL CONTENT
Optimization of crop production is a must needed objective in this present world for maintaining
global food security. Not only the yield, the other features like quality and the nutritional content of
the plant is also necessary. Even though enormous effort was used to boost the crop production
through the conventional agricultural technologies, it is usually time-consuming and costly process.
So, several studies have been performed using CRISPR/Cas9 system for development of new
desirable and heritable traits in various crops. Some of the successful examples are studies here.

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Production of elite varieties of rice (J809, L237, and CNXJ) with improved yield having better grain
yield, width, 1000 grain weight and number which is obtained by simultaneous editing of three genes
related to quantitative trait loci (QTLs) viz. OsGW2, OsGS3 and OsGn1a [44]. CRISPR/Cas9
mediated mutagenesis of starch branching enzyme SBEIIb successfully produced in rice [15]. Early
maturing variety of rice produced using CRISPR-Cas9 system through multiplex genome editing of
three flowering suppressor genes Hd2, Hd4, and Hd5 allowing labour and time efficient breeding as
these genes are involved in photoperiodic flowering pathway [15].
Through successful mutation of fatty acid desaturase 2 (FAD2) genes, OsFAD2-1 and FAD2 genes,
a significant increase in the content of oleic acid content in rice seed and oilseed rape was obtained
[16,17]. In tomato also, lycopene-enriched tomato was developed by altering the genes involved in
lycopene biosynthesis and by altering the genes involved in GABA shunt, the GABA (y-aminobutyric
acid) content was accumulated [18].
CRISPR-Cas9 system was also used to simplify the production of hybrid wheat and maize through
the silencing of wheat male fertility gene (Ms1) and genic male sterile 5 (ZmTMS5) gene which result
in generation of thermo-sensitive male-sterile lines [19,20].
ROLE OF CRISPR/CAS9 IN BREEDING FOR BIOTIC AND ABIOTIC STRESS
RESISTANCE/TOLERANCE
Plants are prone to the threats of both biotic and abiotic stresses which affect the growth and
development of the plant. Traditional and conventional agricultural techniques have been utilized for
enhancing plant’s resistance however there is presence of constraints associated with it because of
fast evolving ability of pathogens and continuous changing environmental condition. Some of the
successful example of implementation of CRISPR-Cas9 is being discussed here. Disruption of
eukaryotic translational initiation factor 4E (eIF4E) gene through CRISPR/Cas9 system provide
resistance to potyviruses Zucchini yellow mosaic virus, immunity to cucumber vein yellowing virus
(Ipomovirus) infection and Papaya ring spot mosaic virus-W providing the generation of virus free
crop plants [21].
Tungro spherical virus (RTVS) resistant rice variety is produced by targeting the translation initiation
factor 4 gamma (Eif4g) gene [22]. Crops resistant to either fungi or bacteria was also developed in
case of orange for canker-resistant Wanjincheng orange and Duncan grapefruit by harbouring
mutation of the promoter region of 1(CsLOB1) gene, which is the susceptible gene for citrus canker
caused by Xanthomonascitri [25]. Gene knockout of ERF transcription factor Oserf922 gene in rice
result in resistance of the plants against rice blast disease caused by the filamentous fungus
Magnaportheoryza [23]. Tomato resistant variety was also generated through CRISPR/Cas9 mediated
mutagenesis of mildew resistant locus O (Mlo) which gives plants resistant to powdery mildew.
Drought resistant maize variety was achieved through replacing of native promoter of ethylene
negative response regulator ARGOS8 gene through HR repair pathway. CRISPR-Cas9 targeted
knockout of gene OsRR22, a salt-related gene which encode transcription factor involved in
metabolism and signal transduction of cytokinin was found to give enhance salinity tolerance in rice
[24]. Several research works are also under process with an aim of obtaining plants with better
tolerance to stress condition.

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FUNCTIONAL CHARACTERIZATION OF GENES AND REGULATORY ELEMENTS
CRISPR-Cas9 system is also used in the study of gene function and regulatory elements through
bottom-up approach or reverse genetics approach. Some of the successful examples are discussed
here. In case of Rice, several genes have been annotated functionally like those of mitogen-activated
protein kinase (MPK) related genes MPK1 and MPK6 for development of rice [26], sugar transporter
OsSWEET11 in releasing sucrose during initial stage of caryopsis development [27], annotation of
rice annexin gene OsAnn3 for cold tolerance in rice [28], discovery of OsMADS3 gene as floral organ
number 2 (FON2) for flower meristem maintenance in rice [29]. CRISPR-Cas9 system also used in
the validation of regulatory function of GT-1 element in promoter region of OsRAV2 gene for salt
induced expression in rice [29]. Gene knockout of SIMAPK3 gene through CRISPR-Cas9 system
result in drought tolerance of the plant. Knockout of SICBF gene results in chilling tolerance of tomato
plant [30]. Validation of SIPHO1:1 gene, a homolog of phosphate 1 (PHO1) gene helps in phosphate
acquisition and transfer. Inactivation of CCT transcription factor (ZmCCT9) in case of Maize
highlight the significance of gene in Maize adapted to higher latitudes [31]. Dek42 in maize encode
RRM_RBM48 type RNA binding protein which affect pre-mRNA splicing during kernel
development through CRISPR-Cas9 mediated mutagenesis [32]. Thus CRISPR-Cas9 system help in
straightforward inactivation of genes for regulatory components and gene function.
CONCLUSION
CRISPR-Cas9 system is a straightforward genome editing tool which revolutionized the genomic
modification in various plants and organisms. This novel platform still has several potential to be used
in several research works in the coming days and several research works is also under process. Despite
its potential and superior ability for genome editing, it is still troubled by certain limitations due to
lack of efficient delivery system like that of off-target effect. So efforts are needed to reduce the off-
target events and obtain precisely tailored mutations like the development of online bioinformatics
tools which help in the selection of highly specific sgRNAs. Also the efficiency of CRISPR/Cas9
system differs with different plant species. Hence, fine tuning of the conditions is needed in a
particular species for its utilization to ensure highly efficient and successful genome editing.
ACKNOWLEDGEMENT
We would like to thanks to Scientist R Academy, Bangalore India.
REFERNCES
1. Montecillo JAV, Chu LL. and Bae H. CRISPR-Cas9 system for Plant Genome Editing: Current
Approaches and Emerging Developments. Agronomy. 2020; 10:10-33;
doi:10.3390/agronomy10071033
2. Ishino Y, Shinagawa H, Makino K, Amemura M, Nakata A. Nucleotide sequence of the iapgene,
responsible for alkaline phosphatase isozyme conversion in Escherichia coli, and
identification of the gene product. J. Bacteriol.1987; 169:5429–5433.
3. Jansen R, Van Embden, JDA, Gaastra W, Schouls LM. (2002). Identification of genes that are
associated with DNA repeats in prokaryotes. Mol. Microbiol. 2002; 43:1565–1575.

15
4.Bolotin A, Quinquis B, Sorokin A, Ehrlich SD. Clustered regularly interspaced short palindrome
repeats (CRISPRs) have spacers of extrachromosomal origin. Microbiology.2005; 151:
2551–2561.
5. Makarova, KS Wolf, YI Alkhnbashi, OS, Costa F, Shah SA, Saunders SJ, Barrangou R, Brouns
SJJ, Charpentier E, Haft DH. et al.An updated evolutionary classification of CRISPR–
Cassystems. Nat. Rev. Microbiol. 2015; 13: 722–736.
6. Makarova K S, Koonin EV. Annotation and Classification of CRISPR-Cas Systems. Methods Mol.
Biol.2015;1311: 47–75.
7. Makarova KS, Wolf YI, Koonin EV. Classification and Nomenclature of CRISPR-Cas Systems:
Where from Here? Cris. J. 2018;1: 325–336.
8. Shmakov S, Abudayyeh O O, Makarova KS, Wolf YI, Gootenberg JS, Semenova E, Minakhin
L,Joung J, Konermann S, Severinov K. et al. Discovery and Functional Characterization of
Diverse Class 2 CRISPR-Cas Systems. Mol. Cell. 2015; 60:385–397.
9. Makarova KS Wolf YI, Iranzo J, Shmakov SA, Alkhnbashi OS, Brouns SJJ, Charpentier E, Cheng
D, Haft DH, Horvath P, et al. Evolutionary classification of CRISPR–Cas systems: A burst
of class 2 and derived variants. Nat. Rev. Microbiol. 2020;18: 67–83.
10. Mali P Esvelt KM Church GM. Cas9 as a versatile tool for engineering biology. Nat.
Methods.2013; 10:957–963.
11. Deltcheva E,Chylinski K, Sharma CM, Gonzales K, Chao Y, Pirzada ZA, Eckert MR, Vogel
J,Charpentier E. CRISPR RNA maturation by trans-encoded small RNA and host factor
RNase III. Nature.2011; 471: 602–607.
12.Horvath P, Romero DA, Coute-Monvoisin AC, Richards M, Deveau H, Moineau S, Boyaval P,
Fremaux C, Barrangou R. Diversity, Activity, and Evolution of CRISPR Loci in
Streptococcus thermophilus. J. Bacteriol. 2008; 190: 1401–1412.
13.Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E. A Programmable Dual-
RNA-Guided DNA Endonuclease in Adaptive Bacterial Immunity. Sci. 2012; 337: 816–821.
14. Schmidt C, Pacher M, Puchta H. DNA Break Repair in Plants and Its Application for Genome
Engineering. Methods Mol. Biol. 2019; 1864: 237–266.
15. Sun Y, Jiao G, Liu Z, Zhang X, Li J, Guo X, Du W, Du J, Francis F, Zhao Y, et al.
(2017).Generation of High-Amylose Rice through CRISPR/Cas9-Mediated Targeted
Mutagenesis of Starch Branching Enzymes. Front. Plant. Sci.2017;8: 298-308.
16. Abe K, Araki E, Suzuki Y, Toki S, Saika H.(2018). Production of high oleic/low linoleic rice by
genome editing. Plant. Physiol. Biochem. 2018; 131: 58–62.
17. Okuzaki A, Ogawa T, Koizuka C, Kaneko K, Inaba M, Imamura J, Koizuka N. CRISPR/Cas9-
mediated genome editing of the fatty acid desaturase 2 gene in Brassica napus. Plant. Physiol.
Biochem. 2018; 131: 63–69.
18. Li R, Li R, Li X, Fu D, Zhu B, Tian H, Luo Y, Zhu H. Multiplexed CRISPR/Cas9-mediated
metabolic engineering of γ-aminobutyric acid levels in Solanumlycopersicum.
Plant.Biotechnol.J.2018; 16: 415–427.

16
19. Li J, Zhang H, Si X, Tian Y, Chen K, Liu J, Chen H, Gao C. Generation of thermosensitive male-
sterile maize by targeted knockout of the ZmTMS5 gene. J. Genet. Genomics.2017;44: 465–
468.
20. Okada A, Arndell T, Borisjuk N, Sharma N, Watson-Haigh NS, Tucker EJ, Baumann U,
Langridge P,Whitford R. CRISPR/Cas9-mediated knockout of Ms1 enables the rapid
generation of male-sterile hexaploid wheat lines for use in hybrid seed production. Plant.
Biotechnol. J. 2019; 17: 1905–1913.
21.Chandrasekaran J, Brumin M, Wolf D, Leibman D, Klap C, Pearlsman M, Sherman A, Arazi T,
Gal-On A.(2016). Development of broad virus resistance in non-transgenic cucumber using
CRISPR/Cas9 technology. Mol. Plant. Pathol. 2016; 17:1140–1153.
22. Macovei A, Sevilla NR, Cantos C, Jonson GB, Slamet-Loedin I, Cerm ˇ ák T, Voytas DF, Choi I-
R, Chadha-Mohanty P. Novel alleles of rice eIF4G generated by CRISPR/Cas9-targeted
mutagenesis confer resistance to Rice tungro spherical virus. Plant. Biotechnol. J. 2018;16:
1918–1927.
23. Wang F, Wang C, Liu P, Lei C, Hao W, Gao Y, Liu Y.-G, Zhao K. Enhanced Rice Blast Resistance
by CRISPR/Cas9-Targeted Mutagenesis of the ERF Transcription Factor Gene OsERF922.
PLoS ONE.2016:11, e0154027.
24. Zhang A, Liu Y, Wang F, Li T, Chen Z, Kong D, Bi J, Zhang F,Luo X, Wang J, et al. Enhanced
rice salinity tolerance via CRISPR/Cas9-targeted mutagenesis of the OsRR22 gene. Mol.
Breed.2019; 39: 47-57.
25. Peng A, Chen S, Lei T, Xu L, He Y, Wu L, Yao L, Zou X. Engineering canker-resistant plants
through CRISPR/Cas9-targeted editing of the susceptibility gene CsLOB1 promoter in citrus.
Plant.Biotechnol. J.2017; 15: 1509–1519.
26. Minkenberg B, Xie K, Yang Y. Discovery of rice essential genes by characterizing a CRISPR-
edited mutation of closely related rice MAP kinase genes. Plant. J.2017; 89: 636–648.
27. Ma L, Zhang D, Miao Q, Yang J, Xuan Y, Hu Y. Essential Role of Sugar Transporter
OsSWEET11 During the Early Stage of Rice Grain Filling. Plant Cell Physiol.2017; 58: 863–
873.
28. Shen C, Que Z, Xia Y, Tang N, Li D, He R, Cao M. Knock out of the annexin gene OsAnn3 via
CRISPR/Cas9-mediated genome editing decreased cold tolerance in rice. J Plant Biol. 2017;
60: 539–547.
29. Duan Y-B, Li J, Qin R-Y, Xu R.-F, Li H, Yang Y-C, Ma H, Li L, Wei PC, Yang JB. Identification
of a regulatory element responsible for salt induction of rice OsRAV2 through ex situ and in
situ promoter analysis. Plant. Mol. Biol. 2016; 90: 49–62.
30. Li R, Zhang L, WangL, Chen L, Zhao R, Sheng J, Shen L. Reduction of Tomato-Plant Chilling
Tolerance by CRISPR–Cas9-Mediated SlCBF1 Mutagenesis. J. Agric. Food Chem. 2018; 66:
9042–9051.
31. Huang C, Sun H, Xu D, Chen Q, Liang Y, Wang X, Xu G,Tian J, Wang C, Li D. et al. ZmCCT9
enhances maize adaptation to higher latitudes. Proc. Natl. Acad. Sci. USA, 2018; 115: E334–
E341.

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32. Zuo Y, Feng F, Qi W, and Song R.Dek42 encode an RNA binding protein that affects alternative
pre-mRNA splicing and maize kernel development. J. Integr. Plant. Biol.2019; 61: 728–748.
Citations
Yengkhom Linthoingambi1*, Rajeev Shrivastava2
APPLICATION OF CRISPR-CAS9 IN BREEDING OF CROP PLANTS. Recent Trends
in Life Sciences. 1st
Edition. 2022. 1-5.
©Scientist R Academy, Bangalore, India
Book is available on https://www.scientistracademy.com/

18
Book Chapter 2
ISBN: 978-93-5593-624-3
ENZYMES AND THEIR APPLICATIONS
Dr. Sujana kamepalli*, Boddepalli Hima Kalyani, Bommisetty Swarna
Lakshmi, Devapatla Greeshma, Surabatttula Roopavathi
Department of Pharmceutical Analysis, University College of Pharmaceutical
Sciences, Acharya Nagarjuna University, Nagarjun nagar, Guntur.
drksujana36@gmail.com
Received: 19.01.2022
Revised: 26.01.2022
Accepted: 30.01.2022
KEYWORDS:
1. Enzymes
2. Biocatalysts
3. Substrate
4. Applications
Enzymes are biocatalysts that speed up the reactions taking place
inside the cell. They are widely used in industries, scientific
research and clinical diagnostics. Enzymes can be classified into
six categories: Oxidoreductases, Transferases, Hydrolases,
Lyases, Isomerases and Ligases. Enzymes are specific for their
substrates. They increase the rate of reaction by lowering the
activation energy required to convert the substrates into the
products. The catalysis by an enzyme is influenced by the nature
of medium, substrate, enzyme concentration, temperature, pH and
the presence of activators and Inhibitors. Enzyme catalysis is an
area of fundamental importance in different areas. This chapter
offers a concise review to the fundamental principles and
mechanism of action, catalysis inhibition and its applications.
Additionally, this section also covers basics information related
with enzymes such as its structure, function and different
properties.
Citations
Dr. Sujana kamepalli*, Boddepalli Hima Kalyani, Bommisetty Swarna Lakshmi,
Devapatla Greeshma, Surabatttula Roopavathi. Enzymes and their applications. Recent
Trends in Life Sciences. 1st
Edition. 2022. 1-5.

19
DEFINITION:
Enzymes can be defined as biological polymers that catalyze biochemical reactions.
Majority of enzymes are proteins with catalytic capabilities crucial to perform different processes. Metabolic
processes and other chemical reactions in the cell are carried out by a set of enzymes that are necessary to
sustain life. The initial stage of metabolic process depends upon the enzymes, which react with a molecule
and is called the substrate. Enzymes convert the substrates into other distinct molecules and are called the
products. The regulation of enzymes has been a key element in clinical diagnosis because of their role in
maintaining life processes. The macromolecular components of all enzymes consist of protein, except in the
class of RNA catalysts called ribozymes. The word ribozyme is derived from the ribonucleic acid enzyme.
Many ribozymes are molecules of ribonucleic acid, which catalyze reactions in one of their own bonds or
among other RNAs. Enzymes are found in all tissues and fluids of the body. Catalysis of all reactions taking
place in metabolic pathways is carried out by intracellular enzymes. The enzymes in plasma membrane
govern the catalysis in the cells as a response to cellular signals and enzymes in the circulatory system regulate
clotting of blood. Most of the critical life processes are established on the functions of enzymes.
There are thousands of individual enzymes in the body. Each type of enzyme only has one job. For example,
the enzyme sucrase breaks down a sugar called sucrose. Lactase breaks down lactose, a kind of sugar found
in milk products.
Some of the most common digestive enzymes are:
• Carbohydrase breaks down carbohydrates into sugars.
• Lipase breaks down fats into fatty acids.
• Protease breaks down protein into amino acids.
Each enzyme has an “active site.” This area has a unique shape. The substance an enzyme works on is a
substrate. The substrate also has a unique shape. The enzyme and the substrate must fit together to work.
ENZYME STRUCTURE:
Enzymes are a linear chain of amino acids, which give rise to a three-dimensional structure. The sequence of
amino acids specifies the structure, which in turn identifies the catalytic activity of the enzyme. Upon heating,
enzyme’s structure denatures, resulting in a loss of enzyme activity that typically is associated with
temperature.
ENZYMES CLASSIFICATION
According to the International Union of Biochemists (I U B), enzymes are divided into six functional classes
and are classified based on the type of reaction in which they are used to catalyze. The six kinds of enzymes
are hydrolases, oxidoreductases, lyases, transferases, ligases and isomerases.
1. Oxidoreductases These catalyze oxidation and reduction reactions, e.g., pyruvate dehydrogenase, catalysing
the oxidation of pyruvate to acetyl coenzyme A.

20
2. Transferases These catalyze transferring of the chemical group from one to another compound. An example
is a transaminase, which transfers an amino group from one molecule to another.
3. Hydrolases They catalyze the hydrolysis of a bond. For example, the enzyme pepsin hydrolyzes peptide
bonds in proteins.
4. Lyases These catalyze the breakage of bonds without catalysis, e.g., aldolase (an enzyme in glycolysis)
catalyzes the splitting of fructose-1, 6-bisphosphate to glyceraldehyde-3-phosphate and dihydroxyacetone
phosphate.
5. Isomerases They catalyze the formation of an isomer of a compound. Example: phosphoglucomutase
catalyzes the conversion of glucose-1-phosphate to glucose-6-phosphate (phosphate group is transferred from
one to another position in the same compound) in glycogenolysis (glycogen is converted to glucose for energy
to be released quickly).
6. Ligases Ligases catalyze the association of two molecules. For example, DNA ligase catalyzes the joining of
two fragments of DNA by forming a phosphodiester bond.
Figure 1 : Enzymes classification
Cofactors
Cofactors are non-proteinous substances that associate with enzymes. A cofactor is essential for the
functioning of an enzyme. An enzyme without a cofactor is called an apoenzyme. An enzyme and its cofactor
together constitute the holoenzyme.
There are three kinds of cofactors present in enzymes:
Prosthetic groups: These are cofactors tightly bound to an enzyme at all times. A fad is a prosthetic group
present in many enzymes.
Coenzyme: A coenzyme binds to an enzyme only during catalysis. At all other times, it is detached from the
enzyme. NAD+ is a common coenzyme.

21
Metal ions: For the catalysis of certain enzymes, a metal ion is required at the active site to form coordinate
bonds. Zn2+ is a metal ion cofactor used by a number of enzymes [4].
CATALYSIS:
The role of a catalyst is to increase the speed of a chemical reaction. When the rate of a chemical reaction is
governed by a soluble catalyst, which may result in a further increase in the rate of chemical reaction, it is
called homogeneous catalysis. In this case catalysis occurs in a solution. When the catalyst is in a separate
phase from the reactants, or when catalysis occurs on an insoluble surface or an immobilized matrix, it is
known as heterogeneous catalysis. Enzymes are also called biological catalysts. These biological catalysts
generally have the properties of homogeneous catalysts; however, a number of enzymes present in
membranes are insoluble, and thus are called heterogeneous catalysts. Enzyme specificity is the absolute
specificity of protein catalysts to identify and bind to only one or a few molecules. In this process the enzyme
carries a defined arrangement of atoms in their active site to bind with the substrate. This active site on the
enzyme should have a shape that accurately matches the substrates. Thus, specificity is achieved when an
enzyme with an active site bind with the chemical reactants (the substrates) at their active sites via weak bond
interactions. To undergo a chemical reaction, this active site carries certain residues that form a temporary
bond with the chemical reactants, termed the binding site, whereas the catalytic site carries the residues that
are responsible for catalysis. Specificity is achieved when a substrate binds to an enzyme that has a defined
arrangement of atoms in the active site. An enzyme always catalyzes a single type of chemical reaction, which
involves the formation and breakdown of covalent bonds. Since they are specific to one particular reaction,
this feature of enzymes is called reaction specificity; also known as absolute reaction specificity, i.e., no by-
products is formed [1].
MECHANISM OF ACTION OF ENZYMES:
The mechanism of action is based on a chemical reaction, in which the enzyme binds to the substrate and
finally forms an enzyme–substrate complex. This reaction take place in a relatively small area of the enzyme
called the active or catalytic site. In other words, the mechanism of enzyme action is based on the nature of
the enzyme–substrate interaction, which accounts for the reaction specificity of the biological catalysts. The
active or catalytic site of an enzyme is constituted by several amino acids, located at some distance from each
other in the peptide chain. These amino acids are brought close together by the folding resulting from the
secondary and tertiary structure of the enzymes. Side chains of amino acid residues at the catalytic site provide
groups for binding with specific groups of the substrate. Co-factors assist the catalysis. The substrate forms
bonds with amino acid residues in the substrate binding domain of the active site. The binding induces a
conformational reaction in the active site. During the reaction, the enzyme forms a transition-state complex.
As the products of the reaction disassociate, the enzyme returns to the original state. Two different models
postulated for the mechanism of enzyme action are given below.
1. The Fisher template model (lock and key model):
This is a rigid model of the catalytic site, proposed by Emil Fischer in 1894. The model explains the
interaction between a substrate and an enzyme in terms of a lock and key analogy. In this model, the catalytic
site is presumed to be reshaped. The substrate fits as a key fit into a lock. The drawback of this model is the
implied rigidity of the catalytic site. The model cannot explain changes in enzyme structure in the presence
of allosteric modulators [1].

22
Figure 2: Lock and Key Model
2. Induced fit model
In contrast to the above method, this model suggests a flexible mode for the catalytic site. To overcome the
problems of the lock and key model owing to the rigid catalytic site, Koshland suggested an induced fit model
in 1963. The important feature of this procedure is the flexibility of the active site. In the induced fit model,
the substrate induces a conformational change in the active site of the enzyme so that the substrate fits into
the active site in the most convenient way so as to promote the chemical reaction. This method suggests
competitive inhibition, allosteric modulation and inactivation of enzymes on denaturation. The Michaelis–
Menten theory of enzyme action [2] offers the basis for most current research on the mechanism of enzyme
action. This concept of the enzyme–substrate complex scheme assumes the combination of the enzyme and
substrate in phase one (occasionally known as the transition phase) of the enzyme activity and liberation of
the enzyme and the products of the catalysis in phase two of the reaction.
Conformational Selection:
In conformational-selection binding, a conformational change occurs prior to the binding of a ligand
molecule, as a conformational excitation from the unbound-ground state conformation of the protein. In this
mechanism, the ligand seems to ‘select’ and stabilize a higher-energy conformation for binding. In induced-
fit binding, the conformational change occurs after ligand binding and is a conformational relaxation into the
bound ground-state conformation that is apparently ‘induced’ by the ligand. The function of proteins is
affected by their conformational dynamics, i.e., by transitions between lower-energy ground-state
conformations and higher-energy excited-state conformations of the proteins [3].
(a) In induced-fit binding, the change between the conformations P1 and P2 of the protein occurs after binding
of the ligand L. The intermediate state P1L relaxes into the bound ground state P2L with r20te kr, and is
excited from the ground state with rate ke.
(b) In conformational-selection binding, the conformational change of the protein occurs prior to ligand binding.
The intermediate state P2 is excited from the unbound ground state P1 with rate ke, and relaxes back into the
ground state with rate kr.
(c)
Figure 3: Characteristic chemical relaxation of induced-fit and conformational-selection
binding.

23
Enzyme inhibition:
Enzyme inhibition decreases the activity of an enzyme without significantly disrupting its three-dimensional
macromolecular structure. Inhibition is therefore distinct from denaturation and is the result of a specific
action by a reagent directed or transmitted to the active site region. When low molecular weight compounds
interfere with the activity of enzymes by partially reducing or completely inhibiting the enzyme activity either
reversibly or irreversibly, it is known as enzyme inhibition. The compounds responsible for such inhibition
are called enzyme inhibitors. To protect the enzyme catalytic site from any change, a ligand binds with a
critical side chain in the enzyme. Chemical modification can be performed to test the inhibitor for any drug
value. The pharmacological action of drugs is mainly based on enzyme inhibition, e.g. sulfonamides and
other antibiotics. In the majority of cases the enzyme inhibited is known. The development of nerve gases,
insecticides and herbicides is based on enzyme inhibition studies. There are two major types of enzyme
inhibition: reversible and irreversible [1].
PROPERTIES OF ENZYMES – CHEMICAL NATURE
Enzymes are protein catalysts that speed up the rate of biochemical reactions but do not change the structure
of the final product. Like a catalyst, without being used up, the enzymes control the speed and specificity of
the reaction, but unlike catalysts, only living cells generate enzymes. The rate of biochemical reaction often
influences enzymes like catalysts, so that they can take place at a relatively low temperature. The enzymes
are thus known to lower the energy of activation. In certain cases, the biological response is initiated by
enzymes. The term enzyme is derived from the Greek word enzymes, meaning 'in yeast' since the enzyme
activity in living organisms was first discovered in the yeast cells. The enzyme term was invented by W.
Kuhne in 1878.
Properties of Enzymes can be classified into:
➢ Physical properties
➢ Chemical Properties
➢ General properties
1. Physical Properties of Enzymes:
➢ Physically, enzymes act as colloids or as high-molecular-weight compounds.
➢ At a temperature below the boiling point of the water, enzymes are killed or inactivated.
➢ Most enzymes in the liquid medium are inactivated at 60°C.
➢ Extracting dried enzymes can withstand temperatures of 100°C to 120°C or even higher. Enzymes are,
therefore, thermos-labile.
➢ The optimum activity of each enzyme is always at a particular temperature, which typically varies from 25°C
to 45°C. At 37°C, enzyme action is strongest and as temperatures rise above 60°C, enzymes become inactive.
2. Chemical Properties of Enzymes:
Catalytic Properties: Biological catalysts are enzymes. The greater amounts of compounds are catalyzed by
a small number of enzymes. This means that enzymes are highly capable of turning giant amounts of the
substrate into a substance. Enzymes improve the reaction rate and remain unaffected by the reaction they
catalyze.

24
Enzyme Specificity: Enzymes are extremely variable in nature, which means that a specific enzyme can
catalyze a specific reaction. For example, only sucrose hydrolysis can be catalyzed by Enzyme sucrase.
3. General Properties of Enzymes:
➢ Enzymes initiate the biochemical reaction rate and accelerate it.
➢ The activity of enzymes depends on the medium acidity of the (pH specific). At a particular pH, each catalyst
is most active. pH 2 for pepsin, pH 8.5 for trypsin, for example. At near neutral pH, most intracellular
enzymes act.
➢ The reaction in either direction can be accelerated by enzymes.
➢ Both enzymes have active sites involved in biochemical reactions.
➢ Enzymes, often soluble in water, dilute glycerol, NaCl, and dilute alcohol, are very unstable compounds.
➢ At the optimum temperature, enzymes work aggressively.
➢ In nature, all enzymes are proteins, but all proteins may not be enzymes.
➢ Enzymes lower the molecule's activation energy so that the biochemical reaction can take place at the normal
temperature of the body, which is 37°C.
Chemical Nature of Enzymes: Proteins are all enzymes, but all proteins are not enzymes. However, there
are several conjugated enzymes bound to the protein portion of the enzyme with a non-protein moiety, which
is called the Apo enzyme. The portion of the non-protein is known as the cofactor. If the co-factor, like
Potassium, calcium, magnesium, manganese, is of an inorganic type, it is known as the prosthetic group. In
general, the prosthetic group is closely bound to the protein portion of the enzyme and it is difficult to separate
it with a simple technique such as diffusion. The enzyme is called a holoenzyme with the prosthetic group
and the Apoenzyme. If organic molds such as NADP, NAD, FAD, etc. are co-factor attached to an enzyme
protein, it is called a coenzyme. In general, a coenzyme is loosely bound to the Apoenzyme and can be
isolated easily from the prosthetic group. Often, coenzymes are heat tolerant [5].
Most Important Properties of an Enzyme
• Catalytic Property
• Specificity: Enzymes are very specific in their action. Particular enzymes act on particular substrates only.
Enzymes are also specific to a particular type of reaction. In some rare cases, the specificity may not be too
strong. Enzymes show different types of specificity as follows: Bond Specificity, Group Specificity, Substrate
Specificity, Optical Specificity, Cofactor Specificity, and Geometric Specificity.
• Reversibility: Most of the enzymes in catalyzed reactions are reversed. Reversal of response depends on cell
requirements. In some cases, there are different enzymes for reaction and regression. Some enzyme-induced
reactions are irreversible [6].
• Sensitiveness to heat and temperature and pH.
FACTORS AFFECTING ENZYME ACTIVITY:
The conditions of the reaction have a great impact on the activity of the enzymes. Enzymes are particular
about the optimum conditions provided for the reactions such as temperature, pH, alteration in substrate
concentration, etc.
1. Temperature Effect:
The enzyme activity gradually lowers as the temperature raises more than the optimal temperature until it
reaches a certain temperature at which the enzyme activity stops completely due to the change of its natural

25
composition. On the other hand, if the temperature lowers below the optimal temperature, the enzyme activity
lowers until the enzyme reaches a minimum temperature at which the enzyme activity is the least. The enzyme
activity stops completely at 0 °C, but if the temperature rises again, and then the enzyme reactivates once
more [7].
2. pH Effect:
Each enzyme has a pH value that it works at with maximum efficiency called the optimal pH. If the pH is
lower or higher than the optimal pH, the enzyme activity decreases until it stops working. For example, pepsin
works at a low pH, i.e., it is highly acidic, while amylase works at a high pH, i.e., it is basic. Most enzymes
work at neutral pH 7.4.
3. Concentration Effect:
a. Substrate concentration:
Increasing substrate concentration also increases the rate of reaction to a certain point. Once all of the
enzymes have bound, any substrate increase will have no effect on the rate of reaction, as the available
enzymes will be saturated and working at their maximum rate.
b. Enzyme concentration:
Increasing enzyme concentration will elevate the chemical reaction rate, as long as there is substrate available
for binding. Once all of the substrate is bound, the reaction will no longer speed up, because there will be
nothing for additional enzymes to bind to.
4. Activators Effect:
Activators they enhance the activity of an enzyme. Some of the enzymes require certain inorganic metallic
cations, like Mg2+, Mn2+, Zn2+, Ca2+, Co2+, Cu2+, Na+, K+ etc., for their optimum activity. Compounds
which are active as prosthetic groups or which provide stabilization of the enzyme’s conformation or of
the enzyme-substrate complex Rarely, anions are also needed for enzyme activity, e.g. a chloride ion (CI–)
for amylase.
5.Water effect:
In preservation of food, it is mandatory to inhibit enzymatic activity completely if the storage temperature
is below the phase transition temperature.[8]
APPLICATIONS:
Currently, enzymes are often utilized for a broad range of applications such as: washing powders (e.g.,
proteases, lipases, amylases); textile manufacture (amylases and catalase to remove the starch); the leather
industry (proteases to hydrolyze proteins); the paper industry; improvement of the environment; food
production (enzyme-modified cheese/butter), processing (glucose oxidase for dough strengthening) and
preservation; and medical applications. According to current reports, several enzymes are produced
industrially and there are significant applications in the food industry (45% of use), detergent industry (35%),
textiles industry (10%) and leather industry (3%).
1. Pharmaceutical applications:

26
1.1 Diagnostic applications of enzymes
Enzymes have been used widely in diagnostic applications varying from immunoassays to biosensors.
Enzyme immunoassay methods hold great promise for application under a wide variety of conditions. Under
laboratory conditions they can be as sensitive as radio-immunoassays, but they can also be adapted as simple
field screening procedures. The examination of enzyme quantity in the extracellular body fluids (blood
plasma and serum, urine, digestive juices, amniotic fluid and cerebrospinal fluid) are vital aids to the clinical
diagnosis and management of disease. Most enzyme-catalyzed reactions occur within living cells, however,
when an energy imbalance occurs in the cells because of exposure to infective agents, bacterial toxins, etc,
enzymes 'leak' through the membranes into the circulatory system. This causes their fluid level to be raised
above the normal cell level. Estimation of the type, extent and duration o
f these raised enzyme activities can then furnish information on the identity of the damaged cell and indicate
the extent of injury. Table 1 includes a number of diagnostically important enzymes which are most often
examined in clinic laboratories [1].
1.1.1Enzyme examinations in diseases of the liver and biliary
The diseases of the liver and gastrointestinal tract were among the first to which serum enzyme tests were
applied. They have proved to be most effective owing to the large size of the organs and the wide range and
abundance of enzymes. The liver-based enzymes GOT (glutamine oxalo-acetic acid transaminase), GPT
(glutamate pyruvate transaminase) and AP (alkaline phosphatase) are examined to evaluate the site and nature
of liver disease. LD (lactate dehydrogenase), GGT (gamma glutamyl transaminase), OCT (ornithine
carbamoyl transferase) and ChE (cholinesterase) are also examined. Several enzymes employed in the
diagnosis of liver diseases along with their respective levels are listed in table 2 [1].
1.1.2. Enzyme applications in heart disease:
The serum glutamine oxalacetic acid transaminase determination (GOT) was considered a significant step
forward in the diagnosis of acute myocardial infarction. A mixture of results from assays of CPK (creatine
phosphokinase), HBD (α-hydroxybutyrate dehydrogenase) and GOT (glutamine oxalacetic acid
transaminase)—each of which has been shown to be elevated in more than 90% of cases—is used for
diagnostic purposes. An enzyme known as hyaluronidase use. 5luronate hydrolysis) has been reported to cure
heart attack. The activity of many enzymes including aldolase, malic dehydrogenase, isomerase and ICD (Iso
citrate dehydrogenase) may increase myocardial infarction.
1.1.3. Diagnosis of muscle disease
Skeletal muscle disorders include diseases of the muscle fibers (myopathies) or of the muscle nerves
(neurogenic disorders) [9]. In myopathies CPK, LD, ALD (aldosterone), GOT and GPT levels are raised. In
the case of neurogenic diseases and hereditary diseases, CPK is occasionally raised (2–3 fold) [9]. In muscular
disorders the level of CPK is elevated in serum with the highest frequency and is assayed in the diagnosis of
these disorders. An additional useful assayed enzyme is acetyl cholinesterase (AChE), which is significant in
regulating certain nerve impulses. Various pesticides affect this enzyme, so farm labors are frequently tested
to be sure that they have not received accidental exposure to significant agricultural toxins. There are number
of enzymes that are characteristically used in the clinical laboratory to diagnose diseases. There are highly
specific markers for enzymes active in the pancreas, red blood cells, liver, heart, brain, prostate gland and
many of the endocrine glands. From the time when these enzymes became comparatively easy to examine

27
using automated techniques, they have been part 13 of the standard blood tests that veterinarians and medical
doctors are likely to need in the diagnosis and treatment/management of diseases.
1.2. Enzymes in therapeutics
Enzymes have two significant features that differentiate them from all other types of drugs. First, enzymes
frequently bind and act on their targeted sites with high affinity and specificity. Second, enzymes are catalytic
and convert numerous target molecules to the desired products. These two important features make enzymes
specific and potent drugs that can achieve therapeutic biochemistry in the body that small molecules cannot.
These features have resulted in the development of many enzyme-based drugs for a wide range of disorders
[10]. Currently, numerous enzymes are used as therapeutic agents, owing to the following features:
High specificity to their substrates.
• Proficient in producing the desired effect without provoking any side effects.
• Water soluble.
• Extremely effective in a biological environment.
Table-1: Diagnostically significant Enzymes
Enzymes as therapeutic agents also have some serious disadvantages which restrict their application. Their
bulky structure, due to their large molecular weight, excludes them from the intracellular domain. Owing to
their high proteinaceous nature they are highly antigenic and are rapidly cleared from blood plasma. Extensive

28
purification from pyrogens and toxins is essential for parenteral enzymes, which increases the cost. Table 3.
lists some therapeutically important enzymes.
Table 2: Liver diseases and enzymes used in diagnosis
1.2.1. Enzyme therapy of cancer
Therapeutically, the use of proteolytic enzymes is partly based on scientific reports and is partly empirical.
Clinical evidence of the use of proteolytic enzymes in cancer studies has typically been obtained with an
enzyme preparation comprising a combination of papain, trypsin and chymotrypsin. Proteases and their
inhibitors have long been studied in several tumor systems. However, out of numerous promising serine and
metalloproteinase inhibitors, not a single one is included in oncology at present. The present exploration for
active antiproteolytic agents is in contrast to the traditional approach, as evidenced by John Beard, who
proposed the management of advanced cancer using fresh pancreatic extracts whose antitumor activity was
based on their proteolytic potential.
The enzymatic treatment of tumours is based on the idea of denying the abnormal cells their essential
metabolic precursors such as amino acids, nucleic acids and folates. A number of enzymes have been
examined and evidenced as antitumor agents. l-serine dehydratase, l-arginase, carboxypeptidase G (folate
depletion), l-asparaginase, l-methioninase, l-phenylalanine ammonia lyase, l-glutaminase, l-tyrosinase and

29
xanthine oxidase have been studied for their anticancer activity. Enzyme preparations such as asparaginase
(amidase), bromelain (protease) and chymotrypsin (protease) have also been studied as cancer treatments.l-
asparaginase is the most widely investigated enzyme. It has been reported in treatment against three neoplastic
diseases, acute lymphoblastic leukemia, leukemic lymphosarcoma and myeloblastic leukemia. It deprives the
cancerous cells of their nutritional asparagine supply. Asparagine is essential for protein synthesis, which
takes place inside the cell, and decreased protein synthesis perhaps accounts for the immunosuppression and
toxic effects of asparaginase-based treatment.
Table 3: Therapeutically important enzymes.

30
In the study published on September 16, 2021, in Molecular Cell, the researchers show that an enzyme
complex named HTC (hydride transfer complex) can inhibit cells from aging.
“HTC protects cells from hypoxia, a lack of oxygen that normally leads to their death,”
HTC is made up of three enzymes: pyruvate carboxylase, malate dehydrogenase 1, and malic enzyme 1. Most
interestingly, inhibition of these enzymes stopped the growth of prostate cancer cells, suggesting that HTC
could be a key target to develop new therapeutics for a variety of cancers, including prostate cancer.
1.2.2. Enzymes in thrombolytic treatment
Enzyme-based thrombolysis for treating massive pulmonary embolism has been considered as an effective
approach to dissolving clots in these large vessels. Since surgical removal raises the chances of new blood
clot formation that can cause another pulmonary embolism at the same or a different site, it is considered a
dangerous practice and thrombolytic therapy is considered the more effective treatment. Nevertheless,
reoccurrence of clot formation or clot re-formation is very common in patients who have undergone enzyme-
based thrombolytic treatment.
According to current research, urokinase (produced in the kidneys and obtained from human urine) is
considered safer than streptokinase.
For the production of urokinase, 2300 l of urine is required to yield only 29 mg of purified urokinase, thus
considering the expense involved in its manufacture; its clinical utilization has been restricted. Other
examples are Arvin and reptilase. Utilization of these has been restricted for several reasons, but they are still
considered as potential replacements for heparin as anticoagulants.
Some researchers have noticed that optimum dose plays an important role and is one of the key factors in
determining re-clot formation. Thorough investigation is required to overcome any shortcomings and increase
the acceptance of these enzymes in therapeutic use.
1.2.3. The role of enzymes in digestive disorders and inflammations
Enzymes play an essential role in the management of various digestive disorders, such as exocrine pancreatic
insufficiency. Supplementation with enzymes may also be advantageous for other conditions associated with
poor digestion, such as lactose intolerance. Generally, pancreatic enzymes such as porcine and bovine have
been the preferred form of supplementation for exocrine pancreatic insufficiency. Utilization of microbe-
derived lipase has presented promise with reports showing benefits alike to pancreatic enzymes, but with a
lower dosage concentration and a broader pH range. The safety and efficacy of enzymes derived from
microbial species in the treatment of conditions such as malabsorption and lactose intolerance are promising.
Plant-derived enzymes, e.g., bromelain from pineapple, serve as active digestive aids in the breakdown of
proteins. Synergistic properties have also been reported using a combination of animal-based enzymes and
microbe-derived enzymes or bromelain. Buccal administration of pancreatin (derived from an alcoholic

31
extract of animal pancreas) enhances the enzymatic digestion of starch and proteins in patients with pancreatic
cysts and pancreatitis. Pancreatin in combination with lipase is used to treat patients with fatty stools.
Hydrolytic enzymes such as papain and fungal extracts (Aspergillus niger and Aspergillus otyzae) are used
to enhance absorption from the small intestine .
These fungal extracts comprise amylases and proteases along with cellulases, which support the breakdown
of the otherwise indigestible fibers of cabbages, etc, and thus reduce dyspepsia and flatulence. Currently,
micro-organisms are used at a large scale for the production of therapeutic enzymes. Among various micro-
organisms Saccharomyces cerevisiae, Saccharomyces fragilis, Bacillus subtilis and two Aspergillus species
are considered safe by the FDA (USA) for obtaining oral β-galactosidase (from A. oryzae) which is often
used by patients suffering from inherited intestinal disease lactose deficiency. Children with these genetic
disorder children are incapable of digesting milk lactose. Enzymatic preparations such as β-galactosidase
catalyze the conversion of lactose to glucose and galactose, which are quickly absorbed by the intestine.
Other enzymatic preparations, e.g., penicillinase (from B. subtilis) are often used to treat hypersensitivity
reactions caused by the antibiotic penicillin. This enzyme catalyzes the conversion of penicillin to penicillanic
acid, which is non-immunogenic. In addition, microbial and plant hydrolases are also used to decrease
inflammation and edema.
Thrombin, trypsin, chymotrypsin, papain, streptokinase, streptodornase and sempeptidase are under clinical
trial investigation. These enzymatic preparations are administered orally and have considerable proteolytic
activity in the serum. Streptodornase has also displayed pain-relieving action on systemic injection.
Preparations have also been used to clean dirty wounds and necrotic tissue and to remove debris from second-
and third-degree burns.
2. Plants and algae enzyme systems
Protease, amylase, lipase and cellulose are the important enzymes and are present in plants. Protease breaks
down protein that can be present in meat, fish, poultry, eggs, cheese and nuts. Amylase assists your body with
the breakdown and subsequent absorption of carbohydrates and starches. Lipase aids the digestion of fat.
When your diet includes lipase-rich foods, it eases the production burden on the gall bladder, liver and
pancreas. Cellulase is present in many fruits and vegetables, and it breaks down food fibers, which increases
their nutritional value to our bodies. The presence of cellulase in plant-based sources is important, because it
is not naturally present in the human body. Fruits and vegetables are an ideal source for enzymes. They are
enzyme-rich and easily consumed without needing to be cooked or processed, ultimately preserving the full
functionality of the enzymes. By using plant biotechnology several enzymes can be produced from plants as
well algal resources [1].
During algal photosynthesis various proteins and enzymes are produced which can be utilized in economic
development and environment management, such as in wastewater treatment, production of fine chemicals,
and biodiesel production. Due to their potential to capture and fix carbon dioxide using solar energy,
photosynthetic marine algae are considered as potential models for the production of proteins. It has been
recently observed that algal chloroplasts can be transformed for the production recombinant proteins. Five
different classes of recombinant enzymes; xylanase, α-galactosidase, phytase, phosphate anhydrolase, and β-
mannanase, D. tertiolecta or C. reinhardtii were in the plastids of D. tertiolecta or C. reinhardtii. Similar
strategies should allow for recombinant protein production in many species of marine algae.

32
3. Enzymes in the food industry
Enzymes are widely used by the food industry as processing aids for the production of numerous and common
products.[11]
Table 4: Enzymes in the food industry
3.1. Enzymes in Dairy Industry
The proteases of lactate bacteria are necessary for its growth in substrate milk and help dramatically in
enhancing flavor of fermented milk products. The proteolytic enzyme system constitutes proteinases that first
break down the protein of the milk into peptides. Peptidases then breaks down the peptides into amino acids
and small peptides, then the transport system takes charge of the uptake of amino acids and small peptides.
Different lipases of animal or microbe origin created clear cheese, low bitterness, improved flavor, and potent
malodors, whereas proteinases, in combination with lipases or/and peptidases, developed cheeses with
excellent flavor and low bitterness levels. In order to accelerate the ripening of cheese content of peptidases
and proteinases we can use attenuate cell-free extract or starter cells to do that. Animal lypases that are
isolated from the epiglottis (throat) of calves, sheep, or goats, greatly contribute to release short chain fatty
acids responsible of sharp and piquant flavors. The result is due to improved fat and moisture retention in
cheese caused by the increased emulsifying propert, that is reduction of fat losses in whey and cooking water.
A further recent application is enhancing flavor and structure in butter and dairy cream. Lipases from Mucor
miehei, or Aspergillus niger are used to give stronger flavors in Italian cheeses from milk before adding the
rennet, by a modest lipolysis, increasing the amount of free butyric acid.
Transglutaminase (TG) has recently become of great interest to food scientists for its ability in strengthening
the structure of protein gels. TG catalyzes the posttranslational modification of proteins by transamidation of
available glutamine residues by the formation of covalent cross-links between glutamine and lysine residues.
Addition of transglutaminase to milk induces of cross-linking of caseins and whey proteins that improves the
strength of milk gels. Rennet cheese with modified textural and nutritional properties and improved yield
could be obtained upon transglutaminase modification but simultaneous addition of rennet and
transglutaminase is recommended. Moreover, transglutaminase cross linking and calcium reduction were

33
investigated as ways to improve the texture and storage stability of high-protein nutrition bars formulated
with milk protein concentrate and micellar casein concentrate. Lactase accelerates the breakdown of lactose
into galactose and glucose. It is used to improve the sweetness, solubility, as well as digestive agent, for milk
products. Lactases are important in reducing and removing lactose in milk products for lactose-intolerant
patients in order to protect them from severe diarrhea, fatal consequences, and tissue dehydration.
3.2. Enzymes in Beverage Industry
Enzymatic treatment of fruit juice has many advantages over traditional processing. These advantages include
an increase in fruit juice yield, enhanced clarification, increased total soluble solids in fruit juice, improved
pulp liquefaction, and decreased turbidity and viscosity. The cloudiness of fruit juice is due to the presence
of pectin, cellulose, starch, proteins, tannins, and lignin. The commercial application of enzyme preparations
containing pectinases, cellulases, and tannases benefits the fruit juice industry. These enzymes are known as
macerating enzymes, which are used in fruit juice extraction and clarification.
Glycoside hydrolases hydrolyze glycosidic bonds and are widely used in syrup, beverage (beer, wine), and
dough production. Amylases have a broad range of applications in the food and brewing industries. The
extensive application of enzymes to brew with high amounts of inexpensive raw materials such as barley
focuses on future aspects of enzymes in the brewing industry. In barley, starch has to be broken down into
fermentable sugars before the yeast can make alcohol. Barley malt is the traditional source of enzymes used
for the conversion of cereals into beer. Pectinase catalyses degradation of pectic substances through de-
esterification (esterases) reactions and depolymerization (hydrolases and lyases). Cellulases have gained
worldwide interest, as they have valuable potential to process cellulosic biomasses and transform them to
useful products. Tannases (tannin acyl hydrolases) are important groups of enzymes that are utilized in several
industrial applications, including the manufacture of fruit juice, and tea.
Tea and coffee are among the main beverages used worldwide. In coffee industry, enzymes from microbial
sources such as cellulases, hemicellulases, galactomannase, pectinases are used from Leuconostoc
mesenteroides, Saccharomyces marscianus, Flavobacterium spp., Fusarium spp.. Tea processing requires
cellulases, glucanases, pectinases and tannase.
3.3. Enzymes in Other Food Industry
Industrial applications of enzymes include food (baking, dairy products, starch conversion) and beverage
processing (beer, wine, fruit and vegetable juices), animal feed, textiles, pulp and paper, detergents,
biosensors, cosmetics, health care and nutrition, wastewater treatment, pharmaceuticals and chemical
manufacture and, more recently, biofuels such as biodiesel and bio-ethanol. Enzymes are needed for cheese
production and a wide variety of other dairy goods. For example, their application keeps breadsoft and fresh
longer, leads to crispy crusts, increases dough vol-ume and can compensate for variations in flour and malt
quality. Additionally, enzymes are used to lower alcohol concentration and calories in beer. In winemaking,
the sulphur content can be reduced, clarity and wine colour can be maintained, flavours can be enhanced and
the filterability can be improved with enzymes. In the production of baked goods, enzymes can be added
individually or in complex mixtures at a very low level that may act in a synergistic way. Baking comprises
the use of enzymes from three different sources:
• The endogenous enzymes in flour.
• Enzymes associated with the metabolic activity of the dominant microorganisms.

34
• Exogenous enzymes added in the dough.
In the baking industry, there is a rising focus on lipolytic enzymes. Because the enzymes degrade polar wheat
lipids to produce emulsifying lipids, recent findings suggest that phospholipases can be used to supplement
or substitute traditional emulsifiers.
Glutaminase an enzyme produced by starter cultures to impart flavor, is important in products like meat
sausage. Di fructose anhydride (DFA) III is a non-cariogenic sweetener and non-digestible disaccharide that
promotes the absorption of calcium magnesium, and other minerals in the intestine. DFA III is produced from
inulin by the exo-acting inulin fructotransferase from Arthrobacter ureafaciencs. Actinidin is obtained from
the kiwi fruit (Actinidia deliciosa). Actinidin hydrolyzes both myofibrillar proteins and connective tissue
proteins but appears to have higher proteolytic activity toward collagen.
A large number of fat clearing enzymatic lipases are produced on an industrial scale. Most of the commercial
lipases produced are utilized for flavour development in dairy products and processing of other foods, such
as meat, vegetables, fruit, baked foods, milk product and beer. The function of phospholipase in egg yolk
treatment is to hydrolyze egg lecithin, iso-lecithin, which improves the emulsifying capacity and heat
stability. The egg yolk thus produced can be useful in the processing of custard, mayonnaise, baby foods,
dressings and in dough preparation.
3.4.Extremozymes
Extremozymes (enzymes produced by organisms living under extreme conditions) have potential application
in food for a number of reasons: (a) extremozymes are hardy enzymes that can survive other-than-usual food
processing conditions, (b) extremozymes are more suitable for substrates whose solubility is enhanced only
under extreme conditions, (c) extremozymes may allow in situ catalysis during food processing, for example,
breaking down acrylamide during the baking of food, (d) extremozymes are more suitable for use in foods
that require aging under extreme conditions (high salt, low temperature, etc.), and (e) the use of extremozymes
helps control microbial contamination by microorganisms that grow under normal conditions. Extremozymes
have great economic potential in many industrial processes. Extremozymes may therefore be useful for (a)
the production of glucose from cellulose in wood chips; (b) the release of bioactive peptides from keratinous
materials such as horns, hoofs, and feathers; and (c) the in-situ degradation of polyacrylamide in foods during
baking, etc.
3.5. Antibiofilm Enzymes
Biofilms can become resistant to the chemical and physical treatments applied during cleaning and sanitizing
procedures in the food industry.
Enzymes and detergents have also been used as synergists to improve disinfectant efficacy. The combination
of surfactants/proteolytic enzymes increased the wet ability of biofilms formed by a thermophilic Bacillus
species, thus enhancing cleaning efficiency also reported the synergistic action of phenolic antimicrobials
and surfactants in combination with enzymes. The specificity of the mode of action of each enzyme makes
this technique complex. It also means that it is extremely difficult to identify enzymes that are effective
against all the different types of biofilms.
3.6. Other Industrial Applications of Enzymes
The by-products of the vanilla extract were not utilized earlier, but in 2010, the enzymatic treatment and

35
reutilization of the exhausted vanilla pods were reported. Exhausted vanilla pods contain only small amounts
of aroma and flavor, but after treating them with various enzymes, the aroma and flavor can be extracted and
potentially be used as food additives.
Mushrooms are known to be rich in proteins while containing significant amounts of free glutamic and
aspartic acid, which are found to elicit umami flavor. In order to improve the taste-contributing free amino
acid contents, these proteins need to be hydrolyzed. Flavourzyme contains both endo- and exo-peptidase
activities that are suitable to yield greater amounts of free amino acids while also producing flavoractive
amino acids and peptides from proteins. Consequently, the treatment of mushrooms with the cell-wall
degrading enzyme β–glucanase and the protease Flavourzyme significantly enhanced the umami and other
taste-contributing free amino acids.
Lipoxygenases are enzymes classified in the class oxidoreductases (linoleate means oxygen, LOXs) present
in plants, fungi, and animals. Lipoxygenases produced by plants have a noteworthy importance in the food
industry.
Lipoxygenases generate approximately 9832 aromas and flavor in several plant products. A new enzyme in
the CSyGT family that are similar in structure to the enzymes producing cellulose in plant cell walls.
Unexpectedly, they showed that the new member of the family was responsible for a key step in saponin
synthesis, where a sugar molecule is attached to the triterpenoid backbone.
This discovery challenged the generally accepted view that a different class of enzyme was probably involved
in this step.
They went on to insert the gene for the newly discovered CSyGT enzyme, along with genes for other steps
in the biochemical pathway, into yeast cells. The engineered cells successfully produced glycyrrhizin from
simple sugars, indicating a potential route for industrial manufacture of valuable saponins by growing yeast
cells on a large scale.
4. Enzymes in Light Industry
Light industry usually produces small consumer goods, such as food, textile, furniture, papermaking, printing,
daily chemicals, sporting goods and so on. It is a raw material-oriented industry.
Most light industrial products are produced for end-users rather than intermediate products for use in other
industries. Compared with heavy industry, light industry usually requires less raw materials, space and
electricity, and causes less pollution. With the development of enzyme engineering, enzyme plays a more
and more important role in the field of light industry.

36
Table 4: Other Industrial Applications of Enzymes

37

38
5. Application of Enzymes in Animal Feed Industry
Enzyme has been used as feed additive in industry for decades. During this period, the feed enzyme industry
has gone through several stages of development. The first phase was the use of enzymes to enhance nutrient
digestibility. The industry then started to advocate enzyme addition to poultry diets based on nonviscous
grains, such as sorghum and corn. The next phase is the application of enzymes to noncereal grain
components of the diet. The enzyme industry today is constantly searching for new areas of application.
6. Application of Enzymes in Detergent Industry
The detergent industry is an important application area where industrial enzymes are utilized to boost cleaning
performance. Enzymes represent environmentally sound alternatives to the use of toxic chemicals and
pollutants, with reduced generation of waste and added performance benefits as the result. Enzymes have
effectively promoted the development and improvement of modern household and industrial detergents. The
main categories of detergent enzymes-protease, lipase, amylase and cellulase-each provide special benefits
for laundry and automatic dishwasher applications.
7. Application of Enzymes in Textile Industry
The enzymes used in textile field are amylase, catalase and laccase, which are mainly used for starch removal,
hydrogen peroxide degradation, textile bleaching and lignin degradation. The application of enzyme in textile
chemical processing is rapidly recognized by all countries in the world because of its non-toxic and
environmental protection characteristics, which puts forward higher and higher requirements for textile
enterprises to reduce pollution in textile production. In addition, the use of enzymes reduces processing time,
saves energy and water, and improves product quality and potential process integration.
8. Application of Enzymes in Leather Processing
Leather processing is one of the highly polluting industrial activities. The traditional chemical method of
depilation is the main cause of pollution in leather processing industry. The extensive use of sulfides not only
has adverse consequences for the environment, but also undermines the efficiency of sewage treatment plants.
Therefore, the systematic use of protease instead of lime and sulfides to rationalize the depilation process has
become a primary problem in leather processing. This will lead to a substantial reduction of effluent load and
toxicity in addition to improvement in leather quality.
9. Application of Enzymes in Pulp and Paper Industry
The application of enzymes in pulping and papermaking industry began in 1986, but their use is relatively
small. However, the publication of an increasing number of papers on the subject demonstrated growing
interest in the issue. Cellulase, xylanase, laccase and lipase are the most important enzymes in pulping and
papermaking. In addition, the main goal of enzyme development is xylanase to promote bleaching and
cellulase modification of fiber. The development trend of enzymes is mainly focused on improving the
thermal stability and alkaline strength of enzymes [12].
10. Enzymes Used In Waste Management
The use of enzymes for waste management can be classified in four categories:
(i) effluent treatment and detoxification;
(ii) renewable energy resources (including the formation of byproducts and the production of hydrogen and
methane);

39
(iii) bio indicators for pollution monitoring; and
(iv) Biosensors.
10.1. Effluent Treatment and Detoxification
Most of the bioremedial procedures utilize the biotransformation capability of living organisms, mainly
bacteria, including members of the genera Pseudomonas, Flavobacterium, Arthrobacter, and Azotobacter.
Tolerance of these organisms to extremely toxic molecules, and to changing loadings of less aggressive
substances, can be increased by enzymatic pretreatment of the wastes, because enzymes increase the available
nutrients without additional demand on enzyme production and secretion mechanisms. This in turn increases
the available energy for growth and detoxification activity. Dilution of toxic wastes with biowastes rich in
organic matter that can be hydrolyzed by added enzymes has been noted to improve the overall detoxification
performance at the bench, pilot and full scales.
Many types of enzyme systems may be involved in detoxification. However, the three common enzyme
detoxification systems are: oxidative-reductive, hydrolytic, and conjugating. Examples of
Phytoremediation—the use of plants for bioremediation—are also available. By synchronized action of
oxidative enzymes—cytochrome P450 containing monooxygenase, peroxidase, and phenoloxidase—the
degradation of the toxicmolecules is achieved.
The enzymes are biodegradable and non-toxic, work faster than most chemical catalysts, and work at room
temperature and atmospheric pressure, hence reducing the risk of explosion or fire. Also, there are no alkaline
solutions to dispose of because the process is performed at a neutral pH. In addition to facilitating the direct
treatment of effluents, enzymes also find applications in improving the waste treatment processes. Many
industries, such as the coffee industry, use an evaporation step to concentrate the incoming effluent solutionso
that its disposal is facilitated. In these situations, as the concentration builds, the increased dissolved materials
contribute to excessive viscosity, which greatly reduces evaporator efficiency. If small amounts of
galactomannases are added to aqueous coffee solubles, this problem can be reduced. Similar economic
benefits have been realizedin the fishmeal industry as well, where the addition of alkaline bacterial
proteinases is used to control the excessive viscosity caused by concentrated proteins
10.2. Renewable Energy Resources
10.2.1. Formation of Byproducts
Wastes rich in carbohydrates and with some protein content, can be readily processed at high temperature
using thermostable α-amylases and glucoamylases for starch conversion, and bacterial proteinases for protein
degradation. The resulting product can then be used as the starting material for yeast production. The
conversion of biomass, i.e., lignocellulosic waste, into useful chemicals is another area where enzymes play
a key role. The application of cellulases and pentosanases to improve the nutritive benefits of straw is being
actively considered at present, as these enzymes are abundantly available at a much cheaper price. Enzymes
like fungal proteinases have applications in processing of protein-rich wastes, such as recovering the
component proteins from cheese whey. They act by making minor modifications in the proteins, which in
turn changes their gelling and foaming properties.
Using supercritical carbon dioxide, hydrogen, methanol, an enzyme, and another catalyst, to convert soybean
oil into hydrogensaturated alcohol mixtures. Such mixtures are used to make soaps, detergents, and related

40
products. Spinach enzymes have been documented to degrade explosives to potentially useful chemicals
compounds.
10.2.2. Alternative Fuel Production
The organic polymers (proteins, starch, cellulose, lipids, etc.) in wastes are hydrolyzed to yield low molecular
weight molecules (amino acids, glucose, glycerol, fatty acids, etc.) in addition to smaller molecules (organic
acids, ethanol, carbon dioxide, hydrogen, etc.). The acids and alcohols are then transformed into acetate,
hydrogen, and carbon dioxide. Finally,acetate and hydrogen are used by anaerobic methanogenic bacteria for
productionof methane. Low-priced industrial grade enzymes, e.g. bacterial amylases and proteases, and
fungal cellulases and lipases, are used in the first of the three steps to accelerate depolymerization and increase
the availability of digestible small molecules to microorganisms.
10.3. Bioindicators for Pollution Monitoring
The induction of hepatic cytochromes in some mammals, e.g. wild rodents, can be used as a bioindicator for
monitoring terrestrial environmental pollution. Induction of specific families of hepatic cytochrome enzymes
such as P450IA and P450IIB in various species of animals has been associated with the presence of various
environmental contaminants like polychlorinated dibenzodioxins (PCDDs), polychlorinated dibenzodifurans
(PCDFs), polyaromatic hydrocarbons (PAHs), and polychlorinated biphenyls (PCBs). A number of
investigations have shown a good correlation between the concentration of various pollutants in the soil, liver
or whole body, and the induction of hepatic cytochrome P-450 in wild rodents and birds. A positive
correlation between antioxidant enzymes, mainly catalase and superoxide dismutase (SOD) in digestive tissue
of barnacle, Balanus balanoides, and tissue concentrations of polyaromatic hydrocarbons (PAHs) has also
been shown, suggesting that antioxidant defense components, catalase and SOD, are sensitive parameters that
can be useful biomarkers for the evaluation of contaminated aquatic ecosystems. Hydrolytic enzymes in
activated sludge can be used as indicators of biodegradation activity of biomass. Enzyme activity has been
shown to be a microbial population indicator, a monitor of active biomass, or an indicator of specific
engineering parameter such as COD or phosphorous removal.
10.4. Biosensors
Biosensors may utilize either whole bacterial cells or specified molecules, e.g. enzymes or biomimetics, as a
detection system. Biosensors have the advantage of being nondestructive and can be located on-line, meaning
that samples do not have to be removed and transported to a laboratory for analysis. Combinations of
biosensors in arrays can be exploited to deal with a diversity of toxicants and pollutants. Amperometric
biosensors can offer a viable, low-cost solution to field monitoring in environmental analysis. The rational
choice of immobilization, transduction, and biorecognition chemistries can be used to yield improved
catalytic and affinity electrochemical biosensors for environmental applications. Some of the use of enzymes
in assisting environmental monitoring are - the analysis of PCBs in waste oil by enzyme immunoassay, an
enzyme inhibition sensor for organophosphorus pesticide analysis, the detection of fecal pollution using
fluorimetric assays, and pesticide testing at trace levels by an enzyme immunoassay. The first type of
biosensor involves linking a gene such as the mercury resistance gene (mer) or the toluene degradation gene
(tol) to genes that code for bioluminescence within living bacterial cells. The biosensor cells can signal that
extremely low levels of inorganic mercury or toluene are present in contaminated waters and soils by emitting
visible light, which can be measured with fiber-optic fluorometers

41
The second type of biosensor employs molecular detectors, which might consist of enzymes, nucleic acids,
antibodies or other “reporter” molecules attached to synthetic membranes. Antibodies can be coupled to
changes in fluorescence to increase sensitivity of detection. Fluorescent or enzyme linked immunoassays
have been derived for a variety of contaminants including pesticides, and PCBs. Multiple molecular arrays
can be constructed on synthetic membranes and other matrices allowing the simultaneous detection of a range
of contaminants. An example of the second type of biosensor is a highly selective enzyme electrode for
phosphate ions, which includes a bi-enzyme membrane with co-immobilized nucleoside phosphorylase and
xanthine oxidase, and a platinum amperometric electrode for the detection of enzymatically generated
hydrogen peroxide. A detection limit of 10−7 M is possible and phosphate assays can be easily performed in
the range of 0.1–10 mM, which are typical concentrations encountered in polluted waters [13].
CONCLUSION
Enzymes are proteins that catalyze biological reaction and make them faster by reducing activation energy.
We can measure enzyme activity by either measuring appearance of products or disappearance of substrates.
Enzyme activity is affected by concentration of enzyme and substrate, temperature and water activity,

42
inhibitors. Enzymes have a lot of applications in pharmaceutical industries, food processing and other
industries. According to current reports, several enzymes are produced industrially and there are significant
applications in the food industry (45% of use), detergent industry (35%), textiles industry (10%) and leather
industry (3%).
ACKNOWLEDGEMENT
We would thanks to Authority of Acharya Nagarjuna University College of Pharmaceutial Sciences for
providing Library Facilities.
REFERNCES
1. Introduction to Pharmaceutical Biotechnology, Volume 2 Enzymes, proteins and bioinformatics Chapter 1
Introduction to enzymes and their applications. By Saurabh Bhatia Published September 2018 • Copyright ©
IOP Publishing Ltd 2018. https://iopscience.iop.org/book/978-0-7503-1302-5/chapter/bk978-0-7503-1302-
5ch1
2. Johnson K A and Goody R S. The original Michaelis constant: translation of the Michaelis–Menten paper
Biochemistry, 1913; 50: 8264–9.
3. How to Distinguish Conformational Selection and Induced Fit Based on Chemical Relaxation Rates; Fabian
Paul ,Thomas R. Weikl ; Published: September 16, 2016 https://doi.org/10.1371/journal.pcbi.1005067
4. Enzymes- Structure, Classification, and Functions; https://byjus.com/biology/enzymes/
5. Properties of Enzymes
https://www.vedantu.com/chemistry/properties-of-enzymes
6. Important Properties of Enzymes Infinita Biotech
https://infinitabiotech.com/blog/properties-of-enzymes/
7. Factors Affecting Enzyme Activity
https://www.researchgate.net/publication/347439618_Factors_Affecting_Enzyme_Activity
8. Hassanien, Mohamed. Factors affecting Enzyme- Catalyzed Reactions, 2012.
10.13140/RG.2.2.36798.64328.
9. Sobel B E and Shell W E. Serum enzyme determinations in the diagnosis and assessment of myocardial
infarction Circulation, 1972; 45: 471–82.
10. Vellard M. The enzyme as drug: application of enzymes as pharmaceuticals Curr. Opin. Biotechnol, 2003;
14: 444–50.
11. Ozatay, S. Recent Applications of Enzymes in Food Industry. Journal of Current Research on Engineering,
Science and Technology, 2020; 6 (1): 17-30.
https://www.researchgate.net/publication/342234868_Recent_Applications_of_Enzymes_in_Food_Industry
12. Utilization of enzymes for environmental applications, Sanjeev K Ahuja 1, Gisela M Ferreira, Antonio R
Moreira, PMID: 15493529 DOI: 10.1080/07388550490493726
13. Enzymes immobilization onto magnetic nanoparticles to improve industrial andenvironmental applications;
Osama M. Darwesha,∗, Sameh S. Alib,c, Ibrahim A. Mattera, Tamer Elsamahyc, Yehia A. Mahmoudb; ISSN
0076-6879; https://doi.org/10.1016/bs.mie.2019.11.006

43
Received:
Revised:
Accepted:
KEYWORDS:
1, Mangroves.
2, Cyanobacteria.
3. Synechococcus,
Gloeocapsa
4. 16SrDNA.
Marine Cyanobacteria were isolated from sediments of Pichavaram
mangrove forest situated in south east coast of India. Seven spices
belonging to order Chorococcales were identified based on the size and
shape and they were Synechococcus elongatus, Gloeocapsa sp.
Synechocystis salina, Gloeobacter sp., Gloeocapsa stegophila,
Snechocystis padalecki and Chroococcus minor. Of the seven species,
Synechococcus elongatus and Gloeocapsa sp., were analyzed for 16S
rDNA, and their phylogeny.
Book Chapter 3
ISBN: 978-93-5593-624-3
MOLECULAR CHARACTERIZATION OF UNICELLULAR
CYANOBACTERIA ISOLATED FROM MANGROVE BIOTOPE
*Dr.R.Anburaj1, 2
, Dr.K.Kathiresan1
, Dr.G. Roseline Jebapriya3
1
Centre of Advanced Study in Marine Biology, Faculty of Marine Sciences,
Annamalai University, Parangipettai, Tamil Nadu, India.
2
PG and Research Department of Microbiology, Vivekanandha College of Arts and
Sciences for Women (Autonomous), Tiruchengode, Tamil Nadu, India.
3
Department of Marine Science, Bharathidasan University, Tiruchirappalli, Tamil Nadu,
India.
anbu_nanthu@rediffmail.com
Citations
*Dr.R.Anburaj1, 2
, Dr.K.Kathiresan1
, Dr.G. Roseline Jebapriya3.
Molecular
characterization of unicellular cyanobacteria isolated from mangrove biotope
Recent Trends in Life Sciences. 1st
Edition. 2022. 1-5.

44
INTRODUCTION
Marine unicellular cyanobacteria are abundant in both coastal and oligotrophic environments [1],
where they contribute substantially to primary production [2, 3]. Among the unicellular forms,
Synechococcus sp., are typically responsible for 5 to 25% of primary productivity in temperate and
tropical oceanic environments [4, 5]. The unicellular cyanobacteria exhibit a great diversity of
physiological properties. Few of them are able to fix nitrogen, either aerobically or anaerobically.
These fixing nitrogen cyanobateria can be grouped into two types: sheathed and sheathless. The
former being enclosed in a glycoprotein sheath (glycocalyx, capsule) external to the cell wall [6, 7,
8]. The first sheathed species was classified in the genus Gloeocapsa [9]; however, it was later
assigned to the genus Gloeothece, based on patterns of cell division [10]. On the other hand, the
sheathless isolates were primarily attributed to the genus Synechococcus [11, 12] or Aphanothece
[13].
The current classification of cyanobacteria relies heavily upon morphological observations
such as cell size, shape and arrangement (filamentous, colonial or single cells), colouration and the
presence of characters such as gas vacuoles and a sheath [14]. A number of unicellular, non-beocyte-
forming species of cyanobacteria, however, lack an abundance of complex morphological
characteristics and have been classified together in a single order, the Chroococcales. Although
morphologically similar, it has been shown that this order contains organisms originating from several
evolutionarily distinct and deeply branching groups within the cyanobacterial phylum [15, 16, and
17]. DNA base composition is a very important genetic character to study the taxonomy of
cyanobacteria. Analysis of the DNA base composition (Mol % G+C) is one of the few molecular
characters that have been determined for almost 200 cyanobacterial strains [18]. Large differences in
DNA base composition indicate that the strains cannot be closely related, whereas similar G+C
percentages give no clue concerning genotyping relationships [19, 20]. Grouping based on the
sequence identity is supported by morphological features (size and morphology of vegetative cells,
heterocyst and akinetes, and diameter and morphology of trichomes) [21, 22]. The present study
attempted to isolate, the unicellular cyanobacteria derived from mangrove biotope, and to identify the
predominant species based on morphology and molecular phylogeny.
MATERIALS AND METHODS
1.Collection of soil samples
Sediment soil samples were collected from Pichavaram mangrove forest (Lat. 11°
25’38.4 N; Long.
79º 47’ 35.5 E) by using a corer (1.5m long stainless steel corer with 50cm dia) and transferred to
sterile plastic polythene bags and transport to the laboratory. The plant roots and other debris were
removed from the sediment samples. The samples were transferred to 4°C and analyzed for microbial
groups within 3-6 hours of sampling. The location of sampling site is shown in the figure.1
A known weight of sediment (1g) was aseptically weighed and transferred to a stopper (150ml) sterile
conical flask containing 99ml of sterile diluents. The sediment- diluents mixture was agitated by
means of mechanical shaking for about 5-10 minutes and later it was subjected to microbial analysis.

45
2.Isolation and maintenance of cyanobacteria
Samples were serially diluted up to 10-5
with sterilized 50% seawater and plated with SN medium for
cyanobacteria [23, 24, and 25].
The Cyanobacteria cultures were grown in SN (natural seawater) medium under laboratory conditions
at a light intensity of 3000 lux and room temperature of 24±2˚C with a 14 h light/10 h dark cycle [26,
27, 28]. After a week, the cultures were picked up from the water surface and from the sides of the
flasks and were examined under a microscope. The cyanobacteria were identified by using the
standard references [29, 30,1] . The cyanobacterial isolates were obtained by pour plating [2, 3, and
4] and isolation of single colonies on Petri dishes. The cultures were sub-cultured once in 7 days for
5 times and pure cultures were obtained. The pure stocks of cyanobacterial cultures were maintained
on agar slants. They were incubated in an inverted position low light at a temperature of 25±2°C. The
successive transfers of stock cultures were made for every month [5] and microscopic examination.
Figure. 1. Map showing the location of sampling site.

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