Life in Extremity: Exploration and Evolution

Prof. Satya P. Singh

UGC BSR Faculty
(Formerly Professor & Head)

UGC-CAS Department of Biosciences

Saurashtra University, Rajkot, Gujarat, India
Email: satyapsingh@yahoo.com  satyapsingh125@gmail.com  spsingh@sauuni.ac.in

LinkedIn: https://www.linkedin.com/in/satya-singh-2285a5144/
ResearchGate:  https://www.researchgate.net/profile/Satya_Singh5
Google Scholar: https://scholar.google.com/citations?hl=en&user=jiAzOcgAAAAJ
Scopus: https://www.scopus.com/authid/detail.uri?authorId=22981831200 
Web of Science: ID AAC-7238-2021 UGC:  https://vidwan.inflibnet.ac.in//profile/68903/Njg5MDM%3D  
ORICID Id         https://orcid.org/0000-0002-7531-2872
 Frame of the Talk
• Saline Habitats-Coastal Gujarat
• Diversity of the Haloalkaliphilic bacteria
• Extracellular Enzymes from Haloalkaliphiles
• Proteases: Catalysis, Stability, Thermodynamics
• Directed Evolution & gene Shuffling
Exploration of Microbial Potential
 Limitations

Microbial activities only under limited set of

conditions

Only fraction (1-5%) of microbial world

known and explored Biocatalysts from
microbes able to function only under
delicate & defined set of Conditions

Applications under Natural Environmental

Conditions are limited
 Need of the Hour
Exploration of newer habitats, particularly
extremes ones for environmental and
Biotechnological applications

Evolving the microbial potential by

molecular approaches such gene shuffling
and Directed evolution

Evolving unique & novel biocatalytic

capabilities for industrial & Environmental
applications
 Extreme Environments & Extremophiles

Evolution-Diversity-Commercial Interest

Living Fossils & Evolutionary Relics

Moderate VS Ultra Extreme Environment

Hyper-extremity & Limits - Life on Other planets

 HALOALAKLIPHILIC BACTERIA & ARCHAEBACTERIA
Molecular Phylogeny:
Extensive work is going on Halalakliphilic bacteria & archaea, mostly from soda lakes
 
Biochemical basis of salt tolerance & dependence: Halobacterium salinarum,
Salinibacter ruber
·       Amino acid composition of bulk protein
·       High intracellular salt levels
·       Intercellular osmolytes
 
Protein stability & salt dependence:
·       Few enzymes are purified and characterized
·       Salt dependence assessed- varies extensively
·        Comparison of crude, purified and recombinant glucoglycerol phosphate synthase
(Synchocystis sp.)
 
Regulation of gene expression
Salt dependent Gene expression Marine Cyanobacterium (Synechococcus sp ) Hegemann ,J.
Bact., 2002
 
Denaturation & Protein folding
·        Susceptibility of salt tolerant proteins to denaturants- some are highly resistant to urea
denaturation
·       Renaturation under in-vitro conditions
·       Role of molecular chaperones in salt stress cellular conditions
 
Saline Habitats & Study Sites
 Sea Water
 Saline Soil- salt enriched
 Saline Desert
Little Rann of Kutch – Unexplored saline desert
Majority of studies: Vegetated soil of desert, biological soil crust, Translucent stones,
hurbs
Scarce information on the microbial diversity of unvegetated/barren soil
Isolation strategies: Major role in cultivation
 Sampling site and sample collection

 Kachchigadh (Shivrajpur), Dwarka (N 22.23o, E 68.97o) and

 Alang-Sosiya ship breaking yard, Bhavnagar (N 21.41o, E 72.20o)

Locations of the sampling sites along the Gujarat coast

 
Saline Desert: LRK

Bhatt, H.B. and Singh S.P. 2022, Frontiers in Marine Science, doi:
10.3389/fmars.2022.769043
 Research Groups
Haloalkaliphilic/Salt-tolerant Alkaliphilic actinomycetes
Dr. Jigna Thumar
Dr. Sangeeta Gohel
Dr. Kruti Dangar
Dr. Amit Sharma
Dr. Foram Thakrar
Ms. Majejbin Sheikh
Dr. Dalip Rathore
Ms. Ankita Dobariya
Ms. Gopi Koladiya
Ms. Hasti Ramavat

Haloalkaliphilic/Salt-tolerant Alkaliphilic Bacteria

Dr. Rajesh Patel
Dr. Mital Dodia
Dr. Rupal Joshi
Dr. Chirantan Rawal
Dr. Vikram Raval
Dr. Megha Purohit
Dr. Sandip Pandey
Dr. Hitarth Bhat
 Dimensions of the Diversity Assessment

Morphological and Growth Characteristics

Biochemical/Metabolic

Enzymatic Profiling

Molecular Characteristics

Studies on the Enzymatic Characteristics

Screening & Production

Purification and Characteristics

Gene Profiling, Cloning and heterologous Expression

Structure & Function Relationship

Cultural characterization of actinomycetes
Media Specific growth of the seasonal actinomycetes of
Sea water
 Light microscopic examinations (1000x)
of isolates form Okha Madhi after
Gram’s staining

SEM analysis of actinomycetes from

different sites demonstrating a) vegetative
mycelia of OM-4; b, c, d)

Sheikh, M., Rathore, D.S., Gohel, S.D. and Singh

S.P. 2019. Indian Journal of Geo-Marine Sciences
(CSIR-NISCARE),

Gohel, S. D. and Singh S.P. 2018. Geomicrobiology

Journal, 35:9, 775-789

Thumar, J.T. and Singh S.P. 2011. Biotechnology

Bioprocess Engineering, 16, 6:1180-1186
 Morphology of marine Actinobacteria

Light microscopic examination of the seasonal isolates of the actinobacteria

after Gram’s staining
In situ observation of the isolates using slide culture technique
 Effect of pH on the protease production in marine
actinobacteria
AlS1 AlS2
160 160
140 140
Enzyme Activity(U/ml/min)

Enzyme Activity(U/ml/min)
120 120
100 100
80
80
60
60
40
40
20
0 20
7 8 9 10 11 0
pH 7 8 9 10 11
pH

Effect of pH on the protease production from Nocardiopsis alba AlS1, Nocardiopsis

synnemataformans AlS2 and Streptomyces sp.AlW3

Mehajbin Sheikh and S P Singh, 2022, PhD Thesis, Saurashtra University

 Purification and Characterization of
Enzymes

 
Effect of Ca2+ on temperature optima and enzyme activity of purified proteases
from both OM-6 and OK-5 isolates at 0 - 100mM concentration
500
OM-6
% Residual activity

400

300

200

100

0
37 50 60 70 80 90
Temp (oC)
0 2 5 7 10 20 50 100 900
800
OK-5

% Residual activity
700
600
500
400
300
200
100
0
37 50 60 70 80 90
Temp (oC)
0 2 5 7 10 20 50 100
 Effect of NaCl on Temperature Optima Ah-6 Alkaline Protease

Dodia M. S., Rawal C.

M., Bhimani H. G.,
Joshi R. H., Khare, S.
K. and Singh, S. P.
2008. Journal of
Industrial
Microbiology &
Biotechnology
35(2):121-132
 Effect of NaCl on Ve2-20-91 Protease Temperature optima

100
90
% R e la t iv e a c t iv it y

80
70
60
50
40

30
20

10
0
37°C 50°C 60°C 70°C 80°C 90°C
Temperature
0 M 0.25 M 0.5 M
1 M 2 M 3 M

Raval, V. Rawal, C.M., Pillai, S. and Singh S.P. 2014. Process Biochemistry 49 (6):
955-962 (IF 2.63)
Effect of salt on Temperature optima
S-20-9 (seawater)
Urea Denaturation(8M) among haloalkaline proteases
 Thermodynamics of the Protease
catalyzed Reactions

 
Characteristics and thermodynamics of thermostable protease from
salt-tolerant alkaliphilic actinomycete
Deactivation rate constant (Kd) and Half life (t1/2) of OM-6 and OK-5 purified proteases
in presence of 0 M NaCl, 2 M NaCl, 4 M NaCl and 30% Na-glutamate at the range of
37oC-80oC temperature
30%
0 M NaCl 2 M NaCl 4 M NaCl
Na-glutamate
Isolates T (oC)
Kd t1/2 Kd t1/2 Kd t1/2 Kd t1/2
( ×10-3) (min) ( ×10-3) (min) ( ×10-3) (min) ( ×10-3) (min)
37 1.24 558.98 0.73 949.51 0.51 1359.11 0.42 1650.35
50 4.94 140.31 2.52 274.62 0.91 761.70 0.73 949.51
OM-6 60 27.51 25.19 10.92 63.47 1.68 412.58 1.14 608.02
70 35.86 19.32 27.29 25.39 10.75 64.47 2.41 287.61
80 41.09 16.86 33.30 20.81 28.50 28.50 8.66 80.04
37 1.55 447.19 1.47 471.52 1.08 641.80 0.99 700.14
50 2.30 301.36 2.01 344.84 1.20 577.62 0.826 839.16
OK-5 60 19.15 36.19 2.37 292.46 1.69 433.21 0.77 900.19
70 22.4 30.94 18.38 37.71 6.23 111.25 2.50 277.25
80 37.2 18.63 24.31 28.51 8.29 83.61 4.11 168.64
1. Decrease in favorable electrostatic repulsion
2. Halophilic enzymes have high –ve charge with hydrated groups shielded by high
salt that avoids unfolding and maintain solubility of proteins
3. Lowering NaCl decrease stability of enzyme suggesting low water activity resulting
from high salt conc. required for conformational stability of enzyme
4. Effect of salt on stabilizing enzyme is related not only to the salt/solute conc but
also to the type of salt/solute
∆H*, ∆S* and activation energy for OM-6 and OK-5 proteases deactivation
Isolates OM-6 OK-5
∆H* ∆S* E ∆H* ∆S* E
Treatment
(KJ/mole) (J/mole) (KJ/mole) (KJ/mole) (J/mole) (KJ/mole)
0 M NaCl 111.26 57.03 113.92 71.33 -69.99 74.08
2 M NaCl 96.91 6.48 99.57 62.59 -152.72 65.34
4 M NaCl 41.34 -175.17 44.01 44.91 -160.12 47.66
30% Na-glutamate 34.47 -196.37 37.14 29.23 -211.83 31.97

∆G* for deactivation of OM-6 and OK-5 proteases

Strains OM-6 OK-5
∆G* (KJ/mole) for deactivation of protease ∆G* (KJ/mole) for deactivation of protease
T (oC) 0M 2M 4M 30% Na- 0M 2M 4M 30% Na-
NaCl NaCl NaCl glutamate NaCl NaCl NaCl glutamate
37 93.27 94.63 95.56 96.06 92.69 92.83 93.62 93.85
50 93.58 94.38 98.12 98.71 95.63 95.99 97.38 98.38
60 91.80 94.36 99.54 101.62 92.81 98.59 99.68 101.70
70 93.89 94.67 97.32 101.59 95.23 95.79 98.88 101.48
80 96.31 96.93 99.85 100.88 96.60 97.85 101.01 103.07
1. Lower ∆H and ∆S reveled higher thermal stability
2. –ve ∆S indicated ordered transition state of protease
3. ↑ salt/solute ----- stable conformation of protease ---- decreased entropy of
unfolding ---- thermodynamic
4. ↑ ∆G ----- thermal stability of protease ---- ↑ free energy --- as enzyme
could resist against unfolding of its transition state
 Effect of NaCl on the catalytic constant Kcat
(sec-1) of purified AH-6 protease

NaCl
Kcat ( sec-1)
(M)
370C 500C 600C 700C 800C
0.0 6.18 3.36 0.45 0.234 0.0
0.5 4.08 9.30 10.26 5.82 3.78
1.0 1.98 5.64 7.62 11.28 9.6
1.5 0.66 4.08 4.92 9.90 4.8
2.0 0.12 2.28 3.00 7.32 3.42
Dodia M. S., Rawal C. M., Bhimani H. G., Joshi R. H., Khare, S. K. and Singh, S. P. 2008.
Journal of Industrial Microbiology & Biotechnology 35(2):121-132
 Immobilization of the protease
• Highly thermostable protease from haloalkaliphilic
actinomycete immobilized by 5 different approaches on 24
carrier systems
• Thermodynamic analysis suggested enhanced stability on
immobilization
– Half life increases
– Deactivation rate constant decreases
• Enzyme bioreactor and applications of the immobilized
enzyme
 FT-IR analysis of the free and immobilized protease A:alkaline
protease, B: immobilized alkaline protease

A B

Structural topographies of the immobilized HTASP-FT-IR

Decrease in Amide A band, suggesting the
Interaction of the carriers with –NH groups in protease molecule while C=O
regions remain less affected

Thakrar, F. J., and Singh, S. P. (2019).  Bioresource Technology. 278 150–158

PROTEASES: PRODUCTION AND CATALYSIS
UNDER NON-AQUEOUS CONDITIONS
 Growth behavior of Mit-1 in the presence of Organic
solvents

Control Xylene

Benzene Butanol
 Comparison of specific enzyme production with complex
medium and with organic solvent as the sole source of carbon
(0.1%)

Medium Specific enzyme Comparative

production fold
(Enzyme activity/growth)

Complex medium 49 1.0

(gelatin broth)
MM* + Butanol 2400 48.9
MM+ Xylene 1083 22.1
MM+ Acetone 268 5.5
MM+ Benzene 73.8 1.5
MM+ Ethanol 20.21 0.412

* MM = Minimal Medium

Thumar, J.T. and Singh, S. P. 2009. Journal of Industrial Microbiology &

Biotechnology 36:211–218
 Solvent concentrations (%) required to reduce
enzyme catalysis to its half
Solvent concentration (%) O.M.A18 O.M.E12 O.M.6.2
metagenome

  Native Recombinant Native Recombinant  

Glycerol 10 10 10 <5 <5

Xylene 10 10 10 <5 <5

Hexane 10 5 10 <5 <5

Methanol 10 5 10 <5 <5

Acetone 10 5 10 <5 <5

Chloroform 10 5 10 <5 <5

Purohit et al 2022. Environmental Science & Pollution Research (Springer), Accepted

07 June 2022
 Stability of the proteases in the presence of different solvents
at 10%. Time required (h) by the proteases to reduce its
activity to 50%.

Solvent O.M.A18 O.M.E12 O.M.6.2

concentration (%) metagenome

  Native Recombinant Native Recombinant  

Methanol 3 6 9 9 3

Glycerol 3 6 6 6 3

n-Hexane 6 6 9 9 3

Purohit et al 2022. Environmental Science & Pollution Research (Springer), Accepted

07 June 2022
 Evolution of
Microbial/Biological Potential
 Nobel Prize-2018: Chemistry
Shared with: George P Smith and
Gregory P Winter 

Frances H. Arnold 
Linus Pauling Professor of Chemical Engineering, Bioengineering
and Biochemistry; Director, Donna and Benjamin M. Rosen
Bioengineering Center

Nobel Prize-2018: Chemistry

B.S., Mechanical and Aerospace Engineering, Princeton University,
1979; Ph.D., Chemical Engineering, University of California,
Berkeley, 1985; Postdoctoral, UC Berkeley, Chemistry, 1985;
Postdoctoral, Caltech, Chemistry, 1986
 Directing the evolution of enzymes
RANDOM GENETIC MUTATIONS

SELECTION OF MUTANTS
“Natural evolution’s billion/million-year process
Now probably less than a week.”

How a protein’s sequence encodes its function by random mutation and artificial selection: New and
useful proteins

How functionally neutral mutations: can set the stage for further adaptation

Molecular Sex/ Coupling: combine 32 parents. Or sequences from monkeys and worms. How to do it,
what it can make, and what we can learn from it.

computational tools: to design chimeric proteins and libraries of such proteins

These libraries are extremely diverse, members that differ by tens or even hundreds of mutations while
still maintaining a high proportion of sequences that fold and function

They can also catalyze reactions better than their parents, or even reactions their parents do not
catalyze.
Directed evolution: Like climbing a hill on a 'fitness landscape'

Elevation represents the desired property. Each round of selection samples mutants on all
sides of the starting template (1) and selects the mutant with the highest elevation, thereby
climbing the hill. This is repeated until a local summit is reached (2).
Thomas Shafee- Thomas, Shafee, (2014). "Evolvability of a viral protease: experimental
evolution of catalysis, robustness and specificity". PhD Thesis. University of Cambridge.
Performing multiple rounds of directed evolution is useful not only because a new library of
mutants is created in each round but also because each new library uses better mutants as
templates.
Directed evolution Vs Natural Evolution

Inner cycle: 3 stages of the directed evolution cycle with

the natural process being mimicked in brackets.
Outer circle: Steps in a typical experiment.
Red symbols indicate functional variants, the
Pale symbols indicate variants with reduced function.…

Ref: Thomas Shafee - Thomas, Shafee, (2014).

"Evolvability of a viral protease: experimental
evolution of catalysis, robustness and specificity". PhD
Thesis. University of Cambridge.
 Approaches
Exploration
Investigating in-situ microbial diversity of a given habitat by
molecular approaches such as In-situ PCR
Exploring the vast genetic diversity & potential of non-
cultivable organisms

Evolution
Site directed mutagenesis: on a gene with known
consequences
Gene shuffling and directed evolution:
Sequence space & sequence optimization
Single gene shuffling
Multigene shuffling
Genome shuffling
Starting gene (left) and library of variants (right).  
Point mutations change single nucleotides. 
Insertions and deletions add or remove sections of DNA
Shuffling recombines segments of two (or more) similar genes.
Gene shuffling & Genetic Heterogeneity
Construction of Chimeric genes based on Amino
acid homology

Creation of truncated genes/deleted genes

Creation of a pool of genetic heterogeneity

PCR based Random mutagenesis

Family shuffling
Random Priming recombination (RPR)
Staggered Extension Process (StEP)
Heteroduplex recombination
Chimeric Genes: Construction and
Structure & Function relationship
 T. maritima, A. tumefaciens and C. gilvus

Searching of homologous regions with the two target enzyme

Gene shuffling by Truncation by

overlapping PCR deletion

Identifying DNA sequences of the chimera mutants

Ligation, transformation and expression of chimera genes

Characterization of chimeras having catalytically enzymes

Procedures for chimeric -glucosidases from parent enzymes

 1 st PCR

2nd PCR

3rd PCR
 

Overlapping PCR for gene shuffling at any site

Gene shuffling of -glucosidases from T. maritima and C. gilvus

D242 E524

128 243 578 638

TM 721

D291

177 292 605 665

CG

Tm578/606Cg 754
Tm128/178Cg Tm243/293Cg Tm638/666Cg

Tm336/382Cg Tm476/506Cg Tm538/564Cg

Catalytic residue
; Activity
; No activity
 Construction of the truncated -glucosidases from A. tumefaciens
 
D222 E616
25 364 535 818
Activity
AT 818

AT-4 814

AT-31 787

AT-62 756

AT-89 729

 
AT-119 699
Construction of Chimeric β-Glucosidases with Improved Enzymatic Properties

Singh and Hayashi

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 270, No. 37, Issue of September
15, pp. 21928 –21933, 1995 (*)
J. Molecular Catalysis , B : Enzyamatic,11, 811-816,

Hayashi, K ,Li, Ying.., Singh, S.P., Kaneko,S.,Nirasawa, S.,

Shimonishi ,S.Kawata,Y., Imoto, T., and Kitaoka, M (2001).

Improving enzyme characteristics by gene shuffling:

application to -glucosidase
Characteristics of chimeric enzymes constructed between Thermotoga maritima
and Agrobacterium tumefaciences -glucosidases: role of C-terminal domain in
catalytic activity

Bong-Jo Kima, b, Satya P. Singha, c and Kiyoshi Hayashia

Catalytic activity
T. maritima 2163 bp

Primer No. 1 Primer No. 3

Tm495/591At 1491 bp 684 bp
Primer No. 4 Primer No. 2

Primer No. 1 Primer No. 5

Tm533/626At 1599 bp 579 bp
Primer No. 6 Primer No. 2

Primer No. 1 Primer No. 7

Tm630/727At 1890 bp 276

bp
Primer No. 8 Primer No. 2

A. tumefaciens 2454 bp
 100
100

80
80
Relative activity (%)

Relative activity (%)

60
60

40
40

20
20

0
0
0 20 40 60 80 100 0 20 40 60 80 100
Temperature ( C) Temperature (C)

100
100

80
80
Relative activity (%)

Relative activity (%)

60
60

40
40

20 20

0 0
2 4 6 8 10 2 4 6 8 10 12
pH pH
 Journal of Fermentation and Bioengineering
Journal of Fermentation & Bioengineering

Volume 85, Issue (4)1998, Pages 433-435

Importance of five amino acid residues at C-terminal region for

the folding and stability of β-glucosidase of Cellvibrio gilvus

DeogKim1§SatyaSingh1SachikoMachida1YoungYu1ChikaAoyagi1YasushiKa
wata2KiyoshiHayashi1
 Advantage
 

• The big advantage of gene shuffling in order to

improve enzyme character is that it is not
necessary to know the three dimensional
structure.
• In general, the characters of chimeric enzymes
constructed by the gene shuffling becomes a
mixture of the parental enzymes.
• The outcomes of gene shuffling will become
easier to predict and gene shuffling will provide a
 
relatively accessible way for altering enzyme
characters.
Creating Genetic Heterogeneity
Figure 3. Cell pellets of E. coli transformants expressing wild-type
and mutant cyclases. (A) JM109 carrying plasmid pUC-crtYEU or
pUCY2, together with pAC-crtEEU-crtBEU-crtIEU or pAC-
crtEEU-crtBEU-I14. (B) JM109 transformants carrying pAC-
crtEEU-crtBEU-I14 and various cyclase mutants.

Arnold Group
NATURE BIOTECHNOLOGY VOL 18 JULY 2000
http://biotech.nature.com
How Some Species Will Survive Climate Change
(Ref: Daniel Rubinoff on August 15, 2022, Scientific American

Hybrids/ the organisms containing DNA from more than one species- Referred as
unlucky reproductive-wise and lacked some Darwinian common sense

New genomic data & analysis suggest that the closely unrelated species- which have
swapped genes defy evolutionary common sense- BUT are NOT vanishingly rare.

Are relatively ecologically stable and better equipped against the climate change: in the
face of adversaries: droughts, floods and heat waves

This shuffle of DNA: Confer serious benefits, like disease resistance or adaptations to
new environments.

Homo sapiens is a great example of this:

Thanks to genomic technology, 2-4% of most Eurasian people’s DNA is 
directly traced to Neandertals
 
Genomes of most of us- equipped with a package of non–Homo sapiens genes-
conferring benefits to our species.

Habit of exchanging genes: An unexpected way to dodge extinction

 FEMS MICROBIOL LEETT
159, 41-46, 1998

Overproduction of -glucosidase as active form by E.coli.

System coexpressing the chaperonin GroELS at 25C.

Machida, S.,Yu, Y., Singh,S.P., Kim ,J.D., Hayashi, K. And

Kawata,Y.
 Concluding Remarks
Exploration of Newer habitats, particularly the extreme
ones, would generate new horizons for their applications
and generation of novel and unknown value based products

  By directed evolution enzymes may acquire capabilities

not found in naturally occurring organisms by exploring
vast sequence space available in proteins 

 Exploration of Non-Cultivable: Vast Genetic resources

By In-vitro approaches, biosynthetic and degradative

pathways could be perfected for better bioremediation and
biotechnological applications
 Gratitude
Vice Chancellor, GGV, Bilaspur
HRDC, GGV, Bilaspur
Director HRDC, GGV Bilaspur
Prof. Bhaskar: Coordinators, Course Program
 Research Team of Prof. Satya P. Singh
Dr. Sangeeta Gohel, Assistant Professor, Saurashtra University
Dr. Vikram Raval, DST Young Scientist (Now Asstt. Prof. at Gujarat
University)
Dr. Aparna Singh, DST Women Scientist ( Now Asstt. Prof, Surat)
Dr. Kalpana Rakholiya, SERB- National Post-Doctoral Fellow
Ms. Kruti Dangar, DST Women Scientist (Now Asstt. prof,
Saurashtra University)
&
25PhD Students
24 M Phil Students
110 MSc Students
Financial Support
DBT, UGC, DST, MoES, GSBTM,
Saurashtra University, Rajkot

Research Collaborations
• IIT Delhi, New Delhi: Prof. S. K.Khare
• DUSC, New Delhi: Prof. Sanjay Kapoor
• NFRI, Tsukuba, Japan: Dr. Kiyoshi Hayashi ( Now at
Toyo University, Japan)
• Griffith University, Australia
• JNTU Hyderabad, Prof. Ch. Sasikala
• Central University of Hyderabad, Prof. Ch. Rama Rao
 Acknowledgements : Ph.D. Students
 Dr. Bharat Joshi (Faculty, UBC, Canada)
 Dr. Manish Bhatt ( Entrepreneur, Canada)
 Dr. Rajesh K. Patel ( Professor, VNUSG, Surat)
 Dr. Anju Mittal ( Scientist, USA)
 Dr. Mital Dodia ( Scientist, Canada)
 Dr.. Jignasha Thumar ( Asstt. Prof. Gandhinagar)
 Dr. Rupal Joshi (Scientist, ZRC, Ahmedabad)
 Dr. Chetna Rajyaguru (Associate Prof. Rajkot)
 Ms. Geera Mankad ( Associate Prof. Rajkot)
 Dr. Chirantan Raval ( Asst Prof., Govt College)
 Dr. Megha Purohit (Scientist and Entrepreneur,
Canada)
 Dr. Himanshu Bhimani ( Assoc Prof. Navsari Ag
Univ,)
 Dr. Bhavtosh Kikani (Asstt Prof. CHARUSAT)
 Dr. Sandeep Pandey (Scientist, Pharma, Daman)
 Dr. Viral Akbari ( Scientist, UK)
 Dr. Rushit Shukla (Asstt Prof. Christ College,
Rajkot)
 Dr. Amit Sharma (Scientist, ZRC, Ahmedabad)
 Dr. Kruti Dangar (Asstt Prof. Saurashtra University)
 Dr. Atman Vaidya ( Biology Teacher & Entrepreneur)
 Dr.r. Hitarth Bhatt (Asstt Prof. Atmiya Univ, Rajkot)
 Dr. Rupal Pandya (Scientist, USA)
 Dr. Foram Thakrar ( Ahmedabad)
 Dr. Dalip Singh Rathore ( GBRC, Gandhinagar)
 Dr. Nirali Raiyani ( Asstt Prof. R. K. Univ Rajkot)
 Dr. Mahejbin Sheikh (Visiting Faculty, Saurashtra
University)
 Recent Publications
( Cumulative Impact factor : 201, H-Index: 31)
2022
Purohit, M.K, Rathore Dalip Singh, Koladiya Gopi, Pandey Sandip and Singh S. P., 2022, Comparative
analysis of the catalysis and stability of the native, recombinant and metagenomic alkaline proteases in
organic solvents, Environmental Science and Pollution Research (IF 4.30) (Springer), accepted for
published, 07 June 2022

Thakur Nagendra, Singh Satya P. and Zhang, Changyi, 2022. Microorganisms under extreme environments
and their applications, Current Research in Microbial Sciences (2022), doi:
https://doi.org/10.1016/j.crmicr.2022.100141

Joshi Rupal, Raval Vikram, Bhatt Hitarth and Singh S. P., 2022 “Phenotypic characteristics, phylogenetic
analysis and characterization of alkaline proteases of marine bacteria Geomicrobium halophilum,
Oceanobacillus oncorhynchi, and Oceanobacillus khimchii”, BIOLOGIA (IF 1.34), 10.1007/s11756-022-
01095-7

Raval Vikram, Rathore Dalip Singh and Singh S. P., 2022 “Comparative studies of the characteristics of
two alkaline proteases from Haloalkaliphilic bacterium D-15-9 and Oceanobacillus onchorynchii Mi-10-
54”, APPLIED BIOCHEMISTRY AND MICROBIOLOGY (IF 0.95) (Springer),

Bhatt, H.B. and Singh S.P. 2022, Diversity of cultivable bacteria in a saline desert of Little Rann of Kutch,
India: a phylogenetic perspective , Frontiers in Marine Science (IF 4.90), doi:
10.3389/fmars.2022.769043
2021
Dwivedi, Purna, Sharma, A. K. and Singh, S.P. 2021. Biochemical properties and repression
studies of an alkaline serine protease from a haloalkaliphilic actinomycete, Nocarpdiopsis
dassonvillei subsp. albirubida OK-14. Biocatalysis and Agricultural Biotechnology,
Accepted. 07 June 2021 (Elsevier; IF: 0.90)

Kikani, B.A. and Singh, S.P. 2021. Amylases from thermophilic bacteria: Structure and
Function Relationship. Critical Reviews in Biotechnology, In Press, 30 April 2021 (Taylor &
Francis; IF: 8.102)

Rathore, D. R., Sheikh, M., Gohel G.D, and Singh, S.P. 2021. Genetic and phenotypic
heterogeneity of the Nocardiopsis alba strains of sea water. Current Microbiology, 78: 1377-
1387 (Springer; IF: 1.75), DOI: 10.1007/s00284-021-02420-0

Chauhan, J.V., Mathukiya, R. Singh, S.P. and Gohel, S.D. 2021. Two steps purification,
biochemical characterization, thermodynamics and structure elucidation of thermostable
alkaline serine protease from Nocardiopsis alba strain OM-5. International Journal of
Biological Macromolecules (IJBIOMAC), 169: 39-50 (Elsevier; IF: 5.16),
https://doi.org/10.1016/j.ijbiomac.2020.12.061 , Available On-Line 12 Dec 2020.

Rathore, D R and Singh, S.P. 2021. Kinetics of growth and co-production of amylase and
protease in novel marine actinomycete, Streptomyces lopnurensis KaM5. Folia Microbiologica
(Springer; IF: 1.70), https://doi.org/10.1007/s12223-020-00843-z
2020
Sharma, A.K. Kikani, B.A. and Singh S.P. 2020, Diversity and Phylogeny of Actinomycetes of
Arabian Sea along the Gujarat Coast. Geomicrobiology Journal (Taylor & Francis, IF 1.90),
DOI: 10.1080/01490451.2020.1860165

Raiyani, Nirali and Singh S.P. 2020, Extraction of environmental DNA, construction of
metagenomic libraries and functional screening of enzymes from salt pan soil, Indian Journal
of Geo-Marine Sciences, Accepted (NISCARE, CSIR, IF 0.50),

Raiyani, Nirali and Singh S.P. 2020, Taxonomic and functional profiling of the microbial
communities of Arabian Sea: A Metagenomics approach 
Journal: Genomics (Elsevier, IF 6.20), 112:4361- 4369
https://doi.org/10.1016/j.ygeno.2020.07.024

Bhatt, H.B. and Singh S.P. 2020,

Cloning, Expression and structural elucidation of a biotechnologically potential alkaline serine
protease from a newly isolated Haloalkaliphilic
Bacillus lehensis JO-26, Frontiers in Microbiology (IF 4.25), 11:1-16,
https://doi.org/10.3389/fmicb.2020.00941
 
 
2020
Sharma, A.K. Kikani, B.A. and Singh S.P. 2020,
Biochemical, thermodynamic and structural characteristics of a biotechnologically compatible a
lkaline protease from a haloalkaliphilic,
Nocardiopsis dassonvillei OK-18 International Journal of Biological Macromolecules
(IJBIOMAC), 153:680-696, DOI: 10.1016/j.ijbiomac.2020.03.006 (IF 5.16)

Pandya, Rupal D. and Singh S.P. 2020, Pigment production by an extreme halophilic archaeon
on Halorubrum sp. J4.2.2 from little Rann of Kutch, Gujarat, India. Research Journal of
Biotechnology, 15(1):88-100. E-ISSN: 2278-4535 Print ISSN: 0973-6263

2019 
Thakrar, F.J. and Singh S.P. 2019. Catalytic, thermodynamic and structural properties of an
immobilized and highly thermostable alkaline protease from a haloalkaliphilic actinobacteria,
Nocardiopsis alba Tata-5. Bioresource Technology, 278:150-158 (IF 5.802)

Sheikh, M., Rathore, D.S., Gohel, S.D. and Singh S.P. 2019. Cultivation and characteristics of
the Marine Actinobacterial from the Sea water of Alang, Bhavnagar. Indian Journal of Geo-
Marine Sciences (CSIR-NISCARE), 48(12), 1896-1901(IF 0.4).

Rathore, D.S., Sheikh, M.A., Gohel, S.D. and Singh, S.P. (2019) Isolation strategies, abundance
and characteristics of the marine actinomycetes of Kachhighadi, Gujarat, India. Journal of
Marine Biological Association of India (JMBAI), CMFRI Cochin, India 61(1): 21-27
2018
 
Gohel, S. D. and Singh S.P. 2018. Thermodynamics of a Ca2+ dependent, highly thermostable
and detergent compatible purified alkaline serine protease from Nocardiopsis xinjiangensis
strain OM-6. International Journal of Biological Macromolecules (IJBIOMAC),
https://doi.org/10.1016/j.ijbiomac.2018.02.157, 113:565-574 (IF 3.00)
 
Gohel, S. D. and Singh S.P. 2018. Molecular phylogeny and diversity of the salt-tolerant
alkaliphilic actinobacteria inhabiting Coastal Gujarat, India. Geomicrobiology Journal, DOI: 
10.1080/01490451.2018.1471107, 35:9, 775-789 (IF 1.5)

Thakrar, F.J., Kikani, B.A., Sharma, A.K. and Singh S.P. 2018. Stability of alkaline proteases
from haloalkaliphilic actinobacteria probed by circular dichroism spectroscopy. Applied
Biochemistry and Microbiology (Russia), 54(6), 591-602 (IF 0.68)
 
Sheikh, M., Rathore, D. S., Gohel, S. D. and Singh S.P. 2018. Marine actinobacteria associated
with the invertebrate hosts: a rich source of bioactive compounds: A Review. (Invited
contribution) Journal of Cell &Tissue Research, 18 (01), 6361-6374.
 2018
 
Dangar, K. G., Kalasava, A. B., Dave, A. V. and Singh S.P. 2018. Molecular diversity of
Nocardiopsis alba sp. isolated from the coastal region of Gujarat, India. Journal of Cell
&Tissue Research, 18(3) 6559-6570

Vaidya A., Nair, V. S., Georrge, J. and Singh S.P. 2018. Comparative analysis of
thermophilic proteases, Research Journal of Life Sciences, Bioinformatics, Pharaceutical
and Chemical Sciences (RJLBPCS) 4(6), P. 66. DOI: 10.26479/2018.0406.05
 
Pandey, S. Sharma, A.K., Solanki, Kiran P. and Singh S.P. January 2018. Catalysis and
stability of an extracellular α- amylase from a haloalkaliphilic bacterium as a function of the
organic solvents at different pH, salt concentrations and temperatures. Indian Journal of
Geo-Marine Sciences (CSIR-NISCARE), 47 (01), 240-248 (IF 0.4).
 

 
 
 2018
 
Dangar, K. G., Kalasava, A. B., Dave, A. V. and Singh S.P. 2018. Molecular diversity of
Nocardiopsis alba sp. isolated from the coastal region of Gujarat, India. Journal of Cell
&Tissue Research, 18(3) 6559-6570

Vaidya A., Nair, V. S., Georrge, J. and Singh S.P. 2018. Comparative analysis of
thermophilic proteases, Research Journal of Life Sciences, Bioinformatics, Pharaceutical
and Chemical Sciences (RJLBPCS) 4(6), P. 66. DOI: 10.26479/2018.0406.05
 
Pandey, S. Sharma, A.K., Solanki, Kiran P. and Singh S.P. January 2018. Catalysis and
stability of an extracellular α- amylase from a haloalkaliphilic bacterium as a function of the
organic solvents at different pH, salt concentrations and temperatures. Indian Journal of
Geo-Marine Sciences (CSIR-NISCARE), 47 (01), 240-248 (IF 0.4).
 

 
 
2017
 
Bhatt, H.B., Gohel, S.D. and Singh, S.P. 2017. Phylogeny, Novel bacterial lineage and
enzymatic potential of haloalkaliphilic bacteria from the saline coastal desert of Little Rann of
Kutch, Gujarat, India. 3 Biotech, 8,53, https://doi.org/10.1007/s13205-017-1075-0 (IF 1.36)
 
Bhatt, H.B., Begum, M.A., Chintalapati, S., Chintalapati, V.R. and Singh, S.P.
2017. Desertibacillus haloalkaliphilus gen. nov.sp. nov., isolated from a salt desert.
International Journal of Systematic & Evolutionary Microbiology (IJSEM), 67(11):4435-
4442 (IF 2.1)
 
Kikani, B.A., Sharma, A.K. & Singh, S.P. 2017. Metagenomic and Culture-Dependent
Analysis of the Bacterial Diversity of a Hot Spring Reservoir as a Function of the Seasonal
Variation. International Journal of Environmental Research, 11: 25-38.
DOI:10.1007/s41742-017-0003-9 (IF 1.0).
 
Datta, A., Sharma, A., Kundu, R.S. and Singh S.P. 2017. Diversity and enzymatic profile of
bacterial flora in the gut of an estuarine fish, Mugil jerdoni. Indian Journal of Geo-Marine
Sciences (CSIR-NISCARE), 46(06): 1116-1127 (IF 0.4)
 
Edited Book & Book Chapters
Assignment
 
Question -1: What are different reasons for the variability in publications
among the scientists/students. (One para or 5-7 Points)

Question-2: In the light of the historical background of the citations, discus its
implications (One para or 5-7 Points)

Question-3 Discuss the Impact factors of the journals in the context of the
assessment of the credentials of the scientists. (One-two para)

Question-4 Highlight the merits of H-Index? (up to 5 Points)

Question-5 List 10 Journals with Impact factors and publishers of your

research areas

THANK YOU ALL