3. The Framework of the Talk
âąExtreme Habitats and Actinomycetes
âąMolecular diversity Actinomycetes
âąBiochemical & Genetic Characteristics of Enzymes
4. Domesticating Microbes
Newer Applications Got into the Way
âąUnderstanding the fundamental Processes of Life (
Central Dogma of Life)
âąAbility to manipulate Genes and Genomes
Biological factories & Expression of Foreign Genes
âąTools to rapidly sequence the DNA and proteins
âąSearch for New Microbial Potential
5. 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
6.
7. Adaptations to the Extremity
At Various levels
ï¶Cell Morphology
ï¶Cell envelops & Appendages: CW, CM, Flagella, Capsule
ï¶Membrane Transport
ï¶Metabolism
ï¶Structure & Stability of Macromolecules
ï¶Thermodynamic adaptations
9. Actinomycetes
âą Gram positive, high G+C %
âą Spore forming bacteria; having thin, long,
branched mycelia.
âą About 40 families, 170 Generas, 2000 species
âą Thermophilic actinomycetes are omnipresent; but
are widely found in compost and hay
âą Halophilic actinomycetes are less explored
11. Cultural characterization of actinomycetes
Sheikh, M., Rathore, D.S., Gohel, S.D. and Singh S.P. 2019. Indian Journal of Geo-Marine Sciences (CSIR-
NISCARE), 48(12), 1896-1901
Rathore, D.S., Sheikh, M.A., Gohel, S.D. and Singh, S.P. (2019) Journal of Marine Biological Association of
India (JMBAI), CMFRI Cochin, India 61(1): 21-27
Gohel, S.D. and Singh, S.P. (2015, 2016, 2018) IJBIOMAC, Geomicrobiology
Thakrar, Foram. and Singh, S.P. (2018, 2019 Bioresource Technology, Applied Biochemistry and
Microbiology (Russia)
13. Cell morphology and Gram reaction Cultural characterization
Pigmentation profile
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, 35:1-7 (Elsevier; IF: 0.90), DOI: https://doi.org/10.1016/j.bcab.2021.102059
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
15. Antibiotic sensitivity profile on the basis of mode of action (a: U- I,
b: U- II and c: U- III)
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
16. 0
5
10
15
20
25
F243 &
R513
F984 &
R1378
U1F & R U2F& R NF & R
Total
no.
of
isolates
Chemotaxonomic features
16S rRNA gene amplification profile
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
17. Molecular Approaches to study Actinomycetes
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
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, 35:1-7 (Elsevier; IF: 0.90), DOI: https://doi.org/10.1016/j.bcab.2021.102059
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
Gohel, S. D. and Singh S.P. 2018. Molecular phylogeny and diversity of the salt-tolerant alkaliphilic actinobacteria
inhabiting Coastal Gujarat, India. Geomicrobiology Journal, 35:9, 775-789
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.
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
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.
19. 0.8% agarose gel show PCR products of isolates from Okha Madhi amplified with (a) U1
primer at 52.3°C 55.3°C 59.4°C: lane-1 Medium range DNA ruler lane-2,3,4 OM-3, lane-5,6,7
OM-4 lane-8,9,10 OM-5 lane-11,12,13 OM-6 lane-14 Super Mix DNA ladder, lane-15,16,17
OM-8 lane-18,19,20 OM-9 lane-21,22,23 OM-11 (b) U2 primer at 52.7°C, 55.9°C, 59.2°C:
lane-1 High range DNA ruler lane-2,3,4 OM-1 lane-5,6,7 OM-3 lane-8,9,10 OM-4 lane-11
Super Mix DNA ladder, lane-12,13,14 OM-6 lane-15,16,17 OM-7 lane-18 High range DNA
ruler lane-19,20,21 OM-8 lane-22,23,24 OM-9 lane-25,26,27 OM-11
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)
20. 0.8% agarose gel show PCR products of isolates Okha Madhi amplified with (a) StrepB/E
(Lane 2-24) at 50.7°C, 53.9°C, 56.7°C and StrepB/F (Lane 26-28) at 54.1°C, 58.1°C, 60.0°C
lane-1 High range marker (10 kb), lane-2,3,4 OM-3 lane-5,6,7 OM-4 lane-8 High range
marker, lane-9,10,11 OM-5 lane-12 High range marker, lane-13,14,15 OM-8 lane-16,17,18
OM-9 lane-19,20,21 OM-10 lane-22,23,24 OM-11 lane-25: High range marker, lane-26,27,28
OM-2 (b) N F/R primer at 53.3°C, 56.4°C, 60.0°C lane-1 high range marker (10 kb), lane-2,3,4
OM-6 lane-5,6,7 OM-8 lane-8,9,10 OM-9 lane-11,12,13 OM-11 lane-14,15,16 OM-12
21. 16S rRNA amplification profile
of isolates from A) Okha Madhi
and B) Okha site
U1
U2
StrepB/E
StrepB/F
N-F/R
OK-1
OK-2
OK-3
OK-4
OK-5
OK-6
OK-7
OK-8
OK-9
OK-10
U1
U2
StrepB/E
StrepB/F
N-F/R
OM-1
OM-2
OM-3
OM-4
OM-5
OM-6
OM-7
OM-8
OM-9
OM-10
OM-11
OM-12
23. Diversity Assessment : Phylogenetic Tree
Sea Water actinomycetes
Pre-monsoon Monsoon
Post-monsoon
Dalip Rathore, Mahejbin Sheikh and S P Singh, 2019, 2020. 2021
27. Extra cellular enzyme
detection
ï± Gohel, S. and Singh S.P. 2015. . International Journal of
Biological Macromolecules (IJBIOMAC). DOI:
10.1016/j.ijbiomac.2014.08.008, Vol 74: 421-429 (IF
3.00).
ï± Gohel, S. and Singh S.P. 2013. International Journal of
Biological Macromolecules (IJBIOMAC) 56: 20â 27 (IF
2.45).
ï± Gohel, S. and Singh S.P. 2012., J Chromatography-
B,889â 890, 61â 68 (IF 2.9).
ï± Gohel, S. and Singh S.P. 2012. International Journal of
Biological Macromolecules (IJBIOMAC) 50: 664â 671
(IF 2.45).
28.
29. 0
20
40
60
80
100
120
0
0.5
1
1.5
2
2.5
3
0 1 2 3 4 5 6 7 8
OM-6
0
100
200
300
400
500
600
700
0
0.5
1
1.5
2
2.5
3
0 1 2 3 4 5 6 7 8
OK-5
Growth (â ) and
protease production
(â ) among OM-6
and OK-5 isolates at
increasing number
of amino acids at
1% concentration of
each
Growth
at
540nm
Protease
activity
(U/ml/min)
Protease
activity
(U/ml/min)
Growth
at
540nm
Increasing number of amino acids
Increasing number of amino acids
1. Phenylalanine,
2. Leucine
3. Methionine,
4. Tyrosine
5. Aspartic acid
6. Arginine
7. Histidine
8. Asparagine
30. Effect of combinations of amino acids on
protease production
0
0.5
1
1.5
2
2.5
0 1 2 3 4 5 6
No. of amino acids
Growth
(A
540
)
0
50
100
150
200
250
Activity
(U/ml)
Growth Activity
1) Phe ala
2) Phe ala + leu
3) Phe ala + leu + met
4) Phe ala + leu + met + tyr
5) Phe ala + leu + met + tyr + asp
6) Phe ala + leu + met + tyr + asp + arg
31. 0
0.5
1
1.5
2
2.5
C
ontrol
M
et
A
la
H
is
Tyr
P
he
A
rg
Leu
A
sn
Amino acids (1%, w/v)
Growth
(A
540
)
0
20
40
60
80
100
120
140
Activity
(U/ml)
0
0.5
1
1.5
2
2.5
Control Molasses Wheat
flour
Whey
Crude source (1%, w/v)
Growth
(A
540
)
0
30
60
90
120
150
Activity
(U/ml)
Growth (â )
Activity
(â )
Effect of amino acids and crude nutritional
sources on growth and protease production
Amino acids
Crude sources
32. Effect of cations and media on protease production
0
1
2
3
4
GB SB CMB SCB AB GA GAB GCB GGB
Media
Growth
(A540)
0
25
50
75
100
125
150
Activity
(U/ml)
0
0.5
1
1.5
2
2.5
KCl MnCl2 MgCl2 CaCl2
Cations (0.5 %, w/v)
Growth
(A540)
0
100
200
300
400
Activity
(U/ml)
Growth (â )
Activity (â )
cations
Media
33. Deproteinization studies using crustaceous crab shell
The crab (a) used in the present study collected from Somnath Beach (Western; Arabian Seawater); (b) Effect of crustaceans crab shell
(0-3.5 % w/v) on the growth and protease production in Nocardiopsis dassonvillei OK-18 at 30°C under shake flask (120 rev/min)
conditions in minimal medium
0
10
20
30
40
50
60
70
80
%DP
a
b
Concentration (%)
Effect of deproteinization on crustaceans crab shell at 30°C
Sharma, A.K. Kikani, B.A. and Singh S.P. 2020, Biochemical, thermodynamic and structural characteristics of a
biotechnologically compatible alkaline 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)
35. International Journal of Biological Macromolecules
2020
Biochemical, thermodynamic and structural characteristics of a biotechnologically
compatible alkaline protease from a haloalkaliphilic, Nocardiopsis dassonvillei OK-18
Amit K. Sharma, Bhavtosh A. Kikani, Satya P. Singhâ
UGC-CAS Department of Biosciences, Saurashtra University, Rajkot 360 005, Gujarat, India
39. 0
100
200
300
400
500
37 50 60 70 80 90
%
Residual
activity
Temp (oC)
OM-6
0 2 5 7 10 20 50 100
0
100
200
300
400
500
600
700
800
900
37 50 60 70 80 90
%
Residual
activity
Temp (oC)
OK-5
0 2 5 7 10 20 50 100
Effect of Ca2+ on temperature optima and enzyme activity of
purified proteases from both OM-6 and OK-5 isolates at 0 - 100mM
concentration
40. Enzyme system ÎH*
(kJ/mol)
ÎS*
(J/mol)
E
(kJ/mol)
Purified enzyme [A] 31.62 -184.75 34.34
Purified enzyme [A] at varying pH
A + pH 8 24.41 -203.61 27.12
A + pH 9 31.62 -184.75 34.34
A + pH 10 36.45 -171.09 36.45
A + pH 11 33.4 -181.31 33.4
Purified enzyme [A] at varying salt concentrations
A + 0.5 M 33.51 -182.08 36.22
A + 1.0 M 30.96 -192.07 33.67
A + 2.0 M 44.87 -143.64 33.67
A + 3.0 M 39.22 -158.60 41.94
A + 4.0 M 44.32 -141.04 47.03
Purified enzyme [A] at Na-glutamate and CaCl2
A + 30 % Na-glutamate 18.64 -201.42 21.35
A + 5 mM CaCl2 32.7 -195.85 35.42
A + 10 mM CaCl2 32.63 -195.69 35.34
Changes in enthalpy, entropy and activation energy
( SEA WATER Actinomycetes)
Sharma, A.K. Kikani, B.A. and Singh S.P. 2020, Biochemical, thermodynamic and structural characteristics of a
biotechnologically compatible alkaline 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)
41. Enzyme system
ÎG* (kJ/mol) for deactivation of protease
30 °C 50 °C 60 °C 70 °C 80 °C
Purified enzyme [A] 87.63 91.33 93.17 95.02 96.87
Purified enzyme [A] at varying pH
A + pH 8 86.14 90.21 92.24 94.28 96.32
A + pH 9 87.63 91.33 93.17 95.02 96.87
A + pH 10 88.32 91.74 93.45 95.16 96.87
A + pH 11 88.36 91.99 93.8 95.62 97.43
Purified enzyme [A] at varying salt concentrations
A + 0.5 M 88.71 92.35 94.17 95.99 97.81
A + 1 M 89.19 93.03 94.95 96.87 98.79
A + 2 M 88.42 91.29 92.73 94.17 95.6
A + 3 M 87.3 90.48 92.06 93.65 95.23
A + 4 M 87.08 89.9 91.31 92.72 94.13
Purified enzyme [A] at Na-glutamate and CaCl2
A + 30 % Na-glutamate 79.7 83.73 85.75 87.76 89.79
A + 5 mM CaCl2 92.07 95.99 97.95 99.91 101.86
A + 10 mM CaCl2 91.95 95.86 97.82 99.78 101.73
Enzyme system
ÎG* (kJ/mol) for deactivation of protease
30 °C 50 °C 60 °C 70 °C
Purified enzyme [A] 88.20 90.93 92.30 93.66
Purified enzyme [A] at varying pH
A + pH 8 88.45 91.31 92.74 94.17
A + pH 9 87.93 91.31 92.99 94.68
A + pH 10 88.20 90.93 92.30 93.66
A + pH 11 86.85 90.43 92.22 94.01
Purified enzyme [A] at varying salt concentrations
A + 0.5 M 87.65 92.0 94.18 96.3
A + 1 M 88.33 93.41 95.94 98.48
A + 2 M 88.12 92.71 95.0 97.29
A + 3 M 87.05 91.62 93.91 96.19
A + 4 M 86.06 90.25 92.35 94.44
Purified enzyme [A] at Na-glutamate and CaCl2
A + 30 % Na-glutamate 79.70 83.73 85.74 87.75
A + 5 mM CaCl2 93.89 97.79 99.73 101.6
A + 10 mM CaCl2 88.72 92.66 94.63 96.60
Determination of the ÎG* values for the thermal deactivation of the purified proteases from
a)Nocardiopsis dassonvillei OK-18 & b) Nocardiopsis sp. D-5
a b
42. Effect of osmolytes, inhibitors, metal ions, oxidizing and
reducing agents and surfactants
Gohel, S. D., & Singh, S. P. (2013). Characteristics and thermodynamics of a thermostable protease from a
salt-tolerant alkaliphilic actinomycete.International journal of biological macromolecules, 56, 20-27.
48. Growth behavior of Mit-1 in the presence of Organic
solvents
Control Xylene
Butanol
Benzene
49. 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
Jignasha Thumar and S P Singh 2010
50. Organisms
NCBI
Accession
number
Gene cloned Host Reference
Haloalkaliphilic bacterium OME12 EU680960 Alkaline protease
BL21
(DE3)
Purohit and Singh
2013
Proc. Biochem
Metagenome from salt enriched
soil
--------------- Alkaline protease
BL21
(DE3)
Purohit and Singh
2013
IJBIOMAC
Oceanobacillus ihyehensis OME18 EU680961 Alkaline protease
BL21
(DE3)
Purohit and Singh
2013
Proc. Biochem
Alkalibacillus haloalkaliphilus C-5 --
Serine alkaline
protease
E.coli
(DH5α)
Rawal et al 2012
Haloakaliphilic bacteria Ve2-20-91 JX296114
Serine alkaline
protease
E.coli
(DH5α)
Raval et al. 2015
Ann Microbiology
Haloalkaliphilic actinomycetes
Serine alkaline
protease
BL21
(DE3)
Gohel & Singh,
2012
IJBIOMAC
Bacillus lehensis JO-26 ----------
Serine alkaline
protease
BL21
(DE3)
Hitarth Bhatt and S
P Singh 2019
( Unpublished)
Cloning, Expression and structure and function
relationship
of proteases from Haloalkaliphilic bacteria & Actinobacteria
52. ïDr. Bharat Joshi (Canada)
ïDr. Manish Bhatt ( Canada)
ïDr. Rajesh K. Patel ( Professor, V N University of
South Gujarat, Surat)
ïDr. Anju Mittal ( USA)
ïDr. Mital Dodia (Scientist, Canada)
ï Dr.. Jignasha Thumar ( Asstt Professor, Gandhinagar)
ïDr. Rupal Joshi (ZRC, Ahmedabad)
ïDr. Chetna Rajyaguru (Asstt Professor, Rajkot)
ïMs. Geera Mankad (Associate Professor, Rajkot)
ïDr. Chirantan Raval ( Asst Prof., Govt College)
ï Dr. Megha Purohit ( Scientist and Entrepreneur,
Canada)
ïDr. Himanshu Bhimani (Assoc Prof, Navsari Ag Univ)
ïDr. Bhavtosh Kikani (Asstt Prof. CHARUSAT)
ïDr. Vikram Raval (Asstt Prof. Gujarat University)
ïDr. Sangeeta Gohel (Asstt Prof. Saurashtra University)
ïDr. Sandeep Pandey (Scientist, Pharma, Daman)
ïDr. Viral Akbari ( Scientist, UK)
ïDr. Rushit Shukla (Asstt Prof. Christ College, Rajkot)
ïDr. Amit Sharma (Scientist, ZRC, Ahmedabad)
ïDr. Kruti Dangar (Asstt Prof. Saurashtra University)
ïDr. Atman Vaidya ( Biology Teacher & Entrepreneur)
ïMr. Hitarth Bhatt (Asstt Prof. Virani College, Rajkot)
ïMs. Rupal Pandya (USA)
ïMs. Foram Thakrar (Ahmedabad)
ïDr. Dalip Rathore ( GBRC, Gandhinagar)
Acknowledgements : Ph.D. Students
53. Research Team
Dr. Sangeeta Gohel, Assistant Professor
Dr. Vikram Raval, DST Young Scientist ( Now 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
&
Ph.D./M.Phil/M.Sc.Students
55. Financial Support
DBT, UGC, DST, MoES, GSBTM,
Saurashtra University, Rajkot
Research Collaborations
âąIIT Delhi, New Delhi: Prof. S. K.Khare
âąDUSC, New Delhi: Prof. Sanjay Kapoor
âąNFRI, Tsukuba, Japan: Dr. Kiyoshi Hayashi ( Now at
Toyo University, Japan)
âąGriffith University, Australia
âąJNTU Hyderabad, Prof. Ch. Sasikala
âąCentral University of Hyderabad, Prof. Ch. Rama Rao