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133

KEYWORDS

ISSN: 0974 - 0376

NSave Nature to Survive

: Special issue, Vol. III:

www.theecoscan.inAN INTERNATIONAL QUARTERLY JOURNAL OF ENVIRONMENTAL SCIENCES

Prof. P. C. Mishra Felicitation Volume

Paper presented in

National Seminar on Ecology, Environment &Development

25 - 27 January, 2013

organised by

Deptt. of Environmental Sciences,

Sambalpur University, Sambalpur

Guest Editors: S. K. Sahu, S. K. Pattanayak and M. R. Mahananda

Amrita K. Panda et al.

Thermophiles

Hot spring

Isolation

Purification

Lipase

Bioremediation

133 - 145; 2013

BIO-CHEMICAL AND MOLECULAR CHARACTERIZATION OF

THERMOPHILES FROM HOT WATER SPRING OF SOUTHERN

ODISHA

134

AMRITA K. PANDA, S. SATPAL BISHT1 AND ASHOK K. PANIGRAHI*1Environmental Science Division, Department of Botany, Berhampur University,

Berhampur - 760 007, Odisha

Department of Biotechnology, Ronald Institute of Pharmaceutical Sciences, Berhampur, Odisha

E-mail:drakpanigrahi@gmail. com

INTRODUCTION

Thermophiles are a group of heat loving microbes which thrive at high temperature

usually more than 45ºC. They are inhabitants of various ecological niches like

deep sea hydrothermal vents, terrestrial hot springs and other extreme geographical/

geological sites including volcanic sites , tectonically active faults as well as

decaying matters such as the compost and deep organic land fills (Panda, 2008).

During last three decades these microbes gained lot of attention from various

scientists and industries all over the world. Life in extreme environments has

been studied extensively with reference to the diversity of organisms on the basis

of molecular and regulatory mechanisms. The products of extremophiles i.e.

proteins, enzymes (extremozymes) are of great interest to the molecular biologists.

Habitats of thermophiles

Natural geothermal areas are widely distributed across the globe and primarily

associated with tectonically active zones at which the movements of the Earth’s

crust occur. Hot springs are distributed all over the world and the countries

renowned for their hot springs are Iceland, New Zealand, Mexico, USA, Chile,

Japan, Indonesia, Russia, Brazil etc. The best known terrestrial sites and biologically

well studied hot springs are the Naples in Italy, Yellowstone National Park in USA,

Kamchatka Peninsula Russia, Beppu Japan, Rincon de la Vieja Costa Rica, El

Tatio Chile, Rotorua sits on the shores of Lake Rotorua New Zealand, Huanglong

China, Pamukkale in Turkey, Dallol in Ethiopia and Blue Lagoon in Iceland . The

hot springs are of following types terrestrial, subterranean and marine.

Terrestrial hot springs

Terrestrial geothermal areas are divided into two classes according to the nature

of the heat source and pH i.e. high-temperature fields and low temperature fields.

Subterranean hot springs

Subterranean hot springs are further divided into deep subsurface environments

and hot geothermal water reservoirs, marine and terrestrial oil reservoir. Deep

subsurface environments and hot geothermal water reservoirs Marine and

terrestrial oil reservoirs

Marine hot springs

The Marine hot springs are subdivided into Coastal, inter-tidal and shallow

submarine hot springs and deep-sea hydrothermal vents.

Other geothermal habitats

Constant hot habitats other than geothermal are very few in nature. Solar-heated

ponds and biologically-heated composts, hay, litter or manure may cause high

temperature but these are very transient ecosystems and mostly inhabited by

rapidly-growing spore formers. Man-made constant hot environments have also

been created by this time; these include hot water pipelines, burning coal refuse

piles, wastes from treatment plants or industrial processes in the food or chemical

NSave Nature to Survive QUARTERLY

The present study was an attempt to

characterize few industrially important

thermophilic bacteria from Taptapani a less

known hot water spring of Odisha, India.

Screening and isolation of three potential

lipolytic bacteria, their identification by

morphological, biochemical and molecular

methods (16S rRNA sequencing), media

optimization for the production of lipase

enzyme, purification of lipase enzyme by

hydrophobic ion exchange chromatography

and partial amplification of lipase gene were

the major exercises of this study. During the

investigation it was observed that the SDS-

PAGE fingerprinting is also an ideal,

economical and less time consuming method

for bacterial identification because the cell

surface proteins act as biochemical marker to

discriminate various bacterial strains. It was

also concluded from the present study that

very less number of bacteria can be isolated

from an environmental sample by changing

the culture condition and media composition.

The Taptapani hot water spring may have many

more commercially important microbes’

special reference to enzyme industry.

Production of lipase by the isolate AK-P2

quantitatively is one of the significant finding

of this study. The present investigation contains

both genomic and proteomic studies to achieve

high degree of accuracy in terms of

characterization, industrial prospecting of few

thermophilic isolates those contributed

interesting and promising results. This

investigation indicates that the Taptapani hot

water spring of South-Eastern India is a rich

source of many thermophilic bacteria and

need to be explored for the industrially

important enzymes by employing meta-

genomics studies.

ABSTRACT

*Corresponding author

135

industry. There are more than 300 known thermal springs in

India. Thermal springs of the Indian subcontinent

(temperature range of about 30-100ºC) occur in groups along

with certain major tectonic trends, plate boundaries,

continental margins and rifted structures (Fig. 3). These hot

springs are mostly of non volcanic type thus form anintermediate to low grade exploitable resources. Geo-

tectonologists have grouped them into the following broad

regions:

NW-SE Himalayan arc system with continuation to Andaman

Nicobar Island

Son-Narmada-Tapti lineament

West coast continental margin end

Parts of Gondwana grabens

Regions of Delhi folding.

In India hot water springs are popularly known as “Agnikunds”

means fire wells and distributed across various states of the

Country i.e. Kashmir, Himachal Pradesh, Uttarakhand, Gujarat,

West Bengal, Orissa, Arunchal Pradesh etc but their microbial

diversity has not been well studied at molecular level.

Well known hot springs of India

Ganeshpuri, Akloli, Vajreshwari: Maharastra.

Manikaran, Khirganga, Tapri, Tattapani, Garam Kund:

Himachal Pradesh.

Bendrutheertha, Irde, Bandaru: Karnataka.

Chavalpani near pachmarhi an evergreen plateau in the

Mahadeo Hills, Dhunipani, Tatapani: Madhya Pradesh.

Suryakund; Gaya, Bihar.

Phurchachu (Reshi), Yumthang, Borang, Ralang, Taram-chu

and Yumey Samdong; Sikkim.

Bakreshwar of Birbhum, Tantloi, Kendughata, Bholeghata,

Tantni: West Bengal.

Gaurikund, Tapt Kund, Surya Kund: Uttarakhand.

Hotspring of Dirang area; West Kameng, Arunachal Pradesh.

Taptapani hot spring in Ganjam District, Atri hot spring in

Khurda , Deulajhari hot spring in Angul , Tarabalo hotspring

in Nayagarh of Orissa.

Tatta hot water spring, Jarom, Brahma Kund, Ram Kund

:Jharkhand

Ushnagudam: Andhra Pradesh

Mannargudi: Tamil Nadu

VarKala: Kerala

Few unnamed hot water springs of Andaman and Nicobar

Taxonomy and identification of microorganisms

Taxonomy is synonym of systematics or biosystematics and is

traditionally divided into three parts: (i) Classification, i.e., the

orderly arrangement of organisms into taxonomic groups on

the basis of similarity; (ii) Nomenclature, i.e., the labeling of

the units defined and (iii) Identification of unknown organisms,

i.e., the process of determining whether an organism belongs

to one of the units defined or not. There are three groups of

taxonomic methods: Numerical Taxonomy, Chemical

Taxonomy and Molecular Taxonomy here the most accepted

method is used to study the bacterial strains from the Taptapani

hot water spring.

Molecular taxonomy and 16S rRNA sequencing

The breakthrough formulation was reached by Carl Woese

during the 1970’s when the ribosomal RNA turned out to be

an excellent evolutionary chronometer. Ribosomal RNA is an

ancient molecule, functionally constant, universally distributed

and moderately well conserved across broad phylogenetic

distances (Madigan et al., 1997). More over there is no

evidence of lateral gene transfer of rRNA genes between

different species; therefore, rRNA genes can bring true

information regarding evolutionary relationships (Pace, 1997).

The 16S rRNA molecule has several advantages like some

Figure 1: Rooted universal phylogenetic tree as determined by

comparative analysis of ribosomal genes sequences. The data supports

the discrimination of three domains, two of which contain

prokaryotic representatives (Bacteria and Archaea). The root

represents the position of a suspected universal ancestor of all cells.

Dashed lines indicate phylogenetic groups which are exclusively

thermophilic or contain few thermophilic representatives (modified

from Madigan et al. 1997). Morphometric characterization can not

determine the evolutionary relationships between the different

microbial groups therefore microbial systematics is now based on

nucleic acid sequences

Figure 2: Relation of temperature and growth rates for a typical

Psychrophilic, Mesophilic, Thermophilic and Hyperthermophilic

microorganism. The respective optimal growth temperatures Topt

are

indicated on the graph (modified from Madigan et al. 1997).

Gro

wth

Rate

-10 0 10 20 30 40 50 60 70 80 90 100 110 120

Temperature (ºC)

PsychrophilesFlovobacterium sp.

MesophilesEscherichia coli

ThermophilesBacillus stearothermophilus

HyperthermophilesPyrodictium brockii

105ºC

60ºC

37ºC

13ºC

CHARACTERIZATION OF THERMOPHILES

136

Figure 3: Geothermal provinces of India. As deduced by geotectonic,

geothermal and terrestrial heat flow data.Distribution of thermal

spring localities (Ravi Shankar 1988, Ravi Shankar et al., 1991) are

shown by solid dots. Son-Narmada-Tapti lineament is represented

by thick broken line

Figure 4: Flowchart showing the characterization of the microbial

diversity by 16S rRNA analysis

regions of the gene are universally conserved and suitable for

phylogenetic studies of distantly related organisms. The other

regions are semi-conserved and useful for the analysis of

phylogenetic relationship between phyla and families;

variable and hyper-variable regions in the 16S rRNA enable

us to discriminate between organisms belonging to the same

genus or even between species, although not between strains

within the same species (Amann et al., 1995). ARDRA, DGGE,

TGGE, T-RFLP are the recent techniques in the field of

microbial systematics. The amplified rDNA restriction analysis

(ARDRA) is a widely used method for the microbial diversity.

ARDRA analysis can identify strains at the genus/ species level

and is faster than 16S rDNA sequencing, therefore it is useful

for the analysis of large number of samples. DGGE (Denaturing

gradient gel electrophoresis) and TGGE (Temperature gradient

gel electrophoresis) of PCR amplified ribosomal DNA is also

used for microbial typing. Terminal restriction fragment length

polymorphism (T-RFLP) involves tagging one end of PCR

amplicons through the use of fluorescent molecule attached

to a primer. The amplified product is then cut with a restriction

enzyme. Terminal restricted fragments (TRF) are separated by

electrophoresis and visualized by excitation of the

fluorochrome and these fingerprints represent the species

composition of the communities in the metagenomic DNA

sample. Metagenomics is an emerging field in which the power

of genomic analysis (the analysis of the entire DNA in an

organism) is applied to entire communities of microbes,

bypassing the need to isolate and culture individual microbial

species. Metagenomic techniques provide researchers to

access millions of microbes that have not previously been

AMRITA K. PANDA et al.,

studied and it transcends the limitations of classical genomics

and microbiology. The adaptive radiation of thermophilic

proteins attributes the greater stability to the thermophilic

proteins by greater hydrophobicity, better packing, deletion

or shortening of loops, smaller and less numerous cavities,

increased surface area buried upon oligomerization, amino

acid substitutions within and outside the secondary structure,

increased occurrence of proline residues, decreased

occurrence of thermolabile residues, increased helical

content, increased polar surface area, better hydrogen

bonding and better salt bridges ( Kumar et al., 2000 and Rainer,

1981). Thermophilic proteins exhibit many structural

modifications, the most consistent modifications are surface

loop deletion, increased occurrence of hydrophobic residues

with branched side chains and an increased proportion of

charged residues at the expense of uncharged polar residues

(Sandeep Kumar et al., 2001). Strategic exchange of various

amino acids like prolines in β turns, Alanine is preferred over

Tyrosine, whereas in others Valine or Glycine is preferred

over Isoleucine in thermophiles and Lysine is preferred in

Archaea but not in Eubacteria (Trivedi et al., 2006). The

percent of glutamate (E) and lysine (K) increased in

thermophiles proteomes and the percent of glutamine (Q)

and histidine (H) decreased. There are reports that the higher

density of packing in hyperthermophilic proteins is also

reflected in the increased number of hydrogen bonds per

residue and in the involvement of 62% of residues into

elements of secondary structure compared with 39 - 40% in

mesophilic proteins (Berezovsky and Shakhnovich, 2005).

Studies have been made on statistical analysis of preferred

amino acids of thermophilic and mesophilic enzymes such

as DNA polymerase 1, glyceraldehydes - 3 - phosphate

137

Figure 5: Mixed plates at 10-5 serial dilution

dehydrogenase, ferridoxin and malate dehydrogenase

(Santosh Kumar, 1998).

Few Industrially important thermozymes

DNA Polymerases (EC 2.7.7.7) Amylolytic enzymes (EC

3.2.1.1), Xylanases (EC 3.2.1.8), Cellulases (EC 3.2.1.4),

Chitinases (EC 3.2.1.14), Proteases (EC 3.4.21 .19), Lipases

(EC 3.1.1.3), Nitrile-degrading enzymes (EC 4.2.1.84)

Other extremozymes

There are many other thermozymes isolated fromthermophiles; the first thermostable ligase was discovered inThermus thermophilus HB8, thermostable type I pullulanasefrom Thermus caldophilus and Fervidobacteriumpennavorans, type II pullulanases from Pyrococcus woeseiand Pyrococcus furiosus, thermostable glucose isomerasefrom Thermus maritime, solvent-stable esterases are formedby the extreme thermophilic bacterium Clostridiumsaccharolyticum and thermostable dehydrogenases fromThermus litoralis have been identified.

MATERIALS AND METHODS

Description of the site

Taptapani hot water spring (19º30' 0N latitude, 84º24' 0E

longitude and 1053 feet altitude) is located in Ganjam District

of the state Odisha, India. It is 56 km from the silk city Behrampur

and 190km from the state capital Bubaneswar, Odisha.

Sample Collection

Water samples (200mL) were collected in sterile capped

collection bottles from the hot water spring. The bottles were

kept in thermos flask to minimize the loss in temperature during

their transportation to the laboratory.

Isolation of Thermophilic bacteria

Dilution plate method was used for the bacterial isolation.The water sample was serially diluted up to 10 -5 withautoclaved double distilled water (Garthright, 1998). Thediluted samples were cultured on thermus agar (ATCC 697)medium and incubated at 48ºC for preliminary screening ofthermophilic isolates. Bacterial growth on each Petriplate wasobserved over next three days.

Culture maintenance and preservation of isolates

All well separated individual colonies were transferred to

nutrient agar slants, cultured, sub-cultured and maintained in

the laboratory. The glycerol stock cultures of potential strains

stored at -20ºC for long term storage and the cultured nutrient

agar plates were preserved at 4ºC.

Phenotypic Characterization

Colony morphology, Gram staining, Examination of

endospores.

Biochemical Characterization

All the biochemical tests performed by KB002 HiAssorted

Biochemical test kit of Himedia Laboratories, Indole, Methyl

Red and voges-Proskauer test, Starch and Gelatin liquefaction

as per (Conn et al., 1957) Catalase (Smibert and Krieg, 1994),

Oxidase (Tarrand and Groschel, 1982), Lipid, Casein

hydrolysis tests (Cappuccino and Sherman, 2007) followed

by Whole Cell Protein analysis by Sodium Dodecyl Sulphate-

Figure 7: Gram stain Photographs

of three potent isolates

AK-P3

AK-P1 AK-P2

Figure 6: Quadrant streaking

photograph of (a) AKP2, (b) AKP4

and (c) AKP5

a b

c


138

Polyacrylamide Gel Electrophoresis (SDS-PAGE). Quantitative

estimation of enzyme activity (i) Estimation of lipase activity (b)

Enzyme assay Estimation of amylase activity Estimation of Total

Protein in culture broth Temperature stability, pH stability,

Culture conditions for maximal production of lipase (a) Carbon

source (b) Nitrogen source.

Purification of the enzyme lipase

(i) Ammonium sulphate fractionation and dialysis

Estimation of molecular weight

Denaturing polyacrylamide gel electrophoresis (SDS-PAGE,

12%) was used to check the protein purity and determine the

molecular mass of the purified enzyme (Laemmli, 1970).

Zymography was done as per (Gupta et al., 2006) with minor

modifications.

Molecular Characterization and Lipase gene amplification

DNA preparation and PCR amplification

Genomic DNA was extracted from three potential isolates using

Chromous Genomic DNA isolation kit (RKT09). Each genomic

DNA used as template was amplified by PCR with the aid of

16SrDNA primers.

PCR mixture

100mg template DNA; 3 U Taq DNA polymerase; 10X Taq

DNA Polymerase assay buffer dNTPs (2.5 mM each) ; 400 ng

(each) of the primer (Volume made up to 100μL with sterile

distilled water).

PCR Reaction programme

(Thermal cycler ABI 2720): Denaturation at 94ºC for 5 min;

Denaturation at 94ºC for 30 sec, Annealing at 55ºC for 30

sec; Extension at 72ºC for 2 min; Go to step 2 Repeat up to 34

cycles. Final extension at 72ºC for 5min.

16S rRNA sequencing and data analysis

Sequencing analysis was performed on a 1500 bp PCR

product. The sequence analysis was performed using the ABI

3130 genetic analyzer and Big Dye Terminator version 3.1

cycle sequencing kit. The three 16SrRNA sequences were

aligned and compared with other 16SrRNA genes in the Gen

Bank by using the NCBI Basic Local alignment search tools

BLAST n program (http://www.ncbi.nlm.nih.gov/BLAST). The

16SrRNA gene sequences have been deposited to Genbank

using BankIt submission tool.

RESULTS AND DISCUSSION

The present study on thermophiles of Taptapani hot water

spring, Orissa, India yielded many significant findings with

respect to the biochemical nature, commercial utility,

systematic position of the strains characterized at molecular

level. The present investigation is the first report on the

thermophiles of this hot water spring at molecular level. The

microbes were cultured, sub cultured and their culture

conditions were optimized for better growth and production

of required enzyme at their best (Panda and Bisht, 2009). The

significant results are summarized under following headings

and subheadings.

Bacterial growth


Figure 9: Catalase test reactions

of potential isolates

AK-P1 AK-P1

AK-P1

Figure 8: Biochemical reaction tests for potential isolates; Citrate

utilization; Lysine utilization; Ornithine utilization; Urease; Pheny-

lalanine deamination; Nitrate reduction; H2S production; Glucose;

Adonitol; Lactose; Arabinose; Sorbitol

1 2 3 4 5 6 7 8 9 10 11 12

Control

AK-P1

AK-P2

AK-P3

Strain Name Lipase Amylase Protease

Substrate

Olive oil Starch Skim Milk

AKP1 + + + - -

AKP 2 (AK-P1) + + + + + +

AKP 3 - + -

AKP 4(AK-P2) + + + + + + + +

AKP 5(AK-P3) + + + + + + +

AKP 6 - - +

AKP 7 + + - -

AKP 8 - - -

AKP 9 - - -

AKP 10 - - -

AKP 11 -*

-

AKP 12 - - -

AKP 13 - -*

AKP 14 -*

-

AKP 15 - -*

Table 1: Extra-cellular enzyme profile of fifteen thermophilic isolates

+ Positive result; - Negative result; *

weak

139

Figure 10: Lipid hydrolysis zone

of AKP 6 and AKP 1


of AKP2 and AKP 3


of AKP4 and AKP 5

Figure 13: Lipid hydrolysis

zone of AKP7 and AKP8

Figure 14: Starch hydrolysis zone of (a) AKP5 (b) AKP4

a b

(i) Primary screening on selective media

A large number of intermixed bacterial colonies appeared

when low dilution samples were plated on petriplatess. On

the other hand very few or number of colonies were detected

in case of very high dilutions. Therefore appropriate plates

were selected for the sample in which sufficiently high numbers

of discrete colonies were present. Serial dilutions were made

up to 10-5.

(ii) Selection of appropriate strain

The strains were selected on the basis of the clearing zonearound the colonies on Tributyrin agar, Starch agar and Milkagar plates, five isolates for lipase, five for amylase and five forprotease were selected. All these isolates were streaked on

Figure 15: Whole-cell Protein pattern (WCP) obtained by SDS-PAGE

electrophoresis of seven thermophilic isolates; WCP of AKP1; WCP

of AKP2; WCP of AKP3; WCP of AKP4; 5WCP of AKP5; 6Protein

Molecular weight marker; (GeNei, PMWB1); 7WCP of AKP6; 8WCP

of AKP7

Figure 16 and 17: (16) Electrophoregram showing Genomic DNA of

three potential isolates; Lane 1: Genomic DNA from AK-P1, Lane 2:

Genomic DNA from AK-P2; Lane 3: Genomic DNA from AK-P3;

(17) PCR amplification of ~1.5kb 16S rDNA fragment of potential

isolates; Lane 1: 500 bp DNA ladder, Lane 2: Amplified 16S rDNA

from AK-P1; Lane 3: Amplified 16S rDNA from AK-P2, Lane 4:

Amplified 16S rDNA from AK-P3; The genomic DNA isolated from

each potential isolate was used as template DNA and amplified by

using consensus 16S rDNA primers having the following sequence

5’→→→→→3’

16 17

Figure18: Blue white colony screening for recombinant clones


140

Nutrient Agar slant and stored at 4ºC for further studies.Glycerol stocks of potent isolates were also prepared and stored

at -20ºC. The isolates AKP1, AKP2, AKP4, AKP5 and AKP7 are

lipase producers; AKP2, AKP3, AKP4, AKP5 and AKP11 are

amylase producers; AKP2, AKP4, AKP5, AKP6 and AKP 13

are protease producers. Out of these fifteen isolates seven

strains have shown the enzyme activity and rest eight were

not the enzyme producers (Table 1).

(iii) Proteotyping of enzyme producing thermophilic strains

(iv) Molecular phylogenetics

16S rRNA gene sequence analysis


16S ribosomal RNA gene sequence of AK-P1

1 gggccggtgc ggcaggcttt aacacatgca agtcgagcgg gggaaggtag cttgctaccg

61 gacctagcgg cggacgggtg agtaatgctt aggaatctgc ctattagtgg gggacaacat

121 ctcgaaaggg atgctaatac cgcatacgtc ctacgggaga aagcagggga tcttcggacc

181 ttgcgctaat agatgagcct aagtcggatt agctagttgg tggggtaaag gcctaccaag

241 gcgacgatct gtagcgggtc tgagaggatg atccgccaca ctgggactga gacacggccc

301 agactcctac gggaggcagc agtggggaat attggacaat ggggggaacc ctgatccagc

361 catgccgcgt gtgtgaagaa ggccttatgg ttgtaaagca ctttaagcga ggaggaggct

421 actctagtta atacctaggg atagtggacg ttactcgcag aataagcacc ggctaactct

481 gtgccagcag ccgcggtaat acagagggtg cgagcgttaa tcggatttac tgggcgtaaa

541 gcgtgcgtag gcggcttatt aagtcggatg tgaaatcccc gagcttaact tgggaattgc

601 attcgatact ggtgagctag agtatgggag aggatggtag aattccaggt gtagcggctg

661 aaatgcgtag agatctggag gaataccgat ggcgaaggca gccatctggc ctaatactga

721 cgctgaggta cgaaagcatg gggagcaaac aggattagat accctggtag tccatgccgt

781 aaacgatgtc tactagccgt tggggccttt gaggctttag tggcgcagct aacgcgataa

841 gtagaccgcc tggggagtac ggtcgcaaga ctaaaactca aatgaattga cgggggcccg

901 cacaagcggt ggagcatgtg gtttaattcg atgcaacgcg aagaacctta cctggccttg

961 acatactaga aactttccag agatggattg gtgccttcgg gaatctagat acaggtgctg

1021 catggctgtc gtcagctcgt gtcgtgagat gttgggttaa gtcccgcaac gagcgcaacc

1081 cttttcctta cttgccagca tttcggatgg gaactttaag gatactgcca gtgacaaact

1141 ggaggaaggc ggggacgacg tcaagtcatc atggccctta cggccagggc tacacacgtg

1201 ctacaatggt cggtacaaag ggttgctaca cagcgatgtg atgctaatct caaaaagccg

1261 atcgtagtcc ggattggagt ctgcaactcg actccatgaa gtcggaatcg ctagtaatcg

1321 cggatcagaa tgccgcggtg aatacgttcc cgggccttgt acacaccgcc cgtcacacca

1381 tgggagtttg ttgcaccaga agtagctagc ctaactgcaa agagggcggt taccccaccg

1441 gtgggccccg agagc


1 tcctgggcgg gcgtgcctaa tacatgcaag tcgagcgagt cccttcgggg gctagcggcg

61 gacgggtgag taacacgtag gcaacctgcc cgtaagctcg ggataacatg gggaaactca

121 tgctaatacc ggatagggtc ttctctcgca tgagaggaga cggaaaggtg gcgcaagcta

181 ccacttacgg atgggcctgc ggcgcattag ctagttggtg gggtaacggc ctaccaaggc

241 gacgatgcgt agccgacctg agagggtgac cggccacact gggactgaga cacggcccag

301 actcctacgg gaggcagcag tagggaattt tccacaatgg acgaaagtct gatggagcaa

361 cgccgcgtga acgatgaagg tcttcggatt gtaaagttct gttgtcagag acgaacaagt

421 accgttcgaa cagggcggta ccttgacggt acctgacgag aaagccacgg ctaactacgt

481 gccagcagcc gcggtaatac gtaggtggca agcgttgtcc ggaattattg ggcgtaaagc

541 gcgcgcaggc ggctatgtaa gtctggtgtt aaagcccggg gctcaacccc ggttcgcatc

601 ggaaactgtg tagcttgagt gcagaagagg aaagcggtat tccacgtgta gcggtgaaat

661 gcgtagagat gtggaggaac accagtggcg aaggcggctt tctggtctgt aactgacgct

721 gaggcgcgaa agcgtgggga gcaaacagga ttagataccc tggtagtcca cgccgtaaac

781 gatgagtgct aggtgttggg ggtttcaata ccctcagtgc cgcagctaac gcaataagca

841 ctccgcctgg ggagtacgct cgcaagagtg aaactcaaag gaattgacgg gggcccgcac

901 aagcggtgga gcatgtggtt taattcgaag caacgcgaag aaccttacca ggtcttgaca

961 tcccgctgac cgtcctagag atagggcttc ccttcggggc agcggtgaca ggtggagcat

1021 ggttgtcgtc agctcgtgtc gtgagatgtt gggttaagtc ccgcaacgag cgcaaccctt

1081 atctttagtt gccagcattc agttgggcac tctagagaga ctgccgtcga caagacggag

1141 gaaggcgggg atgacgtcaa atcatcatgc cccttatgac ctgggctaca cacgtgctac

1201 aatggctggt acaacgggaa gctagctcgc gagagtatgc caatctctta aaaccagtct

1261 cagttcggat tgcaggctgc aactcgcctg catgaagtcg gaatcgctag taatcgcgga

1321 tcagcatgcc gcggtgaata cgttcccggg ccttgtacac accgcccgtc acaccacggg

1381 agtttgcaac acccgaagtc ggtgaggtaa ccgcaaggag ccagccgccg aaggtgggag

1441 aggt

The thermophilic isolates AKP2, AKP4 and AKP5 were found

very promising in terms of the production of all the three i.e.

lipase, protease and amylase enzymes therefore the isolates

AKP2, AKP4 and AKP5 were selected for further characterization

at molecular level. These isolates were named further as AK-P1,

AK-P2 and AK-P3 respectively and deposited to the International

database National Centre for Biotechnology Information (NCBI)

with the same nomenclature. The Genomic DNA was extracted

from all the three potential isolates and separated on 0.8%

agarose gel. The agarose gel product shows high degree of

fluorescence and good quality of intact DNA bands which is an

indicator of purified DNA (Fig. 16).

141

0

5

10

15

20

25

30

35

40

45

50

4 4 5 5 6 6 7 7 8

AK -P1AK -P2AK -P3

Figure 19: Effect of temperature on lipase activity. The optimum pH

for lipase production was determined at various pH i.e. 5 to 11. The

isolate AK-P1 gave maximum enzyme activity at pH 10 (27 U/mL),

AK-P2 has shown maximum activity at the same pH 10 (38 U/mL)

and isolate AK-P3 shown the optimum activity at pH 8 (15 U/mL).

All these three potential strains provided better activity in alkaline

pH (Fig. 20)

Temperature

En

zym

e a

cti

vit

y (μ

/mL)

Figure 20: Effect of pH on lipase activity. Growth curve for the

bacterial isolates AK-P1, AK-P2 and AK-P3

05

10

1520

25

3035

40

3 5 7 9 11

AK -P1 AK - P2 AK -P3

pH

En

zym

e a

cti

vit

y (μ

/mL)

0

2

4

6

8

10

12

14

16

0 20 40 60 80

0

2

4

6

8

10

12

enzyme activity Dry wt. of cell

Figure 21: Growth curve and lipase production by AK-P1

Incubation period (h)

En

zym

e a

cti

vit

y (μ

/mL)

Dw

t cell

s (m

g/m

L)


1 tcgagctagg cgggctgcct aacaaaatgc agtcgaacga tcccttcggg gatagtggcg

61 cacgggtgcg taacgcgtgg gaacctgccc ttaggttcgg aataactcag agaaatttga

121 gctaataccg gataatgtct tcggaccaaa gatttatcgc ctttggatgg gcccgcgttg

181 gattagctag ttggtggggt aaaggcctac caaggcgacg atccatagct ggtctgagag

241 gatgatcagc cacactggga ctgagacacg gcccagactc ctacgggagg cagcagtggg

301 gaatattgga caatgggcga aagcctgatc cagcaatgcc gcgtgagtga tgaaggcctt

361 agggttgtaa agctctttta cccgggatga taatgacagt accgggagaa taagccccgg

421 ctaactccgt gccagcagcc gcggtaatac ggagggggct agcgttgttc ggaattactg

481 ggcgtaaagc gcacgtaggc ggccttttaa gtcaggggtg aaatcccggg gctcaacccc

541 ggaactgccc ttgaaactgg gaggctagaa tcttggagag gcgagtggaa ttccgagtgt

601 agaggtgaaa ttcgtagata ttcggaagaa caccagtggc gaaggcgact cgctggacaa

661 gtattgacgc tgaggtgcga aagcgtgggg agcaaacagg attagatacc ctggtagtcc

721 acgccgtaaa cgatgataac tagctgtccg ggttcatgga acttgggtgg cgcagctaac

781 gcattaagtt atccgcctgg ggagtacggt cgcaagatta aaactcaaag gaattgacgg

841 gggcctgcac aagcggtgga gcatgtggtt taattcgaag caacgcgcag aaccttacca

901 gcctttgaca tcctaggacg gcttctggag acagattcct tcccttcggg gacctagaga

961 caggtgctgc atggctgtcg tcagctcgtg tcgtgagatg ttgggttaag tcccgcaacg

1021 agcgcaaccc tcatccttag ttgccatcat tcagttgggc actttaagga aactgccggt

1081 gataagccgg aggaaggtgg ggatgacgtc aagtcctcat ggcccttaca ggctgggcta

1141 cacacgtgct acaatggcaa ctacagtggg cagctatccc gcgagggtgc gctaatctcc

1201 aaaagttgtc tcagttcgga ttgttctctg caactcgaga gcatgaaggc ggaatcgcta

1261 gtaatcgcgg atcagcatgc cgcggtgaat acgttcccag gccttgtaca caccgcccgt

1321 cacaccatgg gagttggatt cacccgaagg cagtgcgcta accgcaagga ggcagctgac

1381 cacggtggta cgcgggggg

16S forward Primer: 5'-AGAGTRTGATCMTYGCTWAC-3'.

16S reverse Primer: 5'-CGYTAMCTTWTTACGRCT-3'.

The amplified fragment obtained approximately was of 1.5

Incubation period (h)

En

zym

e a

cti

vit

y (μ

/mL)

Dw

t cell

s (m

g/m

L)


0

5

10

15

20

25

30

35

40

0 20 40 60 80

0

5

10

15

2025

30

35

40

45

50

Figure 22: Growth curve and lipase production by AK-P2


142

1

0.8

Dex

trose

Glu

cose

Sucr

ose

Arabi

nose

Malto

se

Galac

tose

Xylos

e

Fruc

tose

Lactos

e

0.6

0.4

0.2

0

Figure 24: Effect of various carbon sources on the production of

lipase by AK-P2

Kbp size (Fig. 17). The PCR amplified fragment was eluted and

cloned into suitable vector which was further transformed

into competent DH5α strain. Selection of the recombinant



0

2

4

6

8

10

12

0 20 40 60 80

0

5

10

15

20

25

30

Figure 23: Growth curve and lipase production by AK-P3Incubation period (h)

En

zym

e a

cti

vit

y (μ

/mL)

Dw

t cell

s (m

g/m

L)

Carbon source

Lip

ase

acti

vit

y (μ

/mL)

1

0.8

Trib

utyr

in

Trio

lein

Palm

oil

Sunf

lower

oil

Cocon

ut o

il

Soya

bean

oil

Oliv

e oi

l

0.6

0.4

0.2

0

Figure 26: Effect of various substrate on the production of lipase by

AK-P2

Substrate

Lip

ase

acti

vit

y (μ

/mL)

1

0.8

Amm

onium

chlo

ride

Amm

onium

Nitr

ate

Ure

a

Amm

onium

sulp

hate

Amm

onium

hepta

moly

bdate

Amm

onium

Fer

ricsu

lfate

Sodiu

m n

itrat

e

0.6

0.4

0.2

0

Figure 25: Effect of various nitrogen sources on the production of

lipase by AK-P2

Nitrogen source

Lip

ase

acti

vit

y (μ

/mL)

Figure 27: SDS-PAGE analysis of purified lipase lane A1 molecular

weight marker and A2 ammonium sulphate fraction, Lane B1 and

B3 molecular weight markers; lane B2 purified lipase

66

43

29

66

43

29

66

43

29

14.3

97.4

20.1

14.3 14.3

70KDa

1 2

A B1 2 3

Figure 28: Native PAGE gel of purified lipase; (a) Gel stained with

Coomassie brilliant blue (Lane 2: purified lipase, Lane 3: nonlipolytic

purified protein); (b) Zymogram showing lipase activity against

tributyrin (Lane 2: Yellow band, lane 3: There is no change in colour)

Purified lipase

Non lipolytic protein

Hydrolytic band

143

clones was done on X-gal IPTG Ampicillin agar medium as

blue and white colony screening (Fig. 18). Colony PCR was

performed to examine the presence of PCR amplified 16S

rRNA gene from white recombinant clones. The gel eluted

colony PCR product was subjected to sequencing reaction by

ABI 3130 genetic analyzer and Big Dye Terminator version

3.1 cycle sequencing kit. The present investigation is focused

on one specific enzyme i.e. lipase due to its very high demand

in various industries like food, leather, detergent, cosmetics,

perfumery and bioremediation.

Lipase production, purification, characterization and

Amplification of lipase gene

Lipases (triacylglycerol acylhydrolases) belong to the class ofserine hydrolases and not need any co-factor. The naturalsubstrates of lipases are triacylglycerols having very lowsolubility in water. Under natural conditions lipases catalyzethe hydrolysis of ester bonds at the interface between aninsoluble substrate phase and the aqueous phase in whichthe enzyme is dissolved. Lipases catalyze the hydrolysis oftriglycerides into diglycerides, monoglycerides, glycerol andfatty acids. Lipases have attracted much attention during thelast decade due to the diversity of their applications. Lipaseproduction from a variety of bacteria, fungi and actinomyceteshas been studied by various scientists (Sztajer et al., 1988,Rapp and Backhaus 1992; Kulkarni and Gadre 2002 andBisht and Panda, 2011).

Figure 29: Amplified Lipase gene (200bp) of AK-P2, lane 1; PCR

amplified fragment from AK-P2 genomic DNA by lipase prospecting

primers, lane 2; 100 bp DNA ladder

200 bp

Lipase Production by AK-P1, AK-P2 and AK-P3

The enzyme activity was measured as amount of enzyme

required to liberate one micromole equivalent fatty acid per

ml/min. The isolate AK-P1 gave maximum enzyme activity at

55ºC (45 U/mL), AK-P2 has shown maximum activity at 60ºC

(45 U/mL) and AK-P3 gave optimum activity at 55ºC (25 U/

mL). Out of these three potential strains the isolate AK-P2 hasshown better activity at the temperature range 60 – 65ºC witha maximum activity at 60ºC (Fig. 19). Though the enzymeproduced by the isolate AK-P3 is a thermostable enzyme but itshows less activity at high temperature. The optimumtemperature for lipase activity produced by other thermophilicBacillus stearothermophilus, Bacillus thermocatenletus andBacillus thermoleovorans ID-1 was 68ºC, 60ºC-70ºC and70ºC-75ºC respectively (Haki and Rakshit, 2003). Similarlymany other reports are also available on other lipases ofAcinetobacter sp. (Kok et al., 1996, Ahmed et al., 2010, Liuand Tsai 2003, Barbaro et al., 2001). Scanty of reports areavailable on the lipase producing ability of Brevibacillus sp.and Porphyrobacter sp. The present investigation added newinformation to the knowledge by reporting lipase producingPorphyrobacter and Brevibacillus from less known Taptapanihot water spring, Orissa, India (Panda and Bisht, 2009 and

Table 3: Biochemical tests of potential isolates

Biochemical tests Results

AK-P1 AK-P2 AK-P3 Carbo

hydrate

fermentation

Glucose +ve -ve -ve

Adonitol -ve -ve +ve, weak

Lactose -ve -ve -ve

Arabinose -ve -ve +ve, weak

Sorbitol -ve -ve +ve

Citrate utilization -ve -ve -ve

Indole -ve -ve +ve

Motility -ve -ve +ve

Nitrate reduction +ve +ve -ve

Lysine utilization -ve -ve -ve

Ornithine utilization -ve -ve -ve

Phenylalanine deamination -ve -ve -ve

Urease production -ve -ve -ve

H2S production -ve -ve +ve

Catalase +ve +ve +ve, weak

Oxidase -ve -ve -ve

Methyl Red -ve -ve -ve

Voges-Proskauer -ve +ve -ve

Starch hydrolysis +ve +ve +ve

Gelatin liquefaction -ve -ve -ve

+ Positive result; - Negative result

Table 2: Colony, Cellular morphology and Gram reaction of potential isolates

Strain Colony size Colony morphology Gram reaction Cellular morphology

(mm)

AK-P1 1 Circular, entire edge, -ve Coccoid

Smooth surface

AK-P2 2 Irregular and spreading +ve Rods

Raised margin

AK-P3 1 Circular, entire edge, -ve Ovoid to short rods

Smooth surface,

Orange pigmented


144

Table 4: the run down of lipase from the isolate AK-P2

Fraction Total Protein (mg) Total Units Specific activity Yield (%) Fold of Purification

(U/mg of protein)

Supernatant 24 17.80 0.74 100 1.00

Ammonium sulphate 17 13.20 0.77 74.1 1.04

Phenyl sepharose 6 12.60 2.10 70.7 2.83

Table 5: Conversion of enzyme unit /mL based on pNP liberation

Fraction pNP liberated Enzyme units/mL

in μM/15 min

Supernatant 31 0.445

Ammonium sulphate 23 0.330

1st elution with 50% EG 5 0.071

2nd elution with 50% EG 7 0.100

3rd elution with 50% EG 22 0.310

1st elution with 80% EG 5 0.071

2nd elution with 80% EG 4 0.057

3rd elution with 80% EG 4 0.057

Bisht and Panda, 2011). Reports are there that Brevibacillusborstelensis strain MH301 produces hydantoinase andcarbamoylase enzymes which are the key biocatalysts for theproduction of optically pure amino acids from dl-5-substitutedhydantoins (Yanzhen et al., 2009). In the present study ofisolate AK-P2 have shown promising results for the productionof lipase and indicates its varied commercial applications.The growth curves plotted for all three isolates revealed themaximum production of enzyme by AK-P1, AK-P2 and AK-P3at 40, 30 and 40h of growth respectively (Fig. 21, 22 and 23).

Purification of AK-P2 Lipase

The purification of the thermostable lipase was confined to

the lipase produced by the isolate AK-P2 on its merit as per the

protocol (Nawani and Kaur, 2000) with minor modifications.

The culture supernatant was concentrated by ammonium

sulphate precipitation followed by subsequent hydrophobic

interaction chromatography on Phenyl Sepharose CL- 4B

column that resulted to the elution of the lipase with maximum

activity in 50% ethylene glycol fraction. A single major band

with molecular mass of 70kDa was observed after its separation

on SDS-PAGE containing 12% resolving and 4% stacking gel,

stained with Coomassie brilliant blue (Fig. 27 A and B). In the

(Fig. 27 A) left to right lane number 1 is a medium range protein

molecular weight marker and lane number 2 is the crude

ammonium sulfate fraction of lipase enzyme and in the (Fig.

27 B) lane number 1 is the broad range Protein molecular

weight marker, lane number 2 purified lipase having the size

70 kDa and lane number 3 medium range Protein molecular

weight marker. Both medium and broad range protein

molecular weight markers were used simultaneously to

determine the molecular weight of the purified lipase with

higher accuracy.

The lipase gene from the isolate AK-P2 was amplified (Fig. 29)

using highly degenerate consensus primers to the oxyanion

hole (Jaeger et al., 1994) and active-site regions of lipase genesto amplify fragments of putative lipases with the following

lipase prospecting primers OXF1 and ACR1.

1. OXF1 Lipase-prospecting primer CCYGT KGTSYTN

GTNCAYGG oxyanion hole

2.ACR1 Lipase-prospecting primer AGGCCNCCCAK

NGARTGNSC active site

Screening and isolation of three potential lipolytic bacteria,

their identification by morphological, biochemical and

molecular methods (16S rRNA sequencing), media optimization

for the production of lipase enzyme, purification of lipase

enzyme by hydrophobic ion exchange chromatography and

partial amplification of lipase gene were the major exercises of

this study. During the investigation it was observed that the

SDS-PAGE fingerprinting is also an ideal, economical and less

time consuming method for bacterial identification because

the cell surface proteins act as biochemical marker to

discriminate various bacterial strains. It was also concluded

from the present study that very less number of bacteria can

be isolated from an environmental sample by changing the

culture condition and media composition. The Taptapani hot

water spring may have many more commercially important

microbes’ special reference to enzyme industry. Production

of lipase by the isolate AK-P2 quantitatively is one of the

significant finding of this study and the commercial scale

optimization of various parameters needs to be addressed by

employing more recent techniques and equipments to

characterize and amplify the full length lipase gene and its

expression. Though the present investigation contains both

genomic and proteomic studies to achieve high degree of

accuracy in terms of characterization, industrial prospecting

of few thermophilic isolates those contributed interesting and

promising results. This investigation indicates that the

Taptapani hot water spring of South-Eastern India is a rich

source of many thermophilic bacteria and need to be explored

for the industrially important enzymes by employing meta-

genomics studies. Simultaneously three 16S rRNA gene

sequences with the accession number HM359120,

HM359119 and HM 359118 submitted to the NCBI,

Maryland, USA is an another relevant finding of the study

which is an addition to the information and knowledge to the

scientific world. The purification of lipase in various fractions

is summarized in Table 4. The Zymography (Fig. 28) was

carried out after separating the purified lipase on native PAGE

with 12% resolving gel to confirm the purity and activity of the

purified lipase from isolate AK-P2.

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