FIELD OF THE INVENTION
The present invention relates to a halotolerant and thermostable lipase from a bacterial halophile Staphylococcus arlettae JPBW-1 (MTCC 5589). The present invention also relates to a process for the production of halotolerant and thermostable lipase from Staphylococcus arlettae JPBW-1 (MTCC 5589).
BACKGROUND OF THE INVENTION
Enzymes play diversified functions in many aspects of everyday life including helping in digestion, the production of food and various industrial applications. Enzymes are biodegradable, show improved use of raw materials and decreased amounts of waste products. Extremophilic microorganisms are adapted to thrive at extreme temperatures, pH, salt concentrations or other extreme conditions. These microorganisms are seen as a promising source of enzymes robust enough for a number of demanding industrial processes. Enzymes that are active at elevated salt concentrations are beneficial for a large variety of processes because running processes at high salt concentrations have many advantages. The increase in salt concentration has a significant effect on the bioavailability and solubility of organic compounds. A considerable amount of effort has been dedicated to the identification of extracellular salt-tolerant enzymes for their use in biotechnological processes (e.g. hydrolysis of starch, laundry detergents, saline fermentation processes involved in the production of various protein rich foods, fish sauce fermentation, textile industry, etc.). Halophiles produce exozymes such as amylases, proteases, lipases and nucleases of potential commercial values. There has been growing interest in scientific research on salt tolerant enzymes derived from halophilic bacteria due to the potential industrial applications of these enzymes. It is generally believed and has been proven that many halophilic enzymes are polyextremophilic. These enzymes not only remain active and stable in high salt environments but are often also thermotolerant and alkaliphilic. These properties made halophilic enzymes attractive for various biotechnological applications as they would be able to catalyze reactions under harsh conditions typical of many industrial processes.

The enzymes produced by halophiles have developed particular features including adequate stability and solubility in high salt concentrations by acquiring a relatively large number of charged amino acid residues on their surfaces to prevent precipitation. Novel hydrolytic catalysts (enzymes) with better catalytic efficiency and specific properties suitable for special reaction conditions are in high demand. Lipases are one type of enzymes which are versatile biocatalysts that can act upon numerous different reactions. Unlike other hydrolases that work in aqueous phase, lipases are unique as they act at the oil/water interface. Besides being lipolytic, lipases also possess esterolytic activity and thus have a wide substrate range.
The current industrial applications of lipases involve food, dairy, pharmaceutical, detergent, textile, pulp and paper, animal feed and leather industry. However, most of the lipases currently in industrial use lack in activities at high temperature (90°C) and salt concentration, which could add not only additional value to the lipases but also broaden their activity horizons.
OBJECTS OF THE INVENTION
The main object of the present invention is to explore the isolation and identification of extremely halotolerant Staphylococcus arlettae JPBW-1 (MTCC 5589) from a rock salt mine.
Another object of the invention is to screen, partially purify and analyse preliminary activity analysis of the thermo-tolerant lipase of the present invention.
Yet another object of the present invention is to produce a thermo-tolerant lipase enzyme capable of retaining its activity at high temperature and salt concentration.
SUMMARY OF THE INVENTION
Accordingly, the present invention provides a halotolerant and thermostable lipase enzyme derived from Staphylococcus arlettae JPBW-1 (MTCC 5589), capable of operation at a salt

concentration in the range of 0 to 19% (w/v) NaCl and over a temperature range of 40°C to
90°C.
In one embodiment of the invention, the lipase is isolated from Staphylococcus arlettae by a
process comprising the steps of:
(i) dissolving soil sample collected from rock salt mines in sterile distilled water in
a ratio of 1:100 by weight followed by its serial dilution (10-1 to 10-5); (ii) plating the dilutions obtained in step (b) on Luria-Bertani agar (LBA); (iii) isolating the microbial colonies growing on LBA and individually streaking
Staphylococcus arlettae JPBW-1 (MTCC 5589) on LBA plates supplemented
with 19% (w/v) salt; (iv) inoculating Staphylococcus arlettae JPBW-1 (MTCC 5589) into 5 ml LB
medium and incubating with shaking at 200 rpm for 16 h at 37.0°C; (v) transferring 5 ml of the culture obtained in step (iv) to 45 ml of LB medium and
incubating again for 16 h at 37.0°C; (vi) transferring 50 ml of the culture obtained in step (v) to 450 ml of LB medium
and incubating with shaking at 200 rpm for 3 h at 37.0°C; (vii) pelleting the cells by centrifugation at 5,000 rpm for 30.0 min. at 4.0°C, and
extracting the supernatant; (viii) precipitating the supernatant obtained in step (vii) with 20.0% (NH4)2S04 with
constant stirring at 4.0°C for 12h to obtain partially purified protein; (ix) recovering the partially purified protein obtained in step (viii) by centrifugation
at 5,000 rpm for 30.0-45.0 min. at 4.0°C; (x) precipitating the supernatant obtained in step (ix) with 60.0% (NH4)2S04 with
constant stirring at 4.0°C for 12h; (xi) recovering the protein by centrifuging the precipitate obtained in step (x) at
5,000 rpm for 30.0-45.0 min. at 4.0°C; (xii) dissolving the precipitate obtained in step (xi) in 0.75 ml of 50.0 mM phosphate
buffer (pH 7.0) followed by dialysis with 10.0 kD membrane to obtain a
partially purified lipase enzyme from Staphylococcus arlettae JPBW-1 (MTCC
5589) with a 16s rRNA sequence which is 99% identical (Coverage 100%) with
16S rRNA gene sequence of Staphylococcus arlettae strain CM 18, (Accession
No. GUl43791.1).

In another embodiment of the invention, the lipase is translated from a nucleotide sequence of SEQ ID 1 cloned by a process comprising the steps of:
a) isolating genomic DNA from Staphylococcus arlettae by Phenol-Chloroform method;
b) designing primers for PCR amplification of lipase gene from the genomic DNA obtained in step (a) from conserved sequence regions of lipase genes of other species to obtain forward primer of SEQ ID 3 and reverse primer of SEQ ID 4;
c) amplifying lipase gene from the genomic DNA obtained in step (a) by PCR using the primers obtained in step (b);
d) cloning the amplified products of step (c) in pGEM-T vector (Promega) and transferring to DH5a chemical-competent cells of E. coli for multiplication;
e) screening the transformants based on blue/white colonies observed on LB agar plates containing IPTG, X-gal and ampicillin;
f) setting up colony PCR of the transformed colonies that were picked randomly using forward primer of SEQ ID 5 and reverse primer of SEQ ID 6 followed by isolating transformed plasmids using AxyPrep™ Plasmid Miniprep Kit;
g) sequencing the isolated plasmids obtained in step (f) by sequence homology (BlastN) analysis to obtain an amplicon having sequence length of 317 bp and 97% identities to putative lipase (Protein id: AAW53417.1) of Staphylococcus epidermidis RP62A;
h) designing forward primer of SEQ ID 7 and reverse primer of SEQ ID 8 by taking 1000 bp upstream and 1000 bp downstream of the putative lipase gene (2046 base pairs) of Staphylococcus epidermidis RP62A followed by PCR amplification of the putative lipase gene using said primers of SEQ ID 7 and SEQ ID 8;
i) cloning the amplified products obtained in step (h) in pGEM-T vector (Promega), transferring to DH5a chemical-competent cells of E. coli for multiplication, and screening transformants based on blue/white colonies observed on LB agar plates containing IPTG, X-gal and ampicillin;

j) setting up colony PCR of the transformed colonies that were randomly picked
up from LB agar plates in step (i) using forward and reverse primers of SEQ
ID 7 and SEQ ID 8 respectively;
k) isolating transformed plasmids using AxyPrep™ Plasmid Miniprep Kit;
1) amplifying and sequencing the amplicon cloned step (i) in split parts using
internal primers designed from the putative lipase gene of Staphylococcus
epidermidis RP62A, said internal primers represented by SEQ ID 9 and 10
(Forward 5'-ATTTAGGTGACACTATAG-3' and Reverse 5'-
GCACACCACATAATGGTACA-3', SEQ ID 11 and 12 (Forward 5'-
CAAAACGTCAAATTCAAACA-3' and Reverse 5'-
GTTGTTTTAATCCCCATTGA-3'), SEQ ID 13 and 14 (Forward 5'-
AAAATACGCAGTGGGAGTAG-3' and Reverse 5'-
CGTCTCCCTTGTTGTACTTC-3'), SEQ ID 15 and 16 (Forward 5'-
GCATTACCTTGTTTTGAAGC-3' and Reverse 5'-
TAATACGACTCACTATAGGG-3'); m) purifying the PCR amplicons obtained in step (1) using AxyPrepTM PCR
clean up kit followed by its sequencing; n) compiling the sequencing data obtained in step (m) using DNA Star, to obtain sequence of lipase gene of Staphylocococcus arlettae of SEQ ID 1 having sequence length of 2046 bp and 99% identities to putative lipase gene of Staphylococcus epidermidis RP62A.
In another embodiment of the invention, translation of gene sequence of SEQ ID 1 yields a
681 amino acid long polypeptide of SEQ ID 2, having 98% identities with putative lipase
protein of Staphylococcus epidermidis RP62A.
In yet another embodiment of the invention, the lipase comprises the following
characteristics:
(i) Molecular Mass = 60KDa;
(ii) Michaelis constant (Km) = 20%;
(iii) Maximum reaction rate (Vmax) = 1666U/ml.
In still another embodiment of the invention, the lipase shows optimal activity at a temperature in the range of 50°C to 60°C.

In yet another embodiment of the invention, the lipase shows optimal activity over a pH range of 8 to 10.
In yet another embodiment of the invention, the lipase is used in post tanning operations in leather industry.
BRIEF DESCRIPTION OF DRAWINGS
The above-mentioned and other features and advantages of this present disclosure, and the manner of attaining them, will become more apparent and the present disclosure will be better understood by reference to the following description of embodiments of the present disclosure taken in conjunction with the accompanying drawings, wherein:
Fig. 1. Identification of lipase activity on LB agar plates containing olive oil and 12 % salt, after overnight incubation at 37°C. A fluorescent halos formed around the colony indicates the production of lipase.
Fig. 2. Effect of substrate on lipase activity produced by Staphylococcus arlettae obtained from rock salt mine.
Fig. 3. Activity of lipase in 20, 60 and 80 % fractions of (NH4)2 S04 precipitation.
Fig. 4. Effect of pH on lipase activity produced by Staphylococcus arlettae.
Fig. 5. Lipase activity produced by Staphylococcus arlettae at different temperatures
Fig. 6. Lipase activity produced by Staphylococcus arlettae in different salt concentrations.
Fig. 7. SDS-PAGE analysis of different fractions taken during the purification process of Lipase. Lane 1: 20% fraction; Lane 2: 60% fraction and Lane 3: 80% fraction.
Fig. 8. Kinetic parameters analysis of lipase produced from Staphylococcus arlettae.

DETAILED DESCRIPTION OF THE INVENTION
Before describing in detail embodiments that are in accordance with the present disclosure, it should be observed that the embodiments reside primarily in combinations of process steps.
In this document, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process or method does not include only those elements but may include other elements not expressly listed or inherent to such process or method. An element proceeded by "comprises ...a" does not, without more constraints, preclude the existence of additional identical elements in the process or method.
It is noted that, as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural references unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as "solely," "only" and the like in connection with the recitation of claim elements, or use of a "negative" limitation
Any embodiment described herein is not necessarily to be construed as preferred or advantageous over other embodiments. All of the embodiments described in this detailed description are illustrative, and provided to enable persons skilled in the art to make or use the disclosure and not to limit the scope of the disclosure, which is defined by the claims.
Lipases isolated from different sources have a wide range of properties depending on their sources with respect to positional specificity, fatty acid specificity, thermostability, pH optimum, etc. One of the important sectors which demand for lipases is leather industry, where lipases are used in post-tanning operations such as de-greasing and de-hairing. One of the advantage and suggested use of lipase isolated from Staphylococus arlettae in the present invention is its stability at a very high temperature (even at 90°C) and its activity at 19% salt concentration which makes this enzyme (lipase) a suitable candidate for application in post-tanning operation without a necessary step of removing all salt (NaCl) normally used in

tanning operation. The use of a lipase capable of working at high salt concentration is expected to save excessive use of water required in tanning of skin. Lipases also improve the production of hydrophobic (waterproof) leather. Other sectors that use lipases are in biochemical reactions to get finished economically viable products in food and pharmaceutical industries. One of the major applications of lipase in food industries is the production of fat-free products such as margarine.
The enzyme of the present invention was isolated from rock salt mine, Darang, Himachal Pradesh. To our knowledge this is the only rock salt mine in India.
Procedure for Production of Lipase from Staphylococcus arlettae JPBW-1 (MTCC
5589)
• The soil samples were collected using a quadrant method from different sites in the rock salt mine and pooled to represent the whole area. For isolation of bacterial strain, 1.0 g of soil was dissolved in 100 ml of distilled sterile water, serially diluted (10-1 to 10-5) and the dilutions were plated on Luria-Bertani agar (LBA).
• The microbial colonies growing on LBA were isolated and individually streaked on LBA plates supplemented with 19% (w/v) salt.
• Staphylococcus arlettae JPBW-1 (MTCC 5589) was inoculated into 5 ml LB medium, incubated with shaking at 200 rpm for 16 h at 37.0°C.
• The culture obtained (5 ml) in the previous step was transferred to 45 ml of LB medium and again grown for 16 h at 37.0°C.
• Further, 50 ml of the culture from the previous step was added to 450 ml of LB medium and incubated with shaking at 200 rpm for 3 h at 37.0°C.
• Cells were pelleted by centrifugation at 5,000 rpm for 30.0 min. at 4.0°C, and the supernatant was extracted.
• Partial protein purification was performed by total protein precipitation. Cell free broth (supernatant obtained in the previous step) was precipitated with 20.0% (NH4)2SO4 with constant stirring at 4.0°C for 12h. Protein was recovered by centrifugation at 5,000 rpm for 30.0-45.0 min. at 4.0°C.

• The supernatant was again precipitated with 60.0% (NH4)2SO4 with constant stirring at 4.0°C for 12h. Protein was recovered by centrifugation at 5,000 rpm for 30.0-45.0 min. at 4.0°C.
• The precipitate obtained was dissolved in 0.75 ml of 50.0 mM phosphate buffer (pH 7.0) followed by dialysis with 10.0 kD membrane.
• The enzyme solution obtained after dialysis was the partially purified enzyme (lipase) from Staphylococcus arlettae JPBW-1 (MTCC 5589).
• The identity of Staphylococcus arlettae was established through 16s rRNA sequences. Sequence similarity search showed that the 16s rRNA sequence was 99% identical (Coverage 100%) with the 16S rRNA gene sequence of Staphylococcus arlettae strain CM18, (Accession No. GU143791.1), i.e., a gram positive bacterial strain used in the treatment of effluents from the textile industry.
Sequencing of Lipase Gene from Staphylococcus arlettae JPBW-l (MTCC 5589)
• Genomic DNA isolation from Staphylococcus arlettae JPBW-1 (MTCC 5589) was done by Phenol-Chloroform method.
• PCR amplification of lipase gene was done using forward (5'-ACTATGACCGTGCCGTTGAA-3') and reverse (5'-GGACGTGTTGACTATGGTGCAGC-3') primers (SEQ ID 3 and SEQ ID 4 respectively). The primers were designed from conserved sequence regions of lipase genes from other species. The PCR was performed in a final reaction mixture (25.0 µl) containing 500 ng of genomic DNA, 0.4µM of each primer, 0.2 µM of dNTPs (Bio Basic Inc.), 1.25 U Taq polymerase (Intron Technologies) and 10x reaction buffer (Intron Technologies). Amplification reactions were performed with the following cycling conditions: initial denaturation for 4.0 min at 95.0°C followed by 30.0 cycles of 30.0 s at 94.0°C, 1 min at 52.0oC and 1 min at 72.0°C with a final extension for 7.0 min at 72.0°C and cooling to 4.0°C.
• The amplified products were cloned in pGEM-T vector (Promega) and transferred to the DH5a chemical-competent cells of E. coli for multiplication.
• The transformants were screened based on blue/white colonies observed on LB agar plates containing IPTG, X-gal and ampicillin.

• Colony PCR of the transformed colonies that were picked randomly was set-up using forward (5'-ACTATGACCGTGCCGTTGAA-3') and reverse (5'-GGACGTGTTGACTATGGTGCAGC-3') primers (SEQ ID 5 and 6 respectively). The PCR was performed in a final reaction mixture (25.0 µl) containing a single transformed colony, 0.4µM of each primer, 0.2 µM of dNTPs (Bio Basic Inc.), 1.25 U Taq polymerase (IntronTechnologies) and 10x reaction buffer (Intron Technologies). Amplification reactions were performed similarly as stated above.
• Transformed plasmids were isolated using AxyPrep™ Plasmid Miniprep Kit. The isolated plasmids were sent for sequencing.
• Sequence homology (BlastN) analysis showed that an amplicon having sequence length of 317 bp has 97% identities to putative lipase {Protein id: AAW53417.1) of Staphylococcus epidermidis RP62A.
• Primer pair (Forward 5'-TATCAGCGCACGAATAGTGG-3' and Reverse 5'-TCCCTATTGAGGGCTTCCTT-3', SEQ ID 7 and 8 respectively) was designed by taking 1000 bp upstream and 1000 bp downstream of the putative lipase gene (2046 base pairs) of Staphylococcus epidermidis RP62A. The PCR was performed in a final reaction mixture (25.0 µl) containing 500 ng of genomic DNA, 0.4µM of each primer, 0.2 µM of dNTPs (Bio Basic Inc.), 1.25 U Taq polymerase (Intron Technologies) and 10x reaction buffer (Intron Technologies). Amplification reactions were performed with the following cycling conditions: initial denaturation for 4.0 min at 95.0°C followed by 30.0 cycles of 30.0 s at 94.0°C, 45.0 s at 56.0°C and lmin at 72.0°C with a final extension for 7.0 min at 72.0°C and cooling to 4.0°C.
• The amplified products were cloned in pGEM-T vector (Promega) and transferred to the DH5α chemical-competent cells of E. coli for multiplication.
• The transformants were screened based on blue/white colonies observed on LB agar plates containing IPTG, X-gal and ampicillin.
• Colony PCR of the transformed colonies that were picked randomly was set-up using forward (5'-TATCAGCGCACGAATAGTGG-3') and reverse (5'-TCCCTATTGAGGGCTTCCTT-3') primers. The PCR was performed in a final reaction mixture of 25.0 µl containing a single transformed colony, 0.4µM of each primer, 0.2 µM of dNTPs (Bio Basic Inc.), 1.25 U Taq polymerase (IntronTechnologies) and 10x reaction buffer (Intron Technologies). Amplification

reactions were performed with the following cycling conditions: initial denaturation for 4.0 min at 95.0°C followed by 30.0 cycles of 30.0 s at 94.0°C, 45.0 s at 56.0°C and lmin at 72.0°C with a final extension for 7.0 min at 72.0°C and cooling to 4.0°C.
• Transformed plasmids were isolated using AxyPrep™ Plasmid Miniprep Kit.
• Internal primers were designed from the putative lipase gene of Staphylococcus epidermidis RP62A to amplify and sequence the above amplicon cloned in pGEM-T vector (Promega) in split parts. The PCR was performed using the primer pairs (Forward 5'-ATTTAGGTGACACTATAG-3' and Reverse 5'-GCACACCACATAATGGTACA-3' of SEQ ID 9 and 10 respectively, Forward 5'-CAAAACGTCAAATTCAAACA-3' and Reverse 5'-GTTGTTTTAATCCCCATTGA-3' of SEQ ID 11 and 12 respectively, Forward 5'-AAAATACGCAGTGGGAGTAG-3' and Reverse 5'-CGTCTCCCTTGTTGTACTTC-3' of SEQ ID 13 and 14 respectively, Forward 5'-GCATTACCTTGTTTTGAAGC-3' and Reverse 5'-TAATACGACTCACTATAGGG-3' of SEQ ID 15 and 16 respectively) in a final reaction mixture (25.0 µl) containing 500 ng of plasmid DNA, 0.4µM of each primer, 0.2 µM of dNTPs (Bio Basic Inc.), 1.25 U Taq polymerase (Intron Technologies) and 10x reaction buffer (Intron Technologies). Amplification reactions were performed with the following cycling conditions: initial denaturation for 4.0 min at 95.0°C followed by 30.0 cycles of 30.0 s at 94.0°C, annealing of 45.0 s at 49.0°C for Forward SP6 and Reverse 5'-GCACACCACATAATGGTACA-3', 45.0°C for Forward 5'-CAAAACGTCAAATTCAAACA-3' and Reverse 5'-GTTGTTTTAATCCCCATTGA-3', 49.0°C for Forward 5'-AAAATACGCAGTGGGAGTAG-3' and Reverse 5'-CGTCTCCCTTGTTGTACTTC-3', 47.0°C for Forward 5'-GCATTACCTTGTTTTGAAGC-3' and Reverse T7, and lmin at 72.0°C with a final extension for 7.0 min at 72.0°C and cooling to 4.0°C.
• The PCR amplicons were purified using AxyPrepTM PCR clean up kit, Axygen Biosciences and sent for sequencing.
• The sequencing data obtained were compiled using DNA Star, DNASTAR, Inc. Sequence homology (BlastN) analysis showed that the amplicon having sequence length of 2046 bp has 99% identities to putative lipase gene of Staphylococcus

epidermidis RP62A. Translation of the gene sequence (Translate Tool, ExPASy) gave a 681 amino acid long polypeptide which has 98% identities with the putative lipase protein of Staphylococcus epidermidis RP62A when analysed through protein sequence homology analysis (BlastP).
• The nucleotide sequence of Lipase encoding gene of Staphylococcus arlettae is represented by SEQ ID 1.
• The translated amino acid sequence of Lipase gene of Staphylococcus arlettae is represented by SEQ ID 2.
WORKING EXAMPLES
The present examples are given by way of illustration and should not be construed so as to limit the scope of the invention.
EXAMPLE 1
The activity of lipase production in Staphylococcus arlettae was demonstrated through qualitative analysis of the bacterial culture on agar plates containing olive oil showing positive response as a zone of hydrolysis around the bacterial colony after overnight incubation at 37°C (Fig. 1). Quantitative estimation of lipase was carried out using substrates, olive oil and soyabean oil. The lipolytic activity of enzyme was more in soyabean oil in comparison to olive oil at an optimum pH of 8.0 and temperature 50°C (Fig. 2). Thus, for further studies, soyabean oil was used. Purification of protein was achieved with (NFL4)2 SO4 fractionation giving a cut-off of 20, 60 and 80%. All the fractions were tested for lipase activity and we found that 60% fraction is giving best activity at pH 8.0 and temperature 50°C (Fig. 3).
EXAMPLE 2
The efficiency of lipase producing bacteria was assayed at different pH, temperature and salt concentrations using 60 % (NH4)2 SO4 fraction. It was found that lipase production was maximum at pH 8.0 i.e., 750 U/ml temperature of 50°C, followed by pH 10.0 at which the

activity was 400 U/ml. The enzyme was active in the pH range of 4 - 11 (Fig. 4). The optimum temperature for lipase activity was found to be 50°C. Lipolytic activity in the halophilic culture was observed at temperature range of 40 - 90°C (Fig. 5), showing that the enzyme is thermotolerant.
EXAMPLE 3
The effect of salt concentration on lipase activity was tested in NaCl (0 - 19 % w/v) at pH of 8.0 and temperature 50°C (Table 2). Highest activity of lipase was observed in the culture grown without salt (Fig. 6). Molecular mass of Lipase measured by SDS-PAGE analysis (Lanes 2-3, Fig. 7) was found to be approximately 60 kDa.
Table 2. Effect of salt (supplemented in Luria broth) on enzyme production by Staphylococcus arlettae during submerged studies in shake flask
(Table Removed)
Amount of enzyme required to liberate 50µM fatty acids at optimum conditions of temperature and pH
EXAMPLE 4
Enzyme Kinetics analysis was done according to Michaelis-Menten and Lineweaver-Burk plot, calculated Km is 20% and Vmax is 1666 U/ml (Fig. 8).
EXAMPLE 5

Table 3. Comparison of the lipase produced from Staphylococcus arlettae with other lipases from commercial manufacturers.
(Table Removed)
ADVANTAGES OF THE PRESENT INVENTION
The enzyme produced from Staphylococus arlettae has shown a versatile operational pH and temperature range making it a most promising enzyme for its probable commercial application in industrial sectors. The lipase produced by a bacterial strain reported in the current invention is expected to have following potential applications:
1. Lipase use in leather tanning industry
2. Performing biocatalytic reactions in the presence of salt or at alleviated
temperature for improving product quality.
3. Lipase in pharmaceutical industries primarily in resolution of racemic drugs.
4. Lipase as a component of finished products in detergent industries that incorporate lipase along with other enzymes as one of the components. The lipase is expected to perform at high temperature (90°C) for better action of detergent and enzyme.

We claim:
1. A halotolerant and thermostable lipase enzyme derived from Staphylococcus arlettae JPBW-1 (MTCC 5589), capable of operation at a salt concentration in the range of 0 to 19% (w/v) NaCl and over a temperature range of 40°C to 90°C.
2. The lipase as claimed in claim 1, wherein the lipase is isolated from staphylococcus arlettae by a process comprising the steps of:
(i) dissolving soil sample collected from rock salt mines in sterile distilled water
in a ratio of 1:100 by weight followed by its serial dilution (10-1 to 10-5);
(ii) plating the dilutions obtained in step (b) on Luria-Bertani agar (LBA);
(iii) isolating the microbial colonies growing on LBA and individually streaking
Staphylococcus arlettae JPBW-1 (MTCC 5589) on LBA plates supplemented with
19% (w/v) salt;
(iv) inoculating Staphylococcus arlettae JPBW-1 (MTCC 5589) into 5 ml LB
medium and incubating with shaking at 200 rpm for 16 h at 37.0°C;
(v) transferring 5 ml of the culture obtained in step (iv) to 45 ml of LB medium
and incubating again for 16 h at 37.0°C;
(vi) transferring 50 ml of the culture obtained in step (v) to 450 ml of LB medium
and incubating with shaking at 200 rpm for 3 h at 37.0°C;
(vii) pelleting the cells by centrifugation at 5,000 rpm for 30.0 min. at 4.0°C, and
extracting the supernatant;
(viii) precipitating the supernatant obtained in step (vii) with 20.0% (NH4)2S04
with constant stirring at 4.0°C for 12h to obtain partially purified protein;
(ix) recovering the partially purified protein obtained in step (viii) by
centrifugation at 5,000 rpm for 30.0-45.0 min. at 4.0°C;
(x) precipitating the supernatant obtained in step (ix) with 60.0% (NH4)2S04
with constant stirring at 4.0°C for 12h;
(xi) recovering the protein by centrifuging the precipitate obtained in step (x) at
5,000 rpm for 30.0-45.0 min. at 4.0°C;
(xii) dissolving the precipitate obtained in step (xi) in 0.75 ml of 50.0 mM
phosphate buffer (pH 7.0) followed by dialysis with 10.0 kD membrane to obtain a

partially purified lipase enzyme from Staphylococcus arlettae JPBW-1 (MTCC 5589) with a 16s rRNA sequence which is 99% identical (Coverage 100%) with 16S rRNA gene sequence of Staphylococcus arlettae strain CM 18, (Accession No. GU143791.1).
3. The lipase as claimed in claim 1, wherein the lipase is translated from a nucleotide sequence of SEQ ID 1 cloned by a process comprising the steps of:
a) isolating genomic DNA from Staphylococcus arlettae by Phenol-Chloroform method;
b) designing primers for PCR amplification of lipase gene from the genomic DNA obtained in step (a) from conserved sequence regions of lipase genes of other species to obtain forward primer of SEQ ID 3 and reverse primer of SEQ ID 4;
c) amplifying lipase gene from the genomic DNA obtained in step (a) by PCR using the primers obtained in step (b);
d) cloning the amplified products of step (c) in pGEM-T vector (Promega) and transferring to DH5a chemical-competent cells of E. coli for multiplication;
e) screening the transformants based on blue/white colonies observed on LB agar plates containing IPTG, X-gal and ampicillin;
f) setting up colony PCR of the transformed colonies that were picked randomly using forward primer of SEQ ID 5 and reverse primer of SEQ ID 6 followed by isolating transformed plasmids using AxyPrep™ Plasmid Miniprep Kit;
g) sequencing the isolated plasmids obtained in step (f) by sequence homology (BlastN) analysis to obtain an amplicon having sequence length of 317 bp and 97% identities to putative lipase (Protein id: AAW53417.1) of Staphylococcus epidermidis RP62A;
h) designing forward primer of SEQ ID 7 and reverse primer of SEQ ID 8 by taking 1000 bp upstream and 1000 bp downstream of the putative lipase gene (2046 base pairs) of Staphylococcus epidermidis RP62A followed by PCR amplification of the putative lipase gene using said primers of SEQ ID 7 and SEQ ID 8;

i) cloning the amplified products obtained in step (h) in pGEM-T vector (Promega), transferring to DH5a chemical-competent cells of E. coli for multiplication, and screening transformants based on blue/white colonies observed on LB agar plates containing IPTG, X-gal and ampicillin;
j) setting up colony PCR of the transformed colonies that were randomly picked up from LB agar plates in step (i) using forward and reverse primers of SEQ ID 7 and SEQ ID 8 respectively;
k) isolating transformed plasmids using AxyPrep™ Plasmid Miniprep Kit;
1) amplifying and sequencing the amplicon cloned step (i) in split parts using
internal primers designed from the putative lipase gene of Staphylococcus
epidermidis RP62A, said internal primers represented by SEQ ID 9 and 10
(Forward 5'-ATTTAGGTGACACTATAG-3' and Reverse 5'-
GCACACCACATAATGGTACA-3', SEQ ID 11 and 12 (Forward 5'-
CAAAACGTCAAATTCAAACA-3' and Reverse 5'-
GTTGTTTTAATCCCCATTGA-3'), SEQ ID 13 and 14 (Forward 5'-
AAAATACGCAGTGGGAGTAG-3' and Reverse 5'-
CGTCTCCCTTGTTGTACTTC-3'), SEQ ID 15 and 16 (Forward 5'-
GCATTACCTTGTTTTGAAGC-3' and Reverse 5'-
TAATACGACTCACTATAGGG-3');
m) purifying the PCR amplicons obtained in step (1) using AxyPrepTM PCR clean up kit followed by its sequencing;
n) compiling the sequencing data obtained in step (m) using DNA Star, to obtain sequence of lipase gene of Staphylocococcus arlettae of SEQ ID 1 having sequence length of 2046 bp and 99% identities to putative lipase gene of Staphylococcus epidermidis RP62A.
4. The lipase as claimed in claim 3, wherein translation of gene sequence of SEQ ID 1 yields a 681 amino acid long polypeptide of SEQ ID 2, having 98% identities with putative lipase protein of Staphylococcus epidermidis RP62A.
5. The lipase as claimed in claim 1, comprising the following characteristics:
(i) Molecular Mass = 60KDa;

(ii) Michaelis constant (Km) = 20%;
(iii) Maximum reaction rate (Vmax) = 1666U/ml.
6. The enzyme as claimed in claim 1, wherein the lipase shows optimal activity at a temperature in the range of 50°C to 60°C.
7. The lipase as claimed in claim 1, wherein the lipase shows optimal activity over a pH range of 8 to 10.
8. The lipase as claimed in claim 1 for use in post tanning operations in leather industry, Performing biocatalytic reactions, pharmaceutical industries and detergent industries.
9. The lipase and the process of producing the same substantially as herein described with reference to foregoing examples and attached drawings.