FIELD OF INVENTION:
The invention relates to the process of preparing thermoactive alkaline lipase from lipolytic strain of Burkholderia multivorans. More particularly it relates to the thermoactive alkaline lipase from Burkholderia multivorans. The invention describes the process for preparing lipase, and industrially important properties followed by the analysis of the lipase gene.
BACKGROUND:
Lipases are an important group of biotechnologically relevant enzymes and they find immense applications in food, dairy, detergent and pharmaceutical industries. Today, lipases outshine other hydrolases, especially in the field of pharmaceuticals where they find increasing usage in drug designing and development due to their enantioselective nature. Bacterial lipases hold a special significance in the biotechnology sector due to their wide substrate specificity, broad range of pH and temperature tolerance as well as chemo-, regio- and enantio-selectivity. The bacterial genera most frequently exploited for lipase production are Bacillus, Staphylococcus and Pseudomonas. Several lipase-based products derived from these bacterial genera have been launched successfully in the market in the past few years. However, there is a still a huge market for novel lipases. The growing arena of lipase applications has triggered interest in isolation of newer and more interesting lipases directly from nature both by the conventional cultural techniques and the modern molecular biology methods like database mining and metagenomics.
The Burkholderia bacteria are human and plant pathogens as well as environmentally important bacteria. Burkholderia funsorum strain LB400 and other closely related Burkholderia strains are good biodegraders of polychlorinated biphenyls (PCBs) with "unparalleled ability to destroy environmentally important PCB congeners" (DOE). Conversely, Burkholderia pseudomallei are the causative agent of melioidosis, as disease that harms both humans and animals, as well as
septicemia and pneumonia in susceptible individuals (Godoy et al. 2003).
Burkholderia bacteria are rod-shaped, motile, Gram-negative bacteria that are capable of both pathogenic characteristics and degrading PCBs. The bacteria are also generally obligately aerobic. Of the bacterial isolates found in the Arctic that are able to degrade PCBs, none used compounds such as 2-chlorobiphenyl, 3-chlorobiphenyl, camphor, citronellol, cymene, dehydroabietic acid, limonene, methanol, n-hexadecane, pentachlorophenol, phenol, or pinene as primary growth substrates (Master and Mohn 1998).
Burkholderia bacteria are commonly found in the soil and in groundwater worldwide. Burkholderia and related bacteria have been found in soils of all tempuratures including Arctic soil of 7°C.
Several strains of Burkholderia glumae; Burkholderia plantarii; Burkholderia cepacia have been found to produce lipase however lipase from Burkholderia multivorans has never been reported.
OBJECT OF THE INVENTION:
The object of the present invention is to provide a process for the preparation of thermostable alkaline lipase form Burkholderia multivorans having higher yields.
Another object of the present invention is characterize the lipase and its gene sequence obtained from Burkholderia multivorans .
SUMMARY OF THE INVENTION:
In order to achieve the said objectives, the present invention provides the process of preparing thermoactive alkaline lipase from lipolytic strain of Burkholderia multivorans, said process comprising the steps of:
isolating lipolytic strain of B. multivorans from compost, culturing the said isolated B. multivorans in nutrient source,
separating lipase by conventional methods,
purifying the said separated lipase to electrophoretic grade,
to obtain lipase that is stable and active in a wide range of temperature and pH, shows stability and activity in organic solvents, exhibits regioselectivity, enantioselectivity, wide substrate specificity and detergent compatibility.
The instant invention further provides a thermoactive alkaline lipase from lipolytic strain of Burkholderia multivorans, wherein said lipase is stable at a wide range of temperature and pH, shows stability and activity in organic solvents, exhibits regioselectivity, enantioselectivity, wide substrate specificity and detergent compatibility.
A lipase from a novel lipolytic strain Burkholderia multivorans has been identified. The N-terminal sequence of the lipase protein shows 80% homology with lipA of Burkholderia cepacia, an important commercial enzyme while the lipase gene sequence shows 86% homology to lipA of B. cepacia.
The alkaline lipase according to the invention has been obtained in high amounts using submerged fermentation using a variety of carbon and nitrogen sources. The lipase is also produced under solid-state fermentation using oil cake as the sole substrate. The lipase is purified to homogeneity using column chromatography. The enzyme displays many industrially important properties like pH and temperature stability, stability and activity in organic solvents, regioselectivity, enantioselectivity, wide substrate specificity and detergent compatibility. The enzyme can catalyze esterification and transesterification reactions.
DESCRITION OF THE DRAWINGS:
Figure 1: Effect of pH on activity and stability of Lipase Figure 2: Effect of temperature on activity and stability of Lipase Figure 3: Stability of Lipase in various organic solvents for 1hr. Figure 4: Lineweaver-Burk plot of the values of the rate of reaction as a function of substrate concentration of determining Km and Vmax values of lipase
from 6. multivorans on [A] Tributyrin [B] Triolein
Figure 5: Effect of inhibitor on lipase activity
DETAILED DESCRIPTION OF THE INVENTION:
The lipase according to the invention is produced by cultivating a novel lipolytic strain Burkholderia multivorans, the lipase from which has not been reported earlier.
The organism was initially isolated from compost using oil enrichment method. The organism was identified by partial 16s rRNA gene sequencing. The partial 16s rRNA gene sequence has been deposited in Genbank with accession no. AY597545. The culture will be deposited at MTCC.
The organism could be grown in the temperature range of 30-45°C, preferably at 37°C and pH 7.0. The production of the lipase is inducible. Any lipid source, fats, fatty acids, hydrocarbons or surfactants could be used for lipase production without additin of sugars or with addition of sugars preferably glucose. The lipase is produced in presence of any organic or inorganic nitrogen source, preferably a combination of ammonium chloride and yeast extract. Addition of some metal ions, preferably Ca2+ enhanced lipase production.
The seed inoculum is prepared either in nutrient broth or in the oil medium itself. The lipase is also produced under solid-state fermentation conditions, using an oil cake with or without supplementation with additional nutritional factors. Following SSF, the enzyme is extracted from the solid support using a variety of solvents, preferably Tween 80.
For downstream processing, the lipase is concentrated by ultra filtration or organic solvent precipitation, preferably methanol or acetone. The lipase is purified to electrophoretic grade by hydrophobic interaction chromatography using octyl-sepharose, phenyl-sepharose or butyl-sepharose as the binding matrix and subsequent elution using any polar solvent, preferably, isoproponaol in 20-50% v/v
concentration.
The purified enzyme showed a single band of approximately 33 kDa on SDS-PAGE after silver staining. The N-terminal sequence analysis (A-D-D-Y-A-A-T-R-Y-P-l-V-L-V-H-G) of the purified lipase shows 80% homology to LipA of Burkholderia cepacia, a very important commercial lipase.
The lipase exhibited activity over a wide pH range of 3-12 with optima at pH 11 and was also stable between pH range of 3-12 for more than 6 h at room temperature (Fig 1). The enzyme was thermoactive showing an increase in activity with increase in temperature. The temperature optima of the purified lipase was found to be 90°C while that of crude lipase was 70°C (Fig 2). The lipase retained 40% of its activity at 60°C after 1 h incubation and exhibits half-life of 15 min and 5 min at 70 and 80°C, respectively. The lipase was completely stable in many organic solvents for a period of 1 h (Fig 3). It catalyzed hydrolysis of a large variety of fats, oils, esters and triglycerides (Table 1).
Table 1: Activity of lipase on various fats and oils, methyl esters, p-nltrophenyl esters, methyl esters and triglycerides

(Table Removed)
It exhibited Km of 71.42 mM and Vmax of 66.66 µmoles/ml/min for tributyrin Km value of 47.61 mM and Vmax of 50.1 µmoles/ml/min for triolein (Fig 4).
The activity of purified enzyme was inhibited in presence of serine inhibitors, PMSF and phenylboronic acid and tryptophan inhibitor, N-bromosuccinimide indicating serine and tryptophan to be important for its catalytic activity (Fig 5).
The enzyme cleaved triolein with some preference for the 1,3-ester bonds indicating the regioselective nature of the lipase (Table 2).
Table 2: Analysis of triolein hydrolysis by 8. multivorans lipase in aqueous system and hexane indicating the regioselcetive nature of the enzyme

(Table Removed)
The enzyme displayed high degree of enantioselectivity for some chiral compounds. The lipase catalyzed a large number of esterification and transesterification reactions (Table 3).
Table 3: Esterification reactions catalyzed by the lipase: Synthesis of sucrose/methanol/ascorbic acid fatty acid esters by the lipase in microwave for 30 seconds and under non-microwave conditions for 24 h

(Table Removed)
aEster synthesis was expressed as the percent molar conversion of the acid to the ester after titrating residual fatty acid against 0.05N KOH using phenolphthalein as an indicator. cParentheses indicates the % conversion obtained by microwave method.
The enzyme was also detergent compatible as evident by the display of its stability in the presence of various commercially available bleaching agents, detergents and surfactants such as H2O2, sodium perborate, tweens, Triton X-100, sodium choleate etc. The lipase also showed its compatibility towards a number of commercial detergents and bleaches used in laundries, such as Ezee, Ariel, Tide, 555, Surf-ultra and Rin Supreme. The enzyme was stable in presence of many proteases (Table 4).
Table 4: Effect of various oxidizing agents, bleaches, surfactants, proteases and commercial detergents on lipase stability

(Table Removed)
The gene sequence of the lipase was 1095bp long encoding 364 amino acid residues. The detailed amino acid sequence was identified to code for 44 amino acid signal peptide and 320 amino acid mature lipase. The gene displayed 86% homology with UpA of B. cepacia. The gene sequence has been deposited at Genbank with accession no. AY845057.
The present invention will now be explained with the help of examples.
It will readily be appreciated by those skilled in the art that the present invention is not limited to the specific embodiments herein shown. Thus variations may be made within the scope and spirit of the accompanying claims without sacrificing the principal advantages of the invention.

We claim:
1. The process of preparing thermoactive alkaline lipase from lipolytic strain of
Burkholderia multivorans, said process comprising the steps of:
- isolating lipolytic strain of B. multivorans from compost, - culturing the said isolated B. multivorans in nutrient source,
- separating lipase by conventional methods,
- purifying the said separated lipase to electrophoretic grade,
to obtain lipase that is stable and active in a wide range of temperature and pH, shows stability and activity in organic solvents, exhibits regioselectivity, enantioselectivity, wide substrate specificity and detergent compatibility
2. The process as claimed in claim 1 wherein the said 6. multivorans is isolated from compost by oil enrichment method.
3. The process as claimed in claim 1 wherein the said B. multivorans was identified by partial 16s rRNA gene sequencing.
4. The process as claimed in claim 1 wherein the said culture is maintained at a temperature in the range of 30 to 45°C at a pH 7.
5. The process as claimed in claim 1 wherein the nutrient medium comprises of lipid source with or without sugar source, nitrogen source and metal ion source.
6. The process as claimed in claim 5 wherein the said lipid source is selected from fats, fatty acids, hydrocarbons or surfactants.
7. The process as claimed in claim 5 wherein the nitrogen source may be organic or inorganic.
8. The process as claimed in claim 5 wherein the nitrogen source is ammonium chloride and yeast extract.
9. The process as claimed in claim 5 wherein the metal ion is Ca+.
10. The process as claimed in claim 1 wherein seed inoculum is prepared in nutrient broth or in oil medium.

10. The process as claimed in claim 1 wherein lipase prepared both by submerged and solid-state fermentation
11. The process as claimed in claim 10 wherein the solid-state fermentation uses oil cake with or without supplementation with additional nutritional factors.
12. The process as claimed in claim 10 wherein lipase is extracted from the solid support using a variety of solvents such as Tween 80.
13. The process as claimed in claim 1 wherein lipase is concentrated by ultra filtration or organic solvent precipitation, preferably methanol or acetone.
14. The process as claimed in claim 1 wherein lipase is purified by hydrophobic interaction chromatography using octyl-sepharose, phenyl-sepharose or butyl-sepharose as the binding matrix and subsequent elution using any polar solvent, preferably, isoproponaol in 20-50% v/v concentration.
15. Thermoactive alkaline lipase from lipolytic strain of Burkholderia multivorans, prepared by the process as claimed in any of the claim 1 to 14 wherein said lipase is stable at a wide range of temperature and pH, shows stability and activity in organic solvents, exhibits regioselectivity, enantioselectivity, wide substrate specificity and detergent compatibility.
16. Thermoactive alkaline lipase as claimed in claim 17 wherein the molecular weight of lipase is 33kDa.
17. Thermoactive alkaline lipase as claimed in claim 17 wherein N-terminal sequence of the purified lipase is A-D-D-Y-A-A-T-R-Y-P-l-V-L-V-H-G.
18. Thermoactive alkaline lipase as claimed in claim 17 wherein the gene sequence of the lipase is 1095 bp long encoding 364 amino acid residues.
19. Thermoactive alkaline lipase as claimed in claim 17 wherein N-terminal sequence of the lipase protein shows 80% homology with lipA of Burkholderia cepacia, while the lipase gene sequence shows 86% homology to lipA of B. cepacia.
20. Thermoactive alkaline lipase as claimed in claim 17 wherein the gene sequence of lipase has an accession no. AY845057.
21. The process of preparing thermoactive alkaline lipase from lipolytic strain of Burkholderia multivorans substantially as herein described with reference to foregoing examples.
22. Thermoactive alkaline lipase from lipolytic strain of Burkholderia multivorans substantially as herein described with reference to foregoing examples.