FIELD OF THE INVENTION
The present invention primarily relates to a novel bacterial strain. The invention also provides a process of producing methyl esters.
BACKGROUND OF THE INVENTION
As we arc well aware of the potential problem associated with the petrodiesel, the world is looking forward to an effective renewable and economic alternative. Lipase catalysed reaction seem to be providing a possible answer for the same.
The team of inventors has tried to address this problem. The inventors provide a novel bacterial strain which is capable of producing methyl esters. Methyl esters are useful for biodicsel production.
Biotransformation of jatropha oil through lipase is preferred over chemical methods for ester production because enzymatic methods of transesterification are simpler, cleaner and more environmental friendly then their counterpart. Also the stability and the reusability of the enzyme are significantly improves in water limiting reaction and hence the cost of the process can be reduced. Although present industrial process for methyl esters production is carried out by using inorganic catalyst like NaOH, KOFI but the major drawback is that the homogenous catalyst are removed with glycerol layers after reaction and cannot be reused. Formation of soap during reaction further leads to the low yield of ester formation and makes it difficult to separate glycerol and ester. Also very high temperature requirement and large quantity of inorganic catalyst further limits the preference of chemical methods for biodicsel production. However, synthesis of methyl esters catalyzed by lipases is an alternative to overcome the problem. Biotransfonnation of oil in different molar ratio with alcohol using lipase for biodicsel is more efficient process because process can take place at room temperature and physiological pH, at low temperature when compared to chemical catalyzed reaction, highly specific and economically feasible. Also biodiesel is clean burning fuel, require no modification in engine design when blended with diesel and release less or negligible particulate matter thus taking care of environmental issues as well. Fuel efficiency of biodicsel is also better in lipase catalyzed trans fonnation reaction. Flash point which is a measure of lowest temperature for volatile liquids to ignite in air mixture is quite high for biodiesel that is 210( C compared to 130°C for diesel.

SUMMARY OF THE INVENTION
The invention provides a novel bacterial strain useful for production of methyl esters. The invention also provides a process of producing the methyl esters using the bacterial strain, further, the invention provides a lipase enzyme obtained from the bacterial strain and a process of producing the methyl esters using the lipase enzyme.
STATEMENT OF INVENTION
The invention provides a novel bacteria strain with accession number MTCC 5618 useful for producing methyl esters.
DETAILED DESCRIPTION OF THE INVENTION
Accordingly, the invention provides a novel bacteria strain with accession number MTCC 5618 useful for producing methyl esters.
The invention also provides a process of producing methyl esters using the bacterial strain with accession number MTCC 5618, said process comprising the steps of:
(i) mixing oil with methanol in a ratio ranging from 25mM to 100mM
along with a suitable solvent and the bacterial strain; (ii) leaving the reaction mixture of step (i) for 3 to 20 hours at a
temperature of 20 °C to 40 °C; and (iii) obtaining the methyl esters that are authenticated using mass spectrometry.
Preferably the ratio of oil and methanol for the purpose of the process of the invention is 1:4M.
The concentration of the bacterial strain in the reaction mixture is 50-150mg.
The oil for the purpose of the invention is either edible oil or is non-edible oil.
The edible oil is selected from the group comprising palm oil, rapeseed oil, sunflower oil, safflower oil.

The process of the invention is capable of utilizing cheap non-edible oil as substrate for the reaction.
The non-edible oil is selected from the group comprising jatropha oil, cotton oil, flax seed oil.
The solvent required in the process of the invention is selected from the group comprising toluene, hexane, ethyl acetate and methanol.
Preferably, the reaction mixture in step (ii) of the process is left for 8 to 10 hours.
Preferably, the process is performed at room temperature.
The bacterial strain of the invention is an organic solvent tolerant lipase producer. The bacterial strain is capable of utilizing cheap non-edible oil for the purpose of production of methyl esters.
In an embodiment of the invention, the invention also provides a lipase enzyme obtained from the bacterial strain with accession number MTCC 5618. The lipase enzyme of the invention is useful in production of methyl esters.
In yet another embodiment the invention provides a process of obtaining methyl esters using the lipase enzyme obtained from the bacterial strain with accession number MTCC 5618, said process comprising the steps of:
(i) mixing oil with methanol in a ratio ranging from 25mM to 150mM
along with a suitable solvent and the lipase enzyme; (ii) leaving the reaction mixture of step (i) for 2 to 20 hours at a
temperature of 20°C to 40 °C; and (iii) obtaining the methyl esters that were authenticated using mass spectrometry .
In a preferred embodiment, the ratio of oil and methanol is 1:4 M.

It is preferred that the concentration of the lipase enzyme in the reaction mixture is 50-150 mg.
The oil used for the process is edible oil or non-edible oil.
The edible oil is selected from the group comprising palm oil, rapeseed oil, sunflower oil, safflower oil.
The process of the invention is capable of utilizing cheap non-edible oil as substrate for the reaction.
The non-edible oil is selected from the group comprising jatropha oil and flax seed.
The solvent required in the process of the invention is selected from the group comprising toluene, hexane ethyl acetate and methanol.
The lipase enzyme of the invention has high temperature, pH and solvent stability. The enzyme is stable at a temperature of 40 °C to 70 °C for 2 to 20 hours. The enzyme is stable in organic solvents for 2 to 50 hours. These properties of the enzyme make the enzyme useful as a catalyst in reactions occurring at high temperature and high percentage of organic solvents.
for the purpose of obtaining the bacterial strain of the invention, liquid and solid samples are obtained from wastes of various alcohol distilleries and oil manufacturing and packaging industries of Punjab. This is followed by the process of enrichment of the strains by suspending the isolates samples in a minimal salt medium containing 2mM to 100mM oil or tributyrin and serially enriching by incrementally increasing the concentrations of methanol, oil or tributyrin. The medium is preferably incubated at a temperature of 30 °C. The pH of the medium is maintained in range of 5 to 10. This is followed by subjecting the strains to primary and secondary screening on a selection medium and obtaining the strain showing the desired lipase activity. Primary screening is perfonned by plating the culture on selection medium containing tributyrin or oil and methanol with nutrient afar or minimal salt medium as base. The plates arc incubated and subcultured every 15 days. The cultures are maintained on nutrient agar. Following the

primary screening, the isolated bacterial strains are subjected to secondary screening by preparing a seed culture of the bacteria by inoculating a loopful of the freshly streaked culture in flasks containing nutrient broth. The flasks are incubated and centrifuged. The cell free suspension and pellet obtained is assessed for lipolytic potential of the bacterial. The enzyme is assessed by spectrophometric method.
The invention is illustrated further by the following examples which are only meant to illustrate the invention and not act as limitations. The embodiments which may be apparent to a person skilled in the art are deemed to fall within the scope of the present invention.
EXAMPLE 1: Purification of the bacterial strains:
One gram soil sample is suspended in 5ml demineralised water to prepare the inoculum.
To the minimal salt medium (MSM), containing triglycerides (2-100mM) as a sole carbon
and nitrogen source, 2% (v/v) inoculum is added and incubated at 30 C at 200 rpm for 1
to 2 weeks. Growth in the selective medium is checked by streaking samples onto the
selective plates, which consists of MSM, triglyceride and methanol containing agar as
gelling agent. Positive isolates are selected on the basis of growth on the selective plates.
Cultures are purified and maintained on nutrient agar plates.
Composition of MSM (g/1)
NH4C1 0.1
NaHPO4 2
KH4PO4 2
MgCl2 0.2
Primary screening is designed based on the substrate tolerance of the isolates and is done by checking the growth of different isolates on selective plates of 2, 5, 10, 20, 25, 50, 100 mM triglyceride plus methanol (0.25-20% v/v). The bacterial strains are purified based on the production of methyl esters.
Methyl ester/s producing bacterial strains as obtained above is maintained on nutrient agar plates of following composition: Composition of nutrient agar (g/1)
Peptic digest of animal tissue 5

NaCl 5
Beef extract 1.5
Yeast extract 1.5
Agar 1.5
The plates are incubated at 30 C for 24 h. the culture is maintained by periodic transfer on nutrient plates and stores at 4°C. The microorganism is inoculated in nutrient broth of following composition and cultured at 30 C for 48 hours. 2% (v/v) inoculum is subsequently transferred to induction medium of the same composition containing triglycerides as the inducer (0.5%)
Composition of Nutrient broth (g/1)
Peptic digest of animal tissue 5
NaCl 5
Beef extract 1.5
Yeast extract 1.5
Triglycerides 0.5%
Both cells and supernatant is obtained from the culture broth at intervals of 20 - 70 h to determine the enzyme activity. The enzyme activity is determined using the method given by Vodcrwiilbecke et al, (Comparision of lipases by different assays. Enzyme Microb. Tcchnol., 14:631-639. 1992).
Analysis was carried through Spectrophotometric by using Voderwuellbecke et al, (1992) method. Assay was comprises of solution I (freshly prepared) containing 3mg of substrate in 1 ml of isopropanol for solubilization and solution II containing 0.5g of Gum Arabic, 2g of Triton X100 in 450 ml 50mM Tris HCI buffer having pH 7.5. Working solution was prepared by drop wise addition of 1ml solution I to 9ml of solution II.
Assay reaction is 3ml in total volume, containing 2ml of buffer, 900 microlitrc of working solution and 100 microlitrc appropriately diluted enzyme sample.
This method involves measuring the release of p-Nitrophenol by the hydrolysis of 4-Nitrophenyl Butyrate (SIGMA). Amount of p- Nitrophenol released was calculated from the standard curve.

One unit of lipase activity has been defined as the amount of enzyme required to liberate
1 µg of p- Nitro phenol per ml/mg under the standard assay conditions.
Reagent used:
Solution I Solution II
Iso-propyl alcohol 10.0 ml Tris-HCI buffer (50 mM) 450 ml
p- Nitrophcnyl Butyrate 0.003 g Gum Arabic 0.5 g
Triton X-100 2.0 g
Working solution
Add 1 ml of solution to 9 ml solution dropwise with continuous stirring. This is always to be prepared a fresh.
Activity of 51Uml-1 mg-1 of wet bacterial cells was obtained after 72 hours of incubation of the cells with jatropha oil in (0.5%w/v).
EXAMPLE 2: Production of methyl esters using the bacterial strain with accession number MTCC 5618
(i) Mixing oil in an amount of 1 with methanol in an amount of 4 along with a suitable solvent in an amount of 1-10ml and the bacterial strain in an amount of 50-150 mg; (ii) leaving the reaction mixture of step (i) for 2-20 hours at a temperature of
20-40 °C; and (iii) obtaining the methyl esters that were authenticated using mass spectrometry.
EXAMPLE 3: Production of methyl esters using the Lipase Enzyme obtained from the bacterial strain with accession number MTCC 5618
1. Mixing oil in an amount of 1 with methanol in an amount of 4 along with a suitable solvent in an amount of l-10ml and the lipase enzyme in an amount of 50-150mg;
2. leaving the reaction mixture of step (i) for 2-20 hours at a temperature of 20-40 °C; and

ADVANTAGES OF THE INVENTION:
1. This application discusses the use of vegetable/non-edible oil transfonncd to alkyl esters. This has an advantage over pure vegetable oils being used as a fuel source in diesel engines, they often cause excessive engine wear and fuel injector coking, and have high smoke values. Further, their viscosity is much higher than petroleum based diesel fuel.
2. This process is an effort to overcome some of the problems associated with using pure vegetable oils, by producing fatty acid methyl esters. These esters have been prepared by transesterifying triglycerides, the major component in fats and oils, with methanol/ethanol, using biocatalyst/s.
3. The biocatalysts have shown certain extremophilic characters like stability in the presence of organic solvents for longer durations of time.

WE CLAIM:
1. A novel bacterial strain with accession number MTCC 5618 useful for producing methyl esters.
2. A process of producing methyl esters using the bacterial strain as claimed in claim 1, said process comprising the steps of:
(i) mixing oil with methanol in a ratio ranging from 1 to 4 along with a suitable solvent and the bacterial strain as claimed in claim 1;
(ii) leaving the reaction mixture of step (i) for 3 to 20 hours at a temperature of 20°C to 40°C;and
(iii) obtaining the methyl esters that were authenticated using mass spectrometry.
3. The process as claimed in claim 2, wherein preferably the ratio of oil and methanol is 1:4.
4. The process as claimed in claim 2, wherein the concentration of the bacterial strain in the reaction mixture is 50-150 mg.
5. The process as claimed in claim 2(ii), wherein preferably the reaction mixture is left for 8 to 10 hours.
6. The process as claimed in claim 2, wherein preferably, the process is performed at room temperature.
7. A lipase enzyme obtained from the bacterial strain as claimed in claim 1, wherein the lipase enzyme is useful in production of methyl esters.
8. A process of producing methyl esters using the lipase enzyme as claimed in claim 10, said process comprising the steps of:
(i) mixing oil with methanol in a ratio ranging from 1:3 to 1:4 along with a suitable solvent and the lipase enzyme as claimed in claim 13;
(ii) leaving the reaction mixture of step (i) for 2 to 20 hours at room temperature; and
(iii) obtaining the methyl esters that were authenticated using mass spectrometry.
9. The process as claimed in claim 8, wherein the concentration of the lipase enzyme
in the reaction mixture is 50-150mg.
10. The process as claimed in claims 2 and 8, wherein the oil is edible oil and is
preferably selected from the group comprising palm oil, rapeseed oil, sunflower
oil, safflowcr oil.
11. The process as claimed in claims 2 and 8, wherein the oil is non-edible oil and is preferably selected from the group comprising jatropha and flax seed oil.
12. The process as claimed in claims 2 and 8, wherein solvent is selected from the group comprising toluene, hexane, ethyl acetate and methanol.
13. A novel bacterial strain with accession number MTCC 5618, a process of producing methyl esters and a lipase enzyme substantially as herein described with reference to the accompanying drawings and as illustrated with the foregoing examples.