Process for the preparation of acidic lipase

Details Process for the preparation of acidic lipase Process for the preparation of acidic lipase

2001-03-13

2003-03-18

Meller, Michael V. (Department: 1651)

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Hydrolase

C435S254300, C435S917000, C424S093500, C424S094600

Reexamination Certificate

active

06534303

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to a process for the preparation of an acidic lipase. More particularly, the present invention relates to the production of thermostable and acid stable lipase using
Aspergillus niger.
BACKGROUND OF THE INVENTION
Research on microbial lipases has increased in recent years because of their practical application in industry in the hydrolysis of fats, production of fatty acids and food additives, synthesis of esters and peptides, resolution of racemic mixtures or as additives for detergents. [Bjorkling F., Godtfredsen S. E., and Kirk O., (1991), Trends Biotechnol. 9, 360-363]. These enzymes are widely distributed in filamentous fungi [Sugihara A., Shimada Y., and Tominaga Y., (1990), J. Biochem. 107, 426-430; Torossian K. and Bell A. W. (1991) Biotechnol. Appl. Biochem. 13, 205-211; Yadav R. P., Saxena R. K., Gupta R., and Davidson W. S., (1998) Biotechnol. Appl. Biochem. 28, 243-249], yeasts [Kalkote U. R., Joshi R. A., Ravindranathan T., Bastawade K. B., Patil S. G., and Gokhale D. V., (1992) Biotechnol. Lett. 14, 785-788; Valero F., Ayats F., Lopez-Santin J., and Poch M. (1998) Biotechnol. Lett. 10, 741-744; Dalmau E., Montesions J. L., Lotti M. and Casas C., (2000), Enzyme Microb. Technol. 26, 657-663] and bacteria [Jaeger K. E., Ransae S., Dijkstra B. W., Colson C., van Heuvel M. S. and Misset O. (1994) FEMS Microbiol. Rev. 15, 29-63; Jaeger K. E., Dijkstra B. W. and Reetz M. T., (1999) Ann. Rev. Microbiol. 53, 315-351].
Filamentous fungi are preferred sources of lipase since they secrete the enzymes extracellularly. The most productive strains known till date belong to the genera Rhizopus, Mucor, Geotrichum, Penicillium and Aspergillus [Bjorkling F., Godtfredsen S. E., and Kirk O., (1991), Trends Biotechnol. 9, 360-363]. An acid resistant lipase preparation active between pH 4.5-5.5 was reported from
Aspergillus niger
[Torossian K. and Bell A. W. (1991) Biotechnol. Appl. Biochem. 13, 205-211]. Lipases active at highly acidic pH's have not been reported so far from microbial sources. Such acidic lipases have potential applications in the food industry. It is therefore desirable to obtain such acidic lipases which are active at highly acidic pH from microbial sources.
OBJECTS OF THE INVENTION
The main object of the invention is to provide a process for the preparation of acidic lipase which is active even at highly acidic pH from microbial sources.
It is another object of the invention to provide a process for the preparation of acidic lipase which is active even at highly acidic pH using
Aspergillis niger.
SUMMARY OF THE INVENTION
Accordingly the present invention provides a process for the preparation of acidic lipase, said process comprising growing
Aspergillus niger
sp. in a conventional fermentation medium containing carbon and nitrogen sources along with conventional nutrients for a period in the range of 72-96 hours at a temperature in the range of 25° C. to 35° C. under agitation, separating the fungal biomass and recovering the culture filtrate/broth and separating the lipase enzyme.
In one embodiment of the invention, the fungal strain used is isolated from decaying wood and is deposited at the National Collection of Industrial Microorganisms (NCIM), Biochemical Sciences Division, National Chemical Laboratory, Pune 411 008, India and designated as
Aspergillus niger
NCIM 1207.
In another embodiment of the invention, the dry mycelium of
Aspergillus niger
is prepared after harvesting the growth of the fungal strain, washing the mycelium with distilled water followed by washing with chilled acetone, drying the acetone treated mycelium under vacuum for 6-10 hours to remove acetone and water.
In a further embodiment of the invention, a CELITE (diatomaceous earth) bound (extracellular) enzyme is prepared by adding CELITE (diatomaceous earth) 545 (1 gm) to culture filtrate (20 ml) with mixing, ice cold acetone (25 ml) added to the suspension over a period of 5 minutes while stirring, the resultant suspension stirred for another 30 minutes using a magnetic stirrer at 0° C., filtered and dried.
DETAILED DESCRIPTION OF THE INVENTION
The dry mycelium of
Aspergillus niger
is prepared after harvesting the growth of the fungal strain, washing the mycelium with distilled water followed by washing with chilled acetone, drying the acetone treated mycelium under vacuum for 6-10 hours to remove acetone and water. The vacuum dried mycelial preparation was used for the estimation of cell bound (intracellular) activity on the basis of formation of esters.
CELITE (diatomaceous earth) bound (extracellular) enzyme is prepared by adding CELITE (diatomaceous earth) 545 (1 gm) to culture filtrate (20 ml) with mixing. Ice cold acetone (25 ml) was then added to the suspension over a period of 5 minutes while stirring, the resultant suspension stirred for another 30 minutes as source of extracellular enzyme. The extracellular enzyme activity was measured on the basis of formation of butyl esters.
Aspergillus niger
NCIM 1207 was used. Fermentation was done under submerged conditions. Lipase enzyme was produced by growing
Aspergillus niger
strain on a conventional growth medium such as MGYP (malt extract 0.3%; glucose 5.0%; yeast extract 0.3%; peptone 0.5% and Agar 2.0%) for 8-10 days at 25-30° C. The fermentation medium used was selected from MGYP liquid medium and basal oil based (BOB) medium (NaNO
3
0.05%; MgSO
4
.7H
2
O 0.05%; KCl 0.05%; KH
2
PO
4
0.2%; yeast extract 0.1%; peptone 0.5%; and olive oil 2.0%). All the media were sterilised at 15 lbs for 20 minutes. The pH of the media was adjusted to 5.5 prior to sterilisation. Resultant fermentation medium was inoculated with spores (10
8
-10
9
) from fully grown agar slope and was incubated at 25-30° C. for 72-96 hours with shaking at 150-180 rpm. Biomass was separated by known methods such as filtration to recover the broth and lipase activity was estimated by pNPP assay or on the basis of formation of butyl esters (indicative of lipase activity).


REFERENCES:
patent: 78201 (1969-07-01), None
NCIM Catalogue of Fungi, 2002, pp. 69-84.*
Hawley's Condensed Chemical Dictionary, Edited by Lewis, 1997, 13th Edition, pp. 225, 350 and 351.*
Manual of Industrial Microbology and Biotechnology, Edited by Demain et al., 1986, p. 103.

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