Cold active enzyme and method thereof
AssigneeUniversity Putra Malaysia
InventorsRAJA NOOR ZALIHA, ABU BAKAR SALLEH, MAHIRAN BASRI, MOHD SHUKURI BIN MOHAMAD ALI
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Abstract
The objective of the present invention is to obtain a low temperature producing enzyme and more particularly, the enzyme provides a bifunctional purpose of varying its enzyme activity into activity of another enzyme. The present invention has overcome problems such as ability of obtaining an enzyme at low temperature.
Claims17
A biologically pure culture of Leucosporodium antarcticum, is isolated from antarctic sea, wherein the Leucosporodium antarcticum is Leucosporodium antarcticum PI12 strain, deposited under the accession number NCYC number 3391 at the National Collection of Yeast Cultures (NCYC).
A process of producing a bifunctional enzyme, wherein the bifunctional enzyme isolated and characterized from Leucosporodium antarcticum PI12 strain, wherein the process includes the following steps:
- a) obtaining Leucosporodium antarcticum PI12 strain;
- b) screening the strain from step (a) for extracellular lipase activity at 4° C.;
- c) conducting a quantitative assay from step (b) for cold active lipase activity;
- d) conducting a quantitative assay from step (b) for protease activity;
- e) identifying and amplifying 16S rDNA sequence by conducting polymerase chain reaction (PCR) technique;
- f) obtaining a cold active lipase (LipPI12) gene by performing cloning (genomic library) technique;
- g) conducting intracellular expression of the cold active lipase gene and obtaining an expressed cold active lipase (LipPI12) gene;
- h) conducting extracellular expression of the cold active lipase gene and obtaining an expressed cold active lipase (LipPI12) gene;
- i) comparing between intracellular and extracellular of the LipPI12 from step (g-h);
- j) purifying intracellular and extracellular of the LipPI12 (i) and obtaining a purified LipPI12 lipase;
- k) determining size of protein;
- l) characterizing the cold active lipase (LipPI12) using biochemical and biophysical methods.
A novel bifunctional enzyme of cold adapted LipPI12 obtained from Leucosporodium antarcticum PI12 strain, wherein the cold adapted LipPI12 includes the following characteristics:
- a. Working temperature range and working temperature working temperature range from 5° C. to 40° C. with an optimum temperature at least 20° C. for lipase and 35-40° C. for protease
- b. Working pH
- having a working pH in the range of pH 4 to 12.
- c. Working metal ions, the metal ions includes Na+, Mg+, Ca 2+, Fe2+, Mn2+, K+, Zn2+ and Cu2+, wherein the metal ions having the capability to treat the LipP112 enzyme for at least 30 min at 20° C.
- d. Working substrate, the substrate includes triglycerides, p-nitrophenol esters and natural oils,
- wherein the triglycerides;
- triacetin, tributyrin, tricaprylin and triolein.
- wherein the p-nitrophenol esters includes;
- p-nitrophenylbutyrate, p-nitrophenylcaprylate, p-nitrophenyllaurate, p-nitrophenylpalmitate.
- wherein the natural oils include:
- olive oil, soy bean oil, corn oil, sun flower oil, rice bran oil and palm oil.
- e. Working inhibitors, the inhibitors includes:
- PMSF, EDTA and pepstatin wherein the inhibitors having the capability to treat the LipP112 enzyme for at least 30 min at 20° C.
- f. Working organic solvents, wherein the organic solvents include:
- hexadecane, benzene, dodecane, heptane, hexane, decane heptanes, dimethylsulphonyl, hexadecane, toluene, ethybenzene, diethylether.
- g. Working surfactant includes Tween 20-80, Triton X-100, SDS and SLS.
Dependencies are inferred from the claim text.