CLONING AND SECRETIVE EXPRESSION OF BACILLUS THERMOCATENULATUS BTL2 LIPASE IN PICHIA PASTORIS USING NATURAL AND YEAST Α FACTOR SIGNAL SEQUENCES

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VAFA M.; YAKHCHALI B.; HAGHNAZARI A.; KARKHANEH A.A.; RASTGAR JAZI F.; AHMADI DANESH H.

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Journal: IRANIAN JOURNAL OF BIOLOGY Year:2011 | Volume:24 | Issue:5 Page(s): 640-647

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Title

CLONING AND SECRETIVE EXPRESSION OF BACILLUS THERMOCATENULATUS BTL2 LIPASE IN PICHIA PASTORIS USING NATURAL AND YEAST Α FACTOR SIGNAL SEQUENCES

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Keywords

BACILLUS THERMOCATENULATUSQ2

PICHIA PASTORISQ2

RECOMBINANT LIPASEQ2

SECRETIONAL EXPRESSIONQ2

Abstract

Lipase (Triacylglycerol acylhydrolases, EC 3.1.1.3) hydrolyzes triacylglycerol to glycerol and fatty acids in the water and fatty acid interface. They have many applications in various industries including food, dairy, pharmaceutical, agrochemical, oleochemical and mainly in detergents. In this study btl2 gene (with and without signal sequence) was amplified from purified genomic DNA of BACILLUS THERMOCATENULATUS using primers designed based on the sequence in the GeneBank data base (GenBank X95309). PCR products were cloned in pGEM5zf vector. Then the genes were sub cloned into the pPIC9 expression vector under alcohol oxidase (AOX1) promoter (pYV128, pYV129). Molecular and restriction enzyme analysis confirmed cloning accuracy. The recombinant plasmids were linearized with BglII enzyme and transferred into PICHIA PASTORIS host cells with electroporation. Finally, recombinant yeasts were cultured in BMGY medium and induced with methanol. Para nitrophenyl palmitate (pNPP) test and Immunoblotting method showed presence of the RECOMBINANT LIPASEs in medium culture. The RECOMBINANT LIPASE was purified by ion exchange chromatography method and the specific activity was measured. The results showed that the RECOMBINANT LIPASE production was 18000U/L in flask culture, which 12000U/L purified RECOMBINANT LIPASE with 5728U/mg specific activity was recovered with this purification method.

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APA: Copy

VAFA, M., YAKHCHALI, B., HAGHNAZARI, A., KARKHANEH, A.A., RASTGAR JAZI, F., & AHMADI DANESH, H.. (2011). CLONING AND SECRETIVE EXPRESSION OF BACILLUS THERMOCATENULATUS BTL2 LIPASE IN PICHIA PASTORIS USING NATURAL AND YEAST Α FACTOR SIGNAL SEQUENCES. IRANIAN JOURNAL OF BIOLOGY, 24(5), 640-647. SID. https://sid.ir/paper/21290/en

Vancouver: Copy

VAFA M., YAKHCHALI B., HAGHNAZARI A., KARKHANEH A.A., RASTGAR JAZI F., AHMADI DANESH H.. CLONING AND SECRETIVE EXPRESSION OF BACILLUS THERMOCATENULATUS BTL2 LIPASE IN PICHIA PASTORIS USING NATURAL AND YEAST Α FACTOR SIGNAL SEQUENCES. IRANIAN JOURNAL OF BIOLOGY[Internet]. 2011;24(5):640-647. Available from: https://sid.ir/paper/21290/en

IEEE: Copy

M. VAFA, B. YAKHCHALI, A. HAGHNAZARI, A.A. KARKHANEH, F. RASTGAR JAZI, and H. AHMADI DANESH, “CLONING AND SECRETIVE EXPRESSION OF BACILLUS THERMOCATENULATUS BTL2 LIPASE IN PICHIA PASTORIS USING NATURAL AND YEAST Α FACTOR SIGNAL SEQUENCES,” IRANIAN JOURNAL OF BIOLOGY, vol. 24, no. 5, pp. 640–647, 2011, [Online]. Available: https://sid.ir/paper/21290/en

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