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Asrul Muhamad Fuad
Indonesia Institute of Sciences
Agriculture, Biological Sciences & Forestry Biochemistry, Genetics & Molecular Biology Chemical Engineering, Chemistry & Bioengineering Chemistry Immunology & microbiology Medicine & Pharmacology

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Constitutive Expression of Candida antarctica Lipase B (CALB) in Pichia pastoris Using pGAPZα Vector Febriana Dwi Wahyuni; Asrul Muhamad Fuad; Suharsono Suharsono
ANNALES BOGORIENSES Vol 20, No 1 (2016): Annales Bogorienses
Publisher : Research Center for Biotechnology - Indonesian Institute of Sciences (LIPI)

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (461.586 KB) | DOI: 10.14203/ann.bogor.2016.v20.n1.31-38

Abstract

The CalBsyn gene was previously constructed synthetically to encode Candida antarctica lipase B (CALB). Lipase from CalBsyn gene is slightly different from that of wild type CALB (CALB-wt) where it has three amino acids substitutions at different positions, i.e. V210I, A281E, and V221D, to improve enzyme’s thermostability and catalytic efficiency. The CalBsyn gene was isolated from pJ912-CalBsyn vector by digestion using XhoI restriction enzyme. The CalBsyn gene then was ligated to pGAPZα expression vector and transformed into E. coli TOP10F’ in order to obtain recombinant vector pGAPZα-CalBsyn. The result showed that pGAPZα-CalBsyn recombinant vector was successfully transformed into E. coli TOP10F’ with transformation efficiency of 4.11 x 103 cfu/µg plasmid DNA. The pGAPZα-CalBsyn recombinant plasmid was successfully introduced into Pichia pastoris SMD1168H using electroporation method with transformation efficiency of 1.01 x 102 cfu/µg DNA. Qualitative lipase activity assays showed that transformed P. pastoris secreted recombinant lipase (CALB) and has lipolytic activity; while quantitative lipase activity assays showed that the lipase activity was 63.5 Units/ml in 48 hours. Analysis using SDS-PAGE showed that CALB protein was expressed successfully and the recombinant protein’s molecular size was approximately 45 kDa.
Co-Authors Febriana Dwi Wahyuni Suharsono Suharsono