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Suharsono Suharsono

Bogor Agricultural University

Author-ID : 3965109

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)

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.

Partial Purification, Characterization, and Application of Extracellular Aspartic Protease from Lactobacillus casei WSP in Producing the Bioactive Peptides with Antibacterial and Antioxidant Activity Akhmad Solikhin; Apon Zaenal Mustopa; Suharsono Suharsono; Wendry Setiyadi Putranto
ANNALES BOGORIENSES Vol 22, No 2 (2018): Annales Bogorienses
Publisher : Research Center for Biotechnology - Indonesian Institute of Sciences (LIPI)

Lactobacillus casei WSP-derived an aspartic protease was sequentially purified by using chromatography gel filtration sephadex G-50. It resulted in a 22.81-fold increase of specific activity (51.5 U/mg) with a final yield of 1.9%. The estimated molecular weight of the purified enzyme was 37 kDa and showed gelatinolytic activity in zymogram assay. The enzyme exhibited optimum activity at 40ºC and pH 6 with casein as the substrate. Enzyme activity was significantly inhibited by pepstatin A (0.5 mM and 1 mM), confirming that this enzyme is a group of aspartic proteases, while other inhibitors such as EDTA, PMSF and iodoacetic acid showed no inhibition effect on the activity of enzyme. The addition of metal ion to the enzyme decreased enzyme activity, indicating the proteolytic enzyme was metal ion- dependent. Denaturant such as DDT tended to increase caseinolytic activity. Furthermore, this enzyme was capable of generating the new peptides from skimmed milk with the size 8 kDa, 10 kDa and 15 kDa. These peptides have potential as antibacterial and antioxidant agents.

Co-Authors Akhmad Solikhin Apon Zaenal Mustopa Asrul Muhamad Fuad Febriana Dwi Wahyuni Wendry Setiyadi Putranto

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