Production and Purification of Recombinant Chitosanase (Csn1) from Bacillus amyloliquefaciens ABBD Expressed in Pichia pastoris GS115

• Main Author: (NIM : 10513015), RENDY
• Format: Final Project
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/30316
• Institution: Institut Teknologi Bandung

<p align="justify">Chitosan is a linear polysaccharide formed from randomly distributed D-glucosamine and N-acetyl-D-glucosamine. Hydrolysis of &#946;-1,4-glycosidic bonds of chitosan using chitosanase (EC 3.2.1.132) produces chitooligosaccharides that have several medical activities, such as to lower blood pressure, to lower blood cholesterol, to boost the immune system, and as an antitumor. The aims of this study were to produce, purify, and characterize chitosanase (Csn1) from the marine bacterium Bacillus amyloliquefaciens ABBD which was expressed in Pichia pastoris GS115. The Csn1 coding gene was under the control of AOX1 promoter requiring methanol as inducer and the coding region was fused with the MF prepro-&#945; sequence signal to produce an extracellular Csn1 protein. The production of recombinant chitosanase in P. pastoris GS115/pPICZ&#945;A-csn1 was conducted for 24 hours by adding 2% (v/v) methanol. The specific activity of recombinant Csn1 was 151 U/mg. The culture supernatant containing Csn1 recombinant was concentrated by precipitation with 80% saturated ammonium sulfate and then purified by gel filtration chromatography using HiLoad 16/60 Superdex 200 prep grade column. The SDS-PAGE analysis showed that the recombinant Csn1 protein had a molecular mass of ~35 kDa. Specific activity of purified Csn1 was 600 U/mg at optimum of pH 5.5, and temperature of 50 oC.<p align="justify">