OPTIMIZATION OF CHITOSAN HYDROLYSIS BY RECOMBINANT CHITOSANASE FROM BACILLUS AMYLOLIUEFACIENS ABBD FOR ANTIBACTERIAL SCREENING

• Main Author: MONICA ANDRIANI NIM:10514036, LUTTYA
• Format: Final Project
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/28682
• Institution: Institut Teknologi Bandung

Indonesia is an archipelago country that has abundant fishery resources. One of them is a <br /> <br /> group of crustaceans such as shrimp and crab. Based on statistical data from the Ministry <br /> <br /> of Marine Affairs and Fisheries of Indonesia, production of shrimp in Indonesia has <br /> <br /> reached 645,000 tons/year and with 56,200 tons/year waste. The wastes of shrimp shell <br /> <br /> contain of 17–20% chitin. Chitosan (derived from deacetylation of chitin) can be utilized <br /> <br /> to be a high-value material for industrial purposes. Hydrolysis of &#946;-1,4-glycosidic bond of <br /> <br /> chitosan using chitosanase (EC 3.2.1.132) produces chitooligosaccharides. This <br /> <br /> compound has many advantages for industrial use such as water-soluble and has various <br /> <br /> bioactivity including antibacterial, antifungal, anti-inflammatory, and antidiabetic. The <br /> <br /> aims of this study are to produce and purify recombinant chitosanase (rek-Csn) from <br /> <br /> marine bacteria Bacillus amyloliquefaciens ABBD on Escherichia coli BL21 (DE3), and <br /> <br /> to analyze hydrolysis chitosan by rec-Csn product. Cell that contain rec-Csn were cultured <br /> <br /> in Lysogeny Broth media with the addition of isopropyl &#946;-D-1-thiogalactopyranoside <br /> <br /> (IPTG) after the cell reached a degree of turbidity (OD600) a number of 0.6–0.8. Cultured <br /> <br /> cells were incubated 37 °C for 4 hours. Cell pellets are then lyzed and the supernatant is <br /> <br /> purified by cation exchange chromatography (CM TOYOPEARL). The analysis result of <br /> <br /> Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) showed the <br /> <br /> rec-Csn that protein produced was pure and had a molecular mass of ~27 kDa. The rec- <br /> <br /> Csn protein has a specific activity of 1594,86 U/mg at pH 6 and a temperature of 50 °C. <br /> <br /> The product of chitosan hydrolysis by rec-Csn produces chitooligosaccharides which has <br /> <br /> antibacterial bioactivity.