PRODUCTION, PURIFICATION, AND IMMOBILIZATION OF RECOMBINANT CHITOSANASE (RCSN1) FROM BACILLUS AMYLOLIQUIFACIENS

• Main Author: Pratiwi, Septiani
• Format: Final Project
• Language: Indonesia
• Subjects: Kimia
• Online Access: https://digilib.itb.ac.id/gdl/view/47517
• Institution: Institut Teknologi Bandung

The shrimp market has occupied ten main export commodities in Indonesia. Based on data by Badan Pusat Statistik in 2002, Indonesia had exported 142,000 tons of shrimps whithin 60,000 tons dumped as a waste. On the other hand, shrimp waste consist of 17 – 20% (m/m) high-selling compounds called chitin. Chitin is polysaccharide of acetyl-D-glucosamine monomers. Chitin can be deacetylated into chitosan which has a high use value because of its characteristics and that it has a higher solubility than chitin. Chitosan can be hydrolized by chitosanase to produce chitooligosaccarides and D-glucosamine which is very beneficial in medical fields. This research aim to purify and immobilize recombinant chitosanase (rCsn1). The gen was obtained from marine bacterial isolate Bacillus amyloliquifaciens ABBD and expressed into the host cell Escherichia coli BL21 (DE3) utilize pET30-a(+) plasmid. Production parameters- optimization has been carried out for lysis buffer type, lysis method, IPTG concentration and induction duration. Optimal performance was shown by 50 mM phosphate buffer pH 6 with an addition of 50 mM NaCl on lysis buffer; combination of homogenizer-sonication on lysis methods; and 0,2 mM of IPTG concentration for induction during 4 hours incubation. Purification of rCsn1 has been carried out by cation exchange chromatography (CM-TOYOPEARL) and single band of ~27 kDa was observed through SDS-PAGE in fraction of 50 mM phosphate buffer pH 6 with an addition of 200 mM NaCl elution. Silica matrix has been modified by 3- Aminopropyl triethoxy silane (APTES) and Glutaraldehyde to immobilize recombinant chitosanase (rCsn1). Measurement of absorption at 500 nm wavelength to rCsn1 unimmobilized chitosanase (0.351); immobilized chitosanase (0.332); and supernatan (0.193) indicate rCsn1 has been integrated into the matrix and has the ability to hydrolize chitosan. This successfull rCsn1 immobilization approach opens up many opportunities for industries producing chitooligosaccaride.