Purification And Characterization Of Chitosanase

This dissertation investigates the whole process of separation, purification, and characterization of chitosanases, as well as the preparation of 50 kDa chitosan by mixed chitosanase and endo-chitosanase respectively.The first part of this dissertation emphasizes on exploring the process to separate and purify chitosanases from Penicillium sp. ZD-Z1 fermentation liquids. The concentration of crude enzyme liquids was carried out using one ultrafiltration membrane reactor with MWCO 2 kDa. The organic solvent precipitation method was then performed using cold ethanol. In the IEC step, strong cation exchange and anion exchange were compared, and the former was chosen as preferable. Two chitosanases ChA and ChB were isolated from the crude enzyme liquids by IEC, and ChA was further purified by Sephadex G-50 gel filtration. At last, the yield of ChA was 10.5%, and purification fold was 20.8 with specific activity of 475.5 U/mg at 50 °C; the yield of ChB was 9.4%, and purification fold was 2.9 with specific activity 67.5 U/mg at 50 ℃.The second part presents the characterization of the two purified chitosanases. The isoelectric points of ChA and ChB were obtained as 5.21 and 5.92 by isoelectrofocusing. The relative molecular weights of ChA and ChB were 64 kDa and 74 kDa by Sephacryl S 100 gel filtration, and 43 kDa and 115 kDa by SDS-PAGE. During 30 minutes reaction time, with the substrate of 1% chitosan solution, ChA represented the highest activity at pH 5.0 and 65 ℃, while ChB showed the highest activity at pH 5.5 and 60 ℃. ChA manifested higher thermal stability under 50 ℃, and could hold more than 75% activity after two hours at 50 ℃. ChB could still retain over 90% activity after two hours at 55 ℃. Eight kinds of metal ions could accelerate ChA hydrolysis process at the concentration of 1 mM and 0.1 mM, while the presence of most metal ions could inhibit ChB action except for Co2+. Increasing the deacetylation (DA) of the chitosan resulted in increasing the activities of both of the two chitosanases. ChA was identified as an endo-chitosanase, and ChB was an exo-chitosanase using paper chromatography and HPLC to test their hydrolysis products.The last part refers to the preparation of 50 kDa chitosan by mixed chitosanase and endo-chitosanase respectively. The method to determine the relative weight average molecular mass was established. The relationship between theintrinsic viscosity of chitosan solution and its relative weight average molecular mass was described by MHS equation as followed: = 3.72 X 10-5 Mw 1.37. The preparation of 50 kDa chitosan was first performed by mixed chitosanase hydrolysis. This method was relatively simple and would cost 50 hours or so, with the yield of 70%. Alternatively, endo-chitosanase ChA was applied to prepare 50 kDa chitosan, and the degradation course could be monitored by rotational viscosimeter. 500 ml of 4% chitosan solution would be degraded into 50 kDa chitosan within 20 hours with the yield of 100%.