壳聚糖酶的分离纯化及性质研究

Purification and Characterization of a Chitosanase from Strain YJ02

以本实验室分离的产壳聚糖酶YJ02菌株出发,制备壳聚糖酶发酵液,经离心、(NH4)2SO4分级盐析、Q-SepharoseFast Flow阴离子交换、Sephacry S-100凝胶过滤分离纯化步骤,SDS-PAGE显示为一条带,壳聚糖酶纯化了28.9倍,收率为47.6%。该酶的性质研究表明,该酶的分子量为66.2KD,最适反应温度为50℃,最适反应pH为6.0;Mn2+对酶活力有明显的促进作用,Cu2+,Fe3+,Hg2+对酶活力有抑制作用;小分子有机物EDTA和SDS对酶活力有抑制作用,而IAA和β-巯基乙醇对酶活力稍有促进作用。该壳聚糖酶对高脱乙酰度壳聚糖底物水解作用强,酶解位点可能为NGlc-NGlc,酶最大反应速度Vmax=4.1μmol/min,常数Km为64mmol/L。此壳聚糖酶水解壳聚糖制备的壳寡糖数均分子量为2 400Da。

The purification of chitosanase was conducted from fermented broth of bacterial strain YJ02. The enzyme purification procedures include centrifugation, Q-sepharose Fast Flow ion exchange and Sephacry S-100 gel filtration chromatography. The purification fold of the chitosanase was 28.9 and the its yield was 47.6 %. The molecular weight of the ehitosanase determined by SDS-PAGE was 66.2kD. The optimum pH and temperature for the enzymatic catalysis activity were pH = 6.0 and 5012, respectively. Mn2 ＋ enhanced the enzyme activity, whereas Cu^2 ＋ , Fe^3 ＋ and Hg^2 ＋ inhibited the activity. The chitosanase activity was strongly inhibited by EDTA and SDS, but IAA and 2-mercaptoethanol enhanced its activity. The chitosanase showed chitosan sub- strate specificity. The kinetic parameter Km was 64mmol/L and Vmax was 4. 1μmol/min. The chitosanase hydrolyzing chitosan to produce chitooligosaecharides was studied. The average molecular weight of the ehitooligosaccharides was 2 400Da.