产壳聚糖酶菌株ncps116发酵条件优化及其酶学性质

Optimization of fermentation conditions of the chitosanase-producing strain ncps116 and enzymatic properties of the chitosanase

通过形态学观察、生理生化实验及16S r DNA序列分析鉴定产壳聚糖酶菌株ncps116为蜡状芽孢杆菌(Bacillus cereus)。通过单因素和正交实验对该菌株发酵产酶条件进行了优化。结果表明其最适产酶条件为:粉末壳聚糖15g/L,硫酸铵30g/L,初始p H 6.0,温度32℃,发酵时间72h,500m L三角瓶装液量120m L,接种量4%,在此条件下该菌株产壳聚糖酶活力达43.89U/m L。经硫酸铵沉淀、DEAE-Sepharose Fast Flow离子交换层析对菌株发酵液中的壳聚糖酶进行了纯化,并对其酶学性质进行了初步研究。结果表明,壳聚糖酶经SDS-PAGE分析,其分子量为4.37万。该酶酶促反应最适p H和最适反应温度分别为5.6和50℃,在低于40℃、p H 3.6~5.6范围内较为稳定,5mmol/L的Mn^(2+)对该酶酶活力有明显的增强作用,Cu^(2+)、Ni^(2+)、Fe^(3+)、Ag^+对该酶酶活力有不同程度的抑制作用。壳聚糖酶酶促反应的米氏常数(K_m)为11.10mg/m L,最大反应速率(V_(max))为1.38μmol/(min·m L),对底物表现出较强的专一性。此外,该酶能够抑制黑曲霉(Aspergillus niger)菌丝的生长。

The chitosanase-producing strain ncps116 was identified as Bacillus cereus based on morphological,physiological and biochemical characteristics and 16S rDNA sequence analysis.The fermentation conditions of ncps116 for chitosanase prodution were optimizied by one-facor-at-a-time and orthogonal array designs.The results showed that the optimal cultivation for ncps116 producing chitosanase were powder chitosan 15g/L,（NH4）2SO4 30g/L,the initial pH 6.0,32℃incubating temperature,culivation for 72h,500m L flask containing 120m L medium and inoculation 4%.The chitosanase activity of the fermented broth is 43.89U/m L when ncps116 was incubated under aforementioned conditions.The chitosanase of the fermented broth was extracted by ammonium sulfate precipitation,and purified by DEAE-Sepharose Fast Flow chromatography.The molecular weight ofthe purified chitosanase was estimated to be 43700 by SDS-PAGE. The best chitosanase activity existed at pH 5.6 and 50℃. The chitosanase was stable below 40℃within the pH range from 3.6—5.6. The addition of 5 mmol/L Mn^2＋ stimulated the chitosanase activity significantly. However,5 mmol/L Cu^2＋,Ni^2＋,Fe^2＋ and Ag^＋ exhibited different degrees of inhibitory activities. The Km and V（max） values were 11.10 mg/m L and 1.38μmol/（min·m L）. The chitosanase showed strong substrate specificity. Furthermore,the ncps116 chitosanase inhibited the mycelial growth of Aspergillus niger.