产壳聚糖酶菌株ncps116发酵条件优化及其酶学性质

摘要

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)2SO430g/L,the initial pH 6.0,32℃ incubating temperature,culivation for 72h,500mL flask containing 120mL medium and inoculation 4%. The chitosanase activity of the fermented broth is 43.89U/mL 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 of the 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 Mn2+stimulated the chitosanase activity significantly. However,5mmol/L Cu2+,Ni2+,Fe3+and Ag+exhibited different degrees of inhibitory activities. The Kmand Vmaxvalues were 11.10mg/mL and 1.38μmol/(min·mL). The chitosanase showed strong substrate specificity. Furthermore,the ncps116 chitosanase inhibited the mycelial growth of Aspergillus niger.%通过形态学观察、生理生化实验及16S rDNA 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pH 6.0,温度32℃,发酵时间72h,500mL三角瓶装液量120mL,接种量4%,在此条件下该菌株产壳聚糖酶活力达43.89U/mL.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pH和最适反应温度分别为5.6和50℃,在低于40℃、pH 3.6~5.6范围内较为稳定,5mmol/L 的 Mn2+对该酶酶活力有明显的增强作用,Cu2+、Ni2+、Fe3+、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(Km)为11.10mg/mL,最大反应速率(Vmax)为1.38μmol/(min·mL),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.