产壳聚糖酶菌株ncps116发酵条件优化及其酶学性质

doi:10.16085/j.issn.1000-6613.2017-1553

化工进展 ›› 2018, Vol. 37 ›› Issue (06): 2354-2363.DOI: 10.16085/j.issn.1000-6613.2017-1553

产壳聚糖酶菌株ncps116发酵条件优化及其酶学性质

张翔, 张彦昊, 刘孝永, 辛雪, 王易芬, 陈蕾蕾, 周庆新, 赵双枝

山东省农业科学院农产品研究所, 山东省农产品精深加工技术重点实验室, 农业部新食品资源加工重点实验室, 山东济南 250100

收稿日期:2017-07-26 修回日期:2017-11-14 出版日期:2018-06-05 发布日期:2018-06-05
通讯作者: 赵双枝,副研究员,从事微生物发酵多糖、寡糖类物质应用研究。
作者简介:张翔(1982-),男,助理研究员。E-mail:modestzhangxiang@163.com。
基金资助:
国家科技支撑计划（2015BAD16B02）、山东省自然科学基金（ZR2016YL031，ZR2016YL022）、山东省农业科学院农业科技创新工程（CXGC2017A01，CXGC2017B06）及山东省农业重大应用技术创新课题（鲁财农指2015[16]）项目。

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

ZHANG Xiang, ZHANG Yanhao, LIU Xiaoyong, XIN Xue, WANG Yifen, CHEN Leilei, ZHOU Qingxin, ZHAO Shuangzhi

Institute of Agro-Food Science and Technology, Shandong Academy of Agricultural Sciences;Key Laboratory of Ago-Products Processing Technology of Shandong Province;Key Laboratory of Novel Food Resources Processing, Ministry of Agriculture, Jinan 250100, Shandong, China

Received:2017-07-26 Revised:2017-11-14 Online:2018-06-05 Published:2018-06-05

摘要/Abstract

摘要： 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（K_m）为11.10mg/mL，最大反应速率（V_max）为1.3μmol/（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。

关键词: 壳聚糖酶, 发酵条件优化, 分离纯化, 酶学性质, 蜡状芽孢杆菌

Abstract: 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,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 K_m and V_maxvalues were 11.10mg/mL and 1.3μmol/(min·mL). The chitosanase showed strong substrate specificity. Furthermore,the ncps116 chitosanase inhibited the mycelial growth of Aspergillus niger.

Key words: chitosanase, optimization of fermentation conditions, separation and purification, enzyme property, Bacillus cereus

中图分类号:

Q936

张翔, 张彦昊, 刘孝永, 辛雪, 王易芬, 陈蕾蕾, 周庆新, 赵双枝. 产壳聚糖酶菌株ncps116发酵条件优化及其酶学性质[J]. 化工进展, 2018, 37(06): 2354-2363.

ZHANG Xiang, ZHANG Yanhao, LIU Xiaoyong, XIN Xue, WANG Yifen, CHEN Leilei, ZHOU Qingxin, ZHAO Shuangzhi. Optimization of fermentation conditions of the chitosanase-producing strain ncps116 and enzymatic properties of the chitosanase[J]. Chemical Industry and Engineering Progress, 2018, 37(06): 2354-2363.

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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

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