一株海洋高产壳聚糖酶菌株Renibacterium sp．QD1的筛选鉴定及产酶研究

Screening,identification and research on enzyme production of the chitosanase producing marine bacteria Renibacterium sp. QD1

目的:获得高产壳聚糖酶菌株。方法:利用唯一碳源法富集产壳聚糖酶菌株,并利用平板水解圈和酶活力检测进行复筛。利用PCR克隆16S rDNA基因进行种属鉴定。利用SDS-PAGE凝胶原位复性鉴定野生菌株产壳聚糖酶的数量和大小。利用单一变量法进行发酵条件的初步优化。结果:获得了一株高产壳聚糖酶菌株,对该菌株的16S rDNA序列进行比对和分析发现该菌株属于Renibacterium属,将该菌命名为Renibacterium sp.QD1。SDS-PAGE电泳和凝胶原位复性结果显示,该菌株能产生一种胞外壳聚糖酶,分子量大小在25ku左右。对其发酵条件进行初步优化,用成分为壳聚糖(0.50%)、KH2PO4(0.10%)、K2HPO4(0.20%)、MgSO4(0.07%)、NaC(l0.10%)、CaCl2(0.01%)、酵母粉(0.05%)、马铃薯浸粉(%)、蛋白胨(0.2%)的培养基发酵,其粗酶活力可达400U/mL,比文献报道的活力最高的Microbacterium sp.OU01高近4倍。结论:获得了一株高产壳聚糖酶菌株,该菌株的发现为壳寡糖的制备提供了新的工具。

Objective：This study was carried out to obtain high yield chitosanase producing strain. Methods： Chitosanase producing strains were enriched by sole carbon source,and rescreened by observing hydrolysis circle on chitosan plate and assaying culture supernatant enzyme activity. The 16S rDNA gene was amplified by polymerase chain reaction（PCR） for strain identification. SDS-PAGE gel in situ refolding was used for crude enzyme analysis. Single-factor method was adopted to optimizing the fermentation parameters to maximize chitosanase activity. Results：Strain QD1 with high chitosanase activity was isolated from marine environment. Based on the 16S rDNA sequence,QD1 was assessed to be Renibacterium sp.,and was thus named Renibacterium sp.QD1. A chitosanase with the molecular weight of about 25ku was observed on refolded SDS-PAGE gel. After optimization,the yield of chitosanase activity reached up to 400U/mL,which was nearly four times of the previous reported chitosanase from Microbacterium sp. QD1. The fermentation medium including chitosan （0.50%）,KH2PO4 （0.10%）,K2HPO4 （0.20%）,MgSO4 （0.07%）,NaCl （0.10%）,CaCl2 （0.01%） ,yeast extract（0.05% ）, potato starch （%） and peptone （0.2%）. Conclusion .A strain with high chitosanase activity was isolated from marine environment which provided a new tool for chitooligosaccharides preparation.