一株地芽胞杆菌属CKYG菌株的分类鉴定及产蛋白酶特性

Classification and Identification of CKYG Strain in Bacillus and its Protease Producing Properties

为了从经过高温烘烤过的烟草叶片中获得功能菌株,采用固体分离培养基,从豫烟10浓香型烟草品种烤烟叶片中分离获得一株内生细菌,编号为CKYG。结果表明:该菌株在牛奶琼脂鉴别培养基上产生明显的透明圈,发酵液的蛋白酶活性测定进一步确认CKYG菌株具有产蛋白酶活性功能。对温度、pH、接种量和种龄四个因素对CKYG产酶的影响进行了测定,结果显示,CKYG菌株最适宜的发酵产酶温度为40℃,适宜pH为7.0,适宜接种量为3%,适宜种龄为36 h;CKYG菌株可以耐受50℃的生长温度,故可定性为中温菌。通过综合CKYG菌株的形态特征、生理生化特征及16S r DNA序列的聚类分析结果,将其确定为土芽胞杆菌属Geobacillus菌株,暂命名为Geobacillus sp. CKYG,保藏于中国典型培养物保藏中心,保藏编号CCTCC:M2015116。RYXP菌株可作为蛋白酶开发、烤烟品质调控、烟杆废物发酵和堆肥发酵等应用领域的备选微生物资源。

To obtain functiona l strains from tobacco leaves that have been roasted over a high temperature,an endogenous bacteria from the tobacco leaves of Yuyan 10 thick aromatic tobacco varieties was isolated and obtained by the solid separation medium,its number was CKYG.Research results showed that the strain produced a clear and transparent ring on the agar identification medium of milk,and the activity of protease of the fermentation liquid was further determined that the CKYG strain had the function of protease-producing activity.The effects of temperature,pH,inoculation and seed age on CKYG enzyme production were measured and the results showed that the optimum fermentation enzyme production temperature for CKYG strain was 40℃,the optimum pH was 7.0,the optimum inoculation amount was 3%,and the optimum seed age was 36 h;CKYG could tolerate growth temperature of 50℃,so it could be identified as medium temperature strain.Based on the morphological characteristics,physiological and biochemical characteristics of CKYG strains and the results of cluster analysis of 16 S rRNA sequence,it was identified as the Geobacillus,temporarily named Geobacillus sp.CKYG,which was preserved in the China Center for typical culture collection,and its preservation number was CCTCC:M2015116.RYXP could be used as alternative microbial resources in the application of protease development,flue-cured tobacco quality control,tobacco rod waste fermentation and compost fermentation.