中温大曲中产淀粉酶芽孢杆菌的筛选鉴定及培养条件优化

Screening,identification and culture conditions optimization of amylase-producing Bacillus strain from medium-temperature Daqu

为了获得高产淀粉酶的芽孢杆菌(Bacillus sp.),该研究采用稀释平板法和划线法从中温大曲中分离纯化芽孢杆菌,通过透明圈法初筛和3,5二硝基水杨酸(DNS)法复筛,获得高产淀粉酶芽孢杆菌,并采用形态学观察和16S r RNA同源序列分析方法进行菌种鉴定。以淀粉酶活力为响应值,采用单因素试验、Plackett-Burman试验、最陡爬坡试验、Box-Behnken试验对筛选菌株的培养条件进行优化。结果表明,共分离纯化出72株芽孢杆菌,从中筛选得到一株淀粉酶活力最高的芽孢杆菌菌株,编号为ZTW17-6,经鉴定为贝莱斯芽孢杆菌(Bacillus velezensis);其最佳培养条件为乳糖50 g/L、蛋白胨60 g/L、接种量5%、发酵温度35℃、转速180 r/min、装液量76 m L/250 m L、初始pH值7。在此优化条件下,淀粉酶活力达到8 352.95 U/m L,是优化前的2.61倍。

To obtain high-yield amylase Bacillus sp.,the Bacillus sp.was isolated and purified from medium-temperature Daqu using dilution plate and streaking methods,and the high-yield amylase Bacillus sp.strain was pre-screened by transparent circle method and re-screened by 3,5 dinitrosalicylic acid(DNS) method.The screened strain was identified as by morphological observation and 16S r RNA homology sequence analysis method.Using amylase activity as response value,the culture conditions of screened strain were optimized by single factor experiments,Plackett-Burman experiments,steepest ascent experiments and Box-Behnken experiments.The results showed that a total of 72 strains of Bacillus sp.were isolated and purified.Among them,a strain of Bacillus sp.with the highest amylase activity was obtained,numbered as ZTW17-6,and identified as Bacillus velezensis.The optimal culture conditions were obtained as follows:lactose 50 g/L,peptone 60 g/L,inoculum 5%,fermentation temperature 35 ℃,rotation speed180 r/min,liquid volume 76 ml/250 ml,and initial pH 7.Under these optimal conditions,the amylase activity reached 8 352.95 U/ml,which was2.61 times that of before optimization.