1株耐高温产蛋白酶细菌的筛选鉴定及其液体发酵条件的优化

Screening and Identification of A High Temperature Resistance Proteaseproducing Bacteria and Optimization of Its Liquid Fermentation Condition

为高效发酵鸡粪,在鸡粪发酵高温时期（≥50℃）筛选到11株菌株,确定其中1株高温细菌GX7具有较高的蛋白酶活力。经16S r DNA测序并进行同源性比较可知,GX7与枯草芽孢杆菌（Bacillus subtilis）的16S r DNA序列相似性达到100%;通过构建系统发育进化树,结合生理生化试验,鉴定GX7为枯草芽孢杆菌。通过绘制细菌生长曲线、测定细菌蛋白酶活力可知,细菌GX7的蛋白酶活力与菌数呈强正相关。从发酵培养基的组成及菌株的培养条件等方面优化枯草芽孢杆菌GX7产蛋白酶的条件,结果显示,最优培养基配方为：酵母浸膏1.5%、玉米粉1.5%、MgSO4 0.5%;最优培养条件为：p H值8.1、培养温度37℃、接菌量2%、装液量50 m L（250 m L）、摇床转速140r/min。在此条件下,活菌数提高1个数量级,达到2.56×10^9cfu/m L,蛋白酶活力提高约3倍,为26 U/m L。

To improve chicken manure fe rme ntation, 11 stra ins were screened in chicken manure fermentat ion during the period of high temperature （ ≥50 ℃） , of which thermophilic bacteria GX7 with high protease activity by 16S rDNA sequencing and homology comparison, the sequence homology of GX7 with Bacillus subtilis of 1 00% . GX7 was identified as Bacillus subtilis by phylogen etic tree and combined with physiological and biochemical tests. By drawing the bacterial growth curve and determining the bacterial protease activity, i t could be known that the protease activity of GX7 was strongly positively correlated with the number of bacteria. By optimization of cul ture medium composition and strain cul ture conditions, the optimal medium was yeast extract of 1.5% , corn powder of 1.5% , MgSO4 of 0. 5% ;The optimum cul ture condition was pH value of 8. 1 ,culture temperature of 37℃ , inoculation amount of 2% , liquid volume of 50 mL （ 250 mL） , shaker speed of 140 r/min. Under these conditions, the number of viable bacteria increased 10 times to 2. 56 ×10^9 cfu/mL ,and the protease activity increased three times to 26 U/mL.