磷脂酶A1高产菌株的筛选及其等离子体诱变  

Screening and plasma mutagenesis of phospholipase A1 high-producing strain

作　　者：马琦亚[1] 薛正莲[1] 苏燕南[1] 王洲[1] 赵世光[1] 

机构地区：[1]安徽工程大学生物与化学工程学院微生物发酵安徽省工程技术研究中心,安徽芜湖241000

出　　处：《中国生物制品学杂志》2013年第11期1656-1662,共7页Chinese Journal of Biologicals

基　　金：安徽省自然科学基金项目(11040606M81);安徽省高校自然科学基金项目(KJ2009A168)

摘　　要：目的从富油土壤中筛选磷脂酶A1高产菌株,优化其发酵条件,并经等离子诱变提高其产酶能力。方法用筛选培养基和发酵培养基分别对富油土壤中磷脂酶A1产生菌进行初筛和复筛,经气相色谱鉴定产酶类型;对获得的高产菌株进行形态、生理生化特征和分子生物学鉴定;优化该菌株的发酵时间,温度,pH值及摇床转速;从该菌株出发进行等离子体诱变,绘制致死率曲线,进行平板初筛及摇瓶复筛,并检测获得菌株的遗传稳定性。结果经初筛和复筛,获得1株磷脂酶A1高产菌株W22,经气相色谱鉴定其产酶为磷脂酶A1;该菌株在普通琼脂培养基上呈圆形,质地软,表面光滑、扁平,乳白色,晕圈明显,为芽孢杆菌属蜡样芽孢杆菌;最佳发酵条件为:发酵时间12 h,初始pH 8.0,温度32℃,摇床转速200 r/min,优化后酶活为9.12 U/ml,较优化前(6.74 U/ml)提高了35.3%;等离子体诱变后,经平板及摇瓶筛选,获得的菌株W22-5酶活最高,为12.80 U/ml,较诱变前(9.12 U/ml)提高了89.91%,且具有良好的遗传稳定性。结论成功获得1株磷脂酶A1高产菌株W22,优化了该菌的发酵条件,且经等离子诱变进一步提高了菌株的产酶能力,为磷脂酶A1的生产和应用奠定了基础。Objective To screen a phospholipase AI high-producing strain from oil-rich soil, optimize its fermentation condition and improve its phospholipase Al-producing ability by plasma mutagenesis. Methods Phospholipase A1 producing-stains in oil-rich soil were primarily and further screened by using selective and fermentation media respectively, and identified for the type of enzyme by gas chromatography, of which the phospholipase A1 high-producing strain was analyzed for morphological, physiological and biological characters. The time, temperature, pH and rotation rate of shaker for fermentation of high-producing strain were optimized, and the strain was subjected to plasma mutagenesis, based on which a lethality curve was plotted. The mutant was preliminarily screened on plate and further screened in shake flask, and evaluated for genetic stability. Results A phospholipase A1 high-producing strain, named as W22, was screened, by which the enzyme produced was phospholipase A1. The strain was round and soft on common agar medium, of which the surface was smooth, flat and milky white in color, with apparent haloes, thus was identified as Bacillus cereus. The optimal time, initial pH value, temperature and rotation rate of shaker for fermentation were 12 b, 8. 0, 32 ℃ and 200 r/min respectively. Under the optimized condition, the phospholipase A1 activity was 9. 12 U/ml, which increased by 35.3% as compared with that before optimization （6. 74 U/ml）. The phopholipase A1 activity of strain W22-5 screened after plasma mutagenesis was 12. 80 U/ml, which increased by 89. 91% as compared with that before mutagenesis （9. 12 U/ml）. The strain also showed high genetic stability. Conclusion A phospholipase A1 high-producing strain W22 was successfully screened, of which the fermentation condition was optimized, and the phospholipase-producing ability was enhanced by plasma mutagenesis. It laid a foundation of production and application of phospholipase A1.

关 键 词：磷脂酶A1 筛选 等离子体 诱变 

分 类 号：Q556.1[生物学—生物化学] Q789