桔青霉产核酸酶P1菌种高效选育方法研究  

Screening of Nuclease P1-Producing Strain with High Efficiency from Penicillium citrinum

作　　者：郑美娟[1] 郭金玲[1] 田毅红[1] 雷生姣[1] 吕育财[1] 余华顺[2] 姚鹃[2] 王栋 龚大春[1] 

机构地区：[1]三峡大学湖北省生物酵素工程技术研究中心,湖北宜昌443002 [2]安琪酵母股份有限公司酶制剂事业部,湖北宜昌443002 [3]车用生物燃料技术国家重点实验室,河南南阳473000

出　　处：《化学与生物工程》2018年第2期33-37,共5页Chemistry & Bioengineering

基　　金：湖北省重大科技攻关项目(2009BCB009);宜昌市科技攻关项目(A2015-001)

摘　　要：为了提高产核酸酶P1菌种的筛选效率,研究了甲苯胺蓝-RNA(TB-RNA)平板筛选方法 ,考察了溶剂体系、甲苯胺蓝浓度、底物RNA浓度等对TB-RNA平板上核酸酶P1酶解圈的影响。结果表明,在甲苯胺蓝浓度为2.0×10^(-4)mol·L^(-1)、底物RNA浓度为0.1%时,于超滤水体系中29℃恒温培养,紫外诱变12h,核酸酶P1的酶活与酶解圈直径呈正相关关系。以酶解圈与菌落直径比1.71为指标,从116株诱变菌株中筛选出10株菌株;经过后期发酵验证,选育出6株优势菌株;通过发酵及遗传稳定性考察,6#菌株的酶活最高,为390U·mL^(-1),比原始菌株提高了29%。为工业化生产快速选育菌株提供了高效方法。In order to improve screening efficiency of nuclease P1-producing strain from Penicillium citrinum,we studied TB-RNA plate screening method,and investigated the effects of the solvent system,toluidine blue concentration,and substrate RNA concentration on the pink enzymolysis circle.The results show that enzyme activity of nuclease P1 is positively correlated with the diameter of the enzymolysis circle under the optimal conditions as follows：toluidine blue concentration is 2.0×10-4 mol·L-1,substrate RNA concentration is 0.1%,incubation temperature is 29 ℃in ultra filtration water system,and UV-mutagenesis time is 12 h.Using the diameter ratio of the enzymolysis circle and the colony of 1.71 as an evaluation index,we screened 10 strains from 116 UV-mutagenesis strains by TB-RNA,screened 6 superior strains by late fermentation,and obtained a strain 6# with the highest enzyme activity of 390 U·mL-1,which increased by 29% compared to that of the original strain.The research provides an efficient method for rapidly screening strains in industrialized production.

关 键 词：桔青霉 核酸酶P1 甲苯胺蓝 高效筛选 紫外诱变 

分 类 号：TQ469[化学工程—制药化工] Q936[生物学—微生物学]