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12 h,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390 U·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 citri-num,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 en-zyme activity of nuclease P1 is positively correlated with the diameter of the enzymolysis circle under the opti-mal conditions as follows:toluidine blue concentration is 2.0×10-4mol ·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 1.7 1 as an evaluation index,we screened 1 0 strains from 1 1 6 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 o-riginal strain.The research provides an efficient method for rapidly screening strains in industrialized produc-tion.