一株产柚苷酶菌株的ARTP诱变育种及培养基的优化

ARTP Mutain Breeding of Naringinase-Producing Strains and Optimization of Fermentation Conditions

利用常温室压等离子体技术对一株产柚苷酶菌株互隔交链孢霉(A. alternate)SK.37001进行快速诱变处理,获得一株高产柚苷酶菌株A. alternate SK.37002,与野生菌株相比,该菌株发酵酶活提高了208%。通过单因素实验确定发酵产柚苷酶的最适碳源为蔗糖,最适诱导物为柚皮苷,最适氮源为硝酸钠,利用Plackett-Burman试验筛选出影响液态摇瓶发酵酶产量的3个重要因素:硝酸钠、氯化钙、磷酸氢二钾,在此基础上运用最陡爬坡实验逼近最大响应值区域,再利用Box-Behnken设计及响应面分析法进行回归分析,获得最佳培养基配方为:1 g/L柚皮苷,5 g/L蔗糖,5 g/L硝酸钠,0.8 g/L磷酸氢二钾,0.7 g/L氯化钙,0.5 g/L氯化钾,0.5 g/L硫酸镁。采用优化后的培养基进行摇瓶发酵产柚苷酶酶活为624.73 U/mL,与模型预测接近,发酵产酶量比优化前提高了90%。

Atmospheric and room temperature plasma(ARTP)was used to mutagenize Alternaria alternate which is a naringinase-producing strain.The naringinase activity of the mutagenized stain SK37.002 reached 327.32 U/mL,which was 208%higher than that of original strain.The optimum carbon sources for naringinase-producing is sucrose.The most suitable inducer is naringin and nitrogen sources being NaNO3.According to Plackett-Burman design,NaNO3,CaCl2 and K2HPO4 were screened out of 8 factors as main affecting variables of naringinase production.Then steepest ascent method was used to approach themaximum response regions and it was followed by Box-Behnken and response surface analysis.As a result,the optimum medium formula(g/L)determined was composed of naringin 1,sucrose 5,NaNO3 5,K2HPO4 0.8,CaCl2 0.7,KCl 0.5,MgCl2 0.5.The resulting naringinase activity in fermentation broth reached up to 624.73±30.33 U/mL and was 90%higher than before which was consistent with the predicted maximum value.