Medium ingredients for neutral proteinase(NPR) production of B.subtilis CNMC-0014 was optimized using stastically-based experimental designs. A single-factor experiment(SFE) method was adopted to choose the carbon and nitrogen sources significantly influcing NPR biosynthesis. Among the six variables of carbon sources we studied, the most significant variables were stastically elucidated for optimization, and included glycerol and corn flour with the corresponding value of NPR of 3955.16±2.15 U/ml and 3939.15±1.87 U/ml. For nitrogen sources, the most significant variable affecting NPR production was soybean flour with maximun NPR production of 4318.12±5.66 U/ml. The optimum concentration of each significant variable was then determined by range analysis (RA) and analysis of variance (ANOVA), the optimun values for the critical nutrients supplements for the maximun NPR production were 2% of corn flour, 1% of glycerol, 3% of soybean flour and 3% of bran, with the maximun NPR activity of 4526.28 U/ml. Results, statistically, indicated that the influences of clycerol and corn flour on NPR prodoction were significant at 5 % (p＜0.05) level, but, the effect of soybean flour was most significant at 1 % (p＜0.01) and bran had no significant influence (p＞0.05) on NPR production. And meanwhile, the effects of metal ions, dissoluble oxygen (DO), initial pH, strain ages and inoculum sizes on NPR production were also investigated. Resulsts shown that Ca2+, Mg2+, Na+, Zn+, Mn2+ could activate enzyme biosynthesis at different levels, especially Ca2+ with a maximun activity of 4552.97 U/ml. And whenas, Fe3+, Fe2+, K+, Ag+ had strongly inhibitory influence on NPR production, particularly Ag+ (NPR activity was only 987.46 U/ml).The most suitable filled amount of SmF medium in 300 ml Erlenmeyer flask was 50 ml. Optimal pH was 7.5, the most suitable strain ages and inoculum sizes for maximun NPR production were 24-h-old strain and 3% culture broth, respectively.%应用统计学方法优化了B.sustilis CNMC-0014中性蛋白酶产生的培养基组分.单因素试验研究发现,在测试的6种碳源中,以甘油和玉米粉对产酶影响显著,酶活力分别达到了3955.16±2.15U/ml和3939.15± 1.87U/ml.氮源试验中发现,大豆粉对产酶影响最为显著,酶活力达到4318.12±5.66U/ml.通过极差分析与正交分析优化了培养基配比,优化方案为:2%玉米粉,1%甘油,3%玉米粉和3%麸皮.方差分析结果发现,甘油和玉米粉对产酶影响显著(p＜0.05),大豆粉对产酶极显著(p＜0.01), 麸皮对产酶影响不显著(p＞0.05).同时,还 研究了金属离子、溶氧、起始pH值、菌龄以及接种量对产酶的影响,研究结果表明,Ca 2+、Mg2+、Na+、Zn+ 、Mn2+可以在不同程度对产酶有激活作用,特别是Ca 2+,酶活力达到了4552.97U/ml,而Fe3+ 、Fe2+、K+、Ag+对产酶有强烈的抑制作用,特别是Ag +(酶活力仅为987.46 U/ml),最适摇瓶装量为50ml/300ml,最佳起始pH值为7.5,最佳菌龄与接种量分别为24h和3%.
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