枯草芽孢杆菌WB600角蛋白酶重组菌发酵条件优化

本试验旨在对已构建枯草芽孢杆菌WB600角蛋白酶重组菌WB600-K进行发酵条件优化，获得最佳产酶条件，以期提高重组菌角蛋白酶的产量；同时通过研究不同种类碳源和氮源间的组合优化获得成本低廉的重组菌发酵培养基配方。结果显示，当发酵液D_{600 nm}=1.0时添加0.5%木糖诱导发酵WB600-K分泌表达角蛋白酶酶活最佳（P<0.05）；山梨醇作为碳源可显著提高WB600-K角蛋白酶酶活（P<0.05），但成本较高不宜作为混合碳源来源；不同种类碳源（葡萄糖、玉米粉、糖蜜、甘油）组合添加能显著提高重组菌角蛋白酶酶活（P<0.05）。不同种类氮源（蛋白胨、酵母粉、豆粕粉、尿素）组合添加对于重组菌提高角蛋白酶酶活效果不如单一氮源（蛋白胨）（P<0.05）。在诱导发酵48 h时WB600-K产角蛋白酶酶活最高（P<0.05）。对发酵配方进行正交试验优化，WB600-K在玉米粉5.1 g/L、葡萄糖5.9 g/L、糖蜜5.9 g/L、甘油3.0 g/L、蛋白胨20 g/L、NaCl 10 g/L，发酵液D_{600 nm}=1.0时加0.5%木糖诱导发酵48 h能显著提高角蛋白酶酶活（P<0.05），达到56.9 U/mL，较初始培养条件提高49.74%。综上，在优化发酵条件下进行发酵及不同种类碳源间组合能提高WB600-K角蛋白酶活性，可为角蛋白酶工业化生产提供试验依据。

Optimization of the Fermentation Conditions of Recombinant B.subtilis WB600 Produced Keratinase

The aim of this experiment was to optimize the fermentation conditions of the constructed keratinase recombinant B.subtilis WB600 strain WB600-K,and obtain the optimal enzyme production conditions in order to increase the yield of recombinant keratinase;At the same time,the combination optimization between different types of carbon sources and different nitrogen sources was studied in order to obtain a low-cost recombinant fermentative medium formula.The results showed that when the fermentation D_{600 nm}=1.0,and 0.5% xylose was added to induce fermentation,the expression of keratinase was best in the recombinant WB600-K (P<0.05).As a carbon source,sorbitol could significantly increase the activity of the recombinant strain WB600-K keratinase (P<0.05),but the cost was too high to be a source of mixed carbon sources.The combination of different carbon sources (glucose,corn flour,molasses and glycerin) could significantly increase the activity of keratinase (P<0.05).The combination of different kinds of nitrogen sources (peptone,yeast powder,soybean meal and urea) significantly increased the activity of keratinase less than single nitrogen source (peptone) (P<0.05).The WB600-K had the highest keratinase activity at 48 h after induction of fermentation (P<0.05).The overall experimental optimization results showed that,at the condition of corn flour 5.1 g/L,glucose 5.9 g/L,molasses 5.9 g/L,glycerol 3.0 g/L,peptone 20 g/L,NaCl 10 g/L,0.5% xylose was added when fermentation D_{600 nm}=1.0,and 48 h fermentation,the activity of keratinase could be increased significantly (P<0.05),which was 56.9 U/mL and 49.74% higher than that in initial culture conditions.In summary,fermentation under optimized fermentation conditions could increase the activity of recombinant strain WB600-K keratinase,which laid a foundation for the industrial production of keratinase.