重组枯草芽孢杆菌产角蛋白酶的发酵培养基及发酵条件优化  

Optimization of fermentation medium and fermentation conditions for keratinase production by recombinant Bacillus subtilis

作　　者：利刚慧 刘俊杰 彭帅英 梁颖茵 鄢陆琪 李昆太 LI Ganghui;LIU Junjie;PENG Shuaiying;LIANG Yingyin;YAN Luqi;LI Kuntai(College of Food Science and Technology,Guangdong Ocean University,Zhanjiang,Guangdong Province 524088,China;College of Biological Sciences and Engineering,Jiangxi Agricultural University,Nanchang,Jiangxi Province 330045,China)

机构地区：[1]广东海洋大学食品科技学院,广东湛江524088 [2]江西农业大学生物科学与工程学院,江西南昌330045

出　　处：《中国饲料》2023年第19期29-36,共8页China Feed

基　　金：广东省普通高校重点领域专项(2021ZDZX4010);广东省大学生创新创业训练计划项目(S202110566013);广东海洋大学科研启动费资助项目(060302042006)。

摘　　要：角蛋白是潜在的优良饲用蛋白资源,其中角蛋白酶作为可特异性降解角蛋白的酶类,在角蛋白饲料化领域展现出广泛的应用潜力和价值。本研究以自主构建的重组角蛋白酶工程菌枯草芽孢杆菌WB600-pMA5-kerJY-23为研究对象,利用单因素和二次通用旋转组合设计试验对其摇瓶发酵产角蛋白酶的发酵培养基和发酵条件进行优化。结果表明:该菌株最佳发酵产酶条件为:葡萄糖25 g/L,蔗糖25 g/L,酵母浸膏20 g/L,硫酸铵10 g/L,磷酸氢二钾1.5 g/L,氯化钠0.3 g/L,氯化钙0.025 g/L,硫酸镁0.025 g/L,硫酸亚铁0.015 g/L,氯化锰0.05 g/L,初始pH 8,装液量45 mL/250 mL三角瓶,接种量6%,培养温度37℃,摇床转速200 r/min,发酵周期36 h。在该优化条件下,重组枯草芽孢杆菌WB600发酵产角蛋白酶的酶活达到(2110±15.011)U/mL,较优化前提高了5.10倍(P<0.05),为该酶在角蛋白降解及资源化利用领域的应用提供了理论依据与研究基础。Keratin is a potential excellent feed protein resource,among which keratinase,as an enzyme that can specifically degrade keratin,shows a wide application potential and value in the field of keratin feederization.In this study,the fermentation medium and fermentation conditions of keratinase production were optimized using single-factor and quadratic generalized rotational combination design tests with the independently constructed recombinant keratinase engineering bacterium Bacillus subtilis WB600-pMA5-kerJY-23 under shake flask fermentation.The optimized results showed that the optimal fermentation conditions for keratinase production were as follows:glucose 25 g/L,sucrose 25 g/L,yeast extract 20 g/L,ammonium sulfate 10 g/L,dipotassium hydrogen phosphate 1.5 g/L,sodium chloride 0.3 g/L,calcium chloride 0.025 g/L,magnesium sulfate 0.025 g/L,ferrous sulfate 0.015 g/L,manganese chloride 0.05 g/L,initial pH 8,medium volume 45 mL/250 mL Erlenmeyer flask,inoculation amount 6%,culture temperature 37℃,rotation speed 200 r/min,and fermentation cycle 36 h.Under the optimized conditions,the enzyme activity of keratinase produced by the recombinant B.subtilis WB600 reached(2110±15.011)U/mL,which was 5.10 times higher than that before optimization(P<0.05).The study provided a theoretical basis and research foundation for the application of this keratinase in the field of keratin degradation and resource utilization.

关 键 词：角蛋白酶 重组枯草芽孢杆菌WB600 发酵培养基 发酵条件 优化 

分 类 号：S816.3[农业科学—饲料科学]