摘要
在实验室成功构建的表达凝乳酶的重组巴斯德毕赤酵母GSl15/pPIC9KG4-3-35的基础上,对其表达外源蛋白的发酵条件进行了研究。经过单因素实验研究表明,培养基发酵产酶的最佳条件为:每隔12 h补加一次浓度为1.3%的甲醇、诱导时间为96 h;诱导过程中是否更换培养基对外源蛋白表达影响不大,重组菌的最佳初始诱导菌浓度为2.0 OD600/mL。对酶的酶学特性的研究结果表明,该酶作用的最适温度和最适pH分别为55℃和5.5。在50℃处理30 min和55℃处理10 min的条件下酶的活性完全丧失,重组凝乳酶的对热不稳定性的特性有利于干酪生产工艺中对乳清处理的要求。该酶在pH6.0时最稳定。金属离子中,Ca2+和Na+对酶凝乳有促进作用,而Mg2+、K+和Cu+则对酶凝乳有抑制作用。
Studies on the factors affecting the production of recombinant chymosin in Pichia pastoris（GS115/pPIC9K） constructed in our laboratory were carried out.By single factor test,the results showed： the inducing time with methanol 96 h,the methanol addition value 1.3%/12 h,the original yeast concentration 2.0 OD600/mL.There was no significant effect on the milk-clotting enzyme（MCE） activity by change of media during fermentation.The properties of the recombinant MCE were studied.The optimal temperature of the enzyme was 55 ℃,The recombinant MCE was inactivated when treated at 50 ℃ for 30 min or 55 ℃ for 10 min.The thermal instability of the recombinant MCE was suitable for the treatment of whey resulted from the process of cheese production.The recombinant MCE was stable at pH 6.0.When the substrate pH5.5,the enzyme showed the best clotting activity.The metal ions such as Ca2＋ and Na＋ could increase the milk-clotting activity,but K＋,Mg2＋ and Cu＋inhibited the activity.
出处
《食品科技》
CAS
北大核心
2010年第11期2-6,共5页
Food Science and Technology
基金
国家高技术研究发展计划(863计划)项目(2006AA10Z306)
现代农业产业技术体系建设专项资金项目(nycytx-0502)
关键词
微小毛霉
重组凝乳酶
优化
酶学特性
Mucor pusillus
recombinant MCE
optimization
enzymatic properties
