利用淀粉产生碱性蛋白酶工程菌的构建  

THE CONSTRUCTION OF ALKALINE PROTEASEPRODU-CING ENGINEERING STRAIN USING STARCH AS RAW MATERIAL FROM BACILLUS PUMILUS C172-60

作　　者：杨文博[1] 冯耀宇[1] 

机构地区：[1]南开大学微生物学系,天津大学化学工程研究所

出　　处：《微生物学通报》1994年第5期273-278,共6页Microbiology China

基　　金：天津市自然科学基金

摘　　要：以不能利用淀粉为碳源、抗ｐｐ系列噬菌体和ｐＬ１噬菌体、产碱性蛋白酶（９４００－９８００ｕ／ｍｌ）的ＢａｃｉｌｌｕｓｐｕｍｉｌｕｓＣ１７２－６０为受体株，采用原生质体转化技术将携带糖化型小淀粉酶基因（Ａｍｖ）、Ｃｍ￣ｒ基因、Ｋｍ￣ｒ基因的ｐＢＸ９６质粒导入到受体株内，经两次利福平抗性筛选，获得一株能直接利用淀粉为发酵碳源、保留噬菌体抗性、产碱性蛋白酶的工程菌Ｃ１７２－３０６（ｐＢＸ９６）。菌体增殖９６．５代质粒丢失率为０．７７％，摇瓶发酵蛋白酶活力１４０１４ｕ／ｍｌ，５００Ｌ罐发酵蛋白酶活力平均１０１９２ｕ／ｍｌ。Bacillus pumilus C 172-60 could produce alkaline protease to the level of 9400-9800u/ml and it was resistant to pp series and pL1 phages as well.However,it couldn’t use starch asraw material, only could use sugar hydrolyzate as carbon source for its fermentation. By themeans of protoplast transformation, pBX 96 which had the saccharifying type α-amylase gene wastransformed to the recipient C172-60. Screened among mutants by twice rifampin disposals,C172-306 (pBX96) which not only could tise starch as carbon source to produce alkaline proteasebut was also resistant to pp1-pp10 and pL1 phages was selected.The enzyme activity offerrnentation in shaking flask and 500L fermenter was 14014u/ml , and 10192u/ml,respectively.Pass generation test indicated that the frequency of plasmid-free cells after 96. 5 generations wasonly 0.77%.

关 键 词：短小芽孢杆菌 质粒 原生质体 转化 碱性蛋白酶 

分 类 号：TQ920.1[轻工技术与工程—发酵工程]