海参i-型溶菌酶在毕赤酵母基因工程菌中优化表达及纯化  

Optimization of fermentation conditions for i-type lysozyme produced by recombinant Pichia pastoris

作　　者：马俊[1] 张洋[1] 丛丽娜[1] 李成[1] 

机构地区：[1]大连工业大学生物工程学院,辽宁大连116034

出　　处：《工业微生物》2016年第2期41-46,共6页Industrial Microbiology

基　　金：辽宁省教育厅重点实验室(LZ2014029);辽宁省研究生教育创新计划项目(2014-154);国家海洋食品工程技术研究中心开放实验室(2012FU125X03)

摘　　要：将实验室已构建的毕赤酵母基因工程菌(p PIC9K-Sj Lys/GS115)作为海参i-型溶菌酶生产菌株,本研究分别从甲醇浓度、培养基p H、温度和诱导时间对其产酶发酵条件进行优化。实验得出甲醇诱导浓度为1.0%,发酵培养基初始p H 6.0,温度30℃,培养96 h为最佳目的蛋白表达条件,其发酵液中海参i-型溶菌酶含量达10.63 mg/L。将发酵液经离心和超滤浓缩后得到上清液,再经离子交换和凝胶过滤层析纯化获得海参i-型溶菌酶产品,其酶活力达826.44 U/mg。经测定该酶对革兰氏阳性菌溶壁微球菌和革兰氏阴性菌副溶血弧菌均具有明显的抑菌作用。In this study,the genetic strain Pichia pastoris p PIC9K-Sj Lys / GS115（ Sj Lys） was used to produce the i-type lysozyme from sea cucumber Apostichopus japonicus. The fermentation conditions were optimized for enhancement of i-type lysozyme production. The results showed that the maximum i-type lysozyme yield of 10. 63 mg / L was achieved under the conditions of initial p H 6. 0,culture temperature 30 ℃,fermentation time 96 h and methanol concentration of 1%. The fermentation culture of Sj Lys was centrifuged and concentrated by ultra-filtration,then purified by cation exchange and gel filtration chromatography. The specific activity of the purified i-type lysozyme was 826. 44 U / mg. The purified Sj Lys product diaplayed inhibitive effects on the growth of the gram-positive bacterium Micrococcus lysodeikticus and gramnegative bacterium Vibrio parahemolyticus.

关 键 词：海参i-型溶菌酶 毕赤酵母 发酵条件优化 酶活性 

分 类 号：TQ920.1[轻工技术与工程—发酵工程]