摘要
木聚糖酶和甘露聚糖酶是两种重要的半纤维素酶,也是两种重要的饲用酶制剂,通过毕赤酵母表达系统中的体外串联表达盒构建多拷贝的方法构建了木聚糖酶DSB和甘露聚糖酶Man A共表达重组质粒p PICZαA/DSB-ManA,将该重组质粒电转化至宿主菌毕赤酵母X33中获得共表达两种酶的重组菌X33/DSB-ManA,实现了两种酶的共分泌表达,经诱导表达后木聚糖酶和甘露聚糖酶的酶活分别为273. 6 U/ml和256. 8 U/ml,为单独表达重组菌X33/DSB和X33/Man A酶活的30. 4%和73. 4%。酶学性质的分析显示DSB和Man A的最适反应温度均为75℃,在45℃～75℃范围内具有较好的温度稳定性,酶活可保持最高酶活的60%以上; DSB最适pH为6. 5,Man A最适pH为6. 0,在pH 3. 0、40℃条件下,Man A处理1h能保持最高酶活的80%以上,DSB处理1 h时能保持最高酶活的50%以上; DSB和Man A对多种金属离子和化学试剂(浓度为1 mM)具有较好的耐受性,均可保留60%以上的酶活力。通过单一菌株成功完成了不同酶的共表达,为复合酶饲料添加剂的生产和应用研究提供了一定的理论依据。
Xylanase and mannanase are two important hemicellulases and feed enzyme preparations.Construction method of in vitro multimers in Pichia pastoris expression system has been used to construct recombinant plasmid pPICZαA/DSB-ManA which contained both DSB gene and Man A gene.Then, pPICZαA/ DSB-ManA was transformed to host cells Pichia pastoris X33 by electroporation to obtain the co-expression recombinant strain X33/DSB-ManA.In shake-flask cultivation, xylanase activity and mannanase activity in the supernatant were 273.6 U/ml and 256.8 U/ml that reached 30.4% and 73.4% activity compared to X33/DSB and X33/ManA, respectively.Properties of the mixed enzyme suggested that the optimal reaction temperature for DSB and ManA is 75℃.Both xylanase and mannanase showed more than 60% activity across temperature ranges 45℃ to 75℃.The optimal reaction pH value of DSB and ManA were 6.5 and 6.0, respectively.After incubation 1 hour under the pH3.0 and 40T treatment condition, mannanase activity of ManA displayed greater than 80% activity and xylanase activity of DSB revealed more than 50% activity.Meanwhile, DSB and ManA have shown good tolerance to a variety of metal ions and chemical reagents.Both enzyme activities could keep over 60% activity when the treatment concentration of different metal ions and chemical reagents was 1 millimole.Xylanase DSB and mannanase ManA have been co-expressed in Pichia pastoris successfully.The successful expression of the two enzymes of the recombinant strain could offer the theory basis for production and application of compound enzyme in the animal feed.
作者
姚银
闵琪
熊海容
张莉
YAO Yin;MIN Qi;XIONG Hai-rong;ZHANG Li(College of Life Science,South-Central University for Nationalities, Wuhan 430074, China)
出处
《中国生物工程杂志》
CAS
CSCD
北大核心
2019年第3期37-45,共9页
China Biotechnology
基金
湖北省技术创新专项重大项目(2018ABA093)资助项目
关键词
木聚糖酶
甘露聚糖酶
毕赤酵母
共表达
酶学特性
Xylanase
Mannanase
Pichia pastoris
Co-expression
Enzymatic characteristics
