植酸酶基因的定点突变及其在巴斯德毕赤酵母表面展示  

Site-directed mutagenesis of Aspergillus niger phytase gene and its cell-surface display on Komagataella phaffii

作　　者：余道兵 程学松 王群 史艳可 张昕[1] 

机构地区：[1]浙江农林大学林业与生物技术学院,杭州311300 [2]浙江大学生命科学学院,杭州310058

出　　处：《浙江大学学报（农业与生命科学版）》2018年第1期10-20,共11页Journal of Zhejiang University:Agriculture and Life Sciences

基　　金：国家自然科学基金(31670110;31470191;41671314);国家重点基础研究发展计划(973计划)(2015CB150502)

摘　　要：为解决植酸酶在制粒过程中易变性失活的问题,以黑曲霉(Aspergillus niger)ZJUY中的植酸酶基因phy A为亲本,借助聚合酶链式反应(polymerase chain reaction,PCR)介导的定点突变,对植酸酶基因的关键位点进行密码子优化、引入氢键以及二硫键Cys196-Cys446的缺失突变;凭借无缝克隆成功将锚定片段FS和携带标签Flag的目的片段phy A插入酵母表达载体pPICZαC中;采用氯化锂转化法,将构建的8个表达载体pPICZαC-FS/phy A通过同源重组的方式整合到巴斯德毕赤酵母(Komagataella phaffii)GS115基因组上。细胞免疫荧光染色和流式细胞仪检测证实,基因改良后的植酸酶在巴斯德毕赤酵母细胞表面成功展示。重组菌株A31、A61和A84的植酸酶活性均可达7 000 U/g。与分泌型植酸酶相比,展示酶具有更好的高温和酸碱耐受性,其中菌株A61在90℃水浴处理1h后仍有18%的酶活,在pH 1.6~4.0范围内,残余酶活保持在80%以上。菌株A61这一特性可以克服制粒过程中(60~90℃)植酸酶变性失活的不足,能够较好地满足动物饲料用酶的需求。A full-length sequence of phytase gene(phy A) from Aspergillus niger ZJUY was cloned and modified based on polymerase chain reaction(PCR)-directed mutagenesis, aiming at addressing phytase denaturalization and deactivation during the process of pelleting. The mutation types included codon optimization for key sites, the introduction of hydrogen bond(T295 S, Q296 R and V43 N) and the deletion mutation of disulfide bond(Cys196-Cys446). The anchored-Flo1 p(FS) and target-phy A were successfully inserted into the yeast expression vector p PICZαC by seamless cloning. Eight kinds of expression vector p PICZαC-FS/phy A were integrated into the Komagataella phaffii GS115 genome by homologous recombination using lithium chloride transformation method, respectively. Phytases were successfully displayed on the surface of K. phaffii GS115, verified by immunofluorescence staining and flow cytometry. The resulted recombinants A31, A61 and A84 all showed a high phytase activity of 7 000 U/g. In addition, phytase-displaying exhibited better thermostability and p H-stability than those of secretory phytase. Among them, A61 remained 18% phytase activity after water bath at 90 ℃ for 1 h, and still maintained more than 80% of phytase activity at a p H range of 1.6 to 4.0. This characteristic of strain A61 can overcome deactivation during the process of granulating at 60 to 90 ℃ and better meet the demand in animal feed and food processing.

关 键 词：植酸酶 定点突变 表达载体pPICZαC-FS/phyA 巴斯德毕赤酵母 表面展示 

分 类 号：Q78[生物学—分子生物学]