摘要
利用RT-PCR技术从黑曲霉(EIM-6)中扩增得到去除信号肽的果胶裂解酶基因A,将其插入到毕赤酵母表达载体pPIC9k上,构建重组表达质粒pPIC9K-pelA,电击转化毕赤酵母GS115,得到了表达成功的工程菌株。用终浓度为1.5%的甲醇对其进行诱导,将发酵上清液浓缩后,用盐酸法测定其酶活可以达到2.3U/mL。通过对重组毕赤酵母诱导表达产物进行SDS-PAGE鉴定,发现重组毕赤酵母分泌了1个约38kD的蛋白,与该酶基因产物的理论值相符,并通过水解圈法测定验证,均说明果胶裂解酶得到正确的分泌表达.
In this study,the mature peptide sequence of a pectin lyase gene A was amplified from Aspergillus niger strain EIM-6 by using RT-PCR reverse transcription technique.The cloned gene was then inserted into a Pichia pastoris expression vector pPIC9k to produce the recombinant expression plasmid pPIC9K-pelA.By using electric shocks,we successfully transformed the recombinant pPIC9K-pelA into Pichia pastoris GS115.The activity of the engineered strain reached to 2.3 U/mL after induction with the final concentration of 1.5% methanol. SDS-PAGE analysis revealed that the pPIC9K-pelA transformant had an additional protein band of approximately 38 kD, which was not present in the control. There were no significant differences between the recombinant and native pectin lyase with regard to their hydrolysis activities.
出处
《生物工程学报》
CAS
CSCD
北大核心
2009年第12期1962-1968,共7页
Chinese Journal of Biotechnology
基金
国家自然科学基金项目(No.30800735)
福建省自然科学基金项目(No.2008F3036)资助~~
关键词
黑曲霉
果胶裂解酶
毕赤酵母
表达
Aspergillus niger
pectin lyase
Pichia pastoris GS115
expression
