用pGAP启动子在P.pastoris中组成型表达漆酶  

Using pGAP Promoter to Express of Laccase Gene from Bacillus subtilis in P. pastoris

作　　者：张泽华[1] 杨穗珊[2] 张爱联[2] 

机构地区：[1]海南大学农学院,海南海口570228 [2]中国热带农业科学院热带生物技术研究所农业部热带作物生物技术重点开放实验室,海南海口571101

出　　处：《生物技术》2011年第6期24-27,共4页Biotechnology

基　　金：中央级公益性科研院所基本科研业务费(ITBB120503);中国热带农业科学院科研基金项目(RKY0623)资助

摘　　要：目的:用pGAP启动子在P.pastoris中组成型表达漆酶。方法:用PCR从枯草芽孢杆菌基因组中扩增漆酶基因lac2。用NotⅠ和EcoRⅠ双酶切将lac2基因重组于表达载体PGAP9K。通过电转法将其转化于P.pastoris基因组,筛选高G418抗性以及高表达Lac2酶的重组子作为工程菌Gs115(pGAP9k-lac2)。用甘油作为碳源在50L生物反应器中表达重组漆酶Lac2。用ABTS法测定发酵液中的漆酶活力。结果:在发酵30h时,其Lac2酶的表达达峰值,其活性为136.67U/L。其峰值的Lac2酶的表达量为50mg/L。表达产物具有分解ABTs的活性。结论:成功克隆了漆酶基因lac2,并首次实现用pGAP启动子在P.pastoris中组成型表达漆酶,为用P.Pastoris规模化生产漆酶奠定了基础。Objective:Using pGAP promoter to express of laccase gene from Bacillus subtilis in P.pastoris.Method:Laccase gene lac2 was amplified from Bacillus subtilis genome by PCR technique and recombinant into expression vector PGAP9K after restriction enzyme digestion With NotⅠ and EcoRⅠ.Transformed it into the P.pastoris Gs115 genome by electroporation method.Then screened with G418-resistant recombinant enzyme and high expression of Lac2 as engineering bacteria Gs115(pGAP9k-lac2).Using glycerol as the carbon source recombinant laccase Lac2 in a 50L bioreactor.Determination laccase activity of fermentation broth with ABTS.Result:In the fermentation of 30 hours,Lac2 enzyme express to peak,activity is 136.67U/L.Its peak Lac2 enzyme expression levels 50mg/L.Expression products showed decomposition ABTs activity.Conclusion:The successful cloning of laccase gene lac2,and to achieve with pGAP promoter in P.pastoris in the constitutive expression of laccase for large-scale production with P.pastoris laccase basis.

关 键 词：漆酶 毕赤酵母 pGAP启动子 基因表达 酶活力 

分 类 号：Q81[生物学—生物工程]