米曲霉果胶酸酯裂解酶(pectin lyase 1)在原核系统中重组表达  

Expression of Pectin Lyase 1 from Aspergillus oryzae in Escherichia coli

作　　者：赵庆新[1,2] 袁生[1] 张宇玲[1] 

机构地区：[1]江苏省生物多样性和生物技术重点实验室,南京师范大学生命科学学院微生物工程重点实验室 [2]盐城师范学院生命科学与技术学院,盐城224002

出　　处：《生物工程学报》2007年第5期873-877,共5页Chinese Journal of Biotechnology

基　　金：国家自然科学基金(No.30170005);江苏省自然科学基金(No.BK2005136);江苏省教育部高校自然科学基金(No.07KJD1180237)资助~~

摘　　要：来自米曲霉(Aspergillus oryzae)和黑曲霉(Aspergillus niger)的果胶酸酯裂解酶(pectinlyase)一直被用于传统发酵食品的生产,但自然条件下A.oryzae和A.niger的果胶酸酯裂解酶产量较低。通过RT-PCR的方法,获得不含信号肽的A.oryzaePel1cDNA,将Pel1cDNA连入pET-28a(+)载体,构建pET-28a(+)-pel1质粒。pET-28a(+)-pel1转化Turner(DE3)placⅠ细胞,得到转化子pET-28a(+)-pel1-Turner(DE3)placⅠ,表达与6个组氨酸融合的Pel1。进一步对Pel1在E.coli系统中表达的条件进行了研究,在37℃,220r/min条件下,培养pET-28a(+)-pel1-Turner(DE3)placⅠ细胞,当OD600至0.8左右时,用500μmol/Lisopropylβ-D-thiogalactogalactop-yranoside(IPTG)进行诱导表达,在15℃和170r/min条件下,继续培养60h后,表达效果最好,产酶可达到400u/mL,是A.oryzae自然条件下产酶量的4000倍,也高于已报道的真菌果胶酸酯裂解酶在真菌体系中重组表达的效果。Pectin lyases from Aspergillus oryzae and Aspergillus niger are usually used for the production of traditional fermented foods,but these fungi produce less pectinases under natural conditions.The cDNA coding mature Pel1（without signal peptide）was amplified from Aspergillus oryzae by RT-PCR.Pel1 cDNA was cloned into pET-28a（＋）expression vector,then was transformed into E.coli Turner（DE3）placⅠcells to express Pel1 with 6-His tag.For improving the efficiency of Pel1 expression in E.coli,the conditions of expressing the Pel1 in E.coli were optimized.E.coli Turner（DE3）placⅠcells with pET-28a（＋）-pel1 was first cultivated at 37℃,220 r/min until OD_ 600 reached about 0.8.Then,cultivation broth was added with 0.05～0.1mmol/L IPTG and continuously incubated at 15℃,at 170 r/min for 60 h for expressing of Pel1.The recombinant expressed Pel1 activity could reach 400u/mL medium,which is 4000-fold of Pel1 produced naturally by A.oryzae and superior than known recombinant amount of pectin lyases expressed in different fungi expression systems.

关 键 词：米曲霉 果胶酸酯裂解酶1 大肠杆菌 表达 

分 类 号：Q78[生物学—分子生物学]