摘要
通过简并引物扩增热球菌(Thermococcus siculi)HJ21高温α-淀粉酶保守区域之间的序列,再利用Site-finding技术获得两端未知序列。构建了在N端添加了His6标签的表达载体pEt-28a-His6-THJA后转化E.coli,在IPTG诱导下表达。进一步纯化后SDS-PAGE电泳检测达到电泳纯,重组酶分子量为50 KDa。重组酶最适作用温度和pH值分别为90℃,pH5.0。重组酶在pH为5.5时最稳定;K+,Sr2+,Mg2+,Na+对α-淀粉酶活力的促进作用不显著,Cu2+,Pb2+,Hg2+,Zn2+,N-溴代丁酰亚胺和三氯乙酸对该酶有显著抑制作用。
The gene sequence between the conserved regions was acquired by PCR using the primers designed based on the sequence of that of Thermococcus siculi.HJ21 deposited in the GenBank.The upstream and downstream of the HJ21 α-amylase gene were acquired by sitefinding PCR.The expression plasmid pEt-28a-His6-THJA was structured.The plasmid was transformed into E.coli stain TOP 10F′ and expressed.The His6-α-amylase was further purified.Its molecular weight was about 54.5 KDa detected by SDS-PAGE.The His6-α-amylase was further purified.The optimal temperature and pH of His6-α-amylase are 90 ℃ and 5.0 respectively.K＋,Sr2＋,Mg2＋,Na＋ improved the activity of His6-α-amylase while Cu2＋,Pb2＋,Hg2＋,Zn2＋ reduced activity of His6-α-amylase.N-bromosuccinimide and TCA significantly inhibited the activity of His6-α-amylase.
出处
《海洋学报》
CAS
CSCD
北大核心
2011年第3期158-164,共7页
基金
国家自然科学基金项目(40746030)
江苏省科技支撑计划(BE2008340)
江苏省高校自然科学研究重大项目(09KJA170001)
关键词
深海古菌
高温酸性α-淀粉酶
基因克隆
表达
酶学性质
archaeon in deep sea
thermostable and acid-stable α-amylase
gene cloning
expression
characterization
