摘要
从土壤中筛选到一能分泌碱性高温淀粉酶的芽孢杆菌属菌株。用质粒pTB522作载体,该淀粉酶基因(amyA)被克隆并在枯草芽孢杆菌ANA-1中得到表达。又通过亚克隆,得到含酶基因的更小重组质粒pTBX32(14.6kb)。携带该重组质粒的枯草芽孢杆菌ANA-1分泌的淀粉酶的特性与供体菌的相同。该酶作用最佳pH为8.5、最适温度为75℃,但在65℃,pH8.5条件下处理1h,该酶活性不降低。通过硫铵沉淀、热处理、DEAE——纤维素色谱和亲和色谱等分离技术,从培养液中部分纯化了该酶,其分子量估计为56000。
A thermostable and alkalophilic Bacillus strain XAL601 (rod, Spore-forming, gram-positive, catalase positive), With the ability to secrere thermostable amylase active at high pH, has been screened from Soil samples. The amylase gene. (amyA) was cloned and expressed in Bacillus subtilis ANA-1 (arg15, hsmM, hsrM, Npr^-, Amy^-, Apr^-)by using p^(TB522(T_c r) as a vector plasmid. Restriction map of the recombinant plasmid pTBX12 vas constructed. Since, amyA is contained in the 7.7 kbp pstl fragment of the plasmid pTBX12 (18.5 kbp), a smaller plasmid pTBX32 (14.6 kbp) catrying the amylase gene was obtained. Bacillus subtilis ANA-1 carrying the recombinant plasmid pTBXI2 secretes the amylase which exhibits the same charateristics with those of enzyme from the-parental strain XAL601. The optimum pH and temperature for the enzyme activity were around 8.5 and 75℃,.(respectively. The enzyme activity was not changed after the heat treatment at 65℃, pH8.5, for one hour. The amylase was partially purified by ammonium sulfate precipitation, heat treatment, DEAE-cellulose column chromatography and affinity column chromatography. According to the patterns of SDS-PAGE, molecular weight of the amylase was estimated to be 56. 000.
关键词
淀粉酶
碱性
高温
分子克隆
thermostable
alkolophilic
amylase
Bacillus
Moleculoning
