摘要
PCR扩增Bacillus licheniformis14580、Bacillus subtilis168、Geobacillus stearothermophilusIFO12589氨肽酶基因,分别酶切连接表达质粒pET-28a,构建重组表达载体pET28a-BLAP、pET28a-BSAP、pET28a-GSAP,酶活力检测表明3个氨肽酶基因均在大肠杆菌宿主BL21（DE3）中获得重组表达。进一步对3株重组菌的氨肽酶粗酶液反应条件进行比较研究,结果表明：重组氨肽酶BSAP与GSAP的粗酶活较高,达到1500U/L以上;BLAP、BSAP、GSAP粗酶的最适酶反应温度分别为50、75、60℃,BSAP温度稳定性最好,在30-70℃时比较稳定;3种重组氨肽酶的最适pH值都是9.0,pH值在8.5-9.0时比较稳定;0.lmmol/L Co2＋对酶活有较强的激活作用,BSAP的相对酶活力最高达到195.6%,其他二价金属离子对酶活均有不同程度的抑制,其中Zn2＋抑制作用最大;重组质粒pET28a-BSAP、pET28a-GSAP在大肠杆菌中较pET28a-BLAP稳定。
The genes encoding aminopeptidase GSAP, BSAP and BLAP were respectively amplified from Geobacillus stearothermophilus IFO12589, Bacillus subtilis 168 and Bacillus licheniformis 14580. These amplified DNA fragments were individually inserted into the expression vector E. coli pET-28a to construct plasmids pET28a-BLAP, pET28a-BSAP and pET28a-GSAP, respectively. The expression of these genes was confirmed by activity analysis, and then the expressed enzymes were characterized. The activities of both recombinant BSAP and GSAP were 1500 U/L, which were higher than that of BLAP. The optimum temperatures were 50, 75℃ and 60 ℃ for BSAP, GSAP and BLAP, respectively, and the optimum pH values were all 9.0. They were stable at pH 8.5-9.0. BSAP was obviously activated by 0.1 mmol/L Co^2＋, reaching a maximum of 195.6% of its original activity, but inhibited to different extents by other divalent metal ions, with Zn^2＋ being the strongest inhibitor. In addition, the recombinant plasmids pET28a-BSAP and pET28a-GSAP were more stable than pET28a-BLAP in E. coil
出处
《食品科学》
EI
CAS
CSCD
北大核心
2013年第23期200-205,共6页
Food Science
基金
国家"863"计划项目(2011AA100905
2012AA021201)
教育部"新世纪优秀人才支持计划"项目(NCET-11-0665)
关键词
氨肽酶
重组表达
酶反应
aminopeptidase
recombinant expression
enzymatic reaction
作者简介
张洁（1988-），女，硕士研究生，研究方向为轻工技术与工程。E-mail：rain880207jie@163．com
通信作者：石贵阳（1963-），男，教授，博士，研究方向为再生资源生物转化、发酵过程控制、酶技术。E-mail：cbbshi@139．com
