文章摘要
引用本文:林 云,林 娟,王国增,林梦丹,叶秀云.温泉来源地芽孢杆菌耐热α-淀粉酶基因的克隆表达及酶学性质研究[J].福州大学学报(自然科学版),2018,46(1):143~150
温泉来源地芽孢杆菌耐热α-淀粉酶基因的克隆表达及酶学性质研究
Cloning,expression and characterization of the thermostable alpha-amylase gene from Geobacillus sp.WQJ-1 isolated from hot springs
  
DOI:10.7631/issn.1000-2243.2018.01.143
中文关键词: 地芽孢杆菌  α-淀粉酶  克隆  表达  酶学性质
英文关键词: Geobacillus  alpha-amylase  cloning  expression  characterization
基金项目:
作者单位
林 云 福州大学生物科学与工程学院福建省海洋酶工程重点实验室福建 福州 350116 
林 娟 福州大学生物科学与工程学院福建省海洋酶工程重点实验室福建 福州 350116 
王国增 福州大学生物科学与工程学院福建省海洋酶工程重点实验室福建 福州 350116 
林梦丹 福州大学生物科学与工程学院福建省海洋酶工程重点实验室福建 福州 350116 
叶秀云 福州大学生物科学与工程学院福建省海洋酶工程重点实验室福建 福州 350116 
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中文摘要:
      Geobacillus sp.WQJ-1基因组DNA为模板,利用PCR扩增获得成熟α-淀粉酶基因amyWQJ,构建重组质粒pET-28a(+)-amyWQJ,转化大肠杆菌BL21(DE3)诱导表达. 用镍柱亲和层析对粗酶液进行分离纯化,获得相对分子质量约为59ku的重组酶rAmyWQJ. 研究表明,该酶最适pH值为6.0,具有较好pH稳定性,最适温度为70℃;Ca2+能提高该酶的热稳定性;Hg2+、EDTA、SDS、Cr3+、Mn2+、Cu2+和Pb2+对该酶活力具有不同程度的抑制作用,而Co2+、Na+β-Mercaptoethanol则具有促进作用. 以可溶性淀粉为底物,该酶动力学参数Km为6.62mg·mL-1vmax为2197.80μmol·(mg·min)-1;降解最适作用底物木薯淀粉的终产物为寡聚糖.
英文摘要:
      The amyWQJ gene encoding alpha-amylase from Geobacillus sp.WQJ-1 was amplified by PCR,and linked with vector pET-28a(+). The constructed recombinant plasmid pET-28a(+)-amyWQJ was transformed into E. coli BL21(DE3),and the recombinant protein was obtained via induction of expression and purified by nickel affinity chromatography,the purified enzyme presented as one protein band on SDS-PAGE with molecular weight of 59ku. The study showed that the optimum pH value was 6.0,and it has good pH stability,optimum temperature of purified alpha-amylase was 70℃. Ca2+ can significantly improve the thermostability of the enzyme. The activity of the enzyme was inhibited by Hg2+,EDTA,SDS,Cr3+,Mn2+,Cu2+ and Pb2+in different degrees,and activated by Co2+,Na+ and β-Mercaptoethanol. By using starch soluble as substrate,the Km was 6.56mg·mL-1 and vmax was 2197.80μmol·(mg·min)-1. Oligosaccharide is the final product of enzymatic hydrolysis of Starch cassava.
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