摘要
以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amy WQJ,构建重组质粒p ET-28a(+)-amy WQJ,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59 ku的重组酶r Amy WQJ.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℃;Ca^(2+)能提高该酶的热稳定性;Hg^(2+)、EDTA、SDS、Cr3+、Mn^(2+)、Cu^(2+)和Pb^(2+)对该酶活力具有不同程度的抑制作用,而Co^(2+)、Na+和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6.62 mg·m L-1,vmax为2 197.80μmol·(mg·min)-1;降解最适作用底物木薯淀粉的终产物为寡聚糖.
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 59 ku. 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 fl-Mercaptoethanol. By using starch soluble as substrate, the Km was 6.62 mg · mL-1 and Vmax was 2 197.80 μmol - （mg ·min） -1. Oligosaccharide is the final product of enzymatic hydroly- sis of Starch cassava.
作者
林云
林娟
王国增
林梦丹
叶秀云
LIN Yun;LIN Juan;WANG Guozeng;LIN Mengdan;YE Xiuyun(Fujian Key Laboratory of Marine Enzyme Engineering, College of Bioiogical Science and Technology, Fuzhou University, Fuzhou, Fujian 350116, China)
出处
《福州大学学报（自然科学版）》
CAS
北大核心
2018年第1期143-150,共8页
Journal of Fuzhou University(Natural Science Edition)
基金
国家863计划资助项目(2013AA102101)
