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南极菌Pseudoalteromonas sp. A211-5产α-淀粉酶Amy3809的表达、性质及其降解特性
引用本文:谷晓倩,李江,潘爱红,林学政.南极菌Pseudoalteromonas sp. A211-5产α-淀粉酶Amy3809的表达、性质及其降解特性[J].海洋科学进展,2020,38(1):81-90.
作者姓名:谷晓倩  李江  潘爱红  林学政
作者单位:自然资源部第一海洋研究所海洋生态环境科学与技术重点实验室,山东青岛,266061,青岛科技大学化工学院,山东青岛,266061
基金项目:海洋公益性行业科研专项项目——海藻寡糖应用关键技术研究;农用系列产品开发
摘    要:对具有高淀粉酶活性的南极菌Pseudoalteromonas sp. A211-5的全基因组数据进行了分析和筛选,筛选获得α-淀粉酶疑似序列amy3809,并采用基因工程手段对该基因的功能和性质进行验证和分析。首先,以淀粉酶Amy3809的完整开放阅读框(ORF)为模板设计特异引物,克隆获得amy3809的全序列并对其进行重组表达,获得的重组蛋白采用镍柱进行分离纯化;DNS-还原糖法测定重组酶的酶学性质;薄层层析(TLC)技术对Amy3809的酶解产物进行分析。实验结果:1)克隆获得的amy3809成功地连接到pET-30a载体,并在工程菌E.coli BL21(DE3)中实现了高效表达,纯化的重组酶Amy3809分子量为67 kDa;2)重组酶Amy3809在10~40℃的范围内仍能保持85%以上的酶活,但随着温度的升高酶活迅速降低,70℃时几乎失活,表明该酶具有良好的低温耐受特性及热敏感性;3)最适pH为7.0,在pH 5.0~10.0的范围内仍能保持50%以上的活性;4)金属离子Na+,K+和Ca2+均能提高Amy3809的活性,而Cu2+,Fe2+,Mg2+和EDTA则能显著降低Amy3809的活性;5)Amy3809的酶解产物主要为麦芽四糖、麦芽三糖、麦芽糖和葡萄糖。由此可知,南极菌产的α-淀粉酶Amy3809,具有良好的低温耐受特性,热敏感性和较广的pH耐受范围,并能够有效地将淀粉降解为低聚糖和葡萄糖,因而具有潜在的工业应用前景。

关 键 词:Α-淀粉酶  表达  酶学性质  酶解特性

Expression and Characterization of α-amylase Amy3809 From Antarctic Bacterium Pseudoalteromonas sp .A211-5
GU Xiao-qian,LI Jiang,PAN Ai-hong,LIN Xue-zheng.Expression and Characterization of α-amylase Amy3809 From Antarctic Bacterium Pseudoalteromonas sp .A211-5[J].Advances in Marine Science,2020,38(1):81-90.
Authors:GU Xiao-qian  LI Jiang  PAN Ai-hong  LIN Xue-zheng
Institution:(Key Laboratory of Marine Eco-Environmental Science and Technology,First Institute of Oceanography,MNR,Qingdao 266061,China;Chemical Engineering Institute,Qingdao University of Science&Technology,Qingdao 266061,China)
Abstract:The complete genome of a starch degradation bacteria Pseudoalteromonas sp. A211-5 from Antarctic sample was analyzed by bioinformatics methods and a putative alpha-amylase amy3809 was screened. Expression and characterization of the putative alpha-amylase amy3809 was investigated in this paper. The alpha-amylase amy3809 was cloned and expressed by genetic engineering method firstly;then, the recombinant enzyme Amy3809 was purified by Ni-NTA chromatography and the characterization of recombinant Amy3809 was determined by dinitrosalicylic acid method;the hydrolysis product of recombinant Amy3809 was analyzed by thin-layer chromatography(TLC) finally. The results are listed as follows: 1) The recombinant expression vectors(pET-30 a+amy3809) was high-efficiency expressed in E.coli BL21(DE3). The purifiedrecombine Amy3809 revealed a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with an apparent molecular weight of 67 kDa;2) The optimum temperature of the recombinant Amy3809 was 50 ℃, and it can maintain more than 85% of the initial activity between 10 ℃ to 40 ℃, but the activity declined rapidly over 50 ℃ and the activity was almost completely lost when the temperature at 70 ℃, the results showed that recombine Amy3809 has good low temperature resistance properties and thermal sensitivity;3) The optimum pH of the recombinant Amy3809 was 7.0, and it maintained as much as 50% of its maximum activity between pH 5.0~10.0;4) Amy3809 was activated by Na+, K+ and Ca2+, but it was significantly inhibited by Cu2+, Fe2+, Mg2+ and EDTA;5) Thin-layer chromatographic analysis indicated that Amy3809 degraded soluble starch generating maltotetraose, maltotriose, maltose and glucose as the final products. The unique characteristics of Amy3809, including its low temperature resistance, thermal sensitivity, a wide range of pH tolerance, implied that this enzyme might be an interesting candidate for industrial processes.
Keywords:alpha-amylase  expression  enzymatic characterization  enzymatic hydrolysis
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