低温、嗜盐α-淀粉酶Amy3的克隆、表达及重组酶性质
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基金项目:

国家自然科学基金(31570064);安徽省高校省级自然科学研究项目(KJ2015A038)


Cloning and characterization of cold-active and salt-tolerant α-amylase Amy3 from Pseudoalteromonas sp. K8
Author:
  • Yi Xue

    Yi Xue

    School of Life Sciences, Anhui University, Hefei 230601, Anhui Province, China;Anhui Key Laboratory of Modern Bio-manufacture, Hefei 230601, Anhui Province, China;Anhui Provincial Engineering Technology Research Center of Microorganisms and Biocatalysis, Hefei 230601, Anhui Province, China
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  • Mei Wang

    Mei Wang

    School of Life Sciences, Anhui University, Hefei 230601, Anhui Province, China;Anhui Key Laboratory of Modern Bio-manufacture, Hefei 230601, Anhui Province, China;Anhui Provincial Engineering Technology Research Center of Microorganisms and Biocatalysis, Hefei 230601, Anhui Province, China
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  • Zemin Fang

    Zemin Fang

    School of Life Sciences, Anhui University, Hefei 230601, Anhui Province, China;Anhui Key Laboratory of Modern Bio-manufacture, Hefei 230601, Anhui Province, China;Anhui Provincial Engineering Technology Research Center of Microorganisms and Biocatalysis, Hefei 230601, Anhui Province, China
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  • Wei Fang

    Wei Fang

    School of Life Sciences, Anhui University, Hefei 230601, Anhui Province, China;Anhui Key Laboratory of Modern Bio-manufacture, Hefei 230601, Anhui Province, China;Anhui Provincial Engineering Technology Research Center of Microorganisms and Biocatalysis, Hefei 230601, Anhui Province, China
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  • Yazhong Xiao

    Yazhong Xiao

    School of Life Sciences, Anhui University, Hefei 230601, Anhui Province, China;Anhui Key Laboratory of Modern Bio-manufacture, Hefei 230601, Anhui Province, China;Anhui Provincial Engineering Technology Research Center of Microorganisms and Biocatalysis, Hefei 230601, Anhui Province, China
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    摘要:

    [目的]从分离自北极海底沉积物Pseudoalteromonas sp.K8菌株克隆、重组表达α-淀粉酶Amy3,并研究其酶学性质。[方法]基于Pseudoalteromonas haloplanktis TAC125基因组分析,从亲缘关系较近的Pseudoalteromonas sp.K8克隆获得α-淀粉酶基因amy3,以大肠杆菌为宿主进行重组表达,经Ni-NTA亲和层析柱纯化获得重组蛋白Amy3。以可溶性淀粉等为底物,研究Amy3的酶学性质。[结果]Amy3最适催化pH为8.5,在pH 6.5-10.0范围内酶活力维持在40%以上;其在pH 7.5-8.5范围的稳定性较好,pH 8.0条件下的半衰期可达4 h。Amy3在低温下较稳定,25℃半衰期为5 h;最适反应温度为25℃,并且在0℃可以保持50%以上酶活力,显示良好的低温催化特性。NaCl能够有效提升Amy3的酶活力及稳定性;荧光光谱分析表明,NaCl并未引起Amy3酶蛋白三级结构的改变。动力学分析显示,NaCl影响了酶催化的Kmkcat,进而提升了酶的催化效率。底物特异性分析表明,Amy3对支链淀粉的水解能力优于直链淀粉,并能够有效地水解小麦淀粉、玉米淀粉和木薯淀粉。[结论]来源于Pseudoalteromonas sp.K8菌株的α-淀粉酶Amy3具有良好的低温催化及嗜盐性,在洗涤、食品、污水处理等行业中有潜在的应用前景。

    Abstract:

    [Objective] The present study was to clone a novel alpha-amylase from marine bacterium Pseudoalteromonas sp. K8 isolated from the sediment of Kongsfjorden and characterize the enzyme.[Methods] We cloned a cold-active and salt-tolerant α-amylase Amy3, from Pseudoalteromonas sp. K8. The protein was expressed in Escherichia coli, purified by Ni-NTA and characterized.[Results] Using soluble starch as substrate, the optimum pH of Amy3 was about pH 8.5, and more than 40% of the maximal activity maintained in the pH range of 6.5 to 10. Amy3 showed the maximum activity at 25℃, and retained more than 50% activity at 0℃. It exhibited improved catalytic activity and thermostability in NaCl solution, with maximal activity occurring at 800 mmol/L NaCl/KCl, and more than 50% of maximal activity retained after incubation in 2 mol/L NaCl for 80 h at 25℃. NaCl did not cause significant change of the global tertiary structure of Amy3, whereas influenced the catalysis efficiency. Amy3 hydrolyzed amylopectin preferentially, and could also hydrolyze wheat starch, corn starch and tapioca starch.[Conclusion] The results indicated that the alpha-amylase Amy3 is a cold-active and salt-tolerant α-amylase with potential use in basic research and industry.

    参考文献
    [1] Gupta R, Gigras P, Mohapatra H, Goswami VK, Chauhan B. Microbial α-amylases:a biotechnological perspective. Process Biochemistry, 2003, 38(11):1599-1616.
    [2] Song QS, Wang Y, Yin C, Zhang XH. LaaA, a novel high-active alkalophilic alpha-amylase from deep-sea bacterium Luteimonas abyssi XH031T. Enzyme and Microbial Technology, 2016, 90:83-92.
    [3] Qin YJ, Huang ZQ, Liu ZD. A novel cold-active and salt-tolerant α-amylase from marine bacterium Zunongwangia profunda:molecular cloning, heterologous expression and biochemical characterization. Extremophiles, 2014, 18(2):271-281.
    [4] Yooseph S, Sutton G, Rusch DB, Halpern AL, Williamson SJ, Remington K, Eisen JA, Heidelberg KB, Manning G, Li WZ, Jaroszewski L, Cieplak P, Miller CS, Li WZ, Mashiyama ST, Joachimiak MP, van Belle C, Chandonia JM, Soergel DA, Zhai YF, Natarajan K, Lee S, Raphael BJ, Bafna V, Friedman R, Brenner SE, Godzik A, Eisenberg D, Dixon JE, Taylor SS, Strausberg RL, Frazier M, Venter JC. The sorcerer Ⅱ global ocean sampling expedition:expanding the universe of protein families. PLoS Biology, 2007, 5(3):e16.
    [5] Lu MS, Wang SJ, Fang YW, Li HZ, Liu S, Liu HF. Cloning, expression, purification, and characterization of cold-adapted α-amylase from Pseudoalteromonas arctica GS230. The Protein Journal, 2010, 29(8):591-597.
    [6] Zhang JW, Zeng RY. Purification and characterization of a cold-adapted α-amylase produced by Nocardiopsis sp. 7326 isolated from Prydz Bay, Antarctic. Marine Biotechnology, 2008, 10(1):75-82.
    [7] Homaei A, Ghanbarzadeh M, Monsef F. Biochemical features and kinetic properties of α-amylases from marine organisms. International Journal of Biological Macromolecules, 2016, 83:306-314.
    [8] Mi ZH, Yu ZC, Su HN, Wang L, Chen XL, Pang XH, Qin QL, Xie BB, Zhang XY, Zhou BC, Zhang YZ. Physiological and genetic analyses reveal a mechanistic insight into the multifaceted lifestyles of Pseudoalteromonas sp. SM9913 adapted to the deep-sea sediment. Environmental Microbiology, 2015, 17(10):3795-3806.
    [9] Li W, Xue Y, Li JJ, Yuan J, Wang XT, Fang W, Fang ZM, Xiao YZ. A cold-adapted and glucose-stimulated type Ⅱ α-glucosidase from a deep-sea bacterium Pseudoalteromonas sp. K8. Biotechnology Letters, 2016, 38(2):345-349.
    [10] Li W, Fan H, He C, Zhang XC, Wang XT, Yuan J, Fang ZM, Fang W, Xiao YZ. PspAG97A:a halophilic α-glucoside hydrolase with wide substrate specificity from glycoside hydrolase family 97. Journal of Microbiology and Biotechnology, 2016, 26(11):1933-1942.
    [11] Liu Y, Lei Y, Zhang XC, Gao Y, Xiao YZ, Peng H. Identification and phylogenetic characterization of a new subfamily of α-amylase enzymes from marine microorganisms. Marine Biotechnology, 2012, 14(3):253-260.
    [12] Feller G, Lonhienne T, Deroanne C, Libioulle C, van Beeumen J, Gerday C. Purification, characterization, and nucleotide sequence of the thermolabile alpha-amylase from the antarctic psychrotroph Alteromonas haloplanctis A23. Journal of Biological Chemistry, 1992, 267(8):5217-5221.
    [13] MacGregor EA, Janeček Š, Svensson B. Relationship of sequence and structure to specificity in the α-amylase family of enzymes. Biochimica et Biophysica Acta (BBA)-Protein Structure and Molecular Enzymology, 2001, 1546(1):1-20.
    [14] Buisson G, Duée E, Haser R, Payan F. Three dimensional structure of porcine pancreatic alpha-amylase at 2.9Å resolution. Role of calcium in structure and activity. The EMBO Journal, 1987, 6(13):3909-3916.
    [15] Chakraborty S, Raut G, Khopade A, Mahadik K, Kokare C. Study on calcium ion independent α-amylase from haloalkaliphilic marine Streptomyces strain A3. Indian Journal of Biotechnology, 2012, 11(4):427-437.
    [16] Malhotra R, Noorwez SM, Satyanarayana T. Production and partial characterization of thermostable and calcium-independent α-amylase of an extreme thermophile Bacillus thermooleovorans NP54. Letters in Applied Microbiology, 2000, 31(5):378-384.
    [17] Shih NJ, Labbé RG. Purification and characterization of an extracellular alpha-amylase from Clostridium perfringens type A. Applied and Environmental Microbiology, 1995, 61(5):1776-1779.
    [18] Ajayi AO, Fagade OE. Heat activation and stability of amylases from Bacillus species. African Journal of Biotechnology, 2007, 6(10):1181-1184.
    [19] Feller G, Payan F, Theys F, Qian MX, Haser R, Gerday C. Stability and structural analysis of α-amylase from the antarctic psychrophile Alteromonas haloplanctis A23. European Journal of Biochemistry, 1994, 222(2):441-447.
    [20] Zhang C, Kim SK. Research and application of marine microbial enzymes:status and prospects. Marine Drugs, 2010, 8(6):1920-1934.
    [21] Hough DW, Danson MJ. Extremozymes. Current Opinion in Chemical Biology, 1999, 3(1):39-46.
    [22] Boutaiba S, Bhatnagar T, Hacene H, Mitchell DA, Baratti JC. Preliminary characterisation of a lipolytic activity from an extremely halophilic archaeon, Natronococcus sp.. Journal of Molecular Catalysis B:Enzymatic, 2006, 41(1/2):21-26.
    [23] Qian MX, Ajandouz EH, Payan F, Nahoum V. Molecular basis of the effects of chloride ion on the acid-base catalyst in the mechanism of pancreatic α-amylase. Biochemistry, 2005, 44(9):3194-3201.
    [24] Maurus R, Begum A, Kuo HH, Racaza A, Numao S, Andersen C, Tams JW, Vind J, Overall CM, Withers SG, Brayer GD. Structural and mechanistic studies of chloride induced activation of human pancreatic α-amylase. Protein Science, 2005, 14(3):743-755.
    [25] Aghajari N, Feller G, Gerday C, Haser R. Structural basis of α-amylase activation by chloride. Protein Science, 2002, 11(6):1435-1441.
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薛毅,王梅,方泽民,房伟,肖亚中. 低温、嗜盐α-淀粉酶Amy3的克隆、表达及重组酶性质[J]. 微生物学报, 2018, 58(2): 336-345

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  • 收稿日期:2017-04-06
  • 最后修改日期:2017-06-14
  • 在线发布日期: 2018-01-26
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