极端嗜热α-淀粉酶ApkA的高温活性和热稳定性的优化研究

doi:10.13560/j.cnki.biotech.bull.1985.2017-0172

摘要/Abstract

摘要： 旨在获得高温活性和热稳定性提高的α-淀粉酶。通过向α-淀粉酶ApkA中引入目前已知的最稳定的α-淀粉酶PFA的Zn²⁺结合位点，获得Zn²⁺结合位点突变体ApkAdsK152H/A166C。酶学性质分析表明，ApkAdsK152H/A166C的高温活性和热稳定性明显提高，最适反应温度由90℃提高至100℃，对应的酶比活力为5 201.08 U/mg。ApkAdsK152H/A166C于90℃的半衰期由5 h延长至10 h，于100℃的半衰期由7.5 min延长至80 min。重组α-淀粉酶中Zn²⁺含量测定结果显示ApkAdsK152H/A166C结合了一个Zn²⁺。结果表明，向ApkA中引入Zn²⁺结合位点有利于提高其高温活性和热稳定性。

关键词: 极端嗜热α-淀粉酶, Zn²⁺-结合位点, 定点突变, 高温活性, 热稳定性

Abstract: This work aims to obtain α-amylase with improved thermal activity and stability. Based on the structure analysis of hyperthermophilic α-amylase ApkA and the most thermo-stable α-amylase PFA，a Zn²⁺-binding site mutant ApkAdsK152H/A166C was constructed by introducing the Zn²⁺-binding site of PFA into ApkA. The mutant ApkAdsK152H/A166C exhibited effective increase in terms of thermal activity and stability. The optimal temperature of the mutant increased from 90℃ to 100℃ and the corresponding specific activity was 5 201.08 U/mg. The half-life of ApkAdsK152H/A166C prolonged from 5 h to 10 h while incubated at 90℃，and from 7.5 min to 80 min at 100℃. The results of Zn²⁺ content measurement in recombinant α-amylases confirmed that ApkAdsK152H/A166C bound with one Zn²⁺ ion. These results suggested that the introduction of Zn²⁺-binding site improved the thermal activity and stability of ApkA.

Key words: hyperthermophilic α-amylase, Zn²⁺-binding site, site-directed mutagenesis, thermo-activity, thermostability

曾静, 郭建军, 袁林, 杨罡, 陈俊. 极端嗜热α-淀粉酶ApkA的高温活性和热稳定性的优化研究[J]. 生物技术通报, 2017, 33(8): 192-198.

ZENG Jing, GUO Jian-jun, YUAN Lin, YANG Gang, CHEN Jun. Optimization of the Thermal Activity and Stability of Hyperthermophilic α-amylase ApkA[J]. Biotechnology Bulletin, 2017, 33(8): 192-198.

参考文献

[1] Jane?ek ?, Svensson B, Macgregor EA. α-Amylase：an enzyme specificity found in various families of glycoside hydrolases[J] . Cellular and Molecular Life Sciences, 2014, 71（7）：1149-1170.
[2] Rana N, Walia A, Gaur A. α-Amylases from microbial sources and its potential applications in various industries[J] . National Academy Science Letters, 2013, 36（1）：9-17.
[3] Zhang Q, Han Y, Xiao H. Microbial α-amylase：a biomolecular overview[J] . Process Biochemistry, 2017, 53：88-101.
[4] Homaei A, Ghanbarzadeh M, Monsef F. Biochemical features and kinetic properties of α-amylases from marine organisms[J] . International Journal of Biological Macromolecules, 2016, 83：306-314.
[5] Souza PM. Application of microbial α-amylase in industry-a review[J] . Brazilian Journal of Microbiology, 2010, 41（4）：850-861.
[6] Kumari A, Singh KM, Kayastha A. α-Amylase：general properties, mechanism and biotechnological applications-a review[J] . Current Biotechnology, 2012, 1（1）：98-107.
[7] Atomi H, Sato T, Kanai T. Application of hyperthermophiles and their enzymes[J] . Current Opinion in Biotechnology, 2011, 22（5）：618-626.
[8] Prakash O, Jaiswal N. α-Amylase：an ideal representative of thermostable enzymes[J] . Applied Biochemistry and Biotechnology, 2010, 160（8）：2401-2414.
[9] Konsoula Z, Liakopoulou-Kyriakides M. Co-production of α-amylase and β-galactosidase by Bacillus subtilis in complex organic substrates[J] . Bioresource Technology, 2007, 98（1）：150-157.
[10] Zhou W, You C, Ma H, et al. One-pot biosynthesis of high-concentration α-glucose 1-phosphate from starch by sequential addition of three hyperthermophilic enzymes[J] . Journal of Agricultural and Food Chemistry, 2016, 64（8）：1777-1783.
[11] Dalmaso GZL, Ferreira D, Vermelho AB. Marine extremophiles：a source of hydrolases for biotechnological applications[J] . Marine Drugs, 2015, 13（4）：1925-1965.
[12] 曾静, 郭建军, 邱小忠, 等. 极端嗜热微生物及其高温适应机制的研究进展[J] . 生物技术通报, 2015（9）：30-37.
[13] Tachibana Y, Leclere MM, et al. Cloning and expression of the α-amylase gene from the hyperthermophilic archaeon Pyrococcus sp. KOD1, and characterization of the enzyme[J] . Journal of Fermentation and Bioengineering, 1996, 82（3）：224-232.
[14] Green MR, Sambrook J. Molecular cloning：a laboratory manual[M] . New York：Cold Spring Harbor Laboratory Press, 2012.
[15] Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4[J] . Nature, 1970, 227：680-685.
[16] Bernfeld P. Amylases, α and β[J] . Methods in Enzymology, 1955, 1：149-158.
[17] Savchenko A, Vieille C, Kang S, et al. Pyrococcus furiosus α-amylase is stabilized by calcium and zinc[J] . Biochemistry, 2002, 41（19）：6193-6201.
[18] Dey TB, Kumar A, Banerjee R, et al. Improvement of microbial α-amylase stability：strategic approaches[J] . Process Biochemistry, 2016, 51（10）：1380-1390.
[19] Linden A, Mayans O, Meyer-Klaucke W, et al. Differential regulation of a hyperthermophilic α-amylase with a novel（Ca, Zn）two-metal center by zinc[J] . Journal of Biological Chemistry, 2003, 278（11）：9875-9884.
[20] Machius M, Declerck N, Huber R, et al. Activation of Bacillus licheniformis α-amylase through a disorder→ order transition of the substrate-binding site mediated by a calcium-sodium-calcium metal triad[J] . Structure, 1998, 6（3）：281-292.
[21] Qin Y N, Fang Z, Pan FG, et al. Significance of Tyr302, His235 and Asp194 in the α-amylase from Bacillus licheniformis[J] . Biotechnology Letters, 2012, 34（5）：895-899.
[22] Machius M, Declerck N, Huber R, et al. Kinetic stabilization of Bacillus licheniformis α-amylase through introduction of hydrophobic residues at the surface[J] . Journal of Biological Chemistry, 2003, 278（13）：11546-11553.
[23] Ghollasi M, Ghanbari-Safari M, Khajeh K. Improvement of thermal stability of a mutagenised α-amylase by manipulation of the calcium-binding site[J] . Enzyme and Microbial Technology, 2013, 53（6）：406-413.
[24] 柯涛, 刘征, 杨建伟, 等. A154C/G155C双点突变对嗜热耐酸淀粉酶酶活性及热稳定性的影响[J] . 食品科学, 2012, 33（3）：207-211.
[25] 曾静, 郭建军, 等. 定点突变提高极端嗜热α-淀粉酶ApkA的高温活性和热稳定性[J] . 食品科学, 2017, 38（2）：20-26.
[26] J?rgensen S, Vorgias Ce, Antranikian G. Cloning, sequencing, characterization, and expression of an extracellular α-amylase from the hyperthermophilic archaeon Pyrococcus furiosus in Escherichia coli and Bacillus subtilis[J] . Journal of Biological Chemistry, 1997, 272（26）：16335-16342.
[27] 曾静, 郭建军, 袁林. N-糖基化修饰对极端嗜热酸性α-淀粉酶ApkA酶学性质的影响[J] . 食品科学, 2017, 38（6）：1-6.