Improvement of Catalytic Activity of Amylase from Bacillus amyloliquefaciens and Its High Expression in Bacillus subtilis

doi:10.13560/j.cnki.biotech.bull.1985.2019-0234

Abstract

Abstract: α-Amylase is a kind of enzymes that catalyzes the random internal hydrolysis of α-1,4 glycosidic linkages in starch chain into dextrin,oligosaccharides and monosaccharides. The enzyme is widely used in bread making and washing detergent etc. Modifying amylase at the molecular level to improve the hydrolysis activity and catalytic efficiency has been the goal for many years and has important research significance. In this study,based on BAMYA,a widely used neutral amylase from Bacillus amyloliquefaciens,the mutation sites（L473K/K474H/N475K）at the C-terminal similar substrate binding region（CBM）were rationally designed as the site-directed mutation was aimed to increase its hydrolysis ability,and its efficient expression in Bacillus subtilis was achieved. The optimal temperature and pH of wild BAMYA were 55℃and 6.0,respectively and the specific activity was 6 835 U/mg. The optimal temperature of mutant BAMYA-L473K/K474H/N475K was 60℃,5℃ higher than that of wild one,while no changes in pH. The specific activity of the mutant was 10 148 U/mg,and it increased by 48% compared to the wild one. The Km and catalytic efficiency of wild BAMYA and mutant BAMYA-L473K/K474H/N475K were 3.58 mg/mL and 2.21 mg/mL,1 577 mL/（s·mg）and 4 760 mL/（s·mg）,respectively. The catalytic efficiency increased over two times than that of wild one. In sum,the application of the mutant in industry will effectively improve the efficiency of hydrolyzing starch and reduce the application cost.

Key words: α-amylase, site-directed mutation, Bacillus subtilis, catalytic efficiency

QIU Jin, HUANG Huo-qing, YAO Bin, LUO Hui-ying. Improvement of Catalytic Activity of Amylase from Bacillus amyloliquefaciens and Its High Expression in Bacillus subtilis[J]. Biotechnology Bulletin, 2019, 35(9): 134-143.

References

[1] Reddy CK, Choi SM, Lee DJ, et al.Complex formation between starch and stearic acid:Effect of enzymatic debranching for starch[J]Food Chem, 2018, 244(1):136-142.
[2] Bentley IS, Williams EC.Starch conversion[C]//Godfrey T, West S(eds. )London, UK:Industrial Enzymology, The Macmillan Press Ltd, 1996:341-357.
[3] 王丽萨. 来源于Pyrococcus furiosus极端嗜热α-淀粉酶的研究[D]. 上海:中国科学院研究生院, 2007.
[4] 段钢. 新型工业酶制剂的进步对生物化学品工业生产过程的影响[J]. 生物工程学报, 2009, 25(12):1808-1818.
[5] Goyal N, Gupta JK, Soni SK.A novel raw starch digesting thermostable a-amylase from Bacillus sp. I-3 and its use in the direct hydrolysis of raw potato starch[J]. Enzyme Microb Tech, 2005, 37(2):723-734.
[6] Jens EN, Torben V.Protein engineering of bacterial amylases[J]. Biochim Biophys Acta, 2000, 1543(2):253-274.
[7] Van der Maarel MJ, Van der Veen B, Uitdehaag JCM, et al. Properties and applications of starch-converting enzymes of the α-amylase family[J]. Journal of Biotechnology, 2002, 94(2):137-155.
[8] Gupta R, Gigras P, Mohapatra H, et al.Microbial α-amylase:A biotechnological perspective[J]. Process Biochemistry, 2003, 38(11):1599-1616.
[9] Kilara A, Desai. M. Enzymes.In:Food Additives[C], III(Eds. ), New York, USA:Marcel Dekker Inc., 2002:661-706.
[10] Lee BH.Other microorganism based products. In:fundamentals of food biotechnology[C]. New York, USA:Wiley-VCH Inc, 1996:291-352.
[11] van der Maarel MJ, van der Veen B, Uitdehaag JCM, et al. Properties and applications of starch-converting enzymes of the α-amylase family[J]. Journal of Biotechnology, 2002, 94(2):137-155.
[12] Swetha S, Dhanya G, Kesavan MN, et al.α-amylase from microbial sources-an overview on recent developments[J]. Food Technol Biotechnol, 2006, 44(2):173-184.
[13] Weemaes C, De Cordt S, Goossens K, et al.High pressure, thermal, and combined pressure-temperature stabilities of α-amylases from Bacillus species[J]. Biotechnol Bioeng, 1996, 50(1):49-56.
[14] Alikhajeh J, Khajeh K, Ranjbar B, et al.Structure of Bacillus amyloliquefaciens alpha-amylase at high resolution:implications for thermal stability[J]. Acta Cryst, 2010, 66(2):121-129.
[15] Hwang KY, Song HK, Chang C, et al.Crystal structure of thermostable alpha-amylase from Bacillus licheniformis refined at 1. 7 A resolution[J]. Mol Cells, 1997, 7(2):251-258.
[16] Shirai T, Igarashi K, Ozawa T, et al.Ancestral sequence evolutionary trace and crystal structure analyses of alkaline alpha-amylase from Bacillus sp. KSM-1378 to clarify the alkaline adaptation process of proteins[J]. Proteins, 2007, 66(3):600-610.
[17] Cornelius B, Jutta S, Karl HM, et al.Directed evolution of a bacterial-amylase:toward enhanced pH-performance and higher specific activity[J]. Protein Science, 2003, 12(10):2141-2149.
[18] Nathalie D, Mischa M, Philippe J, et al.Engineering the thermostability of Bacillus licheniformis α-amylase[J]. Biologia Bratislava, 2002, 57(11):203-211.
[19] Archana S, Satyanarayana T.Cloning and expression of acidstable, high maltose-forming, Ca²+-independent α-amylase from an acidophile Bacillus acidicola and its applicability in starch hydrolysis[J]. Extremophiles, 2012, 16(3):515-522.
[20] Azadeh E, Khosro K, Hossein NM.Thermostabilization of Bacillus amyloliquefaciens amylase by chemical cross-linking[J]. J Biotechnol, 2006, 123(4):434-442.
[21] Janecek S, Svensson B, MacGregor EA. Alpha-amylase:an enzyme specificity found in various families of glycoside hydrolases[J]. Cellular and Molecular Life Sciences, 2014, 71(4):1149-1170.
[22] Sumitani J, Tottori T, Kawaguchi T, et al.New type of starch-binding domain:the direct repeat motif in the C-terminal region of Bacillus sp. no. 195 alpha-amylase contributes to starch binding and raw starch degrading[J]. Biochemical Journal, 2000, 350(2):477-484.
[23] Li Z, Duan X, Chen S, et al.Improving the reversibility of thermal denaturation and catalytic efficiency of Bacillus licheniformis α-amylase through stabilizing a long loop in domain B[J]. PLoS One, 2017, 12(3):1-11.
[24] Nielsen JE, Borchert TV.Protein engineering of bacterial alpha-amylases[J]. Biochimica Et Biophysica Acta-Protein Structure and Molecular Enzymology, 2000, 1543(2):253-274.
[25] Robert X, Haser R, Gottschalk TE, et al.The structure of barley alpha-amylase isozyme reveals a novel role of domain C in substrate recognition and binding:A pair of sugar tongs[J]. Structure, 2003, 11(8):973-984.
[26] Wang H, Yang L, Ping YH, et al.Engineering of a Bacillus amyloliquefaciens strain with high neutral protease producing capacity and optimization of its fermentation conditions[J]. PLoS One, 2016, 11(1):1-8.
[27] Igarashi K, Hatada Y, Hagihara H.Enzymatic properties of a novel liquefying α-amylase from an alkaliphilic Bacillus isolate and entire nucleotide and amino acid sequences[J]. Appl Environ Microbiol, 1998, 64(9):3282-3289.
[28] Prajapati VS, Trivedi UB, Patel KC.A statistical approach for the production of thermostable and alklophilic alpha-amylase from Bacillus amyloliquefaciens KCP2 under solid-state fermentation[J]. 3 Biotech, 2015, 5(2):211-220.
[29] Dowd JE, Riggs DS.A comparison of estimates of michaelis-menten kinetic constants from various linear transformations[J]. Journal of Biological Chemistry, 1965, 240(2):863-869.
[30] Syu LJ, Chen YH.A study on the α-amylase fermentation performed by Bacillus amyloliquefaciens[J]. Chem Eng, 1997, 65(1):237-247.
[31] Lu Z, Wang Q, Jiang S, et al.Truncation of the unique N-terminal domain improved the thermos-stability and specific activity of alkaline α-amylase Amy703[J]. Sci Rep, 2016, 6:1-10.
[32] Yang H, Liu L, Shin HD, et al.Integrating terminal truncation and oligopeptide fusion for a novel protein engineering strategy to improve specific activity and catalytic efficiency:alkaline-amylase as a case study[J]. Appl Environ Microbiol, 2013, 79(20):6429-6438.
[33] Bessler C, Schmitt J, Maurer KH, et al.Directed evolution of a bacterial -amylase:toward enhanced pH-performance and higher specific activity[J]. Protein Sci, 2003, 12(10):2141-2149.
[34] Shirai T, Igarashi K, Ozawa T, et al.Ancestral sequence evolutionary trace and crystal structure analyses of alkaline alpha-amylase from Bacillus sp. KSM-1378 to clarify the alkaline adaptation process of proteins[J]. Proteins, 2007, 66(3):600-610.
[35] Robert X, Haser R, Gottschalk TE, et al.The structure of barley alpha-amylase isozyme reveals a novel role of domain C in substrate recognition and binding:A pair of sugar tongs[J]. Structure, 2003, 11(8):973-984.
[36] Hondoh H, Kuriki T, Matsuura Y.Three-dimensional structure and substrate binding of Bacillus stearothermophilus neopullulanase[J]. J Mol Biol, 2003, 326(1):177-188.
[37] Kim MS, Park JT, Kim YW, et al.Properties of a novel thermostable glucoamylase from the hyperthermophilic archaeon Sulfolobus sol- fataricus in relation to starch processing[J]. Applied Environme-ntal Microbiology, 2004, 70(7):3933-3940.
[38] Kobayashi T, Kanai H, Hayashi T, et al.Haloalkaliphilic maltot-riose-forming amylase from the archae bacterium Natronococcus sp. strain Ah-36[J]. Journal of Bacteriology, 1992, 174(11):3439-3444.
[39] Wang Y, Feng SY, Zhan T, et al.Improving catalytic efficiency of endo-β-1, 4-xylanase from Geobacillus stearothermophilus by directed evolution and H179 saturation mutagenesis[J]. Journal of Biotechnology, 2013, 168(4):341-347.
[40] Goyal N, Gupta JK, Soni SK.A novel raw starch digesting thermostable a-amylase from Bacillus sp. I-3 and its use in the direct hydrolysis of raw potato starch[J]. Enzyme Microb Technol, 2005, 37(7):723-734.
[41] Yang CH, Liu WH.Purification and properties of a maltotriose-producing a-amylase from Thermobifida fusca[J]. Enzyme Microb Technol, 2004, 35(2):254-260.