Recombinant expression of maltotriose-forming alpha-amylases fromThermobifida fusca and its application in preparation of maltotriose

  • HU Fan ,
  • SU Lingqia ,
  • WU Jing
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  • 1(State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi 214122, China)
    2(Key Laboratory of Industrial Biotechnology Ministry of Education,Jiangnan University,Wuxi 214122, China)

Received date: 2019-11-12

  Online published: 2020-04-10

Abstract

The maltotriose-forming α-amylase (AmyA) can produce maltotriose-rich syrup from starch or maltodextrin, and has broad application prospects in food and beverage industry. The maltotriose-forming α-amylase derived from Thermobifida fusca NTU22 (referred as TfAmyA) was recombinantly expressed in Bacillus subtilis WS11 for the first time. The recombinant TfAmyA was further purified and characterized. The optimal temperature of the TfAmyA is 55 ℃, the optimal pH is 6.0. The optimal conditions for maltotriose production from maltodextrin catalyzed by the recombinant TfAmyA were also investigated. The results showed that when 15% (mass concentration) maltodextrin (DE 5-7) was used as the substrate, the optimal conditions for enzymatic conversion were temperature 55 ℃ and pH 5.5. The dosage of TfAmyA is 60 U/g substrate, and the dosage of pullulanase was 32 U/g substrate. Under these conditions, the maximum conversion rate of maltotriose was 44.4% when the reaction proceeded to 11 h. These results indicated that the recombinant TfAmyA has superior properties and provides a theoretical basis for the industrial preparation of maltotriose.

Cite this article

HU Fan , SU Lingqia , WU Jing . Recombinant expression of maltotriose-forming alpha-amylases fromThermobifida fusca and its application in preparation of maltotriose[J]. Food and Fermentation Industries, 2020 , 46(5) : 23 -30 . DOI: 10.13995/j.cnki.11-1802/ts.022793

References

[1] 干昭波,邵先豹,窦光朋,等.一种高纯度麦芽三糖的制备方法:山东,CN103215326A[P].2013-07-24.
[2] 沈微,郭玉婉,黄雯雯,等.一种以淀粉为原料的麦芽三糖制备方法及其专用真菌α-淀粉酶:江苏,CN103014097B[P].2013-04-03.
[3] 杜宇国,贺鹏.一种制备麦芽三糖的新方法:北京,CN105713051A[P].2016-06-29.
[4] 乔长晟,朱明,李文军,等.麦芽三糖形成酶的高产菌株及其筛选和培养方法:天津,CN105647827A[P].2016-06-08.
[5] WAKO K,TAKAHASHI C,HASHIMOTO S,et al.Studies on Maltotriose- and Maltose-forming Amylases from Streptomyces[J].Journal of the Japanese Society of Starch Science,1978,25(2):155-161.
[6] TAKASAKI Y J A,CHEMISTRY B.An amylase producing maltotriose from Bacillus subtilis[J].Agricultural and Biological Chemistry,1985,49(4):1 091-1 097.
[7] TAKASAKI Y,KITAJIMA M,TSURUTA T,et al.Maltotriose-producing Amylase from Microbacterium imperiale [J].Agricultural and Biological Chemistry,1991,55(3):687-692.
[8] RATANAKHANOKCHAI K,KANEKO J,KAMIO Y,et al.Purification and Properties of a Maltotetraose- and Maltotriose-Producing Amylase from Chloroflexus aurantiacus[J].Appl Environ Microb,1992,58(8):2 490-2 494.
[9] KOBAYASHI T,KANAI H,HAYASHI T,et al.Haloalkaliphilic Maltotriose-Forming α-amylase from the Archaebacterium Natronococcus sp. strain Ah-36[J].J Bacteriol,1992,174(11):3 439-3 444.
[10] SATOH E,NIIMURA Y,UCHIMURA T,et al.Molecular cloning and expression of two alpha-amylase genes from Streptococcus bovis 148 in Escherichia coli[J].Appl Environ Microb,1993,59(11):3 669-3 673.
[11] YANG C H,LIU W H.Purification and properties of a maltotriose-producing alpha-amylase from Thermobifida fusca[J].Enzyme Microb Tech,2004,35(2-3):254-260.
[12] DOUKYU N,YAMAGISHI W,KUWAHARA H,et al.Purification and characterization of a maltooligosaccharide-forming amylase that improves product selectivity in water-miscible organic solvents, from dimethylsulfoxide-tolerant Brachybacterium sp. strain LB25[J].Extremophiles,2007,11(6):781-788.
[13] BEN ABDELMALEK-KHEDHER I,URDACI M C,LIMAM F,et al.Purification, characterization, and partial primary sequence of a major-maltotriose-producing alpha-amylase, ScAmy43, from Selerotinia sclerotiorum[J].J Microbiol Biotechn,2008,18(9):1 555-1 563.
[14] 郭玉婉.酵母α-淀粉酶基因克隆、表达及其应用于麦芽三糖的制备[D].无锡:江南大学,2013.
[15] WANG J,LI Y,LU F.Molecular cloning and biochemical characterization of an α-amylase family from Aspergillus niger[J].Electron J Biotechn,2018,32:55-62.
[16] 吴春森.麦芽三糖酶催化作用机制及其应用研究[D].无锡:江南大学,2017.
[17] TAKASAKI Y,KITAJIMA M,TSURUTA T,et al.Studies on enzymatic production of oligosaccharides. Part VI. Maltotriose-producing amylase from Microbacterium imperiale[J].Agricultural and Biological Chemistry,1991,55(3):687-692.
[18] 徐贵华,刘钟栋,陈肇锬.小麦淀粉制备麦芽三糖的研究[J].郑州工程学院学报,2002(3):1-4.
[19] LEE Y S,PARK D J,CHOI Y L.Characterization of maltotriose production by hydrolyzing of soluble starch with alpha-amylase from Microbulbifer thermotolerans DAU221[J].Appl Microbiol Biot,2015,99(9):3 901-3 911.
[20] YANG C H,LIU W H.Cloning and characterization of a maltotriose-producing alpha-amylase gene from Thermobifida fusca[J].J Ind Microbiol Biot (2745),2007,34(4):325-330.
[21] YANG C H,HUANG Y C,CHEN C Y,et al.Expression of Thermobifida fusca thermostable raw starch digesting alpha-amylase in Pichia pastoris and its application in raw sago starch hydrolysis[J].J Ind Microbiol Biot,2010,37(4):401-406.
[22] YANG C H,HUANG Y C,CHEN C Y,et al.Heterologous expression of Thermobifida fusca thermostable alpha-amylase in Yarrowia lipolytica and its application in boiling stable resistant sago starch preparation[J].J Ind Microbiol Biot,2010,37(9):953-960.
[23] WU C,ZHOU X,XU Y,et al.Characterization and mechanism of action of Microbacterium imperiale glucan 1,4-α-maltotriohydrolase[J].Carbohyd Res,2014,384:46-50.
[24] HONG H A,HUANG J M,KHANEJA R,et al.The safety of Bacillus subtilis and Bacillus indicus as food probiotics[J].J Appl Microbiol,2008,105(2):510-520.
[25] 杨亚楠,宿玲恰,吴敬.重组Bacillus subtilis产麦芽四糖淀粉酶的发酵优化及麦芽四糖制备[J].食品与发酵工业,2019,45(08):44-49;56.
[26] 吴世雄,宿玲恰,姚锴琳,等.MTSase和MTHase在Brevibacillus brevis中的克隆表达及应用研究[J].食品与生物技术学报,2018,37(8):838-844.
[27] BRADFORD M M.A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[J].Anal Biochem,1976,72(1):248-254.
[28] MILLER G L.Use of Dinitrosalicylic Acid Reagent for Determination of Reducing Sugar[J].Anal Chem,1959,31(3):426-428.
[29] FIELDS P A.Review: Protein function at thermal extremes: balancing stability and flexibility[J].Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology,2001,129(2):417-431.
[30] ZHOU H-X,CHEN Y-D.Chemically Driven Motility of Brownian Particles[J].Phys Rev Lett,1996,77(1):194-197.
[31] TAWADA K,SEKIMOTO K.Protein friction exerted by motor enzymes through a weak-binding interaction[J].J Theor Biol,1991,150(2):193-200.
[32] DOUKYU N,YAMAGISHI W,KUWAHARA H,et al.A maltooligosaccharide-forming amylase gene from Brachybacterium sp. strain LB25: cloning and expression in Escherichia coli[J].Bioscience, Biotechnology, and Biochemistry,2008,72(9):2 444-2 447.
[33] KASHIWAGI N,MIYAKE M,HIROSE S,et al.Cloning and starch degradation profile of maltotriose-producing amylases from Streptomyces species[J].Biotechnol Lett,2014,36(11):2 311-2 317.
[34] KAMON M,SUMITANI J,TANI S,et al.Characterization and gene cloning of a maltotriose-forming exo-amylase from Kitasatospora sp. MK-1785[J].Appl Microbiol Biot,2015,99(11):4 743-4 753.
[35] NIELSEN J E,BORCHERT T V,VRIEND G.The determinants of α-amylase pH–activity profiles[J].Protein Engineering, Design and Selection,2001,14(7):505-512.
[36] NAKAKUKI T,AZUMA K,KAINUMA K.Action patterns of various exo-amylases and the anomeric configurations of their products[J].Carbohyd Res,1984,128(2):297-310.
[37] 黄金莲,钟振声.普鲁兰酶对不同原料淀粉糖生产的影响[J].安徽农业科学,2011,39(32):20 015-20 017.
[38] 朱荣,宿玲恰,吴敬.重组4-α-糖基转移酶转化淀粉制备大元环糊精的条件优化[J].食品与生物技术学报,2018,37(12):1 248-1 254.
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