嗜热绿色糖单孢菌木聚糖酶XyN10A基因在毕赤酵母中的分泌表达及其酶学性质研究
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摘要
木聚糖酶(Xylanase)可降解自然界中大量存在的木聚糖类半纤维素,由于其在饲料、食品、造纸等领域的广泛应用,越来越受到人们的关注。利用分子生物学技术首次成功构建了一株高效表达嗜热绿色糖单孢菌(Saccharomonospora viridis)木聚糖酶SviXyN10A的重组工程菌,建立了SviXyN10A的毕赤酵母表达体系,实现了SviXyN10A在毕赤酵母(Pichia pastoris)中的分泌表达,并对表达产物进行了诱导表达及纯化,以及酶学性质的研究。结果表明,重组木聚糖酶SviXyn10A用甲醇诱导2 d后酶活即达到7.53IU/mL,最适反应温度为70℃,pH7.0。pH作用范围较广(pH5.0-9.0),具有较强的热稳定性和耐碱性,几乎没有纤维素酶活性,故可以应用在很多工业领域,尤其是在纸浆造纸行业具有很大的潜在应用价值。
Xylanase has attracted extensive attention because of the application potential in feed,food and paper area,capable of degrading hemicellulose which is prevalent in nature.This research is to construct a recombinant engineered bacteria with efficient secretory expression for thermophilic Saccharomonospora viridis xylanase SviXyN10A using molecular biology techniques,and then study the characterization of the gene product expressed in Pichia pastoris.The results indicate that the gene product of Svixyn10A was successfully expressed in Pichia pastoris,showing the highest enzymatic activity of 7.53 IU/mL with methanol induction for 2 days.Recombinant xylanase SviXyN10A showed good activity at wide range of pH effect(pH5.0-9.0),and the optimum reaction temperature is 70℃,pH7.0.What is more,the xylanase showed excellent thermo-alkali-stability and almost no cellulase activity.Thus,it is a potential candidate for future use in industrial areas particularly in the pulp and paper industry.
Xylanase has attracted extensive attention because of the application potential in feed,food and paper area,capable of degrading hemicellulose which is prevalent in nature.This research is to construct a recombinant engineered bacteria with efficient secretory expression for thermophilic Saccharomonospora viridis xylanase SviXyN10A using molecular biology techniques,and then study the characterization of the gene product expressed in Pichia pastoris.The results indicate that the gene product of Svixyn10A was successfully expressed in Pichia pastoris,showing the highest enzymatic activity of 7.53 IU/mL with methanol induction for 2 days.Recombinant xylanase SviXyN10A showed good activity at wide range of pH effect(pH5.0-9.0),and the optimum reaction temperature is 70℃,pH7.0.What is more,the xylanase showed excellent thermo-alkali-stability and almost no cellulase activity.Thus,it is a potential candidate for future use in industrial areas particularly in the pulp and paper industry.
引文
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[16]周蕾,顾建新.N-糖基化位点鉴定方法和非经典N-糖基化序列[J].生命科学,2011,23(6):605-611.
[2]Du Y,Shi P,Huang H,et al.Characterization of three novel thermophilic xylanases from Humicola insolens Y1 with application potentials in the brewing industry[J].Bioresource Technology,2013,130:161-167.
[3]Lin XQ,Han SY,Zhang N,et al.Bleach boosting effect of xylanase A from Bacillus halodurans C-125 in ECF bleaching of wheat straw pulp[J].Enzyme and Microbial Technology,2013,52(2):91-98.
[4]Polizeli ML,Rizzatti AC,Monti R,et al.Xylanases from fungi:properties and industrial applications[J].Appl Microbiol Biotechnol,2005,67(5):577-591.
[5]Gellissen G.Heterologous protein production in methylotrophic yeasts[J].Appl Microbiol Biotechnol,2000,54(6):741-750.
[6]Yurimoto H.Molecular basis of methanol-inducible gene expression and its application in the methylotrophic yeast Candida boidinii[J].Bioscience,Biotechnology,and Biochemistry,2009,73(4):793-800.
[7]Roberts JC,McCarthy AJ,Flynn NJ,Broda P.Modification of paper properties by the pretreatment of pulp with Saccharomonospora viridis xylanase[J].Enzyme and Microbial technology,1990,12(3):210-213.
[8]Wang Z,Jin Y,Wu H,et al.A novel,alkali-tolerant thermostable xylanase from Saccharomonospora viridis:direct gene cloning,expression and enzyme characterization[J].World Journal of Microbiology and Biotechnology,2012,28(8):2741-2748.
[9]Laemmli UK.Cleavage of structural proteins during the assembly of the head of bacteriophage T4[J].Nature,1970,227(5259):680-685.
[10]Bradford MM.A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of proteindye binding[J].Analytical Biochemistry,1976,72(1):248-254.
[11]Miller GL.Use of Dinitrosalicylic acid reagent for determination of reducing sugar[J].Anal Chem,1959,31(3):426-428.
[12]Lineweaver H,Burk D.The determination of enzyme dissociation constants[J].Journal of the American Chemical Society,1934,56(3):658-666.
[13]李宁.链霉菌来源的木聚糖降解酶相关基因的克隆及酶学性质研究[D].北京:中国农业科学院,2009.
[14]Paice M,Jurasek L.Removing hemicellulose from pulps by specific enzymic hydrolysis[J].Journal of Wood Chemistry and Technology,1984,4(2):187-198.
[15]张义生慈元钊.碱性木聚糖酶在麦草浆漂白生产中的应用试验[J].湖北造纸,2002,2:8-9.
[16]周蕾,顾建新.N-糖基化位点鉴定方法和非经典N-糖基化序列[J].生命科学,2011,23(6):605-611.