黑曲霉木聚糖酶在同源宿主中的分泌性表达及酶学特性研究
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摘要
木聚糖酶(Xylanase)，又称β—D—1，4-内切木聚糖酶，是降解木聚糖的关键酶，它能水解木聚糖主链上的β—1，4—糖苷键，将木聚糖降解为低聚糖和木糖，广泛应用于饲料、造纸和食品医药等行业，具有广阔的应用前景。但木聚糖酶的单位产量较低，是限制木聚糖酶应用的主要因素之一。本研究应用同源高效表达策略，在丝状真菌表达系统中构建高产的黑曲霉木聚糖酶基因工程菌。
本课题首先从黑曲霉菌(Aspergillus niger)TS1菌株中提取总RNA，以其为模板运用RT-PCR的方法成功克隆出678bp的木聚糖酶xynB基因，将该基因片段插入融合蛋白基因表达载体pYG1.2葡萄糖淀粉酶编码基因的下游，融合之处设计内胰蛋白酶(KEX2)加工位点，构建融合表达质粒pYG1.2—xynB。测序结果表明，该木聚糖酶基因序列与A.niger IBT-90木聚糖酶(GenBank accession AY536639)基因序列具有99.4％的同源性，有4个碱基的差异，仅一个碱基的突变导致氨基酸改变，编码的蛋白质序列同源性达99.5％。随后运用原生质体转化法将pYG1.2—xynB转化尿嘧啶缺陷型宿主黑曲霉菌M54，获得重组菌株。SDS-PAGE结果表明，重组木聚糖酶在转化菌株中获得了高效分泌性表达，其分子量约为21KD。在1％木聚糖为诱导物的培养条件下，重组菌分泌的木聚糖酶酶活有明显提高，最高酶活达507 IU／ml，分别是原始出发菌和宿主菌的6.7倍和3.89倍。对获得的转化子进行连续传代培养结果表明，重组菌生长特性十分稳定，具有良好遗传稳定性。重组菌最佳产酶时间为培养后72h，最适反应pH为5.2，属酸性木聚糖酶；最适反应温度为50℃，在50℃-60℃之间热稳定性较好，60℃保温30min后残余酶活为63.95％。
本实验首次运用同源表达的策略，将黑曲霉木聚糖酶基因在同源丝状真菌中表达。研究结果表明，木聚糖酶在重组黑曲霉中获得了高效分泌性表达。重组菌发酵周期短，产酶活性高，具有重要的应用前景，为酸性木聚糖酶的广泛应用奠定了一定基础。
Xylan, composed of polysaccharide containingβ-1, 4-linked D-xylopyranosideresidues, is one of the major components of plant cell walls. Endo-β-1,4-xylanase(EC3.2.1.8) degrades the backbone ofβ-1, 4-linkages of xylose.Xylanase is widely applied in animal feed, medicine, food processing and papermanufacturing. However, the low yield of xylanase becomes a limiting factor to theuse of xylanase in various industries. The aim of my study is to construct a high levelxylanase recombinant in filamentous fungal expression system.
The cDNA encoding the acidic xylanase from original A. niger TS1 wasamplified by RT-PCR and cloned into the eukaryotic expression vector pYG1.2 underthe control of the glaA promoter. The recombinant plasmids were transformed intopyrG deficient A. niger M54 host strain by protoplast transformation technology. Thishomologous expression system expressed xylanase activity of 507 IU/ml which wasmeasured in shake-flask cultures containing 1％xylan. This activity was 6.7-fold and3.89-fold higher than the activity for the original strain and host strain respectively.The maximum xylanase activity was obtained after culturing for 72 hours and thexylan induced enzymatic activity was 3.94-fold higher compared to non-inducedcondition. The optimum pH of recombinant xylanase was at 5.2,whereas the optimumtemperature was at 50℃and remained more than 63.95％activity even afterincubation at 60℃for 30min.
It's the first time that high secreting expression system was constructed using ahomologous expression process in filamentous fungus. The promising resultsachieved in this study may have a good industrial perspective especially in the animalfeed.
本课题首先从黑曲霉菌(Aspergillus niger)TS1菌株中提取总RNA，以其为模板运用RT-PCR的方法成功克隆出678bp的木聚糖酶xynB基因，将该基因片段插入融合蛋白基因表达载体pYG1.2葡萄糖淀粉酶编码基因的下游，融合之处设计内胰蛋白酶(KEX2)加工位点，构建融合表达质粒pYG1.2—xynB。测序结果表明，该木聚糖酶基因序列与A.niger IBT-90木聚糖酶(GenBank accession AY536639)基因序列具有99.4％的同源性，有4个碱基的差异，仅一个碱基的突变导致氨基酸改变，编码的蛋白质序列同源性达99.5％。随后运用原生质体转化法将pYG1.2—xynB转化尿嘧啶缺陷型宿主黑曲霉菌M54，获得重组菌株。SDS-PAGE结果表明，重组木聚糖酶在转化菌株中获得了高效分泌性表达，其分子量约为21KD。在1％木聚糖为诱导物的培养条件下，重组菌分泌的木聚糖酶酶活有明显提高，最高酶活达507 IU／ml，分别是原始出发菌和宿主菌的6.7倍和3.89倍。对获得的转化子进行连续传代培养结果表明，重组菌生长特性十分稳定，具有良好遗传稳定性。重组菌最佳产酶时间为培养后72h，最适反应pH为5.2，属酸性木聚糖酶；最适反应温度为50℃，在50℃-60℃之间热稳定性较好，60℃保温30min后残余酶活为63.95％。
本实验首次运用同源表达的策略，将黑曲霉木聚糖酶基因在同源丝状真菌中表达。研究结果表明，木聚糖酶在重组黑曲霉中获得了高效分泌性表达。重组菌发酵周期短，产酶活性高，具有重要的应用前景，为酸性木聚糖酶的广泛应用奠定了一定基础。
Xylan, composed of polysaccharide containingβ-1, 4-linked D-xylopyranosideresidues, is one of the major components of plant cell walls. Endo-β-1,4-xylanase(EC3.2.1.8) degrades the backbone ofβ-1, 4-linkages of xylose.Xylanase is widely applied in animal feed, medicine, food processing and papermanufacturing. However, the low yield of xylanase becomes a limiting factor to theuse of xylanase in various industries. The aim of my study is to construct a high levelxylanase recombinant in filamentous fungal expression system.
The cDNA encoding the acidic xylanase from original A. niger TS1 wasamplified by RT-PCR and cloned into the eukaryotic expression vector pYG1.2 underthe control of the glaA promoter. The recombinant plasmids were transformed intopyrG deficient A. niger M54 host strain by protoplast transformation technology. Thishomologous expression system expressed xylanase activity of 507 IU/ml which wasmeasured in shake-flask cultures containing 1％xylan. This activity was 6.7-fold and3.89-fold higher than the activity for the original strain and host strain respectively.The maximum xylanase activity was obtained after culturing for 72 hours and thexylan induced enzymatic activity was 3.94-fold higher compared to non-inducedcondition. The optimum pH of recombinant xylanase was at 5.2,whereas the optimumtemperature was at 50℃and remained more than 63.95％activity even afterincubation at 60℃for 30min.
It's the first time that high secreting expression system was constructed using ahomologous expression process in filamentous fungus. The promising resultsachieved in this study may have a good industrial perspective especially in the animalfeed.
引文
[1]. Jun HS, Ha 1K, Malburg LM,et al.Characteristics of a cluster of xylanase genes in Fibrobacter succinogenes S85 [J]. Can J Microbiol, 2003, 49(3): 171-80
[2]. Kulkami N, Shendye A, Rao M. Molecular and biotechnological aspects of xylanase[J]. FEMS Microbiol Rev, 1999,23(2) : 411-456.
[3]. Subramaniyan S, Prema P. Biotechnoiogy of microbial xylanases: enzymology, molecular biology and application[J]. Crit Rev Biotechnol, 2002, 22(1):33-64.
[4]. Srivastava P, Mukherjee KJ. Cloning, characterization, and expression of xylanase gene from Bacillus lyticus in Escherichia coli and Bacillus subtilis[J]. Biochem Biotechnol. 2001,31(4):389-400
[5]. Kosugi A, Murashima K, Doi RH. Xylanase and acetyl xylan esterase activities of XynA, a key subunit of the Clostridium cellulovorans cellulosome for xylan degradation[J]. Appl Environ Microbiol, 2002, 68(1):6399-6402
[6]. Beg Q K,Kapoor M,Mahajan L. Microbial xylanase and their industrial applications:a review. Appl Microbiol Biotechnol,2001,56:326～338..
[7]. Bajpai P.Application of enzymes in the pulp and paper industry.Biotechnol Prog,1999,15:147-157
[8]．陈红歌，朱静，梁改芹，．酸性木聚糖酶产生菌的筛选及产酶条件微生物学报，1999，39：89-93
[9]．刘相梅，祁蒙，曲音波．木聚糖酶基因克隆、表达与分泌及定点诱变研究进展．生物工程进展，2001，21(2)：28～31
[10]．朱运平，江正强，李里特。乳糖诱导极耐高温木聚糖酶基因B在大肠杆菌中的表达。食品与发酵工业。2005，31(4)：37-41
[11]．解复红，李文鹏，张克勤．耐碱和耐热木聚糖酶研究进展．生物工程杂志，2003，23(7)：11～13
[12]．李育阳．基因表达技术．北京：科学出版社，2001
[13]. Gouka RJ, Punt PJ, van den Hondel CA et al. Efficient production of secretedproteins by Aspergillus: progress, limitations and prospects. Appl Microbiol Biotechnol,1997, 47(1): 1～11
[14]. Ngiam C , Jeenes D J, Punt PJ,et al. Characterization of a foldase, protein disulfide isomerase A, in the protein secretory pathway of Aspergillus niger.Appl Environ Microbiol., 2000,66(2): 775～82
[15]. M.Asther, E.Record,et.Overproduction of the Aspergillus niger feruloyl esterase for puip bleaching application.Appl Microbiol Biotechnol.2003(62):349～355
[16]. Anthony Levasseur Isabelle Benoit,et.Homologous expression of the feruloyl esterase B gene from Aspergillus niger and characterization of the recombinant enzyme..Protein expression and purification.2004(37): 126～133
[17]．刘伟丰，毛爱军，祝令香．耐碱性木聚糖酶基因在短小芽孢杆菌中高效分泌表达的研究．微生物学报．2004，44(4)：487～490
[18]．刘钟滨，David JJ，David B．A．．黑曲霉菌pyrG缺陷株的建立．微生物学杂志，2001，21(3)：15～16
[19]．刘钟滨，David JJ，David B．A．．黑曲霉菌为宿主菌的重组表达性质粒的构建．同济大学学报(医学版)，2001，22(3)：1～3
[20]．陈三凤，刘德虎．现代微生物遗传学．化学工业出版社．2003
[21]．李敏，洪斌，李元．人可溶性肿瘤坏死因子受体Ⅰ型基因在黑曲霉中的表达。科学通报．2001，46(1)：57～60．
[22]. Calmels T P G, Martin F, Durand H, et al. Proteolytic events in the processing of secreted proteins in fungi. J of Biotechnol,1991(17): 51～66
[23]. Subramaniyan S, Prema P. Biotechnology of microbial xylanases: enzymology, molecular biology and application[J]. Crit Rev Biotechnol, 2002, 22(1):33-64
[24]. Miettinen Oinonen A,Suominen P.Enhanced production of Trichodema reesei endoglucanases and use of the new cellulose preparation in producing the stonewashed effect on denim fabric.Appl Environ Microbiol,2002,68(8):3956-3964
[25]. Hatamoto,O sarnu;Watarai, Teruo;K ikuchi,M amoru;M izusawa, Kiyoshi Sekine ,Hiroshi .Cloning and sequencing of the gene encoding tannase and a structural study of the tannase subunit from Aspergillus oryzae Gene.1997,175 (1-2) :215-22
[26]. Berrin J G, Williamson G, Puigserver A.High level production of recombinant funal endo-beta-1,4-xylanase in the methlotrophic yeast Pichia pastoris.Protein Expr Purif,2000,19(1): 179～187.
[27]．李海燕，祝令香，毛爱军等。黑曲霉木聚糖酶在工业酒精酵母中的分泌表达。微生物学报，2005，45(1)：135～138
[28]．王金华，穆跃林，黄遵锡．基因工程酵母产木聚糖酶(xylA)的性质初探．饲料研究，2003，7：7～9．
[29]. Ball A S and McCarthy A J. Production and properties of xylanase from actinomycetes.J Appl Bacteriol, 1989, 66:439-444
[30]. Kosugi A, Murashima K, Doi RH. Xylanase and acetyl xylan esterase activities of XynA, a key subunit of the Clostridium cellulovorans cellulosome for xylan degradation[J]. Appl Environ Microbiol, 2002, 68(1): 6399-6402
[31]．张志光，李东屏，方芳。丝状真菌原生质体技术研究(Ⅴ)。湖南师范大学自然科学学报。1994，17(3)：41-47
[32]．郝勃．黑曲霉UB4和NB3的原生质体制备及其再生条件的研究．生物学杂志．1997，14(4)：18～20
[33]．郝勃，阎淳泰，陈华癸．2株黑曲霉原生质体的形成和再生过程观察．华中农业大学学报，1997，16(1)
[34]．郑佐兴．米曲霉和黑曲霉营养缺陷型的分离及原生质体的制备．工业微生物．1994，24(1)：18～20
[1] Kulkarni N, Shendye A, Rao M. Molecular and biotechnological aspects of xylanase[J]. FEMS Microbiol Rev, 1999, 23(2): 411-456.
[2] Subramaniyan S, Prema P. Biotechnology of microbial xylanases: enzymology, molecular biology, and application[J]. Crit Rev Biotechnol, 2002, 22(1):33-64.
[3] Kosugi A, Murashima K, Doi RH. Xylanase and acetyl xylan esterase activities of XynA, a key subunit of the Clostridium cellulovorans celluiosome for xylan degradation[J]. Appl Environ Microbiol, 2002, 68(1):6399-6402.
[4] Jun HS, Ha 1K, Malburg LM, et al.Characteristics of a cluster of xylanase genes in Fibrobacter succinogenes S85[J]. Can J Microbiol, 2003, 49(3):171-80
[5] 江正强，李里特．极端嗜热菌海栖热袍菌木聚糖酶B的克隆和表达[J]．应用与环境生物学报，2002，8(3)：280-284
[6] 薛业敏，毛忠贵．极端耐热木聚糖酶基因在大肠杆菌中的高效表达[J]．食品与发酵工业，2004，29(11)：20-25
[7] Srivastava P, Mukherjee KJ. Cloning, characterization, and expression of xylanase gene from Bacillus lyticus in Escherichia coli and Bacillus subtilis[J]. Biochem Biotechnol. 2001,31 (4):389-400.
[8] Walsh D J, Bergquist P L. Expression and secretion of athermostable bacterial xylanase in Kluyveromyces lactis[J]. Appl Environ Microbiol, 1997,63: 3297-3300.
[9] Chavez R, Navarro C et al. Secretion of endoxylanase A from Penicillium purpurogenum by Saccharomyces cerevisiae transformed with genomic fungal DNA[J]. FEMS Microbiol Let, 2002, 212(2):237-241.
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[12] Rose SH, van Zyl WH. Constitutive expression of the Trichoderma reesei beta-1,4-xylanase gene (xyn2) and the beta-1,4-endoglucanase gene (egl) in Aspergillus niger in molasses and defined glucose media.Appl Microbiol Biotechnol[J]. 2002,58(4):461-8.
[13] Faria FP, Te'o VSJ,et al. Expression and processing of a major xylanase(XYN2)from the thermophilic fungus Humicola grisea van thermoidea in Trichoderma reesei[J].Lett App; Microbiol,2002,34(2): 119-123.
[14] Bergquist P, Te'o V, Gibbs M, Cziferszky A, de Faria FPet al. Expression of xylanase enzymes from thermophilic microorganisms in fungal hosts[J]. Extremophiles,2002,6(3): 177-184.
[15] Fontes CM, Ali S, Gilbert HJ et al .Bacterial xylanase expression in mammalian cells and transgenic mice[J]. J Biotechnol ,1999 , 72(1-2):95-101.
[16] Kimura Mizutani T et aLMolecular breeding of ransgenic rice expressing a xylanase domain of the xynA gene from Clostridium thermocellum[J]. Appl Microbiol Biotechnol, 2003, 62 (4): 374-379.
[2]. Kulkami N, Shendye A, Rao M. Molecular and biotechnological aspects of xylanase[J]. FEMS Microbiol Rev, 1999,23(2) : 411-456.
[3]. Subramaniyan S, Prema P. Biotechnoiogy of microbial xylanases: enzymology, molecular biology and application[J]. Crit Rev Biotechnol, 2002, 22(1):33-64.
[4]. Srivastava P, Mukherjee KJ. Cloning, characterization, and expression of xylanase gene from Bacillus lyticus in Escherichia coli and Bacillus subtilis[J]. Biochem Biotechnol. 2001,31(4):389-400
[5]. Kosugi A, Murashima K, Doi RH. Xylanase and acetyl xylan esterase activities of XynA, a key subunit of the Clostridium cellulovorans cellulosome for xylan degradation[J]. Appl Environ Microbiol, 2002, 68(1):6399-6402
[6]. Beg Q K,Kapoor M,Mahajan L. Microbial xylanase and their industrial applications:a review. Appl Microbiol Biotechnol,2001,56:326～338..
[7]. Bajpai P.Application of enzymes in the pulp and paper industry.Biotechnol Prog,1999,15:147-157
[8]．陈红歌，朱静，梁改芹，．酸性木聚糖酶产生菌的筛选及产酶条件微生物学报，1999，39：89-93
[9]．刘相梅，祁蒙，曲音波．木聚糖酶基因克隆、表达与分泌及定点诱变研究进展．生物工程进展，2001，21(2)：28～31
[10]．朱运平，江正强，李里特。乳糖诱导极耐高温木聚糖酶基因B在大肠杆菌中的表达。食品与发酵工业。2005，31(4)：37-41
[11]．解复红，李文鹏，张克勤．耐碱和耐热木聚糖酶研究进展．生物工程杂志，2003，23(7)：11～13
[12]．李育阳．基因表达技术．北京：科学出版社，2001
[13]. Gouka RJ, Punt PJ, van den Hondel CA et al. Efficient production of secretedproteins by Aspergillus: progress, limitations and prospects. Appl Microbiol Biotechnol,1997, 47(1): 1～11
[14]. Ngiam C , Jeenes D J, Punt PJ,et al. Characterization of a foldase, protein disulfide isomerase A, in the protein secretory pathway of Aspergillus niger.Appl Environ Microbiol., 2000,66(2): 775～82
[15]. M.Asther, E.Record,et.Overproduction of the Aspergillus niger feruloyl esterase for puip bleaching application.Appl Microbiol Biotechnol.2003(62):349～355
[16]. Anthony Levasseur Isabelle Benoit,et.Homologous expression of the feruloyl esterase B gene from Aspergillus niger and characterization of the recombinant enzyme..Protein expression and purification.2004(37): 126～133
[17]．刘伟丰，毛爱军，祝令香．耐碱性木聚糖酶基因在短小芽孢杆菌中高效分泌表达的研究．微生物学报．2004，44(4)：487～490
[18]．刘钟滨，David JJ，David B．A．．黑曲霉菌pyrG缺陷株的建立．微生物学杂志，2001，21(3)：15～16
[19]．刘钟滨，David JJ，David B．A．．黑曲霉菌为宿主菌的重组表达性质粒的构建．同济大学学报(医学版)，2001，22(3)：1～3
[20]．陈三凤，刘德虎．现代微生物遗传学．化学工业出版社．2003
[21]．李敏，洪斌，李元．人可溶性肿瘤坏死因子受体Ⅰ型基因在黑曲霉中的表达。科学通报．2001，46(1)：57～60．
[22]. Calmels T P G, Martin F, Durand H, et al. Proteolytic events in the processing of secreted proteins in fungi. J of Biotechnol,1991(17): 51～66
[23]. Subramaniyan S, Prema P. Biotechnology of microbial xylanases: enzymology, molecular biology and application[J]. Crit Rev Biotechnol, 2002, 22(1):33-64
[24]. Miettinen Oinonen A,Suominen P.Enhanced production of Trichodema reesei endoglucanases and use of the new cellulose preparation in producing the stonewashed effect on denim fabric.Appl Environ Microbiol,2002,68(8):3956-3964
[25]. Hatamoto,O sarnu;Watarai, Teruo;K ikuchi,M amoru;M izusawa, Kiyoshi Sekine ,Hiroshi .Cloning and sequencing of the gene encoding tannase and a structural study of the tannase subunit from Aspergillus oryzae Gene.1997,175 (1-2) :215-22
[26]. Berrin J G, Williamson G, Puigserver A.High level production of recombinant funal endo-beta-1,4-xylanase in the methlotrophic yeast Pichia pastoris.Protein Expr Purif,2000,19(1): 179～187.
[27]．李海燕，祝令香，毛爱军等。黑曲霉木聚糖酶在工业酒精酵母中的分泌表达。微生物学报，2005，45(1)：135～138
[28]．王金华，穆跃林，黄遵锡．基因工程酵母产木聚糖酶(xylA)的性质初探．饲料研究，2003，7：7～9．
[29]. Ball A S and McCarthy A J. Production and properties of xylanase from actinomycetes.J Appl Bacteriol, 1989, 66:439-444
[30]. Kosugi A, Murashima K, Doi RH. Xylanase and acetyl xylan esterase activities of XynA, a key subunit of the Clostridium cellulovorans cellulosome for xylan degradation[J]. Appl Environ Microbiol, 2002, 68(1): 6399-6402
[31]．张志光，李东屏，方芳。丝状真菌原生质体技术研究(Ⅴ)。湖南师范大学自然科学学报。1994，17(3)：41-47
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