新型耐热β-1,4-木糖苷酶的重组表达及酶学性质
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摘要
【目的】拟对来源于热解纤维素果汁杆菌的新型β-木糖苷酶基因(CoXyl B)进行重组表达和酶学性质研究。【方法】在大肠杆菌系统中成功表达CoXyl B基因,并通过镍柱亲和层析、强阴离子交换和凝胶层析等纯化方法获得纯酶。【结果】对CoXyl B酶学性质的研究结果显示,在以4-对硝基苯酚-β-D-木糖苷为底物时,该酶的最适反应温度为90℃,最适反应pH为6.0。在40–70℃范围内CoXyl B酶活较高且比较稳定。在pH 5.0–6.0之间,70℃孵育1 h后,CoXyl B的相对酶活仍保留80%以上。Ag~+、高浓度的SDS和PMSF对酶活力的抑制作用较显著,而高浓度Mg~(2+)、Li~+和EDTA对酶活力的激活作用较为明显。CoXyl B的k_(cat)和K_m值分别为5.0×10~(–3)s~(–1)和1.9 mmol/L。薄层层析色谱显示CoXyl B具有降解木二糖、木三糖和木四糖的能力。【结论】本研究鉴定出CoXyl B为一种新型的极端耐热木糖苷酶,CoXyl B的酶学性质研究将为其在食品热加工以及生物降解领域中的应用提供参考。
[Objective] A thermostable β-xylosidase from the thermophile Caldicellulosiruptor owensensis OL(CoXyl B) was characterized. [Methods] Recombinant CoXyl B was heterogeneously expressed in Escherichia coli and then purified using Ni-NTA, anion exchange and Superdex 200 chromatography. Further, the hydrolysis activity of CoXyl B was studied using p-nitrophenyl-D-xyloside(pNPX) and xylooligosaccharide as substrates.[Results] The optimum temperature and pH of CoXyl B was 90 ℃ and pH 6.0, respectively. CoXyl B maintained stable at temperature between 40 ℃ and 70 ℃. After incubation at 70 ℃ for 1 h, CoXyl B retained more than 80%of its initial activity at both pH 5.0 and 6.0. Significantly, Ag~+, SDS and PMSF exhibited negative effect on the activity of CoXyl B, whereas Mg~(2+), Li~+ and EDTA significantly enhanced the activity of CoXyl B. The kinetic parameters of CoXyl B towards pNPX were k_(cat) of 5.0×10~(–3)s~(–1) and K_m of 1.9 mmol/L. Moreover, CoXyl B exhibited efficient hydrolysis activity towards xylobiose, xylotriose and xylotetraose. [Conclusion] Our work suggested the application potential of a new thermostable β-xylosidase(CoXyl B) in the area of xylan degradation at high temperature.
[Objective] A thermostable β-xylosidase from the thermophile Caldicellulosiruptor owensensis OL(CoXyl B) was characterized. [Methods] Recombinant CoXyl B was heterogeneously expressed in Escherichia coli and then purified using Ni-NTA, anion exchange and Superdex 200 chromatography. Further, the hydrolysis activity of CoXyl B was studied using p-nitrophenyl-D-xyloside(pNPX) and xylooligosaccharide as substrates.[Results] The optimum temperature and pH of CoXyl B was 90 ℃ and pH 6.0, respectively. CoXyl B maintained stable at temperature between 40 ℃ and 70 ℃. After incubation at 70 ℃ for 1 h, CoXyl B retained more than 80%of its initial activity at both pH 5.0 and 6.0. Significantly, Ag~+, SDS and PMSF exhibited negative effect on the activity of CoXyl B, whereas Mg~(2+), Li~+ and EDTA significantly enhanced the activity of CoXyl B. The kinetic parameters of CoXyl B towards pNPX were k_(cat) of 5.0×10~(–3)s~(–1) and K_m of 1.9 mmol/L. Moreover, CoXyl B exhibited efficient hydrolysis activity towards xylobiose, xylotriose and xylotetraose. [Conclusion] Our work suggested the application potential of a new thermostable β-xylosidase(CoXyl B) in the area of xylan degradation at high temperature.
引文
[1]Zhang L,Ding CH,Ruan WB.Study on application and product development progress of xylo-oligosaccharides.Cereals&Oils,2015,28(2):9-12.(in Chinese)张玲,丁长河,阮文彬.低聚木糖的应用研究和产品开发进展.粮食与油脂,2015,28(2):9-12.
[2]Ye SC,Xue T,He WJ,Ye BY,Wei F,Chen YQ.Applications of xylanases and its research progress.China Brewing,2013,32(7):8-10.(in Chinese)叶世超,薛婷,何文锦,叶冰莹,魏芳,陈由强.木聚糖酶的应用及其研究进展.中国酿造,2013,32(7):8-10.
[3]Chen HY,Cai J,Lin JG,Wang CG,Du X.Research progress of xylanase.China Brewing,2016,35(11):1-6.(in Chinese)陈洪洋,蔡俊,林建国,王常高,杜馨.木聚糖酶的研究进展.中国酿造,2016,35(11):1-6.
[4]Gao YJ,Ding CH.Research progress on the application of xylanase in food industry.Cereal&Food Industry,2017,24(2):32-36.(in Chinese)高雅君,丁长河.木聚糖酶在食品工业中的应用研究进展.粮食与食品工业,2017,24(2):32-36.
[5]Nie H,Li XJ,Li JB.The research development on bioactivity,preparation and application of xylooligosaccharides.Food Research and Development,2016,37(14):210-213.(in Chinese)聂卉,李晓静,李坚斌.低聚木糖的生物活性、制备及应用研究进展.食品研究与开发,2016,37(14):210-213.
[6]Jia XJ,Mi SF,Wang JZ,Qiao WB,Peng XW,Han YJ.Insight into glycoside hydrolases for debranched xylan degradation from extremely thermophilic bacterium Caldicellulosiruptor lactoaceticus.PLoS ONE,2014,9(9):e106482.
[7]Goswami GK,Krishnamohan M,Nain V,Aggarwal C,Ramesh B.Cloning and heterologous expression of cellulose free thermostable xylanase from Bacillus brevis.SpringerPlus,2014,3(1):20.
[8]Su XY,Han YJ,Dodd D,Moon YH,Yoshida S,Mackie RI,Cann IKO.Reconstitution of a thermostable xylan-degrading enzyme mixture from the bacterium Caldicellulosiruptor bescii.Applied and Environmental Microbiology,2013,79(5):1481-1490.
[9]Jia XJ,Qiao WB,Tian WL,Peng XW,Mi SF,Su H,Han YJ.Biochemical characterization of extra-and intracellular endoxylanse from thermophilic bacterium Caldicellulosiruptor kronotskyensis.Scientific Reports,2016,6:21672.
[10]Xu JF,Zhang JZ.Research advances in microbial endo-1,4-β-xylanase.China Brewing,2014,33(5):15-17.(in Chinese)徐君飞,张居作.微生物β-1,4-内切木聚糖酶研究进展.中国酿造,2014,33(5):15-17.
[11]Knob A,Terrasan CRF,Carmona EC.β-xylosidases from filamentous fungi:an overview.World Journal of Microbiology and Biotechnology,2010,26(3):389-407.
[12]Tsujibo H,Miyamoto K,Kuda T,Minami K,Sakamoto T,Hasegawa T,Inamori Y.Purification,properties,and partial amino acid sequences of thermostable xylanases from Streptomyces thermoviolaceus OPC-520.Applied and Environmental Microbiology,1992,58(1):371-375.
[13]Lorenz WW,Wiegel J.Isolation,analysis,and expression of two genes from Thermoanaerobacterium sp.Strain JW/SLYS485:aβ-xylosidase and a novel acetyl xylan esterase with cephalosporin C deacetylase activity.Journal of Bacteriology,1997,179(17):5436-5441.
[14]Lasrado LD,Gudipati M.Purification and characterization ofβ-D-xylosidase from Lactobacillus brevis grown on xylo-oligosaccharides.Carbohydrate Polymers,2013,92(2):1978-1983.
[15]Espina G,Eley K,Pompidor G,Schneider TR,Crennell SJ,Danson MJ.A novelβ-xylosidase structure from Geobacillus thermoglucosidasius:the first crystal structure of a glycoside hydrolase family GH52 enzyme reveals unpredicted similarity to other glycoside hydrolase folds.Acta Crystallographica Section D:Biological Crystallography,2014,70(5):1366-1374.
[16]Peng X,Su H,Mi S,Han Y.A multifunctional thermophilic glycoside hydrolase from Caldicellulosiruptor Owensensis with potential applications in production of biofuels and biochemicals.Biotechnology for Biofuels,2016,9(1):98.
[17]Blumer-Schuette SE,Kataeva I,Westpheling J,Adams MW,Kelly RM.Extremely thermophilic microorganisms for biomass conversion:status and prospects.Current Opinion in Biotechnology,2008,19(3):210-217.
[18]Meng DD,Zhang KD,Ying Y,Chen XH,Li FL.Research progress in lignocellulose degradation by genus Caldicellulosiruptor.Chinese Journal of Bioprocess Engineering,2014,12(1):37-45.(in Chinese)孟冬冬,张坤迪,英瑜,陈晓华,李福利.极端嗜热厌氧菌Caldicellulosiruptor木质纤维素降解研究.生物加工过程,2014,12(1):37-45.
[19]Mi SF,Jia XJ,Wang JZ,Qiao WB,Peng XW,Han YJ.Biochemical characterization of two thermostable xylanolytic enzymes encoded by a gene cluster of Caldicellulosiruptor owensensis.PLoS ONE,2014,9(8):e105264.
[20]Xue YM,Shao WL.Expression and characterization of a thermostableβ-xylosidase from the hyperthermophile,Thermotoga maritima.Biotechnology Letters,2004,26(19):1511-1515.
[21]Wang YJ,Xia QJ,Zhou P,Zhang Y,Wang F,Li X.Purification and characterization of a thermostableβ-xylosidase from Caldicellulosiruptor saccharolyticus.Chemical Industry and Engineering Progress,2017,36(3):1041-1046.(in Chinese)王月皎,夏乾竣,周佩,张瑜,王飞,李迅.解糖热纤维菌木糖苷酶的分离纯化及其酶学性质.化工进展,2017,36(3):1041-1046.
[22]Huang CY,Patel BK,Mah RA,Baresi L.Caldicellulosiruptor owensensis sp.nov.,an anaerobic,extremely thermophilic,xylanolytic bacterium.International Journal of Systematic Bacteriology,1998,48(1):91-97.
[23]Zhang SY,Wang HM,Shi PJ,Xu B,Bai YG,Luo HY,Yao B.Cloning,expression,and characterization of a thermostableβ-xylosidase from thermoacidophilic Alicyclobacillus sp.A4.Process Biochemistry,2014,49(9):1422-1428.
[24]Teng C,Jia HY,Yan QJ,Zhou P,Jiang ZQ.High-level expression of extracellular secretion of aβ-xylosidase gene from Paecilomycesthermophila in Escherichia coli.Bioresource Technology,2011,102(2):1822-1830.
[25]Shao WL,Xue YM,Wu AL,Kataeva I,Pei JJ,Wu HW,Wiegel J.Characterization of a novelβ-xylosidase,XylC,from Thermoanaerobacterium saccharolyticum JW/SL-YS485.Applied and Environmental Microbiology,2011,77(3):719-726.
[26]Anand A,Kumar V,Satyanarayana T.Characteristics of thermostable endoxylanase andβ-xylosidase of the extremely thermophilic bacterium Geobacillus thermodenitrificans tsaa1and its applicability in generating xylooligosaccharides and xylose from agro-residues.Extremophiles Life under Extreme Conditions,2013,17(3):357-366.
[27]Bravman T,Mechaly A,Shulami S,Belakhov V,Baasov T,Shoham G,Shoham Y.Glutamic acid 160 is the acid-base catalyst ofβ-xylosidase from Bacillus stearothermophilus T-6:a family 39 glycoside hydrolase.FEBS Letters,2001,495(1/2):115-119.
[28]Huang ZP,Liu XS,Zhang SW,Liu ZD.GH52 xylosidase from Geobacillus stearothermophilus:characterization and introduction of xylanase activity by site-directed mutagenesis of Tyr509.Journal of Industrial Microbiology&Biotechnology,2014,41(1):65-74.
[29]Nanmori T,Watanabe T,Shinke R,Kohno A,Kawamura Y.Purification and properties of thermostable xylanase andβ-xylosidase produced by a newly isolated Bacillus stearothermophilus strain.Journal of Bacteriology,1990,172(12):6669-6672.
[30]Shi H,Li X,Gu HX,Zhang Y,Huang YJ,Wang LL,Wang F.Biochemical properties of a novel thermostable and highly xylose-tolerantβ-xylosidase/α-arabinosidase from Thermotoga thermarum.Biotechnology for Biofuels,2013,6(1):27.
[31]Ratnadewi AAI,Fanani M,Kurniasih SD,Sakka M,Wasito EB,Sakka K,Nurachman Z,Puspaningsih NNT.β-D-xylosidase from Geobacillus thermoleovorans IT-08:biochemical characterization and bioinformatics of the enzyme.Applied Biochemistry and Biotechnology,2013,170(8):1950-1964.
[32]Bhalla A,Bischoff KM,Sani RK.Highly thermostable GH39β-xylosidase from a Geobacillus sp.strain WSUCF1.BMCBiotechnology,2014,14(1):963.
[33]Bankeeree W,Akada R,Lotrakul P,Punnapayak H,Prasongsuk S.Enzymatic hydrolysis of black liquor xylan by a novel xylose-tolerant,thermostableβ-xylosidase from a tropical strain of Aureobasidium pullulans CBS 135684.Applied Biochemistry and Biotechnology,2018,184(3):919-934.
[34]Fan YY,Li XT,Teng C.Research progress onβ-xylosidases from microorganism.Food Research and Development,2013,34(12):116-121.(in Chinese)范园园,李秀婷,滕超.微生物产β-木糖苷酶的研究进展.食品研究与开发,2013,34(12):116-121.
[2]Ye SC,Xue T,He WJ,Ye BY,Wei F,Chen YQ.Applications of xylanases and its research progress.China Brewing,2013,32(7):8-10.(in Chinese)叶世超,薛婷,何文锦,叶冰莹,魏芳,陈由强.木聚糖酶的应用及其研究进展.中国酿造,2013,32(7):8-10.
[3]Chen HY,Cai J,Lin JG,Wang CG,Du X.Research progress of xylanase.China Brewing,2016,35(11):1-6.(in Chinese)陈洪洋,蔡俊,林建国,王常高,杜馨.木聚糖酶的研究进展.中国酿造,2016,35(11):1-6.
[4]Gao YJ,Ding CH.Research progress on the application of xylanase in food industry.Cereal&Food Industry,2017,24(2):32-36.(in Chinese)高雅君,丁长河.木聚糖酶在食品工业中的应用研究进展.粮食与食品工业,2017,24(2):32-36.
[5]Nie H,Li XJ,Li JB.The research development on bioactivity,preparation and application of xylooligosaccharides.Food Research and Development,2016,37(14):210-213.(in Chinese)聂卉,李晓静,李坚斌.低聚木糖的生物活性、制备及应用研究进展.食品研究与开发,2016,37(14):210-213.
[6]Jia XJ,Mi SF,Wang JZ,Qiao WB,Peng XW,Han YJ.Insight into glycoside hydrolases for debranched xylan degradation from extremely thermophilic bacterium Caldicellulosiruptor lactoaceticus.PLoS ONE,2014,9(9):e106482.
[7]Goswami GK,Krishnamohan M,Nain V,Aggarwal C,Ramesh B.Cloning and heterologous expression of cellulose free thermostable xylanase from Bacillus brevis.SpringerPlus,2014,3(1):20.
[8]Su XY,Han YJ,Dodd D,Moon YH,Yoshida S,Mackie RI,Cann IKO.Reconstitution of a thermostable xylan-degrading enzyme mixture from the bacterium Caldicellulosiruptor bescii.Applied and Environmental Microbiology,2013,79(5):1481-1490.
[9]Jia XJ,Qiao WB,Tian WL,Peng XW,Mi SF,Su H,Han YJ.Biochemical characterization of extra-and intracellular endoxylanse from thermophilic bacterium Caldicellulosiruptor kronotskyensis.Scientific Reports,2016,6:21672.
[10]Xu JF,Zhang JZ.Research advances in microbial endo-1,4-β-xylanase.China Brewing,2014,33(5):15-17.(in Chinese)徐君飞,张居作.微生物β-1,4-内切木聚糖酶研究进展.中国酿造,2014,33(5):15-17.
[11]Knob A,Terrasan CRF,Carmona EC.β-xylosidases from filamentous fungi:an overview.World Journal of Microbiology and Biotechnology,2010,26(3):389-407.
[12]Tsujibo H,Miyamoto K,Kuda T,Minami K,Sakamoto T,Hasegawa T,Inamori Y.Purification,properties,and partial amino acid sequences of thermostable xylanases from Streptomyces thermoviolaceus OPC-520.Applied and Environmental Microbiology,1992,58(1):371-375.
[13]Lorenz WW,Wiegel J.Isolation,analysis,and expression of two genes from Thermoanaerobacterium sp.Strain JW/SLYS485:aβ-xylosidase and a novel acetyl xylan esterase with cephalosporin C deacetylase activity.Journal of Bacteriology,1997,179(17):5436-5441.
[14]Lasrado LD,Gudipati M.Purification and characterization ofβ-D-xylosidase from Lactobacillus brevis grown on xylo-oligosaccharides.Carbohydrate Polymers,2013,92(2):1978-1983.
[15]Espina G,Eley K,Pompidor G,Schneider TR,Crennell SJ,Danson MJ.A novelβ-xylosidase structure from Geobacillus thermoglucosidasius:the first crystal structure of a glycoside hydrolase family GH52 enzyme reveals unpredicted similarity to other glycoside hydrolase folds.Acta Crystallographica Section D:Biological Crystallography,2014,70(5):1366-1374.
[16]Peng X,Su H,Mi S,Han Y.A multifunctional thermophilic glycoside hydrolase from Caldicellulosiruptor Owensensis with potential applications in production of biofuels and biochemicals.Biotechnology for Biofuels,2016,9(1):98.
[17]Blumer-Schuette SE,Kataeva I,Westpheling J,Adams MW,Kelly RM.Extremely thermophilic microorganisms for biomass conversion:status and prospects.Current Opinion in Biotechnology,2008,19(3):210-217.
[18]Meng DD,Zhang KD,Ying Y,Chen XH,Li FL.Research progress in lignocellulose degradation by genus Caldicellulosiruptor.Chinese Journal of Bioprocess Engineering,2014,12(1):37-45.(in Chinese)孟冬冬,张坤迪,英瑜,陈晓华,李福利.极端嗜热厌氧菌Caldicellulosiruptor木质纤维素降解研究.生物加工过程,2014,12(1):37-45.
[19]Mi SF,Jia XJ,Wang JZ,Qiao WB,Peng XW,Han YJ.Biochemical characterization of two thermostable xylanolytic enzymes encoded by a gene cluster of Caldicellulosiruptor owensensis.PLoS ONE,2014,9(8):e105264.
[20]Xue YM,Shao WL.Expression and characterization of a thermostableβ-xylosidase from the hyperthermophile,Thermotoga maritima.Biotechnology Letters,2004,26(19):1511-1515.
[21]Wang YJ,Xia QJ,Zhou P,Zhang Y,Wang F,Li X.Purification and characterization of a thermostableβ-xylosidase from Caldicellulosiruptor saccharolyticus.Chemical Industry and Engineering Progress,2017,36(3):1041-1046.(in Chinese)王月皎,夏乾竣,周佩,张瑜,王飞,李迅.解糖热纤维菌木糖苷酶的分离纯化及其酶学性质.化工进展,2017,36(3):1041-1046.
[22]Huang CY,Patel BK,Mah RA,Baresi L.Caldicellulosiruptor owensensis sp.nov.,an anaerobic,extremely thermophilic,xylanolytic bacterium.International Journal of Systematic Bacteriology,1998,48(1):91-97.
[23]Zhang SY,Wang HM,Shi PJ,Xu B,Bai YG,Luo HY,Yao B.Cloning,expression,and characterization of a thermostableβ-xylosidase from thermoacidophilic Alicyclobacillus sp.A4.Process Biochemistry,2014,49(9):1422-1428.
[24]Teng C,Jia HY,Yan QJ,Zhou P,Jiang ZQ.High-level expression of extracellular secretion of aβ-xylosidase gene from Paecilomycesthermophila in Escherichia coli.Bioresource Technology,2011,102(2):1822-1830.
[25]Shao WL,Xue YM,Wu AL,Kataeva I,Pei JJ,Wu HW,Wiegel J.Characterization of a novelβ-xylosidase,XylC,from Thermoanaerobacterium saccharolyticum JW/SL-YS485.Applied and Environmental Microbiology,2011,77(3):719-726.
[26]Anand A,Kumar V,Satyanarayana T.Characteristics of thermostable endoxylanase andβ-xylosidase of the extremely thermophilic bacterium Geobacillus thermodenitrificans tsaa1and its applicability in generating xylooligosaccharides and xylose from agro-residues.Extremophiles Life under Extreme Conditions,2013,17(3):357-366.
[27]Bravman T,Mechaly A,Shulami S,Belakhov V,Baasov T,Shoham G,Shoham Y.Glutamic acid 160 is the acid-base catalyst ofβ-xylosidase from Bacillus stearothermophilus T-6:a family 39 glycoside hydrolase.FEBS Letters,2001,495(1/2):115-119.
[28]Huang ZP,Liu XS,Zhang SW,Liu ZD.GH52 xylosidase from Geobacillus stearothermophilus:characterization and introduction of xylanase activity by site-directed mutagenesis of Tyr509.Journal of Industrial Microbiology&Biotechnology,2014,41(1):65-74.
[29]Nanmori T,Watanabe T,Shinke R,Kohno A,Kawamura Y.Purification and properties of thermostable xylanase andβ-xylosidase produced by a newly isolated Bacillus stearothermophilus strain.Journal of Bacteriology,1990,172(12):6669-6672.
[30]Shi H,Li X,Gu HX,Zhang Y,Huang YJ,Wang LL,Wang F.Biochemical properties of a novel thermostable and highly xylose-tolerantβ-xylosidase/α-arabinosidase from Thermotoga thermarum.Biotechnology for Biofuels,2013,6(1):27.
[31]Ratnadewi AAI,Fanani M,Kurniasih SD,Sakka M,Wasito EB,Sakka K,Nurachman Z,Puspaningsih NNT.β-D-xylosidase from Geobacillus thermoleovorans IT-08:biochemical characterization and bioinformatics of the enzyme.Applied Biochemistry and Biotechnology,2013,170(8):1950-1964.
[32]Bhalla A,Bischoff KM,Sani RK.Highly thermostable GH39β-xylosidase from a Geobacillus sp.strain WSUCF1.BMCBiotechnology,2014,14(1):963.
[33]Bankeeree W,Akada R,Lotrakul P,Punnapayak H,Prasongsuk S.Enzymatic hydrolysis of black liquor xylan by a novel xylose-tolerant,thermostableβ-xylosidase from a tropical strain of Aureobasidium pullulans CBS 135684.Applied Biochemistry and Biotechnology,2018,184(3):919-934.
[34]Fan YY,Li XT,Teng C.Research progress onβ-xylosidases from microorganism.Food Research and Development,2013,34(12):116-121.(in Chinese)范园园,李秀婷,滕超.微生物产β-木糖苷酶的研究进展.食品研究与开发,2013,34(12):116-121.
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