深绿木霉嗜酸性阿魏酸酯酶酶学性质及生物质转化分析
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摘要
为实现生物质的高效降解,制备具有重要生理功能的阿魏酸,本实验从深绿木霉XS6发酵液中分离纯化深绿木霉阿魏酸酯酶(Trichoderma atroatroviride feruloyl esterase,TaFAE),并研究酶学特性。发酵液经(NH_4)_2SO_4沉淀、DEAE-Cellulose DE52阴离子交换层析和Sephadex G-200凝胶过滤层析后,得到电泳纯的TaFAE,比活力134.3 U/mg,回收率28.9%,纯化倍数31.52。经十二烷基硫酸钠-聚丙烯酰氨凝胶电泳测得酶分子质量约为66.4 kDa。TaFAE对阿魏酸甲酯亲和力最高,以其为底物,酶K_m值和V_(max)值分别为0.53 mmol/L和16.74 μmol/(min·mg)。以芥子酸甲酯为底物,酶促反应速率最大,达到32.21 μmol/(min·mg),是阿魏酸甲酯的2倍,对咖啡酸甲酯无活性,说明该酶具有严格的底物特异性。TaFAE最适pH值和温度分别为pH 4.0和40℃。在pH 2.0～9.0的范围内,30～40℃温度下都表现出良好的稳定性。金属离子Ca~(2+)和Mg~(2+)对TaFAE活性有显著激活作用,重金属离子Hg~(2+)和Pb~(2+)几乎完全抑制酶活性;β-巯基乙醇、二硫苏糖醇、十二烷基硫酸钠、Trition X-100增强酶活性,苯甲基磺酰氟有较强的抑制作用。木聚糖酶降解生物质的体系中加入TaFAE可显著增加阿魏酸和还原糖产量,表明TaFAE和木聚糖酶有良好协同作用。综上所述,TaFAE优良的耐酸性及其他酶学性质说明其在食品和饲料行业有较好的应用潜力。
In order to realize efficient biomass degradation in the preparation of ferulic acid, exerting important physiological functions in the body, feruloyl esterase(TaFAE) was isolated and purified from the fermentation broth of Trichoderma atroatroviride XS6 and its enzymatic characteristics were described in this experiment. The electrophoretically pure enzyme was obtained by consecutive ammonium sulfate precipitation, DEAE-cellulose DE52 anion exchange chromatography and Sephadex G-200 gel filtration chromatography. SDS-polyacrylamide gel electrophoresis(SDS-PAGE) of the enzyme revealed a single band of about 66.4 kDa. Its specific activity was 134.3 U/mg, with a recovery of 28.9%, and 31.52-fold purification. TaFAE had the highest affinity for methyl ferulate. When methyl ferulate was used as substrate, the K_m and V_(max) values of the enzyme were 0.53 mmol/L and 16.74 μmol/(min·mg), respectively. When methyl sultacide was used as substrate, the highest enzymatic reaction rate of 32.21 μmol/(min·mg) was obtained, which was twice as high as that of methyl ferulate. In addition, no activity was shown toward methyl caffeate, indicating that the enzyme has a stringent substrate specificity. The optimum pH and temperature of TaFAE were pH 4.0 and 40 ℃, respectively. In the pH range of 2.0–9.0 and at 30–40 ℃, TaFAE showed good stability. The metal ions Ca~(2+) and Mg~(2+) promoted TaFAE activity, whereas Hg~(2+) and Pb~(2+) almost completely inhibited the activity of the enzyme; β-mercaptoethanol, dithiothreitol(DTT), SDS, and Trition X-100 promoted the enzymatic activity, whereas PMSF had a strong inhibitory effect. Biomass conversion by xylanase produced significantly more ferulic acid and reducing sugar when TaFAE was added to the system, indicating that Ta FAE and xylanase have a good synergistic effect. In summary, TaFAE has good acid tolerance and enzymatic properties, which make it promising for applications in the feed and food industry.
In order to realize efficient biomass degradation in the preparation of ferulic acid, exerting important physiological functions in the body, feruloyl esterase(TaFAE) was isolated and purified from the fermentation broth of Trichoderma atroatroviride XS6 and its enzymatic characteristics were described in this experiment. The electrophoretically pure enzyme was obtained by consecutive ammonium sulfate precipitation, DEAE-cellulose DE52 anion exchange chromatography and Sephadex G-200 gel filtration chromatography. SDS-polyacrylamide gel electrophoresis(SDS-PAGE) of the enzyme revealed a single band of about 66.4 kDa. Its specific activity was 134.3 U/mg, with a recovery of 28.9%, and 31.52-fold purification. TaFAE had the highest affinity for methyl ferulate. When methyl ferulate was used as substrate, the K_m and V_(max) values of the enzyme were 0.53 mmol/L and 16.74 μmol/(min·mg), respectively. When methyl sultacide was used as substrate, the highest enzymatic reaction rate of 32.21 μmol/(min·mg) was obtained, which was twice as high as that of methyl ferulate. In addition, no activity was shown toward methyl caffeate, indicating that the enzyme has a stringent substrate specificity. The optimum pH and temperature of TaFAE were pH 4.0 and 40 ℃, respectively. In the pH range of 2.0–9.0 and at 30–40 ℃, TaFAE showed good stability. The metal ions Ca~(2+) and Mg~(2+) promoted TaFAE activity, whereas Hg~(2+) and Pb~(2+) almost completely inhibited the activity of the enzyme; β-mercaptoethanol, dithiothreitol(DTT), SDS, and Trition X-100 promoted the enzymatic activity, whereas PMSF had a strong inhibitory effect. Biomass conversion by xylanase produced significantly more ferulic acid and reducing sugar when TaFAE was added to the system, indicating that Ta FAE and xylanase have a good synergistic effect. In summary, TaFAE has good acid tolerance and enzymatic properties, which make it promising for applications in the feed and food industry.
引文
[1] SHIN H, CHEN R R. Production and characterization of a type B feruloyl esterase from Fusarium proliferatum NRRL 26517[J]. Enzyme and Microbial Technology, 2006, 38(3): 478-485. DOI:10.1016/j.enzmictec.2005.07.003.
[2] ZHOU M, HUANG Z, ZHOU B, et al. Construction and expression of two-copy engineered yeast of feruloyl esterase[J]. Electronic Journal of Biotechnology, 2015, 18(5): 338-342. DOI:10.1016/j.ejbt.2015.06.004.
[3] CREPIN V F, FAULDS C B, CONNERTON I F. Functional classification of the microbial feruloyl esterases[J]. Applied Microbiology Biotechnology, 2004, 63(6): 647-652. DOI:10.1007/s00253-003-1476-3.
[4] GAO L, WANG M, CHEN S, et al. Biochemical characterization of a novel feruloyl esterase from Penicillium piceum and its application in biomass bioconversion[J]. Journal of Molecular Catalysis B: Enzymatic, 2016, 133: S388-S394. DOI:10.1016/j.molcatb.2017.02.012.
[5] DILOKPIMOL A, M?KEL? M R, MANSOURI S, et al. Expanding the feruloyl esterase gene family of Aspergillus niger by characterization of a feruloyl esterase, FaeC[J]. New Biotechnology, 2017, 37(Pt B): 200-209. DOI:10.1016/j.nbt.2017.02.007.
[6] GOPALAN N, RODRIGUEZ-DURAN L V, SAUCEDOCASTANEDA G, et al. Review on technological and scientific aspects of feruloyl esterases: a versatile enzyme for biorefining of biomass[J]. Bioresource Technology, 2015, 193: 534-544. DOI:10.1016/j.biortech.2015.06.117.
[7] WANG L, LI Z, ZHU M, et al. An acidic feruloyl esterase from the mushroom Lactarius hatsudake: a potential animal feed supplement[J]. International Journal of Biological Macromolecules, 2016, 93(Pt A): 290-295. DOI:10.1016/j.ijbiomac.2016.08.028.
[8]刘君.阿魏酸酯酶基因在黑曲霉中的同源表达[D].哈尔滨:东北农业大学, 2014.
[9] YANG S, TANG L, YAN Q, et al. Biochemical characteristics and gene cloning of a novel thermostable feruloyl esterase from Chaetomium sp.[J]. Journal of Molecular Catalysis B: Enzymatic, 2013, 97: 328-336. DOI:10.1016/j.molcatb.2013.06.011.
[10] SWAMY S K P, GOVINDASWAMY V. Therapeutical properties of ferulic acid and bioavailability enhancement through feruloyl esterase[J]. Journal of Functional Foods, 2015, 17: 657-666. DOI:10.1016/j.jff.2015.06.013.
[11] SCHROETER H, WILLIAMS R J, MATIN R, et al. Phenolic antioxidants attenuate neuronal cell death following uptake of oxidized low-density lipoprotein[J]. Free Radical Biology and Medicine, 2000, 29(12):1222-1233. DOI:10.1016/S0891-5849(00)00415-9.
[12] SCALBERT A, MONTIES B, LALLEMAND J, et al. Ether linkage between phenolic acids and lignin fractions from wheat straw[J]. Phytochemistry, 1985, 24(6): 1359-1362. DOI:10.1016/S0031-9422(00)81133-4.
[13] DILOKPIMOL A, MAKELA M R, VARRIALE S, et al. Fungal feruloyl esterases: functional validation of genome mining based enzyme discovery including uncharacterized subfamilies[J]. New Biotechnology, 2018, 41: 9-14. DOI:10.1016/j.nbt.2017.11.004.
[14] NIETER A, KELLE S, LINKE D, et al. Feruloyl esterases from Schizophyllumcommuneto treat food industry side-streams[J]. Bioresource Technology, 2016, 220: 38-46. DOI:10.1016/j.biortech.2016.08.045.
[15] WANG X M, BAI Y, CAI Y, et al. Biochemical characteristics of three feruloyl esterases with a broad substrate spectrum from Bacillus amyloliquefaciens H47[J]. Process Biochemistry, 2017, 53: 109-115. DOI:10.1016/j.procbio.2016.12.012.
[16] FAZARY A E, ISMADJI S, JU Y. Biochemical studies on native and cross-linked aggregates of Aspergillus awamori feruloyl esterase[J]. International Journal of Biological Macromolecules, 2009, 44(3): 240-248. DOI:10.1016/j.ijbiomac.2008.12.012.
[17] ZHANG S B, WANG L, LIU Y, et al. Expression of feruloyl esterase A from Aspergillus terreus and its application in biomass degradation[J]. Protein Expression and Purification, 2015, 115: 153-157. DOI:10.1016/j.pep.2015.08.015.
[18] LEVASSEUR A, BENOIT I, ASTHER M, et al. Homologous expression of the feruloyl esterase B gene from Aspergillus niger and characterization of the recombinant enzyme[J]. Protein Expression and Purification, 2004, 37(1): 126-133. DOI:10.1016/j.pep.2004.05.019.
[19]胡泓宇,周燕燕,陈静,等.一株产阿魏酸酯酶丝状真菌的分离鉴定及其粗酶性质研究[J].生物技术通报, 2014(7):162-167.
[20]卢颖欣,张宁,欧仕益,等.产阿魏酸酯酶乳酸菌的乳杆菌筛选以及基因克隆[J].食品工业科技, 2013, 34(20):199-203.
[21] ZENG Y, YIN X, WU M, et al. Expression of a novel feruloyl esterase from Aspergillus oryzae in Pichia pastoris with esterification activity[J]. Journal of Molecular Catalysis B: Enzymatic, 2014, 110: 140-146. DOI:10.1016/j.molcatb.2014.10.002.
[22]黄艳凤,周庆礼,李士炼,等.从黑麦麦麸中提取的阿魏酸的检测[J].药物生物技术,2004,11(1):49-51.DOI:10.3969/j.issn.1005-8915.2004.01.013.
[23] BRADFORD M M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of proteindyebinding[J]. Analytical Biochemistry, 1976, 72: 248-254.
[24] THONGEKKAEW J, IKEDA H, MASAKI K, et al. An acidic and thermostable carboxymethyl cellulase from the yeast Cryptococcus sp. S-2: purification, characterization and improvement of its recombinant enzyme production by high cell-density fermentation of Pichia pastoris[J]. Protein Expression and Purification, 2008, 60(2): 140-146. DOI:10.1016/j.pep.2008.03.021.
[25] OHLHOFF C W, KIRBY B M, VAN ZYL L, et al. An unusual feruloyl esterase belonging to family VIII esterases and displaying a broad substrate range[J]. Journal of Molecular Catalysis B: Enzymatic, 2015, 118:79-88. DOI:10.1016/j.molcatb.2015.04.010.
[26] ZHANG S B, ZHAI H, WANG L, et al. Expression, purification and characterization of a feruloyl esterase A from Aspergillus flavus[J].Protein Expression and Purification, 2013, 92(1): 36-40. DOI:10.1016/j.pep.2013.08.009.
[27] TOPAKAS E, STAMATIS H, MASTIHUBOVA M, et al. Purification and characterization of a Fusarium oxysporum feruloyl esterase (FoFAE-I) catalysing transesterification of phenolic acid esters[J]. Enzyme and Microbial Technology, 2003, 33(5): 729-737. DOI:10.1016/S0141-0229(03)00213-8.
[28] BORNEMAN W S, LJUNGDAHL L G, HARTLEY R D, et al. Purification and partial characterization of two feruloyl esterases from the anaerobic fungus Neocallimastix strain MC-2[J]. Applied and Environmental Microbiology, 1992, 58(11): 3762-3766.
[29] RUMBOLD K, BIELY P, MASTIHUBOVA M, et al. Purification and properties of a feruloyl esterase involved in lignocellulose degradation by Aureobasidiumpullulans[J]. Applied and Environmental Microbiology, 2003, 69(9): 5622-5626. DOI:10.1128/AEM.69.9.5622-5626.2003.
[30]CHANDRASEKHARAIAHM,THULASIA,VIJAYARANIK,etal.Expressionandbiochemicalcharacterizationoftwonovel feruloylesterasesderivedfromfecalsamplesofRusaunicolorand Equusburchelli[J].Gene,2012,500(1):134-139.DOI:10.1016/j.gene.2012.03.027.
[31]VAFIADIC,TOPAKASE,WONGKKY,etal.Mappingthe hydrolyticandsyntheticselectivityofatypeCferuloylesterase(StFaeC)fromSporotrichumthermophileusingalkylferulates[J].Tetrahedron:Asymmetry,2005,16(2):373-379.DOI:10.1016/j.tetasy.2004.11.037.
[32]YAOJ,CHENQL,SHENAX,etal.Anovelferuloylesterase from a soil metagenomic library with tannase activity[J]. Journal of MolecularCatalysisB:Enzymatic,2013,95:55-61.DOI:10.1016/j.molcatb.2013.05.026.
[33]方园,欧仕益,张宁.阿魏酸酯酶的制备及其酶学性质[J].暨南大学学报(自然科学与医学版), 2012(5):481-485.
[34] TOPAKAS E, VAFIADI C, CHRISTAKOPOULOS P. Microbial production, characterization and applications of feruloyl esterases[J]. Process Biochemistry, 2007, 42(4): 497-509. DOI:10.1016/j.procbio.2007.01.007.
[35] KOSEKI T, HANDA H, WATANABE Y Y, et al. An unusual feruloyl esterase from Aspergillus oryzae: two tryptophan residues play a crucial role for the activity[J]. Journal of Molecular Catalysis B: Enzymatic, 2016, 133: 560-568. DOI:10.1016/j.molcatb.2016.11.008.
[2] ZHOU M, HUANG Z, ZHOU B, et al. Construction and expression of two-copy engineered yeast of feruloyl esterase[J]. Electronic Journal of Biotechnology, 2015, 18(5): 338-342. DOI:10.1016/j.ejbt.2015.06.004.
[3] CREPIN V F, FAULDS C B, CONNERTON I F. Functional classification of the microbial feruloyl esterases[J]. Applied Microbiology Biotechnology, 2004, 63(6): 647-652. DOI:10.1007/s00253-003-1476-3.
[4] GAO L, WANG M, CHEN S, et al. Biochemical characterization of a novel feruloyl esterase from Penicillium piceum and its application in biomass bioconversion[J]. Journal of Molecular Catalysis B: Enzymatic, 2016, 133: S388-S394. DOI:10.1016/j.molcatb.2017.02.012.
[5] DILOKPIMOL A, M?KEL? M R, MANSOURI S, et al. Expanding the feruloyl esterase gene family of Aspergillus niger by characterization of a feruloyl esterase, FaeC[J]. New Biotechnology, 2017, 37(Pt B): 200-209. DOI:10.1016/j.nbt.2017.02.007.
[6] GOPALAN N, RODRIGUEZ-DURAN L V, SAUCEDOCASTANEDA G, et al. Review on technological and scientific aspects of feruloyl esterases: a versatile enzyme for biorefining of biomass[J]. Bioresource Technology, 2015, 193: 534-544. DOI:10.1016/j.biortech.2015.06.117.
[7] WANG L, LI Z, ZHU M, et al. An acidic feruloyl esterase from the mushroom Lactarius hatsudake: a potential animal feed supplement[J]. International Journal of Biological Macromolecules, 2016, 93(Pt A): 290-295. DOI:10.1016/j.ijbiomac.2016.08.028.
[8]刘君.阿魏酸酯酶基因在黑曲霉中的同源表达[D].哈尔滨:东北农业大学, 2014.
[9] YANG S, TANG L, YAN Q, et al. Biochemical characteristics and gene cloning of a novel thermostable feruloyl esterase from Chaetomium sp.[J]. Journal of Molecular Catalysis B: Enzymatic, 2013, 97: 328-336. DOI:10.1016/j.molcatb.2013.06.011.
[10] SWAMY S K P, GOVINDASWAMY V. Therapeutical properties of ferulic acid and bioavailability enhancement through feruloyl esterase[J]. Journal of Functional Foods, 2015, 17: 657-666. DOI:10.1016/j.jff.2015.06.013.
[11] SCHROETER H, WILLIAMS R J, MATIN R, et al. Phenolic antioxidants attenuate neuronal cell death following uptake of oxidized low-density lipoprotein[J]. Free Radical Biology and Medicine, 2000, 29(12):1222-1233. DOI:10.1016/S0891-5849(00)00415-9.
[12] SCALBERT A, MONTIES B, LALLEMAND J, et al. Ether linkage between phenolic acids and lignin fractions from wheat straw[J]. Phytochemistry, 1985, 24(6): 1359-1362. DOI:10.1016/S0031-9422(00)81133-4.
[13] DILOKPIMOL A, MAKELA M R, VARRIALE S, et al. Fungal feruloyl esterases: functional validation of genome mining based enzyme discovery including uncharacterized subfamilies[J]. New Biotechnology, 2018, 41: 9-14. DOI:10.1016/j.nbt.2017.11.004.
[14] NIETER A, KELLE S, LINKE D, et al. Feruloyl esterases from Schizophyllumcommuneto treat food industry side-streams[J]. Bioresource Technology, 2016, 220: 38-46. DOI:10.1016/j.biortech.2016.08.045.
[15] WANG X M, BAI Y, CAI Y, et al. Biochemical characteristics of three feruloyl esterases with a broad substrate spectrum from Bacillus amyloliquefaciens H47[J]. Process Biochemistry, 2017, 53: 109-115. DOI:10.1016/j.procbio.2016.12.012.
[16] FAZARY A E, ISMADJI S, JU Y. Biochemical studies on native and cross-linked aggregates of Aspergillus awamori feruloyl esterase[J]. International Journal of Biological Macromolecules, 2009, 44(3): 240-248. DOI:10.1016/j.ijbiomac.2008.12.012.
[17] ZHANG S B, WANG L, LIU Y, et al. Expression of feruloyl esterase A from Aspergillus terreus and its application in biomass degradation[J]. Protein Expression and Purification, 2015, 115: 153-157. DOI:10.1016/j.pep.2015.08.015.
[18] LEVASSEUR A, BENOIT I, ASTHER M, et al. Homologous expression of the feruloyl esterase B gene from Aspergillus niger and characterization of the recombinant enzyme[J]. Protein Expression and Purification, 2004, 37(1): 126-133. DOI:10.1016/j.pep.2004.05.019.
[19]胡泓宇,周燕燕,陈静,等.一株产阿魏酸酯酶丝状真菌的分离鉴定及其粗酶性质研究[J].生物技术通报, 2014(7):162-167.
[20]卢颖欣,张宁,欧仕益,等.产阿魏酸酯酶乳酸菌的乳杆菌筛选以及基因克隆[J].食品工业科技, 2013, 34(20):199-203.
[21] ZENG Y, YIN X, WU M, et al. Expression of a novel feruloyl esterase from Aspergillus oryzae in Pichia pastoris with esterification activity[J]. Journal of Molecular Catalysis B: Enzymatic, 2014, 110: 140-146. DOI:10.1016/j.molcatb.2014.10.002.
[22]黄艳凤,周庆礼,李士炼,等.从黑麦麦麸中提取的阿魏酸的检测[J].药物生物技术,2004,11(1):49-51.DOI:10.3969/j.issn.1005-8915.2004.01.013.
[23] BRADFORD M M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of proteindyebinding[J]. Analytical Biochemistry, 1976, 72: 248-254.
[24] THONGEKKAEW J, IKEDA H, MASAKI K, et al. An acidic and thermostable carboxymethyl cellulase from the yeast Cryptococcus sp. S-2: purification, characterization and improvement of its recombinant enzyme production by high cell-density fermentation of Pichia pastoris[J]. Protein Expression and Purification, 2008, 60(2): 140-146. DOI:10.1016/j.pep.2008.03.021.
[25] OHLHOFF C W, KIRBY B M, VAN ZYL L, et al. An unusual feruloyl esterase belonging to family VIII esterases and displaying a broad substrate range[J]. Journal of Molecular Catalysis B: Enzymatic, 2015, 118:79-88. DOI:10.1016/j.molcatb.2015.04.010.
[26] ZHANG S B, ZHAI H, WANG L, et al. Expression, purification and characterization of a feruloyl esterase A from Aspergillus flavus[J].Protein Expression and Purification, 2013, 92(1): 36-40. DOI:10.1016/j.pep.2013.08.009.
[27] TOPAKAS E, STAMATIS H, MASTIHUBOVA M, et al. Purification and characterization of a Fusarium oxysporum feruloyl esterase (FoFAE-I) catalysing transesterification of phenolic acid esters[J]. Enzyme and Microbial Technology, 2003, 33(5): 729-737. DOI:10.1016/S0141-0229(03)00213-8.
[28] BORNEMAN W S, LJUNGDAHL L G, HARTLEY R D, et al. Purification and partial characterization of two feruloyl esterases from the anaerobic fungus Neocallimastix strain MC-2[J]. Applied and Environmental Microbiology, 1992, 58(11): 3762-3766.
[29] RUMBOLD K, BIELY P, MASTIHUBOVA M, et al. Purification and properties of a feruloyl esterase involved in lignocellulose degradation by Aureobasidiumpullulans[J]. Applied and Environmental Microbiology, 2003, 69(9): 5622-5626. DOI:10.1128/AEM.69.9.5622-5626.2003.
[30]CHANDRASEKHARAIAHM,THULASIA,VIJAYARANIK,etal.Expressionandbiochemicalcharacterizationoftwonovel feruloylesterasesderivedfromfecalsamplesofRusaunicolorand Equusburchelli[J].Gene,2012,500(1):134-139.DOI:10.1016/j.gene.2012.03.027.
[31]VAFIADIC,TOPAKASE,WONGKKY,etal.Mappingthe hydrolyticandsyntheticselectivityofatypeCferuloylesterase(StFaeC)fromSporotrichumthermophileusingalkylferulates[J].Tetrahedron:Asymmetry,2005,16(2):373-379.DOI:10.1016/j.tetasy.2004.11.037.
[32]YAOJ,CHENQL,SHENAX,etal.Anovelferuloylesterase from a soil metagenomic library with tannase activity[J]. Journal of MolecularCatalysisB:Enzymatic,2013,95:55-61.DOI:10.1016/j.molcatb.2013.05.026.
[33]方园,欧仕益,张宁.阿魏酸酯酶的制备及其酶学性质[J].暨南大学学报(自然科学与医学版), 2012(5):481-485.
[34] TOPAKAS E, VAFIADI C, CHRISTAKOPOULOS P. Microbial production, characterization and applications of feruloyl esterases[J]. Process Biochemistry, 2007, 42(4): 497-509. DOI:10.1016/j.procbio.2007.01.007.
[35] KOSEKI T, HANDA H, WATANABE Y Y, et al. An unusual feruloyl esterase from Aspergillus oryzae: two tryptophan residues play a crucial role for the activity[J]. Journal of Molecular Catalysis B: Enzymatic, 2016, 133: 560-568. DOI:10.1016/j.molcatb.2016.11.008.
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