黄曲霉产β-1,4-木聚糖酶发酵条件的优化及其酶学特性
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摘要
研究发现一株高产β-1,4-木聚糖酶的黄曲霉,拟优化其产酶条件并分析该酶的酶学特性。采用单因素法优化其摇瓶发酵条件,聚丙烯酰胺凝胶电泳(SDS-PAGE)结合酶谱法判定酶的特性以及薄层色谱分析法(TLC)判定酶的水解特异性。结果表明,产酶最佳培养基为:麸皮3.5%、磷酸氢二铵3.0%、吐温-60 1.0%、NaCl 0.5%、MgSO_4·7H_2O 0.05%和KH_2PO_4 0.075%;黄曲霉在35℃下培养5 d达到最高酶活力115.08 U/mL,为未优化时的9.4倍。该菌能分泌两种β-1,4-木聚糖酶,分子量约为20.1和31.0 kDa,均不同于已有报道。粗酶的最适pH和最适温度分别为MOPS 7.5和55℃,在pH5.5～9.0及30～50℃范围内能保持酶活力的稳定。该酶不仅能水解榉木木聚糖产生低聚木糖,还能微弱降解大麦葡聚糖,有助于它在木质纤维素降解领域中的应用。
This study was aimed at optimization of the fermentation conditions for β-1,4-xylanase production by Aspergillus flavus and characterization of its enzymatic properties. The fermentation conditions in shake flask were optimized by a single-factor experiment. And the enzymatic properties of the enzyme were analyzed by SDS-PAGE,zymogram and TLC. The results indicated that the optimal culture medium components for β-1,4-xylanase production were 3.5% wheat bran,3.0%(NH_4)_2HPO_4,1.0% Tween-60,0.5% NaCl,0.05% MgSO_4·7 H_2O and 0.075% KH_2PO_4.The highest enzyme activity of 115.08 U/mL was obtained when the strain was cultured at 35 ℃ for 5 d in the optimized culture medium,which was 9.4 times compared with the initial activity. Two β-1,4-xylanases were secreted by A. flavus,and their molecular mass were different from all previous studies. The crude enzyme's optimal temperature and pH were determined to be 55 ℃ and 7.5,respectively. It was stable at 30～50 ℃and within pH5.5～9.0. The enzyme could not only efficiently hydrolyze beechwood xylan to produce xylooligosaccharides,but also weakly degrade the barley glucan,suggesting it was a good candidate for lignocelluloses' degradation.
This study was aimed at optimization of the fermentation conditions for β-1,4-xylanase production by Aspergillus flavus and characterization of its enzymatic properties. The fermentation conditions in shake flask were optimized by a single-factor experiment. And the enzymatic properties of the enzyme were analyzed by SDS-PAGE,zymogram and TLC. The results indicated that the optimal culture medium components for β-1,4-xylanase production were 3.5% wheat bran,3.0%(NH_4)_2HPO_4,1.0% Tween-60,0.5% NaCl,0.05% MgSO_4·7 H_2O and 0.075% KH_2PO_4.The highest enzyme activity of 115.08 U/mL was obtained when the strain was cultured at 35 ℃ for 5 d in the optimized culture medium,which was 9.4 times compared with the initial activity. Two β-1,4-xylanases were secreted by A. flavus,and their molecular mass were different from all previous studies. The crude enzyme's optimal temperature and pH were determined to be 55 ℃ and 7.5,respectively. It was stable at 30～50 ℃and within pH5.5～9.0. The enzyme could not only efficiently hydrolyze beechwood xylan to produce xylooligosaccharides,but also weakly degrade the barley glucan,suggesting it was a good candidate for lignocelluloses' degradation.
引文
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[18]刘文玉,史应武,王杏芹,等. 低温β-半乳糖苷酶产生菌14-1摇瓶发酵研究[J]. 中国乳品工业,2007,35(8):19-22.
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[21]Deshmukh R A,Jagtap S,Mandal M K,et al. Purification,biochemical characterization and structural modelling of alkali-stable β-1,4-xylan xylanohydrolase from Aspergillus fumigayus R1 isolated from soil[J]. BMC Biotechonology,2016,16(1):11.
[22]Carmona E C,Fialho M B,Buchgnani é B,et al. Production,purification and characterization of a minor form of xylanase from Aspergillus versicolor[J]. Process Biochemistry,2005,40(1):359-364.
[23]Fan G S,Yang S Q,Yan Q J,et al. Characterization of a highly thermostable glycoside hydrolase family 10 xylanase from Malbranchea cinnamomea[J]. International Journal of Biological Macromolecules,2014,70(8):482-489.
[24]Nair S G,Sindhu R,Shashidhar S. Purification and biochemical characterization of two xylanases from Aspergillus sydowii SBS 45[J]. Applied Microbiology and Biotechnology,2008,149(3):229-243.
[25]Bajaj B K,Abbass M. Studies on an alkali-thermostable xylanase from Aspergillus fumigates MA28[J]. Biotech,2011,1(3):161-171.
[26]Linares-Pastén J A,Aronsson A,Karlsson E N. Strucyural considerations on the use of endo-xylanases for the production of prebiotic xylooligosaccharides from biomass[J]. Current Protein & Peptide Science,2018,19:48-67.
[27]Hong S,Kyung M,Jo I,et al. Structure-based protein engineering of bacterial β-xylosidase to increase the production yield of xylobiose from xylose[J]. Biochemical and Biophysical Research Communication,2018,501:703-710.
[28]Maeda R,Ida T,Ihara H,et al. Induction of apoptosis in MCF-7 cells by β-1,3-xylooligosaccharides prepared from Caulerpa lentillife[J]. Bioscience,Biotechnology and Biochemistry,2012,76(5):1032-1034.
[2]Loureiro D B,Braia M,Romanini D,et al. Partitioning of xylanase from Thermomyces lanuginosus in PEG/NaCit aqueous two-phase systems:Structural and functional approach[J]. Protein Expression and Purification,2017,129:25-30.
[3]Sanjivkumar M,Siambarasan T,Palavesam A,et al. Biosynthesis,purification and characterization of β-1,4-xylanase from a novel mangrove associated actinobacterium Streptomyces olivaceus(MSU3)and its applications[J]. Protein Expression and Purification,2017,130:1-12.
[4]Aachary A A,Prapulla S G. Corncob-Induced endo-1,4-β-D-xylanase of Aspergillus oryzae MTCC 5154:production and characterization of xylobiose from glucuronoxylan[J]. Journal of Agricultral and Food Chemistry,2008,56(11):3981-3988.
[5]Wu X Y,Tian Z N,Jiang X K,et al. Enhancement in catalytic activity of Aspergillus niger XynB by selective site-directed mutagenesis of activity site amino acids[J]. Applied Microbiology and Biotechnology,2017,102(6):1-12.
[6]Sajith S,Sreedevi P,Priji K N,et al. Production and partial purification of cellulose from a novel fungus,Aspergillus flavus BS1[J]. Annals of Microbiology,2014,64(2):763-771.
[7]Karim K M R,Husaini A,Sing N N,et al. Purification of an alpha amylase from Aspergillus flavus NSH9 and molecular characterization of its nucleotide gene sequence[J]. 3 Biotech,2018,8(4):227.
[8]Gao G,Mao R Q,Xiao Y,et al. Efficient yeast cell-surface display of an endoglucanase of Aspergillus flavus and functional characterization of the whole-cell enzyme[J]. World Journal of Microbiology and Biotechnology,2017,33(6):114.
[9]Bharat B,Ajay P,Satish K. Production and partial characterization of extracellular xylanase from acidophilic Aspergillus flavus MTCC 9390 grown in SSF mode[J]. Research Journal of Biotechnology,2014,9:(5):97-105.
[10]Mellon J E,Cotty P J,Callicott A,et al. Identification of a major xylanase from Aspergillus flavus as a 14-kD protein[J]. Mycopathologia,2011,172(4):299-305.
[11]Bhushan B,Pal A,Kumar S,et al. Biochemical characterization and kinetic comparison of encapsulated haze removing acidophilic xylanase with partially purified free xylanase isolated from Aspergillus flavus MTCC 9390[J]. Journal of Food Science and Technology,2015,52(1):191-200.
[12]Kim J D. Production of xylanolytic enzyme complex from Aspergillus flavus using agricultural wastes[J]. Mycobiology,2005,33(2):84-89.
[13]李科,孙彦平,蔡小波,等. 黄曲霉产木聚糖酶条件优化及酶解产物初步分析[J]. 生物技术通报,2009,s1:348-351.
[14]Bharat B,Ajay P,Jain K. Isolation,screening and optimized production of extracellular xylanase under submerged condition from Aspergillus flavus Mtcc 9390[J]. Enzyme Engineering,2012,1:103.
[15]Pal A,Khanum F. Identification and optimization of critical medium components using statistical exprerimental designs for enhanced production of xylanase from Aspergillus flavus DFR-6[J]. Food Technology and Biotechnology,2011,49(2):228-236.
[16]Liu X Q,Liu Y,Jiang Z Q,et al. Biochemical characterization of a novel xylanase from Paenibacillus barengoltzii and its application in xylooligosaccharides production from corncobs[J]. Food Chemistry,2018,264:310-318.
[17]Laemmli U K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4[J]. Nature,1970,277(5259):680-685.
[18]刘文玉,史应武,王杏芹,等. 低温β-半乳糖苷酶产生菌14-1摇瓶发酵研究[J]. 中国乳品工业,2007,35(8):19-22.
[19]Jiang Z Q,Cong Q Q,Yan Q J,et al. Characterization of a thermostablexylanase from Chaetomium sp. and its application in Chinese steamed bread[J]. Food Chemistry,2010,120(2):457-462.
[20]Sudan R,Bajaj B K. Production and biochemical characterization of xylanase from an alkalitolerant novel species Aspergillus niveus RS2[J]. World Journal of Microbiology and Biotechnology,2007,23(4):491-500.
[21]Deshmukh R A,Jagtap S,Mandal M K,et al. Purification,biochemical characterization and structural modelling of alkali-stable β-1,4-xylan xylanohydrolase from Aspergillus fumigayus R1 isolated from soil[J]. BMC Biotechonology,2016,16(1):11.
[22]Carmona E C,Fialho M B,Buchgnani é B,et al. Production,purification and characterization of a minor form of xylanase from Aspergillus versicolor[J]. Process Biochemistry,2005,40(1):359-364.
[23]Fan G S,Yang S Q,Yan Q J,et al. Characterization of a highly thermostable glycoside hydrolase family 10 xylanase from Malbranchea cinnamomea[J]. International Journal of Biological Macromolecules,2014,70(8):482-489.
[24]Nair S G,Sindhu R,Shashidhar S. Purification and biochemical characterization of two xylanases from Aspergillus sydowii SBS 45[J]. Applied Microbiology and Biotechnology,2008,149(3):229-243.
[25]Bajaj B K,Abbass M. Studies on an alkali-thermostable xylanase from Aspergillus fumigates MA28[J]. Biotech,2011,1(3):161-171.
[26]Linares-Pastén J A,Aronsson A,Karlsson E N. Strucyural considerations on the use of endo-xylanases for the production of prebiotic xylooligosaccharides from biomass[J]. Current Protein & Peptide Science,2018,19:48-67.
[27]Hong S,Kyung M,Jo I,et al. Structure-based protein engineering of bacterial β-xylosidase to increase the production yield of xylobiose from xylose[J]. Biochemical and Biophysical Research Communication,2018,501:703-710.
[28]Maeda R,Ida T,Ihara H,et al. Induction of apoptosis in MCF-7 cells by β-1,3-xylooligosaccharides prepared from Caulerpa lentillife[J]. Bioscience,Biotechnology and Biochemistry,2012,76(5):1032-1034.