Aspergillus niger NL-1来源的木聚糖酶的耐热性能改造及其在水解木聚糖中的应用
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摘要
为提高来源于Aspergillus niger NL-1的木聚糖酶MxynB的热稳定性,通过定点突变技术在木聚糖酶MxynB的相应位置引入二硫键(Cys116-Cys135),获得突变酶MynxB-116-135;通过基因融合技术在木聚糖酶MxynB的N端融合了具有较高热稳定性的来源于嗜热菌Thermotoga thermarum DSM5069的纤维素结合结构域(CBD),获得突变酶CBD-MxynB和CBD-MxynB-116-135。分别将原酶及突变酶在E.coli BL21 (DE3)中表达,经纯化后对比其酶学性质。结果显示,突变酶MxynB-116-135的最适温度为50℃,较原酶MxynB提高了5℃;突变酶CBDMxynB和CBD-MxynB-116-135的温度稳定性明显提高,其中CBD-MxynB-116-135尤为突出,在70℃下保温2h仍能保持50%以上的酶活力,而原酶基本失活。研究表明,二硫键的引入和CBD的融合对提高MxynB的热稳定性具有重要的作用。此外,研究了突变酶CBD-MxynB-116-135水解玉米芯木聚糖的产物,结果显示玉米芯木聚糖质量浓度为2.5mg/mL,酶用量为40U/g,在50℃、pH 5.0条件下水解10h后,水解得率为39.6%,水解产物XOS2-3含量占87.9%。结果表明,利用该突变木聚糖酶酶解碱提玉米芯木聚糖可产生以木二糖及木三糖为主要成分的低聚木糖。
To improve the thermostability of family GH11 xylanase MxynB from Aspergillus niger NL-1, a disulfide bridge(Cys116-Cys135) was introduced into the corresponding region of MxynB by site-directed mutagenesis. Then, xylanases MxynB and MxynB-116-135 were fused with the thermophilic cellulose binding domain(CBD) from Thermotoga thermarum DSM5069 at N-terminal of xylanases using PCR. The wild-type enzyme MxynB and the mutated enzymes MxynB-116-135,CBD-MxynB and CBD-MxynB-116-135 were expressed in Escherichia coli BL21(DE3),separately.After purification,their enzymatic properties were analyzed and compared.The optimal temperature of the recombinant MxynB-116-135was 50℃,which was 5℃higher than that of MxynB.Moreover,the thermostabilities of two recombinant mutants CBD-MxynB and CBD-MxynB-116-135 were clearly increased as compared with the wild-type enzyme under 70℃.Between them,CBD-MxynB-116-135 increased notably.After 2h,the relative activity of the mutant CBD-MxynB-116-135 was over 50%,and the wild-type MxynB was basically inactivated.All the results indicated that the introduction of a disulfide bridge and cellulose binding domain(CBD)could improve the thermostability of the MxynB notably.In addition,2.5mg/mL corncob xylan was treated with 40U/g mutant xylanase CBD-MxynB-116-135 for hydrolysis.After 10h,the xylooligosaccharides yield was 39.6%,and the content of XOS2-3 reached 87.9%.All these results indicated that this mutant xylanase could be suitable for potential applications in the xylobiose and xylotriose production.
To improve the thermostability of family GH11 xylanase MxynB from Aspergillus niger NL-1, a disulfide bridge(Cys116-Cys135) was introduced into the corresponding region of MxynB by site-directed mutagenesis. Then, xylanases MxynB and MxynB-116-135 were fused with the thermophilic cellulose binding domain(CBD) from Thermotoga thermarum DSM5069 at N-terminal of xylanases using PCR. The wild-type enzyme MxynB and the mutated enzymes MxynB-116-135,CBD-MxynB and CBD-MxynB-116-135 were expressed in Escherichia coli BL21(DE3),separately.After purification,their enzymatic properties were analyzed and compared.The optimal temperature of the recombinant MxynB-116-135was 50℃,which was 5℃higher than that of MxynB.Moreover,the thermostabilities of two recombinant mutants CBD-MxynB and CBD-MxynB-116-135 were clearly increased as compared with the wild-type enzyme under 70℃.Between them,CBD-MxynB-116-135 increased notably.After 2h,the relative activity of the mutant CBD-MxynB-116-135 was over 50%,and the wild-type MxynB was basically inactivated.All the results indicated that the introduction of a disulfide bridge and cellulose binding domain(CBD)could improve the thermostability of the MxynB notably.In addition,2.5mg/mL corncob xylan was treated with 40U/g mutant xylanase CBD-MxynB-116-135 for hydrolysis.After 10h,the xylooligosaccharides yield was 39.6%,and the content of XOS2-3 reached 87.9%.All these results indicated that this mutant xylanase could be suitable for potential applications in the xylobiose and xylotriose production.
引文
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[8]何敏超,张宇,许敬亮,等.木聚糖酶高产菌筛选研究进展[J].化工进展, 2013, 32(1):145-150.HE M C, ZHANG Y, XU J L, et al. Research progress of screening ofhigh xylanase-producing strain[J]. Chemical Industry and EngineeringProgress, 2013, 32(1):145-150.
[9]裴建军,李杰,李琦,等. Thermoanaerobacterium saccharolyticum JW/SL-YS485来源的GH11家族木聚糖酶的克隆表达及其应用[J].化工进展, 2018, 37(4):1544-1551.PEI J J, LI J, LI Q, et al. Expression and characterization of a GH11xylanase from Thermoanaerobacterium saccharolyticum JW/SL-YS485and its application in xylooligosaccharide production[J]. ChemicalIndustry and Engineering Progress, 2018, 37(4):1544-1551.
[10] SUBRAMANIYAN S, PREMA P. Biotechnology of microbialxylanases:enzymology, molecular biology, and application[J]. Crit.Rev. Biotechnol., 2002, 22(1):33-64.
[11]李同彪,周晨妍,王丹丹.木聚糖酶热稳定性分子改造的研究进展[J].中国生物制品学杂志, 2014, 27(11):1493-1496.LI T B, ZHOU C Y, WANG D D. Progress in research on molecularmodification of xylanase thermostability[J]. Chin. J. Biologicals, 2014,27(11):1493-1496.
[12] SUN J Y, LIU M Q, XU Y L, et al. Improvement of the thermostabilityand catalytic activity of a mesophilic family 11 xylanase by N-terminus replacement[J]. Protein Expression and Purification, 2005, 42(1):122-130.
[13] FONSECAMALDONADO R, VIEIRA D S, ALPONTI J S, et al.Engineering the pattern of protein glycosylation modulates thethermostability of a GH11 xylanase[J]. Journal of Biological Chemistry,2013, 288(35):25522-25534.
[14]刘晓彤,邬敏辰,殷欣,等.二硫键对提高木聚糖酶AoXyn11A热稳定性的作用[J].食品与生物技术学报, 2014, 33(10):1038-1043.LIU X T, WU M C, YIN X, et al. Effect of disulfide bridge onthermostability improvement of the xylanase AoXyn11A[J]. Journal ofFood Science and Biotechnology, 2014, 33(10):1038-1043.
[15] XUE H, ZHOU J, YOU C, et al. Amino acid substitutions in the N-terminus, cord andα-helix domains improved the thermostability of afamily 11 xylanase XynR8[J]. Journal of Industrial Microbiology andBiotechnology, 2012, 39(9):1279-1288.
[16] LIU L W, CHENG J, CHEN H, et al. Directed evolution of amesophilic fungal xylanase by fusion of a thernmophilic bacterialcarbohydrate-binding module[J]. Process Biochem., 2011, 46(1):395-398.
[17] LI F, YANG S Y, ZHAO L G, et al. Synonymous condon usage biasand overexpression of a synthetic xynB gene from Aspergillus niger NL-1 in Pichia pastoris[J]. Bioresources, 2012, 7(2):2330-2343.
[18] MILLER G L. Use of dinitrosalicylic acid reagent for determination ofreducing sugar[J]. Analytical Chem., 1959, 31(3):426-428.
[19]李琦,陈明真,赵林果.热木聚糖酶基因的克隆表达及其在酶解玉米芯木聚糖中的应用[J].林业工程学报, 2017, 2(4):63-69.LI Q, CHEN M Z, ZHAO L G. Cloning and expression of a thermophileGH11 xylanase and its application in xylooligosaccharide production[J]. Journal of Forestry Engineering, 2017, 2(4):63-69.
[20]许正宏,熊筱晶,陶文沂.低聚木糖的生产及应用研究进展[J].食品与发酵工业, 2002, 28(1):56-59.XU Z H, XIONG X J, TAO W Y. Research progress of production andapplication of xylooligosaccharide[J]. Food and FermentationIndustries, 2002, 28(1):56-59.
[2] COLLINS T, GERDAY C, FELLER G. Xylanases, xylanase familiesand extremophilic xylanases[J]. FEMS Microbiol. Rev., 2005, 29(1):3-23.
[3]唐伟,王立东,张丽萍.超声波前处理去麸皮中木聚糖提取率的影响[J].食品科技, 2010, 35(5):191-194.TANG W, WANG L D, ZHANG L P. Promotional effect of ultrasonicwave on extraction of xylan from wheat bran[J]. Food Science andTechnology, 2010, 35(5):191-194.
[4] BEG Q K, KAPOOR M, MAHAJAN L, et al. Microbiol xylanases andtheir industrial applications:a review[J]. Appl. Microbiol. Biotechnol.,2001, 56(3/4):326-338.
[5] LIAB K, AZADI P, COLLINS R, et al. Relationships between activitiesof xylanases and xylan structures[J]. Enzyme Microb. Technol., 2000,27(1/2):89-94.
[6] LOMBARD V, GOLACONDA R H, DRULA E, et al. Thecarbohydrate-active enzymes database(CAZy)in 2013[J]. NucleicAcids Research, 2013, 42(D1):D490-D495.
[7] JUTURU V, WU J C. Microbial xylanases:engineering, production andindustrial applications[J]. Biotechnology Advances, 2012, 30(6):1219-1227.
[8]何敏超,张宇,许敬亮,等.木聚糖酶高产菌筛选研究进展[J].化工进展, 2013, 32(1):145-150.HE M C, ZHANG Y, XU J L, et al. Research progress of screening ofhigh xylanase-producing strain[J]. Chemical Industry and EngineeringProgress, 2013, 32(1):145-150.
[9]裴建军,李杰,李琦,等. Thermoanaerobacterium saccharolyticum JW/SL-YS485来源的GH11家族木聚糖酶的克隆表达及其应用[J].化工进展, 2018, 37(4):1544-1551.PEI J J, LI J, LI Q, et al. Expression and characterization of a GH11xylanase from Thermoanaerobacterium saccharolyticum JW/SL-YS485and its application in xylooligosaccharide production[J]. ChemicalIndustry and Engineering Progress, 2018, 37(4):1544-1551.
[10] SUBRAMANIYAN S, PREMA P. Biotechnology of microbialxylanases:enzymology, molecular biology, and application[J]. Crit.Rev. Biotechnol., 2002, 22(1):33-64.
[11]李同彪,周晨妍,王丹丹.木聚糖酶热稳定性分子改造的研究进展[J].中国生物制品学杂志, 2014, 27(11):1493-1496.LI T B, ZHOU C Y, WANG D D. Progress in research on molecularmodification of xylanase thermostability[J]. Chin. J. Biologicals, 2014,27(11):1493-1496.
[12] SUN J Y, LIU M Q, XU Y L, et al. Improvement of the thermostabilityand catalytic activity of a mesophilic family 11 xylanase by N-terminus replacement[J]. Protein Expression and Purification, 2005, 42(1):122-130.
[13] FONSECAMALDONADO R, VIEIRA D S, ALPONTI J S, et al.Engineering the pattern of protein glycosylation modulates thethermostability of a GH11 xylanase[J]. Journal of Biological Chemistry,2013, 288(35):25522-25534.
[14]刘晓彤,邬敏辰,殷欣,等.二硫键对提高木聚糖酶AoXyn11A热稳定性的作用[J].食品与生物技术学报, 2014, 33(10):1038-1043.LIU X T, WU M C, YIN X, et al. Effect of disulfide bridge onthermostability improvement of the xylanase AoXyn11A[J]. Journal ofFood Science and Biotechnology, 2014, 33(10):1038-1043.
[15] XUE H, ZHOU J, YOU C, et al. Amino acid substitutions in the N-terminus, cord andα-helix domains improved the thermostability of afamily 11 xylanase XynR8[J]. Journal of Industrial Microbiology andBiotechnology, 2012, 39(9):1279-1288.
[16] LIU L W, CHENG J, CHEN H, et al. Directed evolution of amesophilic fungal xylanase by fusion of a thernmophilic bacterialcarbohydrate-binding module[J]. Process Biochem., 2011, 46(1):395-398.
[17] LI F, YANG S Y, ZHAO L G, et al. Synonymous condon usage biasand overexpression of a synthetic xynB gene from Aspergillus niger NL-1 in Pichia pastoris[J]. Bioresources, 2012, 7(2):2330-2343.
[18] MILLER G L. Use of dinitrosalicylic acid reagent for determination ofreducing sugar[J]. Analytical Chem., 1959, 31(3):426-428.
[19]李琦,陈明真,赵林果.热木聚糖酶基因的克隆表达及其在酶解玉米芯木聚糖中的应用[J].林业工程学报, 2017, 2(4):63-69.LI Q, CHEN M Z, ZHAO L G. Cloning and expression of a thermophileGH11 xylanase and its application in xylooligosaccharide production[J]. Journal of Forestry Engineering, 2017, 2(4):63-69.
[20]许正宏,熊筱晶,陶文沂.低聚木糖的生产及应用研究进展[J].食品与发酵工业, 2002, 28(1):56-59.XU Z H, XIONG X J, TAO W Y. Research progress of production andapplication of xylooligosaccharide[J]. Food and FermentationIndustries, 2002, 28(1):56-59.
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