摘要
木聚糖酶能催化木聚糖分解为低聚木糖和木单糖,对于木聚糖高效利用具有重要意义。该研究将黑曲霉(Aspergillus niger)AG11来源的β-D-1,4内切木聚糖酶(β-D-1,4 endoxylanase,xynA)分泌表达于大肠杆菌(Escherichia coli)BL21(DE3),并进行了表达条件优化和酶学性质研究。最终确定xynA最佳表达条件为:诱导剂(异丙基硫代半乳糖苷)浓度0.1 mmol/L、诱导温度20℃、诱导起始OD_(600)值0.5。在该条件下,胞外xynA活力达到15.8 U/mL,较初始条件提高85.9%。重组xynA经镍柱纯化至单一条带后进行酶学性质分析。酶学性质分析表明,重组xynA比酶活力、最适反应温度及最适反应pH分别为175.1 U/mg、50℃和4.0。重组xynA在40℃半衰期为182 min,且在pH为2.0~9.0内孵育1 h保持在70%以上活性。以桦木木聚糖为底物,重组xynA的K_(m)和k_(cat)分别为3.45 g/L和58.51 s^(-1)。研究结果为xynA优良突变体的筛选和在工业生产中的应用奠定了基础。
Xylanase catalyzes the decomposition of xylan into xylooligosaccharides and xylomaccharides,which is of great significance for the efficient utilization of xylan.In this study,secretory expression ofβ-D-1,4 endoxylanase(xynA)from Aspergillus niger AG11 was achieved in Escherichia coli BL21(DE3).And the optimal expression conditions of xynA were determined as follows:the concentration of inducer(isopropylthiogalactoside)0.1 mmol/L,induction temperature of 20℃,and induction initiation OD_(600) of 0.5.Under these conditions,the extracellular xynA activity reached 15.8 U/mL,85.9%higher than that of the initial condition.The recombinant xynA was purified to a single band by nickel column and its enzymatic properties were analyzed.It was showed that the specific enzyme activity,optimum reaction temperature,and pH were 175.1 U/mg,50℃and 4.0,respectively.The half-life of recombinant xynA was 182 min at 40℃,and the activity of recombinant xynA remained above 70%when incubated at pH 2.0-9.0 for 1 h.Using birchwood xylan as a substrate,the K_(m) and k_(cat) values of recombinant xynA were 3.45 g/L and 58.51 s^(-1),respectively.The results lay a foundation for the screening of excellent mutants and industrial applications.
作者
李阳阳
刘松
尹小强
堵国成
LI Yangyang;LIU Song;YIN Xiaoqiang;DU Guocheng(National Engineering Laboratory for Cereal Fermentation Technology,Jiangnan University,Wuxi 214122,China;School of Biotechnology,Jiangnan University,Wuxi 214122,China)
出处
《食品与发酵工业》
CAS
CSCD
北大核心
2022年第6期1-7,共7页
Food and Fermentation Industries
基金
国家重点研发计划(2019YFA0905300)
国家自然科学基金项目(31771913,32071474)。
关键词
黑曲霉
木聚糖酶
胞外表达
表达优化
酶学性质
Aspergillus niger
xylanase
extracellular expression
expression optimization
enzymatic properties
