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OD600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Km和kcat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 of β-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 OD600 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 Km and kcat 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.
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