Liu Ming-Qi, Liu Guang-Fu
Department of Food Science, College of Life Science, China JiLiang University, Hangzhou 310018, China.
Protein Expr Purif. 2008 Feb;57(2):101-7. doi: 10.1016/j.pep.2007.10.020. Epub 2007 Nov 4.
The mature peptide of Bacillus licheniformis xylanase A (BlxA) was successfully expressed in Pichia pastoris under the control of AOX1 promoter. After 96-h 0.25% methanol induction, the activity of recombinant B. licheniformis xylanase A (reBlxA) in culture supernatant was 122.9 U/mg. Enzymatic properties assays showed that the optimum temperature and pH for reBlxA were 60 degrees C and pH 6.0, respectively. When treated at 70 degrees C, pH 6.0 for 2 min, the residual activities of the reBlxA were 76%. Over 80% of reBlxA activity was retained after treatment of the enzyme by preincubation over a pH range of 5.0-9.0 for 1h at 25 degrees C. High performance liquid chromatography (HPLC) analysis revealed that xylotriose (X3) was the main hydrolysis product released from birchwood xylan and wheat bran insoluble xylan by reBlxA. The mode of action studies showed that reBlxA was an endo-acting xylanase and xylobiose (X2), xylotriose, xylotetraose (X4), xylopentaose (X5), and xylohexaose (X6) could be hydrolyzed by it. This is the first report on the expression of reBlxA in yeast and on determining and quantifying the hydrolysis products released from xylans by reBlxA.
地衣芽孢杆菌木聚糖酶A（BlxA）的成熟肽在AOX1启动子的控制下于毕赤酵母中成功表达。经过96小时0.25%甲醇诱导后，培养上清液中重组地衣芽孢杆菌木聚糖酶A（reBlxA）的活性为122.9 U/mg。酶学性质分析表明，reBlxA的最适温度和pH分别为60℃和pH 6.0。在70℃、pH 6.0处理2分钟后，reBlxA的残余活性为76%。在25℃下于pH 5.0 - 9.0范围内预孵育1小时处理该酶后，reBlxA的活性保留超过80%。高效液相色谱（HPLC）分析显示，木三糖（X3）是reBlxA从桦木木聚糖和麦麸不溶性木聚糖释放的主要水解产物。作用方式研究表明，reBlxA是一种内切型木聚糖酶，木二糖（X2）、木三糖、木四糖（X4）、木五糖（X5）和木六糖（X6）都可被其水解。这是关于reBlxA在酵母中的表达以及确定和定量reBlxA从木聚糖释放的水解产物的首次报道。
