通过基因克隆方法获得枯草芽孢杆菌木聚糖酶Xyn A,考察经镍离子亲和柱纯化后的Xyn A分别在桦木木聚糖和毛榉木木聚糖中的酶解情况,利用薄层色谱法(thin layer chromatography,TLC)及基质辅助激光解吸/电离飞行时间质谱(matrix-assisted laser desorption/ionization time-of-flight mass spectrometry,MALDI-TOF/MS)法鉴定木聚糖酶Xyn A的酶解产物。运用MALDI-TOF/MS分析枯草芽孢杆菌木聚糖酶Xyn A酶解桦木木聚糖和毛榉木木聚糖产物不同的聚合度寡糖的分布情况。结果表明:在桦木木聚糖酶解液中,产生的中性木寡糖主要为木二糖(X2)和木三糖(X3),酸性木寡糖聚合度为412,并且每一个酸性木寡糖上仅连接着一个甲基葡萄糖醛酸侧链(Me G)。在毛榉木木聚糖酶解液中,产生的中性木寡糖与在桦木木聚糖酶解液中相同,酸性木寡糖的结构相似,聚合度为416。因此木聚糖酶Xyn A具有生产木二糖(X2)和木三糖(X3)以及酸性木寡糖(Me GXn)的功能。
Matrix assisted laser desorption ionization time-of-flight mass spectrometry(MALDI-TOF/MS) was applied to analyze molecular weight distribution of hydrolysis products from birchwood xylan and beechwood xylan digested by Bacillus subtilis xylanase(Xyn A), obtained through recombinant expression and purified by Ni+-IDA column chromatography. The hydrolysis products were identified by thin layer chromatography(TLC) and MALDI-TOF/MS. Results showed the enzymatic hydrolysis of birchwood xylan yielded mainly xylobiose(X2) and xylotriose(X3) as well as acidic xylooligosacchrides with a degree of polymerization(DP) of 4–12 each including a single methyl-glucuronic acid side chain. Both xylobiose and xylotriose were found in the hydrolysate of beechwood xylan as well as acidic xylooligosacchrides with a DP of 4–16 structurally similar to those from birchwood xylan. Therefore, the recombinant Xyn A has the potential to produce X2, X3 and acidic xylooligosacchrides.