枯草芽孢杆菌木聚糖酶的克隆表达及其酶解两种木聚糖的产物分析  

Cloning and Expression of Bacillus subtilis Xylanase and Determination of Hydrolysis Products of Two Different Xylans by It

作　　者：韦露莎[1] 吴一飞[1] 陈辉[1] 

机构地区：[1]西北农林科技大学林学院,陕西杨凌712100

出　　处：《食品科学》2016年第5期108-113,共6页Food Science

基　　金：国家自然科学基金面上项目(31170607)

摘　　要：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.

关 键 词：木聚糖内切酶 低聚木糖 基质辅助激光解吸/电离飞行时间质谱 

分 类 号：Q819[生物学—生物工程]