Xue Yemin, Wu Ailian, Zeng Hongyan, Shao Weilan
Jiangsu Key Laboratory for Biodiversity and Bio-resources, The Key Laboratory of Microbial Engineering, Nanjing Normal University, Nanjing, P.R. China.
Biotechnol Lett. 2006 Mar;28(5):351-6. doi: 10.1007/s10529-005-5934-0.
To efficiently produce xylobiose from xylan, high-level expression of an alpha-L-arabinofuranosidase gene from Thermotoga maritima was carried out in Escherichia coli. A 1.5-kb DNA fragment, coding for an alpha-L-arabinofuranosidase of T. maritima, was inserted into plasmid pET-20b without the pelB signal sequence leader, and produced pET-20b-araA1 with 8 nt spacing between ATG and Shine-Dalgarno sequence. A maximum activity of 12 U mg(-1) was obtained from cellular extract of E. coli BL21-CodonPlus (DE3)-RIL harboring pET-20b-araA1. The over-expressed alpha-L-arabinofuranosidase was purified 13-fold with a 94% yield from the cellular extract of E. coli by a simple heat treatment. Production of xylooligosaccharides from corncob xylan by endoxylanase and alpha-L-arabinofuranosidase was examined by TLC and HPLC: xylobiose was the major product from xylan at 90 degrees C and its proportion in the xylan hydrolyzates increased with the reaction time. Hydrolysis with in the xylanase absence of alpha-L-arabinofuranosidase gave only half this yield.
为了从木聚糖高效生产木二糖，在大肠杆菌中对来自嗜热栖热菌的α-L-阿拉伯呋喃糖苷酶基因进行了高水平表达。将一个编码嗜热栖热菌α-L-阿拉伯呋喃糖苷酶的1.5 kb DNA片段插入没有pelB信号序列前导肽的质粒pET-20b中，构建出ATG和Shine-Dalgarno序列之间间隔8个核苷酸的pET-20b-araA1。从携带pET-20b-araA1的大肠杆菌BL21-CodonPlus(DE3)-RIL细胞提取物中获得的最大活性为12 U mg(-1)。通过简单的热处理，从大肠杆菌细胞提取物中以94%的产率将过量表达的α-L-阿拉伯呋喃糖苷酶纯化了13倍。通过薄层色谱法(TLC)和高效液相色谱法(HPLC)检测了内切木聚糖酶和α-L-阿拉伯呋喃糖苷酶从玉米芯木聚糖生产低聚木糖的情况：在90℃下，木二糖是木聚糖的主要产物，并且其在木聚糖水解产物中的比例随反应时间增加。在没有α-L-阿拉伯呋喃糖苷酶的情况下用木聚糖酶水解，产量仅为前者的一半。
