来源于橄榄绿链霉菌A1的高比活木聚糖酶XYNB的高效表达及性质的比较分析  

Overexpression of Streptomyces olivaceoviridis A1 Xylanase with High Specific Activity and Analysis of Enzymic Properties

作　　者：何永志[1] 姚斌[1] 王亚茹[1] 袁铁峥[1] 罗会颖[1] 史秀云[1] 伍宁丰[1] 范云六[1] 

机构地区：[1]中国农业科学院饲料研究所,北京100081

出　　处：《微生物学报》2004年第3期340-344,共5页Acta Microbiologica Sinica

基　　金：国家"8 63计划" ( 2 0 0 1AA2 14 0 41)~~

摘　　要：高效表达高比活木聚糖酶是进一步提高木聚糖酶发酵效价、降低其生产成本的有效途径。将橄榄绿链霉菌 (Streptomycesolivaceoviridis)A1的高比活木聚糖酶成熟蛋白编码基因xynB克隆到毕赤酵母表达载体pPIC9中 ,转化毕赤酵母得到重组酵母 ,在重组酵母中木聚糖酶基因得到了高效分泌表达 ,且表达产物具有生物学活性。在 3L发酵罐中蛋白表达量约 1 4mg mL ,酶活性 (效价 )为 1 2 0 0IU mL。SDS PAGE分析表明 ,表达的木聚糖酶XYNBa为糖基化蛋白 ,分子量为 31kD ,经脱糖基化处理得到 2 1kD的XYNBb ,与橄榄绿链霉菌A1所产原酶XYNB大小一致。通过对XYNB、XYNBa及XYNBb酶学性质的比较发现 :三者在比活性、Vmax及热稳定性方面有较大差异。该酶对不同木聚糖的酶解产物的糖份分析表明 :酶解产物的主要成分为木二糖、木三糖和木四糖 ,占总糖含量的 95High-level expression of xylanase with high specific activity is an effective way to improve xylanase fermentation potency in recombinant host and decrease its production costs. The gene xynB encoding the mature protein of xylanase with high specific activity from Streptomyces olivaceoviridis A1 was cloned into Pichia pastoris expression vector pPIC9 and introduced into the host Pichia pastoris by electroporation. The results of SDS-PAGE and activity assay of the xylanase expressed by recombinant P. pastoris showed that the xylanase gene xynB was overexpressed and secreted, and the expressed xylanase had normal bioactivity. In 3L fermentor, the expression level of xylanase protein in recombinant P. pastoris was 1 4mg/mL, and the xylanase activity exceeded 1200IU/mL. The results of SDS-PAGE showed that the expressed xylanase(XYNBa) could be modified by glycosylation, had a single apparent molecular weight of about 31kD, while the deglycosylated xylanase(XYNBb) treated with Endo H had an apparent molecular weight of about 21kD, which was as same as that of original xylanase(XYNB) from Streptomyces olivaceoviridis A1 The results of the researches on enzymatic properties revealed that there were remarkable differences on specific activity,V max and thermal stability among XYNB,XYNBa,XYNBb. The analysis of enzymatic hydrolysate for different xylans revealed that the main constitutions of enzymatic hydrolysate were xylobiose,xylotriose and xyloquaiose, which account for more than 95% of all hydrolysate.

关 键 词：高比活木聚糖酶 高效表达 毕赤酵母 酶学性质 

分 类 号：Q55[生物学—生物化学]