橄榄绿链霉菌Streptomyces olivaceoviridis A1木聚糖酶基因xynB在毕赤酵母中的高效表达
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摘要
木聚糖酶是可将木聚糖降解为低聚糖和木糖的一类酶的总称。它广泛地存在于各种微生物中，在饲料、造纸、食品医药以及能源工业中有着广阔的应用前景。我们将来源于橄榄绿链霉菌A1(Streptomyces olivaceoviridis A1)的木聚糖酶基因xynB的成熟蛋白编码序列克隆到毕赤酵母表达载体pPIC9α中，转化Pichia pastoris GS115得到重组子，实现了木聚糖酶基因xynB的高效表达，表达产物能有效分泌且具有正常的生物学活性。在3升发酵罐中木聚糖酶的表达量达1200IU／mL。SDS-PAGE分析表明，毕赤酵母表达产物XYNBa的分子量为31kD，大于橄榄绿链霉菌A1所产原酶XYNB的23kD。进一步对XYNBa进行了脱糖基化处理得到XYNBb,其分子量恢复到23kD，证明XYNBa是糖基化蛋白。通过对毕赤酵母重组表达的木聚糖酶XYNBa、脱糖基化的木聚糖酶XYNBb以及橄榄绿链霉菌A1所产原酶XYNB之间酶学性质的比较发现：三种酶的最适pH差异不大，XYNB和XYNBa均为5.2，XYNBb为5.0；XYNB和XYNBa的最适温度均为60℃，XYNBb降为50℃：在耐热性上，XYNBa由于糖基化作用热稳定性明显高于未糖基化的XYNB和XYNBb；XYNBa和XYNBb的比活性分别为883.88IU／mg和832.51IU／mg，明显低于原酶的比活2814.45IU／mg；XYNB和XYNBa的Km值相当，分别为21.56(g／Kg)和20.87(g／Kg)，而XYNBb的Km值较大为27.10(g／Kg)；XYNBa和XYNBb的Vmax相差不大，分别为4568μmol／mg·min和5329μmol／mg·min，明显低于XYNB的27623μmol／mg·min此外三种酶均无纤维素酶活性，对胃蛋白酶和胰蛋白酶有很好的抗性，且对作用环境中的各种离子、表面活性剂、螯合剂不敏感。通过对不同木聚糖的酶解产物的糖份分析发现：以桦木木聚糖为底物时，酶解产物主要为木三糖和木四糖，含量分别为68.43％和16.50％，另外还含有11.79％的木二糖；以玉米芯木聚糖为底物时，酶解产物主要为木二糖和木三糖，含量分别为81.78％和11.55％。证实该酶为内切β—1—4木聚糖酶，非常适用于低聚木糖的工业化生产。
Xylanase refers to a type of enzyme which can hydrolyze xylans into xylooligosaccharides and D-xylose.It broadly exists in microorganism and has wide commerical application in industrial processes,such as feed, paper, foodstuff, medicine and energy industries.The xylanase gene xynB encoding the native protein of xylanase from Streptomyces olivaceoviridis A1 was cloned into Pichia pastoris expression vector pPIC9 a. The recombinants were constructed by transforming pPIC9 a -xynB into P. pastoris GS115.The assay results revealed that the xylanase gene xynB was overexpressed and secreted effectually in P. pastoris.In 3L fermentor the expression level of xylanase XYNBa exceeded 1200IU/ml and the expressed xylanase had normal bioactivity.The molecule weight of XYNBa was determined as about 31kD which is higher than 23kD of original enzyme XYNB from Streptomyces olivaceoviridis A1.XYNBb was gotten by deglycasylation of XYNBa,whose molecule weight returned to 23kD.We comparised the enzymatic properties of XYNBa expressed in P. pastoris, XYNBb deglycasylated from XYNBa and XYNB produced from Streptomyces olivaceoviridis Al :There was little difference among the three enzymes on optimal pH,the optimal pH of XYNB and XYNBa were both 5.2,the optimal pH of XYNBb was 5.0;The optimal temperature of XYNB and XYNBa were both 60 C,while the optimal temperature of XYNBb was 50癈;Because of glycosylation the thermal stability of XYNBa was better than XYNB and XYNBb;The specific activity of XYNBa and XYNBb were 883.88IU/mg and 832.5HU/mg respectively,which were both lower than 2814.45IU/mg of XYNB;The Km values of XYNB and XYNBa were similar to each other which were 21.56(g/kg) and 20.87(g/kg),while the Km value of XYNBb was 27.10(g/kg);The Fmax of XYNBa and XYNBb were 4568umol/mg.min and 5329umol/mg.min respectively which were lower than 27623 umol/mg.min of XYNB; Additionally all of the three enzymes did not display cellulase activity.They all had well resistance to pepsion and trypsin,and were not sensitive to metal iron, surface active agent and chelating agent.The analysis of different xylans enzymatic hydrolysate revealed:By XYNBa,that the main constitutions of enzymatic hydrolysate of birch wood xylans were xylotriose and xyloquaiose,which account for 68.43% and 16.50% respectively,additionally there was 11.79% of xylobiose; The main constitutions
of enzymatic hydrolysate of corncobs xylans were xylobiose and xylotriose, which account for 81.78% and 11.55%.The result indicated that this xylanase was a kind of 1,4- b -D-xylanohydrolase and was fit to used in industrial procession of xylooligosacc harides.
Xylanase refers to a type of enzyme which can hydrolyze xylans into xylooligosaccharides and D-xylose.It broadly exists in microorganism and has wide commerical application in industrial processes,such as feed, paper, foodstuff, medicine and energy industries.The xylanase gene xynB encoding the native protein of xylanase from Streptomyces olivaceoviridis A1 was cloned into Pichia pastoris expression vector pPIC9 a. The recombinants were constructed by transforming pPIC9 a -xynB into P. pastoris GS115.The assay results revealed that the xylanase gene xynB was overexpressed and secreted effectually in P. pastoris.In 3L fermentor the expression level of xylanase XYNBa exceeded 1200IU/ml and the expressed xylanase had normal bioactivity.The molecule weight of XYNBa was determined as about 31kD which is higher than 23kD of original enzyme XYNB from Streptomyces olivaceoviridis A1.XYNBb was gotten by deglycasylation of XYNBa,whose molecule weight returned to 23kD.We comparised the enzymatic properties of XYNBa expressed in P. pastoris, XYNBb deglycasylated from XYNBa and XYNB produced from Streptomyces olivaceoviridis Al :There was little difference among the three enzymes on optimal pH,the optimal pH of XYNB and XYNBa were both 5.2,the optimal pH of XYNBb was 5.0;The optimal temperature of XYNB and XYNBa were both 60 C,while the optimal temperature of XYNBb was 50癈;Because of glycosylation the thermal stability of XYNBa was better than XYNB and XYNBb;The specific activity of XYNBa and XYNBb were 883.88IU/mg and 832.5HU/mg respectively,which were both lower than 2814.45IU/mg of XYNB;The Km values of XYNB and XYNBa were similar to each other which were 21.56(g/kg) and 20.87(g/kg),while the Km value of XYNBb was 27.10(g/kg);The Fmax of XYNBa and XYNBb were 4568umol/mg.min and 5329umol/mg.min respectively which were lower than 27623 umol/mg.min of XYNB; Additionally all of the three enzymes did not display cellulase activity.They all had well resistance to pepsion and trypsin,and were not sensitive to metal iron, surface active agent and chelating agent.The analysis of different xylans enzymatic hydrolysate revealed:By XYNBa,that the main constitutions of enzymatic hydrolysate of birch wood xylans were xylotriose and xyloquaiose,which account for 68.43% and 16.50% respectively,additionally there was 11.79% of xylobiose; The main constitutions
of enzymatic hydrolysate of corncobs xylans were xylobiose and xylotriose, which account for 81.78% and 11.55%.The result indicated that this xylanase was a kind of 1,4- b -D-xylanohydrolase and was fit to used in industrial procession of xylooligosacc harides.
引文
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