长野芽胞杆菌(Bacillus naganoensis)普鲁兰酶在大肠杆菌中的活性表达与分泌调控

Expression and secretion regulation of Bacillus naganoensis pullulanase in recombinant Escherichia coli

摘要:

摘要:【目的】从长野芽胞杆菌(Bacillus naganoensis)JNB-1中克隆出普鲁兰酶基因并在大肠杆菌系统中表达，通过优化诱导条件和使用化学添加剂提高了胞外酶活。【方法】采用PCR方法，从B.naganoensis 基因组中扩增出普鲁兰酶基因pul，构建重组菌E.coli BL21/pET-20b-pul。通过优化，确定优化后的IPTG诱导条件以及甘氨酸、Na+的最佳添加参数。【结果】普鲁兰酶在大肠杆菌中得到有效表达，其相对分子质量为119kDa。在优化后的诱导条件(诱导温度20℃，IPTG 终浓度0.4 mmol/L，在菌体OD600至1. 2时诱导)下，普鲁兰酶的总酶活达到10.8U/mL。添加甘氨酸和Na+均能有效促进普鲁兰酶的分泌。在诱导时添加终浓度0.08 mol/L的甘氨酸和0.2 mol/L Na+，胞外酶活提高至8.1 U/mL，是不加任何添加剂的10.3倍。【结论】该重组菌的构建为普鲁兰酶制剂的工业生产提供了有价值的菌株，对化学添加剂促进分泌的研究也为重组酶的高水平胞外生产提供了有效的方法。

Abstract:

Abstract:［Objective］To increase extracellular productivityof pullulanase，pullulanase gene from Bacillus naganoensis JNB-1 was expressed in recombinant Escherichia coli，followed by optimizing induction conditions and applying chemical additives．［Methods］ We amplified pullulanase gene pul from B．naganoensis genome by PCR and constructed recombinant E.coli BL21/pET-20b-pul． Optimal induction conditions and additive parameters of glycine and Na + were determined by measuring the extracellular pullulanase activity． ［Results］Pullulanase was expressed in E． coli with the molecular weight of 119 kDa． Under optimal induction conditions，i．e．induction was initiated with 0.4 mmol/L isopropyl β-D-1-Thiogalactopyranoside (IPTG) at 20℃ when OD600 of bacteria culture reached 1.2，total pullulanase activity including intracellular and extracellular enzyme reached 10.8 U/mL．Addition of glycine and Na+ enhanced the secretion of pullulanase．With the supplementation of 0.08 mol /L glycine and 0.2 mol/L Na+ ，extracellular pullulanase activity was increased up to 8.1 U/mL，10.3 times of that without additives． ［Conclusion］A promising resource of pullulanase was achieved by construction of recombinant E．coli for industrial production of pullulanase，and additionally the efficient regulation method with chemical additives was developed for pullulanase secretion，which would also be useful for highlevel extracellular production of recombinant enzymes.