作　　者： ; ; ; ; ;

机构地区： 清华大学化学工程系

出　　处： 《微生物学报》 2004年第3期336-339,共4页

摘　　要： 从三角酵母中提取总RNA ,反转录后进行PCR扩增得到D 氨基酸氧化酶 (D AminoAcidOxidase ,DAAO)基因 ,经测序可知 ,与文献中三角酵母的DAAO基因序列的同源性在 99%以上。将DAAO基因用NcoⅠ和BamHⅠ双酶切后 ,与相同酶切的大肠杆菌表达载体pET 2 8a连接 ,转化大肠杆菌TOP 1 0F′,并筛选得到重组质粒pET DAAO ,转化BL2 1 (DE3)感受态细胞 ,得到重组大肠杆菌BL2 1 (DE3) pET DAAO。对重组大肠杆菌中的D 氨基酸氧化酶进行了诱导表达 ,考察了诱导温度、菌浓度、诱导剂IPTG用量以及溶氧等因素对酶活的影响。结果表明 ,在 2 8℃、菌浓度 (OD6 0 0 ) 1 0、IPTG浓度 1mmol L时 ,DAAO酶活最高达 2 3 3U mL。研究进一步显示 ,用廉价无毒的乳糖可以替代IPTG进行诱导 ,当乳糖浓度为 2mmol L ,DAAO酶活可达 2 2 7U mL。经过补料分批培养和乳糖诱导 ,DAAO酶活可以达到 1 75U mL。 A gene of DAAO was obtained by isolating total RNA from Trignoposis variabilis and then amplified by reverse transcription (RT)-PCR. Comparing its nucleotide sequence with other DAAO genes from Trignoposis variabilis reported in literature, considerable homology (more than 99%) was observed. The DAAO gene, digested with NcoⅠ and BamHⅠ, was inserted into a prokaryotic expression vector, pET-28a. By colony-PCR method screening, a recombinant plasmid pET-DAAO was obtained and then transformed into the expression host BL21 (DE3). The influences of induction conditions such as IPTG concentration, the time of induction,the induction temperature and dissolved oxygen condition on expression of the recombinant protein were investigated. Under optimal condition, the enzyme activity could reach 23 3U/mL. The possibility of using lactose as an alternative inducer of IPTG was also studied. After induction of 2mmol/L lactose, the DAAO enzyme activity amounts to 22 7U/mL. A high DAAO activity of 175 U/mL was obtained by fed-batch culture and lactose induction.

关 键 词： 氨基酸氧化酶 三角酵母 大肠杆菌 乳糖

领　　域： [生物学]