CAJ | 학술논문

借助生物信息学对已克隆的枯草杆菌脂肪酶LipB2全长基因序列进行比对分析.结果显示该脂肪酶基因全长635 bp,编码包括31个氨基酸分泌型信号肽在内的211个氨基酸,与NCBI GenBank中已报道的枯草杆菌属脂肪酶核苷酸序列有94.0%的一致性.将该基因克隆到pET-28a(+)表达载体上,转化大肠杆菌BL21(DE3),利用枯草杆菌脂肪酶的信号肽序列进行了分泌表达.SDS-PAGE电泳显示分泌表达的脂肪酶分子质量约为21 kD.对表达条件优化后,在30℃、大肠杆菌菌液OD_(600)值为1 8、乳糖诱导浓度为1 5 mM、摇瓶发酵10 h后大肠杆菌分泌表达26 0 U/mL重组脂肪酶,相比较IPTG的诱导,既实现了脂肪酶的高效表达,又节省了成本.
차조생물신식학대이극륭적고초간균지방매LipB2전장기인서렬진행비대분석.결과현시해지방매기인전장635 bp,편마포괄31개안기산분비형신호태재내적211개안기산,여NCBI GenBank중이보도적고초간균속지방매핵감산서렬유94.0%적일치성.장해기인극륭도pET-28a(+)표체재체상,전화대장간균BL21(DE3),이용고초간균지방매적신호태서렬진행료분비표체.SDS-PAGE전영현시분비표체적지방매분자질량약위21 kD.대표체조건우화후,재30℃、대장간균균액OD_(600)치위1 8、유당유도농도위1 5 mM、요병발효10 h후대장간균분비표체26 0 U/mL중조지방매,상비교IPTG적유도,기실현료지방매적고효표체,우절성료성본.
Bioinformatics analysis results showed that, the sequence length of the target lipase gene is 635 bp, encoding 211 AA which contain 31 AA secretion signal peptide,with the indentify of 94.0% by comparing with Bacillus subtili lipase reported in NCBI GenBank. The lipase gene was inserted into the expression vector pET-28a(+), which was finally expressed in Escherichia coli BL21(DE3) by its secretory signal peptide. SDS-PAGE detected the molecular weight of lipase was 21 kD. With the optimal induction temperature 30℃, lactose concentration 1.5 mmol/L, strain density OD_(600) 1.8 and induction time 10 h, the specific activity of the recombinant lipase was up to 26.0 U/mL, whcich showed the high efficiency and low cost compare with IPTG induction.