α-L-鼠李糖苷酶以催化活性包涵体形式异源表达及其酶学性质
合集下载
- 1、下载文档前请自行甄别文档内容的完整性，平台不提供额外的编辑、内容补充、找答案等附加服务。
- 2、"仅部分预览"的文档,不可在线预览部分如存在完整性等问题,可反馈申请退款(可完整预览的文档不适用该条件!)。
- 3、如文档侵犯您的权益，请联系客服反馈,我们会尽快为您处理(人工客服工作时间：9:00-18:30)。
Heterologous Expression and Enzymatic Properties of α-L-Rhamnosidase as Catalytically Active Inclusion Bodies
LI Binchun, ZHANG Tian, JI Yaru, LI Yanqin, DING Guobin (Key Laboratory of Chemical Biology and Molecular Engineering, Ministry of Education, Institute of Biotechnology,
and 0.39 g/g bacteria, respectively. Enzymatic activity assay showed that both insoluble inclusion bodies could catalyze the
hydrolysis of p-nitrophenol-α-L-rhamnopyranoside (PNPR). These results revealed that PhRha78E and PhRha78G were
successfully expressed heterologously as catalytically active inclusion bodies (CatIBs) in E. coli. The optimum pH valuess of
PhRha78E and PhRha78G CatIBs were 6.5 and 7.0, respectively. PhRha78E maintained 62% activity at acidic pH 4.8, while
※生物工程
食品科学
2018, Vol.39, No.14 79
α-L-鼠李糖苷酶以催化活性包涵体形式异源 丁国斌
（山西大学生物技术研究所，化学生物学与分子工程教育部重点实验室，山西 太原 030006）
摘 要：从解肝磷脂土地杆菌（Pedobacter heparinus）基因组DNA扩增获得两个α-L-鼠李糖苷酶基因PhRha78E和 PhRha78G，构建于表达载体pET-28a；将重组表达质粒转入大肠杆菌BL21（DE3）进行异源表达，PhRha78E和 PhRha78G以不溶性包涵体形式大量表达于沉淀中，每克菌体可分别获得0.42 g和0.39 g含目的酶包涵体的沉淀。酶 活实验显示二者的不溶性包涵体具有催化活性，可以催化水解底物对硝基苯酚-α-L-吡喃鼠李糖苷（p-nitrophenolα-L-rhamnopyranoside，PNPR），表明二者在大肠杆菌中以催化活性包涵体形式异源表达。PhRha78E和PhRha78G 的最适反应pH值相近，分别为6.5和7.0，PhRha78E在酸性pH 4.8仍能保持62%酶活力，而PhRha78G在碱性pH 8.6 依然保持72%酶活力；PhRha78E和PhRha78G的最适反应温度却相差很大，分别为60 ℃和40 ℃，PhRha78E在高温 70 ℃条件下仍能保持69%酶活力，而PhRha78G在低温20 ℃条件下仍有43%酶活力；动力学常数kcat分别为0.18 s－1和 0.12 s－1，Km分别为0.55 mmol/L和0.40 mmol/L。本研究揭示新型α-L-鼠李糖苷酶PhRha78E和PhRha78G在大肠杆菌 中以催化活性包涵体形式异源表达，蛋白表达量大，二者酶学性质差异较大，可应用于不同的生物催化领域，并丰 富了现有α-L-鼠李糖苷酶资源库。 关键词：α-L-鼠李糖苷酶；催化活性包涵体；解肝磷脂土地杆菌；异源表达
were transformed into Escherichia coli BL21(DE3) for heterologous expression, respectively. PhRha78E and PhRha78G were
expressed in the precipitate as the form of insoluble inclusion bodies. The production of target inclusion bodies were 0.42 g/g
Shanxi University, Taiyuan 030006, China)
Abstract: Two α-L-rhamnosidase genes, PhRha78E and PhRha78G, were amplified from the genome of Pedobacter
heparinus by polymerase chain reaction (PCR), and ligated into the expression vector pET-28a. The recombinant plasmids
PhRha78G retained 72% activity at alkaline pH 8.6. The optimum temperatures of PhRha78E and PhRha78G CatIBs differed remarkably, which were 60 and 40 ℃, respectively. PhRha78E retained 69% activity at high temperature (70 ℃), but PhRha78G displayed 43% activity at low temperature (20 ℃). The enzyme kinetic parameters kcat of both α-L-rhamnosidases were 0.18 and 0.12 s-1, respectively, and Km values were 0.55 and 0.40 mmol/L, respectively. In this study, the two enzymes had different properties and could be used in different biotransformation fields to enrich
LI Binchun, ZHANG Tian, JI Yaru, LI Yanqin, DING Guobin (Key Laboratory of Chemical Biology and Molecular Engineering, Ministry of Education, Institute of Biotechnology,
and 0.39 g/g bacteria, respectively. Enzymatic activity assay showed that both insoluble inclusion bodies could catalyze the
hydrolysis of p-nitrophenol-α-L-rhamnopyranoside (PNPR). These results revealed that PhRha78E and PhRha78G were
successfully expressed heterologously as catalytically active inclusion bodies (CatIBs) in E. coli. The optimum pH valuess of
PhRha78E and PhRha78G CatIBs were 6.5 and 7.0, respectively. PhRha78E maintained 62% activity at acidic pH 4.8, while
※生物工程
食品科学
2018, Vol.39, No.14 79
α-L-鼠李糖苷酶以催化活性包涵体形式异源 丁国斌
（山西大学生物技术研究所，化学生物学与分子工程教育部重点实验室，山西 太原 030006）
摘 要：从解肝磷脂土地杆菌（Pedobacter heparinus）基因组DNA扩增获得两个α-L-鼠李糖苷酶基因PhRha78E和 PhRha78G，构建于表达载体pET-28a；将重组表达质粒转入大肠杆菌BL21（DE3）进行异源表达，PhRha78E和 PhRha78G以不溶性包涵体形式大量表达于沉淀中，每克菌体可分别获得0.42 g和0.39 g含目的酶包涵体的沉淀。酶 活实验显示二者的不溶性包涵体具有催化活性，可以催化水解底物对硝基苯酚-α-L-吡喃鼠李糖苷（p-nitrophenolα-L-rhamnopyranoside，PNPR），表明二者在大肠杆菌中以催化活性包涵体形式异源表达。PhRha78E和PhRha78G 的最适反应pH值相近，分别为6.5和7.0，PhRha78E在酸性pH 4.8仍能保持62%酶活力，而PhRha78G在碱性pH 8.6 依然保持72%酶活力；PhRha78E和PhRha78G的最适反应温度却相差很大，分别为60 ℃和40 ℃，PhRha78E在高温 70 ℃条件下仍能保持69%酶活力，而PhRha78G在低温20 ℃条件下仍有43%酶活力；动力学常数kcat分别为0.18 s－1和 0.12 s－1，Km分别为0.55 mmol/L和0.40 mmol/L。本研究揭示新型α-L-鼠李糖苷酶PhRha78E和PhRha78G在大肠杆菌 中以催化活性包涵体形式异源表达，蛋白表达量大，二者酶学性质差异较大，可应用于不同的生物催化领域，并丰 富了现有α-L-鼠李糖苷酶资源库。 关键词：α-L-鼠李糖苷酶；催化活性包涵体；解肝磷脂土地杆菌；异源表达
were transformed into Escherichia coli BL21(DE3) for heterologous expression, respectively. PhRha78E and PhRha78G were
expressed in the precipitate as the form of insoluble inclusion bodies. The production of target inclusion bodies were 0.42 g/g
Shanxi University, Taiyuan 030006, China)
Abstract: Two α-L-rhamnosidase genes, PhRha78E and PhRha78G, were amplified from the genome of Pedobacter
heparinus by polymerase chain reaction (PCR), and ligated into the expression vector pET-28a. The recombinant plasmids
PhRha78G retained 72% activity at alkaline pH 8.6. The optimum temperatures of PhRha78E and PhRha78G CatIBs differed remarkably, which were 60 and 40 ℃, respectively. PhRha78E retained 69% activity at high temperature (70 ℃), but PhRha78G displayed 43% activity at low temperature (20 ℃). The enzyme kinetic parameters kcat of both α-L-rhamnosidases were 0.18 and 0.12 s-1, respectively, and Km values were 0.55 and 0.40 mmol/L, respectively. In this study, the two enzymes had different properties and could be used in different biotransformation fields to enrich