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

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 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 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 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 α-L-rhamnosidase resource.

Key words: α-L-rhamnosidase, catalytically active inclusion bodies, Pedobacter heparinus, heterologous expression