Cloning and prokaryotic expression of β-glucuronidase from Penicillium purpurogenum Li-3

One new gene of β-glucuronidase (EC 3.2.1.31) which could biosynthesize glycyrrhetinic acid monoglucuronide(GAMG)from glycyrrhizin,was cloned from Penicillium purpurogenum Li-3 by degenerated PCR using primers designed from the conserved amino acid sequences.It is the first time that a β-glucuronidase gene (pgus) (GenBank Accession No.EU095019) was cloned from Penicillium species other than Penicillium canescens.Sequence analysis indicated that the gene of pgus has 1815 base pairs,encoding 604 amino acids with the putative potential molecular weight of 66.7×103 and 4 potential N-glycosylation sites.The prokaryotic expression system was constructed with pET-28a (+),as the vector.The fusion protein(PGUS-E)with activity was overexpressed in E.coli BL21 (DE3) after IPTG induction.The specific activity of enzyme,purified with Ni2+-NTA column to homogeneity (accounting for 95% of soluble protein),was up to 6368 U·mg-1.The production of soluble PGUS-E was about 25.6 mg·L-1 culture medium.The molecular weight of sub-unit was estimated to be about 70×103 by SDS-PAGE.