Inducible and constitutive expression of glutaryl-7-aminocephalosporanic acid acylase by fusion to maltose-binding protein

Glutaryl-7-aminocephalosporanic acid acylase (GL-7-ACA acylase, GLA) from Pseudomonas was expressed by E.coli BL21(DE3)/pET- Acy grown in a 5 L fermentor. The activity accumulation of GLA stopped at 11 h due to the formation of the excessive insoluble and inactive precursor. Therefore, maltose-binding protein (MBP) was introduced to improve the solubility of the target enzyme. Recombinant E. coli TB1/pMAL- Acy and TB1/pMKL- Acy, in which the MBP fusion GLA (MBP-GLA) were inducible expression, as well as recombinant E. coli TB1/pMKC- Acy and TB1/pMKC- AS, in which MBP-GLA were constitutive expression by deleting the gene lac I q, were constructed to assess the function of MBP, respectively. Results showed that the ratio of soluble GLA to total GLA increased from approximately 15–60% to 75–100%, when E. coli BL21 (DE3)/pET- Acy was substituted by TB1/pMKL- Acy grown at 37–28 °C. Meanwhile, MBP-GLA of almost completely soluble form could be harvested in both constitutive expression strains E. coli TB1/pMKC- Acy and TB1/pMKC- AS grown at 28 °C. It can be inferred that MBP-GLAs are more soluble than single GLA in both inducible and constitutive expression. Further researches on constitutive expression of MBP-GLA were performed and the optimal activity of acylase in recombinant E. coli TB1/pMKC- AS reached 4682 U/L at 28 °C in a 5 L fermentor after 31 h culture, in the absence of any inducer. The productivity of the functional active fusion GLA reached as high as 151.0 U/(L h), which is very promising in the industrial enzymatic production of 7-aminocephalosporanic acid.