Effects of Induction Starting Time and Ca2+ on Expression of Active Penicillin G Acylase in Escherichia coli

Abstract

Formation of inclusion bodies is an important obstacle to the production of active recombinant protein in Escherichia coli. Thus, soluble expression of penicillin G acylase from Kluyvera citrophila was investigated in BL21(DE3). In this study, the yield of active enzyme was significantly enhanced by the composition of the medium and induction opportunity. When 0.5 mmol/L IPTG was added to complex medium at 15 h after incubation, the volumetric and specific activities of penicillin G acylase both achieved the highest values, respectively. However, aggravation of intracellular proteolysis and decline of enzyme expression were also observed if induction occurred too much later. Ca2+ ion was another critical factor in cell growth and protein expression. When 24 mmol/L Ca2+ ion was adding to the medium at the beginning of fermentation, a greater than 2-fold increase in cell density and a 7-fold increase in volumetric activity of penicillin G acylase were reached. Nevertheless, no significant benefit for recombination protein expression was found when excess Ca2+ was added after induction time. This study demonstrates that the induction starting time and Ca2+ ion are two critical factors for the expression of active penicillin G acylase.

Functional expression of a penicillin acylase from the extreme thermophile Thermus thermophilus HB27 in Escherichia coli

“…Regarding the auto-proteolytic processing of PAC, we observed that the size of both α- and β-subunits matched those of PAC obtained from Tth cells, suggesting a correct maturation of the pro-protein. Calcium proved to be a critical factor when producing functional Tth PAC, in the same way it was reported for the Eco PGA and Afae PGA [32,33]. The yield in active Tth PAC could be increased 13-fold when culture media was supplemented with 10 mM of CaCl 2 .…”

“…Calcium ions have been suggested to stabilize the PGA native state allowing its maturation to take place [30,31]. Indeed, the production of properly matured PGA proteins in E. coli cells has been improved in cultures supplemented with CaCl 2 [32,33]. Hence, the influence of Ca +2 ions on the expression and maturation of Tth PAC was tested.…”

Exploitation of E. coli for the production of penicillin G amidase: a tool for the synthesis of semisynthetic β-lactam antibiotics

“…The augmentation of translation can be done by increasing the stability of pac mRNA, modifying the region of ribosome binding site, etc., leading to a mature PGA giving higher levels of activity [ 15 , 51 ]. The pac genes from bacterial strains other than E. coli have also been heterologously expressed in E. coli like genes from Arthrobacter viscosus [ 52 ], B. megaterium [ 53 ], Achromobacter xylosoxidans [ 54 ], P. rettgeri [ 55 ], A. faecalis [ 56 ], K. cryocrescens [ 57 ], and Thermus thermophilus [ 43 ]. Their PGAs can surpass EcPGA when expressed in E. coli in terms of particular enzymatic properties like wide operation range, molecular stability, and environmental tolerance [ 2 ].…”

Protein Engineering of Penicillin Acylase

“…This approach allows to increase the portion of the periplasmic enzyme nearly 100-fold, relative to the intracellular enzyme. High enzyme yields were also achieved using the richest medium, YE, of all those studied (YE, TH, MR, M9), as described in [60]. …”

“…Ca 2+ is important both for cellular growth [61] and for the formation of active soluble PA, since calcium ions are present in molecules of active PA [24,60]. According to some authors, Ca 2+ plays an important role in the translocation of the protein precursor across the membrane, as well as in the correct folding and maturation of the enzyme in the periplasm [31].…”