Medium Optimization for Penicillin Acylase (PAc) Production by Recombinant B. megaterium MS941 Containing pac Gene from B. thuringiensis BGSC BD1 Using Response Surface Methodology

Abstract

Penicillin G acylase (PAc) hydrolyses of the amide bond of benzylpenicillin (Pen-G) releasing PAA and 6-APA, key intermediate in the production of various semisynthetic penicillins. In this study, we optimised the production medium of PAc by RSM using two variables (xylose as inducer and CaCl 2 as divalent cations) to obtain the optimum PAc specific activity from Bacillus megaterium bt pac BD1. For this purpose, combinations of five different xylose concentrations (0.13 – 0.87 %) and five different CaCl 2 concentrations (0.64 – 4.36 mM) were analysed, in a total of 22 experiments. CCD used for the analysis showed that in shake flask cultivations, xylose and CaCl 2 showed significant effects on PAc volumetric activity and the quadratic model was in good agreement with the experimental results (R 2 = 0.86 (p-value < 0.0001)). The maximum specific activity (130.669 ± 50.241 units mg protein -1 ) was reached when xylose and CaCl 2 concentrations were 0.49% and 2.4 mM, respectively, and medium pH was around 7. Under such conditions, the activity of PAc and protein concentration achieved were 1.318 ± 0.406 units mL -1 and 0.0101 ± 0.01 mg mL -1 . The shake flask validation experiments demonstrated that with such medium composition the volumetric activity, protein concentration and specific activity achieved were 1.294 ± 0.171 units mL -1 , 0.0102 ± 0.0003 mg mL -1 and 125.91 ± 13.309 units mg -1 , respectively. When the optimum medium composition was applied in 10 L bioreactor, the optimum volumetric activity (2.0687 ± 0.0820 units mL -1 ) and protein concentration (0.0078 ± 0.0008 mg mL -1 ) were achieved 48 h after the start of the cultivation. However, the optimum PAc specific acivity (1260.52 ± 27.5711 units mg protein -1 ) was achieved 18 h after the start of the cultivation.