Inorganic compounds dual effect on recombinant protein production: influence of anlons and cations on serine alkaline protease production

摘要

Aims: Investigation of concerted effects of cations, i.e. Mg~(2+) and Mn~(2+), in combination with their anions, i.e. sulphate, chloride and acetate (Ac), on the physiology of Bacillus lichenifonnis carrying pHV1431::rubC to improve the fermentation medium for serine alkaline protease (SAP) production, whereupon, determination of the acid that can be used in pH control. Methods and Results: The cell concentrations increased with the increase in MnSO_4 and Mn(Ac)_2 concentrations, and the highest values were obtained at C_(MnSO_4)~o = 0·20 mmol l~(-1) and C_(Mn(CH_3COO)_2)~o = 4·0 mmol l~(-1), as 2·3 and 2·2 g l~(-1), respectively. However, C_(MnCl_2)~o did not influence biomass concentration. SAP production was inhibited with MnCl_2 after C_(MnCl_2) = 0·60 mmol l~(-1) but with MnSO_4 SAP production was inhibited drastically. Whereas, at high concentrations of Mn(Ac)_2 SAP production increased and the highest activity was obtained as A_(SAP) = 1285 U ml~(-1) at t = 65 h. With the Mg compounds, cell concentrations increased with the increase in the concentrations of MgSO_4, MgCl_2 and Mg(Ac)_2; and the anions did not show any influence on the cell growth. Similar to the results of Mn compounds, the glucose consumption rate increased with the increase in MgSO_4 and MgCl_2 concentrations; contrariwise, decreased with the increase in Mg(Ac)_2 concentrations, due to the use of acetate as the second carbon source. C_(MgSO_4)~o = 0·40 mmol l~(-1), C_(MgCl_2)~o = 1·60 mmol l~(-1) and C_(Mg(Ac)_2)~o = 0-40 mmol l~(-1) were the optimum concentrations separately, and the highest SAP activity was obtained with Mg(Ac)_2 as ASfiP = 1338 U ml~(-1) at t = 47 h. Consequently, ion acetate and its acid HAc appear, respectively, as the superior anion for the essential cations and the control agent for pH control in the bioreactor. Finally, optimum initial concentrations and the concerted effects of Mg(Ac)_2 and Mn(Ac)_2 were investigated, and the optimum concentrations were found respectively as 0·40 and 0·80 mmol l~(-1), while the maximum activity was obtained as A_(SAP) = 1010 U ml~(-1) at a shortened cultivation time of t = 39 h. Conclusions: Mn(Ac)_2 and Mg(Ac)_2 together enhanced the cell formation and SAP synthesis rates, moreover, SAP synthesis started at an earlier cultivation time. Significance and Impact of the Study: Each inorganic compound with its cation and anion has dual effect on the metabolism. Mg~(2+) and Mn~(2+) at their specific concentrations influence the regulation of the pathways that might cause better coupling of supply and demand for the amino acids on the basis of the amino acid composition of the enzyme molecule.