Inorganic compounds have dual effect on recombinant protein production: influence of anions and cations on serine alkaline protease production.
OBJECTIVE
Investigation of concerted effects of cations, i.e. Mg2+ and Mn2+, in combination with their anions, i.e. sulphate, chloride and acetate (Ac), on the physiology of Bacillus licheniformis carrying pHV1431::subC to improve the fermentation medium for serine alkaline protease (SAP) production, whereupon, determination of the acid that can be used in pH control.


RESULTS
The cell concentrations increased with the increase in MnSO4 and Mn(Ac)2 concentrations, and the highest values were obtained at Co(MnSO4) = 0.20 mmol l-1 and Co(Mn(CH3COO)2) = 4.0 mmol l-1, as 2.3 and 2.2 g l-1, respectively. However, Co(MnCl2) did not influence biomass concentration. SAP production was inhibited with MnCl2 after Co(MnCl2) = 0.60 mmol l-1, but with MnSO4 SAP production was inhibited drastically. Whereas, at high concentrations of Mn(Ac)2 SAP production increased and the highest activity was obtained as ASAP = 1285 U ml-1 at t = 65 h. With the Mg compounds, cell concentrations increased with the increase in the concentrations of MgSO4, MgCl2 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 MgSO4 and MgCl2 concentrations; contrariwise, decreased with the increase in Mg(Ac)2 concentrations, due to the use of acetate as the second carbon source. Co(MgSO4) = 0.40 mmol l-1, Co(MgCl2) = 1.60 mmol l-1 and Co(Mg(Ac)2) = 0.40 mmol l-1 were the optimum concentrations separately, and the highest SAP activity was obtained with Mg(Ac)2 as ASAP = 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 ASAP = 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.


CONCLUSIONS
Each inorganic compound with its cation and anion has dual effect on the metabolism. Mg2+ and Mn2+ 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.
Calik
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P
Industrial Biotechnology Laboratory, Department of Chemical Engineering, Middle East Technical University, Ankara, Turkey. pcalik@metu.edu.tr
Bilir
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Ozçelik
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Calik
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Ozdamar
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D001407:Bacillus / Q000187:drug effects* / Q000378:metabolism; D018533:Biomass; D019149:Bioreactors; D003470:Culture Media; D004305:Dose-Response Relationship, Drug; D005285:Fermentation / Q000187:drug effects; D017616:Magnesium Compounds / Q000494:pharmacology*; D017895:Manganese Compounds / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000096:biosynthesis; D012697:Serine Endopeptidases / Q000096:biosynthesis*
D003470:Culture Media; D017616:Magnesium Compounds; D017895:Manganese Compounds; D011994:Recombinant Proteins; D012697:Serine Endopeptidases