Overexpression of serine alkaline protease encoding gene in Bacillus species: performance analyses

Bacillus species carrying subC gene encoding serine alkaline protease (SAP) enzyme were developed in order to increase the yield and selectivity in the bioprocess for SAP production. For this aim, subC gene was cloned into pHV1431 Escherichia coli–Bacillus shuttle vector, and transferred into nine host Bacillus species, i.e. B. alvei, B. amyloliquefaciens, B. badius, B. cereus, B. coagulans, B. firmus, B. licheniformis, B. sphaericus and B. subtilis. The influence of the host Bacillus species on SAP production on a defined medium with glucose was investigated in bioreactor systems. For each of the recombinant (r-) Bacillus species, effects of initial glucose concentration on cell growth and SAP production were investigated; and, physiological differences and similarities between the wild-type and r-Bacillus species are discussed. The highest biomass concentration was obtained with r-B. coagulans as 3.8 kg m⁻³ at the initial glucose concentration of C_Go=20 kg m⁻³ and the highest volumetric SAP activity was obtained with r-B. amyloliquefaciens as 1650 U cm⁻³ at C_Go=20 kg m⁻³. Overall SAP activity per amount of substrate consumed was the highest for r-B. sphaericus (137 U g⁻¹ cm⁻³) and r-B. licheniformis (130 U g⁻¹ cm⁻³). Among the r-Bacillus species the highest activity increase compared to the wild types was obtained with r-B. sphaericus while the lowest increase was obtained with r-B. amyloliquefaciens and r-B. licheniformis due to high SAP production potential of the wild-type strains. During storage of the host microorganisms, r-B. alvei and r-B. amyloliquefaciens were not able to bear the recombinant plasmid, probably, due to the restriction enzymes synthesized. Due to the highest stable volumetric activities r-B. licheniformis (950 U cm⁻³) and r-B. sphaericus (820 U cm⁻³) appear to be the favorable hosts for the production of SAP. All the r-Bacillus species excreted organic acids oxaloacetic and succinic acids, but, none excreted the amino acid valine. The variations in by-product distributions with each recombinant organism were also discussed.