Intracellular expression of Vitreoscilla hemoglobin (VHb) enhances total protein secretion and improves the production of alpha-amylase and neutral protease in Bacillus subtilis

In an attempt to alleviate oxygen limitation during batch cultivations, a heterologous bacterial hemoglobin gene (vhb) of Vitreoscilla was introduced into Bacillus subtilis. Biochemically active VHb, as demonstrated by immunoblot analysis and carbon monoxide binding assay, was intracellularly expressed in B. subtilis from an inducible promoter-repressor (spac-lacI). Expression of VHb in oxygen-limited B. subtilis batch bioreactor cultivations enhanced cell growth, decreased accumulation of acetate, and increased the total protein secreted into the culture medium by approximately 1.5-fold. In addition, VHb-expressing B. subtilis cultures exhibited increases of approximately 30% and 5-17% in neutral protease activity and alpha-amylase activity, respectively, relative to the parental, VHb-free strain.