INTRACELLULAR EXPRESSION OF VITREOSCILLA HEMOGLOBIN (VHB) ENHANCES TOTAL PROTEIN SECRETION AND IMPROVES THE PRODUCTION OF ALPHA-AMYLASE ANDNEUTRAL PROTEASE IN BACILLUS-SUBTILIS

In an attempt to alleviate oxygen limitation during batch cultivations , a heterologous bacterial hemoglobin gene (vhb) of Vitreoscilla was i ntroduced into Bacillus subtilis. Biochemically active VHb, as demonst rated by immunoblot analysis and carbon monoxide binding assay, was in tracellularly expressed in B. subtilis from an inducible promoter-repr essor (spac-lacI). Expression of VHb in oxygen-limited B. subtilis bat ch bioreactor cultivations enhanced cell growth, decreased accumulatio n of acetate, and increased the total protein secreted into the cultur e medium by approximately 1.5-fold. In addition, VHb-expressing B. sub tilis cultures exhibited increases of approximately 30% and 5-17% in n eutral protease activity and alpha-amylase activity, respectively, rel ative to the parental, VHb-free strain.