BackgroundNattokinases/Subtilisins (EC 3.4.21.62) belong to the second large family of serine proteases, which gain significant attention and play important role in many biotechnology processes. Thus, a number of nattokinases/subtilisins from various Bacillus species, especially from B. subtilis strains, extensively have been investigated to understand their biochemical and physical properties as well as to improve the production for industrial application. The purpose of this study was to clone a nattokinase gene from Bacillus subtilis strain VTCC-DVN-12-01, enhance its production in B. subtilis WB800, which is deficient in eight extracellular proteases and characterize its physicochemical properties for potential application in organic synthesis and detergent production.ResultsA gene coding for the nattokinase (Nk) from B. subtilis strain VTCC-DVN-12-01 consisted of an ORF of 1146 nucleotides, encoding a pre-pro-protein enzyme (30-aa pre-signal peptide, 76-aa pro-peptide and 275-aa mature protein with a predicted molecular mass of 27.7 kDa and pI 6.6). The nattokinase showed 98-99% identity with other nattokinases/subtilisins from B. subtilis strains in GenBank. Nk was expressed in B. subtilis WB800 under the control of acoA promoter at a high level of 600 mg protein per liter culture medium which is highest yield of proteins expressed in any extracellular-protease-deficient B. subtilis system till date. Nk was purified to homogeneity with 3.25 fold purification, a specific activity of 12.7 U/mg, and a recovery of 54.17%. The purified Nk was identified by MALDI-TOF mass spectrometry through three peptides, which showed 100% identity to corresponding peptides of the B. subtilis nattokinase (CAC41625). An optimal activity for Nk was observed at 65°C and pH 9. The nattokinase was stable at temperature up to 50°C and in pH range of 5–11 and retained more than 85% of its initial activity after incubation for 1 h. Mg2+ activated Nk up to 162% of its activity. The addition of Triton X-100, Tween 20, and Tween 80 showed an activation of Nk up to 141% of its initial activity but SDS strongly inhibited. The enzyme was highly resistant to organic solvents.ConclusionsOur findings demonstrated that an eight-protease-gene-deficient Bacillus subtilis WB800 could overproduce the nattokinase from B. subtilis VTCC-DVN-12-01. Due to high resistance to detergents and organic solvents of this nattokinase, it could be potentially applied in organic synthesis and detergent production.

Gram-negative Serratia marcescens is a potential biological control agent of fungal plant diseases in many tropical regions. Its antifungal activity is due to the production of prodigiosin, protease, chitinase, and other substances that act to weaken the fungal cell wall and cause lysis. In the following investigation, a novel extracellular protein with antifungal activity against Rhizoctonia solani and Fusarium oxysporum was optimally produced, purified, and characterized from a native Serratia marcescens DT3. Under optimal conditions (medium containing 1.25% peptone, 0.5% yeast extract; temperature of 28 °C; initial pH of 7; culture time of 28 h), the antifungal activity of the extracellular extract was increased by a factor of 1.6-2.9 in comparison with the initial condition. The purified protein had an apparent molecular mass of 55 kDa. Its antifungal activity was retained at 80 °C for 30 min and after having been treated with proteinase K (0.5-4 µg/mL). The results of protein identification using a MALDI-TOF/TOF mass spectrometer suggested that the purified protein is indeed a serine 3-dehydrogenase.