Cloning and enhancing production of a detergent- and organic-solvent-resistant nattokinase from Bacillus subtilis VTCC-DVN-12-01 by using an eight-protease-gene-deficient Bacillus subtilis WB800

Abstract

Background: Nattokinases/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.

Results: A 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.

Conclusions: Our 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.

Figure 1

SDS-PAGE of expressed and purified Nk by ProBond™ Resin. A. Lane 1–5, culture supernatants; lane 6–9, cell lysates; lane 1,6, WB800/pAC7, lane 2–4, 7–9, WB800/pANk. Lane M, molecular standards indicated in kDa. B. Lane 1, purified Nk; Lane M, molecular standards indicated in kDa. The gel was stained by using Coomassie Briliant Blue R250. The band of lane 1 was cut off and used for LC-ESI-MS/MS analysis.

Figure 2

Monoisotopic mass of three neutral identified peptides. A. AQSVPYGISQIK position 1–12 (A); B. VAVIDSGIDSSHPDLNVR position 28–45; C. YPSTIAVGAVNSSNQR position 171–186 found in gi|14422313, nattokinase from Bacillus subtilis (GenBank, CAC41625) corresponding to ion scores of 35, 96 and 85 with p < 0.05, respectively. D. Alignment of three neutral identified peptides (Nk_Frag) with nattokinase from B. subtilis CAC41625 (gi14422313) and putative Nk from B. subtilis VTCC-DVN-12-01 (Nk).

Figure 3

Effect of temperature and pH. A. Temperature optimum and stability. B. pH optimum and stability. Temperature and pH optimum (■) and stability (□) of Nk from B. subtilis VTCC-DVN-12-01. For temperature and pH optimum, the activity of 0.6 μg of Nk was measured with 1 mM Suc-AAPF-pNA as a substrate at pH 8 and various temperatures 30-70°C and at 65°C and various pH 5 to 11, respectively. For thermal and pH stability, 0.6 μg of Nk was incubated in 50 mM Na-phosphate buffer at various temperatures 30-70°C for 1 h and in 50 mM acetate (pH 5), phosphate (pH 6–8) and Tris–HCl (pH 9–11) at 30°C for 1 h, respectively. The residual activity was determined at pH 9 and 65°C with 1 mM Suc-AAPF-pNA as a substrate.

Figure 4

Construction of expression plasmid. A. Vetor pAC7 map. B. Construction of expression cassette acoA-nk-T7. Fragment A: acoA promoter plus amyE, fragment B, subtilisin encoding gene plus T7 terminator; fragment C, acoA promoter + amyE + nk + T7 terminator. C. Starch hydrolysis of B. subtilis WB800 (1), the transformants B. subtilis WB800/pANk (2-3) and WB800/pAC7 (4).