Dataset Information

High-level extracellular production of recombinant nattokinase in Bacillus subtilis WB800 by multiple tandem promoters.

ABSTRACT:

Background

Nattokinase (NK), which is a member of the subtilisin family, is a potent fibrinolytic enzyme that might be useful for thrombosis therapy. Extensive work has been done to improve its production for the food industry. The aim of our study was to enhance NK production by tandem promoters in Bacillus subtilis WB800.

Results

Six recombinant strains harboring different plasmids with a single promoter (P_P43, P_HpaII, P_BcaprE, P_gsiB, P_yxiE or P_luxS) were constructed, and the analysis of the fibrinolytic activity showed that P_P43 and P_HpaII exhibited a higher expression activity than that of the others. The NK yield that was mediated by P_P43 and P_HpaII reached 140.5?±?3.9 FU/ml and 110.8?±?3.6 FU/ml, respectively. These promoters were arranged in tandem to enhance the expression level of NK, and our results indicated that the arrangement of promoters in tandem has intrinsic effects on the NK expression level. As the number of repetitive P_P43 or P_HpaII increased, the expression level of NK was enhanced up to the triple-promoter, but did not increase unconditionally. In addition, the repetitive core region of P_P43 or P_HpaII could effectively enhance NK production. Eight triple-promoters with P_P43 and P_HpaII in different orders were constructed, and the highest yield of NK finally reached 264.2?±?7.0 FU/ml, which was mediated by the promoter P_HpaII-P_HpaII-P_P43. The scale-up production of NK that was promoted by P_HpaII-P_HpaII-P_P43 was also carried out in a 5-L fermenter, and the NK activity reached 816.7?±?30.0 FU/mL.

Conclusions

Our studies demonstrated that NK was efficiently overproduced by tandem promoters in Bacillus subtilis. The highest fibrinolytic activity was promoted by P_HpaII-P_HpaII-P_P43, which was much higher than that had been reported in previous studies. These multiple tandem promoters were used successfully to control NK expression and might be useful for improving the expression level of the other genes.

SUBMITTER: Liu Z

PROVIDER: S-EPMC6505213 | biostudies-literature | 2019 May

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

High-level extracellular production of recombinant nattokinase in Bacillus subtilis WB800 by multiple tandem promoters.

Liu Zhongmei Z Zheng Wenhui W Ge Chunlei C Cui Wenjing W Zhou Li L Zhou Zhemin Z

BMC microbiology 20190507 1

<h4>Background</h4>Nattokinase (NK), which is a member of the subtilisin family, is a potent fibrinolytic enzyme that might be useful for thrombosis therapy. Extensive work has been done to improve its production for the food industry. The aim of our study was to enhance NK production by tandem promoters in Bacillus subtilis WB800.<h4>Results</h4>Six recombinant strains harboring different plasmids with a single promoter (P<sub>P43</sub>, P<sub>HpaII</sub>, P<sub>BcaprE</sub>, P<sub>gsiB</sub>, ...[more]

PMID: 31064343

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Project description:BackgroundEven though tofu is a traditional Chinese food loved by Asian people the wastewater generated during the production of tofu can pollute the environment, and the treatment of this generated wastewater can increase the operating cost of the plant. In this study, the production of nattokinase could be achieved by using the nitrogen source in tofu processing wastewater (TPW) instead of using the traditional nattokinase medium. This meets the need for the low-cost fermentation of nattokinase and at the same time addresses the environmental pollution concerns caused by the wastewater. Bacillus subtilis 13,932 is, a high yielding strain of nattokinase, which is stored in our laboratory. To increase the activity of nattokinase in the tofu process wastewater fermentation medium, the medium components and culture parameters were optimized. Nattokinase with high enzymatic activity was obtained in 7 L and 100 L bioreactors when TPW was used as the sole nitrogen source catalyzed by Bacillus subtilis. Such a result demonstrates that the production of nattokinase from TPW fermentation using B. subtilis can be implemented at an industrial level.ResultsThe peptide component in TPW is a crucial factor in the production of nattokinase. Box-Behnken design (BBD) experiments were designed to optimize various critical components, i.e., Glucose, TPW, MgSO4·7H2O, CaCl2, in nattokinase fermentation media. A maximum nattokinase activity was recorded at 37 °C, pH 7.0, 70 mL liquid medium, and 200 rpm. The highest nattokinase activities obtained from 7 to 100 L bioreactors were 8628.35 ± 113.87 IU/mL and 10,661.97 ± 72.47 IU/mL, respectively.ConclusionsBy replacing the nitrogen source in the original medium with TPW, there was an increase in the enzyme activity by 19.25% after optimizing the medium and culture parameters. According to the scale-up experiment from conical flasks to 100 L bioreactors, there was an increase in the activity of nattokinase by 47.89%.

Project description: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.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.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.

Dataset Information

High-level extracellular production of recombinant nattokinase in Bacillus subtilis WB800 by multiple tandem promoters.

Background

Results

Conclusions

Publications

High-level extracellular production of recombinant nattokinase in Bacillus subtilis WB800 by multiple tandem promoters.

Similar Datasets

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