A novel esterase from Ralstonia sp. M1: Gene cloning, sequencing, high-level expression and characterization

Quyen, Dinh Thi; Dao, Thi Tuyet; Nguyen, Sy Le Thanh

doi:10.1016/j.pep.2006.06.009

Cited by 21 publications

(20 citation statements)

References 33 publications

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“…Genomic and plasmid DNA isolation was carried out by the method as previously described [36]. DNA fragments and PCR products were excised from a 0.8% agarose gel and purified by a gel extraction kit (Qiagen, Venlo, The Netherlands) according to manufacturer’s instruction.…”

Section: Methodsmentioning

confidence: 99%

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

Nguyen

Quyen

2013

Microb Cell Fact

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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.

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Section: Methodsmentioning

confidence: 99%

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

Nguyen

Quyen

2013

Microb Cell Fact

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“…The optimal reaction temperature reported here falls into the range of optimum temperatures observed in some thermophilic esterases such as those from Ralstonia sp. M1 or Thermotoga maritima (tm0053) (Kakugawa, Fushinobu, Wakagi, & Shoun, 2007;Quyen, Dao, & Nguyen, 2007). The Arrhenius plot of the data (Fig.…”

Section: Influence Of Temperature On Esterase Activity and Stabilitymentioning

confidence: 99%

“…The pH range of activity and stability of the esterase from B. licheniformis S-86 is clearly on the alkaline side, similar to that for some alkalo-stable esterases, such as the enzymes from Ralstonia sp. M1 (Quyen et al, 2007), B. circulans (Kademi et al, 2000) and Kluyveromyces marxianus CBS 1553 (Monti, Ferrandi, Righi, Romano, & Molinari, 2008).…”

Section: Influence Of Ph On Esterase Activity and Stabilitymentioning

confidence: 99%

Enzymatic synthesis of banana flavour (isoamyl acetate) by Bacillus licheniformis S-86 esterase

Torres

Baigorí

Swathy

et al. 2009

Food Research International

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“…Genomic and plasmid DNA isolation was carried out by methods which have been previously described [15]. DNA fragments and PCR products were excised from a 0.8% agarose gel and purified by a gel extraction kit (Qiagen, Venlo, The Netherlands) according to the manufacturer's instructions.…”

Section: Methodsmentioning

confidence: 99%

“…DNA sequencing was performed on an ABI PRISM 3100 Avant Genetic Analyzer (Applied Biosystems Inc., Foster City, USA). E. coli DH5 α and BL21 were transformed using heat shock method that has been previously described [15]. …”

Section: Methodsmentioning

confidence: 99%

Highly Effective Renaturation of a Streptokinase fromStreptococcus pyogenesDT7 as Inclusion Bodies Overexpressed inEscherichia coli

Nguyen

Quyen

2014

BioMed Research International

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The streptokinase (SK) is emerging as an important thrombolytic therapy agent in the treatment of patients suffering from cardiovascular diseases. We reported highly effective renaturation of a SK from S. pyogeness DT7 overexpressed in E. coli, purification, and biochemical characterization. A gene coding for the SK was cloned from S. pyogeness DT7. Because accumulation of active SK is toxic to the host cells, we have expressed it in the form of inclusion bodies. The mature protein was overexpressed in E. coli BL21 DE3/pESK under the control of the strong promoter tac induced by IPTG with a level of 60% of the total cell proteins. The activity of the rSK, renatured in phosphate buffer supplemented with Triton X-100 and glycerol, was covered with up to 41 folds of its initial activity. The purified of protein was identified with MALDI-TOF mass spectrometry through four peptide fragments, which showed 100% identification to the corresponding peptides of the putative SK from GenBank. Due to overexpression and highly effective renaturation of large amounts of inclusion bodies, the recombinant E. coli BL21 DE3/pESK system could be potentially applied for large-scale production of SK used in the therapy of acute myocardial infarction.

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A novel esterase from Ralstonia sp. M1: Gene cloning, sequencing, high-level expression and characterization

Cited by 21 publications

References 33 publications

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

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

Enzymatic synthesis of banana flavour (isoamyl acetate) by Bacillus licheniformis S-86 esterase

Highly Effective Renaturation of a Streptokinase fromStreptococcus pyogenesDT7 as Inclusion Bodies Overexpressed inEscherichia coli

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