High level expression of a recombinant phospholipase C from Bacillus cereus in Bacillus subtilis

Durban, Markus A.; Silbersack, Jörg; Schweder, Thomas; Schauer, Frieder; Bornscheuer, Uwe T.

doi:10.1007/s00253-006-0712-z

Cited by 41 publications

(24 citation statements)

References 21 publications

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“…For nattokinases/subtilisins, several efforts have been invested to enhance the production of recombinant proteins by elimination of limiting factors, using expression vectors with high structure stability, medium optimization using response surface methodology, and promoter optimization [5]. More efficiently, B. subtilis strains were engineered to serve as extracellular-protease-deficient strains for the overproduction of heterologous proteins such as fibrinolytic enzyme/nattokinase/subtilisin in B. subtilis WB600 [6-8], xylanase, interleukin 3, staphylokinase in B. subtilis WB700 [9-11] and phospholipase C, interleukin 3, xylanase in B. subtilis WB800 [10-12]. …”

Section: Introductionmentioning

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: Introductionmentioning

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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“…All chemicals were obtained at the highest purity available from common commercial suppliers. The PLC used was isolated from the culture supernatant of the B. cereus strain 516 (provided by Prof. F. Schauer, Institute of Microbiology, Greifswald University) as described [13].…”

Section: Methodsmentioning

confidence: 99%

An improved assay for the determination of phospholipase C activity

Durban

Bornscheuer

2007

Euro J Lipid Sci & Tech

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Phospholipases C (PLC, EC 3.1.4.3) are enzymes that specifically hydrolyze the C-O-P bond in phospholipids, yielding sn-1,2(2,3)-diacylglycerides and the phosphate residue bearing the corresponding headgroup. The biochemical characterization of PLC requires methods for the reliable determination of their activity. Here, an assay is described in which the phosphate residue released by the PLC is cleaved with an alkaline phosphatase. The phosphate formed is then extracted with n-butanol and quantified as phosphomolybate complex. The applicability of this method is demonstrated for a concentration range from 10 nM to 10 mM for a range of phospholipids bearing different headgroups in an aqueous and a two-phase system. The method has the additional advantage that the crude enzyme can be used without the need for purification.

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“…Also our group discovered several PLCs after screening of different Bacillus sp. strains that could be functionally expressed in Bacillus subtilis for use in degumming [73,74]. Concomitantly, an assay for the reliable determination of their activity was developed in which the phosphoester residue released by the PLC is cleaved with an alkaline phosphatase.…”

Section: Phospholipasesmentioning

confidence: 99%

Engineering and application of enzymes for lipid modification, an update

Zorn

Oroz‐Guinea

Brundiek³

et al. 2016

Progress in Lipid Research

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This review first provides a brief introduction into the most important tools and strategies for protein engineering (i.e. directed evolution and rational protein design combined with high-throughput screening methods) followed by examples from literature, in which enzymes have been optimized for biocatalytic applications. This covers engineered lipases with altered fatty acid chain length selectivity, fatty acid specificity and improved performance in esterification reactions. Furthermore, recent achievements reported for phospholipases, lipoxygenases, P450 monooxygenases, decarboxylating enzymes, fatty acid hydratases and the use of enzymes in cascade reactions are treated.

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High level expression of a recombinant phospholipase C from Bacillus cereus in Bacillus subtilis

Cited by 41 publications

References 21 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

An improved assay for the determination of phospholipase C activity

Engineering and application of enzymes for lipid modification, an update

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