Molecular and structural characterization of a surfactant-stable high-alkaline protease AprB with a novel structural feature unique to subtilisin family

Deng, Aihua; Wu, Jie; Zhang, Guoqiang; Wen, Tingyi

doi:10.1016/j.biochi.2011.01.011

Cited by 25 publications

(19 citation statements)

References 48 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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“…Therefore, the excellent tolerance to solvent of the protease from Bacillus sp. B001 might be attributed to its high ratio of negative charged amino acids which accounts for 21.24% surface [33]. This remarkable property substantiates promising application of enzyme as a biocatalyst for peptide synthesis in low water systems.…”

Section: Effects Of Organic Solvents On Protease Stabilitymentioning

confidence: 66%

A salt, detergent, and solvent tolerant protease from <i>Bacillus</i> sp. B001: Low-cost, easy-purified, and enhanced production by raw material based culture strategy

Wu¹,

Deng²,

Shi³

et al. 2013

ABB

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To enhance the protease production and decrease cost, corn flour and soy peptone were screened as cheap raw materials for the production of extracellular proteases by Bacillus strains. Their compositions in the medium suitable for enzyme production of Bacillus sp. B001 were optimized using statistical experiment designs. Under the optimized conditions, the protease production of Bacillus sp. B001 was stable at the stationary stage and reached to 63,200 U/mL, approximately 1.84-fold increase compared with that using the original medium. These improvements could be attributed to the release of the catabolite repression by crude materials corn flour and soy peptone which contained low level of available nutrients. Additionally, a highly pure protease which displayed excellent stability and compatibility with high salinity, commercial laundry detergents, and organic solvents, was rapidly obtained by two-step procedure involving ammonium sulphate precipitation and anion exchange from the fermentation cultures of B001 in the optimized medium. When the culture method applied to other Bacillus strains, their protease yields were all remarkably increased approximately 2.9 to 8.5 folds. In conclusion, a low-cost, easy-purified, and effective producing strategy using the cheap raw materials was developed here, representing a potential application for protease production in various Industrial processes.

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“…Numerous studies confirm the positive effect of calcium ions (3–10 mM) on serine protease activity isolated from B. circulans (Benkiar et al., ; Rao, Sathish, Ravichandra, & Prakasham, ); S. koyangensis TN650 (Elhoul et al., ); Bacillus sp. strain B001 (Deng, Wu, Zhang, & Wen, ); Bacillus laterosporus ‐AK1 (Arulmani et al., ); and Bacillus caseinilyticus (Mothe & Sultanpuram, ). Many of these authors believe that the removal of Ca 2+ from the strong binding site is associated with a significant decrease in stability, especially at high temperatures.…”

Section: Discussionmentioning

confidence: 99%

Purification and characterization of trypsin produced by gut bacteria from Anticarsia gemmatalis

Pilon

Silva

Visôtto

et al. 2017

Arch Insect Biochem Physiol

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Purification of active trypsin in the digestive process of insects is essential for the development of potent protease inhibitors (PIs) as an emerging pest control technology and research into insect adaptations to dietary PIs. An important aspect is the presence of proteolytic microorganisms, which contribute to host nutrition. Here, we purified trypsins produced by bacteria Bacillus cereus, Enterococcus mundtii, Enterococcus gallinarum, and Staphylococcus xylosus isolated from the midgut of Anticarsia gemmatalis. The trypsins had a molecular mass of approximately 25 kDa. The enzymes showed increased activity at 40°C, and they were active at pH values 7.5-10. Aprotinin, bis-benzamidine, and soybean Kunitz inhibitor (SKTI) significantly inhibited trypsin activity. The l-1-tosyl-amido-2-phenylethylchloromethyl ketone (TPCK), pepstatin A, E-64, ethylenediamine tetraacetic acid, and calcium ions did not affect the enzyme activity at the concentrations tested. We infer the purified trypsins do not require calcium ions, by which they differ from the trypsins of other microorganisms and the soluble and insoluble trypsins characterized from A. gemmatalis. These data suggest the existence of different isoforms of trypsin in the velvetbean caterpillar midguts.

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Molecular and structural characterization of a surfactant-stable high-alkaline protease AprB with a novel structural feature unique to subtilisin family

Cited by 25 publications

References 48 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

A salt, detergent, and solvent tolerant protease from <i>Bacillus</i> sp. B001: Low-cost, easy-purified, and enhanced production by raw material based culture strategy

Purification and characterization of trypsin produced by gut bacteria from Anticarsia gemmatalis

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Molecular and structural characterization of a surfactant-stable high-alkaline protease AprB with a novel structural feature unique to subtilisin family

Cited by 25 publications

References 48 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

A salt, detergent, and solvent tolerant protease from &lt;i&gt;Bacillus&lt;/i&gt; sp. B001: Low-cost, easy-purified, and enhanced production by raw material based culture strategy

Purification and characterization of trypsin produced by gut bacteria from Anticarsia gemmatalis

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A salt, detergent, and solvent tolerant protease from <i>Bacillus</i> sp. B001: Low-cost, easy-purified, and enhanced production by raw material based culture strategy