Identification of novel halotolerant bacillopeptidase F-like proteinases from a moderately halophilic bacterium, Virgibacillus sp. SK37

Phrommao, E.; Rodtong, Sureelak; Yongsawatdigul, Jirawat

doi:10.1111/j.1365-2672.2010.04871.x

Cited by 28 publications

(13 citation statements)

References 48 publications

(73 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…SK37, which was isolated from Thai fish sauce that was saturated with 26-28% of NaCl. This bacterium produced numerous NaCl-activated subtilisin-like proteinases, three of them having been partially purified and characterized as extracellular halotolerant bacillopeptidase F-like proteinases [30]. Their activities did not depend on calcium, showed maximum activity at pH 8, 55-60°C, 25-30% NaCl and 70-100 mMol l -1 CaCl 2 , and stability at 0-30% NaCl and <20 mMol l -1 CaCl 2 [30].…”

Section: Discussionmentioning

confidence: 99%

“…This bacterium produced numerous NaCl-activated subtilisin-like proteinases, three of them having been partially purified and characterized as extracellular halotolerant bacillopeptidase F-like proteinases [30]. Their activities did not depend on calcium, showed maximum activity at pH 8, 55-60°C, 25-30% NaCl and 70-100 mMol l -1 CaCl 2 , and stability at 0-30% NaCl and <20 mMol l -1 CaCl 2 [30]. The NaCl-activated property of AprX-SK37 was not unexpected as it has been shown that the cell membrane of halophilic bacteria is incapable to completely prevent an increased flux of sodium into the cells [31].…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

A novel subtilase with NaCl-activated and oxidant-stable activity from Virgibacillussp. SK37

et al. 2011

View full text Add to dashboard Cite

BackgroundMicrobial proteases are one of the most commercially valuable enzymes, of which the largest market share has been taken by subtilases or alkaline proteases of the Bacillus species. Despite a large amount of information on microbial proteases, a search for novel proteases with unique properties is still of interest for both basic and applied aspects of this highly complex class of enzymes. Oxidant stable proteases (OSPs) have been shown to have a wide application in the detergent and bleaching industries and recently have become one of the most attractive enzymes in various biotechnological applications.ResultsA gene encoding a novel member of the subtilase superfamily was isolated from Virgibacillus sp. SK37, a protease-producing bacterium isolated from Thai fish sauce fermentation. The gene was cloned by an activity-based screening of a genomic DNA expression library on Luria-Bertani (LB) agar plates containing 1 mM IPTG and 3% skim milk. Of the 100,000 clones screened, all six isolated positive clones comprised one overlapping open reading frame of 45% identity to the aprX gene from Bacillus species. This gene, designated aprX-sk37 was cloned into pET21d(+) and over-expressed in E. coli BL21(DE3). The enzyme product, designated AprX-SK37, was purified by an immobilized metal ion affinity chromatography to apparent homogeneity and characterized. The AprX-SK37 enzyme showed optimal catalytic conditions at pH 9.5 and 55°C, based on the azocasein assay containing 5 mM CaCl2. Maximum catalytic activity was found at 1 M NaCl with residual activity of 30% at 3 M NaCl. Thermal stability of the enzyme was also enhanced by 1 M NaCl. The enzyme was absolutely calcium-dependent, with optimal concentration of CaCl2 at 15 mM. Inhibitory effects by phenylmethanesulfonyl fluoride and ethylenediaminetetraacetic acid indicated that this enzyme is a metal-dependent serine protease. The enzyme activity was sensitive towards reducing agents, urea, and SDS, but relatively stable up to 5% of H2O2. Phylogenetic analysis suggested that AprX-SK37 belongs to a novel family of the subtilase superfamily. We propose the name of this new family as alkaline serine protease-X (AprX).ConclusionsThe stability towards H2O2 and moderately halo- and thermo-tolerant properties of the AprX-SK37 enzyme are attractive for various biotechnological applications.

show abstract

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

A novel subtilase with NaCl-activated and oxidant-stable activity from Virgibacillussp. SK37

et al. 2011

View full text Add to dashboard Cite

show abstract

“…Researchers have deployed matrix‐assisted laser desorption/ionization time of flight mass spectrometry and liquid chromatography‐tandem mass spectrometry to obtain peptide mass fingerprint and short de novo sequenced peptides to disclose homology with related proteases ; the ratios are comparable to the BLAST search results (from NCBI). In the current study, peptides obtained from LC‐LTQ Orbitrap furnished evidence that the isolated protease belonged to protease family and it showed high sequence identity to other published proteases.…”

Section: Discussionmentioning

confidence: 99%

Biochemical characteristics of a novel protease from the basidiomycete Amanita virgineoides

Yuan

Zhu

Gao

et al. 2017

Biotech and App Biochem

View full text Add to dashboard Cite

The characterization of a novel protease from Amanita virgineoides is described. The A. virgineoides protease was purified to homogeneity using Q-Sepharose, carboxymethyl-cellulose, diethylaminoethyl-cellulose, and a gel filtration step on Superdex 75. The molecular mass of the purified protease was estimated to be 16.6 kDa. The protease was purified 32.1-fold, and its specific activity was 301.4 U/mg. The optimum pH was 4.0, and the optimum temperature was 50 °C. Kinetic constants (K , V ) were determined under the optimum reaction conditions, with K and V , being 3.74 mg/mL and 9.98 μg mL Min , respectively. The activity of the protease was curtailed by Cu , Pb , Fe , Cd , and Hg ions but enhanced by Mg , Ca , and K ions at low concentrations. The protease activity was adversely affected by ethylene diamine tetraacetic acid, suggesting that it is a metalloprotease. Four peptide sequences were obtained from liquid chromatography-tandem mass spectrometry, including KQALSGIR, TIAMDGTEGLVR, VALTGLTVAEYFR, and AGAGSATLSMAYAGAR, which showed 86%, 64%, 60%, and 75% identity with peptides of Hypsizygus marmoreus, Dacryopinax sp. DJM-731 SS1, Trametes versicolor FP-101664 SS1, and Paxillus involutus ATCC 200175, respectively. The newly isolated protease showed good hydrolytic activity and biochemical characteristics, which expanded the knowledge of biologically active proteins and provided further insight on this poisonous fungus.

show abstract

“…The major secreted proteases from Virgibacillus sp. SK37 showed an acidic pI, implying the presence of numerous negative charges in their structures (23). The repulsive forces between the negative charges of secretory proteases and the cell wall may be minimized during protein translocation by the presence of Mg 2+ , resulting in an improvement in protease secretion.…”

Section: Selection Of Physical and Chemical Factorsmentioning

confidence: 97%

Statistical Optimization and Secretion of NaCl-Tolerant Proteinases by a Moderate Halophile, Virgibacillus sp. SK37

Sinuwan¹,

Jangchud²,

Rodtong³

et al. 2015

Food Technol. Biotechnol.

View full text Add to dashboard Cite

SummaryThe objectives of this study are to optimize the conditions for providing high yield of NaCl-tolerant extracellular protease from Virgibacillus sp. SK37 based on a fi sh-based medium and to investigate the eff ects of the key factors (mass per volume ratios of dried anchovy, yeast extract and NaCl, and initial pH of the medium) on the secretion patt ern of proteases. Based on the predicted response model, the optimized medium contained 1.81 % of dried anchovy, 0.33 % of yeast extract and 1.25 % of NaCl at pH=7.8. Under these conditions, a 5.3-fold increase in protease production was achieved, compared with the broth containing only 1.2 % of dried anchovy (5 % of NaCl at pH=7). The cubic regression adequately described the protease production. Protease activity was determined using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) on the synthetic substrate (Suc-Ala-Ala-Pro-Phe-AMC). Proteases of molecular masses of 19, 34, 35 and 44 kDa were secreted in the presence of NaCl, whereas those of 22 and 42 kDa were the main proteases detected in the absence of NaCl. In addition, no secreted proteases were detected when initial pH of the medium was pH=6. The peptide mass fi ngerprint of the medium cultured with 10 % NaCl showed a higher abundance of peptides with lower mass of 500-1000 m/z compared with the medium containing 0 % NaCl, indicating the higher proteolytic activity of the high-salt medium. The Virgibacillus sp. SK37 proteases showed a marked preference towards Lys, Arg and Tyr in the presence of NaCl and towards Lys and Arg in the absence of NaCl.

show abstract

Identification of novel halotolerant bacillopeptidase F-like proteinases from a moderately halophilic bacterium, Virgibacillus sp. SK37

Cited by 28 publications

References 48 publications

A novel subtilase with NaCl-activated and oxidant-stable activity from Virgibacillussp. SK37

A novel subtilase with NaCl-activated and oxidant-stable activity from Virgibacillussp. SK37

Biochemical characteristics of a novel protease from the basidiomycete Amanita virgineoides

Statistical Optimization and Secretion of NaCl-Tolerant Proteinases by a Moderate Halophile, Virgibacillus sp. SK37

Contact Info

Product

Resources

About