Purification and characterization of an eggshell membrane decomposing protease from Pseudomonas aeruginosa strain ME-4

Cheng, Minyi; Takenaka, Shinji; Aoki, Shunsuke; Murakami, Shinichiro; Aoki, Kenji

doi:10.1016/j.jbiosc.2008.12.010

Cited by 27 publications

(27 citation statements)

References 24 publications

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“…Bacterial strains, plasmids, and culture conditions Pseudomonas aeruginosa strain ME-4 was cultivated as described previously (Cheng et al 2009). E. coli XL-1 Blue and E. coli BL21(DE3)pLysS were used for plasmid construction and gene expression of the P. aeruginosa metalloprotease, respectively.…”

Section: Methodsmentioning

confidence: 99%

“…Cloning of the protease gene The ME4 N primer (5 0 -ATCAGTGGGAAGGCCTGG CCCACG-3 0 ) was designed based on the N-terminal amino acid sequence (AEAGGPGGNQKIGAY) of the ME4 protease (Cheng et al 2009). The primers ME4F (5 0 -GATCGTCGGCGAGCGTCACCTGAA G-3 0 ) and ME4R (5 0 -TACAACGCGCTCGGGCAGG TCACG-3 0 ) were designed based on the conserved region of Pseudomonas metalloproteases (DDBJ/ EMBL-Bank/GenBank accession no.…”

Section: Methodsmentioning

confidence: 99%

“…For purification of the eggshell-membrane-decomposing enzyme (native ME-4 protease), strain ME-4 was cultured in a 500 ml flask containing 70 ml medium as reported previously (Cheng et al 2009). The protease in the culture supernatant from ten flasks (700 ml total) was purified by ammonium sulfate fractionation and CM52 and DE52 column chromatography as reported previously (Cheng et al 2009).…”

Section: Enzyme Purificationmentioning

confidence: 99%

“…The protease in the culture supernatant from ten flasks (700 ml total) was purified by ammonium sulfate fractionation and CM52 and DE52 column chromatography as reported previously (Cheng et al 2009). …”

Section: Enzyme Purificationmentioning

confidence: 99%

“…We previously purified and characterized an eggshell-membrane-degrading enzyme classified as a metalloprotease from Pseudomonas aeruginosa strain ME-4 (Cheng et al 2009). The ME-4 protease solubilizes eggshell membrane and is expected to partially hydrolyze this membrane, which is then more suitable for the production of soluble proteins and/or peptides than chemical hydrolysis.…”

mentioning

confidence: 99%

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Organic solvent-tolerant elastase efficiently hydrolyzes insoluble, cross-linked, protein fiber of eggshell membranes

et al. 2012

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Eggshell membrane is a mechanically stable and insoluble cross-linked fibrous protein. Pseudomonas aeruginosa strain ME-4 synthesizes a metalloprotease that degrades the eggshell membrane. We cloned the encoding gene in Escherichia coli. The recombinant protease, over-expressed in E. coli, was inactive but addition of acetone to crude cell extracts restored the activity and removed many E. coli proteins. We purified the active, acetone-treated protease to homogeneity in a single chromatography step with 57% recovery. The recombinant protease partially hydrolyzed eggshell membrane and produced more soluble peptides and proteins than commercial elastase, α-chymotrypsin, and collagenase. The soluble peptides produced from hydrolyzed eggshell membrane inhibited angiotensin-I-converting enzyme activity. The degradation of eggshell membrane by the recombinant elastase could be applied to the production of soluble bioactive peptides.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Enzyme Purificationmentioning

confidence: 99%

Section: Enzyme Purificationmentioning

confidence: 99%

mentioning

confidence: 99%

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Organic solvent-tolerant elastase efficiently hydrolyzes insoluble, cross-linked, protein fiber of eggshell membranes

et al. 2012

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Novel extremophilic proteases from Pseudomonas aeruginosa M211 and their application in the hydrolysis of dried distiller's grain with solubles

Flores-Fernández

Cárdenas-Fernández

Dobrijevic

et al. 2018

Biotechnology Progress

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Proteases are the most important group of industrial enzymes and they can be used in several fields including biorefineries for the valorization of industrial byproducts. In this study, we purified and characterized novel extremophilic proteases produced by a Pseudomonas aeruginosa strain isolated from Mauritia flexuosa palm swamps soil samples in Peruvian Amazon. In addition, we tested their ability to hydrolyze distillers dried grains with solubles (DDGS) protein. Three alkaline and thermophilic serine proteases named EI, EII, and EIII with molecular weight of 35, 40, and 55 kDa, respectively, were purified. EI and EIII were strongly inhibited by EDTA and Pefabloc being classified as serine‐metalloproteases, while EII was completely inhibited only by Pefabloc being classified as a serine protease. In addition, EI and EII exhibited highest enzymatic activity at pH 8, while EIII at pH 11 maintaining almost 100% of it at pH 12. All the enzymes demonstrated optimum activity at 60°C. Enzymatic activity of EI was strongly stimulated in presence of Mn2+ (6.9‐fold), EII was stimulated by Mn2+ (3.7‐fold), while EIII was slightly stimulated by Zn2+, Ca2+, and Mg2+. DDGS protein hydrolysis using purified Pseudomonas aeruginosa M211 proteases demonstrated that, based on glycine released, EIII presented the highest proteolytic activity toward DDGS. This enzyme enabled the release 63% of the total glycine content in wheat DDGS protein, 2.2‐fold higher that when using the commercial Pronase®. Overall, our results indicate that this novel extremopreoteases have a great potential to be applied in DDGS hydrolysis. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2728, 2019

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Enzymatic valorization process of yellow cocoon waste for production of antioxidative sericin and fibroin film

Yakul

Kaewsalud

Techapun

et al. 2020

J of Chemical Tech & Biotech

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BACKGROUND Cocoon waste, solid waste from the silk industry, is currently utilized as low‐cost spun silk yarn. In this study, the enzymatic valorization process of yellow cocoon waste to produce antioxidative sericin hydrolysate and fibroin film was demonstrated. RESULTS The enzymatic process involving thermostable alkaline protease from Bacillus halodurans SE5 (protease_SE5) was superior to the conventional high temperature and high pressure and 0.5% Na2CO3 processes. Protease_SE5 produced sericin hydrolysate with high peptide concentration (0.335 mg mL–1) and provided degummed fibroin with a complete structure. Sericin hydrolysate from protease_SE5‐assisted hydrolysis showed remarkable radical scavenging activities on ABTS, DPPH and FRAP assays with 602, 3.90 and 24.8 μmol Trolox equivalents (TE) g−1 protein, respectively. After ultrafiltration and size‐exclusion chromatography, two active fractions (F1 and F2) were obtained from sericin hydrolysate with ABTS radical scavenging activity of 2120 and 3289 μmol TE g−1 protein, respectively. Identification of bioactive peptides by de novo sequencing was conducted, and seven candidate peptides were identified with high content of key antioxidative amino acids (His, Phe, Trp, Tyr and Arg) in the sequences. Moreover, bioactivities of several of the peptides were predicted by bioinformatics databases. Besides, the mechanical properties of enzyme‐derived fibroin film were also improved, with tensile strength and elongation at break of 62.1 MPa and 3.6% in the dry state, and 8.5 MPa and 140% in the wet state. CONCLUSION This enzymatic process could be an effective way for valorizing cocoon waste. The value‐added products from agricultural waste have promising applications in food, pharmaceutical and biomedical materials. © 2020 Society of Chemical Industry (SCI)

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Purification and characterization of an eggshell membrane decomposing protease from Pseudomonas aeruginosa strain ME-4

Cited by 27 publications

References 24 publications

Organic solvent-tolerant elastase efficiently hydrolyzes insoluble, cross-linked, protein fiber of eggshell membranes

Organic solvent-tolerant elastase efficiently hydrolyzes insoluble, cross-linked, protein fiber of eggshell membranes

Novel extremophilic proteases from Pseudomonas aeruginosa M211 and their application in the hydrolysis of dried distiller's grain with solubles

Enzymatic valorization process of yellow cocoon waste for production of antioxidative sericin and fibroin film

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