Isolation and characterization of AStreptomyces C5 mutant strain hyperproductive of extracellular protease

Gibb, Gregory D.; Dekleva, Michael L.; Lampel, J S; Strohl, William R.

doi:10.1007/bf01033195

Cited by 14 publications

(6 citation statements)

References 11 publications

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“…The azocasein hydrolysis assay was carried out as previously described (Gibb et al 1987;Gibb and Strohl 1988). The purified enzyme was also tested for its ability to hydrolyze the following synthetic proteinase substrates (all obtained from Sigma, St. Louis, Mo., USA): N-succinyl-alanyl-alanyl-prolyl-phenylalanine p-nitroanilide (SAAPF-pNA), a substrate for chymotrypsin (DelMar et al 1979); N-c~-benzoyl-I.-arginine p-nitroanilide (BA-pNA), a substrate for trypsin (Erlanger et al 1961); and benzyloxycarbonyl-glycyl-glycl-leucine p-nitroanilide (BOC-GGL-pNA), a substrate for subtilisin (Lyublinskaya et al 1974).…”

Section: Methodsmentioning

confidence: 99%

“…5.7, 7.3, 8.2, and 9.2 kb pairs (Vinci 1988). Thus, it could be expected that at least four different serine proteinases are produced by this strain and its derivatives, of which C5-A13 is one (Gibb et al 1987). It appears from our data, however, that only one of the four serine proteinase genes of C5 is highly expressed in C5-A13.…”

Section: Production O F Serine Proteinase By C5-a13mentioning

confidence: 99%

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Purification and properties of the chymotrypsin-like serine proteinase overproduced by Stremptomyces sp. strain C5-A13

Vinci

Aphale

Gibb

et al. 1993

Appl Microbiol Biotechnol

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The major serine proteinase of Streptomyces sp. strain C5-A13, a proteinase-overproducing mutant strain, was purified to homogeneity. It has a relative molecular mass (Mr) of 19500 as determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Superose-6 gel filtration chromatography, a low isoelectric point, optimal activity at pH 8.5-9.5, and an optimal temperature of approx. 55 ° C. This purified enzyme has a high specific activity on azocasein of 11000 units'rag -1 of protein, a Km of 2.7 X 10 -5 M and a Vmax of 2.0 gmol. rain -1. m g -1 of protein when succinyl-alanyl-alanyl-prolyl-phenylalanine p-nitroanilide was used as substrate. It was inhibited by the serine proteinase inhibitors diisopropyl fluorophosphate and phenylmethylsulfonyl fluoride but not by ethylenediaminetetraacetate (EDTA) or N-tosyl-L-phenylalanine chloromethyl ketone. Thus, this serine proteinase appears to be a chymotrypsin-like enzyme and represents nearly 90% of the total extracellular azocasein-hydrolyzing activity produced by strain C5-A13. Since CaCO3 did not stimulate activity of the purified enzyme itself, the carbonate effect may be a transcriptional or translational rather than a post-translational one.

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Section: Methodsmentioning

confidence: 99%

Section: Production O F Serine Proteinase By C5-a13mentioning

confidence: 99%

Purification and properties of the chymotrypsin-like serine proteinase overproduced by Stremptomyces sp. strain C5-A13

Vinci

Aphale

Gibb

et al. 1993

Appl Microbiol Biotechnol

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“…Kole et al , (1988) Azocasein 5 mglmi --c Average RD, % ----, RSD, % Frankena et al , (1985) Azoalbumin 25 mglml Manachini et al , (1988) Casein 2.5 mg/ml 5 Battaglino et al , (1991) Casein 25 mglml Malathi et al , (1991) Caaein 25 mg/ml Sarath ef al. , (1989)* Gibb et al , (1987 Azocasein j GF& AzocaseirJazoalbumin 30 mglml -5 70 mglml Fig. 4.…”

Section: Reagentmentioning

confidence: 99%

Azocasein assay for alkaline protease in complex fermentation broth

Iversen

Jørgensen

1995

Biotechnol Tech

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An azocasein assay has been developed for determination of alkaline protease in fermentation broth from a complex substrate containing ca. 50 g/l protein which to a high degree interferes with azocasein. Methods described in the literature have been found inaccurate as results deviate ca. 50% from true activity, so one of the methods is modified in order to eliminate interference. Proportionality between the relative azocasein concentration and the deviation from true activity is found. An azocasein concentration of 350 mg azocasein per ml sample gives satisfactory results with an accuracy of f5 % . Application of a standard addition method also improves the accuracy, but is laborious and less precise.Abbreviations a: true enzyme activity; as: activity determined from standard curve; AU: Anson unit; cNT: relative azocasein concentration (mg azocasein per mg total protein); r : ratio between measured and true enzyme activity; RD: relative deviation from true value (%); RSD: relative standard deviation (%); TCA: trichloroacetic acid.

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“…The enzymatic hydrolysis of the specific collagenase substrates N-(3-[2-furyl]acryloyl)-leucylglycyl-prolyl-alanine and 4-phenylazobenzyloxycarbonylprolyl-leucyl-glycyl-prolyl-D-arginine were measured as described by Van Wart and Steinbrink (55) and Wunsch and Heidrich (59), respectively. The enzymatic hydrolysis of azocasein was assayed as described previously (20,21), and the hydrolysis of azocoll was carried out as described by Rufo et al (46).…”

Section: Materuils and Methodsmentioning

confidence: 99%

“…strain C5, a mutagenized strain isolated as an overproducer of anthracycline antibiotics, produces negligible extracellular proteinase activity against azocasein but strong extracellular proteolytic activity against the proteins in Carnation dry milk (20,21). In the present study, we used the respective differences in proteolysis profiles of Streptomyces sp.…”

mentioning

confidence: 99%

Cloning and sequencing of a gene encoding a novel extracellular neutral proteinase from Streptomyces sp. strain C5 and expression of the gene in Streptomyces lividans 1326

et al. 1992

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The gene encoding a novel milk protein-hydrolyzing proteinase was cloned on a 6.56-kb SstI fragment from Streptomyces sp. strain C5 genomic DNA into Streptomyces lividans 1326 by using the plasmid vector pU702. The gene encoding the small neutral proteinase (snpA) was located within a 2.6-kb BamHI-SstI restriction fragment that was partially sequenced. The molecular mass of the deduced amino acid sequence of the mature protein was determined to be 15,740, which corresponds very closely with the relative molecular mass of the purified protein (15,500) determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The N-terminal amino acid sequence of the purified neutral proteinase was determined, and the DNA encoding this sequence was found to be located within the sequenced DNA. The deduced amino acid sequence contains a conserved zinc binding site, although secondary ligand binding and active sites typical of thermolysinlike metalloproteinases are absent. The combination of its small size, deduced amino acid sequence, and substrate and inhibition profile indicate that snpA encodes a novel neutral proteinase.

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Isolation and characterization of AStreptomyces C5 mutant strain hyperproductive of extracellular protease

Cited by 14 publications

References 11 publications

Purification and properties of the chymotrypsin-like serine proteinase overproduced by Stremptomyces sp. strain C5-A13

Purification and properties of the chymotrypsin-like serine proteinase overproduced by Stremptomyces sp. strain C5-A13

Azocasein assay for alkaline protease in complex fermentation broth

Cloning and sequencing of a gene encoding a novel extracellular neutral proteinase from Streptomyces sp. strain C5 and expression of the gene in Streptomyces lividans 1326

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