Molecular characterization of alkaline protease of Bacillus amyloliquefaciens SP1 involved in biocontrol of Fusarium oxysporum
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Bacillus amyloliquefaciens
Protease production and its characteristics were investigated with Bacillus subtilis PCA20-3 which was isolated from Korean traditional meju. The optimum culture conditions of Bacillus subtilis PCA20-3 for the production of the protease were as follow: 0.2% soytone, 2% starch, 0.1% and 20 hrs. The optimum pH and temperature for enzyme activity of protease producing Bacillus subtilis PCA20-3 were pH 8.0-10.0 and , respectively. The enzyme was relatively stable at pH and at temperature below . The activity of the enzyme was inhibited by . 2 mM phenymethanesulfonyl fluoride inhibited 89.2% of enzyme activity. This indicates that the enzyme is serine protease. The value was . This enzyme hydrolyzed casein more rapidly than bovine serum albumin.
Bovine serum albumin
Neutral protease
Bacillaceae
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An alkaline serine protease producing strain was isolated from local soil samples and identified based on morphological and biochemical characteristics as Bacillus subtilis NR18. The enzyme was purified in three step procedure involving ammoniumsulfate precipitation, followed by gel filtration and ion-exchange chromatography. Through the process 13.7-fold increase in puritywith a specific activity of 283.1 U/mg proteins was obtained. The molecular weight of the purified enzyme was found to be 21 kDa by SDS-PAGE. The enzymewasmost active at 60Cand pH 9.0. It was relatively stable between pH 7.0-10.0 and temperature between 40 and 60°C Influence of metal ions on enzyme activity revealed that, Ca2+, Mg2+ and Mn2+ slightly enhanced the enzyme activity; whereas Co2+, Fe2+, Hg2+ and Zn2+ strongly inhibited the enzyme activity.Among the protease inhibitors that were tested, the PMSF and DFP completely inhibited the enzyme activity, indicating that the protease is a serine protease. The enzyme retained more than 50%activity after incubation at different time intervals at 60°C in the presence of commercial detergents indicating its suitability for application in detergent industry.
Specific activity
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Phenylmethylsulfonyl Fluoride
Subtilisin
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Objective To detect the partial molecular characteristics of a novel fibrinolytic protease from the coelomic fluid of Nereis virens identified and purified to homogeneity.Methods The fibrinolytic protease was purified with anion and cation exchange chromatography and gel filtration chromatography.The identification of fibrinolytic activity and active distributed curve had been assessed by method of fibrin plate.Its apparent molecular weight and isoelectric point were analysed by two-dimensional gel electrophoresis(2-DE).The effects of several protease inhibitors on the protease activity were examined,and its protease type was identified.Results A novel and single chain fibrinolytic protease was purified effectively.Its apparent molecular weight and isoelectric point were 29 000 and 4.5,respectively.The proteolytic activity peaked at pH 7.8 and 45℃.The protease was completely inhibited by DFP and PMSF,assessing as a serine protease.Conclusion From the coelomic fluid of Nereis virens,a novel and single chain serine protease with more strong fibrinolytic activity is discovered.The fibrinolytic protease has a medical value for preventing and treating thrombosis.
Molecular mass
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Sephadex
Ammonium sulfate precipitation
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Bacillus subtilis 1A20 transformed with a hybrid plasmid, pNP150, to which a DNA fragment from Bacillus amyloliquefaciens F was attached, produced a large amount of a neutral protease. To identify the origin of the gene specifying this neutral protease, neutral proteases from B. amyloliquefaciens F, B. subtilis NP58 (a derivative of Marburg 6160), and B. subtilis 1A20 transformed with pNP150 were purified. We investigated their immunological properties and primary structures. The proteases from these two species were indistinguishable by chromatography, but they were distinguishable from each other by SDS-polyacrylamide gel electrophoresis and double immunodiffusion. Amino acid sequencing of these two proteases by Edman degradation showed that there were four substitutions in the 20-residue amino acid sequence from the N-termini. Neutral protease from the transformant had the same immunological characteristics and N-terminal amino acid sequence as that from B. amyloliquefaciens. These results meant that the gene in question was derived from a gene specifying the neutral protease in this bacterium.
Bacillus amyloliquefaciens
Edman degradation
Bacillaceae
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Two proteases isolated from senescent oat (Avena sativa) leaves have been subjected to further study. One of these, an acid protease active at pH 4.2, is inhibited by phenylmethylsulfonyl fluoride (PMSF) but not by iodoacetamide (IAc). The other, active at pH 6.6, is inhibited by both PMSF and IAc. These results, together with previously reported evidence that mercaptoethanol stimulates the activity of only the neutral protease, are taken to indicate that the acid protease is probably of the serine type, whereas the neutral enzyme is of the sulfhydryl type. Both enzymes are inhibited by irradiation in the presence of rose bengal, a selective histidine modification reagent. The acid protease was completely unaffected by chelators, but data on the neutral protease were equivocal.All protein substrates tested were attacked by both enzymes, though at strikingly different rates. Characterization of the digestion products, with denatured hemoglobin as substrate, indicated that the acidic enzyme is an endoprotease, while the neutral one is an exoprotease. Evidence is presented that these proteases undergo autolysis in vitro.
Autolysis (biology)
Iodoacetamide
Phenylmethylsulfonyl Fluoride
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Pepstatin
Protease inhibitor (pharmacology)
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