bromochloroacetic-acid and 1-10-phenanthroline

Serratia proteamaculans HY-3 isolated from the digestive tract of a spider produces an extracellular protease named arazyme, with an estimated molecular mass of 51.5 kDa. The purified enzyme was characterized as having high activities at wide pH and temperature ranges. We further characterized biochemical features of the enzymatic reactions under various reaction conditions. The protease efficiently hydrolyzed a broad range of protein substrates including albumin, keratin, and collagen. The dependence of enzymatic activities on the presence of metal ions such as calcium and zinc indicated that the enzyme is a metalloprotease, together with the previous observation that the proteolytic activity of the enzyme was not inhibited by aspartate, cysteine, or serine protease inhibitors, but strongly inhibited by 1,10-phenanthroline and EDTA. The araA gene encoding the exoprotease was isolated as a 5.6 kb BamHl fragment after PCR amplification using degenerate primers and subsequent Southern hybridization. The nucleotide sequence revealed that the deduced amino acid sequences shared extensive similarity with those of the serralysin family of metalloproteases from other enteric bacteria. A gene (inh) encoding a putative protease inhibitor was also identified immediately adjacent to the araA structural gene.

Topics: Albumins; Amino Acid Sequence; Animals; Cations, Divalent; Coenzymes; Collagen; DNA, Bacterial; Edetic Acid; Enzyme Stability; Gastrointestinal Tract; Hydrogen-Ion Concentration; Keratins; Metalloproteases; Metals; Molecular Sequence Data; Molecular Weight; Phenanthrolines; Protease Inhibitors; Sequence Analysis, DNA; Sequence Homology, Amino Acid; Serratia; Spiders; Substrate Specificity; Temperature

This study was developed to purify and to characterize a keratinolytic protease from the bacterium Microbacterium sp. strain kr10.. Enzyme purification was carried out by sequential liquid chromatography on Sephadex G-100 and Q-Sepharose columns. The purification was about 255-fold, with a yield of 34%, as determined with azocasein as substrate. The molecular weight of the enzyme was estimated as 42,000 Da by SDS-PAGE. The enzyme had pH and temperature optima of 7.5 and 50 degrees C respectively. This keratinase was inhibited by EDTA and 1,10-phenanthroline, and analysis of metal content indicates that Zn(2+) and Mg(2+) are present. A 2(2) factorial design was developed to investigate the effect of keratinase and mercaptoacetate concentration on feather keratinolysis. Statistical analysis showed that both variables have a significant effect on hydrolysis of keratin.. A new keratinase produced by Microbacterium sp. was purified and characterized.. This keratinolytic enzyme offers an interesting potential for the hydrolysis of keratin wastes to be used as feed supplement or bioconversion to added-value products.

Topics: Animals; Chromatography, Liquid; Edetic Acid; Electrophoresis, Polyacrylamide Gel; Enzyme Inhibitors; Feathers; Hydrogen-Ion Concentration; Industrial Microbiology; Keratins; Magnesium; Molecular Weight; Mycobacterium; Peptide Hydrolases; Phenanthrolines; Temperature; Thioglycolates; Zinc