Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.25.1 (proteasome)
 28,817 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

An artificially inserted extra peptide (21 amino acid peptide) between the B. subtilis alpha-amylase signal peptide and the mature thermostable alpha-amylase was completely cleaved by B. subtilis alkaline protease in vitro. The cleavage to form a mature enzyme was observed between pH 7.5 and 10, but not between pH 6.0 and 6.5, although a similar protease activity toward Azocall was observed between pH 6.0 and 7.5. To analyze the effects of pH on the cleavage, CD spectra at pH 6, 8, and 11 of the NH2-terminally extended thermostable alpha-amylase were analyzed and the results were compared with those of the mature form of the alpha-amylase. It is suggested that the cleavage of the NH2-terminally extended peptide is controlled by the secondary and tertiary structure of the precursor enzyme. Similar cleavage of different NH2-terminally extended peptides by the alkaline protease was also found in other hybrid thermostable alpha-amylases obtained.
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PMID:Maturation of an NH2-terminally extended thermostable alpha-amylase in Bacillus subtilis: a possible mechanism examined by in vitro experiments. 136 50

In order to obtain a large quantity of glutamic-acid-specific endopeptidase of Staphylococcus aureus ATCC12600 (SPase) without cultivating its pathogenic host bacterium, expression plasmids enabling secretion of SPase from Bacillus subtilis were constructed by inserting the SPase gene into B. subtilis-Escherichia coli shuttle vectors. B. subtilis harbouring a simple recombinant plasmid containing the coding and the 5'-flanking regions of SPase in the shuttle vector pHY300PLK secreted 22 mg/l of SPase into the medium. As this level was lower than that of the natural strain (45 mg/l), we tried to increase the expression level by constructing a series of hybrid plasmids with the following features: (1) the terminator sequence of the alkaline protease gene from B. subtilis, (2) the promoter and the leader sequences of the alpha-amylase gene or of alkaline protease gene from B. amyloliquefaciens, (3) the vector pHY300PLK and the fused vector of pHY300PLK and pUB110. By using a variety of hybrid plasmids, the resulting transformants secreted SPase at levels of 33-120 mg/l. The recombinant SPase isolated from the medium was indistinguishable from the natural one with respect to its behaviour on sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Western blotting as well as its enzyme activity.
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PMID:Secretory expression of a glutamic-acid-specific endopeptidase (SPase) from Staphylococcus aureus ATCC12600 in Bacillus subtilis. 136 43

To analyze the processing of extracellular enzymes of Bacillus subtilis, an NH2-terminally extended hybrid alpha-amylase [pTUBE638-alpha-amylase (E24)] was purified from the periplasm of E. coli(pTUBE638) as the substrate for the in vitro processing reaction, in which a 21-amino-acid extra-peptide was added at the NH2-terminus of the mature thermostable alpha-amylase. The extended peptide in pTUBE638-alpha-amylase (E24) was completely processed by the extracellular alkaline protease of B. subtilis alone at pH 7.5 to 10.0. The processing was inhibited by 2 mM PMSF. In contrast, the neutral protease did not process the extended peptide. The processing activity of the purified alkaline protease was fully active in 100 mM phosphate and glycine-NaCl-NaOH buffer while it was partially active in 100 mM Tris-HCl or MOPS buffer. The optimum pH of the activity ranged from 8.0 to 9.0, although the optimum pH of the alkaline protease activity toward casein and Azocoll was 10.5. The NH2-terminal amino acid sequences of the enzymes processed in vitro coincided with those of the mature extracellular thermostable alpha-amylases in the culture medium of B. subtilis (pTUBE638). The appearance of the processing activity of alkaline protease was correlated with the changes of the pH in the culture medium.
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PMID:Processing of an NH2-terminally extended thermostable alpha-amylase by Bacillus subtilis alkaline protease. 212 90

The sacQ gene from Bacillus licheniformis was cloned and expressed in Bacillus subtilis. Deletion analysis shows that it encodes a 46-amino-acid polypeptide homologous to the B. subtilis sacQ gene product. The polypeptide, when it is overexpressed, activates the expression of a number of target genes in B. subtilis, all encoding secreted enzymes: alkaline protease, levansucrase, beta-glucanase(s), xylanase, and alpha-amylase. The maximum stimulations measured for alkaline protease and levansucrase were by a factor of 70 and 50, respectively, when the sacQ gene from B. licheniformis was present on a multicopy plasmid in B. subtilis. The sacQ genes from B. subtilis and B. licheniformis, cloned in the same multicopy plasmid, were compared under the same conditions. The sacQ gene from B. licheniformis was more efficient than the sacQ gene from B. subtilis in producing the hypersecretion phenotype. The sacQ structural genes from B. subtilis and B. licheniformis were placed under the control of the same inducible promoter. Hypersecretion was specifically obtained under conditions of full induction of the promoter. The target site of levansucrase regulation by sacQ was identified as a 440-base-pair fragment located in the 5' noncoding region of sacB, suggesting transcriptional control.
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PMID:Characterization of the sacQ genes from Bacillus licheniformis and Bacillus subtilis. 309 32

Studies were performed on the prtR gene which enhances the production of the Bacillus subtilis extracellular proteases and levansucrase, but not the alpha-amylase, RNase, and alkaline phosphatase. To investigate the mode of action of prtR, the Escherichia coli bla gene was placed under the control of two promoters. One was the promoter of the alkaline protease gene (aprE), and the other was the promoter of B. subtilis dihydrofolate reductase gene (dfrA). Expression of the bla gene was enhanced by prtR only when the apr promoter was used. From these results, it was concluded that the apr promoter or its vicinity was the target of prtR and that prtR does not affect the process after transcription. The mRNA levels of aprE and nprE (the neutral protease gene) were significantly increased by prtR, but the half-life of the aprE mRNA was not affected. These results show that the prtR gene product enhances protease production by increasing the rate of transcription initiation.
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PMID:prtR enhances the mRNA level of the Bacillus subtilis extracellular proteases. 311 Jan 32

Some regularities of the enzyme immunoassay (EIA) of whole bacterial cells have been studied on one of the bacillary species of contaminant microflora. Early detection of this microorganism is highly important for the microbiological production of alpha-amylase and alkaline protease (produced by Bacillus subtilis). The effective kinetic and equilibrant parameters of the interaction of peroxidase-labeled antibodies with the cells of the contaminant microflora in the solution and on the surface of the polystyrene plates have been defined. Two methods for the separation of cells after their interaction with peroxidase-labeled antibodies have been optimized: filtration involving the use of special filter plates and centrifugation in plates. The method for the immobilization of cells in the wells of standard assay plates by centrifugation has been proposed. Four EIA methods for measurement of contaminant microflora have been developed and optimized. These methods permit the determination of the microflora at concentration of 5 X 10(5)-5 X 10(4) cells/ml, depending on the scheme of the assay, within 1-3.5 minutes.
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PMID:[Patterns in the immunoenzyme analysis of bacterial cells]. 312 Apr 44

A transformable strain of Bacillus subtilis 6160, a derivative of B. subtilis 168, produces three kinds of casein hydrolytic enzymes (alkaline protease, neutral protease, and esterase) in a culture medium. B. natto IAM 1212 produces 15 to 20 times as much total proteolytic activity as does B. subtilis. Extracellular proteases produced by the two strains were separated into each enzyme fraction by diethylaminoethyl-Sephadex A-50 column chromatography. The difference in the total protease activities of extracellular proteases between the two strains was due to the amount of neutral protease. The ratios of neutral protease activity to alkaline protease activity (N/A) were 1.1 in B. subtilis 6160 and 13.0 in B. natto IAM 1212. Enzymological and immunological properties of alkaline protease and neutral protease obtained from the two strains were quite similar or identical, respectively. Specific activities measured by an immunological analysis of the two neutral proteases against casein were also equal. A genetic character of high protease productivity in B. natto IAM 1212 was transferred to B. subtilis 6160 by the deoxyribonucleic acid-mediated transformation. Among 73 transformants that acquired high protease productivity, 69 produced a higher amount of neutral protease and the ratios of N/A were changed to 15 to 60. Three other strains were transformed in the productivity of neutral protease and alpha-amylase simultaneously, and one showed considerable change in the production of alkaline protease and neutral protease. The specific activities (casein hydrolytic activities/enzyme molecules) of neutral proteases from the representative four transformants were equal to those of the two parental strains. These results suggested the presence of a specific gene(s) that participated in the productivity of neutral protease in B. subtilis.
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PMID:Regulation of neutral protease productivity in Bacillus subtilis: transformation of high protease productivity. 420 70

A deletion of the spoIIAC gene of Bacillus licheniformis was prepared in vitro by using the splicing-by-overlap-extension technique. This gene was introduced into B. licheniformis on a temperature-sensitive plasmid, and following integration and excision from the chromosome, a precisely located deletion on the chromosomal gene was prepared. The mutated bacterium was totally asporogenous and formed abortively disporic cells characterized by asymmetric septa at the poles of the cells. Qualitative plate tests revealed that the bacterium synthesized normal levels of DNase, polygalacturonate lyase, protease, RNase, and xylanase, but the hydrolysis zones due to beta-1,3-glucanase and carboxymethyl cellulase activity were smaller in the mutant than in the parent strain. The synthesis of alkaline protease was the same in batch cultures of the mutant and the parent during prolonged incubation for 72 h, but the alpha-amylase yields were reduced by about 30% by the mutation.
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PMID:Extracellular enzyme synthesis in a sporulation-deficient strain of Bacillus licheniformis. 852 85

Thirty-six employees who produced industrial enzymes from selected strains of bacteria and fungi were evaluated by epicutaneous threshold testing and enzyme-linked immunosorbent assays (ELISA) for specific IgE and IgG antibodies. The workers complained of 'asthma- and flu-like' symptoms, which generally lessened away from work. The enzymes evaluated were: alpha-amylase (1,4-alpha-d-glucan glucanohydrolase) from Bacillus licheniformis (alpha ABl), B. subtilis formation 1 (alpha A1Bs) and B. subtilis formation 2 (alpha A2Bs); purified alpha-amylase from B. licheniformis (C alpha ABl) and A. oryzae (C alpha AAo); alkaline protease from B. licheniformis (APBl) and purified alkaline protease (CAPBl); amyloglucosidase (1,4-alpha-d-glucan glucohydrolase) from A. niger (AGAn) and purified amyloglucosidase (CAGAn). Statistically significant increases (P > 0.05) in the proportion of workers having positive skin tests to CAPBl, AGAn and CAGAn were found. Significantly elevated (P > 0.05) mean specific IgE results were observed for C alpha AAo CAGAn and AGAn, and elevated (P > 0.05) mean specific IgGs were observed for C alpha AAo, CAGAn, AGAn, alpha A1Bs, alpha AB1 and alpha A2Bs. These results indicate that occupational exposure to some industrial enzymes can cause immediate-onset cutaneous hypersensitivity reactions, pulmonary function deficits and significantly elevated specific antibody levels. Our results are equivocal as to whether work-related respiratory and cutaneous hypersensitivity reactions are antibody mediated, as there was no statistically significant association between these reactions and specific IgE or IgG levels.
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PMID:Hypersensitivity reactions and specific antibodies in workers exposed to industrial enzymes at a biotechnology plant. 893 88

A cold alkaline protease, isolated from an Antarctic Pseudomonas aeruginosa strain, has been purified and crystallized. Large crystals were obtained in the presence of PEG 6000 at pH 7 and pH 8. They belong to the space group P2(1)2(1)2(1). A complete data set to 2.1 A resolution has been measured. The structure has been determined by the molecular replacement method using the coordinates of the mesophilic alkaline protease as a model. The molecular replacement solution displays a correlation coefficient of 0.39 and an R-factor of 0.48. Subsequent inspection of the electron density map in the active site region has confirmed the correctness of the solution. Model building and structure refinement will be initiated when the protease sequence becomes fully available. This is the second report, following one on an alpha-amylase, of the preliminary crystallographic characterization of a psychrophilic enzyme.
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PMID:Preliminary crystal structure determination of the alkaline protease from the Antarctic psychrophile Pseudomonas aeruginosa. 938 50


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