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Query: UMLS:C0272170 (SDS)
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Extracellular chitinases of Streptomyces peucetius and a chitinase overproducing mutant, SPVI, were purified to homogeneity by ion exchange and gel filtration chromatography. The purified enzyme has a molecular mass of 42 kDa on SDS-PAGE, and the N-terminal amino acid sequence of the protein from the wild type showed homology to catalytic domains (Domain IV) of several other Streptomyces chitinases such as S. lividans 66, S. coelicolor A3(2), S. plicatus, and S. thermoviolaceus OPC-520. Purified SPVI chitinase cross-reacted to anti-chitinase antibodies of wild-type S. peucetius chitinase. A genomic library of SPVI constructed in E. coli using lambda DASH II was probed with chiC of S. lividans 66 to screen for the chitinase gene. A 2.7 kb fragment containing the chitinase gene was subcloned from a lambda DASH II clone, and sequenced. The deduced protein had a molecular mass of 68 kDa, and showed domain organization similar to that of S. lividans 66 chiC. The N-terminal amino acid sequence of the purified S. peucetius chitinase matched with the N-terminus of the catalytic domain, indicating the proteolytic processing of 68 kDa chitinase precursor protein to 42 kDa mature chitinase containing the catalytic domain only. A putative chiR sequence of a two-component regulatory system was found upstream of the chiC sequence.
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PMID:Purification, cloning, and DNA sequence analysis of a chitinase from an overproducing mutant of Streptomyces peucetius defective in daunorubicin biosynthesis. 1131 8

A novel goose-type lysozyme was purified from egg white of cassowary bird (Casuarius casuarius). The purification step was composed of two fractionation steps: pH treatment steps followed by a cation exchange column chromatography. The molecular mass of the purified enzyme was estimated to be 20.8 kDa by SDS-PAGE. This enzyme was composed of 186 amino acid residues and showed similar amino acid composition to reported goose-type lysozymes. The N-terminal amino acid sequencing from transblotted protein found that this protein had no N-terminal. This enzyme showed either lytic or chitinase activities and had some different properties from those reported for goose lysozyme. The optimum pH and temperature on lytic activity of this lysozyme were pH 5 and 30 degrees C at ionic strength of 0.1, respectively. This lysozyme was stable up to 30 degrees C for lytic activity and the activity was completely abolished at 80 degrees C. The chitinase activity against glycol chitin showed dual optimum pH around 4.5 and 11. The optimum temperature for chitinase activity was at 50 degrees C and the enzyme was stable up to 40 degrees C.
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PMID:Purification and characterization of goose type lysozyme from cassowary (Casuarius casuarius) egg white. 1133 Jun 72

A G561 mutant of the Aeromonas caviae chitinase ChiA was made by PCR site-directed deletion mutagenesis in order to study the role of the 304 C-terminal amino acid residues of ChiA in the enzymatic hydrolysis of chitin. The recombinant ChiAG561 encoded on a 1.6-kb DNA fragment of A. caviae chiA was expressed in a heterologous Escherichia coli host using the pET20b(+) expression system. The His-Tag-affinity-purified recombinant ChiAG561 had a calculated molecular mass of 63,595 Da, which was consistent with the 67,000 Da estimated by SDS-PAGE. The G561 deletion mutant enzyme had the same optimum pH (6.5) as the full-length ChiA and a lower optimum temperature (37 degrees C instead of 42.5 degrees C). Biochemical properties of the recombinant ChiAG561 suggested that deletion of the 304 C-terminal amino acid residues of ChiA did not significantly affect ChiA enzyme activity. However, compared to the full-length ChiA, the mutant chitinase had a ten-fold higher relative activity with 4-methylumbelliferyl-N-N'-N"-triacetylchitotriose [4-MU-(GlcNAc)3] as a substrate, and higher rates of hydrolysis with both chitin and colloidal chitin substrates. Results obtained from this study suggest that the active region of A. caviae ChiA is located in the region before G561 of the protein molecule.
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PMID:G561 site-directed deletion mutant chitinase from Aeromonas caviae is active without its 304 C-terminal amino acid residues. 1135 14

The mechanisms of incorporation of two antigens have been determined using a monoclonal antibody (3A10) raised against the material released from the mycelial cell wall by zymolyase digestion and retained on a concanavalin A column. One of the hybridomas secreted an IgG that reacted with two bands in Western blots. Indirect immunofluorescence showed that the antigens were located on the surfaces of mycelial cells, but within the cell walls of yeasts. These antigens were detected in a membrane preparation, in the SDS-soluble material and in the material released by a 1,3-beta-glucanase and chitinase from the cell walls of yeast and mycelial cells. In the latter three samples, an additional high-molecular-mass, highly polydispersed band was also detected. Beta-elimination of each fraction resulted in the disappearance of all antigen bands, suggesting that they are highly O-glycosylated. In addition, the electrophoretic mobility of the high-molecular-mass, highly polydispersed bands increased after digestion with endoglycosidase H, indicating that they are also N-glycosylated. New antigen bands were released when remnants of the cell walls extracted with 1,3-beta-glucanase or chitinase were digested with chitinase or 1,3-beta-glucanase. These results are consistent with the notion that, after secretion, parts of the O-glycosylated antigen molecules are transferred to an N-glycosylated protein(s). This molecular complex, as well as the remaining original 70 and 80 kDa antigen molecules, next bind to 1,3-beta-glucan or chitin, probably via 1,6-beta-glucan, and, in an additional step, to chitin or 1,3-beta-glucan. This process results in the final molecular product of each antigen, and their distribution in the cell walls.
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PMID:Secretion, interaction and assembly of two O-glycosylated cell wall antigens from Candida albicans. 1142 75

Chitinases are produced throughout the growth process of fungi and are thought to play important roles in morphogenesis. Aspergillus fumigatus, is an important pathogen of immunocompromised individuals in which it causes pneumonia and invasive disseminated disease with high mortality; it is also known to produce chitinase. We have induced an exceptionally stable extracellular chitinase in A. fumigatus YJ-407, which could be isolated readily in a homogeneous form by using ammonium sulfate precipitation followed by DEAE-cellulose chromatography and preparative PAGE. The molecular mass of this chitinase was estimated to be 46 000 by SDS/PAGE, and its isoelectric point was pH 5.6. The enzyme was most active at pH 5.0 and 60 degrees C, and was inhibited strongly by Hg2+, Pb2+, Ag+, Fe2+, Mn2+ and Zn2+. The enzyme was stable over a broad pH range 4-8 and below 45 degrees C. Tryptophan and carboxyl groups were found to be essential for the enzyme activity. The Michaelis constants for swollen chitin and chitosan were 1.12 mg.mL-1 and 1.84 mg.mL-1, respectively. The enzyme showed maximum activity towards glycol chitin and partially deacetylated chitosan, and lower activity towards colloidal chitin. Analysis of the hydrolysis product showed that the enzyme has both endo- and exo-hydrolytic activities. In addition, a transglycosyl activity was also observed.
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PMID:A novel chitinase having a unique mode of action from Aspergillus fumigatus YJ-407. 1145 2

The chitinase gene (chiA71) from Bacillus thuringiensis subsp. pakistani consists of an open reading frame of 1,905 nucleotides encoding 635 amino acid residues with an estimated molecular mass of 71 kDa. Comparison of the deduced amino acid sequence of the mature enzyme to other microbial chitinases shows a putative catalytic domain and a region with conserved amino acids similar to that of the type III module of fibronectin and a chitin-binding domain. By activity detection of chitinase on SDS-PAGE after renaturation, the molecular mass of protein bands with chitinase activity were 66, 60, 47, and 32 kDa. The N-terminal amino acid sequence of each chitinase activity band was the same (Asp-Ser-Pro-Lys-Gln), suggesting that the 60-, 47-, and 32-kDa chitinases were derived from the 66-kDa chitinase by processing step(s) at the C-terminus. The enzyme was identified as an exochitinase, since it generated N-acetylglucosamine from early stage of colloidal chitin hydrolysis. The crude protein (2.3-18.4 mg/ml), containing chitinase at final activities of 8, 16, 32, and 64 mU/ml, was toxic to Aedes aegypti larvae and caused mortalities of 7.5, 15.0, 51.3, and 70.0% respectively, but the same amount of crude protein from a B. thuringiensis subsp. pakistani mutant lacking chitinase was not toxic.
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PMID:Chitinase from Bacillus thuringiensis subsp. pakistani. 1154 8

Protein extracts from soybean (Glycine max [L.] Merr) seed hulls were fractionated by isoelectric focusing and SDS-PAGE analysis and components identified by peptide microsequencing. An abundant 32 kDa protein possessed an N-terminal cysteine-rich hevein domain present in class I chitinases and in other chitin-binding proteins. The protein could be purified from seed coats by single step binding to a chitin bead matrix and displayed chitinase activity by an electrophoretic zymogram assay. The corresponding cDNA and genomic clones for the chitinase protein were isolated and characterized, and the expression pattern determined by RNA blot analysis. The deduced peptide sequence of 320 amino acids included an N-terminal signal peptide and conserved chitin-binding and catalytic domains interspaced by a proline hinge. An 11.3 kb EcoRI genomic fragment bearing the 2.4 kb chitinase gene was fully sequenced. The gene contained two introns and was flanked by A+T-rich tracts. Analysis by DNA blot hybridization showed that this is a single or low copy gene in the soybean genome. The chitinase is expressed late in seed development, with particularly high expression in the seed coat. Expression was also evident in the late stages of development of the pod, root, leaf, and embryo, and in tissues responding to pathogen infection. This study further illustrates the differences in protein composition of the various seed tissues and demonstrates that defence-related proteins are prevalent in the seed coat.
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PMID:A class I chitinase from soybean seed coat. 1170 78

Several chitinases were expressed in a rice cell suspension culture and detected in the medium. One of them, designated as RCB4, was isolated 248 fold from the culture filtrate to homogeneity by 70% ammonium sulfate precipitation, DEAE-cellulose, CM-cellulose, Sephadex G-75 column chromatography, and native gel slicing. RCB4 had a molecular mass of 32 kDa by SDS-PAGE. The optimum temperature was 40 degrees C, and 96% of its activity still remained at 60 degrees C. The optimum pH was 4, and 95% of its activity was maintained at pH 2. Using a substrate (GlcNAc)6, the Km and Vmax values of RCB4 were 0.53 mM and 11.1 mM/min, respectively. The N-terminal and internal amino acid sequences of RCB4 were determined to be VNSNLFRDYIGA and MALWA, respectively. A cDNA (C12523) clone that contained the N-terminal and internal amino acid sequences of RCB4 was obtained, sequenced, and renamed RCB41. RCB41 encoded 307 amino acid protein with a signal peptide of 25 amino acids and showed a 45% similarity to gladiolus chitinase GBC-a, one of the class III chitinase family. The expression of RCB4l in E. coli showed that RCB41 encodes a chitinase.
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PMID:Purification, characterization, and cDNA cloning of rice class III chitinase. 1191 77

Transglutaminase activity, which forms the interpeptidic cross-link N(epsilon)-(gamma-glutamyl)-lysine, was demonstrated in cell-free extracts of Saccharomyces cerevisiae by incorporation of [(14)C]lysine into an exogenous acceptor, N,N'-dimethylcasein. Higher levels of the activity were present in the cell wall, which also contained endogenous acceptors. The enzyme activity in the wall was inhibited by cystamine, a known inhibitor of transglutaminase, and by EDTA, indicating a cation-dependent activity. After the endogenous wall acceptors were labelled radioactively by transglutaminase, extraction with SDS solubilized about 50% of the total radioactivity, while Zymolyase and chitinase each released a further 3%. The proteins solubilized by SDS had molecular masses less than 50 kDa, whereas the material released by Zymolyase or chitinase had molecular masses greater than 180 kDa, suggesting a precursor-product relationship. Cystamine inhibited the growth of several strains of S. cerevisiae. Treated cells showed increased sensitivity to Zymolyase and appeared as protoplasts, indicating gross alterations in the cell wall. These data suggest that transglutaminase may be involved in the formation of covalent cross-links between wall proteins during wall construction.
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PMID:Transglutaminase activity is involved in Saccharomyces cerevisiae wall construction. 1198 6

Isolates of five species of the yeast-like fungus Tilletiopsis Derx (Tilletiopsis albescens Gokhale, Tilletiopsis fulvescens Gokhale, Tilletiopsis minor Nyland, Tilletiopsis pallescens Gokhale, and Tilletiopsis washingtonensis Nyland) were screened for exo- and endo--beta-1,3-glucanase and chitinase production in a liquid broth used to produce inoculum for biological control studies. There were significant differences among the species, and highest overall enzyme activity was present in T albescens and T. pallescens and lowest in T. washingtonensis. A time-course study of beta-1,3-glucanase and chitinase production in T pallescens ATCC 96155 in broth culture with 2.5% glucose as the carbon source showed that enzyme activity gradually increased over a 3- to 21-day period. Maximum enzyme activity was found between pH 4.0 and 5.0. SDS-PAGE of beta-1,3-glucanase isozymes revealed a range of molecular masses from 18 to 29 kDa. Five isozymes were present in both T albescens and T. pallescens and two in T washingtonensis. Antifungal compounds were also detected in ethyl acetate extracts of culture filtrates of T. pallescens ATCC 96155 after 6 days of incubation, while no activity was detected at 14 days. One active fraction was selected following fractionation and preparative chromatography and was bioassayed against Podosphaera (sect. Sphaerotheca) xanthii (Castagne) U. Braun & N. Shishkoff and a number of other fungi. A concentration of 130 microg/mL inhibited germ tube development in P. xanthii, and mildew spores appeared plasmolyzed. Other fungi were inhibited at higher concentrations. Collapse of hyphae and conidiophores was also observed on mildewed leaves treated with the active fraction. Proton NMR analysis indicated that the inhibitory compound was a fatty acid ester. In 3- to 6-day-old cultures of T pallescens ATCC 96155 demonstrating biological control activity, antifungal compound production may have a primary role in restricting growth of mildew fungi and other competitors when applied to leaves.
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PMID:Hydrolytic enzymes and antifungal compounds produced by Tilletiopsis species, phyllosphere yeasts that are antagonists of powdery mildew fungi. 1198 66


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