Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.2.1.17 (lysozyme)
 21,489 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A new method for determining the specificity of hydrolysis of the linear binary heteropolysaccharide chitosan composed of (1-->4)-linked 2-acetamido-2-deoxy-beta-D-glucopyranose (GlcNAc; A-unit) and 2-amino-2-deoxy-beta-D-glucopyranose (GlcN; D-unit) residues is described. The method is based on the assignments of the 13C chemical shifts of the identity (A- or D-units) of the new reducing and non-reducing ends and the variation in their nearest neighbours, using low molecular weight chitosans with known random distribution of A- and D-units as substrate. A highly N-acetylated chitosan with fraction of acetylated units (FA) of 0.68 and a number-average degree of polymerization (DPn) of 30 was hydrolysed with hen egg-white lysozyme, showing that both the new reducing and non-reducing ends consisted exclusively of A-units, indicating a high specificity for A-units in subsites DL and EL on lysozyme. Our data suggests that the preceding unit of the reducing A-units, is invariable, and based on earlier studies, most probably an A-unit, while the unit following the non-reducing A-units can be either an A- or a D-unit. A more detailed study of the specificity of lysozyme at subsite DL was performed by hydrolyzing a more deacetylated chitosan (FA = 0.35 and DPn of 20) to a DPn of 9, showing that even for this chitosan more than 90% of the new reducing ends were acetylated units. Thus, lysozyme depolymerizes partially N-acetylated chitosans by preferentially hydrolyzing sequences of acetylated units bound to site CL, DL and EL of the active cleft, while there is no specificity between acetylated and deacetylated units to site FL. In addition, a moderately N-acetylated chitosan with fraction of acetylated units (FA) of 0.35 and a DPn of 20 was hydrolysed with Bacillus sp. No. 7-M chitosanase, showing that both the new reducing and non-reducing ends consisted exclusively of D-units. Our data suggests that the nearest neigbour to the D-unit at the reducing end is invariable, and based on earlier studies, most probably a D-unit, while the unit following the non-reducing D-units can be either an A- or a D-unit. We conclude that the Bacillus chitosanase hydrolyzes partially N-acetylated chitosan by preferentially attacking sequences of three consecutive deacetylated units, hypothetical subsites CC, DC and EC, where the cleavage occur between sugar units bound to subsites DC and EC. A hypothetical subsite FC on the chitosanase show no specificity with respect to A- and D-units. The new NMR method described herein offers a time and labour-saving alternative to the procedure of extensive hydrolysis of the binary heteropolysaccharide chitosan and subsequent isolation and characterization of the oligosaccharides.
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PMID:Determination of enzymatic hydrolysis specificity of partially N-acetylated chitosans. 878 19

Seven endochitinases (EC 3.2.1.14) (relative molecular masses 23,000-28,000 and isoelectric points 10.3-10.4) were purified from nonembryogenic Citrus sinensis L. Osbeck cv. Valencia callus tissue. The basic chitinase/lysozyme from this tissue (BCLVC) exhibited lysozyme, chitinase and chitosanase activities and was determined to be a class III chitinase. While BCLVC acted as a lysozyme at pH 4.5 and low ionic strength (0.03) it acted as a chitinase/chitosanase at high ionic strengths (0.2) with a pH optimum of ca. 5. The lysozyme activity of BCLVC was inhibited by histamine, imidazole, histidine and the N-acetyl-D-glucosamine oligosaccharide (GlcNAc)3. The basic chitinase from cv. Valencia callus, BCVC-2, had an N-terminal amino acid sequence similar to tomato and tobacco AP24 proteins. The sequences of the other five chitinases were N-terminal blocked. Whereas BCLVC was capable of hydrolyzing 13.8-100% acetylated chitosans and (GlcNAc)4-6 oligosaccharides, BCVC-2 hydrolyzed only 100% acetylated chitosan, and the remaining enzymes expressed varying degrees of hydrolytic capabilities. Experiments with (GlcNAc)2-6 suggest that BCLVC hydrolysis occurs in largely tetrasaccharide units whereas hydrolysis by the other chitinases occurs in disaccharide units. Cross-reactivities of the purified proteins with antibodies for a potato leaf chitinase (AbPLC), BCLVC, BCVC-3, and tomato AP24 indicate that these are separate and distinct proteins.
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PMID:Characterization of seven basic endochitinases isolated from cell cultures of Citrus sinensis (L.). 893 50

Novel information on the structure and function of chitosanase, which hydrolyzes the beta-1,4-glycosidic linkage of chitosan, has accumulated in recent years. The cloning of the chitosanase gene from Streptomyces sp. strain N174 and the establishment of an efficient expression system using Streptomyces lividans TK24 have contributed to these advances. Amino acid sequence comparisons of the chitosanases that have been sequenced to date revealed a significant homology in the N-terminal module. From energy minimization based on the X-ray crystal structure of Streptomyces sp. strain N174 chitosanase, the substrate binding cleft of this enzyme was estimated to be composed of six monosaccharide binding subsites. The hydrolytic reaction takes place at the center of the binding cleft with an inverting mechanism. Site-directed mutagenesis of the carboxylic amino acid residues that are conserved revealed that Glu-22 and Asp-40 are the catalytic residues. The tryptophan residues in the chitosanase do not participate directly in the substrate binding but stabilize the protein structure by interacting with hydrophobic and carboxylic side chains of the other amino acid residues. Structural and functional similarities were found between chitosanase, barley chitinase, bacteriophage T4 lysozyme, and goose egg white lysozyme, even though these proteins share no sequence similarities. This information can be helpful for the design of new chitinolytic enzymes that can be applied to carbohydrate engineering, biological control of phytopathogens, and other fields including chitinous polysaccharide degradation.
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PMID:Chitosanase from Streptomyces sp. strain N174: a comparative review of its structure and function. 959 57

Soluble chitosan and poly-L-lysine are readily hydrolysed using lysozyme or chitosanase for chitosan, and trypsin, chymotrypsin or proteinase K for poly-L-lysine. For similar amounts of enzyme, chitosanase hydrolysed 57% of the chitosan, compared to 35% for lysozyme. In the case of poly-L-lysine, chymotrypsin and trypsin exhibited similar activities, hydrolysing approximately 41% of the polymer compared to proteinase K at only 16%. In contrast, chitosan and poly-L-lysine membranes, coating alginate beads, were almost totally inert to the respective hydrolytic enzymes. Less than 2% of the membrane weight was hydrolysed. It appears that either membrane material would be stable for in vivo application, and in particular in the protection of DNA during gastrointestinal transit. At chitosanase concentrations of 1.4 mg/ml and in the presence of sodium ions, 20% of the total double-stranded DNA was released from chitosan coated beads. An exchange of calcium for sodium within the bead liquefied the alginate core releasing DNA. The presence of calcium stabilized the alginate bead, retaining all the DNA. Highly pure DNA was recovered from beads through mechanical membrane disruption, core liquefaction in citrate and use of DNA spin-columns to separate DNA/alginate mixtures in a citrate buffer. DNA recovery efficiencies as high as 94% were achieved when the initial alginate/DNA weight ratio was 1000.
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PMID:Stability of chitosan and poly-L-lysine membranes coating DNA-alginate beads when exposed to hydrolytic enzymes. 997 4

This article describes the preparation of porous chitosan, a hydroxyapatite hybrid, by partial enzymatic degradation. Two enzymes, chitosanase and lysozyme, were selected to hydrolyze a chitosan-reinforced matrix and create pores within the chitosan-hydroxyapatite composite. The degree of enzymatic hydrolysis of the chitosan-hydroxyapatite composite was determined by measuring the % weight loss of the chitosan matrix and the hydroxyapatite component. Hydroxyapatite loss from the chitosan matrix increased with the degree of enzymatic hydrolysis of the chitosan-reinforced matrix. After hydrolysis, the composite was further characterized by FTIR. Quantitative analysis revealed a decrease in the characteristic pyranose ring peak (1072 cm(-1)), compared with Po4(2-) (1110 cm(-1)), showing that the chitosan matrices were enzymatically hydrolyzed. The surface of the porous chitosan-hydroxyapatite composite, prepared by controlling enzymatic hydrolysis, was also observed by SEM.
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PMID:Inorganic-organic polymer hybrid scaffold for tissue engineering--II: partial enzymatic degradation of hydroxyapatite-chitosan hybrid. 1246 61

Two types of biocompatible nanoparticles with an average diameter of around 200 nm were formed only by mixing hydrolysates of chitosan and carboxymethyl cellulose (CMC). Nanoparticle A was produced from chitosanase hydrolysate of chitosan and cellulase hydrolysate of carboxymethyl cellulose, and nanoparticle B was produced from lysozyme hydrolysate of chitosan and the carboxymethyl cellulose hydrolysate. Negatively charged or amphoteric compounds were first mixed with chitosan hydrolysate and then added to carboxymethyl cellulose hydrolysate to effectively entrap them in the particles. Positively charged compounds could also be effectively entrapped by mixing the hydrolysates and the compound in the reverse order. Negatively charged compounds with high molecular weights were maintained in the particles even at the higher pH levels than the pK(a) of the amino groups of chitosan. Entrapped compounds were gradually released from nanoparticle A by lysozyme treatment. In contrast, there was no release from nanoparticle B. These results indicate that nanoparticle A can be applied to controlled-release drug delivery systems, and that nanoparticle B is stably retained in the body without releasing the entrapped compounds.
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PMID:Entrapment of some compounds into biocompatible nano-sized particles and their releasing properties. 1583 66

According to published DNA sequence of Aspergillus fumigatus chitosanase(Csn) gene, 8 long single DNA strands each about 100bp and 4 DNA primers were designed and synthesised. By PCR, 8 DNA strands were connected into a complete chitosanase gene of 624bp. This chitosanase gene was not identical with its wild type, some point mutations were introduced into its DNA sequence by special design of those 8 DNA strands. These mutaions did not change amino acid composition of the chitosanase, however, the codens were changed into E. coli favorites. The Csn gene was cloned into plasmid pGEM-Teasy and verified by DNA sequence analysis. Thereafter, Csn gene was subcloned into a fusion-protein expressing vector pGEX-3X. Recombinant plasmid pGEX-Csn was transformed into E. coli DH5alpha and the transformant was induced expressing with 0.5 mmol/L IPTG. Expressing product was analyzed by SDS-PAGE, fusion protein GST-Csn was purified by affinity chromatography. By factor X a digestion GST-Csn was cleaved and GST was taken out by another chromatography. The biological activity of recombinant chitosanase(rCsn) was also detected, as a result the recombinant Csn had a strong ability of degrading chitosan, which was much higher than lysozyme. Its chitosan-degradation activity could be influenced by pH and temperature.
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PMID:[Cloning and expression of an Aspergillus fumigatus chitosanase gene]. 1624 66

A group of chitin-binding proteins were isolated from tuberous roots of Raphanus sativus by affinity chromatography with deaminated regenerated chitin (Fig. 1). SDS-PAGE showed that there are at least five proteins in the sample (Fig. 2-b). Through carboxyl methyl-cellulose chromatography, two chitin-binding proteins with lysozyme activity, named as CBP1 and CBP2 (Fig. 3), were purified to homogeneity with the molecular weights of 26.9 kD and 24.8 kD respectively (Fig. 2-d, e). CBP1 and CBP2 were found to be bifunctional enzymes with activities of lysozyme and chitinase (Figs. 4, 5), but without chitosanase activity (Table 1). The CBP1 and CBP2 could be specifically absorbed by various forms of chitin, such as powdered, regenerated and colloidal forms chitin (Fig. 6). No disulfide bridge was observed in CBP1 and CBP2 by reduced/nonreduced one-dimensional SDS-PAGE (Fig. 7).
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PMID:[Purification and characterization of two chitin-binding proteins with lysozyme activity from roots of Raphanus sativus]. 1695 96

Besides the specific chitinase, chitosanase and lysozyme, chitosan also could be hydrolyzed by some non-specific enzymes such as cellulase, protease, lipase and pepsin, especially cellulase, which show high activity on chitosan. Almost all the cellulases produced by different kinds of microorganisms could degrade chitosan to chitooligomers. The existence of bifunctional enzymes with cellulase and chitosanase activity is one of the reasons for cellulase on chitosan hydrolysis. The bifunctional cellulase-chitosanases mainly belong to glycoside hydrolase family 8 (GH-8), few belong to GH-5 and GH-7, according to the homogeneity analysis of amino acids sequences. Their three dimensional structures however have not been clearly determined. This paper may serve as a guide for a further study on the relationship between structure and function of chitosanolytic cellulases.
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PMID:Advance in chitosan hydrolysis by non-specific cellulases. 1832 93

Chitosan has been shown to be effective in regulating progenitor salivary tissue morphogenesis, however, the specificity of chitosan effects remains unclear. To assess the regulatory ability of chitosan in salivary gland morphogenesis, progenitor salivary tissue from embryonal submandibular gland (SMG) was cultured in chitosan-containing medium. It was found that soluble chitosan was able to promote SMG branching in a dose-dependent manner. The effect was chitosan-specific and was not reproduced by substrates with similar chemical structures or other polymeric molecules of natural or synthetic origin. Furthermore, the branch-promoting effects were molecular weight-dependent. In addition, following digestion with lysozyme, chitinase, or chitosanase, digested chitosan was unable to reproduce the similar effects. In all, this study clarifies the specificity and preferential activity of chitosan in enhancing branching morphogenesis of progenitor salivary tissue and highlights its potential utility for application in salivary tissue regeneration.
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PMID:The specificity of chitosan in promoting branching morphogenesis of progenitor salivary tissue. 1898 27


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