Substrate chitin

Figure 4. Hydrolysis of pendant urethane groups as a function of medium. Pendant CH, —NHCOO— substrate chitin (A) basic medium (pH - 11.3) acidic medium (pH - 3.1) (9) deionized HtO (pH = 7.0)...

Chitin is the most abundant biomacromolecule in the animal field, which is found normally in invertebrates as a structural component. This important polysaccharide was synthesized for the first time by the enzymatic polymerization using chitinase and a chitobiose oxazoline derivative (Scheme 14).131 The latter activated monomer has a distorted structure with an a configuration at Cl, which resembles a transition-state structure of substrate chitin at the active site during a hydrolysis process (Scheme 15).3b 131132 The ring-opening polyaddition of the chitobiose oxazoline derivative was exclusively promoted by chitinase at pH 10.6, where the hydrolytic activity of chitinase was very much lowered. 

A number of modification products were isolated by ion-exchange chromatography for a mixture of 7V-carboxymethylated derivatives (CM derivatives) of lysozyme, produced by treatment of the protein with mono-iodoacetic acid. In each CM derivative the position of the modified amino-acid residues was established. Possible causes of such selectivity of the process of carboxymethyla-tion are discussed. It was also shown that salt activation of CM derivatives of lysozyme proceeds more readily, the higher the degree of their modification. As a result of this, the enzymatic activity of CM derivatives of lysozyme is higher than that of the native enzyme, which is correlated with their increased affinity for the substrate (chitin). 

Various modification products have been isolated by chromatography on Amberlite CG-50 of a mixture of A-carboxymethylated derivatives of lysozyme. In each carboxymethyl derivative the position of the modified amino-acid was established. It was found that L-histidine-15, L-lysines-1, -33, -96, or -97 are blocked, whereas L-lysines-13 and -116 remain unaffected. Possible causes of this selectivity were discussed. Salt activation of carboxy-methyl derivatives of lysozyme proceeds more readily the higher the degree of modification, and the enzymatic activity of carboxymethyl derivatives of lysozyme is higher than that of the native enzyme owing to their increased affinity for substrate (chitin). 

Fungal chitin synthases are found as integral proteins of the plasma membrane and in chitosomes a divalent cation, Mg(II), is necessary for enzyme activity but neither primers nor a hpid intermediate are required. The substrate and free GlcNAc activate the allosteric enzyme. UDP, the byproduct of the enzymatic activity, is strongly inhibitory to chitin synthase however, it may be metabohzed readily to UMP by a diphosphatase. 

In concentrated NaOH, chitin becomes alkali chitin which reacts with 2-chloroethanol to yield 0-(2-hydroxyethyl) chitin, known as glycol chitin this compoimd was probably the first derivative to find practical use (as the recommended substrate for lysozyme). Alkali chitin with sodium monochloroacetate yields the widely used water-soluble 0-carboxymethyl chitin sodium salt [118]. The latter is also particularly susceptible to lysozyme, and its oUgomers are degraded by N-acetylglucosaminidase, thus it is convenient for medical appHcations, including bone regeneration. 

Poly(HASCL) depolymerases are able to bind to poly(3HB)-granules. This ability is specific because poly(3HB) depolymerases do not bind to chitin or to (crystalline) cellulose [56,57]. The poly(3HB)-binding ability is lost in truncated proteins which lack the C-terminal domain of about 60 amino acids, and these modified enzymes do not hydrolyze poly(3HB). However, the catalytic domain is unaffected since the activity with water-soluble oligomers of 3-hy-droxybutyrate or with artificial water-soluble substrates such as p-nitrophenyl-esters is unaffected [55, 56, 58, 59]. Obviously, the C-terminal domain of poly(3HB) depolymerases is responsible and sufficient for poly(3HB)-binding [poly(3HB)-binding domain]. These results are in agreement ... 

There are several reports on the coating of bone-like hydroxyapatite onto natural polymer substrates. Kawashita et at. [57] reported that carboxymethylated chitin and gellan gum gels, which have carboxyl groups, can form hydroxyapatite on their surfaces in SBF if they are treated with a saturated Ca(OH)2 solution in advance, while curdlan gel, which has no carboxyl group, does not form hydroxyapatite in SBF, even if it is treated with Ca(OH)2 solution. These results support the hypothesis that carboxyl groups induce hydroxyapatite nucleation. Kokubo et at. [58,59] reported that non-woven fabrics of carboxymethylated chitin and alginate fibers also form hydroxyapatite on their surfaces in SBF if they are treated with Ca(OH)2 solution. 

Affinity chromatography was carried out on columns prepared with lightly carboxymethylated chitin, which is known to be a poor substrate for lysozyme. Both native lysozyme and regenerated 13-105 were bound to the column at pH 7 and eluted at pH 3. As controls, the basic proteins cytochrome c and pancreatic RNase A, as well as concanavalin A and a-amylase, were not bound from the same solvent at pH 7. These findings constitute a third line of evidence for formation of native-like structure in regenerated 13-105. 

The crystal structure of a pentamer of GlcNAc residues, representing the chitin polymer (poly-/l-(l-4)-GlcNAc), boimd to the chitinase enzyme ChiB from Serratia marcescens, revealed a narrow, timnel-like active site in the center of the TIM barrel fold [167]. Several conserved residues near the center of the site, which are important in catalysis, interact with the substrate via hydrogen bonds, while interactions farther from the center depend on van der Waals interactions. The sugar in the - 1 subsite adopts a boat conformation, presumably due to interactions with these critical active-site residues. 

Thus in this example the tetramer serves as both the donor and the acceptor. Usui et al, (43) propose this reaction for the synthesis of hexa-N-acetylchitohexaose, an oligosaccharide with reported antitumour activity (24), They dso observed formation of heptamer by incubating the pentamer wiA the Nocardia enzyme, but no chain elongation was observed with tiie hexamer as initial substrate. Similar activities with the formation of chitin oligosaccharides have also been observed for... 

Characterization of Enzyme-Substrate Complex by use of CM-Chitin (Carboxymethyl Chitin). CM-chitinwas prepared by carboxymethylation of chitin according to the method of Imoto, Hayashi and Funatsu (13). The ozonized lysozyme (1.3 mg) solutions at different pHs were neutralized with NaOH or HCl to pH 8.0 and the poured into the column (1.5 X 4 cm.) containing white cotton-like Cm-chitin ( 65 mg.), which was equilibrated with 0.1 M Tris-Cl buffer pH 8.0. Aliquots were eluted first with 0.1 M Tris-Cl pH 8.0 and then with 0.2 M HAc. The absorbance of the fractions was measured spectrophotometrically at 280 nm. 

Estimation of the Binding Site. Tryptophan-108 shows a specific reaction with iodine, distinguishing it from other tryptophan residues of lysoz3mie. When try-108 is selectively oxidized by iodine, lysozyme completely loses its activity. Nevertheless, the lysozyme still shows the ability to form an enzyme-substrate complex with CM-chitin. This observation contributes to the conclusion that try-62 is an essential binding site for a complex formation (13). All ozonized lysozymes formed strong complexes with CM-chitin and could only be eluted by 0.2N HA (Fig. 6). This further confirms that two tryptophan residues (108 and 111) are indispensible for the hydrolytic action of lysozyme, and that inactivation by ozone cannot be attributed to inhibition of substrate binding capability. 

High-molecular-weight solid substrates such as hide powder azure, and cellulose, chitin, and agar stained with remazol brilliant blue R have been used in enzyme assays to investigate activities in extracts from sediments (Reichardt, 1986). This technique is one of the few means of examining the particle — dissolved transition in relationship to sedimentary enzyme activity, but the necessity of extracting enzymes from sediments complicates interpretation of the results, because extraction efficiency, as well as the... 

Enzyme-Degradable Hydrogel. Because lysozyme is a well characterized enzyme, our first choice was a lysozyme-degradable hydrogel (11, 12). The natural substrate for lysozyme is chitin (13). but because chitin is a rigid, hydrophobic material, it is clearly not suitable for this work. The other natural substrates for lysozyme are certain bacterial cell-wall peptidoglycans (13. 

It is well known that well-ordered (3-chitin (a polysaccharide) associated with a less ordered protein in the (3-sheet conformation is the main component of nacreous organic matrix in shell. The amino acid sequence of such proteins is very similar to those of silk fibroins. Indeed, the amino acid sequence of a major protein from the nacreous shell layer of the pearl oyster resembles that of spidroin (Sudo et al., 1997 Weiner and Traub, 1980). The question of whether silk-like proteins play an important role in shell formation is raised. When Falini et al. (1996) did the experiment with the proteins from the shell, they assembled a substrate in vitro that contained (3-chitin and natural silk fibroin and concluded that the silk fibroin may influence ion diffusion or the accessibility to the chi tin surface or both. Furthermore, cryo-TEM study of the structure of the Atrina shell nacreous organic matrix without dehydration... 

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