Showing papers on "Chitin published in 1996"

Applications of chitin and chitosan for biomaterials.

Abstract: (1996). Applications of Chitin and Chitosan for Biomaterials. Biotechnology and Genetic Engineering Reviews: Vol. 13, No. 1, pp. 383-420.

Differential trafficking and timed localization of two chitin synthase proteins, Chs2p and Chs3p.

Abstract: The deposition of the polysaccharide chitin in the Saccharomyces cerevisiae cell wall is temporally and spatially regulated. Chitin synthase III (Chs3p) synthesizes a ring of chitin at the onset of bud emergence, marking the base of the incipient bud. At the end of mitosis, chitin synthase II (Chs2p) deposits a disk of chitin in the mother-bud neck, forming the primary division septum. Using indirect immunofluorescence microscopy, we have found that these two integral membrane proteins localize to the mother-bud neck at distinct times during the cell cycle. Chs2p is found at the neck at the end of mitosis, whereas Chs3p localizes to a ring on the surface of cells about to undergo bud emergence and in the mother-bud neck of small-budded cells. Cell synchronization and pulse-chase experiments suggest that the timing of Chs2p localization results from cell cycle-specific synthesis coupled to rapid degradation. Chs2p degradation depends on the vacuolar protease encoded by PEP4, indicating that Chs2p is destroyed in the vacuole. Temperature-sensitive mutations that block either the late secretory pathway (sec1-1) or the internalization step of endocytosis (end4-1) also prevent Chs2p degradation. In contrast, Chs3p is synthesized constitutively and is metabolically stable, indicating that Chs2p and Chs3p are subject to different modes of regulation. Differential centrifugation experiments show that a significant proportion of Chs3p resides in an internal compartment that may correspond to a vesicular species called the chitosome (Leal-Morales, C.A., C.E. Bracker, and S. Bartnicki-Garcia. 1988, Proc. Natl. Acad. Sci. USA. 85:8516-8520; Flores Martinez, A., and J. Schwencke. 1988. Biochim. Biophys. Acta. 946:328-336). Fractionation of membranes prepared from mutants defective in internalization (end3-1 and end4-1) indicate that the Chs3p-containing vesicles are endocytically derived. Collectively, these data suggest that the trafficking of Chs2p and Chs3p diverges after endocytosis; Chs3p is not delivered to the vacuole, but instead may be recycled.

Chitin hydrogels, methods of their production and use

Abstract: This invention is directed to the preparation and utilization of supplemented chitin hydrogels, such as chitosan hydrogels. Further provided are biomaterials comprising same. The particular supplement delivered by the chitin hydrogel is selected as a function of its intended use. In one embodiment, this invention provides a composition of matter, comprising a chitin hydrogel or chitin-derived hydrogel, wherein the hydrogel does not inhibit full-thickness skin wound healing.

Chs1p and Chs3p, two proteins involved in chitin synthesis, populate a compartment of the Saccharomyces cerevisiae endocytic pathway.

Abstract: In Saccharomyces cerevisiae, the synthesis of chitin, a cell-wall polysaccharide, is temporally and spatially regulated with respect to the cell cycle and morphogenesis. Using immunological reagents, we found that steady-state levels of Chs1p and Chs3p, two chitin synthase enzymes, did not fluctuate during the cell cycle, indicating that they are not simply regulated by synthesis and degradation. Previous cell fractionation studies demonstrated that chitin synthase I activity (CSI) exists in a plasma membrane form and in intracellular membrane-bound particles called chitosomes. Chitosomes were proposed to act as a reservoir for regulated transport of chitin synthase enzymes to the division septum. We found that Chs1p and Chs3p resided partly in chitosomes and that this distribution was not cell cycle regulated. Pulse-chase cell fractionation experiments showed that chitosome production was blocked in an endocytosis mutant (end4-1), indicating that endocytosis is required for the formation or maintenance of chitosomes. Additionally, Ste2p, internalized by ligand-induced endocytosis, cofractionated with chitosomes, suggesting that these membrane proteins populate the same endosomal compartment. However, in contrast to Ste2p, Chs1p and Chs3p were not rapidly degraded, thus raising the possibility that the temporal and spatial regulation of chitin synthesis is mediated by the mobilization of an endosomal pool of chitin synthase enzymes.

Homologs of the Xenopus developmental gene DG42 are present in zebrafish and mouse and are involved in the synthesis of Nod-like chitin oligosaccharides during early embryogenesis

Abstract: The Xenopus developmental gene DG42 is expressed during early embryonic development, between the midblastula and neurulation stages. The deduced protein sequence of Xenopus DG42 shows similarity to Rhizobium Nod C, Streptococcus Has A, and fungal chitin synthases. Previously, we found that the DG42 protein made in an in vitro transcription/translation system catalyzed synthesis of an array of chitin oligosaccharides. Here we show that cell extracts from early Xenopus and zebrafish embryos also synthesize chitooligosaccharides. cDNA fragments homologous to DG42 from zebrafish and mouse were also cloned and sequenced. Expression of these homologs was similar to that described for Xenopus based on Northern and Western blot analysis. The Xenopus anti-DG42 antibody recognized a 63-kDa protein in extracts from zebrafish embryos that followed a similar developmental expression pattern to that previously described for Xenopus. The chitin oligosaccharide synthase activity found in extracts was inactivated by a specific DG42 antibody; synthesis of hyaluronic acid (HA) was not affected under the conditions tested. Other experiments demonstrate that expression of DG42 under plasmid control in mouse 3T3 cells gives rise to chitooligosaccharide synthase activity without an increase in HA synthase level. A possible relationship between our results and those of other investigators, which show stimulation of HA synthesis by DG42 in mammalian cell culture systems, is provided by structural analyses to be published elsewhere that suggest that chitin oligosaccharides are present at the reducing ends of HA chains. Since in at least one vertebrate system hyaluronic acid formation can be inhibited by a pure chitinase, it seems possible that chitin oligosaccharides serve as primers for hyaluronic acid synthesis.

Role of three chitin synthase genes in the growth of Candida albicans.

Abstract: The CHS2 and CHS3 genes of Candida albicans were disrupted. The double disruptant was still viable. Assessment of chitin and of calcofluor white resistance shows that CHS1 is responsible for septum formation and CHS3 is responsible for overall chitin synthesis otherwise. There were only small differences in virulence to immunocompromised mice of homozygous chs2 delta amd chs3 delta null mutants.

Determination of chitin in fungi and mycorrhizal roots by an improved HPLC analysis of glucosamine

Abstract: A method to measure chitin content in fungi and ectomycorrhizal roots with high-performance liquid chromatography (HPLC) was developed. Measurements of fluorescence of 9-fluorenylmethylchloroformate (FMOC-CI) derivatives of glucosamine were made on acid hydrolysates of pure chitin, chitin-root mixtures and fungal-root mixtures. The method was applied on 5 isolates of ectomycorrhizal fungi, and ectomycorrhizal and non-mycorrhizal Pinus sylvestris roots. Interference from amino acids was removed by pre-treatment of samples with 0.2 N NaOH. This pre-treatment did not reduce the recovery of chitin, nor did plant material affect the recovery of chitin. The HPLC method was compared with a colorimetric chitin-method by measurements on root-fungal mixtures, with known fungal content. The HPLC method gave estimates of fungal biomass which were equal to the expected while the colorimetric method showed values significantly (p<0.001) lower than the expected. The present chitin method offers a sensitive and specific tool for the quantification of chitin in fungi and in ectomycorrhizal roots.

Purification and Characterization of Extracellular Chitin Deacetylase from Colletotrichum lindemuthianum

Abstract: Chitin deacetylase, active in the presence of acetate (96% of the enzymatic activity was retained in the presence of 100 mM sodium acetate), was purified to electrophoretic homogeneity from a culture filtrate of Colletotrichum lindemuthianum (944-fold with a recovery of 4.05%). The enzyme was induced in the medium after the eighth day of incubation simultaneously with the blackening of the medium. The molecular mass of the enzyme was 31.5 kDa and 33 kDa as judged by SDS-PAGE and gel filtration, respectively, suggesting that the enzyme is a single polypeptide. The optimum temperature was 60 degrees C and the optimum pH was 11.5-12.0 when glycol chitin was used as substrate. The enzyme was active toward glycol chitin, partially N-deacetylated water soluble chitin, and chitin oligomers the degrees of polymerization of which were more than four, but was less active with chitin trimer and dimer, and inactive with N-acetylglucosamine. The Km and kcat for glycol chitin were 2.55 mM and 27.1 s-1, respectively, and those for chitin pentamer were 414 microM and 83.2 s-1, respectively. The reaction rates of the enzyme toward glycol chitin and chitin oligomers seemed to follow the Michaelis-Menten kinetics.

Use of chitin and chitosan in lobster shell wastes for color removal from aqueous solutions

Abstract: Chitin and chitosan prepared from lobster shell wastes were used as adsorbents for the removal of various dyes from aqueous solutions. It was shown that high adsorption capacities were observed for reactive dyes. The adsorption equilibrium data could be well described by Langmuir equation under the concentration range investigated (50–500 mg/L). On the basis of Langmuir adsorption capacity, chitosan obtained in this work was found to be more effective compared to chitin and a commercial chitosan.

Chitin derivatives. I. Kinetics of the heat-induced conversion of chitosan to chitin

Abstract: The water-soluble solids comprised of the ionic complex between chitosan and acetic acid, chitosonium acetate, are converted into chitin by heating. The thermally-induced conversion of a water-soluble chitosonium acetate in film form into a water-insoluble chitin film was examined by thermal analysis (DMTA, TGA, DSC, and TMA) and by solid state 13C-NMR spectroscopy. Results indicate that tan δ-transitions occur at increasingly high temperatures, and over progressively wider temperature ranges, as the transformation progresses. Likewise, the storage modulus, log E′, increases as the chitosonium acetate film undergoes “cure” and converts to chitin. Cure kinetic parameters are obtained using the model proposed by Provder et al. modified for glass transition temperature (Tg). The results suggest the existence of two sequential first order reactions, an initial and a late cure reaction, having activation energies of approximately 15 and 21 kcal/mol, respectively. © 1996 John Wiley & Sons, Inc.

chs-4, a class IV chitin synthase gene from Neurospora crassa.

Abstract: In Saccharomyces cerevisiae, most of the cel- lular chitin is produced by chitin synthase III, which requires the product encoded by the CSD2/CAL1/ DIT101/KT12 gene. We have identified, isolated and structurally characterized a CSD2/CAL1/DIT101/KT12 homologue in the filamentous ascomycete Neurospora crassa and have used a ''reverse genetics'' approach to determine its role in vivo. The yeast gene was used as a heterologous probe for the isolation of a N. crassa gene (designated chs-4) encoding a polypeptide belong- ing to a class of chitin synthases which we have desig- nated class IV. The predicted polypeptide encoded by this gene is highly similar to those of S. cerevisiae and Candida albicans. N. crassa strains in which chs-4 had been inactivated by the Repeat-Induced Point muta- tion (RIP) process grew and developed in a normal manner under standard growth conditions. However, when grown in the presence of sorbose (a carbon source which induces morphological changes accompanied by elevated chitin content), chitin levels in the chs-4RIP strain were significantly lower than those observed in the wild type. We suggest that CHS4 may serve as an auxiliary enzyme in N. crassa and that, in contrast to yeasts, it is possible that filamentous fungi may have more than one class IV chitin synthase.

Does DG42 synthesize hyaluronan or chitin?: A controversy about oligosaccharides in vertebrate development.

Abstract: Science generally progresses in slow but deliberate increments, which are punctuated by major advances in concept or fact. However, the latter are rare and not infrequently go unrecognized when they first occur. Another event that can add spice to a field, and attract the attention of scientists from outside the discipline, is a genuine controversy. One such controversy is presented by two reports in this issue of the Proceedings (1, 2). To be asked to referee such a controversy is an interesting but difficult task, since both groups have significant data to back their claims. The story begins in 1983, when Igor Dawid and colleagues (1) reported the isolation of several genes that are Differentially expressed at Gastrulation (DG) in embryos of the frog, Xenopus laevis (3). One of these, the endoderm-specific DG42, is expressed in a short window during embryogenesis, being first detected after the midblastula stage, peaking at late gastrula, and decaying by the end of neuralation (4-6). Appropriate probes were used to show that the messenger RNA and predicted protein product move in a wave or gradient through the embryo, with the last remnants seen in the ventral regions of the gut at the tailbud stage. For a while thereafter, DG42 remained an interesting gene in search of a function. As often happens, the first clues came from unexpected sequence homology information. When it was first cloned, DG42 showed no obvious homologies to any previously known protein or gene. Subsequently, some similarities were found with fungal chitin synthases (7) and with the rhizobium NodC gene that is known to synthesize chitin oligomers (8-10). What is chitin? It is a repeating ,B1-4-linked homopolymer of the monosaccharide GlcNAc (see Fig. 1) that is one of the most widespread and abundant molecules in the biosphere, providing, for example, a major component of the cell walls of fungi and the shells of crustaceans and arthropods (11, 12). This important structural role for the extended polysaccharide may seem of little relevance to vertebrate development. However, shorter oligomers of the same repeating sequence are known to be soluble "oligosaccharins," mediating short range hormonal responses between Rhizobium bacteria and leguminous plants during the process of nitrogen-fixing root-nodule formation (13-15). Indeed, complex structural variations on the theme of the basic chitin backbone are well known to mediate a variety of specific interactions between bacteria and plants (for some examples, see refs. 16-20). Intrigued by these homologies, Semino and Robbins (21) then showed that when generated in an in vitro transcription/ translation system, the DG42 gene product was capable of synthesizing both short chitin oligomers and some larger products. The required sugar nucleotide donor was UDPGlcNAc; the products had the correct chromatographic properties, and they were degraded appropriately by a bacterial chitinase. Thus, DG42 was proposed to be the first recognized vertebrate chitooligosaccharide synthase (21). However, another interesting homology had also appeared between DG42 and the hasA gene of Streptococci (22, 23). The latter is responsible for the synthesis of another repeating polymer of sugars called hyaluronan. What is hyaluronan? It is a polymer consisting of alternating units of ,B1-4-linked GlcNAc and ,B1-3-linked glucuronic acid (GlcA, see Fig. 1). At first glance, these may seem to be very similar structures. Indeed, the linkages are very similar, and the donor nucleotides for both units are based on UDP (UDP-GlcNAc and UDP-GlcA). However, the similarity ends there (24, 25). Partly by virtue of its carboxylate groups, hyaluronan has physical properties that are almost diametrically opposite to those of chitin, being capable of retaining large amounts of water to form a gel. Furthermore, unlike chitin, hyaluronan expression is primarily reported in vertebrates, and in a few pathogenic bacteria such as group A and C Streptococci (24, 25). In view of these homologies, Semino and Robbins had also checked to see if the DG42 protein had hyaluronan synthase activity in vitro, but did not find any (21). This seemed to settle the issue that the DG42 gene product was primarily a chitin synthase. Enter the new study of Meyer and Kreil (1), which shows that rabbit kidney and human osteosarcoma cells induced to express the DG42 gene with a vaccinia virus system synthesize increased amounts of hyaluronan. Lysates and membranes from such transfected cells showed markedly increased hyaluronan synthase activity, which required the addition of both UDP-GlcNAc and UDP-GlcA donors. The product of the reaction was sensitive to hyaluronidases, but not to chitinases, and appropriate controls showed that the overexpression of hyaluronan synthesis was clearly related to DG42 expression (1). These authors conclude that their results are at variance with the earlier report of Semino and Robbins (21). Meanwhile, the latter group have an update to their story that is also published in this issue (2). They now show that DG42 homologues and their protein products are expressed in early embryos of zebrafish and mouse during the gastrula-early neuralation stages, and that chitin-oligosaccharide synthesis can be detected in extracts from these sources as well. Furthermore, this activity was immunoprecipitated by a DG42specific antibody (4) provided by Dawid. Also, overexpression of DG42 in a different cell type (mouse 3T3 cells) gives the synthesis of chitooligosaccharides, but no increase in background levels of hyaluronan synthesis. Finally, these authors show a physical separation of chitin synthase activity from most (but not all) of the hyaluronan synthase activity in embryo extracts (2). How can one reconcile the findings of the two studies and determine the true role of DG42? Semino et al. do make one preliminary attempt to do so (2). They state that commercial preparations of hyaluronan have chitin oligomers at their reducing end core region (further details are evidently to be published elsewhere). They suggest that DG42 might function to produce chitin oligomers that act as templates for hyaluronan synthesis (see Fig. 1). In this regard, it is interesting that Meyer and Kreil note a requirement for high concentrations of UDP-GlcNAc in their reactions (1). To consider this possibility further, let us review what is known about hyaluronan synthesis in vertebrate systems. The biosynthesis of this polysaccharide is peculiar, in that it follows a route different from that taken by most other molecules

Chitinase levels in guinea pig blood are increased after systemic infection with Aspergillus fumigatus.

Abstract: The presence of chitinase activity in human serum has recently been described by us On that occasion we speculated on the possible role of mammalian chitinases as a defense mechanism against chitin-containing pathogens The results of the present study substantiate our hypothesis We demonstrate and partially characterize the chitinase activities that are present in plasma of guinea pigs and in homogenates of Afumigatus with the aid of the substrates MU-[GlcNAc]2,3 and also with glycol [3H]chitin Upon infection with Afumigatus the serum chitinase activity levels in the circulation of pathogen-free guinea pigs increased in a time-dependent manner The increase was also dependent on the size of the infecting fungal inoculum Antifungal treatment diminished the increases The increased chitinase activity was of guinea pig origin The activity of beta-hexosaminidase showed a very slight increase subsequent to the infection The activities of three other enzymes of lysosomal origin (alpha-mannosidase, beta-galactosidase and beta-glucosidase) did not increase

Attachment of Vibrio alginolyticus to chitin mediated by chitin-binding proteins

Abstract: Vibrio alginolyticus is the only culturable vibrio associated with the chitinaceous carapace of the copepod Tigriopus fulvus (Fisher 1860) living in Ligurian coastal rock pools (Tyrrhenian Sea). The characteristics of the interaction between chitin particles and V. alginolyticus were studied by analysing strains isolated both from the copepod surface and from rock-pool water. The highest degree of attachment to chitin was observed at 20°, in the presence of 3% NaCI. Bacterial treatment with N-acetylglucosamine and pronase E caused a reduction in attachment of 52-62% and 77-94%, respectively. Chitin pretreatment with either wheat germ agglutinin or membrane proteins (MPs) from V. alginolyticus caused a reduction in attachment, of 50-57% and 53-70%, respectively. No inhibition was observed when bacteria were pretreated with D-glucose, D-fucose or D-fructose, or when chitin was pretreated with concanavalin A and Escherichia coli DH5α MPs. V. alginolyticus MPs able to bind chitin were isolated and analysed by SDS-PAGE. Four chitin-binding proteins were visualized in all tested strains (53, 35, 20 and 14 kDa); in vivo these peptides may efficiently mediate V. alginolyticus attachment to chitin-containing substrates.

Cloning and characterization of chsD, a chitin synthase-like gene of Aspergillus fumigatus.

Abstract: A chitin synthase-like gene (chsD) was isolated from an Aspergillus fumigatus genomic DNA library. Comparisons with the predicted amino acid sequence from chsD reveals low but significant similarity to chitin synthases, to other Nacetylglucosaminyltransferases (NodC from Rhizopus spp., HasA from Streptococcus spp. and DG42 from vertebrates. A chsD− mutant strain constructed by gene disruption has a 20% reduction in total mycelial chitin content; however, no differences between the wild-type strain and the chsD− strain were found with respect to morphology, chitin synthase activity or virulence in a neutropenic murine model of aspergillosis. The results show that the chsD product has an important but inessential role in the synthesis of chitin in A. fumigatus.

Biological activities of biodegradable polysaccharide

Abstract: A low toxicity of chitin was demonstrated to be mostly due to biodegradability and the fast metabolization of hydrolysate in animal body. Chitosan, a deacetylated derivative of chitin, is also known to be a biocompatible polymer in spite of a slight immunoadjuvant activity in animal body. Chitosan is easily regenerated to fiber, film, beads and non woven fabrics owing to its high solubility toward diluted aqueous organic acids such as formic acid, acetic acid, glutamic acid and ascorbic acid. The regeneration of chitin was achieved into fibers, gels, porous foams and non woven fabrics following to dissolution of chitin with formic acid or calcium chloride dihydrate saturated methanol. Chitin and its derivatives have been applied as biomedical materials due to remarkable advantages such as antimicrobial activity, acceleration of epidermal cell assembly, low toxicity, and biodegradability.

Chitin-binding proteins from cowpea (Vigna unguiculata) seeds.

Abstract: Vicilins (7S storage proteins) from cowpea (Vigna unguiculata) and other legume seeds were shown to bind to chitin, to regenerated chitin (fully acetylated chitin) and to chitosan (deacetylated chitin). Adsorbed vicilins were desorbed from these matrices by acetic and hydrochloric acids and by highly polymerized soluble chitosan. Proteins such as the lectin of common bean (PHA), soybean trypsin inhibitor (Kunitz), a beta-1,3-glucanase from cowpea seeds, bovine pancreatic alpha-chymotrypsin, chicken ovalbumin, serum albumin and rabbit gamma-globulin did not bind. The present result is the first description of vicilin binding to chitin but other proteins, such as wheat germ agglutinin (WGA), a lectin that contains the so called "chitin-binding domain", and a chitinase isolated from cowpea seeds, which are involved in the defense mechanisms of plants against insects and fungi, were also shown to bind to chitin as previously reported. The binding of vicilins to chitin is probably effected not through a "chitin-binding domain" because they do not share this sequence with the defense-related proteins cited above. We propose that this association of vicilins with chitin may be related to the effect of variant vicilins on the development of Callosobruchus maculatus (bruchid) in resistant cowpea seeds.

Production of chitinolytic enzymes from a novel species of Aeromonas

Abstract: A bacterial strain secreting potent chitinolytic activity was isolated from shrimp-pond water by enrichment culture using colloidal crab-shell chitin as the major carbon source. The isolated bacterium, designated asAeromonas sp No. 16 exhibited a rod-like morphology with a polar flagellum. Under optimal culture conditions in 500-ml shaker flasks, it produced a chitinolytic activity of 1.4 U ml−1. A slightly higher enzymatic activity of 1.5 U ml−1 was obtained when cultivation was carried out in a 5-liter jar fermentor using a medium containing crystalline chitin as the carbon source. The secretion of the enzyme(s) was stimulated by several organic nitrogenous supplements. Most carbon sources tested (glucose, maltose, N-acetylglucosamine, etc) enhanced cell growth, but they slightly inhibited enzyme secretion. Glucosamine (0.5% w/v) severely inhibited cell growth (16% of the control), but it did not significantly affect enzyme secretion. The production of chitinolytic enzymes was pH sensitive and was enhanced by increasing the concentration of colloidal chitin to 1.5%. The observed chitinolytic activity could be attributed to the presence of β-N-acetylglucosaminidase and chitinase. Chitinase was purified by ammonium sulfate fractionation and preparative gel electrophoresis to three major bands on SDS-PAGE. An in-gel enzymatic activity assay indicated that all three bands possessed chitinase activity. Analysis of the enzymatic products indicated that the purified enzyme(s) hydrolyzed colloidal chitin predominantly to N,N-diacetyl-chitobiose and, to a much lesser extent, the mono-, tri, and tetramer of N-acetylglucosamine, suggesting that they are mainly endochitinases.

Chitin Biosynthesis and Morphogenetic Processes

Abstract: Chitin, one of the most abundant substances of biological origin, is an important component of cell walls and septa of many fungi (Wessels and Sietsma 1981; Ruiz-Herrera 1992). Because walls and septa have a well-defined shape and an essential role in cell maintenance and growth, there has been increasing interest in chitin biogenesis. Thus, the synthesis of chitin in fungi has served as a useful model for morphogenesis (Cabib 1987; Cabib et al. 1982b, 1988; Bulawa 1993).

Characterization of chitin synthase 2 of Saccharomyces cerevisiae. II: Both full size and processed enzymes are active for chitin synthesis.

Abstract: When chitin synthase 2 of Saccharomyces cerevisiae was overexpressed in yeast cells using GAL1 promoter, deletion of the N-terminal 193 amino acids significantly increased the level of the protein without affecting its characteristics. We partially purified N-terminally truncated chitin synthase 2 by product entrapment and ion exchange column chromatography, and found that it was active even without trypsin treatment when appropriate divalent cations were present in the reaction mixture. This chitin synthase activity was independent of the N-terminal 193 amino acid truncation, because partially purified full length enzyme also exhibited the activity without trypsin treatment in the presence of appropriate cations. Furthermore, the molecular weights of these two forms of chitin synthase 2 were coincident with those estimated from the deduced amino acid sequence, and most of the chitin synthase 2 in the yeast membrane was present as an unprocessed form, as judged from its molecular weight. Treatment of either full length or truncated enzyme with trypsin, however, further increased the enzyme activity by four to fivefold, and produced a 35 kDa polypeptide that specifically reacted with monoclonal antibody raised against the region containing the putative active site of chitin synthase 2. Thus, it appears that predominant native (unprocessed) chitin synthase 2 is active, but the 35 kDa region encompassing the active site is sufficient for the catalytic activity.

Limited proteolysis and reduction-carboxymethylation of rye seed chitinase-a: role of the chitin-binding domain in its chitinase action.

Abstract: By a limited proteolysis with thermolysin, rye seed chitinase-a (RSC-a) was separated into a N-terminal cysteine-rich chitin-binding (CB-) domain (48 residues) and a catalytic (Cat-) domain (254 residues). The hydrolytic activity of the isolated Cat-domain toward soluble glycolchitin, was similar to that of RSC-a, but that toward insoluble colloidal chitin was 28% of that of RSC-a. Five disulfide bonds in the CB-domain were reduced with 2-mercaptoethanol (2-ME) in the absence of denaturing agents by an “all-or-none” process, that is, once the disulfide bond between Cysl5 and Cys42 in the CB-domain was cleaved, the remaining four disulfide bonds were reduced very easily. The reduced and carboxymethylated RSC-a completely lost the chitin-binding ability, but retained 50% of the hydrolytic activity toward colloidal chitin of RSC-a.From these results, it was shown that RSC-a consists of a CB-domain and a Cat-domain connected by a flexible linker, and it was suggested that the CB-domain increases the hydrolyti...