Biosynthesis of chitin by particulate fractions from Cunnighamella elegans.

The enzyme chitin synthetase (UDP-acetylaminodeoxyglucosyl transferase, EC 2.4.1.16) in Cunninghamella elegans has been investigated. The enzyme was present in the microsomal, cell wall, mitochondrial and the soluble cytoplasmic fraction of the mycelium, with the former having the highest specific activity. The properties of the enzyme in this fraction were investigated; the Km for UDP GlcNAc was 1.23 mM and 2.08 mM GlcNAc in the presence of 1 mM UDP GlcNAc. The temperature optimum was between 26 degrees and 29 degrees C and maximal activity was at pH 6.25. Mg++ ions had no effect on chitin synthesis, but soluble chitodextrins inhibited the enzyme. The production of chitin synthetase was correlated with the growth of the fungus, maximum activity being found during the late exponential phase of growth. Chitin was confirmed as the sole product of enzyme action, by digestion with chitinase.     

Chitin biosynthesis by a fungal membrane preparation. Evidence for a transient non-crystalline state of chitin

Chitin synthase activity of membrane preparations from hyphae of Schizophyllum commune was strongly inhibited by added chitinase because chitin immediately after its synthesis was highly susceptible to chitinase. In the absence of synthesis, chitin became more resistant to chitinase with time. Chitin synthesized in the presence of the optical brightener Calcofluor White M2R was extremely susceptible to degradation by chitinase and this susceptibility was maintained for a long time. X-ray diffraction analysis of chitin synthesized in the presence of Calcofluor revealed the absence of crystallinity as long as the material was kept in wet conditions. After drying, discrete deflections characteristic for alpha-chitin appeared concomitant with a decrease in the susceptibility for chitinase. These results strongly suggest the existence of a gap between polymerization and crystallization of chitin chains.     

The effect of monensin on chitin synthesis in Candida albicans blastospores

Monensin, a monovalent cation ionophore, was used to investigate some steps of the wall synthesis and morphogenesis in Candida albicans blastospores. In the presence of the drug, the pathogenic yeast developed enormous wall and septum thickenings that reacted intensely and specifically with wheat germ agglutinin and chitinase coupled to colloidal gold and fluorescein isothiocyanate. Therefore, the aberrant zones are interpreted as sites of chitin accumulation. The increased production of this homopolymer, also demonstrated by the chemical analysis of cell wall preparations, implies that monensin interferes in some way with the regulatory factors that normally control, in space and time, chitin synthetase activity.     

Chitin in the peritrophic membrane of Acarus siro (Acari: Acaridae) as a target for novel acaricides

The peritrophic membrane in Acarus siro L. (Acari: Acaridae) is produced by distinct cells located in the ventriculus. In this study, the chitin inside the peritrophic membrane was detected using wheat germ-lectin conjugated with colloidal gold (10 nm). The chitin fibrils of the peritrophic membrane were a target for chitin effectors, including 1) chitinase, which hydrolyzes chitin fibers inside the peritrophic membrane; 2) calcofluor, which binds to chitin and destroys the peritrophic membrane mesh structure; and 3) diflubenzuron, which inhibits chitin synthesis. In addition, soybean trypsin protease inhibitor (STI) and cocktails of chitinase/calcofluor, diflubenzuron/calcofluor and chitinase/STI were tested. These compounds were supplemented in diets and an increase of population initiated from 50 individuals was observed after 21 d of cultivation. Final A. siro densities on experimental and control diets were compared. The chitin in the peritrophic membrane was determined to be a suitable target for novel acaricidal compounds for suppressing the population growth of A. siro. The most effective compounds were calcofluor and diflubenzuron, whereas the suppressive effects of chitinase and STI were low. The failure of chitinase could be due to its degradation by endogenous proteases. The combination of chitinase and STI suppressed A. siro population growth more effectively than when they were tested in oral admission separately. The combinations of calcofluor/chitinase or calcofluor/difluorbenzuron showed no additive effects on final A. siro density. The presence of chitin in peritrophic membrane provides a target for novel acaricidal compounds, which disrupt peritrophic membrane structure. The suitability of chitin effectors and their practical application in the management of stored product mites is discussed.     

Stability of chitin synthetase in cell-free preparations of a wild-type strain and a ''slime'' variant of Neurospora crassa

Chitin synthetase activity in cell-free preparations from a wild-type strain and a 'slime' variant of Neurospora crassa was monitored over many days in samples stored at 0 degrees C. Total activity in whole-cell-free extracts and low-speed supernatants from both organisms was very unstable, losing more than 90% of the initial activity on storage at 0 degrees C for 96 h. Chitin synthetase detection was not masked by chitinase activity present in the preparations. Gel-filtration chromatography of these preparations increased the stability of the activity from the 'slime' variant, whereas removal of particulate structures by high-speed centrifugation stabilized the chitin synthetase activity in the supernatant, particularly in the wild type. These results suggest that factor(s) involved in the regulation of chitin synthetase may be differentially located or altered in 'slime' cells.     

The co-ordination of chitosan and chitin synthesis in Mucor rouxii

Chitin synthetase preparations from cell walls and chitosomes of the fungus Mucor rouxii were tested for their ability to synthesize chitosan when incubated with uridine diphosphate N-acetyl-D-glucosamine in the presence of chitin deacetylase. The most effective chitin synthetase preparation was one dissociated from cell walls with digitonin. The rate of chitosan synthesis by the wall-dissociated chitin synthetase was about three times that of an equivalent amount of cell walls. The chitosan-synthesizing ability of chitosomes was relatively low, but was more than tripled by treatment with digitonin. Presumably, digitonin improves chitosan yields of dissociating chitin synthetase. The dissociated enzyme would produce dispersed chitin chains that could be attacked by chitin deacetylase before they have time to crystallize into microfibrils. The regulation of chitin and chitosan syntheses in vivo may be determined by the organization of chitin synthetase molecules at the cell surface. Those molecules that remain organized as a complex, similar if not identical to that found in chitosomes, would produce mainly chitin. Chitosan would be preferentially produced by chitin synthetase molecules which are dispersed upon reaching the cell surface.     

Regulation of chitinase synthesis in Trichoderma harzianum.

The production of chitinase by Trichoderma species is of interest in relation to their use in biocontrol and as a source of mycolytic enzymes. Fourteen isolates of the genus were screened to identify the most effective producer of chitinase. The best strain for chitinase was Trichoderma harzianum 39.1, and this was selected for study of the regulation of enzyme synthesis. Washed mycelium of T. harzianum 39.1 was incubated with a range of carbon sources. Chitinase synthesis was induced on chitin-containing medium, but repressed by glucose and N-acetylglucosamine. Production of the enzyme was optimal at a chitin concentration of 0.5%, at 28 degrees C, pH 6.0 and was independent of the age of the mycelium. The synthesis of chitinase was blocked by both 8-hydroxyquinoline and cycloheximide, inhibitors of RNA and protein synthesis, respectively. The mode of chitinase synthesis in this fungus is discussed.     

Identification and characterization of a chitinase antigen from Pseudomonas aeruginosa strain 385

A chitinase antigen has been identified in Pseudomonas aeruginosa strain 385 using sera from animals immunized with a whole-cell vaccine. The majority of the activity was shown to be in the cytoplasm, with some activity in the membrane fraction. The chitinase was not secreted into the culture medium. Purification of the enzyme was achieved by exploiting its binding to crab shell chitin. The purified enzyme had a molecular mass of 58 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a pI of 5.2. NH2-terminal amino acid sequencing revealed two sequences of M(I/L)RID and (Q/M/V)AREDAAAAM that gave an exact match to sequences in a translated putative open reading frame from the P. aeruginosa genome. The chitinase was active against chitin azure, ethylene glycol chitin, and colloidal chitin. It did not display any lysozyme activity. Using synthetic 4-methylumbelliferyl chitin substrates, it was shown to be an endochitinase. The Km and kcat for 4-nitrophenyl-beta-D-N,N'-diacetylchitobiose were 4.28 mM and 1.7 s(-1) respectively, and for 4-nitrophenyl-beta-D-N,N',N"-triacetylchitotriose, they were 0.48 mM and 0.16 s(-1) respectively. The pH optimum was determined to be pH 6.75, and 90% activity was maintained over the pH range 6.5 to 7.1. The enzyme was stable over the pH range 5 to 10 for 3 h and to temperatures up to 50 degrees C for 30 min. The chitinase bound strongly to chitin, chitin azure, colloidal chitin, lichenan, and cellulose but poorly to chitosan, xylan, and heparin. It is suggested that the chitinase functions primarily as a chitobiosidase, removing chitobiose from the nonreducing ends of chitin and chitin oligosaccharides.     

Role of Chitin-Binding Proteins in the Specific Attachment of the Marine Bacterium Vibrio harveyi to Chitin

We examined the mechanism of attachment of the marine bacterium Vibrio harveyi to chitin. Wheat germ agglutinin and chitinase bind to chitin and competitively inhibited the attachment of V. harveyi to chitin, but not to cellulose. Bovine serum albumin and cellulase do not bind to chitin and had no effect on bacterial attachment to chitin. These data suggest that this bacterium recognizes specific attachment sites on the chitin particle. The level of attachment of a chitinase-overproducing mutant of V. harveyi to chitin was about twice as much as that of the uninduced wild type. Detergent-extracted cell membranes inhibited attachment and contained a 53-kDa peptide that was overproduced by the chitinase-overproducing mutant. Three peptides (40, 53, and 150 kDa) were recovered from chitin which had been exposed to membrane extracts. Polyclonal antibodies raised against extracellular chitinase cross-reacted with the 53- and 150-kDa chitin-binding peptides and inhibited attachment, probably by sterically hindering interactions between the chitin-binding peptides and chitin. The 53- and 150-kDa chitin-binding peptides did not have chitinase activity. These results suggest that chitin-binding peptides, especially the 53-kDa chitin-binding peptide and chitinase and perhaps the 150-kDa peptide, mediate the specific attachment of V. harveyi to chitin.     

Studies on the Chitinolytic Enzymes of Black-koji Mold

It was found that the purified chitinase preparation acts upon glycol chitin resulting in the decomposition to constituent aminosugar, the saccharifying activity being determined by application of the Morgan-Elson reaction. The enzymatic properties of the mold chitinase were investigated by measuring liquefying activity and saccharifying activity. Distinct differences were observed between the two activities, and especially liquefying activity was more stable than saccharifying activity against heat treatment. The chitinase preparation whose saccharifying activity was inactivated by heating was able to decrease the viscosity of glycol chitin solution, with an insignificant production of aminosugar.     

Purification, Characterization, and Antifungal Activity of Chitinase from Streptomyces venezuelae P10

Streptomyces venezuelae P₁₀ could produce extracellular chitinase in a medium containing 0.6% colloidal chitin that was fermented for 96 hours at 30°C. The enzyme was purified to apparent homogeneity with 80% saturation of ammonium sulfate as shown by chitin affinity chromatography and DEAE-cellulose anion-exchange chromatography. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) of the enzyme showed a molecular weight of 66 kDa. The chitinase was characterized, and antifungal activity was observed against phytopathogens. Also, the first 15 N-terminal amino-acid residues of the chitinase were determined. The chitin hydrolysed products were N-acetylglucosamine and N, N’-diacetylchitobiose.     

免疫亲和层析法纯化苦瓜几丁酶

用扁豆几丁酶免疫家兔,获得抗扁豆几丁酶的抗体,将此抗体与Ｓｅｐｈａｒｏｓｅ ４Ｂ偶联,制备免疫亲和吸附剂,用以纯化苦瓜几丁酶．苦瓜叶片的粗提液经过免疫亲和吸附柱后,可获得电泳纯的几丁酶,其分子量为３５ ｋＤ,与用几丁质凝胶为亲和吸附剂的纯化结果一致．表明利用植物几丁酶在结构上的保守性,用免疫亲和法可纯化不同植物的同类几丁酶．与几丁质凝胶亲和柱相比,免疫亲和法纯化植物几丁酶具有快速、亲和柱可重复使用等的优点．利用免疫亲和层析获得的纯化样品,研究了苦瓜几丁酶对真菌的抑制试验,研究结果表明,苦瓜几丁酶能分解棉花枯萎病菌的菌丝体细胞壁制备物,并对其孢子芽管的伸长有一定抑制作用．     

Production and purification of a chitinase from Ewingella americana, a recently described pathogen of the mushroom, Agaricus bisporus

The chitinolytic properties of Ewingella americana, a recently described pathogen of the mushroom, Agaricus bisporus, are reported. E. americana was shown to produce chitinolytic activity in the absence of chitin and in the presence of glucose and N-acetylglucosamine, indicating constitutive synthesis by these strains. A single 33-kDa protein with chitinolytic activity was purified to homogeneity from culture filtrates, by hydrophobic interaction chromatography using a phenyl-group substituted matrix. This enzyme, by virtue of differential activity against chromogenic chitooligosaccharides and against dye-labelled soluble carboxymethylated chitin (CM-chitin-RBV), was demonstrated to be an endochitinase. Our data suggest this 33-kDa chitinase appeared to be the only chitinolytic enzyme produced by E. americana, strains of which do not grow using chitin as a carbon source. The significance of these findings in the context of mushroom disease is discussed.     

Chitinase and chitin synthase 1: counterbalancing activities in cell separation of Saccharomyces cerevisiae.

Previous results [E. Cabib, A. Sburlati, B. Bowers & S. J. Silverman (1989) Journal of Cell Biology 108, 1665-1672] strongly suggested that the lysis observed in daughter cells of Saccharomyces cerevisiae defective in chitin synthase 1 (Chs1) was caused by a chitinase that partially degrades the chitin septum in the process of cell separation. Consequently, it was proposed that in wild-type cells, Chs1 acts as a repair enzyme by replenishing chitin during cytokinesis. The chitinase requirement for lysis has been confirmed in two different ways: (a) demethylallosamidin, a more powerful chitinase inhibitor than the previously used allosamidin, is also a much better protector against lysis and (b) disruption of the chitinase gene in chs1 cells eliminates lysis. Reintroduction of a normal chitinase gene, by transformation of those cells with a suitable plasmid, restores lysis. The percentage of lysed cells in strains lacking Chs1 was not increased by elevating the chitinase level with high-copy-number plasmids carrying the hydrolase gene. Furthermore, the degree of lysis varied in different chs1 strains; lysis was abolished in chs1 mutants containing the scs1 suppressor. These results indicate that, in addition to chitinase, lysis requires other gene products that may become limiting.     

细菌几丁质酶结构、功能及分子设计的研究进展

几丁质是仅次于纤维素的第二大天然多糖，由N-乙酰-D-氨基葡萄糖聚合而成，具有重要的应用价值。自然界中几丁质可被细菌高效降解。细菌可分泌多种几丁质降解酶类，主要分布在GH18家族和GH19家族中。细菌中几丁质降解酶基因存在明显的基因扩增及多结构域组合现象，不同家族、不同作用模式的几丁质酶系协同作用打破复杂的抗降解屏障，完成结晶几丁质的高效降解。因此，深入分析细菌几丁质酶结构与功能，对几丁质高效降解与高值转化应用具有重要意义。本文介绍了细菌几丁质酶的分类、结构特点与催化作用机制；总结了不同细菌胞外几丁质降解酶系的协同降解模式；针对几丁质酶家族分子改造的研究进展，展望了以结构生物信息学及大数据深度学习为基础的蛋白质工程设计策略在今后改造中的作用，为几丁质酶的设计与理性改造提供新的视角与思路。     

Chitinolytic activities in the gut of Aedes aegypti (Diptera:Culicidae) larvae and their role in digestion of chitin-rich structures

Mosquito larvae are believed to be capable of digesting chitin, an insoluble polysaccharide of N-acetylglucosamine, for their nutritional benefit. Studies based on physiological and biochemical assays were conducted in order to detect the presence of chitinase activities in the gut of the detritus-feeding Aedes aegypti larvae. Larvae placed for 24 h in suspensions of chitin azure were able to digest the ingested chitin. Semi-denaturing PAGE using glycol chitin and two fluorogenic substrate analogues showed the presence of two distinct chitinase activities: an endochitinase that catalyzed the hydrolysis of chitin and an endochitinase that cleaved the short substrates [4MU(GlcNAc)(3)] and [4MU(GlcNAc)(2)] that hydrolyzed the chitobioside [4MU(GlcNAc)(2)]. The endochitinase had an extremely broad pH-activity against glycol chitin and chitin azure, pH ranging from 4.0 to 10.0. When the substrate [4MU(GlcNAc)(3)] was used, two activities were observed at pH ranges 4.0-6.0 and 8.0-10.0. Chitinase activity against [4MU(GlcNAc)(3)] was detected throughout the gut with the highest specific activity in the hindgut. The pH of the gut contents was determined by observing color changes in gut after feeding the larvae with color indicator dyes. It was observed a correlation between the pH observed in the gut of feeding larvae (pH 10-6.0) and the optimum pH for gut chitinase activities. In this work, we report that gut chitinases may be involved in the digestion of chitin-containing structures and also in the partial degradation of the chitinous peritrophic matrix in the hindgut.     

Inhibition of chitin synthetase from Saccharomyces cerevisiae by a new UDP-GlcNAc analogue

The synthesis and biological evaluation of a new UDP-GlcNAc competitor (I), designed to mimic the transition state of the sugar donor in the enzymatic reaction catalysed by chitin synthetase, is described. Compound (I) was found to competitively inhibit chitin synthetase from Saccharomyces cerevisiae with respect to UDP-GlcNAc, but displayed minimal antifungal activity.     

Designing a new chitinase with more chitin binding and antifungal activity

Chitinases have the ability of chitin digestion that constitutes a main compound of the cell wall in many of the phytopathogens such as fungi. Chitinase Chit42 from Trichoderma atroviride PTCC5220 is considered to play an important role in the biocontrol activity of this fungus against plant pathogens. Chit42 lacks a chitin binding domain (ChBD). We have produced a chimeric chitinase with stronger chitin-binding capacity by fusing to Chit42 a ChBD from Serratia marcescens Chitinase B. The fusion of ChBD improved the affinity to crystalline and colloidal chitin and also the enzyme activity of the chimeric chitinase when compared with the native Chit42. The chimeric chitinase showed higher antifungal activity toward phytopathogenic fungi.     

Chitin synthase 1, an auxiliary enzyme for chitin synthesis in Saccharomyces cerevisiae

Previously, we showed that chitin synthase 2 (Chs2) is required for septum formation in Saccharomyces cerevisiae, whereas chitin synthase 1 (Chs1) does not appear to be an essential enzyme. However, in strains carrying a disrupted CHS1 gene, frequent lysis of buds is observed. Lysis occurs after nuclear separation and appears to result from damage to the cell wall, as indicated by osmotic stabilization and by a approximately 50-nm orifice at the center of the birth scar. Lysis occurs at a low pH and is prevented by buffering the medium above pH 5. A likely candidate for the lytic system is a previously described chitinase that is probably involved in cell separation. The chitinase has a very acidic pH optimum and a location in the periplasmic space that exposes it to external pH. Accordingly, allosamidin, a specific chitinase inhibitor, substantially reduced the number of lysed cells. Because the presence of Chs1 in the cell abolishes lysis, it is concluded that damage to the cell wall is caused by excessive chitinase activity at acidic pH, which can normally be repaired through chitin synthesis by Chs1. The latter emerges as an auxiliary or emergency enzyme. Other experiments suggest that both Chs1 and Chs2 collaborate in the repair synthesis of chitin, whereas Chs1 cannot substitute for Chs2 in septum formation.