Serratia marcescens chitinase: One-step purification and use for the determination of chitin

The extracellular chitinase produced by Serratia marcescens was obtained in highly purified form by adsorption-digestion on chitin. After gel electrophoresis in a nondenaturing system, the purified preparation exhibited two major protein bands that coincided with enzymatic activity. A study of the enzyme properties showed its suitability for the analysis of chitin. Thus, the chitinase exhibited excellent stability, a wide pH optimum, and linear kinetics over a much greater range than similar enzymes from other sources. The major product of chitin hydrolysis was chitobiose, which was slowly converted into free N-acetylglucosamine by traces of β-N-acetylglucosaminidase present in the purified preparation. The preparation was free from other polysaccharide hydrolases. Experiments with radiolabeled yeast cell walls showed that the chitinase was able to degrade wall chitin completely and specifically.     

Role of the N-terminal polycystic kidney disease domain in chitin degradation by chitinase A from a marine bacterium, Alteromonas sp. strain O-7

AIMS: The aim of study was to clarify whether the polycystic kidney disease (PKD) domain of chitinase A (ChiA) participates in the hydrolysis of powdered chitin. METHODS AND RESULTS: Site-directed mutagenesis of the conserved aromatic residues of PKD domain was performed by PCR. The aromatic residues, W30, Y48, W64 and W67, were replaced by alanine, and single- and double-mutant chitinases were produced in Escherichia coli XL10 and purified with HisTrap column. Single mutations were not quite effective on the hydrolysing activities against chitinous substrates when compared with wild-type ChiA. However, mutations of W30 and W67 decreased the activities against powdered chitin by 87.6%. Wild-type and mutant PKD domains were produced in E. coli TOP10 and purified with glutathione-Sepharose 4B column. Wild-type PKD domain showed significant binding activity to powdered chitin, whereas mutations of W30 and W67 reduced the binding activity to powdered chitin drastically. These results suggest that PKD domain of ChiA is essential for effective hydrolysis of powdered chitin through the interaction between two aromatic residues and chitin molecule. CONCLUSIONS: PKD domain of ChiA participates in the effective hydrolysis of powdered chitin through the interaction between two aromatic residues (W30 and W67) and chitin molecule. SIGNIFICANCE AND IMPACT OF THE STUDY: The findings of this study provide important information on chitin degradation by microbial chitinases.     

Chitin synthesis in a gas1 mutant of Saccharomyces cerevisiae

The existence of a compensatory mechanism in response to cell wall damage has been proposed in yeast cells. The increase of chitin accumulation is part of this response. In order to study the mechanism of the stress-related chitin synthesis, we tested chitin synthase I (CSI), CSII, and CSIII in vitro activities in the cell-wall-defective mutant gas1 delta. CSI activity increased twofold with respect to the control, a finding in agreement with an increase in the expression of the CHS1 gene. However, deletion of the CHS1 gene did not affect the phenotype of the gas1 delta mutant and only slightly reduced the chitin content. Interestingly, in chs1 gas1 double mutants the lysed-bud phenotype, typical of chs1 null mutant, was suppressed, although in gas1 cells there was no reduction in chitinase activity. CHS3 expression was not affected in the gas1 mutant. Deletion of the CHS3 gene severely compromised the phenotype of gas1 cells, despite the fact that CSIII activity, assayed in membrane fractions, did not change. Furthermore, in chs3 gas1 cells the chitin level was about 10% that of gas1 cells. Thus, CSIII is the enzyme responsible for the hyperaccumulation of chitin in response to cell wall stress. However, the level of enzyme or the in vitro CSIII activity does not change. This result suggests that an interaction with a regulatory molecule or a posttranslational modification, which is not preserved during membrane fractionation, could be essential in vivo for the stress-induced synthesis of chitin.     

In vitro effect of chitin particles on the innate cellular immune system of gilthead seabream (Sparus aurata L.)

The interaction between chitin particles and gilthead seabream (Sparus aurata L.) head-kidney leucocytes, as well as their effects on the main innate cellular immune responses were studied. Three different chitin particle-sizes were tested: unfiltered, <10 microM and >10 microM. Leucocytes were able to phagocytose only the chitin particles of <10 microM but not the >10 microM ones. Leucocytes were incubated with different concentrations (0 to 1000 microg ml(-1)) of the above chitin particles for 1, 4, 24 or 48 h and their effects on leucocyte viability and the innate cellular immune system were evaluated. Leucocytes incubated with chitin for 48 h maintained their viability as determined by the MTT viability test. Leucocyte phagocytosis of bacteria after chitin incubation for 1 or 4 h was enhanced by the highest chitin concentration tested of each of the chitin fractions studied, while the respiratory burst activity was unaffected. As regards leucocyte natural cytotoxic activity against tumour cells, prior incubation of leucocytes with chitin particles for 1 or 4 h increased while incubation for 24 or 48 h reduced the cytotoxic activity in a dose dependent manner. Statistically significant differences between the different chitin concentrations and between the three chitin particle-size fractions were detected. To conclude, gilthead seabream head-kidney leucocytes were able to phagocytose chitin particles smaller than 10 microM, and the main cellular innate immune activities were enhanced as a consequence of prior incubation with chitin particles.     

Purification and Characterization of Extracellular Chitin Deacetylase from Colletotrichum lindemuthianum

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°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 K_m and k_cat for glycol chitin were 2.55 mm and 27.1s^?1, respectively, and those for chitin pentamer were 414 μm and 83.2s^?1, respectively. The reaction rates of the enzyme toward glycol chitin and chitin oligomers seemed to follow the Michaelis–Menten kinetics.     

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.     

Analysis of expression and chitin‐binding activity of the wing disc cuticle protein BmWCP4 in the silkworm,Bombyx mori

The insect exoskeleton is mainly composed of chitin filaments linked by cuticle proteins. When insects molt, the cuticle of the exoskeleton is renewed by degrading the old chitin and cuticle proteins and synthesizing new ones. In this study, chitin‐binding activity of the wing disc cuticle protein BmWCP4 in Bombyx mori was studied. Sequence analysis showed that the protein had a conservative hydrophilic “R&R” chitin‐binding domain (CBD). Western blotting showed that BmWCP4 was predominately expressed in the wing disc‐containing epidermis during the late wandering and early pupal stages. The immunohistochemistry result showed that the BmWCP4 was mainly present in the wing disc tissues containing wing bud and trachea blast during day 2 of wandering stage. Recombinant full‐length BmWCP4 protein, “R&R” CBD peptide (CBD), non‐CBD peptide (BmWCP4‐CBD^?), four single site‐directed mutated peptides (M₁, M₂, M₃ and M₄) and four‐sites‐mutated peptide (M_F) were generated and purified, respectively, for in vitro chitin‐binding assay. The results indicated that both the full‐length protein and the “R&R” CBD peptide could bind with chitin, whereas the BmWCP4‐CBD^? could not bind with chitin. The single residue mutants M₁, M₂, M₃ and M₄ reduced but did not completely abolish the chitin‐binding activity, while four‐sites‐mutated protein M_F completely lost the chitin‐binding activity. These data indicate that BmWCP4 protein plays a critical role by binding to the chitin filaments in the wing during larva‐to‐pupa transformation. The conserved aromatic amino acids are critical in the interaction between chitin and the cuticle protein.     

Substrate specificities of tobacco chitinases

Ten tobacco chitinases (1,4-N-acetyl-β-D-glucosaminide glycanhydrolase, EC 3.2.1.14) were purified from tobacco leaves hypersensitively reacting to tobacco mosaic virus. The 10 enzymes, which belong to five distinct structural classes of plant chitinases, were incubated with several potential substrates such as chitin, a β-1,4 N-acetyl-D-glucosamine (GlcNAc) polymer, chitosan (partially deacetylated chitin), chitin oligomers of variable length and bacterial cell wall. Tobacco chitinases are all endo-type enzymes that liberate oligomers from chitin and are capable of processing the chito-oligomers further at differential rates. Chitin reaction products were separated and quantified by HPLC and differential kinetics of oligomer accumulation and degradation were observed with the distinct classes of chitinases. Depending on the substrate to be hydrolysed, each isoform displayed a different spectrum of activity. For example, class I isoforms were the most active on chitin and (GlcNAc)_4–6 whereas class III basic isoforms were the most efficient in inducing bacterial lysis. Class V and class VI chitinases were shown to more readily hydrolyse chitin oligomers than the chitin polymer itself. Together, these data indicate that the 10 tobacco chitinases represent complementary enzymes which may have synergistic effects on their substrates. This paper discusses their implication in plant defense by attacking pathogen's structural components and in plant development by maturing signal molecules.     

Chemical analysis of cell wall regeneration and reversion of protoplasts from Schizophyllum commune.

In the presence of MgSo4 as osmotic stabilizer, nucleated protoplasts of Schizophyllum commune developed a large vacuole and could be isolated on the basis of their low buoyant density. All these protoplasts were capable of wall regeneration and about 50 percent reverted to the hyphal mode of growth in liquid medium. The kinetics of the formation of three main cell-wall components, S-glucan (alpha-1,3-glucan), R-glucan (beta-1,3, beta-1,6-glucan) and chitin were studied from the onset of regeneration. S-glucan and chitin accumulation as well as RNA and protein synthesis started simultaneously after a short lag, but R-glucan formation was delayed. The reversion of hyphal tubes only began after several hours of rapid R-glucan synthesis. Cycloheximide (0.5 mug/ml), inhibiting protein synthesis by 98% inhibited the formation of R-glucan and the reversion to hyphal growth but the formation of chitin and S-glucan did start and continued seemingly unimpaired for several hours. This indicates that the enzymes responsible for the synthesis of S-glucan and chitin remained intact during protoplast preparation. Polyoxin D inhibited both the synthesis of chitin and R-glucan and also the reversion to hyphal growth. However, the synthesis of S-glucan was not suppressed. These inhibitor studies as well as the kinetics of R-glucan formation during normal regeneration suggest that the synthesis of R-glucan is required for the initiation of hyphal morphogenesis.     

Relationship between the nitrogenase activity and dark respiration rate of Synechococcus RF-1

Growth of three different anaerobic rumen fungi Neocallimastix frontalis, Piromonas communis and Sphaeromonas communis was assessed in vitro at regular intervals by measurements of protein and chitin content and of chitin synthase activity of the cell free extracts. Similar trends and a comparable amount of protein and chitin were observed in the three species. However, chitin synthase activity was higher in S. communis and contrary to the activity of the other two strains did not decrease after maximum enzyme activity was reached. There were positive correlations between chitin content, protein content and chitin synthase activity during the active growth phase of the fungi indicating that they could be confidently used to determine in vitro growth phase and biomass concentration.     

Purification and characterization of three separate bacteriolytic enzymes excreted by Staphylococcus aureus, Staphylococcus simulans, and Staphylococcus saprophyticus.

As a further development of previous investigations showing that different staphylococcal species display different bacteriolytic activity patterns (lyogroups), the bacteriolytic enzymes excreted by three different Staphylococcus species, Staphylococcus aureus (lyogroup I), S. simulans (lyogroup II), and S. saprophyticus (lyogroup IV); have been purified and characterized. A representative strain from each species was grown in a preselected medium made of fully dialyzable products. Culture supernatants were collected in the appropriate growth phase. Two different affinity adsorbents were used for enzyme purification. One was obtained by coupling lysozyme-digested pure peptidoglycan from Micrococcus luteus to cyanogen bromide-activated Sepharose 4B. The second affinity adsorbent used was chitin. The S. aureus bacteriolytic enzyme bound to the solubilized peptidoglycan but not to chitin, whereas the opposite was true for the S. simulans enzyme. The bacteriolytic enzyme from S. saprophyticus did not bind to either the Sepharose 4B-peptidoglycan resin or to chitin, and its purification was achieved by two ion-exchange chromatography steps combined with gel filtration. All three enzymes were purified to apparent homogeneity. Their subsequent characterization indicated that all acted as endo-beta-N-acetylglucosaminidases. However, the three glucosaminidases differed significantly in their kinetics of activity and bacteriolytic spectrum against heat-killed cells of a variety of microorganisms. Very different values also resulted from molecular weight determinations: 80,000 for the S. aureus enzyme, 45,000 for the S. simulans enzyme, and 31,000 for the S. saprophyticus enzyme. Other important differences were observed in their stability, optimal pH and ionic strength for their activity, and their responses to temperature and divalent cations. These results confirmed the previous proposal that different staphylococcal species excrete different lytic enzymes.     

Effect of chitin heparinoids on the activation of peritoneal macrophages and on the production of monokines in mice

Sulfonated derivatives of chitin which showed anticoagulant activity (chitin heparinoids) were studied with regard to the activation of mouse peritoneal macrophages and the production of monokines. In comparison with 70% deacetylated chitin (DAC-70), which was the most adjuvant-active derivative of chitin, all chitin heparinoids were less effective for the augmentation of cytolytic activity of peritoneal macrophages. The number of macrophages was hardly increased or decreased by intraperitoneal injection of chitin heparinoids, and the activity of circulating colony-stimulating factor was not changed by their treatment. Only N-sulfonated DAC-70 stimulated the production of interleukin-1 by thioglycolate-induced peritoneal macrophages in vitro. However, its effect was weaker than that of DAC-70. Chitin heparinoids showed no or weak mitogenic activity on normal mouse spleen cells.     

The activity of immobilized enzymes on different krill chitin preparations

The suitability of krill chitin, prepared by using different concentrations of KOH and HCI for deproteinization and demineralization, respectively, was investigated. The activity of enzymes immobilized on such supports depends on the degree of deproteinization of chitin, availability of amino groups, content of minerals, mesh size, structure of the surface, and conformation of the chitin molecules. It was found that invertase and amyloglucosidase achieved high activity after immobilization on chitin obtained at not too rigorous conditions of deproteinization. However, the activity of immobilized α-amylase and diastase increased significantly with the increase in concentration of KOH used for deproteinization. High content of minerals and proteins in chitin preparation causes a loss of immobilized enzyme activity.     

A Septin-based Hierarchy of Proteins Required for Localized Deposition of Chitin in the Saccharomyces cerevisiae Cell Wall

Just before bud emergence, a Saccharomyces cerevisiae cell forms a ring of chitin in its cell wall; this ring remains at the base of the bud as the bud grows and ultimately forms part of the bud scar marking the division site on the mother cell. The chitin ring seems to be formed largely or entirely by chitin synthase III, one of the three known chitin synthases in S. cerevisiae. The chitin ring does not form normally in temperature-sensitive mutants defective in any of four septins, a family of proteins that are constituents of the “neck filaments” that lie immediately subjacent to the plasma membrane in the mother-bud neck. In addition, a synthetic-lethal interaction was found between cdc12-5, a temperature-sensitive septin mutation, and a mutant allele of CHS4, which encodes an activator of chitin synthase III. Two-hybrid analysis revealed no direct interaction between the septins and Chs4p but identified a novel gene, BNI4, whose product interacts both with Chs4p and Cdc10p and with one of the septins, Cdc10p; this analysis also revealed an interaction between Chs4p and Chs3p, the catalytic subunit of chitin synthase III. Bni4p has no known homologues; it contains a predicted coiled-coil domain, but no other recognizable motifs. Deletion of BNI4 is not lethal, but causes delocalization of chitin deposition and aberrant cellular morphology. Overexpression of Bni4p also causes delocalization of chitin deposition and produces a cellular morphology similar to that of septin mutants. Immunolocalization experiments show that Bni4p localizes to a ring at the mother-bud neck that lies predominantly on the mother-cell side (corresponding to the predominant site of chitin deposition). This localization depends on the septins but not on Chs4p or Chs3p. A GFP-Chs4p fusion protein also localizes to a ring at the mother-bud neck on the mother-cell side. This localization is dependent on the septins, Bni4p, and Chs3p. Chs3p, whose normal localization is similar to that of Chs4p, does not localize properly in bni4, chs4, or septin mutant strains or in strains that accumulate excess Bni4p. In contrast, localization of the septins is essentially normal in bni4, chs4, and chs3 mutant strains and in strains that accumulate excess Bni4p. Taken together, these results suggest that the normal localization of chitin synthase III activity is achieved by assembly of a complex in which Chs3p is linked to the septins via Chs4p and Bni4p.     

枯草芽孢杆菌甲壳素脱乙酰酶的酶学性质

研究了甲壳素脱乙酰酶的热稳定性及酶的反应体系作用条件:酶(干重)添加量为40 mg.L-1,甲壳素底物(干重)质量浓度为75 mg.L-1,反应时间为90 m in,金属离子Mg2+对酶活有激活作用,在最适宜反应条件下的酶活为2250 U.L-1。甲壳素脱乙酰酶的酶解方式为外切酶型,酶降解终产物对酶活力有抑制作用,酶对甲壳素有一定的降解作用。     

The roles of the C-terminal domain and type III domains of chitinase A1 from Bacillus circulans WL-12 in chitin degradation.

The mature form of chitinase A1 from Bacillus circulans WL-12 comprises a C-terminal domain, two type III modules (domains), and a large N-terminal domain which contains the catalytic site of the enzyme. In order to better define the roles of these chitinase domains in chitin degradation, modified chiA genes encoding various deletions of chitinase A1 were constructed. The modified chiA genes were expressed in Escherichia coli, and the gene products were analyzed after purification by high-performance liquid chromatography. Intact chitinase A1 specifically bound to chitin, while it did not show significant binding activity towards partially acetylated chitosan and other insoluble polysaccharides. Chitinases lacking the C-terminal domain lost much of this binding activity to chitin as well as colloidal chitin-hydrolyzing activity. Deletion of the type III domains, on the other hand, did not affect chitin-binding activity but did result in significantly decreased colloidal chitin-hydrolyzing activity. Hydrolysis of low-molecular-weight substrates, soluble high-molecular-weight substrates, and insoluble high-molecular-weight substrates to which chitinase A1 does not bind were not significantly affected by these deletions. Thus, it was concluded that the C-terminal domain is a chitin-binding domain required for the specific binding to chitin and that this chitin-binding activity is important for efficient hydrolysis of the sufficiently acetylated chitin. Type III modules are not directly involved in the chitin binding but play an important functional role in the hydrolysis of chitin by the enzyme bound to chitin.     

Role of sterol structure in the thermotropic behavior of plasma membranes of Saccharomyces cerevisiae

Plasma membranes from Saccharomyces cerevisiae were prepared by a new procedure involving lyticase treatment of the yeast cells. The plasma membranes were right-side-out, closed vesicles of uniform appearance with a sterol to phospholipid molar ratio of 0.365. The thermotropic behavior of these plasma membranes from wild-type yeast and from sterol mutants was examined by differential scanning calorimetry, fluorescence anisotropy and Arrhenius kinetics of plasma membrane enzymes. While differential scanning calorimetry failed to demonstrate any lipid transition, fluorescence anisotropy data indicated that lipid transitions were occurring in the plasma membranes of the yeast sterol mutants but not the sterol wild-type. The temperature dependence of the plasma membrane enzymes, chitin synthase and Mg²⁺-ATPase, was also investigated. The Arrhenius kinetics of chitin synthase did not reveal any transitions in either the sterol mutant or wild-type plasma membranes, yet the Arrhenius kinetics of the Mg²⁺-ATPase suggested that lipid transitions were occurring in both cases.     

The influence of supplemental components in nutrient medium on chitosan formation by the fungus Absidia orchidis

Chitosan, a derivative of chitin, is a natural component of some fungus cell walls. It is formed by the complex action of chitin synthase and chitin deacetylase. The in vitro activity of these two enzymes is known to be influenced by several factors. We investigated the influence of ferrous ions, manganese ions, cobalt ions, trypsin, and chitin, as individual supplements to the nutrient medium, on the in vivo activity of chitin synthase and chitin deacetylase to form chitosan in the fungus Absidia orchidis. Manganese and ferrous ions gave the most significant results. These ions increase chitosan yields through an increase in biomass production rather than an increase of chitosan content in cell walls. Manganese and ferrous ions lowered the activity of chitin deacetylase; however, their influence on the activity of chitin synthase was more complex. The effects of trypsin and chitin on biomass and cell wall chitosan content were negligible, while cobalt ions completely inhibited the growth of fungi.     

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.