Chitin Degradation Products Research Articles

Comparison of the protective effects of chitosan oligosaccharides and chitin oligosaccharide on apoptosis, inflammation and oxidative stress.

Chitin degradation products, especially chitosan oligosaccharides (COSs), are highly valued in various industrial fields, such as food, medicine, cosmetics and agriculture, for their rich resources and high cost-effectiveness. However, little is known about the impact of acetylation on COS cellular bioactivity. The present study aimed to compare the differential effects of COS and highly N-acetylated COS (NACOS), known as chitin oligosaccharide, on H2O2-induced cell stress. MTT assay showed that pretreatment with NACOS and COS markedly inhibited H2O2-induced RAW264.7 cell death in a concentration-dependent manner. Flow cytometry indicated that NACOS and COS exerted an anti-apoptosis effect on H2O2-induced oxidative damage in RAW264.7 cells. NACOS and COS treatment ameliorated H2O2-induced RAW264.7 cell cycle arrest. Western blotting revealed that the anti-oxidation effects of NACOS and COS were mediated by suppressing expression of proteins involved in H2O2-induced apoptosis, including Bax, Bcl-2 and cleaved PARP. Furthermore, the antagonist effects of NACOS were greater than those of COS, suggesting that acetylation was essential for the protective effects of COS.

Chaperone-assisted soluble expression and characterization of chitinase chiZJ408 in Escherichia coli BL21 and the chitin degradation by recombinant enzyme

The chaperone-assisted soluble expression and characterization of high molecular weight chitinase chiZJ408 in Escherichia coli BL21 were investigated. Chitin degradation products by chitinase chiZJ408 were analyzed. The results indicated that the introduction of the chaperone GroELS promoted the correct folding of chitinase chiZJ408 and increased its soluble expression by 14.9% (p < 0.05) in E. coli BL21. The optimal pH and temperature of chitinase chiZJ408 were respectively 6.0 and 50 °C. Chitinase chiZJ408 was stable at pHs of 4.0 ∼ 7.0 and at below 40 °C. Mg2+and Ca2+ had a significant impact on improving the activity of chitinase chiZJ408. Chitinase chiZJ408 was demonstrated to be exochitinase that cleaved the β-1,4-glycosidic bond of the chitin chain to generate only N,N’-diacetylchitobiose. This study broadens our understanding of chitinase and provides a basis for solving the problem of inclusion body formed by long fragment gene expression in E. coli.

Effects of chitin degradation products N-acetylglucosamine and N,Nʹ-diacetylchitobiose on chitinase activity and bacterial community structure in an incubated upland soil

ABSTRACT Chitin, which is the polymer of N-acetylglucosamine (GlcNAc) linked by β1,4 glycoside bonds, has been reported as a soil amendment to mitigate plant soil diseases, increasing the population of chitin-degrading bacteria, and chitinolytic enzymatic activity in the soil. In some chitin-degrading bacteria, whose chitinolytic systems have been intensively studied, the chitin degradation product N,Nʹ-diacetylchitobiose {(GlcNAc)2} induces expression of genes for chitinases whereas GlcNAc does not. To evaluate the effects of these mono- and disaccharides on the population and activity of chitinolytic bacteria in soil, we investigated the chitinolytic enzyme activity and bacterial community structure in an incubated upland soil supplemented with GlcNAc or (GlcNAc)2. The added GlcNAc and (GlcNAc)2 (2 mg g−1) were consumed within 1 d when incubated at 25°C. Chitinase activity was induced by (GlcNAc)2 and chitin after 1-d and 7-d incubation, respectively, but not by GlcNAc. N-acetylglucosaminidase (GlcNAcase) activity was induced by GlcNAc but was lower than those by (GlcNAc)2 and chitin. Amplicon sequencing analysis targeting 16S rRNA genes demonstrated that both GlcNAc and (GlcNAc)2 significantly increased the rate of the order Bacillales, but the compositions of Bacillales differed from each other: the family Planococcaceae significantly increased in either GlcNAc- or (GlcNAc)2-added soil, but the genera Bacillus and Paenibacillus were increased mainly by GlcNAc and (GlcNAc)2, respectively. The family Streptomycetaceae of the order Actinomycetales was significantly increased by (GlcNAc)2 and chitin, but GlcNAc did not. Thus, GlcNAc and (GlcNAc)2, which were promptly consumed in the incubated soil, indicated partly similar but distinctive effects on chitinolytic enzyme activity and bacterial communities. Both aminosugars increased GlcNAcase activity and the population size of Planococcaceae. GlcNAc increased Bacillus. Chitinase activity and the populations of Paenibacillus and Streptomycetaceae, a number of strains of which are known as potent chitin-degraders, were increased by (GlcNAc)2, but not by GlcNAc.

Contribution of chitooligosaccharides to biofilm formation, antibiotics resistance and disinfectants tolerance of Listeria monocytogenes

Listeria monocytogenes is a food-borne pathogen present in various environmental reservoirs. It exhibits resistance and tolerance to antibiotics and sanitizing agents used in several food processing industries. It has been reported that L. monocytogenes chitinase can catalyze hydrolysis of chitin polymeric carbohydrate present in the environment and act as a virulence factor that support its survival in mammalian host cells. By taking advantage of chitinase, L. monocytogenes has both saprophytic and pathogenic lifestyles in the soil and the living host, respectively. The objective of the present study was to determine the involvement of chitin degradation products such as chitooligosaccharides (COS) in biofilm formation of L. monocytogenes. Results showed that different concentrations of COS with various molecular weight enhanced biofilm formation of L. monocytogenes. Such enhancement in biofilm formation contributed to the development of antibiotics resistance and disinfectants tolerance of cells present in the biofilm. The present article also described diverse roles of chitin, chitinase, and degradation of chitin and chitin-like substrates in saprophytic and pathogenic lifestyles of L. monocytogenes. This study offers a new direction for further exploration of the mechanisms of pathogenesis caused by L. monocytogenes.

Chitosugar translocation by an unexpressed monomeric protein channel.

The outer membrane protein channel EcChiP, associated with a silent gene in E. coli, is a monomeric chitoporin. In a glucose-deficient environment, E. coli can express the ChiP gene to exploit chitin degradation products. Single-channel small ion current measurements, which reveal the dynamics of single sugar molecules trapped in channel, are used here to study the exotic transport of chitosugars by E. coli. Molecules escape from the channel on multiple timescales. Voltage-dependent trapping rates observed for charged chitosan molecules, as well as model calculations, indicate that the rapid escape processes are those in which the molecule escapes back to the side of the membrane from which it originated. The probability that a sugar molecule is translocated through the membrane is thus estimated from the current data and the dependence of this translocation probability on the length of the chitosugar molecule and the applied voltage analyzed. The described method for obtaining the translocation probability and related molecular translocation current is applicable to other transport channels.

Systematic genetic dissection of chitin degradation and uptake in Vibrio cholerae.

Vibrio cholerae is a natural resident of the aquatic environment, where a common nutrient is the chitinous exoskeletons of microscopic crustaceans. Chitin utilization requires chitinases, which degrade this insoluble polymer into soluble chitin oligosaccharides. These oligosaccharides also serve as an inducing cue for natural transformation in Vibrio species. There are 7 predicted endochitinase-like genes in the V. cholerae genome. Here, we systematically dissect the contribution of each gene to growth on chitin as well as induction of natural transformation. Specifically, we created a strain that lacks all 7 putative chitinases and from this strain, generated a panel of strains where each expresses a single chitinase. We also generated expression plasmids to ectopically express all 7 chitinases in our chitinase deficient strain. Through this analysis, we found that low levels of chitinase activity are sufficient for natural transformation, while growth on insoluble chitin as a sole carbon source requires more robust and concerted chitinase activity. We also assessed the role that the three uptake systems for the chitin degradation products GlcNAc, (GlcNAc)2 and (GlcN)2 , play in chitin utilization and competence induction. Cumulatively, this study provides mechanistic details for how this pathogen utilizes chitin to thrive and evolve in its environmental reservoir.

Bacterial chitinase: nature and perspectives for sustainable bioproduction

Concurrent advances in a number of fields have fostered the development of bioprocesses for biochemical production. Ideally, future bioprocesses will meet the demands of commercial chemical markets in an economical fashion while being sustainable through the use of renewable starting materials. A number of different renewable and abundant biopolymers (e.g., cellulose, hemicelluloses, lignin, and chitin) are potential starting material for sustainable bioprocesses, but a broad challenge remains on how to efficiently depolymerize these biopolymers to generate monomeric sugars that can be metabolized by industrial microorganisms or other useful building block chemicals. Indeed, a variety of specialty chemicals may be able to be generated from these various monomers. This review focuses on the biopolymer chitin and discusses research and knowledge relevant to chitin degradation and potential chemical products that can be made from chitin degradation products.

Fightin’ for chitin: Wie Bakterien beim Abbau von Polymeren konkurrieren

Interactions between bacteria from different phylogenetic groups are ubiquitous in the environment but have so far been underexplored. Synthetic bacterial communities can serve as model systems for investigating such interspecific interactions on the molecular level. We have established model systems in which chitin-degrading bacteria are forced to interact with opportunistic bacteria that consume chitin degradation products. These interactions range from commensal to parasitic relationships.

The Regulatory Network of Natural Competence and Transformation of Vibrio cholerae

The human pathogen Vibrio cholerae is an aquatic bacterium frequently encountered in rivers, lakes, estuaries, and coastal regions. Within these environmental reservoirs, the bacterium is often found associated with zooplankton and more specifically with their chitinous exoskeleton. Upon growth on such chitinous surfaces, V. cholerae initiates a developmental program termed “natural competence for genetic transformation.” Natural competence for transformation is a mode of horizontal gene transfer in bacteria and contributes to the maintenance and evolution of bacterial genomes. In this study, we investigated competence gene expression within this organism at the single cell level. We provide evidence that under homogeneous inducing conditions the majority of the cells express competence genes. A more heterogeneous expression pattern was observable on chitin surfaces. We hypothesize that this was the case due to the heterogeneity around the chitin surface, which might vary extensively with respect to chitin degradation products and autoinducers; these molecules contribute to competence induction based on carbon catabolite repression and quorum-sensing pathways, respectively. Therefore, we investigated the contribution of these two signaling pathways to natural competence in detail using natural transformation assays, transcriptional reporter fusions, quantitative RT–PCR, and immunological detection of protein levels using Western blot analysis. The results illustrate that all tested competence genes are dependent on the transformation regulator TfoX. Furthermore, intracellular cAMP levels play a major role in natural transformation. Finally, we demonstrate that only a minority of genes involved in natural transformation are regulated in a quorum-sensing-dependent manner and that these genes determine the fate of the surrounding DNA. We conclude with a model of the regulatory circuit of chitin-induced natural competence in V. cholerae.

Chitinases Are Essential for Sexual Development but Not Vegetative Growth in Cryptococcus neoformans

Cryptococcus neoformans is an opportunistic pathogen that mainly infects immunocompromised individuals. The fungal cell wall of C. neoformans is an excellent target for antifungal therapies since it is an essential organelle that provides cell structure and integrity. Importantly, it is needed for localization or attachment of known virulence factors, including melanin, phospholipase, and the polysaccharide capsule. The polysaccharide fraction of the cryptococcal cell wall is a complex structure composed of chitin, chitosan, and glucans. Chitin is an indispensable component of many fungal cell walls that contributes significantly to cell wall strength and integrity. Fungal cell walls are very dynamic, constantly changing during cell division and morphogenesis. Hydrolytic enzymes, such as chitinases, have been implicated in the maintenance of cell wall plasticity and separation of the mother and daughter cells at the bud neck during vegetative growth in yeast. In C. neoformans we identified four predicted endochitinases, CHI2, CHI21, CHI22, and CHI4, and a predicted exochitinase, hexosaminidase, HEX1. Enzymatic analysis indicated that Chi2, Chi22, and Hex1 actively degraded chitinoligomeric substrates. Chi2 and Hex1 activity was associated mostly with the cellular fraction, and Chi22 activity was more prominent in the supernatant. The enzymatic activity of Hex1 increased when grown in media containing only N-acetylglucosamine as a carbon source, suggesting that its activity may be inducible by chitin degradation products. Using a quadruple endochitinase deletion strain, we determined that the endochitinases do not affect the growth or morphology of C. neoformans during asexual reproduction. However, mating assays indicated that Chi2, Chi21, and Chi4 are each involved in sexual reproduction. In summary, the endochitinases were found to be dispensable for routine vegetative growth but not sexual reproduction.

Identification and characterization of genes underlying chitinolysis in Collimonas fungivorans Ter331

Through a combinatorial approach of plasposon mutagenesis, genome mining, and heterologous expression, we identified genes contributing to the chitinolytic phenotype of bacterium Collimonas fungivorans Ter331. One of five mutants with abolished ability to hydrolyze colloidal chitin carried its plasposon in the chiI gene coding for an extracellular endochitinase. Two mutants were affected in the promoter of chiP-II coding for an outer-membrane transporter of chitooligosaccharides. The remaining two mutations were linked to chitobiose/N-acetylglucosamine uptake. Thus, our model for the Collimonas chitinolytic system assumes a positive feedback regulation of chitinase activity by chitin degradation products. A second chitinase gene, chiII, coded for an exochitinase that preferentially released chitobiose from chitin analogs. Genes hexI and hexII showed coding resemblance to N-acetylglucosaminidases, and the activity of purified HexI protein towards chitin analogs suggested its role in converting chitobiose to N-acetylglucosamine. The hexI gene clustered with chiI, chiII, and chiP-II in one locus, while chitobiose/N-acetylglucosamine uptake genes colocalized in another. Both loci contained genes for conversion of N-acetylglucosamine to fructose-6-phosphate, confirming that C. fungivorans Ter331 features a complete chitin pathway. No link could be established between chitinolysis and antifungal activity of C. fungivorans Ter331, suggesting that the bacterium's reported antagonism towards fungi relies on other mechanisms.

The regulator CdsS/CdsR two-component system modulates expression of genes involved in chitin degradation of Pseudoalteromonas piscicida strain O-7

Pseudoalteromonas piscicida strain O-7 (formerly Alteromonas sp. strain O-7) is an efficient degrader of chitin in the marine environment. The chitinolytic system of the strain consists of many enzymes induced by N-acetylglucosamine (GlcNAc). This paper reports that CdsR, which is a response regulator of CdsS/CdsR two-component signal transduction system, is bound to near the promoter region of GlcNAc-induced aprIV gene. The CdsR protein as a response regulator was transphosphorylated by the CdsS protein as a sensor kinase. Furthermore, the transphosphorylation from CdsS to CdsR was promoted by chitin degradation products and a metabolite. The CdsR protein was also phosphorylated by acetyl phosphate which is an indicator of nutritive conditions of cells. Gel mobility shift assays demonstrated that phosphorylated CdsR (CdsR-P) was bound to not only near the promoter region of aprIV gene but also those of chiA, chiB, chiC, chiD and cbp1 genes which are induced in the presence of GlcNAc. Footprinting analysis demonstrated that CdsR-P was bound to the sequences around the transcriptional start sites of aprIV and chiD genes. These results indicate that CdsR is one of the common regulators of these genes involved in chitin degradation of the strain.

Identification of two group A chitinase genes in Botrytis cinerea which are differentially induced by exogenous chitin

Chitin-degrading enzymes represent potential targets for pesticides in the control of plant pathogenic fungi. Here we describe the cloning, molecular characterization, and expression analysis of two putative chitinases of Botrytis cinerea, a pathogenic fungus infecting a wide range of plants. On the basis of conserved motifs from family 18 of the glycosyl hydrolases and group A of the fungal chitinases, two fragments ( BcchiA and BcchiB) were cloned and sequenced. Expression of BcchiA and BcchiB chitinase genes upon growth under different conditions was analysed using RT-PCR. We observed that BcchiA expression was suppressed by glucose, whereas it was strongly stimulated in the presence of chitin or chitin degradation products. Conversely, BcchiB expression was not suppressed by glucose and was not stimulated by chitin or chitin degradation products. The difference in expression regulation is indicative of a functional divergence between the two chitinase paralogous genes.

Differentiation of Chitinase-Active and Non-Chitinase-Active Subpopulations of a Marine Bacterium during Chitin Degradation

The ability of marine bacteria to adhere to detrital particulate organic matter and rapidly switch on metabolic genes in an effort to reproduce is an important response for bacterial survival in the pelagic marine environment. The goal of this investigation was to evaluate the relationship between chitinolytic gene expression and extracellular chitinase activity in individual cells of the marine bacterium Pseudoalteromonas sp. strain S91 attached to solid chitin. A green fluorescent protein reporter gene under the control of the chiA promoter was used to evaluate chiA gene expression, and a precipitating enzyme-linked fluorescent probe, ELF-97-N-acetyl-beta-D-glucosaminide, was used to evaluate extracellular chitinase activity among cells in the bacterial population. Evaluation of chiA expression and ELF-97 crystal location at the single-cell level revealed two physiologically distinct subpopulations of S91 on the chitin surface: one that was chitinase active and remained associated with the surface and another that was non-chitinase active and released daughter cells into the bulk aqueous phase. It is hypothesized that the surface-associated, non-chitinase-active population is utilizing chitin degradation products that were released by the adjacent chitinase-active population for cell replication and dissemination into the bulk aqueous phase.

Method for the detection and quantitation of chitinase inhibitors in fermentation broths; isolation and insect life cycle effect of A82516.

A new method of screening for chitinase inhibitors in crude fermentation broths as a means of discovering new insecticidal leads has been developed. In this procedure soluble Remazol brilliant violet 5R dye-coupled chitin degradation products released from insoluble chitin azure substrate by hydrolysis with Streptomyces griseus chitinase are filtered in 0.45 micron Millititer HA 96 well filtration plates and collected in 96 well microtiter plates. Inhibitors of this reaction are detected by a decrease in absorbance (570 nm) of the filtrate. A chitinase inhibitor, designated A82516, produced by culture A82516 was discovered using this screen. Purified A82516 was found to have an IC50 of 3.7 X 10(-6) M for S. griseus chitinase. At a test concentration of 0.27 mg/ml, A82516 was 100% effective in preventing development of house fly larvae to pupae. Allosamidin, a recently reported chitinase inhibitor in vitro, has spectral properties identical to A82516.