Chitobiase Activities Research Articles

Effect of chronic UVR exposure on zooplankton molting and growth

Molting is a crucial physiological process in arthropods development, growth, and adult reproduction, where the chitinolytic enzyme chitobiase (CB) and the apoptosis process (caspase-3 activity) play crucial roles. Both molecular endpoints have been observed to be affected by different toxics that may be present in aquatic environments. However, the role of ultraviolet radiation (UVR) in the molting process remains with poor evidence and the possible effect of the previous exposure on F1 generation is unknown. Here, we conducted laboratory experiments with chronic UVR exposure to test the effect on the molting process of Daphnia commutata. Our results showed a clear negative effect of the UVR that affected the molting process with a reduction in individual growth. This trend was also observed in CB and caspase-3 activities. Our results also suggest that the UV dose received by the mother and eggs has an additive effect with the dose received by the offspring. These results imply that the cumulative impact of small UVR doses (2 h per day, daily dose: 2520 J m−2 of 340 nm) on mothers and eggs (which cannot be discriminated in our experiments) can have an additive or synergistic effect along with the generations through a potential increase in lethal effect. Finally, the observed desynchronization in the molting process by UVR will affect the fitness of individuals and population dynamics.

Inverse relationship between chitobiase and transglycosylation activities of chitinase-D from Serratia proteamaculans revealed by mutational and biophysical analyses.

Serratia proteamaculans chitinase-D (SpChiD) has a unique combination of hydrolytic and transglycosylation (TG) activities. The TG activity of SpChiD can be used for large-scale production of chito-oligosaccharides (CHOS). The multiple activities (hydrolytic and/or chitobiase activities and TG) of SpChiD appear to be strongly influenced by the substrate-binding cleft. Here, we report the unique property of SpChiD substrate-binding cleft, wherein, the residues Tyr28, Val35 and Thr36 control chitobiase activity and the residues Trp160 and Trp290 are crucial for TG activity. Mutants with reduced (V35G and T36G/F) or no (SpChiDΔ30–42 and Y28A) chitobiase activity produced higher amounts of the quantifiable even-chain TG product with degree of polymerization (DP)-6, indicating that the chitobiase and TG activities are inversely related. In addition to its unprecedented catalytic properties, unlike other chitinases, the single modular SpChiD showed dual unfolding transitions. Ligand-induced thermal stability studies with the catalytically inactive mutant of SpChiD (E153A) showed that the transition temperature increased upon binding of CHOS with DP2–6. Isothermal titration calorimetry experiments revealed the exceptionally high binding affinities for E153A to CHOS with DP2–6. These observations strongly support that the architecture of SpChiD substrate-binding cleft adopted to control chitobiase and TG activities, in addition to usual chitinase-mediated hydrolysis.

Toxicity assessments with Daphnia magna of Guadipyr, a new neonicotinoid insecticide and studies of its effect on acetylcholinesterase (AChE), glutathione S-transferase (GST), catalase (CAT) and chitobiase activities

Guadipyr is a novel neonicotinoid insecticide, developed by the China Agricultural University. This work investigated its aquatic toxicity on Daphnia magna. The acute immobilization test showed that guadipyr was slightly toxic to daphnids, with a 48h EC50 of 13.01mg/L. In addition, guadipyr significantly enhanced the acetylcholinesterase (AChE) and glutathione S-transferase (GST) activity (per gram of protein), but had no obvious impact on catalase (CAT) activity within 48h. The 21d chronic exposure of D. magna to guadipyr induced a significant decrease in body growth and reproduction; both share the same lowest observed effect concentration (LOEC) at 0.10mg/L. In the 14d chronic test, a significant increase in chitobiase activity in test media was observed at day 8 (days to the first breeding), while a significant decrease was observed from days 10 to 14, which might be due to the endocrine imbalance resulting from guadipyr stress. These results demonstrated that guadipyr can induce notable negative ecotoxicological impacts on the aquatic system in long-term exposure at a sub-lethal dose. Further research in environmental behaviors is needed to regulate guadipyr use in the future.

A comparative structural bioinformatics analysis of inherited mutations in β-D-Mannosidase across multiple species reveals a genotype-phenotype correlation

BackgroundLysosomal β-D-mannosidase is a glycosyl hydrolase that breaks down the glycosidic bonds at the non-reducing end of N-linked glycoproteins. Hence, it is a crucial enzyme in polysaccharide degradation pathway. Mutations in the MANBA gene that codes for lysosomal β-mannosidase, result in improper coding and malfunctioning of protein, leading to β-mannosidosis. Studying the location of mutations on the enzyme structure is a rational approach in order to understand the functional consequences of these mutations. Accordingly, the pathology and clinical manifestations of the disease could be correlated to the genotypic modifications.ResultsThe wild-type and inherited mutations of β-mannosidase were studied across four different species, human, cow, goat and mouse employing a previously demonstrated comprehensive homology modeling and mutational mapping technique, which reveals a correlation between the variation of genotype and the severity of phenotype in β-mannosidosis. X-ray crystallographic structure of β-mannosidase from Bacteroides thetaiotaomicron was used as template for 3D structural modeling of the wild-type enzymes containing all the associated ligands. These wild-type models subsequently served as templates for building mutational structures. Truncations account for approximately 70% of the mutational cases. In general, the proximity of mutations to the active site determines the severity of phenotypic expressions. Mapping mutations to the MANBA gene sequence has identified five mutational hot-spots.ConclusionAlthough restrained by a limited dataset, our comprehensive study suggests a genotype-phenotype correlation in β-mannosidosis. A predictive approach for detecting likely β-mannosidosis is also demonstrated where we have extrapolated observed mutations from one species to homologous positions in other organisms based on the proximity of the mutations to the enzyme active site and their co-location from different organisms. Apart from aiding the detection of mutational hotspots in the gene, where novel mutations could be disease-implicated, this approach also provides a way to predict new disease mutations. Higher expression of the exoglycosidase chitobiase is said to play a vital role in determining disease phenotypes in human and mouse. A bigger dataset of inherited mutations as well as a parallel study of β-mannosidase and chitobiase activities in prospective patients would be interesting to better understand the underlying reasons for β-mannosidosis.

Chitinase and apparent digestibility of chitin in the digestive tract of juvenile cobia, Rachycentron canadum

A study was designed to determine the presence of chitinolytic enzymes in cobia. Additionally, the source of the chitinolytic enzymes (bacterial or endogenous) and apparent digestibility of chitinous waste meals were investigated to determine the viability of crustacean processing waste products in juvenile cobia diets. Antibiotics were used to eliminate potential chitinolytic gut flora and chitinolytic enzyme levels were compared to control fish fed an identical diet without antibiotic. Chitinase and chitobiase were measured in the stomach, pyloric caeca, and anterior intestine but were only detected in the stomach. Control stomach chitinase and chitobiase activities were 3075 ± 709 (mean ± SD) and 2076 ± 208 μg N-acetylglucosamine (NAG) g − 1 h − 1, respectively, and not significantly different from antibiotic treatments. This suggests substantial endogenous production and that the activity, if any, from chitinolytic bacteria is not significant. Additionally, high chitobiase levels indicate breakdown of chitin to potentially nutritive NAG. To determine the apparent digestibility coefficient (ADC) of shrimp processing waste meal (shrimp meal) and crab processing waste meal (crab meal) in vivo, diets containing a 70:30 mixture of a reference diet and test ingredient with the inclusion of the non-digestible marker yttrium oxide were fed. Chitin ADC for both crab (mean = 66.8 ± 4.5% SD) and shrimp (78.2 ± 8.0%) meal was high and not significantly different. Organic matter digestion was significantly higher in crab meal than either shrimp meal or the reference diet. However, crude protein, lipid, and gross energy digestion for crab and shrimp meal were not significantly different from the reference diet. Both enzymatic and in vivo results indicate the capability for juvenile cobia to digest chitin through strong endogenous chitinolytic enzymes. The addition of crustacean processing wastes in cobia diets has the potential to reduce the cost of feed production and increase the sustainability of cobia aquaculture by reducing inclusion rates of fish meal and other more expensive ingredients.

Digestive chitinolytic activity in marine fishes of Monterey Bay, California

Chitinolytic activities, both chitinase (EC 3.2.1.14) and minimum chitobiase (β- N-acetyl- d-glucosaminidase; EC 3.2.1.30), were measured in stomach and intestinal tissues and their contents, from 13 fish species. Higher activities were found in the tissues than in the gut contents, and higher activities were seen in the stomachs than in the intestines. Demersal species exhibited chitobiase activities very close to their chitinase activities, suggesting that these fishes can degrade chitin completely to its soluble, absorbable monomer, N-acetyl-glucosamine. This suggests that these species may catabolize chitin not just to penetrate prey exoskeletons but also to derive nutrients from the chitin itself. In contrast, three mesopelagic species exhibited low chitobiase but high chitinase activities. This chitobiase limitation correlated strongly with gastrointestinal tract morphology, with the myctophids having the greatest chitobiase limitation and the shortest alimentary tracts. The high chitinase activities measured in the myctophids reflect their ability to rapidly disrupt prey exoskeletons ingested during their nightly feeding in surface waters. Their chitobiase activities are greatly reduced because with rapid meal evacuation through a short gut there is little time for processing and limited energetic advantage in the complete degradation of chitin. These results suggest multiple roles for chitinolytic enzymes in marine fishes and that feeding habits and frequency may have a bearing on the evolution of their digestive enzymes systems.

Chitinolytic activity of actinomycetes from a cerrado soil and their potential in biocontrol.

The crude enzyme extracts from five actinomycetes selected from a cerrado soil presented very good endochitinolytic activity when compared to a commercial chitinase. Exochitinase and chitobiase activities were also detected. They were identified as Streptomyces, but could not be characterized to species level, probably corresponding to new ones. The crude extracts, obtained from growth on fungal mycelium plus chitin of three of the strains, have shown a very pronounced activity against phytopathogenic fungi. In tests using growing cells, all five strains were active. These data suggest that these strains are potential biocontrol agents.

Chitobiase activity in the epidermis and hepatopancreas of the fiddler crab Uca pugilator during the molting cycle

The activity of chitobiase, also known as N-acetyl-β-glucosaminidase, in the epidermis and hepatopancreas of the fiddler crab Uca pugilator (Bosc, 1802), during the molting cycle, was investigated. A pH optimum of 5 to 6 was found for the enzymatic activity in both the epidermis and hepatopancreas. The temperature optimum for epidermal and hepatopancreatic chitobiase activities was 50 to 60 °C. The K m values for epidermal and hepatopancreatic chitobiase activities at 19 °C were 0.190 ± 0.027 and 0.203 ± 0.016 mM 4-methylumbelliferyl-N-acetyl-β-glucosaminide, respectively. Hepatopancreatic chitobiase activity was significantly higher than epidermal enzymatic activity in all the molt cycle stages tested except Postmolt Stage A-B. Chitobiase activity varied significantly during the molting cycle, with the epidermal enzymatic activity in Premolt Stage D3–4 significantly higher than in Stage C (intermolt) and Premolt Stage D0, whereas hepatopancreatic chitobiase activity in Premolt Stage D3–4 was significantly higher than in all other molt stages tested. The patterns of chitobiase activity in the epidermis and hepatopancreas correlate well with the hemolymph titer of ecdysteroids in U. pugilator during the molting cycle; this suggests that chitobiase activity in both tissues is regulated at least in part by the steroid molting hormones.

An investigation of aquatic bacteria capable of utilizing chitin as the sole source of nutrients

R. OSAWA AND T. KOGA. 1995. A total of 48 bacterial strains capable of utilizing chitin (a polymer of N‐acetyl‐D‐glucosamine [NAG]) as a sole source of nutrients were isolated from river and marine waters in Tokushima. These bacteria were identified as Vibrio fluvialis, V. parahaemolyticus, V. alginolyticus, V. mimicus, Listonella anguillarum and Aeromonas hydrophila. All strains were found positive for chitinase and chitobiase activities, and capable of utilizing NAG as a sole source of carbon and nitrogen.

Characterization of Chitobiase from Daphnia magna and Its Relation to Chitin Flux

Chitobiase (N-acetyl-β-D-glucosaminidase) activity has been measured and partially characterized in Daphnia magna. The pH optimum is approximately 5.5, and the temperature optimum is approximately 45°dg-5° dgC, as determined from crude extract. The enzyme remains stable even after a 2-h incubation at 20° or 40°C. The Km determined for methylumbelliferyl-N-acetyl-β-D-glucosaminide and p-nitrophenyl-N-acetyl-β-D-glucosaminide is 61.5 μM and 359.6 μM, respectively. Activation energy is significantly less for lower temperatures (5°-18°C) than for higher temperatures (18°-40°C). Investigation of the effect of acclimation temperature on chitobiase activities reveals no significant differences in activities between 10° and 18°C for populations raised at different temperatures. A similar pattern in the Q10's was also observed. There was no evidence of any isozyme effect for populations raised at 6°, 10°, 18°, or 25°C Attempts to protect the enzyme with various osmotic solutions and the results of differential centrifugation suggest that at least some of the enzyme is packaged in vesicles and/or lysosomes. Maximal chitobiase activity of asynchronous populations fed ad lib. overestimates the flux of N-acetyl-β-D-glucosaminide (NAG) from the old to the new cuticle by approximately 100×. Chitobiase activity modulates significantly over the molt cycle in Daphnia, with a fivefold increase in activity 6-0 h before molt. This suggests that chitobiase activity may provide an index of molt rate in asynchronously molting populations.

Discrimination between chitinase and chitobiase activities in Candida albicans.

Measurement of chitinase activity in crude homogenates of Candida albicans with the artificial substrates 4-methylumbelliferyl β-D-N, N′, N"-triacetylchitotrioside and 4-methylumbelliferyl-β-D-N, N′-diacetylchitobioside (MU-[GlcNAc]3, 2) showed that both chitobiase and chitinase were involved in the liberation of the fluorophore methylumbelliferone (MU). The substrate MU-[GlcNAc]3 appeared to be digested by means of two separate mechanisms, resulting in a lag-phase in the time curve of the MU-release, and thus making this release non-linear with time. The first mechanism is the action of an endochitinase that released MU in one step; this reaction could be inhibited by allosamidin. The second probable mechanism involves the digestion of the substrate by the stepwise release of the outer two G1cNAc residues by a chitobiase, followed either by a third chitobiase-catalyzed release of the final GlcNAc or by the action of a β-N-acetylhexosaminidase, that is also present in C. albicans. Hydrolysis of the substrate MU-[GlcNAc]2 was not inhibited by allosamidin, suggesting that the two GlcNAc residues in this substrate are probably released in two steps, either by the action of chitobiase alone, or by the successive action of chitobiase (step 1) and β-N-acetylhexosaminidase (step 2). Our results show that the activities of the individual enzymes involved in chitin degradation cannot be determined on the basis of MU release in unfractionated homogenates of C. albicans.

Characterization of chitinase and chitobiase produced by the entomopathogenic fungus Metarhizium anisopliae

Extracellular fluids from Metarhizium anisopliae grown on chitin as the sole carbon source contained distinct chitinase ( pH optimum 5.3, MW 33 kDa) and chitobiase ( pH optimum 5, MW 110 kDa, pI 6.4) activities. Chitinase activity was stabilized against extremes of pH and temperature by the presence of its chitin substrate. Chitinase fractions eluted from a sephadex column had activity against chitosan as well as locust cuticle chitin, crystalline chitin, and colloidal chitin. Products of enzymolysis were analyzed by descending paper chromatography. Chitinase had no activity against chitobiose, only trace activity against chitotriose, but preferentially cleaved chitotetraose in the middle bond releasing chitobiose. N-Acetyglucosamine was the major (vs colloidal chitin) or only (vs crystalline chitin) product detected following chitin hydrolysis. The chitobiase (also called β- N-acetylglucosaminidase) possessed simple glycosidase activity against p-nitrophenyl-acetylglucosaminide and hydrolyzed di-, tri-, and tetrasaccharides to N-acetylglucosamine. Chitobiase activity was significantly inhibited by its reaction product, N-acetylglucosamine. The mechanism of action of chitinolytic enzymes is discussed.

Chitinase production byTalaromyces emersonii

Talaromyces emersonii, when grown on medium containing chitin, yielded extracellular chitinase and chitobiase activities of 0.45 μmol.h−1.ml−1 culture fluid and 1.4 μmol. min−1.ml−1, respectively, after 2–4 days of growth under pH-controlled conditions. The enzyme system was optimally active at pH 5.0–5.5, c. 65°C and the least stable components had half-lives of 20 min at 76°C, pH 5.0.

Chitinolytic properties of Streptomyces lividans

Streptomyces lividans TK24, an established host for genetic and molecular studies in actinomycetes, is able to use chitin as sole carbon and nitrogen source. Extracellular chitinase and N-acetyl-β-d-glucosamidinase (chitobiase) activities were detected in liquid cultures. Chitinase production was inducible by chitin and its low molecular weight derivatives. Low levels of chitinase were also produced in the absence of chitin. Production of extracellular N-acetylglucosaminidase was correlated with the beginning of the stationary phase of growth and was independent of the presence of chitin. Beside highly N-acetylated chitin, supernatants of chitin-induced cultures were able to hydrolyse chitosans with a wide range of degrees of N-acetylation.

Role ofCristispira sp. and other bacteria in the chitinase and chitobiase activities of the crystalline style ofCrassostrea virginica (Gmelin)

Activity was found for chitinase and chitobiase in the crystalline styles of American oysters (Crassostrea virginica Gmelin) collected from the Chesapeake Bay (Maryland, USA). The oysters were maintained in tanks on natural food from a constant flow of unfiltered estuarine water. Chitinase and chitobiase specific activities were compared with total, viable, and chitinoclastic bacterial counts andCristispira counts. Regression analyses revealed that one correlation, chitobiase vsCristispira, was significant (P < 0.05). Several oysters were fed chitin in the presence or absence of chloramphenicol. Although no chitinoclasts were present in the antibiotic-treated oysters, the treatment means did not differ significantly (P > 0.05) for either chitinase or chitobiase activity. In several cases with both chitin-fed and naturally fed oysters, enzyme activity was found when noCristispira were present. The results of the investigations suggest that the oyster produces chitinase and chitobiase endogenously.

The growth of rainbow trout ( Salmo gairdneri) given diets containing chitin and its relationship to chitinolytic enzymes and chitin digestibility

The growth of rainbow trout fed diets containing 4, 10 and 25% chitin, over a 12-week period, was significantly depressed ( P < 0.001) when compared with controls fed diets containing 25% starch. There was no difference in growth rate between control fish and those fed diets containing 10% N-acetyl glucosamine (GlcNAc). Isotopically labelled amino sugars were shown to be oxidized when injected intraperitoneally into rainbow trout. Relatively high levels were found of chitinase activity in the stomachs and of chitobiase in the intestines. These enzyme activities were similar in all the trout, irrespective of the amount of chitin in their diets, except that chitobiase in the intestines of fish fed diets containing GlcNAc showed higher levels of activity than the controls ( P < 0.05). Chitin was not significantly digested when fed at 10 and 30% of the diet but the apparent digestibility of pre-cooked starch was 50% when fed at either 15 or 25%, on the basis of the inert indicator (Cr 2O 3) method. Chitinase and chitobiase activities were not reduced in fish fed diets containing 10% chitin and an antibiotic (Tribrissen ®) although the alimentary microflora were completely eliminated. Enzyme activities were not enhanced when live chitinolytic bacteria ( Vibrio alginolyticus) were incorporated into diets with 10% chitin; these bacteria were only recovered from the intestine. The evidence indicates an endogenous origin of chitinolytic enzymes in the trout gastro-intestinal tract. The presence of either antibiotic or bacteria in the diet had no effect on the digestibility of chitin.

Metabolism in marine flatfish—I. Carbohydrate digestion in Dover sole (Solea solea L.)

Abstract 1. 1. A survey of the carbohydrases in the digestive tract of juvenile and adult Dover sole indicated the presence of strong amylase and maltase activities. 2. 2. The highest levels of these enzymes were present in the foregut region. 3. 3. Chitinase and chitobiase activities were also detected and shown to be optimal at pH 9.5 and 5.0 respectively. 4. 4. Other carbohydrases were either absent from gut homogenates or were only present at very low levels. 5. 5. The relationship between these results and the nutritional requirements of Dover sole is discussed and comparisons made with studies on other fish.