Mouse Acidic Chitinase Effectively Degrades Random-Type Chitosan to Chitooligosaccharides of Variable Lengths under Stomach and Lung Tissue pH Conditions

Chitooligosaccharides exhibit several biomedical activities, such as inflammation and tumorigenesis reduction in mammals. The mechanism of the chitooligosaccharides’ formation in vivo has been, however, poorly understood. Here we report that mouse acidic chitinase (Chia), which is widely expressed in mouse tissues, can produce chitooligosaccharides from deacetylated chitin (chitosan) at pH levels corresponding to stomach and lung tissues. Chia degraded chitin to produce N-acetyl-d-glucosamine (GlcNAc) dimers. The block-type chitosan (heterogenous deacetylation) is soluble at pH 2.0 (optimal condition for mouse Chia) and was degraded into chitooligosaccharides with various sizes ranging from di- to nonamers. The random-type chitosan (homogenous deacetylation) is soluble in water that enables us to examine its degradation at pH 2.0, 5.0, and 7.0. Incubation of these substrates with Chia resulted in the more efficient production of chitooligosaccharides with more variable sizes was from random-type chitosan than from the block-type form of the molecule. The data presented here indicate that Chia digests chitosan acquired by homogenous deacetylation of chitin in vitro and in vivo. The degradation products may then influence different physiological or pathological processes. Our results also suggest that bioactive chitooligosaccharides can be obtained conveniently using homogenously deacetylated chitosan and Chia for various biomedical applications.     

Microbial reclamation of squid pens and shrimp shells

Byproducts generated in high levels by marine processes have been recognized for their value as recyclable or reclaimable waste. Among marine byproducts, shrimp shells, crab shells, and squid pens have the highest chitin content. The chemical treatments of these chitin-containing byproducts for preparing chitin and chitosan create waste disposal problems because neutralization and detoxification of the discharged wastewater are necessary. Therefore, the cost of chitin and chitosan preparations was far higher than those of their raw materials, marine chitin-containing byproducts. Chitin and chitosan have been widely used as the major carbon source of bacteria for producing chitinolytic enzymes. In 1997, the bifunctional chitinase/lysozymes from Pseudomonas aeruginosa K-187 using shrimp and crab shells as the sole carbon/nitrogen (C/N) source was first reported. Thereafter, the use of squid pens as the only C/N source for producing enzymes and bioactive materials had also been studied. The use of shellfish chitin waste as the sole C/N source not only solves environmental problems, it decreases the production costs for microbial conversion. This review summarizes our recent research of microbial reclamation of these marine byproducts for producing enzymes and bioactive materials; the characterization and applications of these products were also studied.     

Purification and Characterization of Thermostable Chitinase from <Emphasis Type="Italic">Bacillus licheniformis</Emphasis> SK-1

Chitinase was purified from the culture medium of Bacillus licheniformis SK-1 by colloidal chitin affinity adsorption followed by diethylamino ethanol-cellulose column chromatography. The purified enzyme showed a single band on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The molecular size and pI of chitinase 72 (Chi72) were 72 kDa and 4.62 (Chi72) kDa, respectively. The purified chitinase revealed two activity optima at pH 6 and 8 when colloidal chitin was used as substrate. The enzyme exhibited activity in broad temperature range, from 40 to 70°C, with optimum at 55°C. It was stable for 2 h at temperatures below 60°C and stable over a broad pH range of 4.0–9.0 for 24 h. The apparent K _m and V _max of Chi72 for colloidal chitin were 0.23 mg ml⁻¹ and 7.03 U/mg, respectively. The chitinase activity was high on colloidal chitin, regenerated chitin, partially N-acetylated chitin, and chitosan. N-bromosuccinamide completely inhibited the enzyme activity. This enzyme should be a good candidate for applications in the recycling of chitin waste.     

Squid chitin as a potential alternative chitin source: Deacetylation behavior and characteristic properties

β-Chitin was isolated from squid pens, and the characteristic chemical and physical properties were elucidated in comparison with those of shrimp chitin, α-chitin. Deacetylation behavior of the squid chitin was first studied to look into the reactivity of β-chitin and also to establish an efficient procedure for preparing squid chitosan. The squid chitin proved to show much higher reactivity in alkaline deacetylation than shrimp chitin. Although it was deacetylated quite easily, the product assumed a dark brown color under the ordinary reaction conditions for shrimp chitosan. Squid chitosan was successfully prepared by repeated alkaline treatments under mild conditions, particularly with high concentration alkali at low temperatures, without appreciable discoloration. The structural characteristics of the squid chitin were discussed on the basis of the IR and x-ray analysis data. The crystalline structure of squid chitin was destroyed easily on deacetylation compared to that of shrimp chitin, and moreover, the resulting squid chitosan was amorphous unlike crystalline shrimp chitosan. The squid chitin was characterized by the remarkable affinity for organic solvents and water. Squid chitin and chitosan also showed much higher hygroscopicity and retention of the absorbed water than shrimp chitin and chitosan and are considered to be useful as highly hydrophilic materials. © 1993 John Wiley & Sons, Inc.     

Purification and Characterisation of a 31-kDa Chitinase from the <Emphasis Type="Italic">Myzus Persicae</Emphasis> Aphid: A Target for Hemiptera Biocontrol

Hydrolytic enzymes involved in chitin degradation are important to allow moulting during insect development. Chitinases are interesting targets to disturb growth and develop alternative strategies to control insect pests. In this work, a chitinase from the aphid Myzus persicae was purified with a 36-fold purification rate in a three step procedure by ammonium sulphate fractionation, anion-exchange chromatography on a DEAE column and on an affinity Concanavalin A column. The purified chitinase purity assessed by 1D and 2D SDS–PAGE revealed a single band and three spots at 31 kDa, respectively. Chitinases were found to have high homologies with Concanavalins A and B, two chitinase-related proteins, a fungal endochitinase and an aphid acetylhydrolase by peptide identification by Maldi-Tof-Tof. The efficiency of two potent chitinase inhibitors, namely allosamidin and psammaplin A, was tested and showed significant rate of enzymatic inhibition.     

The Characteristics of Chitinase Expression in Aeromonas schubertii

In this study, chitinase activity in an incubation broth of Aeromonas schubertii was measured using colloidal chitin azure as the substrate. More specifically, the induction of chitinases due to amendment with various carbon sources was examined. The highest chitinase activity was found following amendment with 0.5–1.0 % chitin powder, whereas the activity increased negligibly due to amendment with other carbon sources, such as glucose, GlcNAc, GlcN, sorbitol, sucrose, cellulose, or starch. The chitinase activity induced by the chitin powder was suppressed when the glucose, GlcNAc, GlcN, or starch was added simultaneously to the medium but was not suppressed by the addition of sorbitol, sucrose, or cellulose. The activity of chitinase in the crude extract was also not directly inhibited by glucose. Taken together, these findings suggest that the induction of chitinase activity depends on the acquisition of suitable carbon sources from the environment and that induction occurs at a regulatory level.     

Role of Tyr-435 of <Emphasis Type="Italic">Vibrio harveyi</Emphasis> Chitinase A in Chitin Utilization

Vibrio harveyi chitinase A or VhChiA (EC.3.2.1.14) is a member of GH-18 chitinases that catalyzes chitin degradation from marine biomaterials. Our earlier structural data of VhChiA suggested that Tyr-435 marks the ending of subsite +2 and may influence binding of the interacting substrate at the aglycone binding sites. This study reports the effects of Tyr-435 using site-directed mutagenesis technique. Mutation of Tyr-435 to Ala (mutant Y435A) enhanced both binding and catalytic efficiency of VhChiA, whereas substitution of Tyr-435 to Trp (mutant Y435W) lessened the ability of the enzyme to bind and hydrolyze chitin substrates. The increased activity of Y435A can be explained by partial removal of a steric clash around subsite (+2), thereby allowing a chitin chain to move beyond or to access the enzyme’s active site from the aglycone side more straightforwardly.     

Self-assembled chitin nanofibers and applications

Self-assembled natural biomaterials offer a variety of ready-made nanostructures available for basic science research and technological applications. Most natural structural materials are made of self-assembled nanofibers with diameters in the nanometer range. Among these materials, chitin is the second most abundant polysaccharide after cellulose and is part of the exoskeleton or arthropods and mollusk shells. Chitin has several desirable properties as a biomaterial including mechanical strength, chemical and thermal stability, and biocompatibility. However, chitin insolubility in most organic solvents has somewhat limited its use. In this research highlight, we describe recent developments in producing biogenic chitin nanofibers using self-assembly from a solution of squid pen β-chitin in hexafluoroisopropanol. With this solution based assembly, we have demonstrated chitin-silk composite self-assembly, chitin nanofiber fabrication across length-scales, and manufacturing of chitin nanofiber substrates for tissue engineering.     

Large-Scale Preparation of N,N'-Diacetylchitobiose by Enzymic Degradation of Chitin and its Chemical Modifications

Abstract

Hundred gram quantities of peracetylated chitobiose were prepared by degradation of colloidal chitin with commercially available chitinase (EC 3.2.1.14) from Streptomyces griseus and subsequent acetylation, the product being readily convertible into N, N'-diacetylchitobiose by conventional de-O-acetylation. These chitobiose derivatives were subjected to further chemical modifications to give novel disaccharide derivatives composed of a pair of 2-acetamido-2-deoxy-D-allopyranose moieties that are potential intermediates for the synthesis of an enzyme inhibitor.     

Preparation of tough hydrogels based on β-chitin nanofibers via NaOH treatment

Cellulose and chitin exist in nature as highly crystalline nanofibers. Previously, we reported preparing unique hydrogels from cellulose nanofibers by a simple NaOH treatment without use of any specific solvents or cross-linking agents. In the present study, a similar gel preparation was applied to β-chitin nanofibers extracted from purified squid pen powder. The crystal structure of chitin nanofibers was transformed from β-chitin to α-chitin by NaOH(aq) treatment above 30 wt%. The crystal conversion involving the interdigitation among adjacent nanofibers caused the formation of stable hydrogels with a α-chitin nanofiber network. The use of ethanol voided the dissolution during neutralization and enabled preparation of a higher crystalline hydrogel with high mechanical strength. It achieved a Young’s modulus of 16.6 MPa, a tensile strength of 7 MPa and a strain at break of 52.2 %, on average. Finally, we note that the shrinkage of the cellulose I and β-chitin nanofibers in aqueous NaOH solutions was caused by the release of tensile residual stress due to the intracrystalline swelling in NaOH solutions.     

Microbial synthesis of chitinase in solid cultures and its potential as a biocontrol agent against phytopathogenic fungus Colletotrichum gloeosporioides

Antifungal activity of chitinase can be effectively utilized in biologic pest control strategies. Because solid-state cultivation has been termed a cost-effective means for fungal growth and metabolite production, chitinase production by Trichoderma harzianum was studied using wheat bran-based solid medium containing 1% colloidal chitin. Chitinase synthesis was found to be growth associated because maximum enzyme (5.4 U/g of dry substrate) and biomass production occurred at 72h. Substrate moisture had a critical impact on chitinase production; five grams of medium having an initial moisture content of 68.4% when incubated for 72 h increased the enzyme yield to 9.3 U/g of dry substrate. Optimization of colloidal chitin concentration showed that improvements in chitinase yield and maximum activity were attained with a 2% (w/w) concentration. Supplementation of additional nitrogen sources also influenced enzyme production, and the best yield was obtained with yeast extract. The effect of crude chitinase on hyphal morphology of the phytopathogenic fungus Collelotrichum gloeosporioides was swelling as well as lysis of hyphal wall, depending on the age of the mycelium. Studies of pH and thermal stability showed that crude culture filtrate was active over pH 4.0–6.0 and retained about 48.2% activity after 40 min of incubation at 40°C.     

Preparation of Chitin Hydrogel Under Mild Conditions

Both the amount of water and the number of calcium ions are main factors affecting the dissolution of chitin in calcium chloride dihydrate-saturated methanol (calcium solvent). The higher degree of N-acetylation of the chitin was also indicated by its higher solubility in calcium solvent. The chitin hydrogel was prepared by adding a large excess of water to the chitin solution with vigorous stirring, followed by extensive dialysis against water or by filtration to remove the methanol and calcium ions. The water content of the chitin hydrogel was approximately 94–96% (w/v) and could be controlled by centrifugation. The chitin gel was also prepared by the addition of a large excess of alcohol, such as ethanol and iso-propanol, and these protocols were found to be effective under anhydrous conditions because the alcohols were exchangeable with other organic solvents in solution. The chitin hydrogel was more susceptible to lysozyme than to chitinase, and showed and a poor susceptibility to chitosanase. A α-chitin-type crystalline structure was regenerated from chitin sheets prepared from both α-chitin and β-chitin solutions in calcium solvent, but the β-chitin-type sheet was formed from the β-chitin hydrogel prepared by mechanical agitation in water. The α-chitin hydrogel solidified when thawed after freezing, but the β-chitin hydrogel prepared by mechanical agitation maintained its gel form even after prolonged freezing. Animal studies revealed a low toxicity for the chitin sheet and an acceleration of epidermal cell regeneration.     

Chitinase‐Catalyzed Synthesis of a Chitin‐Xylan Hybrid Polymer: A Novel Water‐Soluble β(1 → 4) Polysaccharide Having an N‐Acetylglucosamine‐Xylose Repeating Unit

Summary: A chitin‐xylan hybrid polysaccharide having β(1 → 4)‐linked alternating structure of N‐acetyl‐D ‐glucosamine and D ‐xylose was synthesized via chitinase‐catalyzed polymerization. An oxazoline derivative of D ‐xylosyl‐β(1 → 4)‐N‐acetyl‐D ‐glucosamine ( 1 ) was effectively polymerized by the catalysis of chitinase from Bacillus sp., giving rise to a water‐soluble chitin‐xylan hybrid polysaccharide ( 2 ) in good yields. Molecular weights ( ) of 2 reached 1 500, which corresponds to 8–10 saccharide units.

A chitin‐xylan hybrid polysaccharide ( 2 ) synthesized via chitinase‐catalyzed polymerization.     

The pyrroloquinoline quinone biosynthesis pathway revisited: A structural approach

Background

Vibrio carchariae chitinase A (EC3.2.1.14) is a family-18 glycosyl hydrolase and comprises three distinct structural domains: i) the amino terminal chitin binding domain (ChBD); ii) the (α/β)₈ TIM barrel catalytic domain (CatD); and iii) the α + β insertion domain. The predicted tertiary structure of V. carchariae chitinase A has located the residues Ser33 & Trp70 at the end of ChBD and Trp231 & Tyr245 at the exterior of the catalytic cleft. These residues are surface-exposed and presumably play an important role in chitin hydrolysis.

Results

Point mutations of the target residues of V. carchariae chitinase A were generated by site-directed mutagenesis. With respect to their binding activity towards crystalline α-chitin and colloidal chitin, chitin binding assays demonstrated a considerable decrease for mutants W70A and Y245W, and a notable increase for S33W and W231A. When the specific hydrolyzing activity was determined, mutant W231A displayed reduced hydrolytic activity, whilst Y245W showed enhanced activity. This suggested that an alteration in the hydrolytic activity was not correlated with a change in the ability of the enzyme to bind to chitin polymer. A mutation of Trp70 to Ala caused the most severe loss in both the binding and hydrolytic activities, which suggested that it is essential for crystalline chitin binding and hydrolysis. Mutations varied neither the specific hydrolyzing activity against pNP-[GlcNAc]₂, nor the catalytic efficiency against chitohexaose, implying that the mutated residues are not important in oligosaccharide hydrolysis.

Conclusion

Our data provide direct evidence that the binding as well as hydrolytic activities of V. carchariae chitinase A to insoluble chitin are greatly influenced by Trp70 and less influenced by Ser33. Though Trp231 and Tyr245 are involved in chitin hydrolysis, they do not play a major role in the binding process of crystalline chitin and the guidance of the chitin chain into the substrate binding cleft of the enzyme.     

Anomalous electrophoretic behavior of a chitinase isoform from grape berries and wine in glycol chitin-containing sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels

An anomalous electrophoretic behavior of a chitinase isoform present in both grape (Vitis vinifera L.) berries and wine was observed in glycol chitin-containing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels. A progressive shift of the relative molecular mass M(r) of the enzyme (from approximately 30,500 up to approximately 57,700) with increasing glycol chitin concentration in the gels up to 0.1% was revealed when samples were electrophoresed under nonreducing conditions, whereas the presence of glycol chitin had no effects when samples were reduced before SDS-PAGE separation. The M(r) of other grape and wine chitinase isoforms as well as that of the chitinase from pomegranate (Punica granatum L.) fruit was unaffected by the presence of the substrate in the gel under both reducing and nonreducing conditions. Since the enzymes were inactive during the electrophoretic separation, it is likely that the retarding effect of glycol chitin observed specifically for the unreduced chitinase band from grape and wine was due to an interaction between the substrate and a chitin-binding domain different from the catalytic site, such as that typical of class I and class IV chitinases.     

WATER-SOLUBLE CHITIN OF LOW DEGREE OF DEACETYLATION

It was found that regenerated chitin obtained by a concentrated alkali treatment at a low temperature is water soluble. Chitin with 38% deacetylation, obtained by treatment with 15 wt.% NaOH at 10°C for four days, showed very good solubility in water at room temperature; whereas, eight days at 3°C were needed to prepare soluble chitin with 25% deacetylation. For this low-temperature deacetylation, two conditions were necessary to make α-chitin water soluble; first, an extended alkali treatment (e.g., at least four days in 15% alkali solution at 3°C) was required; and second, the degree of deacetylation required was more than 25%. The structural difference in regenerated chitin samples prepared at 3 and 25°C with the same degree of deacetylation (30%) were examined by X-ray diffraction and deamination analyses suggesting that the distribution of N-acetyl groups in the former chitin molecule was more random than those in the latter. This conclusion was supported by enzymatic analyses with chitinase or lysozyme.     

Purification and Characterization of Chitinase from <Emphasis Type="BoldItalic">Paenibacillus</Emphasis> sp. D1

A 56.56-kDa extracellular chitinase from Paenibacillus sp. D1 was purified to 52.3-fold by ion exchange chromatography using SP Sepharose. Maximum enzyme activity was recorded at pH 5.0 and 50 °C. MALDI-LC-MS/MS analysis identified the purified enzyme as chitinase with 60% similarity to chitinase Chi55 of Paenibacillus ehimensis. The activation energy (E _a) for chitin hydrolysis and temperature quotient (Q ₁₀) at optimum temperature was found to be 19.14 kJ/mol and 1.25, respectively. Determination of kinetic constants k _m, V _max, k _cat, and k _cat/k _m and thermodynamic parameters ΔH*, ΔS*, ΔG*, ΔG*_E–S, and ΔG*_E–T revealed high affinity of the enzyme for chitin. The enzyme exhibited higher stability in presence of commonly used protectant fungicides Captan, Carbendazim, and Mancozeb compared to control as reflected from the t _1/2 values suggesting its applicability in integrated pest management for control of soil-borne fungal phytopathogens. The order of stability of chitinase in presence of fungicides at 80 °C as revealed from t _1/2 values and thermodynamic parameters E _a(d) (activation energy for irreversible deactivation), ΔH*, ΔG*, and ΔS* was: Captan > Carbendazim > Mancozeb > control. The present study is the first report on thermodynamic and kinetic characterization of chitinase from Paenibacillus sp. D1.     

Crab-Eating Monkey Acidic Chitinase (CHIA) Efficiently Degrades Chitin and Chitosan under Acidic and High-Temperature Conditions

Chitooligosaccharides, the degradation products of chitin and chitosan, possess anti-bacterial, anti-tumor, and anti-inflammatory activities. The enzymatic production of chitooligosaccharides may increase the interest in their potential biomedical or agricultural usability in terms of the safety and simplicity of the manufacturing process. Crab-eating monkey acidic chitinase (CHIA) is an enzyme with robust activity in various environments. Here, we report the efficient degradation of chitin and chitosan by monkey CHIA under acidic and high-temperature conditions. Monkey CHIA hydrolyzed α-chitin at 50 °C, producing N-acetyl-d-glucosamine (GlcNAc) dimers more efficiently than at 37 °C. Moreover, the degradation rate increased with a longer incubation time (up to 72 h) without the inactivation of the enzyme. Five substrates (α-chitin, colloidal chitin, P-chitin, block-type, and random-type chitosan substrates) were exposed to monkey CHIS at pH 2.0 or pH 5.0 at 50 °C. P-chitin and random-type chitosan appeared to be the best sources of GlcNAc dimers and broad-scale chitooligosaccharides, respectively. In addition, the pattern of the products from the block-type chitosan was different between pH conditions (pH 2.0 and pH 5.0). Thus, monkey CHIA can degrade chitin and chitosan efficiently without inactivation under high-temperature or low pH conditions. Our results show that certain chitooligosaccharides are enriched by using different substrates under different conditions. Therefore, the reaction conditions can be adjusted to obtain desired oligomers. Crab-eating monkey CHIA can potentially become an efficient tool in producing chitooligosaccharide sets for agricultural and biomedical purposes.     

甲壳素/甲壳胺的聚集态结构及性能

制备了不同脱乙酰度的甲壳素,并对脱酰化反应进行了研究,找出了适合不同脱酰度甲壳素的溶剂,探讨了制样温度与甲壳胺膜的结晶形态和力学性能之间的关系,比较了甲壳素、甲壳胺及不同来源甲壳素的结晶形态.     

Chitinase inhibits growth of human breast and colorectal cancer cells in vitro and Ehrlich ascites carcinoma cells in vivo

Trichosanthes dioica seed extract was loaded on a QA-cellulose column and the unbound fraction with the chitinase activity was run on SDS-PAGE. Multiple bands were observed and were separated by a Sephadex G-50 column. The combination of the 6 and 33 kDa masses supported the degradation of chitinase as purified earlier. Only the 33 kDa fraction contained sugar and showed chitinase activity. The chitinase was also isolated by using a chitin column. At 200 µg/ml protein concentration, the chitinase inhibited 49.1 %, 48.8 % and 38.12 % of Ehrlich ascites carcinoma, HCT-116 and MCF-7 cells growth, respectively, in a dose-dependent manner. Exactly, 46 % and 82 % EAC cell growth inhibition were observed after treating the EAC cells bearing Swiss albino mice with the chitinase at the doses of 1.0 and 2.0 mg/Kg/day respectively. EAC, HCT-116 and MCF-7 cells growth inhibitions were due to the induction of apoptosis. ROS was accumulated in HCT-116 and MCF-7 cells. After treatment of HCT-116 cells, the expression level of p53 and TNFα genes increased and PARP gene decreased. On the other hand, elevated expression was observed for PARP, MAPK, NFκB, FAS, FADD, and Caspase-8 genes in MCF-7 cells. The induction of apoptosis in HCT-116 was further confirmed by caspase protein expression. The chitinase causes ‘S’ cell cycle arrest in MCF-7 and HCT-116 cells. T. dioica seed chitinase inhibited EAC, HCT-116 and MCF-7 cells by inducing apoptosis in vitro and EAC in vivo in mice. These promising results indicated that T. dioica seed chitinase can be an anticancer agent.