Chitosan Depolymerization by Enzymes from the Hepatopancreas of the Crab Paralithodes camtschaticus

An enzyme preparation was isolated from the hepatopancreas of Paralithodes camtschaticus thatexhibited chitinase and chitosanase activities. Treatment of chitin and chitosan with this preparation decreased their viscosity-average molecular weights by 96 and 41%, respectively. The chromatographic profiles of the products of chitin and chitosan hydrolysis suggested that the crab hepatopancreas is rich in endochitinases. Enzymatic digestion of chitosan increased its solubility and moderately reduced the extent of its acetylation. A mathematical approach was proposed for calculating the molecular weights of chitosan fractions from weight-average molecular weights determined viscometrically.     

Properties of chitinolytic enzymes from the hepatopancreas of the red king crab (Paralithodes camtschaticus)

Our study confirms the presence of chitinolytic, chitosanolytic, and deacetylase activities in the hepatopancreas of the red king crab, related to the specific diet of this species. The maximum rate of chitin/chitosan hydrolysis by an enzyme preparation from crab hepatopancreas occurs at 36.5-37.0 degrees C. Two pH optimums have been found for the enzymatic reaction under mildly alkaline and acidic conditions for both exo- and endochitinase activities. The enzyme preparation is most affine to partly deacetylated chitin with an acetylation degree within 40-50%.     

Properties of chitinolytic enzymes from the hepatopancreas of the red king crab (<Emphasis Type="Italic">Paralithodes camtschaticus</Emphasis>)

Our study confirms the presence of chitinolytic, chitosanolytic, and deacetylase activities in the hepatopancreas of the red king crab, related to the specific diet of this species. The maximum rate of chitin/chitosan hydrolysis by an enzyme preparation from crab hepatopancreas occurs at 36.5–37.0°C. Two pH optimums have been found for the enzymatic reaction under mildly alkaline and acidic conditions for both exo-and endochitinase activities. The enzyme preparation is most affine to partly deacetylated chitin with an acetylation degree within 40–50%.     

Glycolytic activity of enzyme preparation from the red king crab (Paralithodes camtschaticus) hepatopancreas

Enzyme preparation exhibiting glycolytic activity yielding chitooligosaccharides along with N-acetyl-D-glucosamine was obtained from the red king crab (Paralithodes camtschaticus) hepatopancreas. The results of the analysis confirmed the presence of endo- and exochitinase activities in the preparation. HPLC showed that the hydrolysis products of chitin and chitosan did not contain D(+)-glucosamine, which is indicative of the absence of deacetylase and, apparently, exochitosanase activities. A comparison of the dependence of the enzyme preparation activity on temperature and pH of the incubation medium suggests that chitinase and protease activities are exhibited by different enzymes.     

Synthesis of exo-β-glucosaminidase BY FUNGUS <Emphasis Type="Italic">Penicillium</Emphasis> sp. IB-37-2

A new strain Penicillium sp. IB-37-2, which actively hydrolyzes chitosan (SD ～80–85%) but possesses low activity against colloidal chitin, was isolated. The fungus was observed to have a high level chitosanase biosynthesis (1.5–3.0 U/mL) during submerged cultivation at 28°C, with a pH of 3.5–7.0 and 220 rpm in nutrient media containing chitosan or chitin from shells of crabs. Purification of the chitosanase enzyme complex from Penicillium sp. IB-37-2 by ultrafiltration and hydrophobic chromatography, followed by denaturing electrophoresis, revealed two predominant proteins with molecular weights of 89 and 41 kDa. The purified enzyme complex demonstrated maximal activity (maximal rate of hydrolysis of dissolved chitosan) and stability at 50–55°C and a pH of 3.5–4.0. The enzyme preparation also hydrolyzed laminarin, β-(1,3)-(1,4)-glycan, and colloidal chitin. Exohydrolysis of chitosan by the preparation isolated from Penicillium sp. IB-37-2 resulted in the formation of single product, D-glucosamine.     

Solid-state characterization of chitosans derived from lobster chitin

Two samples of chitosan (CH1 and CH2) of different molecular weights and degrees of deacetylation were prepared from lobster chitin under two different processes. Solid-state properties of CH1 and CH2 were characterized and compared with four commercial chitosans prepared from crab and fresh shrimp shells. Infrared spectroscopy (IR), solid-state CP-MAS ¹³C NMR, powder X-ray diffraction and differential scanning calorimetric techniques were used to characterize the molecular structure and solid-state properties of the materials. Changes in the crystallinity and polymorphic forms of CH1 and CH2 were attributable to the different process conditions used. The differences in crystallinity were confirmed by powder X-ray diffraction data. The methods of preparation of CH1 and CH2 did not significantly influence the bulk, tap and true densities of the bulk material, but they affected the flow properties of CH1 and CH2. In conclusion, the physicochemical properties of the present chitosans prepared from lobster chitin (CH1 and CH2) are comparable with those of commercial chitosan materials of crab or shrimp shell origin.     

Fabrication and properties of a porous chitin/chitosan conduit for nerve regeneration

A porous, biodegradable, natural chitin/chitosan nerve conduit was constructed. Scanning electron microscopy confirmed that it was homogeneous and highly porous. FT-IR spectra showed that there were no residues arising from the preparation process in the conduit. Addition of chitin to the chitosan solution increased the mechanical strength and maximum tensile strength from 7.2 to 9.6 MPa. Preliminary animal tests indicated that porous chitin/chitosan conduits did not swell in vivo and were compatible with surrounding tissue.     

Glycolytic activity of enzyme preparation from the red king crab (<Emphasis Type="Italic">Paralithodes camtschaticus</Emphasis>) hepatopancreas

Enzyme preparation exhibiting glycolytic activity yielding chitooligosaccharides along with N-acetyl-D-glucosamine was obtained from the red king crab (Paralithodes camtschaticus) hepatopancreas. The results of the analysis confirmed the presence of endo- and exochitinase activities in the preparation. HPLC showed that the hydrolysis products of chitin and chitosan did not contain D(+)-glucosamine, which is indicative of the absence of deacetylase and, apparently, exochitosanase activities. A comparison of the dependence of the enzyme preparation activity on temperature and pH of the incubation medium suggests that chitinase and protease activities are exhibited by different enzymes.     

Preparative methods of phosphorylated chitin and chitosan--an overview

Biomaterials such as chitin, chitosan and their derivatives have a significant and rapid development in recent years. Chitin and chitosan have become cynosure of all party because of an unusual combination of biological activities plus mechanical and physical properties. However, the applications of chitin and chitosan are limited due to its insolubility in most of the solvents. The chemical modification of chitin and chitosan are keen interest because of these modifications would not change the fundamental skeleton of chitin and chitosan but would keep the original physicochemical and biochemical properties. They would also bring new or improved properties. The chemical modification of chitin and chitosan by phosphorylation is expected to be biocompatible and is able to promote tissue regeneration. In view of rapidly growing interest in chitin and chitosan and their chemical modified derivatives, we are here focusing the recent developments on preparation of phosphorylated chitin and chitosan in different methods.     

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

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

Effect of molecular weight of chitosan with the same degree of deacetylation on the thermal, mechanical, and permeability properties of the prepared membrane

Different molecular weight, 90% deacetylated chitosans were obtained by ultrasonic degradation on 90% deacetylated chitosan at 80 °C for various times.

Ninety percent deacetylated chitosan was prepared from alkali treatment of chitin that was obtained from red shrimp waste. Number average-, viscosity average- molecular weights were measured by gel permeation chromatography and the viscometric method, respectively. Degree of deacetylation was measured by the titration method. Enthalpy, maximum melting temperature, tensile strength and elongation of the membranes, flow rate of permeates and water are properties measured to elucidate the effect of molecular weight of chitosan on the above thermal, mechanical, and permeation properties, respectively of the prepared membranes. Results show tensile strength, tensile elongation, and enthalpy of the membrane prepared from high molecular weight chitosans were higher than those from low molecular weight. However, the permeability show membranes prepared from high molecular weight chitosans are lower than that from those of low molecular weight.     

几丁质及壳聚糖固定淀粉酶的方法探讨

洗净干燥捣碎后过筛(20目)的蟹壳经稀盐酸常温处理脱钙盐,稀碱保温处理脱蛋白,制得几丁质。几丁质再经浓碱保温处理数小时脱乙酰基,制得壳聚糖。分别以几丁质和壳聚糖为载体,各自与三种交联剂——戊二醛、环氧氯丙烷、一氯醋酸分别作用后用于淀粉酶的固定化。固定化淀粉酶活力测定表明,作为载体壳聚糖优于几丁质;作为交联剂,戊二醛最好,环氧氯丙烷次之,一氯醋酸较差。     

Extracellular proteinases and chitinases, produced by a Streptomyces kurssanovii culture

Extracellular enzymes--a chitinase and a protease with molecular weights 22 and 32 kDa, respectively--were isolated from Streptomyces kurssanovii cells. After purification on modified regenerated chitin, the enzymes were virtually homogeneous according to denaturing PAGE. Both enzymes were found to degrade chitosan.     

壳聚糖酶结构与功能的研究进展

壳聚糖酶是一类对壳聚糖具有较高催化活性而几乎不水解几丁质的糖苷水解酶,其可将高分子量的壳聚糖转化为低分子量的功能性壳寡糖。近年来,对壳聚糖酶的相关研究取得了显著进展,因此,本文对其生化性质、晶体结构、催化机制和蛋白质工程改造进行总结和探讨,并对酶法制备壳寡糖纯品进行展望,这将加深研究者对壳聚糖酶作用机制的认识,推动壳聚糖酶的工业应用。     

Deacetylation of chitin under homogeneity

Chitin isolated enzymatically from Antarctic krill shells was dissolved in aqueous NaOH by freezing and thawing to create homogenous conditions. Deacetylation was performed at room temperature or heating. The degree of deacetylation, molecular weight, and dynamic viscosity of solutions were estimated in chitosan samples. Deacetylation of chitin under homogenous conditions was optimized. Chitosans with molecular weights of 180-220 and 250-300 kDa were obtained from the chitins of Antarctic krill and Northern shrimps, respectively.     

Preparation and physico-chemical characterization of chitin and chitosan from the pens of the squid species, Loligo lessoniana and Loligo formosana

β-chitin and its chitosan from the pens of Loligo lessoniana and Loligo formosana has been isolated, prepared, and physico-chemically characterized to demonstrate a potential chitin source. Without deminerization due to negligible ash content, only deproteinization was used in the chitin isolation with an yield of 35–38%, without significant difference either between the two species or the collection seasons. Reducing step not only saves production cost but also obviates acid pollutant. Mild alkaline deacetylation with various time periods was employed in the chitosan preparation. Optical rotation and thermal transition of chitin from both species suggested the weak intermolecular forces compared with shrimp chitin. The results of nitrogen contents indicate the effectiveness of the deproteinization method used. The samples were categorized as a Class III: moderate hygroscopicity. Traces of elements presented in pens markedly decreases but are incapable to be got rid of within the step of chitin–chitosan preparation. In addition, a small amount of cadmium, as the contamination, was detected in the samples from L. formosana.     

Extraction and characterization of chitin and chitosan from marine sources in Arabian Gulf

Chitin in the α and the β forms has been extracted from different marine crustacean from the Arabian Gulf. The contents of the various exoskeletons have been analyzed and the percent of the inorganic salt (including the various elements present), protein and the chitin was determined. Deacetylation of the different chitin produced was conducted by the conventional thermal heating and by microwave heating methods. Microwave heating has reduced enormously the time of heating from 6–10 h to 10–15 min, to yield the same degree of deacetylation and higher molecular weight chitosan. This technique can save massive amount of energy when implemented on a semi-industrial or industrial scale. The chitin and the obtained chitosan were characterized by elemental analysis, XRD, NMR, FTIR and thermogravimetric measurements. XRD analysis showed that chitosan has lower crystallinity than its corresponding chitin; meanwhile its thermal stability is also lower than chitin.     

Deacetylation of Chitin under Homogeneous Conditions

Chitin isolated enzymatically from Antarctic krill shells was dissolved in aqueous NaOH by freezing and thawing to create homogeneous conditions. Deacetylation was performed at room temperature or under heating. The degree of deacetylation, molecular weight, and dynamic viscosity of solutions were estimated in chitosan samples. Deacetylation of chitin under homogeneous conditions was optimized. Chitosans with molecular weights of 180–220 and 250–300 kDa were obtained from the chitins of Antarctic krill and northern shrimp, respectively.     

Purification and some properties of chitinase produced by Vibrio sp.

Chitinase was purified from the culture filtrate of a Vibrio sp. isolated from soil and its enzymatic properties were examined. The molecular weight measured by SDS-gel electrophoresis was approximately 100,000. The chitinase hydrolyzed colloidal chitin, chitin powder, chitosan and chitin oligosaccharides more than chitotriose but did not hydrolyze glycolchitin and chitobiose.     

The extracellular constitutive production of chitin deacetylase in Metarhizium anisopliae: possible edge to entomopathogenic fungi in the biological control of insect pests

The possible contribution of extracellular constitutively produced chitin deacetylase by Metarhizium anisopliae in the process of insect pathogenesis has been evaluated. Chitin deacetylase converts chitin, a beta-1,4-linked N-acetylglucosamine polymer, into its deacetylated form chitosan, a glucosamine polymer. When grown in a yeast extract-peptone medium, M. anisopliae constitutively produced the enzymes protease, lipase, and two chitin-metabolizing enzymes, viz. chitin deacetylase (CDA) and chitosanase. Chitinase activity was induced in chitin-containing medium. Staining of 7.5% native polyacrylamide gels at pH 8.9 revealed CDA activity in three bands. SDS-PAGE showed that the apparent molecular masses of the three isoforms were 70, 37, and 26 kDa, respectively. Solubilized melanin (10microg) inhibited chitinase activity, whereas CDA was unaffected. Following germination of M. anisopliae conidia on isolated Helicoverpa armigera, cuticle revealed the presence of chitosan by staining with 3-methyl-2-benzothiazoline hydrazone. Blue patches of chitosan were observed on cuticle, indicating conversion of chitin to chitosan. Hydrolysis of chitin with constitutively produced enzymes of M. anisopliae suggested that CDA along with chitosanase contributed significantly to chitin hydrolysis. Thus, chitin deacetylase was important in initiating pathogenesis of M. anisopliae softening the insect cuticle to aid mycelial penetration. Evaluation of CDA and chitinase activities in other isolates of Metarhizium showed that those strains had low chitinase activity but high CDA activity. Chemical assays of M. anisopliae cell wall composition revealed the presence of chitosan. CDA may have a dual role in modifying the insect cuticular chitin for easy penetration as well as for altering its own cell walls for defense from insect chitinase.