几丁质酶和壳聚糖酶对部分乙酰化壳聚糖作用方式的比较

通过对几丁质酶和壳聚糖酶降解部分乙酰化壳聚糖的作用方式的比较,得到几丁质酶切断壳聚糖的ＧｌｃＮＡｃ－ ＧｌｃＮＡｃ 和ＧｌｃＮＡｃ－ ＧｌｃＮ 或ＧｌｃＮ－ ＧｌｃＮＡｃ 糖苷键,而壳聚糖酶切断壳聚糖的ＧｌｃＮ－ ＧｌｃＮ 和ＧｌｃＮ－ ＧｌｃＮＡｃ 或ＧｌｃＮＡｃ－ ＧｌｃＮ 糖苷键,为得到较高聚合度的壳寡糖提供理论基础。     

Purification and characterization of a chitosanase from Serratia marcescens TKU011

A chitosanase was purified from the culture supernatant of Serratia marcescens TKU011 with shrimp shell wastes as the sole carbon/nitrogen source. Zymogram analysis revealed the presence of chitosanolytic activity corresponding to one protein, which was purified by a combination of ion-exchange and gel-filtration chromatography. The molecular weight of the chitosanase was 21 kDa and 18 kDa estimated by SDS–PAGE and gel-filtration, respectively. The optimum pH, optimum temperature, pH stability, and thermal stability of the chitosanase were 5, 50 °C, pH 4–8, and <50 °C, respectively. The chitosanase was inhibited completely by EDTA, Mn²⁺, and Fe²⁺. The results of peptide mass mapping showed that three tryptic peptides of the chitosanase were identical to a chitin-binding protein Cbp21 from S. marcescens (GenBank accession number gi58177632) with 63% sequence coverage.     

Production of chitinases by Aphanocladium album grown on crystalline and colloidal chitin

Abstract The deuteromycete Aphanocladium album produced two endochitinases (EC 3.2.1.14) with apparent isoelectric points of 7.1 and 7.6 and seven exochitinases (EC 3.2.1.30) with apparent isoelectric points ranging from 3.8 to 6.4 when grown on a colloidal chitin preparation. With crystalline chitin as carbon source, two endochitinases (p I 7.1 and 7.6) and only one exochitanase (p I 4.9) were detected. The exochitinase p I 4.9, which was produced with both substrates, has a relative molecular mass of 44 000. The different chitinases could be separated by chromatofocusing and their specific activities were determined.     

Purification and characterization of chitinases and chitosanases from a new species strain Pseudomonas sp. TKU015 using shrimp shells as a substrate

A chitinase (CHT1) and a chitosanase (CHS1) were purified from the culture supernatant of Pseudomonas sp. TKU015 with shrimp shell wastes as the sole carbon and nitrogen source. The optimized conditions of this new species strain (Gen Bank Accession Number EU103629) for the production of chitinases were found to be when the culture was shaken at 30 degrees C for 3 days in 100 mL of medium (pH 8) containing 0.5% shrimp shell powder (SSP) (w/v), 0.1% K2HPO4, and 0.05% MgSO(4).7H2O. The molecular weights of CHT1 and CHS1 determined by SDS-PAGE were approximately 68 kDa and 30 kDa, respectively. The optimum pH, optimum temperature, pH stability, and the thermal stability of CHT1 and CHS1 were pH 6, 50 degrees C, pH 5-7, <50 degrees C and pH 4, 50 degrees C, pH 3-9, <50 degrees C, respectively. CHT1 was inhibited completely by Mn2+ and Fe2+, and CHS1 was inhibited by Mn2+, Cu2+, and PMSF. CHT1 was only specific to chitin substrates, whereas the relative activity of CHS1 increased when the degree of deacetylation of soluble chitosan increased.     

一种源于蜗牛的壳聚糖水解酶的纯化和定性

目的研究属于蜗牛的壳聚糖水解酶的纯化方法,得到壳聚糖水解酶的纯品,从而为氨基酸序列分析、基因克隆及工业菌制备奠定前期基础。方法建立检测蜗牛壳聚糖水解酶活性的手段并考察影响酶活性的各种因素,比较现有层析方法纯化蜗牛壳聚糖水解酶的实际效果,确定纯化的最佳条件,从而设计出最合理的纯化方案。结果经苯基琼脂糖柱层析,DEAE-Sepharose离子交换层析和Sephacryl S-300凝胶过滤分离,得到高纯高活性蛋白质,在SDS-PAGE上用银染的方法呈单一蛋白质条带,比活性提高33.333倍,纯化倍数为18.272,得率为0.15。结论实验建立了1种从蜗牛中分离高效高纯度壳聚糖水解酶的方法,为壳寡糖的酶解工业生产提供了新思路、新方法。     

Purification and characterization of extracellular chitinase from Aeromonas schubertii

A locally isolated stain Aeromonas schubertii was cultured and induced by powdered chitin for the production of chitinases. Extracellular proteins were purified by ammonium sulfate precipitation, dialysis to remove salts, and then preparative isoelectric focusing (IEF) to yield several chitinases. The purified enzymes were analyzed by SDS–PAGE (sodium dodecyl sulfate–polyacrylamide gel electrophoresis) with and without glycol chitin and were found to be SDS-resistant. The chitinase present in the highest abundance was the one with an estimated molecular weight of 75 kDa. The Michaelis constant and turnover number were determined to be 0.29 mM and 1 s⁻¹, respectively, for this enzyme using colloidal chitin azure as the substrate. However, the ethanol treatment of this enzyme could significantly increase its chitinolytic activity. Other chitinases obtained in the same IEF fraction were determined to have molecular weights of ca. 30, 38, and 110 kDa. Since the proteins with highest chitinase activity were collected from IEF fraction tube with pH value of 4.8, those chitinase were believed to be acidic. An activity assay method using colloidal chitin azure as the substrate was recommended since it possessed a broader range of linearity in comparison with conventional reducing sugar equivalent method.     

Entamoeba invadens: Cloning and molecular characterization of chitinases

Entamoeba histolytica, the causative agent of amebiasis infects through its cyst form and this transmission may be blocked using encystation specific protein as drug target. In this study, we have characterized the enzyme chitinase which express specifically during encystation. The reptilian parasite Entamoeba invadens, used as a model for encystation study contain three chitinases. We report the molecular cloning, over-expression and biochemical characterization of all three E. invadens chitinase. Cloned chitinases were over-expressed in bacterial system and purified by affinity chromatography. Their enzymatic profiles and substrate cleaving patterns were characterized. All of them showed binding affinity towards insoluble chitin though two of them lack the chitin binding domain. All the chitinases cleaved and released dimmers from the insoluble substrate and act as an exochitinase. Homology modeling was also done to understand the substrate binding and cleavage pattern.     

Current views on fungal chitin/chitosan, human chitinases, food preservation, glucans, pectins and inulin: A tribute to Henri Braconnot, precursor of the carbohydrate polymers science, on the chitin bicentennial

Two hundred years ago, Henri Braconnot described a polysaccharide containing a substantial percent of nitrogen, later to be called chitin: that discovery stemmed from investigations on the composition of edible mushrooms and their nutritional value. The present interdisciplinary article reviews the major research topics explored by Braconnot, and assesses their importance in the light of our most advanced knowledge. Thus, the value of fungi, seafoods and insects is described in connection with the significance of the presence of chitin itself in foods, and chitinases in the human digestive system. The capacity of chitin/chitosan to depress the development of microbial pathogens, is discussed in terms of crop protection and food preservation. Other topics cherished by Braconnot, such as the isolation of pectin from a large number of plants, and inulin from the Helianthus tubers, are presented in up-to-date terms. Acids isolated from plants at that early time, led to enormous scientific advancements, in particular the glyoxylic acid and levulinic acid used for the preparation of soluble chitosan derivatives that paved the way to a number of applications. An opportunity to trace the origins of the carbohydrate polymers science, and to appreciate the European scientific heritage.     

Preparation of low molecular weight chitosan using solution plasma system

The solution plasma system was introduced to treat chitosan solution in order to prepare low molecular weight chitosan. The plasma treatment time was varied from 0 min to 300 min. The plasma-treated chitosan was characterized including viscosity, molecular weight by GPC, and chemical characteristics by FT-IR. The results showed that after treated with plasma for 15-60 min, the viscosity of chitosan solution and apparent molecular weight of chitosans were remarkably decreased, compared to those of untreated sample. Longer treatment time had less effect on both viscosity and molecular weight of samples. Eventually, long treatment time (≥180 min) showed no influence on both viscosity and apparent molecular weight. This suggested that the degradation process of chitosan occurred during plasma treatment. FT-IR analysis revealed that chemical structure of chitosan was not affected by solution plasma treatment. TOF-MS results showed that chitooligosaccharides with the degree of polymerization of 2-8 were also generated by solution plasma treatment. The results suggested that solution plasma system could be a potential method for the preparation of low molecular weight chitosan and chitooligosaccharides.     

Recombinant expression of a chitosanase and its application in chitosan oligosaccharide production

Recently, considerable attention has been focused on chitosan oligosaccharides (COSs) due to their various biological activities. COSs can be prepared by enzymatic degradation of chitosan, which is the deacetylation product of chitin, one of the most abundant biopolymers in nature. In the current study, we recombinantly expressed a chitosanase and used it for COS preparation. A bacillus-derived GH8 family chitosanase with a 6×His tag fused at its N-terminal was expressed in the Escherichia coli strain BL21(DE3) as a soluble and active form. Its expression level could be as high as 500 mg/L. Enzymatic activity could reach approximately 140,000 U/L under our assay conditions. The recombinant chitosanase could be purified essentially to homogeneity by immobilized metal-ion affinity chromatography. The enzyme could efficiently convert chitosan into monomer-free COS: 1 g of enzyme could hydrolyze about 100 kg of chitosan. Our present work has provided a cheap chitosanase for large-scale COS production in industry.     

Purification,characterization, and molecular cloning of chitinases from the stomach of the threeline grunt Parapristipoma trilineatum

Two chitinase isozymes, PtChiA and PtChiB, were purified from the stomach of the threeline grunt, Parapristipoma trilineatum. The molecular masses of PtChiA and PtChiB were estimated to be 50 and 60 kDa by SDS-PAGE, respectively. Both chitinases were stable at pH 3.0–6.0 (acidic) and showed the optimum pH toward both short and long substrates in the acidic region (pH 2.5–5.0). PtChiA and PtChiB preferentially degraded the second and third glycosidic bonds from the non-reducing end of N-acetylchitooligosaccharides, respectively. PtChiA and PtChiB exhibited wide substrate specificities toward crystalline chitin. Moreover, 2 cDNAs encoding PtChiA and PtChiB, PtChi-1 and PtChi-2, respectively, were cloned. The deduced amino acid sequences of both chitinase cDNAs comprised N-terminal signal peptides, glycoside hydrolase 18 catalytic domains, linker regions, and C-terminal chitin-binding domains. Phylogenetic tree analysis of vertebrate chitinases revealed that fish stomach chitinases including PtChi-1 and PtChi-2 form unique chitinase groups, acidic fish chitinase-1 (AFCase-1) and acidic fish chitinase-2 (AFCase-2), which differ from the acidic mammalian chitinase (AMCase) group. The present results suggest that fish have a chitin-degrading enzymatic system in which 2 different chitinases, AFCase-1 and AFCase-2, with different degradation patterns are expressed in the stomach.     

甲壳素脱乙酰酶的研究概况及展望

甲壳素脱乙酰酶(chitin deacetylase,CDA,E.C.3.5.1.41)是一种能催化脱去甲壳素分子中N-乙酰葡糖胺链上的乙酰基,使之变成壳聚糖的酶。而壳聚糖因其独特的性质被广泛应用于医药、食品、化工、化妆品等行业。对CDA的来源、分离纯化和酶学性质、结构和催化机制、基因的克隆表达及应用前景等方面的研究进行了综述,并分析出今后的主要研究方向应在CDA基因的克隆表达、CDA底物的改造及CDA的结构和催化机制等方面。     

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--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.Abbreviations MS minimal salts - GlcNAc N-acetyl--d-glucosamine - pNP-GlcNAc p-nitrophenyl-2-acetamido-2-deoxy--d-glucopyranoside - d.a. degree of N-acetylation - TLC thin-layer chromatography     

Structural features of plant chitinases and chitin-binding proteins

Structural features of plant chitinases and chitin-binding proteins are discussed. Many of these proteins consist of multiple domains, of which the chitin-binding hevein domain is a predominant one. X-ray and NMR structures of representatives of the major classes of these proteins are available now, and are used to describe the structures of the other ones. Conserved positions of Cys residues can be taken as evidence for identically located disulfide bridges or cysteine residues. The current classification of chitinases is unsatisfactory and needs to be replaced by an evolutionarily more correct one. As the currently known three-dimensional structures of chitinases are those from barley and the rubber tree, Hevea brasiliensis, it is proposed to adopt the designation b-type (classes I, II and IV) and h-type (classes III and V) chitinases, respectively.     

细菌几丁质酶研究进展

真菌病害是影响作物产量的重要原因 ,而几丁质酶能有效抑制其生长、水解其细胞壁。对研究较多的细菌几丁质酶及几丁质酶基因的分子生物学研究进展进行了综述 ,并对细菌几丁质酶基因利用存在的问题进行了探讨。     

Natrarchaeobius chitinivorans gen. nov., sp. nov., and Natrarchaeobius halalkaliphilus sp. nov., alkaliphilic,chitin-utilizing haloarchaea from hypersaline alkaline lakes

Two groups of alkaliphilic haloarchaea from hypersaline alkaline lakes in Central Asia, Egypt and North America were enriched and isolated in pure culture using chitin as growth substrate. These cultures, termed AArcht, were divided into two groups: group 1 which includes eleven isolates from highly alkaline soda lakes and group 2 which contains a single isolate obtained from the alkaline hypersaline Searles Lake. The colonies of chitin-utilizing natronoarchaea were red-pigmented and surrounded by large zones of chitin hydrolysis. The free cells of both groups were mostly flat nonmotile rods, while the cells that attached to chitin or formed colonies on chitin plates were mostly coccoid. The isolates are obligate aerobic saccharolytic archaea utilizing chitin and chitosane (less actively) as the only sugar polymers as well as a few hexoses as their carbon and energy source. Both groups are extremely halophilic, growing optimally at 3.5–4 M total Na⁺, but they differ in their pH profiles: the main group 1 isolates are obligately alkaliphilic, while the single group 2 strain (AArcht-Sl^T) is alkalitolerant. The core archaeal lipids in both groups are dominated by C₂₀–C₂₀ and C₂₀–C₂₅ dialkyl glycerol ethers (DGE) in approximately equal proportion. Phylogenetic analysis indicated that the isolates form an independent genus-level lineage within the family Natrialbaceae with 3 species-level subgroups. The available genomes of the closest cultured relatives of the AArcht strains, belonging to the genera Natrialba and Halopiger, do not encode any chitinase-related genes. On the basis of their unique phenotypic properties and distinct phylogeny, we suggest that the obligate alkaliphilic AArcht isolates (group 1) with an identical phenotype are classified into a new genus and species Natrarchaeobius chitinivorans gen. nov., sp. nov., with strain AArcht4^T as the type strain (JCM 32476^T = UNIQEM U966^T), while the facultatively alkaliphilic strain AArcht-Sl^T (group 2) — as a new species Natrarchaeobius halalkaliphilus sp. nov. (JCM 32477^T = UNIQEM U969^T).     

The effect of ultrasonication on enzymatic hydrolysis of chitin to N-acetyl glucosamine via sequential and simultaneous strategies

In this study, sequential and simultaneous strategies of ultrasonication (ultrasonic bath) were investigated to enhance enzymatic production of N-acetyl glucosamine (GlcNAc) from chitin powder. For the sequential strategy, the ultrasonic caused chitin powder to a visible fleecy structure, and Scanning electron microscopy (SEM) showed the surface of the treated chitin had a fiber-like structure with a diameter of 50 − 200 nm. Moreover, Fourier transform infrared spectroscopy (FTIR), Element analysis (EA), and X-ray diffraction (XRD) revealed that the crystallinity of the chitin decreased with little deacetylation. The simultaneous strategy is a one-pot treatment and enzymatic hydrolysis of chitin. The concentration of GlcNAc was 2.65 g/L for the strategy, which was 1.18- and 5.0-fold higher than the sequential strategy (2.25 g/L) and untreated chitin (0.53 g/L), respectively. In conclusion, this approach provided an efficient and environmentally friendly method for reducing the crystallinity of chitin and enhancing its enzymatic hydrolysis.     

Purification and partial characterization of two chitinases from the mycoparasitic fungus <Emphasis Type="Italic">Talaromyces flavus</Emphasis>

Chitinases were produced by Talaromyces flavus CGMCC 3.4301 when it was grown in the presence of chitin. Two chitinases from the culture filtrate of T. flavus were purified to homogeneity by fractional ammonium sulphate precipitation, ion-exchange chromatography on DEAE–Sepharose and Phenyl–Sepharose hydrophobic interaction chromatography. By SDS–PAGE, the molecular weight of the two enzymes was estimated to be 41 and 32 kDa, respectively. The 41 kDa chitinase (CHIT41) had a 4.0 pH optimum; the 32 kDa chitinase (CHIT32) optimum activity was at pH 5.0. The optimum temperature for the two chitinase activities was 40 °C. The two chitinases had activity against cell wall of Verticillium dahliae, Sclerotinia sclerotiorum and Rhizoctonia solani, and inhibited spore germination and germ tube elongation of Alternaria alternata, Fusarium moniliforme, and Magnaporthe grisea.     

Molecular cloning, expression and characterization of a chitosanase from Microbacterium sp.

A gene encoding a chitosanase (mschito) was cloned from Microbacterium sp. OU01. The ORF consists of 801 bp which encoded a polypeptide of 266 amino acid residues. The deduced amino acid sequence shows 98% identity to that of the chitosanase reported in Pseudomonas sp. A-01. In addition, the fusion protein containing MSCHITO was expressed in E. coli and purified using Ni-NTA affinity chromatography. The purified rMSCHITO protein degraded the chitosan (the degree of deacetylation of 99%) and produced a mixture of chitooligosaccharides. The MSCHITO is thus an endo-chitosanase.     

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.