Isolation and substrate specificities of five chitinases from the hepatopancreas of Northern shrimp, Pandalus borealis

Five enzymes designated chitinase I, IIa, IIb, III, and IV have been isolated from the hepatopancreas of Pandalus borealis in a procedure including column chromatography on Q-Sepharose, Sephacryl S-200, phenyl-Superose and Superdex 75. The isolated enzymes were analysed by SDS PAGE. Chitinase I, III, and IV gave only one major band corresponding to 54–55 kDA. Chitinase IIa showed one major band at 61 kDA and two diminutive bands at 17 and 55 kDa, while chitinase IIb gave two major bands at 17 and 44 kDa. Estimated by gel filtration, the native molecular weights of chitinase I, IIa, IIb, III, and IV were 61, 69, 39, 57, and 54 kDa, respectively. The substrate and reaction specificities of the isolated chitinases were investigated, and the results show that the isolated enzymes are true chitinases. They do not hydrolyse N,N′-diacetylchitobiose or p-Nitrophenyl-N-acetyl-β-D-glucosaminide, but express activities when longer chitooligosaccharides or nitrophenylated chitooligosaccharides are used as substrates. Chitinase I and IIa gave an initial random cleavage pattern and might be classified as endochitinases, while chitinase III and IV released dimeric units from the substrates and might be termed chitobiosidases.     

Bifunctional plant defence enzymes with chitinase and ribosome inactivating activities from Trichosanthes kirilowii cell cultures

Three bifunctional plant enzymes (named TKC 15, TKC 28-I, and TKC 28-II) with both chitinase and RIP activity were purified from the medium of Trichosanthes kirilowii plant cell cultures. Highly purified preparations of these proteins exhibited endochitolytic activity as well as the specific 28S rRNA N-glycosidase activity characteristic of ribosome inactivating proteins (RIPs). In addition to providing a competitive advantage to the plant, these enzymes offer the possibility of improved antifungal protection when expressed in transgenic plants.     

粘帚霉几丁质酶GcCHI1的结构鉴定及其抑菌作用

【目的】对分离纯化自链孢粘帚霉(Gliocladium catenulatum)HL-1-1菌株的几丁质酶Gc CHI1进行化学结构鉴定,并测定该酶对几种病原真菌的抑菌机制。【方法】几丁质酶Gc CHI1化学结构鉴定采用Nano-ESI-MS/MS技术。该酶对病原真菌菌丝生长、病原菌孢子萌发和病原菌菌核萌发的抑制作用采用牛津杯法等方法。【结果】获得几丁质Gc CHI1胰蛋白酶水解肽段的肽质量指纹谱图,较好的MS/MS图谱,以及3个肽段的氨基酸序列(均15个氨基酸),分别为LYNSNDAIEAFISR、VIGYFTQWGIYGR、LNLGIGYYGR。经Mascot数据库检索认为与来自Stenotrophomonas maltophilials 34S1的几丁质酶A具有高度的相似性。几丁质酶Gc CHI1能明显抑制立枯丝核菌、油菜菌核病菌、番茄灰霉病菌等多种植物病原真菌的菌丝生长、孢子萌发和菌核萌发。【结论】几丁质酶Gc CHI1对多种植物病原菌有抑制作用,因此几丁质酶Gc CHI1是HL-1-1菌株抑菌作用的机制之一。     

一种几丁质酶基因的克隆表达及酶解产物分析

【目的】克隆耐冷菌假交替单胞菌(Pseudoalteromonas sp.DL-6)的几丁质酶基因并进行原核表达,纯化重组蛋白并研究其酶解产物。【方法】采用PCR扩增法从Pseudoalteromonas sp.DL-6中克隆几丁质酶基因(chi A),连接到表达载体p ET28a,导入Escherichia coli BL21(DE3)进行诱导表达。SDS-PAGE检测几丁质酶Chi A的分子量与纯度,4-甲基伞形酮荧光底物4MU-(Glc NAc)2测定酶活,电喷雾质谱(ESI-MS)检测酶解产物。【结果】chi A基因(Gen Bank登录号KF234015)在大肠杆菌中高效表达,Ni-NTA亲和层析柱纯化几丁质酶Chi A的总活力可达168.68 U。ESI-MS检测结果表明重组蛋白酶解1%胶体几丁质的产物为几丁寡糖。【结论】利用内切几丁质酶Chi A水解几丁质生产几丁寡糖,为其在食品、医药和农业等领域的潜在应用提供有利参考。     

The mycorrhizal fungus Amanita muscaria induces chitinase activity in roots and in suspension-cultured cells of its host Picea abies

A cell-wall fraction of the mycorrhizal fungus Amanita muscaria increased the chitinase activity in suspension-cultured cells of spruce (Picea abies (L.) Karst.) which is a frequent host of Amanita muscaria in nature. Chitinase activity was also increased in roots of spruce trees upon incubation with the fungal elicitor. Non-induced levels of chitinase activity in spruce were higher in suspension cells than in roots whereas the elicitorinduced increase of chitinase activity was higher in roots. Treatment of cells with hormones (auxins and cytokinin) resulted in a severalfold depression of enzyme activity. However, the chitinase activity of hormone-treated as well as hormone-free cells showed an elicitor-induced increase. Suspension cells of spruce secreted a large amount of enzyme into the medium. It is postulated that chitinases released from the host cells in an ectomycorrhizal system partly degrade the fungal cell walls, thus possibly facilitating the exchange of metabolites between the symbionts.     

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     

Structure of full-length bacterial chitinase containing two fibronectin type III domains revealed by small angle X-ray scattering

Chitinase A1 (ChiA1) from Bacillus circulans WL-12 consists of an N-terminal catalytic domain, two fibronectin type III domains (FnIIIDs), and a C-terminal chitin-binding domain. The full-length structure of ChiA1 was studied by small angle X-ray scattering. The obtained low-resolution structure showed that ChiA1 is an elongated molecule with a length of approximately 145 A composed of a large globular head and a rod-like tail. Combination with known high-resolution structures of individual ChiA1 domains provided a model of the domain arrangement. In this model, two FnIIIDs connect to each other in an extended rod-like shape without large bending between the FnIIIDs, and contribute largely to the length of ChiA1.