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.     

Production and purification of extracellular chitinases from Penicillium aculeatum NRRL 2129 under solid-state fermentation

Fourteen Penicillium strains have been screened on wheat bran–crude chitin mixture medium for extracellular chitinase production in solid-state fermentation. Under the experimental conditions tested, Penicillium aculeatum NRRL 2129 (=ATCC 10409) was selected as the best enzyme producer. The optimum incubation period for chitinase production by the potent organism was found to be 72 h. Chromatofocusing was performed as the first step in the purification scheme, but high amount of contaminating proteins interfered with the method. Hence, ion-exchange chromatography experiments were carried out followed by gel filtration to separate and isolate chitinase isoenzymes. Four major chitinase peaks of molecular weight 82.7, 44.6, 28.2 and 26.9 kDa were observed after gel filtration chromatography while, on SDS-PAGE, three protein bands of molecular weights 82.6, 33.9 and 29.1 kDa were identified. The purified enzyme showed optimal temperature and pH at 50 and 5.5 °C, respectively.     

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。     

Bacterial community composition and chitinase gene diversity of vermicompost with antifungal activity

Bacterial communities and chitinase gene diversity of vermicompost (VC) were investigated to clarify the influence of earthworms on the inhibition of plant pathogenic fungi in VC. The spore germination of Fusarium moniliforme was reduced in VC aqueous extracts prepared from paper sludge and dairy sludge (fresh sludge, FS). The bacterial communities were examined by culture-dependent and -independent analyses. Unique clones selected from 16S rRNA libraries of FS and VC on the basis of restriction fragment length polymorphism (RFLP) fell into the major lineages of the domain bacteria Proteobacteria, Bacteroidetes, Verrucomicrobia, Actinobacteria and Firmicutes. Among culture isolates, Actinobacteria dominated in VC, while almost equal numbers of Actinobacteria and Proteobacteria were present in FS. Analysis of chitinolytic isolates and chitinase gene diversity revealed that chitinolytic bacterial communities were enriched in VC. Populations of bacteria that inhibited plant fungal pathogens were higher in VC than in FS and particularly chitinolytic isolates were most active against the target fungi.     

Effects of Paecilomyces lilacinus protease and chitinase on the eggshell structures and hatching of Meloidogyne javanica juveniles

A serine protease and an enzyme preparation consisting of six chitinases, previously semi-purified from a liquid culture of Paecilomyces lilacinus strain 251, were applied to Meloidogyne javanica eggs to study the effect of the enzymes on eggshell structures. Transmission electron microscopic studies revealed that the protease and chitinases drastically altered the eggshell structures when applied individually or in combination. In the protease-treated eggs, the lipid layer disappeared and the chitin layer was thinner than in the control. The eggs treated with chitinases displayed large vacuoles in the chitin layer, and the vitelline layer was split and had lost its integrity. The major changes in the eggshell structures occurred by the combined effect of P. lilacinus protease and chitinases. The lipid layer was destroyed; the chitin layer hydrolyzed and the vitelline layer had lost integrity. The effect of P. lilacinus protease and chitinase enzymes on the hatching of M. javanica juveniles was also compared with a commercially available bacterial chitinase. The P. lilacinus protease and chitinase enzymes, either individually or in combination, reduced hatching of M. javanica juveniles whereas a commercial bacterial chitinase had an enhancing effect. Some juveniles hatched when the eggs were exposed to a fungal protease and chitinase mixture. We also established that P. lilacinus chitinases retained their activity in the presence of endogenous protease activity.     

Characterization of Antifungal Chitinase from Marine Streptomyces sp. DA11 Associated with South China Sea Sponge Craniella Australiensis

The gene cloning, purification, properties, kinetics, and antifungal activity of chitinase from marine Streptomyces sp. DA11 associated with South China sponge Craniella australiensis were investigated. Alignment analysis of the amino acid sequence deduced from the cloned conserved 451 bp DNA sequence shows the chitinase belongs to ChiC type with 80% similarity to chitinase C precursor from Streptomyces peucetius. Through purification by 80% ammonium sulfate, affinity binding to chitin and diethylaminoethyl-cellulose anion-exchange chromatography, 6.15-fold total purification with a specific activity of 2.95 Umg⁻¹ was achieved. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed a molecular weight of approximately 34 kDa and antifungal activities were observed against Aspergillus niger and Candida albicans. The optimal pH, temperature, and salinity for chitinase activity were 8.0, 50°C, and 45 g‰ psu, respectively, which may contribute to special application of this marine microbe-derived chitinase compared with terrestrial chitinases. The chitinase activity was increased by Mn²⁺, Cu²⁺, and Mg²⁺, while strongly inhibited by Fe²⁺ and Ba²⁺. Meanwhile, SDS, ethyleneglycoltetraacetic acid, urea, and ethylenediaminetetraacetic acid were found to have significantly inhibitory effect on chitinase activity. With colloidal chitin as substrates instead of powder chitin, higher V _max (0.82 mg product/min·mg protein) and lower K _m (0.019 mg/ml) values were achieved. The sponge’s microbial symbiont with chitinase activity may contribute to chitin degradation and antifungal defense. To our knowledge, it was the first time to study sponge-associated microbial chitinase.     

转几丁质酶和葡聚糖酶基因水稻对土壤三种弹尾目昆虫和一种环节动物的影响

采用 3个试验研究转几丁质酶和苜蓿 1,3- β -葡聚糖酶双价基因水稻对土壤弹尾目昆虫和环节动物线蚓的影响。①平皿实验 :观察弹尾目与转基因水稻组织间的营养关系及其在无土壤情况下对弹尾目昆虫繁殖率的短期影响 ;②土壤 -植物残留物单物种微宇宙试验 :观察土壤中不同转基因水稻组织含量对土壤动物繁殖率的短期影响 ;③土壤 -植物残留物多物种微宇宙试验 :模拟大田状况 ,观察多物种共存较长时间后转基因水稻对土壤动物密度的影响。结果表明 :Folsomiacandida取食新鲜转基因水稻叶片和根组织 2 1d后 ,繁殖率比野生型水稻组分别降低 4 5 1% (F1,8=9 9,P <0 0 5 )和 30 5 % (F1,8=5 8,P <0 0 5 ) ;在掺有转基因水稻组织 (30g/kg)的土壤中 ,F .candida 2 1d后的繁殖率比野生型水稻组下降 19 3% (F3 ,16=3 6 ,P <0 0 5 ) ;在掺有转基因水稻组织土壤 (30g/kg)的多物种实验中 ,转基因水稻组中的F .candida、Enchytraeuscrypticus16周后的密度比野生型水稻组分别降低 19 4 % (F1,48=6 8,P <0 0 5 )和 38 0 % (F1,48=6 4 2 ,P <0 0 0 1) ,而Sinellacurviseta的密度升高 84 8% (F1,48=6 4 2 ,P <0 0 0 1)。因此 ,转几丁质酶和葡聚糖酶双价基因水稻对土壤弹尾目和线蚓都是有影响的 ,对某些种类表现     

番茄灰霉病拮抗菌株初步鉴定及通过导入几丁质酶基因提高其生防效果

实验室保存菌Fh对番茄灰霉病有明显的抑制作用,通过形态特征、生理生化特征进行初步鉴定归属于芽孢杆菌属（Bacillus circulans）。从质粒pUC1965中得到含有几丁质酶基因的6.5kb DNA片段,将该基因片段与大肠杆菌 枯草芽孢杆菌穿梭质粒pBE2连接,获得重组质粒pBE2-chib。将重组质粒转入芽孢杆菌Fh中获得工程菌株Fh-chib。几丁质酶基因的PCR检测和几丁质平板实验表明几丁质酶基因被成功转入,工程菌株Fh-chib的原始粗酶液几丁质酶活为4.06U/ml。与野生菌相比,Fh-chib工程菌株对番茄灰霉病（Botrytis cinerea）抑菌效果提高34.46%。     

Chitinase in roots of mycorrhizal Allium porrum: regulation and localization

Chitinase (EC 3.2.1.14) activity was measured in roots of Allium prorrum L. (leek) during development of a vesicular-arbuscular mycorrhizal symbiosis with Glomus versiforme (Karst.) Berch. During the early stages of infection, between 10 and 20 d after inoculation, the specific activity of chitinase was higher in mycorrhizal roots than in the uninfected controls. However, 60–90 d after inoculation, when the symbiosis was fully established, the mycorrhizal roots contained much less chitinase than control roots. Chitinase was purified from A. porrum roots. An antiserum against beanleaf chitinase was found to cross-react specifically with chitinase in the extracts from non-mycorrhizal and mycorrhizal A. porrum roots. This antiserum was used for the immunocytochemical localization of the enzyme with fluorescent and gold-labelled probes. Chitinase was localized in the vacuoles and in the extracellular spaces of non-mycorrhizal and mycorrhizal roots. There was no immunolabelling on the fungal cell walls in the intercellular or the intracellular phases. It is concluded that the chitin in the fungal walls is inaccessible to plant chitinase. This casts doubts on the possible involvement of this hydrolase in the development of the mycorrhizal fungus. However, fungal penetration does appear to cause a typical defense response in the first stages that is later depressed.     

Purification and properties of a chitinase from Penicillium sp. LYG 0704

The chitinase producing Penicillium sp. LYG 0704 was procured from soil of the Chonnam National University crop field. The chitinase activity was detected after the first day which increased gradually and reached its maximum after 3 days of cultivation. The chitinase was purified from a culture medium by precipitation with isopropanol and column chromatography with Mono Q and Butyl-Sepharose. The molecular mass of chitinase was estimated to be 47 kDa by SDS–PAGE. Optimal pH and temperature were 5.0 and 40 °C, respectively. The N-terminal amino acid sequence of the enzyme was determined to be ¹AGSYRSVAYFVDWAI¹⁵. The fully cloned gene, 1287 bp in size, encoded a single peptide of 429 amino acids. BLAST search of the chitinase gene sequence showed similarity with chitinase of Aspergillus fumigatus Af293 chitinase gene (58%) and A. fumigatus class V chitinase ChiB1 gene (56%).