Purification and anti-fungal activity of chitinase against Pyricularia grisea in finger millet

Pseudomonas fluorescens isolate 1 (Pfl) protected finger millet plants treated with the ragi blast fungus, Pyricularia grisea, by upto 27% depending on the cultivar. Induction of pathogenesis-related proteins, viz., chitinase by Pfl isolate, was studied against Py. grisea. The activity of chitinase from plants treated with Pfl was significantly higher than the control plant after pathogen inoculation in all cultivars tested. Chitinase in the cultivars, with and without challenge by Py. grisea, revealed changes in the isoform pattern by western blot analysis. Chitinase was purified by affinity chromatography from the Pfl-treated leaves. It showed a single band at 57 kDa after SDS-PAGE. Western blot analysis using barley chitinase antiserum confirmed a 57 kDa chitinase. The chitinase had anti-fungal activity against Py. grisea in vitro. This revised version was published online in August 2006 with corrections to the Cover Date.     

豇豆几丁质酶部分酶学特性的研究

该文测定了纯化的豇豆几丁质酶部分酶学特性。结果表明,该酶在pH5-8,温度低于60℃的范围内稳定性较好,酶活力最适pH为65,最适温度为50℃。10mmol/L浓度的Hg2 、Mn2 、Mg2 、Co2 等金属离子对酶活力有一定抑制作用,其中Hg2 离子抑制率最高(6883%)。Km(胶状几丁质)值为1662mg/ml;以SDS-PAGE电泳和SephadexG-100柱层析两种方法分别测得分子量为34kD、325kD;IEF电泳测得等电点为83。     

Role of the N-terminal polycystic kidney disease domain in chitin degradation by chitinase A from a marine bacterium, Alteromonas sp. strain O-7

AIMS: The aim of study was to clarify whether the polycystic kidney disease (PKD) domain of chitinase A (ChiA) participates in the hydrolysis of powdered chitin. METHODS AND RESULTS: Site-directed mutagenesis of the conserved aromatic residues of PKD domain was performed by PCR. The aromatic residues, W30, Y48, W64 and W67, were replaced by alanine, and single- and double-mutant chitinases were produced in Escherichia coli XL10 and purified with HisTrap column. Single mutations were not quite effective on the hydrolysing activities against chitinous substrates when compared with wild-type ChiA. However, mutations of W30 and W67 decreased the activities against powdered chitin by 87.6%. Wild-type and mutant PKD domains were produced in E. coli TOP10 and purified with glutathione-Sepharose 4B column. Wild-type PKD domain showed significant binding activity to powdered chitin, whereas mutations of W30 and W67 reduced the binding activity to powdered chitin drastically. These results suggest that PKD domain of ChiA is essential for effective hydrolysis of powdered chitin through the interaction between two aromatic residues and chitin molecule. CONCLUSIONS: PKD domain of ChiA participates in the effective hydrolysis of powdered chitin through the interaction between two aromatic residues (W30 and W67) and chitin molecule. SIGNIFICANCE AND IMPACT OF THE STUDY: The findings of this study provide important information on chitin degradation by microbial chitinases.     

Cloning, expression, and characterization of a highly thermostable family 18 chitinase from Rhodothermus marinus

A family 18 chitinase gene chiA from the thermophile Rhodothermus marinus was cloned and expressed in Escherichia coli. The gene consisted of an open reading frame of 1,131 nucleotides encoding a protein of 377 amino acids with a calculated molecular weight of 42,341 Da. The deduced ChiA was a non-modular enzyme with one unique glycoside hydrolase family 18 catalytic domain. The catalytic domain exhibited 43% amino acid identity with Bacillus circulans chitinase C. Due to poor expression of ChiA, a signal peptide-lacking mutant, chiAsp, was designed and used subsequently. The optimal temperature and pH for chitinase activity of both ChiA and ChiAsp were 70°C and 4.5–5, respectively. The enzyme maintained 100% activity after 16 h incubation at 70°C, with half-lives of 3 h at 90°C and 45 min at 95°C. Results of activity measurements with chromogenic substrates, thin-layer chromatography, and viscosity measurements demonstrated that the chitinase is an endoacting enzyme releasing chitobiose as a major end product, although it acted as an exochitobiohydrolase with chitin oligomers shorter than five residues. The enzyme was fully inhibited by 5 mM HgCl₂, but excess ethylenediamine tetraacetic acid relieved completely the inhibition. The enzyme hydrolyzed 73% deacetylated chitosan, offering an attractive alternative for enzymatic production of chitooligosaccharides at high temperature and low pH. Our results show that the R. marinus chitinase is the most thermostable family 18 chitinase isolated from Bacteria so far.     

Induced chitinase activity in resistant wheat leaves inoculated with an incompatible race of Puccinia striiformis f. sp. tritici,the causal agent of yellow rust disease

Chitinase specific activity was measured spectrophotometrically in wheat leaf tissues during the compatible and incompatible interactions with Puccinia striiformis f. sp. tritici, the causal agent of yellow rust disease. The wheat cultivar, Federation^* 4/Kavkaz, was inoculated with virulent (134E134A+) or avirulent (4EOA+) races of P. striiformis f. sp. tritici in the first leaf stage. The results showed that chitinase activity pattern was similar in both compatible and incompatible interactions up to 72 hrs after inoculation. However, the specific activity increased rapidly in the incompatible reaction thereafter. In susceptible reaction, chitinase activity gradually declined after 72 hrs post-inoculation reaching a level similar to that in the control plants two weeks after inoculation. Chitinase specific activity in resistance response was at least three times greater than that in the susceptible reaction two weeks following the inoculation. Electrophoresis of native polyacrylamide gel impregnated with 0.1% (w/v) glycol chitinas the substrate revealed the presence of eight chitinase isoforms with relative electrophoretic mobility (R_m) values ranging from 0.11 to 0.64 in the resolving gel. This revised version was published online in June 2006 with corrections to the Cover Date.     

Purification and characterization of a serine protease and chitinases from Paecilomyces lilacinus and detection of chitinase activity on 2D gels

The filamentous fungus Paecilomyces lilacinus is currently developed as a biocontrol agent against plant parasitic nematodes. Nematode eggs and cuticles are the infection sites for biocontrol agents that penetrate by the production of lytic enzymes. P. lilacinus was cultured in liquid media and proteases and chitinases were induced by the introduction of egg yolk and chitin, respectively. A serine protease was purified from a culture medium using Sepharose-bacitracin affinity column. The protease occurred in three forms, two of which were C-terminally truncated. Chitinase activity was also observed in the culture supernatant, and after separation by isoelectric focusing six proteins were detected that showed activity. Chitinase activity was further confirmed on non-denaturing one-dimensional (1D) and two-dimensional (2D) gels using a sandwich assay with glycol chitin as a substrate. Two of the proteins had similarities with endochitinases as shown by their N-terminal amino acid sequences.     

An extracellular Bacillus sp. chitinase for the production of chitotriose as a major chitinolytic product

An extracellular chitinase of Bacillus sp. WY22 was purified by 9.6-fold. It had a Mr of 35 kDa, an apparent K _m value for colloidal chitin of 3 mg ml^–1 and was optimally active at 37 °C and pH 5.5 over 1 h. The enzyme could also hydrolyse swollen chitin, glycol chitin and chitosan with relative activities of 76%, 34% and 23% compared with colloidal chitin. It formed chitotriose as a major product from colloidal chitin and glycol chitin.