Antifungal Chitinase Research Articles

Production of an Antifungal Chitinase from Enterobacter sp. NRG4 and its Application in Protoplast Production

In this study flake chitin, crab shell chitin, mushroom stalk, fungal cell wall, wheat bran and rice bran were used as substrate for chitinase production by Enterobacter sp. NRG4 under submerged and solid state fermentation (SSF) conditions. Enterobacter sp. NRG4 produced 72 and 49.7 U/ml of chitinase in presence of cell walls of Candida albicans and Fusarium moniliforme in submerged fermentation. Under SSF, maximum chitinase production was 965 U/g solid substrate with flake chitin and wheat bran (1:3 ratio) at 75% moisture level after 144 h. The purified chitinase inhibited hyphal extension of Fusarium moniliforme, Aspergillus niger, Mucor rouxi and Rhizopus nigricans. The chitinase was effective in release of protoplasts from Trichoderma ressei, Pleurotus florida, Agaricus bisporus and Aspergillus niger. Protoplasts yield was maximum with 60 mg of 24 h old fungal mycelium incubated with 60 U of chitinase and 60 U of cellulase.

Identification of an Antifungal Chitinase from a Potential Biocontrol Agent, Bacillus cereus 28-9

Bacillus cereus 28-9 is a chitinolytic bacterium isolated from lily plant in Taiwan. This bacterium exhibited biocontrol potential on Botrytis leaf blight of lily as demonstrated by a detached leaf assay and dual culture assay. At least two chitinases (ChiCW and ChiCH) were excreted by B. cereus 28-9. The ChiCW-encoding gene was cloned and moderately expressed in Escherichia coli DH5alpha. Near homogenous ChiCW was obtained from the periplasmic fraction of E. coli cells harboring chiCW by a purification procedure. An in vitro assay showed that the purified ChiCW had inhibitory activity on conidial germination of Botrytis elliptica, a major fungal pathogen of lily leaf blight.

Antifungal Activity of Bacillus thuringiensis Chitinase and Its Potential for the Biocontrol of Phytopathogenic Fungi in Soybean Seeds

ABSTRACT: Bacillus thuringiensis var israelensis was used to produce chitinase on shrimp wastes by fermentation at 30°C and 250 rpm for 120 h. The enzyme was concentrated by ultrafiltration and was adjusted to pH 5.8. Antifungal chitinase activity on phytopathogenic fungi was investigated in growing cultures and on soybean seeds infested with Sclerotium rolfsii. Fungal inhibition was found to be 100% for S. rolfsii; 55% to 82% for A. terreus, A. flavus, Nigrospora sp, Rhizopus sp, A. niger, Fusarium sp, A. candidus, Absidia sp, and Helminthosporium sp; 45% for Curvularia sp; and 10% for A. fumigatus (P < 0.05). When soybean seeds were infected with S. rolfsii, germination was reduced from 93% to 25%; the addition of chitinase (0.8 U/mg protein) increased germination to 90%. B. thuringiensis chitinase may contribute to the biocontrol of S. rolfsii and other phytopathogenic fungi in soybean seeds in Integrated Pest Management programs.

Purification and characterization of an antifungal chitinase in jelly fig (Ficus awkeotsang) achenes.

A method was developed to purify a 30-kDa protein from jelly fig (Ficus awkeotsang) pericarp, including preparation of jelly curd from achenes, extraction of proteins from the curd, and isolation of the 30-kDa protein by anion-exchanger and gel filtration. Chitinase activity was detected in the purified 30-kDa protein by activity staining in both non-denaturing gel electrophoresis and SDS-PAGE. Isoelectrofocusing showed that the isoelectric point of the 30-kDa protein was lower than pH 3.5. The K(m), k(cat), optimal pH and temperature of this putative chitinase were determined to be 0.076 mM, 0.089 s(-1), pH 4, and 60 degrees C, respectively. The purified 30-kDa protein was thermostable (retaining activity up to 65 degrees C for several hours) and could be stored at 4 degrees C for a year without apparent loss of chitinase activity. Antifungal activity of this putative chitinase was measured in terms of inhibition of Colletotrichum gloeosporioides spore germination.

Process optimization for antifungal chitinase production by Trichoderma harzianum

Single-parameter optimization was carried out for the production of chitinase using a soil isolate belonging to Trichoderma harzianum in solid-state fermentation. Maximum chitinase activity (3.18 U/gds) was obtained after 96 h of incubation at 30 °C when wheat bran moistened (65.7%) with salt solution was supplemented with colloidal chitin (1%, w/w) and yeast extract (2%, w/w) used as the substrate. The inoculum contains 4×10 7 spores of T. harzianum (TUBF 781). The growth of the fungus on wheat bran particle was visualized by a scanning electron microscope. Chitinase activity was measured as the amount of N-acetyl glucosamine (NAG) liberated in μmol/min under reaction conditions. The crude extract showed antifungal activity against a wide range of fungal strains belonging to Aspergillus, Rhizopus and Mucor sp., and was found significant against Aspergillus niger (NCIM 563).

An antifungal chitinase produced by Bacillus cereus with shrimp and crab shell powder as a carbon source.

The production of inexpensive chitinolytic enzymes is an element in the utilization of shellfish processing wastes. In this study, shrimp and crab shell powder prepared by treating shrimp and crab processing wastes with boiling and crushing was used as a substrate for the isolation of an antifungal chitinase-producing microorganism. Bacillus cereus YQ 308, a strain isolated from the soil samples, excreted one chitinase when cultured in a medium containing 2% (wt/vol) shrimp and crab shell powder as major carbon source. The chitinase, purified by sequential chromatography, had an Mr of 48 kDa and pI of 5.2. The purified chitinase (2 mg/ml) inhibited the hyphal extension of the fungi Fusarium oxysporum and Pythium ultimum.

Purification and characterization of two antifungal chitinases extracellularly produced by Bacillus amyloliquefaciens V656 in a shrimp and crab shell powder medium.

A Gram-positive bacterium with antagonistic activity was isolated from the soil. It has been identified as Bacillus amyloliquefaciens strain V656 on the basis of 16S ribosomal DNA analysis and standard bacteriological tests. B. amyloliquefaciens V656 produced antifungal enzymes when it was grown in a medium containing shrimp and crab shell powder (SCSP) of marine waste. The antifungal enzymes displayed chitinase activities. Two extracellular antifungal chitinases (FI and FII) were purified and characterized, and their molecular weights, isoelectric points, pH and thermal stabilities, and antifungal activities were determined. The characteristics of V656 chitinases are similar to those of the known bacterial chitinases in terms of their isoelectric points, thermal instabilities, and lack of lysozyme activity. In contrast to other known bacterial chitinases, the unique characteristics of V656 chitinases include extremely low molecular weights and nearly neutral optimum pH. Furthermore, this is the first report of the isolation of chitinases from B. amyloliquefaciens that are active against fungi.

Purification of an Elicitor-Inducible Antifungal Chitinase from Suspension-Cultured Rice Cells

Suspension-cultured rice cells showed an appreciable amount of chitinase activity when the cultured cells were treated with an elicitor isolated fromRhizoctonia solani, the rice sheath blight pathogen. A fivefold increase in chitinase activity was observed 24 h after elicitor treatment. The elicitor-inducible chitinase was purified by ammonium sulfate fractionation, chitin affinity chromatography and gel filtration. Its molecular weight is 35 kDa and it has an isoelectric point of 8.3. The 35 kDa basic chitinase inhibited mycelial growth ofR. solani in vitro. Morphological changes appeared within 1 h following exposure of mycelium to the chitinase. The hyphal tip showed marked swelling and subsequently lysis was also observed.

Kinetic Analysis of Barley Chitinase

The endochitinase from barley is the archetypal enzyme for a large class of plant-derived antifungal chitinases. The X-ray structure was solved previously in our laboratory and a mechanism of action proposed based on structural considerations. In this manuscript we report the use of a defined soluble substrate, 4-methylumbelliferyl β-N,N′,N″-triacetylchitotrioside, to characterize kinetic parameters of the enzyme. The pH profile shows that activity is controlled by a base with a pKaof 3.9 (Glu 89) and an acid with a pKaof 6.9 (Glu 67). TheKmusing the synthetic substrate is 33 μm, and thekcatis 0.33 min−1, while theKmfor (GlcNAc)4is 3 μmandkcatis 35 min−1. Binding constants were measured for β-linked oligomers ofN-acetylglucosamine. The monomer does not bind and dissociation constants for the dimer, trimer, and tetramer are 43, 19, and 6 μm, respectively. Analysis of kinetic and dissociation constants proves the mechanism of barley chitinase is consistent with a Bi–Bi kinetic model for hydrolysis, with (GlcNAc)4and water as substrates and (GlcNAc)2as products. Substrate cleavage patterns show that (GlcNAc)6is cleaved in half to (GlcNAc)3as well as into (GlcNAc)4and (GlcNAc)2with almost equal efficiency. NMR analysis of cleavage products confirms that the enzyme proceeds with anomeric inversion of products.

Novel osmotically induced antifungal chitinases and bacterial expression of an active recombinant isoform.

NaCl (428 mM)-adapted tobacco (Nicotiana tabacum L. var Wisconsin 38) cells accumulate and secrete several antifungal chitinases. The predominant protein secreted to the culture medium was a 29-kD peptide that, based on internal amino acid sequence, was determined to be a class II acidic chitinase with similarity to PR-Q. The four predominant chitinases (T1, T2, T3, and T4) that accumulated intracellularly in 428 mM NaCl-adapted cells were purified. Based on N-terminal sequence analyses, two of these were identified as class I chitinase isoforms, one similar to the N. tomentosiformis (H. Shinshi, J.M. Neuhaus, J. Ryals, F. Meins [1990] Plant Mol Biol 14:357-368) protein (T1) and the other homologous to the N. sylvestris (Y. Fukuda, M. Ohme, H. Shinshi [1991] Plant Mol Biol 16:1-10) protein (T2). The other two proteins (T3 and T4) were determined to be novel chitinases that have sequence similarity with class I chitinases, but each lacks a chitin-binding domain. All four chitinases inhibited Fusarium oxysporum f. sp. lycopersici and Trichoderma longibrachiatum hyphal growth in vitro, although the isoforms containing a chitin-binding domain were somewhat more active. Conditions were established for the successful expression of soluble and active bacterial recombinant T2. Expression of soluble recombinant T2 was achieved when isopropyl beta-D-thiogalactopyranoside induction occurred at 18 degrees C but not at 25 or 37 degrees C. The purified recombinant protein exhibited antifungal activity comparable to a class I chitinase purified from NaCl-adapted tobacco cells.

Purification and Characterization of an Antifungal Chitinase from Arabidopsis thaliana

Plants exhibit an altered pattern of protein synthesis in response to pathogen invasion and abiotic stress. One of these ;pathogenesis-related' proteins has been identified as chitinase, which is capable of inhibiting fungal growth in vitro. This observation has led to the suggestion that the in vivo role of chitinases is to protect plants against fungal invasion. Here, we report the purification and characterization of a basic chitinase from Arabidopsis thaliana (L.) Heynh. Columbia wild type. The purified enzyme has a molecular mass of approximately 32 kilodaltons as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, and an apparent pl of approximately 8.7 as determined by isoelectric focusing. The purified protein is an effective inhibitor of the growth of Trichoderma reesei in vitro but does not affect the growth of several other fungi. Amino acid composition analysis of the intact protein as well as amino acid composition analysis and automatic Edman degradation of isolated tryptic fragments of the enzyme indicate that it may be identical to the product of a chitinase gene isolated from an Arabidopsis genomic library (Samac DA, Hironaka CM, Yallaly PE, Shah DM [1990] Plant Physiol 93: 907-914).