Chitinase Bands Research Articles

Purification of Chitinase 33kDa and Expression Pattern of chit33 in Trichoderma longibrachiatum T28

The chitinolytic system of Trichoderma sp. is complex with several types of chitinase working together simultaneously to enhance its mycoparasitism. However, chitinase enzyme has not been studied extensively for Trichoderma longibrachiatum although it has been proven to have significant effect as biocontrol. This study was conducted in the interest to purify chitinase 33kDA from T. longibrachiatum T28 and also to assess chit33 expression patterns. Chitinase activity was determined and the peak of chitinase activity was observed at 36 hours. Chitinase enzyme from the culture filtrate of T. longibrachiatum was precipitated with 80% acetone followed by anion- exchange chromatography using Neobar AQ. Only one chitinase band at 33kDa in size has been detected and analysis of Southern Blotting has confirmed the present of at least a single copy of chitinase 33kDA gene (chit33) for T. longibrachiatum strain T28. Assessment of chit33 expression showed that the expression was strongly affected by substrate specificity; where

Conidial surface proteins of Metarhizium anisopliae: Source of activities related with toxic effects, host penetration and pathogenesis

Conidial contact with an arthropod surface is the first step of the fungal penetration and infection process. However, conidia of Metarhizium anisopliae have associated components, like enzymes that could be involved in triggering the penetration process and toxic effects that have not yet been well characterized. Fungi produce many enzymes that also are toxic components found in bacteria and animal venoms and thus may be considered as potential virulence factors. In this work, we report several enzymatic activities from spore surface protein extracts. The major proteolytic activities observed in spore surface proteins (SSP) were Pr1 and Pr2 activities, in that order. According to the zymograms obtained, SSP contain different proteases. SSP contain trehalase, exo- and endo-chitinase activities, and seven different chitinase bands which have been observed in zymograms. Activities involved in protection against reactive oxygen species (ROS) were also detected. Two lipolytic enzymes were also detected in lipase zymograms. Phospholipase C activity, closely related to microbial pathogenesis, was detected for the first time in M. anisopliae conidia. These activities described could be an initial step towards understanding the mechanisms involved in the first stage of M. anisopliae infection process and its toxic effects against arthropod hosts.

BACILLUS THURINGIENSIS FERMENTATION OF WASTEWATER AND WASTEWATER SLUDGE – PRESENCE AND CHARACTERIZATION OF CHITINASES

This study investigated the presence of chitinases in Bacillus thuringiensis ssp kurstaki HD‐1(Bt) fermented broths of wastewater sludge (non‐hydrolyzed and hydrolyzed); starch industry wastewater and soyameal. Chitinase activity was absent in soyameal and present in others. Chitinase demonstrated peaks at pH 4.0 and temperatures 40 and 50°C with higher activity between pH 4–5 and 10–11. The chitinase band on SDS‐PAGE was found to be between 36 and 45 kDa for non‐hydrolyzed (NH) and hydrolyzed sludge (TH) and starch industry wastewater. The chitinase profile during fermentation showed peaks at 15 and 30 h for non‐hydrolyzed and hydrolyzed sludge and 15 and 24 h for starch industry wastewater. Chitinase retained 96–99 % activity after two weeks incubation at room temperature and pH 4. Bioassays with supplementation of Bt chitinases showed 1.2 fold increase in entomotoxicity of wastewater sludge and a small increase in starch industry wastewater. This study sheds light on production of Bt chitinases in alternative media which will have a long term effect on entomotoxicity of these formulations.

Application of DNA Bar Codes for Screening of Industrially Important Fungi: the Haplotype of Trichoderma harzianum Sensu Stricto Indicates Superior Chitinase Formation

Selection of suitable strains for biotechnological purposes is frequently a random process supported by high-throughput methods. Using chitinase production by Hypocrea lixii/Trichoderma harzianum as a model, we tested whether fungal strains with superior enzyme formation may be diagnosed by DNA bar codes. We analyzed sequences of two phylogenetic marker loci, internal transcribed spacer 1 (ITS1) and ITS2 of the rRNA-encoding gene cluster and the large intron of the elongation factor 1-alpha gene, tef1, from 50 isolates of H. lixii/T. harzianum, which were also tested to determine their ability to produce chitinases in solid-state fermentation (SSF). Statistically supported superior chitinase production was obtained for strains carrying one of the observed ITS1 and ITS2 and tef1 alleles corresponding to an allele of T. harzianum type strain CBS 226.95. A tef1-based DNA bar code tool, TrichoCHIT, for rapid identification of these strains was developed. The geographic origin of the strains was irrelevant for chitinase production. The improved chitinase production by strains containing this haplotype was not due to better growth on N-acetyl-beta-D-glucosamine or glucosamine. Isoenzyme electrophoresis showed that neither the isoenzyme profile of N-acetyl-beta-glucosaminidases or the endochitinases nor the intensity of staining of individual chitinase bands correlated with total chitinase in the culture filtrate. The superior chitinase producers did not exhibit similarly increased cellulase formation. Biolog Phenotype MicroArray analysis identified lack of N-acetyl-beta-D-mannosamine utilization as a specific trait of strains with the chitinase-overproducing haplotype. This observation was used to develop a plate screening assay for rapid microbiological identification of the strains. The data illustrate that desired industrial properties may be an attribute of certain populations within a species, and screening procedures should thus include a balanced mixture of all genotypes of a given species.

Anomalous electrophoretic behavior of a chitinase isoform from grape berries and wine in glycol chitin-containing sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels

An anomalous electrophoretic behavior of a chitinase isoform present in both grape (Vitis vinifera L.) berries and wine was observed in glycol chitin-containing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels. A progressive shift of the relative molecular mass M(r) of the enzyme (from approximately 30,500 up to approximately 57,700) with increasing glycol chitin concentration in the gels up to 0.1% was revealed when samples were electrophoresed under nonreducing conditions, whereas the presence of glycol chitin had no effects when samples were reduced before SDS-PAGE separation. The M(r) of other grape and wine chitinase isoforms as well as that of the chitinase from pomegranate (Punica granatum L.) fruit was unaffected by the presence of the substrate in the gel under both reducing and nonreducing conditions. Since the enzymes were inactive during the electrophoretic separation, it is likely that the retarding effect of glycol chitin observed specifically for the unreduced chitinase band from grape and wine was due to an interaction between the substrate and a chitin-binding domain different from the catalytic site, such as that typical of class I and class IV chitinases.

Biological control of damping-off caused by Rhizoctonia solani using chitinase-producing Paenibacillus illinoisensis KJA-424

A bacterium having strong chitinolytic activity was isolated from a coastal soil in Korea and identified as Paenibacillus illinoisensis KJA-424 on the basis of the nucleotide sequence of a 16S rRNA gene. By activity staining after SDS–PAGE, three major chitinase bands with chitinolytic activity, approximate molecular weight of 63, 54 and 38 kDa were detected. On co-culture Rhizoctonia solani with KJA-424, abnormal swelling and deformation of R. solani hyphae were observed, where the release of N-acetyl- d-glucosamine was detected. The bacterium suppressed the symptom of damping-off cucumber seedlings caused by R. solani, in greenhouse trial.

Multiple chitinase enzymes from a single gene of Bacillus licheniformis TP-1

Three chitinase activity bands in a culture supernatant of Bacillus licheniformis TP-1 were detected by non-denaturing PAGE. They were designated chitinase bands 1, 2, and 3 in order from the gel origin. Analysis by immunodiffusion and immunoelectrophoresis using polyclonal antibody raised against chitinase band 3 indicated that these chitinases were antigenically similar. B. licheniformis TP-1 and Escherichia coli harboring the cloned chitinase gene (pCHIL3) from strain TP-1 expressed 3 chitinase bands by non-denaturing PAGE and SDS-PAGE which gave estimated molecular masses of 68, 62, and 50 kDa (named Chi68, Chi62, and Chi50, respectively). All three chitinases had the same N-terminal amino acid sequences, strongly suggesting that Chi62 and Chi50 were derived from Chi68 by a processing step(s) at the C-terminus. The deduced C-terminal amino acid sequence of Chi68 showed homology to amino acid sequences of known chitin and cellulose binding domains. Chi62 and Chi50 lacked this domain (judging from their deduced amino acid sequences and calculated molecular masses) and they were unable to bind chitin, suggesting that they were generated from Chi68 by cleavage of the chitin binding domain at the C-terminus. Comparison of chitinase activities indicated that this binding domain was important for complete hydrolytic activity towards colloidal chitin.

Characterization of chitinases and β‐1,3‐glucanases in grapefruit flavedo during fruit development

Chitinase (EC 3.2.1.14) and β‐1,3‐glucanase (EC 3.2.1.39) activities in the flavedo of grapefruit (Citrus paradisi cv. Marsh) were determined at 17 times during the course of fruit development. Chitinase activity is initially high in flavedo, but drops rapidly and is low, although fairly constant throughout the remainder of fruit development. In contrast to chitinase, β‐1,3‐glucanase activity is lowest in young fruit and increases during development. Western blots of crude flavedo extracts following SDS‐PAGE were probed with antibodies raised against purified citrus chitinase and β‐1,3‐glucanase. Results of immunostaining revealed that changes in the activities of chitinase and β‐1,3‐glucanase were reflected in the amount of chitinase and glucanase protein present in the extracts. Only a single chitinase band was detected on western blots of crude flavedo extracts, whereas one glucanase band was present in young fruit and a second one appeared later in older fruit. Partial purification of flavedo chitinases and glucanases was performed using extracts prepared from immature and mature fruit for the two enzymes, respectively. Acidic and basic forms of both enzymes were present in the extracts; acidic and basic forms of chitinase were present in nearly equal amounts whereas basic glucanases predominated (91% of total activity). Acidic and basic chitinases differed in substrate specificity as well as products of degradation indicating the heterogeneous nature of the enzymes. Both acidic and basic glucanases required the presence of β‐1,3 linkages for activity, were active against both soluble and insoluble β‐1,3 glucans and generated similar products.

Chitinase profiles in mature carrot (Daucus carota) roots and purification and characterization of a novel isoform

The profile of chaitinases (EC 3.2.1.14) in mature carrot (Daucus carota L. cv. Eagle) roots was studied. Multiple chitinase bands (8–10) were observed in native and sodium dodecylsulfate‐denaturing polyacrylamide gels. The molecular masses of these chitinases were estimated to be from 20 000 to 40 000. One major chitinase was purified and found to be an acidic protein with pI at 4.3 and a molecular mass of 39 500. The optimum pH for enzymatic activity was around 5 and the optimum temperature was 25°C. The enzyme was stable at pHs below 8 and temperatures below 60°C. The protein did not have a chitin‐binding domain, but showed some similarity to the amino acid composition of tobacco class I chitinase. The N‐terminal amino acid sequence did not resemble any of the described classes of chitimases. This chitinase did not possess lysozyme activity and showed antifungal activity when tested against Trichoderma sp.

Chitinase activities are induced in Eucalyptus globulus roots by ectomycorrhizal or pathogenic fungi, during early colonization

A comparative study of root chitinase activities induced by the ectomycorrhizal basidimycete Pisolithus tinctorius Coker and Couch and the pathogenic fungus Phytophthora cinnamomi, has been carried out. Two chitinases were constitutively present in Eucalyptus globulus ssp. bicostata (Maid et al.) Kirkp. roots. When 7‐day‐old seedlings were challenged with the ectomycorrhizal fungus, root chitinase activity was stimulated already after 6 h, during the very early stages of ectomycorrhizal colonization. Comparing chitinase electrophoretic patterns induced by symbiotic strains more or less compatible with Eucalyptus, a strong stimulation of chitinase activity indicated a successful interaction, which evolved quickly towards root infection and mature mycorrhizae formation. Root chitinase activity remained constant over 7 days during the establishment of the symbiosis. No new chitinase band was induced by the pathogen, when compared with the symbiotic fungi. Chitinase activity was only stimulated quantitatively after pathogenic infection. Root chitinase activities were also stimulated by fungal cell extracts applied in vitro. Such stimulation mimicked precisely the stimulation by living fungi. The intensity of the plant response to fungal extracts was related to fungal strain aggressiveness.

Variability in antifungal proteins in the grains of maize, sorghum and wheat

Crude protein extracts were made from kernels of 12 cultivars each of maize, sorghum and wheat. These preparations were fractionated on sodium dodecyl sulfate (SDS)‐polyacrylamide gels and subjected to Western blot analyses. Bands corresponding to chitinases and β‐glucanases were identified immunologically (Western blots) and on activity gels. Ribosome Inactivating Protein(s) (RIP) and permatins were identified immunologically. In maize, two chitinase bands (25–29 kDa) were seen in all cultivars, and a third band of about 23 kDa was detected in 7 of the 12 cultivars. Two or three β‐glucanase bands of sizes between 24 and 36 kDa (probably a mixture of 1,3–β‐ and 1,3–1,4‐β‐glucanases) were detected in blots of SDS gels, and one band was detected in activity gels (1,3‐β‐glucanase). In sorghum, one chitnase band of approximately 29 kDa, and two or three additional bands ranging in size from 21–24 kDa were observed. Only one β‐glucanase band was identified, with an estimated molecular weight of 30 kDa. All bands that appeared on Western blots of SDS gels corresponded to bands detected on activity gels. In wheat, one chitmase band of around 20 kDa, one β‐glucanase band of about 30 kDa and one RIP band of about 30 kDa were identified. Permatins (molecular weight about 22 kDa) were identified in maize, sorghum and wheat, with the different cultivars having varying amounts of permatins.

Bacterial Chitinase Is Modified and Secreted in Transgenic Tobacco

The chiA gene of Serratia marcescens codes for a secreted protein, bacterial chitinase (ChiA). We have investigated the modifications and the cellular location of ChiA when it is expressed in transgenic tobacco plants. Immunoblots on total leaf protein probed with antibody to ChiA showed that when the bacterial chitinase is expressed in plants, it migrates as a series of discrete bands with either the same or a slower mobility than the secreted bacterial protein. Analysis of the vacuum infiltrate of leaves expressing ChiA showed that the modified forms of the protein are enriched in the intercellular fluid. Media recovered from suspension cultures of cell lines expressing the chiA gene were also enriched for the modified forms of ChiA. Washed protoplasts, however, contained only the nonmodified form. The molecular weight of these polypeptides is reduced by treatment with glycopeptidase F but not with endoglycosidase H. Treatment of the suspension cultures with tunicamycin also leads to reduction in the molecular weight of the chitinase bands. We suggest that some of the ChiA protein is N-glycosylated and secreted when expressed in plants, and that the modifications are complex glycans. These results show that a bacterial signal sequence can function in plant cells, and that protein secretion from plant cells probably operates by a default pathway.