Purification and characterization of an extracellular exochitinase,beta-N-acetylhexosaminidase,from the fungal mycoparasite Stachybotrys elegans

The mycoparasite Stachybotrys elegans produces two exo- and one endo-acting chitinases when grown on chitin. We purified to homogeneity one of the exo-acting chitinases, beta-N-acetylhexosaminidase and partially characterized its physical and biochemical properties. The native enzyme has a molecular mass of 120 kDa when determined by gel filtration and 68 kDa by sodium dodecyl sulfate - polyacrylamide gel electrophoresis indicating that the native protein probably occurs as a dimer in solution. The purified beta-N-acetylhexosaminidase is most active at pH 5.0 and 40 degrees C and hydrolyzes the p-nitrophenyl-N-acetyl-beta-D-glucosaminide with apparent Km of 84.6 microM. Polyclonal antibodies raised against the 68-kDa beta-N-acetylhexosaminidase (NAG-68) indicated that the antibody is highly specific and recognizes the protein in crude filtrate preparation. This suggests that the protein is a not a proteolytic product of another protein. Western blot analysis showed that the activity of NAG-68 was induced when S. elegans was grown on purified cell wall fragments of its host, Rhizoctonia solani, as well as during antagonistic interaction of the mycoparasite and host when both were grown on synthetic medium with or without supplemental carbon source.     

Cloning of the 52-kDa chitinase gene from Serratia marcescens KCTC2172 and its proteolytic cleavage into an active 35-kDa enzyme

A chitinase gene (pCHI52) encoding the 52-kDa chitinase was isolated from a Serratia marcescens KCTC2172 cosmid library. This chitinase gene consists of 2526 bp with an open reading frame that encodes 485 amino acids. Escherichia coli harboring the pCHI52 gene secreted not only a 52-kDa but also a 35-kDa chitinase into the culture supernatant. We purified both 52-kDa and 35-kDa chitinases using a chitin affinity column and Sephacryl-S-300 gel filtration chromatography. We determined that the 17 N-terminal amino acid sequences of the 52-kDa and the 35-kDa chitinase are identical. Furthermore, a protease obtained from S. marcescens KCTC2172 cleaved the 52-kDa chitinase into the 35-kDa protein with chitinase activity. These results suggest that the 35-kDa chitinase derives from the 52-kDa chitinase by post-translational proteolytic modification. The optimal reaction temperature of 45°C and the optimal pH of 5.5 were identical for both enzymes. The specific activities of the 52-kDa and 35-kDa chitinases on natural swollen chitin were 67 μmol min⁻¹ mg⁻¹ and 60 μmol min⁻¹ mg⁻¹, respectively.     

Purification and characterization of a Bacillus cereus exochitinase

Five extracellular chitinases of Bacillus cereus 6E1 were detected by a novel in-gel chitinase assay using carboxymethyl-chitin-remazol brilliant violet 5R (CM-chitin-RBV) as a substrate. The major chitinase activity was associated with a 36-kDa (Chi36) gel band. Chi36 was purified by a one-step, native gel purification procedure derived from the new in-gel chitinase assay. The purified Chi36 has optimal activity at pH 5.8 and retains some enzymatic activity between pH 2.5-8. The temperature optimum for Chi36 was 35 degrees C, but the enzyme was active between 4-70 degrees C. Based on its ability to hydrolyze mainly p-nitrophenyl-(N-acetyl-beta-D-glucosaminide)(2), Chi36 is characterized as a chitobiosidase, a type of exochitinase. The N-terminal amino acid sequence of mature Chi36 was determined (25 amino acids). Alanine is the first N-terminal amino acid residue indicating the cleavage of a signal peptide from a Chi36 precursor to form the mature extracellular Chi36. The N-terminal sequence of Chi36 demonstrated highest similarity with Bacillus circulans WL-12 chitinase D and significant similarity with several other bacterial chitinases.     

<Emphasis Type="Italic">Aeromonas caviae</Emphasis> CB101 Contains Four Chitinases Encoded by a Single Gene <Emphasis Type="Italic">chi1</Emphasis>

Aeromonas caviae CB101 secretes four chitinases (around 92, 82, 70, and 55 kDa) into the culture supernatant. A chitinase gene chi1 (92 kDa) was previously studied. To identify the genes encoding the remaining three chitinases, a cosmid library of CB101 was constructed to screen for putative chitinase genes. Nine cosmid clones were shown to contain a chitinase gene on chitin plates. Surprisingly, all the positive clones contained chi1. In parallel, we purified the 55-kDa chitinase (Chi55) from the CB101 culture supernatant by continuous DEAE-Sepharose and Mono-Q anion exchange chromatography. The N-terminal amino acid sequence of the purified chitinase exactly matched the N-terminal sequence of mature Chi1, indicating that the purified chitinase (Chi55) is a truncated form of Chi1. The N- and C-terminal domains of chi1 were cloned, expressed, and purified, separately. Western blots using anti-sera to the N- and C-terminal domains of chi1 on the chitinases of CB101 showed that the four chitinases in the culture supernatant are either chi1 or C-terminal truncations of Chi1. In addition, the CB101 chi1 null mutant showed no chitinolytic activity, while CB101 chi1 null mutant complemented by pUC19chi1 containing chi1 showed all four chitinases in gel activity assay. These data indicated that all four chitinases secreted by CB101 in the culture supernatant are the product of one chitinase gene chi1.     

The 92-kDa chitinase from Streptomyces olivaceoviridis contains a lysine-C endoproteinase at its N-terminus

Abstract Serine proteinases of 42, 22 and 14 kDa were purified from the culture fluid of Streptomyces olivaceoviridis by FPLC. The first 14 amino acids at their N-termini were identical and coincide with the N-terminal amino acid sequence of 92-kDa chitinase, which was found to hydrolyse casein. The four proteins hydrolyse synthetic substrates at the carboxyl group of lysine and (more slowly) arginine. The 14-kDa endoproteinase releases only two fragments of 42 and 43 kDa from β-galactosidase. When the pure 92-kDa chitinase was incubated at 37°C in Tris·HCl buffer, it was cleaved into a 70-kDa chitinase and a 22-kDa proteinase which in its part is rapidly degraded to a 14-kDa proteinase.     

A novel strain of Brevibacillus laterosporus produces chitinases that contribute to its biocontrol potential

A novel strain exhibiting entomopathogenic and chitinolytic activity was isolated from mangrove marsh soil in India. The isolate was identified as Brevibacillus laterosporus by phenotypic characterization and 16S rRNA sequencing and designated Lak1210. When grown in the presence of colloidal chitin as the sole carbon source, the isolate produced extracellular chitinases. Chitinase activity was inhibited by allosamidin indicating that the enzymes belong to the family 18 chitinases. The chitinases were purified by ammonium sulfate precipitation followed by chitin affinity chromatography yielding chitinases and chitinase fragments with 90, 75, 70, 55, 45, and 25 kDa masses. Mass spectrometric analyses of tryptic fragments showed that these fragments belong to two distinct chitinases that are almost identical to two putative chitinases, a 89.6-kDa four-domain chitodextrinase and a 69.4-kDa two-domain enzyme called ChiA1, that are encoded on the recently sequenced genome of B. laterosporus LMG15441. The chitinase mixture showed two pH optima, at 6.0 and 8.0, and an optimum temperature of 70 °C. The enzymes exhibited antifungal activity against the phytopathogenic fungus Fusarium equiseti. Insect toxicity bioassays with larvae of diamondback moths (Plutella xylostella), showed that addition of chitinases reduced the time to reach 50 % mortality upon infection with non-induced B. laterosporus from 3.3 to 2.1 days. This study provides evidence for the presence of inducible, extracellular chitinolytic enzymes in B. laterosporus that contribute to the strain’s antifungal activity and insecticidal activity.     

Molecular characterization of four chitinase cDNAs obtained fromCladosporium fulvum-infected tomato

Complementary DNA clones encoding acidic and basic isoforms of tomato chitinases were isolated fromCladosporium fulvum-infected leaves. The clones were sequenced and found to encode the 30 kDa basic intracellular and the 26 and 27 kDa acidic extracellular tomato chitinases previously purified (M.H.A.J. Joostenet al., in preparation). A fourth truncated cDNA which appears to encode an extracellular chitinase with 82% amino acid similarity to the 30 kDa intracellular chitinase was also isolated. Characterization of the clones revealed that the 30 kDa basic intracellular protein is a class I chitinase and that the 26 and 27 kDa acidic extracellular proteins which have 85% peptide sequence similarity are class II chitinases. The characterized cDNA clones represent four from a family of at least six tomato chitinases. Southern blot analysis indicated that, with the exception of the 30 kDa basic intracellular chitinase, the tomato chitinases are encoded by one or two genes. Northern blot analysis showed that the mRNA encoding the 26 kDa acidic extracellular chitinase is induced more rapidly during an incompatibleC. fulvum-tomato interaction than during a compatible interaction. This difference in timing of mRNA induction was not observed for the 30 kDa basic intracellular chitinase.     

Purification and properties of a thermostable chitinase from Streptomyces thermoviolaceus OPC-520.

A chitinase was purified from the culture filtrate of Streptomyces thermoviolaceus OPC-520. The enzyme showed a high optimum temperature (70 to 80 degrees C), a high optimum pH level (8.0 to 10.0), and heat stability. This enzyme showed high sequence homology with chitinases from Serratia marcescens QMB1466 and Bacillus circulans WL-12.     

Evidence that platelet and skeletal sarcoplasmic reticulum Ca2+-ATPase are structurally distinct

Proteolytic digestion and indirect immunostaining were used to compare the platelet and sarcoplasmic reticulum Ca2+-ATPase proteins. When the platelet and sarcoplasmic reticulum Ca2+-ATPase proteins were digested in the native state with trypsin, the platelet Ca2+-ATPase, which had an apparent undigested molecular mass of 103 kDa, yielded 78-kDa and 25-kDa fragments. Calcium transport activity depended on the integrity of the 103-kDa protein, while the digested protein had residual ATPase activity. Tryptic digestion of the sarcoplasmic reticulum pump protein, which also had an undigested molecular mass of 103 kDa, yielded products with apparent molecular masses of 55 kDa, 36 kDa, and 26 kDa. Distinct patterns were also observed when the platelet and sarcoplasmic reticulum calcium pump proteins were digested with chymotrypsin and Staphylococcus aureus protease in the presence of sodium dodecyl sulfate. Chymotrypsin digestion of the platelet protein resulted in the appearance of products with apparent molecular masses of 70 kDa, 39 kDa, and 31 kDa, while a similar digestion of the sarcoplasmic reticulum calcium pump protein yielded 54-kDa, 52.5-kDa, 46-kDa, 41-kDa, and 36-kDa fragments. Exposure of the sarcoplasmic reticulum and platelet Ca2+-ATPase proteins to S. aureus protease also yielded dissimilar fragmentation patterns. These results indicate that the Ca2+-ATPases from platelets and sarcoplasmic reticulum are distinct proteins.     

Production and partial characterization of chitinase from a halotolerant <Emphasis Type="Italic">Planococcus rifitoensis</Emphasis> strain M2-26

This paper is the first to investigate the production and partial characterization of the chitinase enzyme from a moderately halophilic bacterium Planococcus rifitoensis strain M2-26, earlier isolated from a shallow salt lake in Tunisia. The impact of salt, salinity concentration, pH, carbon and nitrogen sources on chitinase production and activity have been determined. This is the first report on a high salt-tolerant chitinase from P. rifitoensis, since it was active at high salinity (from 5 to 30% NaCl) as well as in the absence of salt. This enzyme showed optimal activity at 70°C and retained up to 82 and 66% of its original activity at 80 or 90°C, respectively. The activity of the enzyme was also shown over a wide pH range (from 5 to 11). For characterization of the enzyme activity, the chitinase secreted in the culture supernatant was partially purified. The preliminary study of the concentrated dialysed supernatant on native PAGE showed at least three chitinases produced by strain M2-26, with highest activity approximately at 65 kDa. Thus, the thermo-tolerant and high salt-tolerant chitinases produced by P. rifitoensis strain M2-26 could be useful for application in diverse areas such as biotechnology and agro-industry.     

Chitinase from Bacillus thuringiensis subsp. pakistani

The chitinase gene (chiA71) from Bacillus thuringiensis subsp. pakistani consists of an open reading frame of 1,905 nucleotides encoding 635 amino acid residues with an estimated molecular mass of 71 kDa. Comparison of the deduced amino acid sequence of the mature enzyme to other microbial chitinases shows a putative catalytic domain and a region with conserved amino acids similar to that of the type III module of fibronectin and a chitin-binding domain. By activity detection of chitinase on SDS-PAGE after renaturation, the molecular mass of protein bands with chitinase activity were 66, 60, 47, and 32 kDa. The N-terminal amino acid sequence of each chitinase activity band was the same (Asp-Ser-Pro-Lys-Gln), suggesting that the 60-, 47-, and 32-kDa chitinases were derived from the 66-kDa chitinase by processing step(s) at the C-terminus. The enzyme was identified as an exochitinase, since it generated N-acetylglucosamine from early stage of colloidal chitin hydrolysis. The crude protein (2.3-18.4 mg/ml), containing chitinase at final activities of 8, 16, 32, and 64 mU/ml, was toxic to Aedes aegypti larvae and caused mortalities of 7.5, 15.0, 51.3, and 70.0% respectively, but the same amount of crude protein from a B. thuringiensis subsp. pakistani mutant lacking chitinase was not toxic.     

Characterization of chitinases excreted by Bacillus cereus CH

Bacillus cereus CH was shown to excrete chitinases into the culture supernatant when cultivated in a medium containing 0.2% colloidal chitin, whereas the removal of colloidal chitin resulted in a low activity. After concentration of the culture supernatant by precipitation with ammonium sulfate, the induced chitinases were purified by sequential chromatography. Four different chitinases, A, B1, B2, and B3 with molecular masses of 35, 47, 58, and 64 kDa, respectively, were separated. All chitinases showed similarities in their kinetic parameters when observed with colloidal chitin, including an optimal pH of 5.0-7.5, and an optimal temperature between 50-60 degrees C. Chitinase A hydrolyzed glycol chitin and p-nitrophenyl-di-N-acetyl-beta-chitobioside at similar rates to that of colloidal chitin, whereas group B chitinases hydrolyzed both substrates in much lower rates. From analyses of the reaction products, it is most likely that chitinase A and all group B chitinases hydrolyze the substrates tested in an endo-fashion. However, group B chitinases were distinct from chitinase A in possessing high transglycosylation activity. From amino terminal sequencing, chitinases B1, B2, and B3 were shown to have almost identical sequences, which differed from that of chitinase A. The similarities in the reaction modes and amino terminal sequences among chitinases B1, B2, and B3 suggest that these chitinases may be derived from a presumptive precursor protein through C-terminal processing.     

Extracellular Chitinases of Fluorescent Pseudomonads Antifungal to Fusarium oxysporum f. sp. dianthi Causing Carnation Wilt

Vascular wilt of carnation caused by Fusarium oxysporum f. sp. dianthi (Prill. & Delacr.) W. C. Synder & H.N. Hans inflicts substantial yield and quality loss to the crop. Mycolytic enzymes such as chitinases are antifungal and contribute significantly to the antagonistic activity of fluorescent pseudomonads belonging to plant-growth-promoting rhizobacteria. Fluorescent pseudomonads antagonistic to the vascular wilt pathogen were studied for their ability to grow and produce chitinases on different substrates. Bacterial cells grown on chitin-containing media showed enhanced growth and enzyme production with increased anti-fungal activity against the pathogen. Furthermore, the cell-free bacterial culture filtrate from chitin-containing media also significantly inhibited the mycelial growth. Both the strains and their cell-free culture filtrate from chitin-amended media showed the formation of lytic zones on chitin agar, indicating chitinolytic ability. Extracellular proteins of highly antagonistic bacterial strain were isolated from cell-free extracts of media amended with chitin and fungal cell wall. These cell-free conditioned media contained one to seven polypeptides. Western blot analysis revealed two isoforms of chitinase with molecular masses of 43 and 18.5 kDa. Further plate assay for mycelial growth inhibition showed the 43-kDa protein to be antifungal. The foregoing studies clearly established the significance of chitinases in the antagonism of fluorescent pseudomonads, showing avenues for possible exploitation in carnation wilt management.     

Characterization of a chitinase and an endo-beta-1,3-glucanase from Trichoderma harzianum Rifai T24 involved in control of the phytopathogen Sclerotium rolfsii

Of 24 Trichoderma isolates, T harzianum Rifai (T24) showed a potential for control of the phytopathogenic basidiomycete Sclerotium rolfsii. When T24 was grown on different carbon sources, growth inhibition of S. rolfsii by the T24 culture filtrate correlated with the activity of extracellular chitinase and beta-1,3-glucanase. The 43-kilodalton (kDa) chitinase and the 74-kDa beta-1,3-glucanase were purified from the T24 culture filtrate in two and three steps, respectively, using ammonium sulphate precipitation followed by hydrophobic interaction chromatography (phenyl-Sepharose) and gel filtration (beta-1,3-glucanase). Km and Kcat were 3.8 g l(-1) and 0.71 s(-1) for the chitinase (chitin) and 1.1 g(-1) and 52 s(-1) for the beta-1,3-glucanase (laminarin). The chitinase showed higher activity on chitin than on less-acetylated substrate analogues (chitosan), while the beta-1,3-glucanase was specific for beta-1,3-linkages in polysaccharides. Both enzymes were stable at 30 degrees C, while at 60 degrees C the chitinase and the beta-1,3-glucanase were rapidly inactivated, showing half-lives of 15 and 20 min, respectively. The enzymes inhibited growth of S. rolfsii in an additive manner showing a promising ED50 (50% effective dose) value of 2.7 microg/ml.     

Purification and characterization of a 54 kDa chitinase from Bombyx mori

The 54 kDa protein that was suggested to be processed from the 65 kDa and 88 kDa chitinases of Bombyx mori [Koga et al., Insect Biochem. Mol. Biol. 27, 757–767 (1997)] was purified and proved to be a third chitinase (EC 3.2.1.14). This chitinase was purified from the fifth larval instar of B. mori by chromatography on DEAE-Cellulofine A–500, hydroxylapatite, Butyl-Toyopearl 650M, and Fractogel EMD DEAE 650(M) columns. The apparent molecular mass was confirmed to be 54 kDa by SDS–PAGE. Its optimum pH was 6.0 toward a short substrate, N-acetylchitopentaose (GlcNAc₅), while in its reaction with a longer substrate, glycolchitin, the enzyme showed a wide pH-range between 4.0 and 10. Kinetic parameters for the chitinase could be obtained in the hydrolysis of glycolchitin but not in that of N-acetylchitooligosaccharides (GlcNAc_n, n=2–6) because of substrate inhibition. The chitinase hydrolyzed N-acetylchitooligosaccharides except for dimer as follows: trimer to monomer plus dimer, tetramer to two molecules of dimer, pentamer to dimer plus trimer, and hexamer to dimer plus tetramer as well as two molecules of trimer. These results suggest that the 54 kDa chitinase is an endo-type hydrolase and preferred the longer-chain N-acetylchitooligosaccharides. Moreover, the anomeric forms of N-acetylchitooligosaccharides were analyzed in the reaction with the 54-kDa chitinase. It was revealed that this enzyme cleaves the substrate to produce the β anomeric product. With respect to inhibition of the 54 kDa chitinase, it was specifically inhibited by allosamidin in a competitive way with K_i values depending on the pH of the reaction mixture (K_i=0.013−0.746 μM). Comparing the properties and kinetic behavior of this chitinase with those of the 88 and 65 kDa chitinases from B. mori, regarding the specific activity of the three enzymes, the 65-kDa chitinase was 2.15 and 2.8 times more active than the 88 and 54-kDa chitinases, respectively. However, in the overall reaction of glycolchitin (k_cat/K_m), the 88-kDa enzyme was 4 and 40 times more active than the 65-kDa and the 54-kDa enzymes, respectively. Concerning the affinity (1/K_m) to glycolchitin, the 88 kDa chitinase affinity (at pH 6.5) was 5.8 times higher than that of the 65 kDa chitinase (at pH 5.5) and 4.0 times higher than that of the 54 kDa chitinase (at pH 6.0). These kinetic results suggest that B. mori chitinases are processed during ecdysis from the larger chitinase to smaller ones that leads to changes in their kinetic properties such as K_m, k_cat and k_cat/K_m successively.     

Isolation and characterization of the 54-kDa and 22-kDa chitinase genes of Serratia marcescens KCTC2172

A DNA fragment (pCHI5422) containing two genes encoding a 54-kDa and a 22-kDa chitinase was isolated from a cosmid DNA library of Serratia marcescens KCTC2172. The complete nucleotide sequence of pCHI5422 consisting of 4581 bp was determined. The nucleotide sequence of the 22-kDa chitinase consists of 681 bp of open reading frame encoding 227 amino acids and is located 1422 bp downstream of the translation termination codon of the 54-kDa chitinase sequence. The 54-kDa chitinase gene consisted of 1497 bp in a single open reading frame encoding 499 amino acids. The genes encoding the 54-kDa and 22-kDa chitinase were separately subcloned in Escherichia coli and the individual chitinases were expressed and purified from the culture broth using chitin affinity chromatography. When chitohexaose was used as substrate, the major product of the enzymatic reaction of both the 54-kDa and 22-kDa chitinases was a (GlcNAc)₂ dimer with a minor amount of monomer. The specific activity of the 54-kDa and 22-kDa chitinases were 300 μM (min)⁻¹ mg⁻¹ and 17 μM (min)⁻¹ mg⁻¹ on the natural swollen chitin, respectively.     

Isolation and characterization of thermostable chitinases from Bacillus licheniformis X-7u

Four kinds of thermostable chitinase were isolated from the cell-free culture broth of Bacillus licheniformis X-7u by successive column chromatographies on Butyl-Toyopearl, Q-Sepharose, and Sephacryl S-200. We named the enzymes chitinases I(89 kDa), II(76 kDa), III(66 kDa) and IV(59 kDa). Chitinases II, III and IV possessed extremely high optimum temperatures (70-80 degrees C), showing remarkable heat stability. Chitinases II, III and IV produced (GlcNAc)2 and GlcNAc from colloidal chitin and chitinase I predominantly produced (GlcNAc)2. The action pattern of chitinase I on PN-(GlcNAc)4 also showed a stronger propensity to cleave off the (GlcNAc)2 unit from the non-reducing end than the other three chitinases. Chitinases II, III and IV catalyzed a transglycosylation reaction that converted (GlcNAc)4 into (GlcNAc)6.     

A constitutively expressed 36 kDa exochitinase from Bacillus thuringiensis HD-1

A 36 kDa chitinase was purified by ion exchange and gel filtration chromatography from the culture supernatant of Bacillus thuringiensis HD-1. The chitinase production was independent of the presence of chitin in the growth medium and was produced even in the presence of glucose. The purified chitinase was active at acidic pH, had an optimal activity at pH 6.5, and showed maximum activity at 65 degrees C. Of the various substrates, the enzyme catalyzed the hydrolysis of the disaccharide 4-MU(GlnAc)(2) most efficiently and was therefore classified as an exochitinase. The sequence of the tryptic peptides showed extensive homology with Bacillus cereus 36 kDa exochitinase. The 1083 bp open reading frame encoding 36 kDa chitinase was amplified with primers based on the gene sequence of B. cereus 36 kDa exochitinase. The deduced amino-acid sequence showed that the protein contained an N-terminal signal peptide and consisted of a single catalytic domain. The two conserved signature sequences characteristic of family 18 chitinases were mapped at positions 105-109 and 138-145 of Chi36. The recombinant chitinase was expressed in a catalytically active form in Escherichia coli in the vector pQE-32. The expressed 36 kDa chitinase potentiated the insecticidal effect of the vegetative insecticidal protein (Vip) when used against neonate larvae of Spodoptera litura.     

Purification, characterization, and cDNA cloning of rice class III chitinase

Several chitinases were expressed in a rice cell suspension culture and detected in the medium. One of them, designated as RCB4, was isolated 248 fold from the culture filtrate to homogeneity by 70% ammonium sulfate precipitation, DEAE-cellulose, CM-cellulose, Sephadex G-75 column chromatography, and native gel slicing. RCB4 had a molecular mass of 32 kDa by SDS-PAGE. The optimum temperature was 40 degrees C, and 96% of its activity still remained at 60 degrees C. The optimum pH was 4, and 95% of its activity was maintained at pH 2. Using a substrate (GlcNAc)6, the Km and Vmax values of RCB4 were 0.53 mM and 11.1 mM/min, respectively. The N-terminal and internal amino acid sequences of RCB4 were determined to be VNSNLFRDYIGA and MALWA, respectively. A cDNA (C12523) clone that contained the N-terminal and internal amino acid sequences of RCB4 was obtained, sequenced, and renamed RCB41. RCB41 encoded 307 amino acid protein with a signal peptide of 25 amino acids and showed a 45% similarity to gladiolus chitinase GBC-a, one of the class III chitinase family. The expression of RCB4l in E. coli showed that RCB41 encodes a chitinase.