Purification and characterization of a novel isozyme of chitinase from Bombyx mori

75-kDa chitinase, which showed potential as a biocontrol agent against Japanese pine sawyer, was characterized after purification from the integument of the fifth instar larvae of Bombyx mori by chromatography on diethylaminoethyl (DEAE)-Toyoperal 650 (M), hydroxylapatite, and Fractogel EMD DEAE 650 (M) columns. The optimum pH was 6.0 toward N-acetylchitopentaose (GlcNAc5) and 10 toward glycolchitin. The optimum temperature was 60 degrees C toward GlcNAc5 and 25 degrees C toward glycolchitn. The enzyme was stable at pH 7-10 and below 40 degrees C. Kinetic analysis and reaction-pattern analysis using glycolchitin and N-acetylchitooligosacchraides as substrates indicated that 75-kDa chitinase is an endo- or random-type hydrolytic enzyme to produce the beta anomeric product and that it prefers the longer N-acetylchitooligosaccharides, suggesting, together with the N-terminal amino acid sequence, that the 75-kDa chitinase belongs to family 18 of glycosyl hydrolases.     

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.     

Kinetic analysis of a chitinase from red sea bream, Pagrus major

Kinetic analysis was done on the 46-kDa chitinase (EC 3.2.1.14) purified from the stomach of red sea bream, Pagrus major, using glycolchitin and N-acetylchitooligosaccharides (GlcNAc(n), n=2-6) as substrates. High activity was observed at two pHs, such as 2.5 and 9.0, toward glycolchitin as seen in other insect chitinases, and also at both pH 2.5 and 5.0 even toward a short substrate, N-acetylchitopentasaccharide. Allosamidin competitively inhibited chitinase with Ki value of 0.0214 microM at pH 2.5 and 0.0024 microM at pH 9.0 in the reaction of glycolchitin. Substrate inhibition was observed in the reaction of N-acetylchitopentasaccharide. The anomeric forms of the products from N-acetylchitooligosaccharides were analyzed to be beta anomer by the high pressure liquid chromatography (HPLC) method. The data for both beta-anomer formation and allosamidin inhibition suggest that red sea bream chitinase belongs to family 18 of glycosyl hydrolases. This suggestion is also supported by the results for the N-terminal amino acid sequence.     

Purification and characterization of a 56 kDa chitinase isozyme (PaChiB) from the stomach of the silver croaker, Pennahia argentatus

A 56 kDa chitinase isozyme (PaChiB) was purified from the stomach of the silver croaker Pennahia argentatus. The optimum pH and pH stability of PaChiB were observed in an acidic pH range. When N-acetylchitooligosaccharides ((GlcNAc)n, n=2 -6) were used as substrates, PaChiB degraded (GlcNAc)4 -6 and produced (GlcNAc)2,3. It degraded (GlcNAc)5 to produce (GlcNAc)2 (23.2%) and (GlcNAc)3 (76.8%). The ability to degrade p-nitrophenyl N-acetylchitooligosaccharides (pNp-(GlcNAc)n, n=2 -4) fell in the following order: pNp-(GlcNAc)3? pNp-(GlcNAc)2 pNp-(GlcNAc)4. Based on these results, we concluded that PaChiB is an endo-type chitinolytic enzyme, and that it preferentially hydrolyzes the third glycosidic bond from the non-reducing end of (GlcNAc)n. Activity toward crystalline α- and β-chitin was activated at 124%-185% in the presence of 0.5 M NaCl. PaChiB exhibited markedly high substrate specificity toward crab-shell α-chitin.     

Identification of a novel acidic mammalian chitinase distinct from chitotriosidase

Chitinases are ubiquitous chitin-fragmenting hydrolases. Recently we discovered the first human chitinase, named chitotriosidase, that is specifically expressed by phagocytes. We here report the identification, purification, and subsequent cloning of a second mammalian chitinase. This enzyme is characterized by an acidic isoelectric point and therefore named acidic mammalian chitinase (AMCase). In rodents and man the enzyme is relatively abundant in the gastrointestinal tract and is found to a lesser extent in the lung. Like chitotriosidase, AMCase is synthesized as a 50-kDa protein containing a 39-kDa N-terminal catalytic domain, a hinge region, and a C-terminal chitin-binding domain. In contrast to chitotriosidase, the enzyme is extremely acid stable and shows a distinct second pH optimum around pH 2. AMCase is capable of cleaving artificial chitin-like substrates as well as crab shell chitin and chitin as present in the fungal cell wall. Our study has revealed the existence of a chitinolytic enzyme in the gastrointestinal tract and lung that may play a role in digestion and/or defense.     

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.     

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.     

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.     

The roles of the C-terminal domain and type III domains of chitinase A1 from Bacillus circulans WL-12 in chitin degradation.

The mature form of chitinase A1 from Bacillus circulans WL-12 comprises a C-terminal domain, two type III modules (domains), and a large N-terminal domain which contains the catalytic site of the enzyme. In order to better define the roles of these chitinase domains in chitin degradation, modified chiA genes encoding various deletions of chitinase A1 were constructed. The modified chiA genes were expressed in Escherichia coli, and the gene products were analyzed after purification by high-performance liquid chromatography. Intact chitinase A1 specifically bound to chitin, while it did not show significant binding activity towards partially acetylated chitosan and other insoluble polysaccharides. Chitinases lacking the C-terminal domain lost much of this binding activity to chitin as well as colloidal chitin-hydrolyzing activity. Deletion of the type III domains, on the other hand, did not affect chitin-binding activity but did result in significantly decreased colloidal chitin-hydrolyzing activity. Hydrolysis of low-molecular-weight substrates, soluble high-molecular-weight substrates, and insoluble high-molecular-weight substrates to which chitinase A1 does not bind were not significantly affected by these deletions. Thus, it was concluded that the C-terminal domain is a chitin-binding domain required for the specific binding to chitin and that this chitin-binding activity is important for efficient hydrolysis of the sufficiently acetylated chitin. Type III modules are not directly involved in the chitin binding but play an important functional role in the hydrolysis of chitin by the enzyme bound to chitin.     

Chitinases of Streptomyces olivaceoviridis and significance of processing for multiplicity.

Five extracellular chitinases of 20.5, 30, 47, 70, and 92 kDa purified from the culture filtrate of Streptomyces olivaceoviridis ATCC 11238 differed in their sequences at the amino termini of the protein chains. In the native state, the chitinases were found to be resistant to proteolysis by trypsin, papain, and Staphylococcus aureus V8 protease. The latter produced several fragments of identical molecular mass from chitinases denaturated with sodium dodecyl sulfate. Five proteases were detected in the protein concentrate from the culture filtrate, and two of them showing ability to cleave chitinases in the native state were purified. One, a protease of 42 kDa, released a 30-kDa protein from the 70-kDa chitinase that reacts with anti-30 kDa chitinase antibodies; the other, a protease of 29 kDa, split the 30-kDa chitinase into 20.5-, 18-, and 16-kDa fragments. From these results, it was deduced that the 70-kDa chitinase is the precursor protein of the 30- and 20.5-kDa chitinases.     

Crystal structure and enzymatic properties of a bacterial family 19 chitinase reveal differences from plant enzymes

We describe the cloning, overexpression, purification, characterization and crystal structure of chitinase G, a single-domain family 19 chitinase from the Gram-positive bacterium Streptomyces coelicolor A3(2). Although chitinase G was not capable of releasing 4-methylumbelliferyl from artificial chitooligosaccharide substrates, it was capable of degrading longer chitooligosaccharides at rates similar to those observed for other chitinases. The enzyme was also capable of degrading a colored colloidal chitin substrate (carboxymethyl-chitin-remazol-brilliant violet) and a small, presumably amorphous, subfraction of alpha-chitin and beta-chitin, but was not capable of degrading crystalline chitin completely. The crystal structures of chitinase G and a related Streptomyces chitinase, chitinase C [Kezuka Y, Ohishi M, Itoh Y, Watanabe J, Mitsutomi M, Watanabe T & Nonaka T (2006) J Mol Biol358, 472-484], showed that these bacterial family 19 chitinases lack several loops that extend the substrate-binding grooves in family 19 chitinases from plants. In accordance with these structural features, detailed analysis of the degradation of chitooligosaccharides by chitinase G showed that the enzyme has only four subsites (- 2 to + 2), as opposed to six (- 3 to + 3) for plant enzymes. The most prominent structural difference leading to reduced size of the substrate-binding groove is the deletion of a 13-residue loop between the two putatively catalytic glutamates. The importance of these two residues for catalysis was confirmed by a site-directed mutagenesis study.     

An endochitinase A from Vibrio carchariae: cloning, expression, mass and sequence analyses, and chitin hydrolysis

We provide evidence that chitinase A from Vibrio carchariae acts as an endochitinase. The chitinase A gene isolated from V. carchariae genome encodes 850 amino acids expressing a 95-kDa precursor. Peptide masses of the native enzyme identified from MALDI-TOF or nanoESIMS were identical with the putative amino acid sequence translated from the corresponding nucleotide sequence. The enzyme has a highly conserved catalytic TIM-barrel region as previously described for Serratia marcescens ChiA. The Mr of the native chitinase A was determined to be 62,698, suggesting that the C-terminal proteolytic cleavage site was located between R597 and K598. The DNA fragment that encodes the processed enzyme was subsequently cloned and expressed in Escherichia coli. The expressed protein exhibited chitinase activity on gel activity assay. Analysis of chitin hydrolysis using HPLC/ESI-MS confirmed the endo characteristics of the enzyme.     

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.     

Molecular analysis of the gene encoding a new chitinase from the marine psychrophilic bacterium <Emphasis Type="Italic">Moritella marina</Emphasis> and biochemical characterization of the recombinant enzyme

The marine psychrophilic bacterium Moritella marina, isolated from a sample raised from a depth of 1,200 m in the northern Pacific Ocean, secretes several chitinases in response to chitin induction. A gene coding for an extracellular chitinolytic enzyme was cloned and its nucleotide sequence was determined. The chitinase gene consists of an open reading frame of 1,650 nucleotides and encodes a protein of 550 amino acids with a calculated molecular weight of 60.788 kDa, named MmChi60. MmChi60 has a modular structure consisting of a glycosyl-hydrolase family 18 N-terminal catalytic region as well as a C-terminal chitin-binding domain (ChBD). The new chitinase was purified to homogeneity from the intracellular fraction of Escherichia coli. The optimum pH and temperature of the recombinant MmChi60 were 5.0 and 28 degrees C, respectively. The mode of action of the new enzyme on N-acetylchitooligomers, chitin polymers, and other substrates was examined, and MmChi60 was classified as an endochitinase. Thermal unfolding of MmChi60 was studied using differential scanning microcalorimetry and revealed that the protein unfolds reversibly at 65 degrees C. On the basis of the crystal structure of the chitinase C of Streptomyces griseus, a homology-based 3-D model of the ChBD of the MmChi60 was calculated.     

Chitinase from Autographa californica multiple nucleopolyhedrovirus: rapid purification from Sf-9 medium and mode of action

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) chitinase is involved in the final liquefaction of infected host larvae. We purified the chitinase rapidly to homogeneity from Sf-9 cells infected with AcMNPV by a simple procedure using a pepstatin-aminohexyl-Sepharose column. In past studies, a recombinant AcMNPV chitinase was found to exhibit both exo- and endo-chitinase activities by analysis using artificial substrates with a fluorescent probe. In this study, however, we obtained more accurate information on the mode of action of the chitinase by HPLC analysis of the enzymatic products using natural oligosaccharide and polysaccharide substrates. The AcMNPV chitinase hydrolyzed the second β-1,4 glycosidic linkage from the non-reducing end of the chitin oligosaccharide substrates [(GlcNAc)(n), n=4, 5, and 6], producing the β-anomer of (GlcNAc)?. The mode of action was similar to that of Serratia marcescens chitinase A (SmChiA), the amino acid sequence of which is 60.5% homologous to that of the AcMNPV enzyme. The enzyme also hydrolyzed solid β-chitin, producing only (GlcNAc)?. The AcMNPV chitinase processively hydrolyzes solid β-chitin in a manner similar to SmChiA. The processive mechanism of the enzyme appears to be advantageous in liquefaction of infected host larvae.     

Purification and characterization of a novel chitinase from the entomopathogenic fungus, Metarhizium anisopliae

A novel chitinase was detected in extracellular culture fluids of the entomopathogenic fungus Metarhizium anisopliae (ATCC 20500) grown in liquid medium containing chitin as a sole carbon source. A chitinase was purified to near homogeneity from culture broth of M. anisopliae by DEAE-Sephacel, CM-Sepharose CL-6B ion-exchange chromatography, and gel filtration with Superose 12HR. The molecular mass of the enzyme determined by SDS-polyacrylamide gel electrophoresis was approximately 60 kDa and the optimum pH of the enzyme was 5.0. This molecular mass is different from values of 33, 43.5, and 45 kDa for endochitinases and 110 kDa for an exochitinase (N-acetylglucosaminidase) from M. anisopliae ME-1 published previously. In addition, N-terminal sequences of 60-kDa chitinase are different from those of 43.4- and 45-kDa endochitinases. The purified enzyme showed high chitinolytic activity against colloidal, crystalline chitin of crab shells as well as against p-nitrophenyl-beta-d-N-acetylglucosamide, p-nitrophenyl-beta-d-N, N'-diacetylchitobiose, and p-nitrophenyl-N, N'-N"-triacetylchitotriose, indicating that this enzyme has both endo- and exochitinase activity.     

Expression of chitinase in Adenanthera pavonina seedlings

Chitinases (EC 3.2.1.14) are hydrolytic enzymes found in different organisms. In plants, they have been described in different tissues and organs, including seeds. This study was triggered by the isolation of a 30-kDa thermostable chitinase from Adenanthera pavonina L. seeds. The enzyme was submitted to N-terminal amino acid sequencing, and the analysis revealed a high degree of homology with class III chitinases. Bidimensional electrophoresis of the 30-kDa band showed the presence of three isoforms with pIs of 5.2, 5.5 and 5.8. A chitinase was also found in exudates released from the same seeds, which was seen to be immunorelated to the above 30-kDa protein. It was also submitted to N-terminal amino acid sequencing and seen as highly homologous to class III chitinases. In addition, the expression of chitinases during A. pavonina L. seed germination and seedling development was investigated. Seeds were allowed to germinate in the absence of light for approximately 5 days and were grown, for different times, in the absence or presence of light. After each seedling developmental time, samples of exudates, roots and cotyledonary leaves were collected and submitted to protein extraction. The presence of proteins immunorelated to the 30-kDa chitinase was detected in all analyzed samples. Further analyses showed that light significantly interfered with the chitinase expression in some organs. The tissue and subcellular chitinase location in seedling roots was also investigated, and it was majorly localized in the cell wall and in the intercellular spaces of the root hair zone.     

Family 19 chitinase from Aeromonas sp. No.10S-24: role of chitin-binding domain in the enzymatic activity

Family 19 chitinase from Aeromonas sp. No.10S-24 (72.6 kDa) is composed of two chitin-binding domains (ChBDs), two proline- and threonine-rich (PT-rich) linkers, and a catalytic domain. The purified enzyme was labile in a standard buffer condition and spontaneously degraded into a 46-kDa fragment upon storage at 4 degrees C. The N-terminal sequence of the 46-kDa fragment was found to correspond to the sequence of the C-terminal region of the second PT-rich linker, indicating that the 46-kDa fragment is produced by truncation of the two ChBDs and the two PT-rich linkers from the mature protein, and consists only of the catalytic domain. The hydrolytic activities toward insoluble and soluble substrates were significantly reduced by the truncation of two ChBDs. In addition, antifungal activity determined from the digestion rate of haustoria of powdery mildew was reduced by the ChBD truncation. Although the profile of the time-course of N-acetylglucosamine hexasaccharide [(GlcNAc)6] degradation catalyzed by the ChBD-truncated enzyme was similar to that of the mature enzyme protein, the specific activity of the ChBD-truncated enzyme determined from the rate of hexasaccharide degradation was lower than that of the mature enzyme. The two CBDs appear to be responsible for facilitating the hydrolytic reaction. The sugar residue affinities (binding free energy changes) at the individual subsites, (-2) (-1) (+1) (+2) (+3) (+4), were estimated by modeling the hexasaccharide hydrolysis by the mature and ChBD-truncated enzymes. The truncation of ChBDs was found to strongly affect the affinity at the (-1) site. This situation seems to result in the lower enzymatic activity of the ChBD-truncated enzyme toward the chitinous substrates.     

Appearance of chitinolytic enzymes in integument of Bombyx mori during the larval-pupal transformation. Evidence for zymogenic forms

The appearance of chitinolytic enzymes, chitinase and β-N-acetylglucosaminidase, involved in ecdysis of the silkworm, Bombyx mori, was investigated using integuments prepared from fifth instar larvae during and after spinning behavior just before the larval-pupal transformation. β-N-Acetylglucosaminidase activity appeared a day after the beginning of spinning (SP1) and gradually increased for 2 more days (SP3), while chitinase activity appeared later at the SP3 stage (1 day before the ecdysis). It was shown by immunoblotting that the changes in activity were due to increases in the amounts of enzymes present. A probable zymogenic form of chitinase, whose molecular weight was about 215 kDa, was detected during spinning period by immunoblotting using anti-65-kDa chitinase antibody. The zymogen was observed 2 days before the appearance of enzyme activity. High molecular proteins (120–190 kDa) related to β-N-acetylglucosaminidase were also observed throughout the spinning period by immunoblotting, but this appearance pattern was different from that of chitinase. The results support, at least in the case of chitinase the hypothesis, that insect chitinolytic enzymes are synthesized as inactive precursors which are activated by limited proteolysis.