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.     

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.     

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.     

Characterization of chitinase from Shewanella inventionis HE3 with bio-insecticidal effect against granary weevil,Sitophilus granarius Linnaeus (Coleoptera: Curculionidae)

A 40 kDa chitinase from Shewanella inventionis HE3 was purified (ChiA-Si40) and characterized. Using fermentor with an optimized medium for 48 h at 37 °C, enzyme activity was enhanced by 10-times compared to those using shaking-flask-culture. Purified chitinase is a homogenous monomer with molecular mass of 40 kDa. Its N-terminal residues revealed significant identity with glycoside hydrolase family 18 (GH18) chitinases from Gammaproteobacteria. Using colloidal chitin as a substrate, its finest activity was accomplished at pH 4 and a temperature of 70 °C. Its catalytic efficiency (k_cat/K_m) was superior to that of some bacterial GH18 chitinases and commercial enzyme, Chitodextrinase®. For scale-up and with regards to the improvement of ChiA-Si40 with PEG 6000 storage stability (6 months), the atomizing process was more pronounced than that of lyophilizing. Bio-assay of ChiA-Si40 against grain weevil Sitophilus granarius, indicates that it had an efficient insecticidal effect. About 10–100 % mortality rates were obtained 1-h after insect came in contact with ChiA-Si40. Histological study clearly demonstrated that luxury larval mid-gut, peritrophic-membrane, and epithelial-cells have been affected considerably after ChiA-Si40 treatment. These properties make ChiA-Si40 a potential bio-insecticidal agent for the biological control of S. granarius that is popular among insect pests of stored grains in Algeria.     

Directed evolution of a Bacillus chitinase

Chitinases have potential in various industrial applications including bioconversion of chitin waste from crustacean shells into chito-oligosaccharide-based value-added products. For industrial applications, obtaining suitable chitinases for efficient bioconversion processes will be beneficial. In this study, we established a straightforward directed evolution method for creating chitinase variants with improved properties. A library of mutant chitinases was constructed by error-prone PCR and DNA shuffling of two highly similar (99% identical) chitinase genes from Bacillus licheniformis. Activity screening was done in two steps: first, activity towards colloidal chitin was screened for on culturing plates (halo formation). This was followed by screening activity towards the chitotriose analogue p-nitrophenyl-β-1,4-N, N'-diacetyl-chitobiose at various pH in microtiter plates. From a medium-throughput screening (517 colonies), we were able to isolate one mutant that demonstrated improved catalytic activity. When using p-nitrophenyl-β-1,4-N, N'-diacetyl-chitobiose as substrate, the overall catalytic efficiency, k_cat/K_m of the improved chitinase was 2.7- and 2.3-fold higher than the average k_cat/K_m of wild types at pH 3.0 and 6.0, respectively. The mutant contained four residues that did not occur in either of the wild types. The approach presented here can easily be adopted for directed evolution of suitable chitinases for various applications.     

Characterization of an intradiol dioxygenase involved in the biodegradation of the chlorophenoxy herbicides 2,4-D and 2,4,5-T

Hydroxyquinol 1,2-dioxygenase, an intradiol dioxygenase, which catalyzes the cleaving of the aromatic ring of hydroxyquinol, a key intermediate of 2,4-D and 2,4,5-T degradation, was purified from Nocardioides simplex 3E cells grown on 2,4-D as the sole carbon source. This enzyme exhibits a highly restricted substrate specificity and is able to cleave hydroxyquinol (K_m for hydroxyquinol as a substrate was 1.2 μM, V_max 55 U/mg, K_cat 57 s⁻¹ and K_cat/K_m 47.5 μM s⁻¹), 6-chloro- and 5-chlorohydroxyquinol. Different substituted catechols and hydroquinones are not substrates for this enzyme. This enzyme appears to be a dimer with two identical 37-kDa subunits. Protein and iron analyses indicate an iron stoichiometry of 1 iron/65 kDa homodimer, α₂ Fe. Both the electronic absorption spectrum which shows a broad absorption band with a maximum at 450 nm and the electron paramagnetic resonance spectra are consistent with a high-spin iron(III) ion in a rhombic environment typical of the active site of intradiol cleaving enzymes.     

Three Extracellular Chitinases in Suspension-cultured Rice Cells Elicited by N-Acetylchitooligosaccharides

In rice suspension culture, a large part (about 90% of total activity in the culture) of the chitinase activity was found in the medium. Two extracellular chitinases (which we named RCH-A and -B) were separated from the cell suspension by DEAE-cellulofìne column chromatography. When cells were treated with N-acetylchitooligosaccharides (chitin oligosaccharides) for 3 days, extracellular chitinase activity increased about 3-fold over the control culture. After the treatment, another extracellular chitinase (named RCH-C) appeared in addition to increases in the levels of RCH-A and -B. Partial amino acid sequences of these enzymes indicated that RCH-A (33.5 kDa) and -B (34kDa) were class Ib chitinases but RCH-C (27kDa) was a class III chitinase. RCH-A and -B were capable of actively degrading water-insoluble chitin with high affinities, while RCH-C had less affinity for the substrate. However, when a water-soluble chitin derivative, 6–O-hydroxyethylchitin (glycolchitin) was used, RCH-C as well as RCH-A and -B degraded actively with a high affinity. A synergistic effect was observed when these three chitinases acted simultaneously in the hydrolysis of chitin.     

Characterization of Chitinase Produced by the Alkaliphilic <Emphasis Type="Italic">Bacillus mannanilyticus</Emphasis> IB-OR17 B1 Strain

The paper reports on the isolation of an extracellular chitinase produced by the alkaliphilic Bacillus mannanilyticus IB-OR17 B1 strain grown in media containing crab shell and bee chitin at a pH of 8–11. The enzyme was 860-fold purified by ultrafiltration and chitin sorption. The molecular weight of the purified chitinase was shown by denaturing electrophoresis to be 56 kDa. The enzyme showed maximum activity at a pH of 7.5–8.0 and 65°C and was stable within a pH range of 3.5–10.5 and temperature range of 75–85°C. With colloidal chitin as substrate, the kinetic characteristics of the chitinase were determined as follows: K_M ~ 1.32 mg/mL and V_max ~ 5.05 μM min^–1. N-acetyl-D-glucosamine and its dimer were the main products of enzymatic chitin cleavage, while the trisaccharide was detected just in minor quantities. The chitinase actively hydrolyzed p-nitrophenyl-GlcNAc₂ according to the exo-mechanism of substrate hydrolysis characteristic of chitobiosidases.     

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.     

Comparative Characterization of Chitinases from Silkworm (<Emphasis Type="Italic">Bombyx mori</Emphasis>) and Bollworm (<Emphasis Type="Italic">Helicoverpa armigera</Emphasis>)

Chitinases play an important role in the degradation of the cuticular chitin during the process of ecdysis. In this study, we compared the chitinases of two insect species, Bombyx mori (silkworm) and Helicoverpa armigera (bollworm), to assess the relation between characteristics and chitinase patterns. Differences between two chitinases were observed after purification using ammonium sulfate precipitation, affinity chromatography, and sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) assay. Although the specific activities of the purified enzymes were different, the purification yields were similar. One band of 88 kDa was observed for B. mori, and the other band of 75 kDa was detected for H. armigera. When a range of properties was tested, it was found that the optimum temperatures of B. mori and H. armigera chitinases were 45 and 50°C, respectively; the optimum pH value was 6.0 for both chitinases. Mn²⁺ played catalytic role while Cu²⁺ and SDS strongly inhibited activities of both enzymes. Between two chitinases, differences in K _M were also observed. K _M of chitinase from silkworm and bollworm was found to be 22.3 and 41.0 μmol/l, respectively. Both the chitinases significantly inhibited the spore germination of two fungal species, Saccharomyces cerevisiae and Penicillium.     

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.     

Purification and characterization of a chitinase from Serratia proteamaculans

A chitinase gene from Serratia proteamaculans 18A1 was cloned, sequenced, and expressed in Escherichia coli M15. Recombinant enzyme (ChiA) was purified by Ni-NTA affinity column chromatography. The ChiA gene contains an open reading frame (ORF), encoding an endochitinase with a deduced molecular weight 60 kDa and predicted isoelectric point of 6.35. Comparison of ChiA with other chitinases revealed a modular structure containing an N-terminal PKD-domain, a family 18 catalytic domain and a C-terminal putative chitin-binding domain. Turn over rate (K _cat) of the enzyme was determined using colloidal chitin (49.71 ± 1.15 S^?1) and crystalline β-chitin (17.20 ± 0.83 S^?1) as substrates. The purified enzyme was active over a broad range of pH (pH 4.5–9.0) and temperature (4–70°C) with a peak activity at pH 5.5 and 55°C. However, enzyme activity was found to be stable up to 45°C for longer incubation periods. Purified enzyme was shown to inhibit fungal spore germination and hyphal growth of pathogenic fungi Fusarium oxysporum and Aspergillus niger.     

Cloning and high-level production of a chitinase from <Emphasis Type="Italic">Chromobacterium</Emphasis> sp. and the role of conserved or nonconserved residues on its catalytic activity

A gene encoding an alkaline (pI of 8.67) chitinase was cloned and sequenced from Chromobacterium sp. strain C-61. The gene was composed of 1,611 nucleotides and encoded a signal sequence of 26 N-terminal amino acids and a mature protein of 510 amino acids. Two chitinases of 54 and 52 kDa from both recombinant Escherichia coli and C-61 were detected on SDS-PAGE. Maximum chitinase activity was obtained in the culture supernatant of recombinant E. coli when cultivated in TB medium for 6 days at 37°C and was about fourfold higher than that from C-61. Chi54 from the culture supernatants could be purified by a single step based on isoelectric point. The purified Chi54 had about twofold higher binding affinity to chitin than to cellulose. The chi54 encoded a protein that included a type 3 chitin-binding domain belonging to group A and a family 18 catalytic domain belonging to subfamily A. In the catalytic domain, mutation of perfectly conserved residues and highly conserved residues resulted in loss of nearly all activity, while mutation of nonconserved residues resulted in enzymes that retained activity. In this process, a mutant (T218S) was obtained that had about 133% of the activity of the wild type, based on comparison of K _cat values.     

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.     

Gene Cloning, Purification, and Characterization of a Phosphodiesterase from Delftia acidovorans

A novel phosphodiesterase (PdeA) was purified from Delftia acidovorans, the gene encoding the enzyme was cloned and expressed in Escherichia coli, and the recombinant enzyme was purified to apparent homogeneity and characterized. PdeA is an 85-kDa trimer that exhibits maximal activity at 65°C and pH 10 even though it was isolated from a mesophilic bacterium. Although PdeA exhibited both mono- and diesterase activity, it was most active on the phosphodiester bis(p-nitrophenyl)phosphate with a K_m of 2.9 ± 0.1 mM and a k_cat of 879 ± 73 min⁻¹. The enzyme showed sequence similarity to cyclic AMP (cAMP) phosphodiesterase and cyclic nucleotide phosphodiesterases and exhibited activity on cAMP in vivo when the gene was expressed in E. coli. The IS1071 transposon insertion sequence was found downstream of pdeA.     

Purification, characterization, and antifungal activity of chitinases from pineapple (Ananas comosus) leaf

Three chitinases, designated pineapple leaf chitinase (PL Chi)-A, -B, and -C were purified from the leaves of pineapple (Ananas comosus) using chitin affinity column chromatography followed by several column chromatographies. PL Chi-A is a class III chitinase having a molecular mass of 25 kDa and an isoelectric point of 4.4. PL Chi-B and -C are class I chitinases having molecular masses of 33 kDa and 39 kDa and isoelectric points of 7.9 and 4.6 respectively. PL Chi-C is a glycoprotein and the others are simple proteins. The optimum pHs of PL Chi-A, -B, and -C toward glycolchitin are pH 3, 4, and 9 respectively. The chitin-binding ability of PL Chi-C is higher than that of PL Chi-B, and PL Chi-A has lower chitin-binding ability than the others. At low ionic strength, PL Chi-B exhibits strong antifungal activity toward Trichoderma viride but the others do not. At high ionic strength, PL Chi-B and -C exhibit strong and weak antifungal activity respectively. PL Chi-A does not have antifungal activity.     

Purification,characterization, and molecular cloning of chitinases from the stomach of the threeline grunt Parapristipoma trilineatum

Two chitinase isozymes, PtChiA and PtChiB, were purified from the stomach of the threeline grunt, Parapristipoma trilineatum. The molecular masses of PtChiA and PtChiB were estimated to be 50 and 60 kDa by SDS-PAGE, respectively. Both chitinases were stable at pH 3.0–6.0 (acidic) and showed the optimum pH toward both short and long substrates in the acidic region (pH 2.5–5.0). PtChiA and PtChiB preferentially degraded the second and third glycosidic bonds from the non-reducing end of N-acetylchitooligosaccharides, respectively. PtChiA and PtChiB exhibited wide substrate specificities toward crystalline chitin. Moreover, 2 cDNAs encoding PtChiA and PtChiB, PtChi-1 and PtChi-2, respectively, were cloned. The deduced amino acid sequences of both chitinase cDNAs comprised N-terminal signal peptides, glycoside hydrolase 18 catalytic domains, linker regions, and C-terminal chitin-binding domains. Phylogenetic tree analysis of vertebrate chitinases revealed that fish stomach chitinases including PtChi-1 and PtChi-2 form unique chitinase groups, acidic fish chitinase-1 (AFCase-1) and acidic fish chitinase-2 (AFCase-2), which differ from the acidic mammalian chitinase (AMCase) group. The present results suggest that fish have a chitin-degrading enzymatic system in which 2 different chitinases, AFCase-1 and AFCase-2, with different degradation patterns are expressed in the stomach.     

Cloning, purification, and characterization of galactomannan-degrading enzymes from Myceliophthora thermophila

Genes of β-mannosidase 97 kDa, GH family 2 (bMann9), β-mannanase 48 kDa, GH family 5 (bMan2), and α-galactosidase 60 kDa, GH family 27 (aGal1) encoding galactomannan-degrading glycoside hydrolases of Myceliophthora thermophila C1 were successfully cloned, and the recombinant enzymes were purified to homogeneity and characterized. bMann9 displays only exo-mannosidase activity, the K _m and k _cat values are 0.4 mM and 15 sec^?1 for p-nitrophenyl-β-D-mannopyranoside, and the optimal pH and temperature are 5.3 and 40°C, respectively. bMann2 is active towards galac-tomannans (GM) of various structures. The K _m and k _cat values are 1.3 mg/ml and 67 sec^?1 for GM carob, and the optimal pH and temperature are 5.2 and 69°C, respectively. aGal1 is active towards p-nitrophenyl-α-D-galactopyranoside (PNPG) as well as GM of various structures. The K _m and k _cat values are 0.08 mM and 35 sec^?1 for PNPG, and the optimal pH and temperature are 5.0 and 60°C, respectively.     

Comparison of chitinase isozymes from yam tuber--enzymatic factor controlling the lytic activity of chitinases

To evaluate the anti-pathogen activity of chitinases, we developed a new method for measuring the lytic activity, and investigated the correlation of the lytic activity with the enzymatic properties by using four chitinase isozymes, Chitinases E, F, H1 and G, which had been purified from yam tubers by column chromatography. Chitinases E, F and H1 had high lytic activity against the plant pathogen, Fusarium oxysporum, but Chitinase G did not. Chitinase E, which is the family 19 chitinase, was similar to Chitinases F and G in its antigenecity, but not to Chitinase H1 or H2. Chitinases H1 and H2 were recognized by the anti-Bombyx mori chitinase antibody, suggesting that Chitinases H1 and H2 are family 18 chitinases like B. mori chitinases. Chitinases E, F and H1 had two optimum pH ranges of 3-4 and 7.5-9 toward glycolchitin, but Chitinase G had only one optimum pH value of 5. Chitinases E, F and H1 had higher affinity to the polymer substrate, glycolchitin, than Chitinase G. These results suggest that the lytic activity of plant chitinases may be related to the chitin affinity and probably to the characteristic optimum pH value, or two values, but not related to its classification. The correlation of the lytic activity of a chitinase isozyme with its elicitor specificity is also discussed.     

Promising properties of a formate dehydrogenase from a methanol-assimilating yeast Ogataea parapolymorpha DL-1 in His-tagged form

The cDNA gene coding for formate dehydrogenase (FDH) from Ogataea parapolymorpha DL-1 was cloned and expressed in Escherichia coli. The recombinant enzyme was purified by nickel affinity chromatography and was characterized as a homodimer composed of two identical subunits with approximately 40 kDa in each monomer. The enzyme showed wide pH optimum of catalytic activity from pH 6.0 to 7.0. It had relatively high optimum temperature at 65 °C and retained 93, 88, 83, and 71 % of its initial activity after 4 h of exposure at 40, 50, 55, and 60 °C, respectively, suggesting that this enzyme had promising thermal stability. In addition, the enzyme was characterized to have significant tolerance ability to organic solvents such as dimethyl sulfoxide, n-butanol, and n-hexane. The Michaelis–Menten constant (K _m), turnover number (k _cat), and catalytic efficiency (k _cat/K _m) values of the enzyme for the substrate sodium formate were estimated to be 0.82 mM, 2.32 s^?1, and 2.83 mM^?1 s^?1, respectively. The K _m for NAD⁺ was 83 μM. Due to its wide pH optimum, promising thermostability, and high organic solvent tolerance, O. parapolymorpha FDH may be a good NADH regeneration catalyst candidate.