Chitinase of Bacillus licheniformis from oyster shell as a probe to detect chitin in marine shells

Bacillus licheniformis CBFOS-03 is a chitinase producing bacteria isolated from oyster (Crassostrea gigas) shell waste. We have cloned and expressed the chi18B gene of B. licheniformis CBFOS-03, which encodes a glycohydrolase family 18 chitinase (GH18). Chi18B is a predicted 598 amino acid protein that consists of a catalytic domain (GH18), a fibronectin type III domain (Fn3), and a chitin binding domain (CBD). Purified Chi18B showed optimum chitinase activity at pH 9 and 55 °C, and activity was stimulated with 25 mM Mn²⁺. In kinetic analysis, Chi18B showed Km values of 9.07?±?0.65 μM and 129.27?±?0.38 μM with the substrates 4-methylumbelliferyl-N-N′-diacetylchitobiose and α-chitin, respectively. Studies of C-terminal deletion constructs revealed that the GH18 domain with one amino acid in C-terminal region was sufficient for chitinase activity; however, fusions of full length and CBD-deleted constructs to green florescent protein (GFP) and yellow florescent protein (YFP) suggest that the C-terminus is supposedly important in binding to shell powder. Full length Chi18B with GFP showed green fluorescence with oyster shell powder, but GH18+Fn3 with GFP did not. Similarly, full length Chi18B with YFP showed yellow fluorescence with clam (Chamelea gallina) shell and disk abalone (Haliotis discus) shell powder, but GH18+Fn3 with YFP construct did not. So, the CBD domain of Chi18B appears to play an important role in binding of oyster and other marine shells. It is likely to be used as a probe to identify the presence of chitin in marine shells like oyster shell, clam shell, and disk abalone shell using fusions of Chi18B with fluorescent proteins.     

Cloning and Expression of a Novel Chitinase chi58 from Chaetomium cupreum in Pichia pastoris

A gene encoding a novel chitinase chi58 was cloned from the fungus Chaetomium cupreum by using inverse PCR. The DNA sequence of chi58 contains a 1,602 bp open reading frame and two introns that are 52 and 201 bp in length. Regarding our in silico analysis, chi58 is a modular enzyme composed of a family-18 catalytic domain, which is responsible for chitinase activity, and a chitin-binding domain containing several cysteines. Apparently, the function of these domains is to anchor the enzyme tightly onto the large insoluble polymeric substrate. Chi58 has a pI of 4.47 and a deduced molecular mass of 58 kDa. The optimal pH and temperature conditions were determined to be 5.8 and 45°C, respectively, when colloidal chitin was used as the substrate. SDS-PAGE and zymogram analyses indicated the presence of a single active chitinase. Cells with pPIC9K-chi58 produced an extracellular chitinase that had an activity of 39 U/ml protein. Metal ions such as Ba²⁺, Mg²⁺, K⁺, Cu²⁺, Fe³⁺, Zn²⁺, and Co²⁺ also influenced the activity of the recombinant enzyme.     

Biochemical characterization of chitinase 2 expressed during the autolytic phase of the inky cap, Coprinellus congregatus

Fungal cell walls consist of various glucans and chitin. The inky cap, Coprinellus congregatus, produces mushrooms at 25°C in a regime of 15 h light/9 h dark, and then the mushroom is autolyzed rapidly to generate black liquid droplets in which no cell walls are detected by microscopy. Chitinase cDNA from the mature mushroom tissues of C. congregatus, which consisted of 1,622 nucleotides (chi2), was successfully cloned using the rapid amplification of cDNA ends polymerase chain reaction technique. The deduced 498 amino acid sequence of Chi2 had a conserved catalytic domain as in other fungal chitinase family 18 enzymes. The Chi2 enzyme was purified from the Pichia pastoris expression system, and its estimated molecular weight was 68 kDa. The optimum pH and temperature of Chi2 was pH 4.0 and 35°C, respectively when 4-nitrophenyl N,N′-diacetyl-β-D-chitobioside was used as the substrate. The K _m value and V _max for the substrate A, 4-nitrophenyl N,N′-diacetyl-β-D-chitobioside, was 0.175 mM and 0.16 OD min^?1unit^?1, respectively.     

Purification and characterization of chitinases from <Emphasis Type="Italic">Paecilomyces</Emphasis><Emphasis Type="Italic">variotii</Emphasis> DG-3 parasitizing on <Emphasis Type="Italic">Meloidogyne incognita</Emphasis> eggs

Two extracellular chitinases were purified from Paecilomyces variotii DG-3, a chitinase producer and a nematode egg-parasitic fungus, to homogeneity by DEAE Sephadex A-50 and Sephadex G-100 chromatography. The purified enzymes were a monomer with an apparent molecular mass of 32 kDa (Chi32) and 46 kDa (Chi46), respectively, and showed chitinase activity bands with 0.01% glycol chitin as a substrate after SDS-PAGE. The first 20 and 15 N-terminal amino acid sequences of Chi32 and Chi46 were determined to be Asp-Pro-Typ-Gln-Thr-Asn-Val-Val-Tyr-Thr-Gly-Gln-Asp-Phe-Val-Ser-Pro-Asp-Leu-Phe and Asp-Ala-X-X-Tyr-Arg-Ser-Val-Ala-Tyr-Phe-Val-Asn-Trp-Ala, respectively. Optimal temperature and pH of the Chi32 and Chi46 were found to be both 60°C, and 2.5 and 3.0, respectively. Chi32 was almost inhibited by metal ions Ag⁺ and Hg²⁺ while Chi46 by Hg²⁺ and Pb²⁺ at a 10 mM concentration but both enzymes were enhanced by 1 mM concentration of Co²⁺. On analyzing the hydrolyzates of chitin oligomers [(GlcNAc) _n , n = 2–6)], it was considered that Chi32 degraded chitin oligomers as an exo-type chitinase while Chi46 as an endo-type chitinase.     

Molecular Docking and Site-directed Mutagenesis of a Bacillus thuringiensis Chitinase to Improve Chitinolytic,Synergistic Lepidopteran-larvicidal and Nematicidal Activities

Bacterial chitinases are useful in the biocontrol of agriculturally important pests and fungal pathogens. However, the utility of naturally occurring bacterial chitinases is often limited by their low enzyme activity. In this study, we constructed mutants of a Bacillus thuringiensis chitinase with enhanced activity based on homology modeling, molecular docking, and the site-directed mutagenesis of target residues to modify spatial positions, steric hindrances, or hydrophilicity/hydrophobicity. We first identified a gene from B. thuringiensis YBT-9602 that encodes a chitinase (Chi9602) belonging to glycosyl hydrolase family 18 with conserved substrate-binding and substrate-catalytic motifs. We constructed a structural model of a truncated version of Chi9602 (Chi9602_35-459) containing the substrate-binding domain using the homologous 1ITX protein of Bacillus circulans as the template. We performed molecular docking analysis of Chi9602_35-459 using di-N-acetyl-D-glucosamine as the ligand. We then selected 10 residues of interest from the docking area for the site-directed mutagenesis experiments and expression in Escherichia coli. Assays of the chitinolytic activity of the purified chitinases revealed that the three mutants exhibited increased chitinolytic activity. The ChiW50A mutant exhibited a greater than 60 % increase in chitinolytic activity, with similar pH, temperature and metal ion requirements, compared to wild-type Chi9602. Furthermore, ChiW50A exhibited pest-controlling activity and antifungal activity. Remarkable synergistic effects of this mutant with B. thuringiensis spore-crystal preparations against Helicoverpa armigera and Caenorhabditis elegans larvae and obvious activity against several plant-pathogenic fungi were observed.     

Purification and partial characterization of intact and truncated chitinase from <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> HZP7 expressed in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Objective

To ascertain the effect of chitin-binding domain (ChBD) and fibronectin type III domain (FN3) on the characterization of the intact chitinase from Bacillus thuringiensis.

Results

An intact chitinase gene (chi74) from B. thuringiensis HZP7 and its truncated genes (chi54, chi63 and chi66) were expressed in Escherichia coli BL21. The expression products were analyzed after purification. All chitinases were active from pH 4–7.5 and from 20 to 80 °C with identical optimal: pH 5.5 and 60 °C. The activity of colloid chitin degradation for Chi74 was the highest, followed by Chi66, Chi63 and Chi54. Ag⁺ reduced the activity of Chi74, Chi54, Chi63 and Chi66, but Mg²⁺ enhanced them. The effect of Ag⁺ and Mg²⁺ was more significant on the activity of Chi54 than on the activities of Chi63, Chi66 and Chi74.

Conclusion

ChBD_Chi74 and FN3_Chi74 domains play a role in exerting enzymatic activity and can improve the stability of chitinase.

     

Production of Antifungal Chitinase by Aspergillus niger LOCK 62 and Its Potential Role in the Biological Control

Aspergillus niger LOCK 62 produces an antifungal chitinase. Different sources of chitin in the medium were used to test the production of the chitinase. Chitinase production was most effective when colloidal chitin and shrimp shell were used as substrates. The optimum incubation period for chitinase production by Aspergillus niger LOCK 62 was 6?days. The chitinase was purified from the culture medium by fractionation with ammonium sulfate and affinity chromatography. The molecular mass of the purified enzyme was 43?kDa. The highest activity was obtained at 40?°C for both crude and purified enzymes. The crude chitinase activity was stable during 180?min incubation at 40?°C, but purified chitinase lost about 25?% of its activity under these conditions. Optimal pH for chitinase activity was pH 6–6.5. The activity of crude and purified enzyme was stabilized by Mg²⁺ and Ca²⁺ ions, but inhibited by Hg²⁺ and Pb²⁺ ions. Chitinase isolated from Aspergillus niger LOCK 62 inhibited the growth of the fungal phytopathogens: Fusarium culmorum, Fusarium solani and Rhizoctonia solani. The growth of Botrytis cinerea, Alternaria alternata, and Fusarium oxysporum was not affected.     

Chi18A, the Endochitinase in the Cellulosome of the Thermophilic, Cellulolytic Bacterium Clostridium thermocellum

The chitinase gene chiA was identified on the Clostridium thermocellum genome downstream of the endoglucanase gene celA. It contains a catalytic module of glycosyl hydrolase family 18 and a cellulosomal dockerin module. Chi18A hydrolyzes aryl-acetyl-chito-oligosaccharides preferentially. In denaturing electrophoresis of purified cellulosomes, a single chitinase activity band was identified in zymograms and Western blots, indicating that Chi18A is the only chitinase in the cellulosome.     

Characteristics of cold-adaptive endochitinase from Antarctic bacterium Sanguibacter antarcticus KOPRI 21702

The psychrotrophic Sanguibacter antarcticus KOPRI 21702^T, isolated from Antarctic seawater, produced a cold-adapted chitinolytic enzyme that is a new 55 kDa family 18 chitinase (Chi21702). Chi21702 exhibited high activities toward pNP-(GlcNAc)₂ and pNP-(GlcNAc)₃ with no activity for pNP-GlcNAc, indicating that it prefers chitin chains longer than dimers, just as endochitinases do. A mixture of GlcNAc and GlcNAc₂ was produced as a main product by Chi21702 activity from chitin oligosaccharides and swollen chitin, while less GlcNAc₃ was produced. These results show that Chi21702 has an endochitinase activity, randomly hydrolyzing chitin at internal sites. Chi21702 displayed chitinase activity at 0–40 °C (optimal temperature of 37 °C), maintained its activity at pH 4–11 (optimal pH of 7.6). Interestingly, Chi21702 exhibited relative activities of 40% and 60% at 0 and 10 °C, respectively, in comparison to 100% at 37 °C, which is higher than those of the previously characterized, cold-adapted, chitinases from bacterial strains.     

A highly thermoactive and salt-tolerant α-amylase isolated from a pilot-plant biogas reactor

Aiming at the isolation of novel enzymes from previously uncultured thermophilic microorganisms, a metagenome library was constructed from DNA isolated from a pilot-plant biogas reactor operating at 55 °C. The library was screened for starch-degrading enzymes, and one active clone was found. An open reading frame of 1,461 bp encoding an α-amylase from an uncultured organism was identified. The amy13A gene was cloned in Escherichia coli, resulting in high-level expression of the recombinant amylase. The novel enzyme Amy13A showed the highest sequence identity (75 %) to α-amylases from Petrotoga mobilis and Halothermothrix orenii. Amy13A is highly thermoactive, exhibiting optimal activity at 80 °C, and it is also highly salt-tolerant, being active in 25 % (w/v) NaCl. Amy13A is one of the few enzymes that tolerate high concentrations of salt and elevated temperatures, making it a potential candidate for starch processing under extreme conditions.     

Protein Engineering of Chit42 Towards Improvement of Chitinase and Antifungal Activities

The antagonism of Trichoderma strains usually correlates with the secretion of fungal cell wall degrading enzymes such as chitinases. Chitinase Chit42 is believed to play an important role in the biocontrol activity of Trichoderma strains as a biocontrol agent against phytopathogenic fungi. Chit42 lacks a chitin-binding domain (ChBD) which is involved in its binding activity to insoluble chitin. In this study, a chimeric chitinase with improved enzyme activity was produced by fusing a ChBD from T. atroviride chitinase 18–10 to Chit42. The improved chitinase containing a ChBD displayed a 1.7-fold higher specific activity than chit42. This increase suggests that the ChBD provides a strong binding capacity to insoluble chitin. Moreover, Chit42-ChBD transformants showed higher antifungal activity towards seven phytopathogenic fungal species.     

Degradation of fungal cell walls taking into consideration the polysaccharide composition

Differences in polysaccharide composition of various fungal cell walls were indicated by their susceptibility to enzymatic digestion. This information was used to optimize the enzymatic extraction of intracellular enzymes or the preparation of fungal protoplasts in high yield. Bacterial glucanase and chitinase specially purified were used for this study. Mycelium of Aspergillus niger grown on uric acid was treated with mixtures of glucanase and chitinase. Cell wall breakdown products were analysed and the ratio of chitin to glucan was estimated to be 1:1.4. A. niger protoplast formation was optimized using this information. When the mixture of chitinase to glucanase was 1:1.4, similar to the fungal cell wall composition, a 95% yield of protoplasts was obtained after 30 min and their mean size was 7 μm. However, a ratio of 1.5 to 1 (chitinase to glucanase) was needed for the maximum extraction of uricase. Yield was 10.5 μ g⁻¹cells after 1.5 h incubation at 28°C. Glucanase alone resulted in a maximum yield of 1.9 μ g⁻¹while chitinase alone yielded 6.0 μ g⁻¹under the same conditions.     

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.     

The 1.8 Å Resolution Structure of Hevamine,a Plant Chitinase/Lysozyme,and Analysis of the Conserved Sequence and Structure Motifs of Glycosyl Hydrolase Family 18

The three-dimensional structure of hevamine, a plant enzyme with chitinase and lysozyme activity, has been refined at 1.8 Å resolution to an R-factor of 14.9% and a freeR-factor of 19.6%. The final model consists of all 273 amino acid residues and 206 ordered water molecules. Two non-prolinecis-peptides were identified, involving Phe32 and Trp255, both of which are implicated in substrate binding.Other glycosyl hydrolase family 18 proteins with known three-dimen sional structure are bacterial chitinase A, endo-β-N-acetylglucosaminidase F₁, endo-β-N-acetylglucosaminidase H, and the two plant proteins concanavalin B and narbonin, which have no known enzymatic activity. All these structures contain a (βα)₈barrel fold, with the two family 18 consensus regions roughly corresponding to the third and fourth barrel strands. This confirms the grouping of these proteins into family 18, which was only based on weak and local sequence similarity. The substrate specificity of the enzymes is determined by the loops following the barrel strands that form the substrate binding site. All enzymes have an aspartic acid and a glutamic acid residue in positions identical with Asp 125 and the catalytic Glu127 of hevamine. The lack of chitinase activity of concanavalin B and narbonin can be explained by the absence of one of these carboxylate groups, and by differences in the loops that form the substrate-binding cleft in hevamine.     

Use of chitinolytic Bacillus atrophaeus strain S2BC-2 antagonistic to Fusarium spp. for control of rhizome rot of ginger

An antagonistic bacillus S2BC-2 isolated from apple rhizosphere soil was identified to be highly chitinolytic on chitinase detection agar. Standard bacteriological tests and sequencing of 16S rRNA, and gyrA and rpoB genes, indicated a taxonomic affiliation of the strain to Bacillus atrophaeus. The strain was studied for its ability to grow and produce chitinase on different substrates. Bacterial cells grown on chitin-containing media showed enhanced growth and chitinase production with increased anti-fungal activity against vascular wilt pathogens. Extracellular proteins of cell-free extracts of media amended with chitin and fungal cell wall contained 4–10 novel polypeptides. In polyhouse (bamboo structures that provide protective shade made of polyvinyl sheet) studies, a chitin-supplemented talc-based formulation of the S2BC-2 challenge inoculated with Fusarium oxysporum f. sp. zingiberi recorded low percent disease indices of 84.9 % and 79.2 % for yellows and rhizome rot, respectively, over the non-bacterised pathogen control. The low disease incidences correlated with 113.3 % maximum rhizome production and 2-fold higher chitinase induction over the pathogen control. In native gel activity assays, upon challenge-inoculation, S2BC-2 expressed more chitinase isoforms than the pathogen control. The results suggest that chitinolytic B. atrophaeus can be used in the biocontrol of rhizome rot of ginger.     

A new approach for discovering cold-active enzymes in a cell mixture of pure-cultured bacteria

To overcome the intrinsic problems of conventional approaches, such as the unavailability of source microorganisms in metagenomic libraries and the production of inactive aggregates, a new method was tested for discovering new enzymes (e.g. cold-active chitinase). A metagenome-like library was constructed using genomes extracted from a cell mixture of pure-cultured chitinolytic bacteria, followed by activity-based screening for Escherichia coli clones that exhibit chitinase activity on selective medium. Within one positive chitinolytic clone, one chitinase gene (chi22718_III) was detected and assigned to the arctic marine bacterium, Pseudoalteromonas issachenkonii PAMC 22718, by colony-PCR with chi22718_III-specific primers. When expressed in E. coli, recombinant R-Chi22718_III lost 85 % of its enzyme activity when pre-incubated at 40 °C for 1 h, whereas its mesophilic counterpart R-ChiK only lost 10 % of its activity under the same conditions indicating that R-Chi22718_III is thermolabile, a characteristic of cold-active enzymes.     

Molecular characterization of an endochitinase from <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> subsp. konkukian

A chitinase gene from Bacillus thuringiensis serovar konkukian S4 was cloned, sequenced, and heterologously expressed in Escherichia coli M15. Recombinant enzyme (Chi74) was purified by Ni-NTA affinity column chromatography. The chi74 gene contains an open reading frame (ORF), with a capacity to encode an endochitinase with a deduced molecular weight 74 kDa and predicted isoelectric point of 5.67. Comparison of Chi74 with other chitinases has shown that it contains a modular structure with an N-terminal family 18 catalytic-domain, a Fibronectin-III like domain and a C-terminal carbohydrate binding module (CBM-II). Turn over rate (K _cat ) of the enzyme was determined using colloidal chitin (28.3 ± 0.70 S⁻¹) as substrate. The Purified enzyme was active at a broad range of pH (pH 3.5–7.5) and temperature (20–70°C) with a peak activity at pH 5.5 and 55°C. However, the enzyme was found to be stable up to 30°C for longer incubation periods. Moreover, the purified enzyme was shown to inhibit fungal spore germination and hyphal growth in the pathogenic fungi Fusarium oxysporum and Aspergillus niger. These studies will lead us to develop broad spectrum resistance in the crop plants via co-expression of the chitinases and the insecticidal proteins.     

Chitinase levels in guinea pig blood are increased after systemic infection with Aspergillus fumigatus

The presence of chitinase activity in human serum has recentlybeen described by us. On that occasion we speculated on thepossible role of mammalian chitinases as a defense mechanismagainst chitin-containing pathogens. The results of the presentstudy substantiate our hypothesis. We demonstrate and partiallycharacterize the chitinase activities that are present in plasmaof guinea pigs and in homogenates of A.fumigatus with the aidof the substrates MU-[GlcNAc]_2,3 and also with glycol [³H]chitin.Upon infection with A.fumigatus the serum chitinase activitylevels in the circulation of pathogen-free guinea pigs increasedin a time-dependent manner. The increase was also dependenton the size of the infecting fungal inoculum. Antifungal treatmentdiminished the increases. The increased chitinase activity wasof guinea pig origin. The activity of ß-hexosaminidaseshowed a very slight increase subsequent to the infection. Theactivities of three other enzymes of lysosomal origin (