Characterization and optimization of heterologous expression in Escherichia coli of the chitinase encoded by the chiA gene of Bacillus halodurans C-125

The diversity of the biotechnological applications of chitinolytic enzymes requires different enzyme-producing strains with different properties suitable for each process. In this work the chitinase encoded by the chiA gene of Bacillus halodurans has been studied. The protein shows a modular structure characterized by the catalytic domain of glycosyl hydrolases family 18 (GH18), fibronectin type III domain (FnIIID) and a carbohydrate-binding module family 5 (CBM5). The expression of the gene in Escherichia coli has made it possible to demonstrate the functionality of the protein which is active in the temperature range of 5–55 °C and pH values of 5.5–8.5 while maintaining a high stability under suboptimal conditions. The enzyme hydrolyzes colloidal chitin and different p-NP(GlcNAc)_n (n = 1–3) by an “-exo” type mechanism according to the information deduced from its sequence. The production of the protein was optimized by constructing recombinant strains, and the effect of the expression vector used, the cell density of the culture, the concentration of inducer and the induction time were studied. Based on its spectrum of activity, stability and mechanism of action, it arises as an enzyme of potential interest for production of N-acetyglucosamine or conversion of chitin into biologically active chito-oligosaccharides.     

NMR assignments and ligand-binding studies on a two-domain family GH19 chitinase allergen from Japanese cedar (<Emphasis Type="Italic">Cryptomeria japonica</Emphasis>) pollen

A two-domain family GH19 chitinase from Japanese cedar (Cryptomeria japonica) pollen, CJP-4, which consists of an N-terminal CBM18 domain and a GH19 catalytic domain, is known to be an important allergen, that causes pollinosis. We report here the resonance assignments of the NMR spectrum of CJP-4. The backbone resonances were almost completely assigned, and the secondary structure was estimated based on the chemical shift values. The addition of a chitin dimer to the enzyme solution perturbed the chemical shifts of the resonances of amino acid residues forming a long extended binding site spanning from the CBM18 domain to the GH19 catalytic domain.     

Characterization of the chitinase gene in Bacillus thuringiensis Mexican isolates

The chitinase gene was molecularly characterized in five Bacillus thuringiensis Mexican isolates, MR10, MR11, MR21, MR33, and RN52. The proteins derived from these genes were tested for their chitinase activity using fluorogenic chitin derivatives. In order to verify if chitinase genes were functional, they were cloned, and enzymatic activity of recombinant chitinases was also tested. Results indicated that enzymes exhibited endochitinase activity. The highest hydrolytic activity shown against the chitin tetrameric derivative occurred at pH value of 6.5, and the optimum activity temperature was around 60 °C. The recombinant endochitinases showed a molecular mass of ～77 kDa with isoelectric points from 6.5 to 7.0. Analysis of the nucleotide sequences showed highly conserved sequences among all isolates (97–99 %). Gene sequence analysis revealed a putative promoter (?35 TTGAGA and ?10 TTAATA) and a Shine–Dalgarno sequence (5´-AGGAGA-3´) upstream from the open reading frame. The deduced amino acid sequence revealed that the proteins are modular enzymes composed by a family 18 glycosyl hydrolase domain located between amino acids 134 and 549, a fibronectin-binding domain (580 through 656), and a chitin-binding domain (664 through 771). The deduced amino acid sequences of our isolates showed a similarity close to 100 % respect to the sequences reported in the GenBank database.     

Unique GH18 chitinase from Euglena gracilis: full-length cDNA cloning and characterization of its catalytic domain

A cDNA of putative chitinase from Euglena gracilis, designated EgChiA, encoded 960 amino acid residues, which is arranged from N-terminus in the order of signal peptide, glycoside hydrolase family 18 (GH18) domain, carbohydrate binding module family 18 (CBM18) domain, GH18 domain, CBM18 domain, and transmembrane helix. It is likely that EgChiA is anchored on the cell surface. The recombinant second GH18 domain of EgChiA, designated as CatD2, displayed optimal catalytic activity at pH 3.0 and 50 °C. The lower the polymerization degree of the chitin oligosaccharides [(GlcNAc)_4–6] used as the substrates, the higher was the rate of degradation by CatD2. CatD2 degraded chitin nanofibers as an insoluble substrate, and it produced only (GlcNAc)₂ and GlcNAc. Therefore, we speculated that EgChiA localizes to the cell surface of E. gracilis and is involved in degradation of chitin polymers into (GlcNAc)₂ or GlcNAc, which are easily taken up by the cells.     

Molecular detection of bacterial and streptomycete chitinases in the environment

Sets of PCR primers were designed to amplify bacterial chitinases at different levels of specificity. The bacterial chitinase group primers were successful in targeting enzymes classified within the group A glycosyl hydrolases of family 18. The widespread occurrence of group A bacterial chitinases in actinomycetes was demonstrated. Streptomycete chitinase specific primers were designed and a collection of type strains of species changed in the genes Streptomyces were screened and shown to have at least one and usually multiple chitinase genes. The presence of the gene for the chitin binding protein was also demonstrated within the streptomycete type strains. These data indicate that streptomycetes are well equipped to degrade chitin. The detection of group A chitinases in total community DNA is described and a sandy soil shown to contain more than 10 different genes using DGGE to indicate genetic diversity.     

Structure of chitinase D from Serratia proteamaculans reveals the structural basis of its dual action of hydrolysis and transglycosylation

Chitinases are known to hydrolyze chitin polymers into smaller chitooligosaccharides. Chitinase from bacterium Serratia proteamaculans (SpChiD) is found to exhibit both hydrolysis and transglycosylation activities. SpChiD belongs to family 18 of glycosyl hydrolases (GH-18). The recombinant SpChiD was crystallized and its three-dimensional structure was determined at 1.49 Å resolution. The structure was refined to an R-factor of 16.2%. SpChiD consists of 406 amino acid residues. The polypeptide chain of SpChiD adopts a (β/α)₈ triosephosphate isomerase (TIM) barrel structure. SpChiD contains three acidic residues, Asp149, Asp151 and Glu153 as part of its catalytic scheme. While both Asp149 and Glu153 adopt single conformations, Asp151 is observed in two conformations. The substrate binding cleft is partially obstructed by a protruding loop, Asn30 - Asp42 causing a considerable reduction in the number of available subsites in the substrate binding site. The positioning of loop, Asn30 - Asp42 appears to be responsible for the transglycosylation activity. The structure determination indicated the presence of sulfone Met89 (SMet89). The sulfone methionine residue is located on the surface of the protein at a site where extra domain is attached in other chitinases. This is the first structure of a single domain chitinase with hydrolytic and transglycosylation activities.     

Characterization of a Chitinase Gene from Stenotrophomonas maltophilia Strain 34S1 and Its Involvement in Biological Control

A chitinase gene was cloned on a 2.8-kb DNA fragment from Stenotrophomonas maltophilia strain 34S1 by heterologous expression in Burkholderia cepacia. Sequence analysis of this fragment identified an open reading frame encoding a deduced protein of 700 amino acids. Removal of the signal peptide sequence resulted in a predicted protein that was 68 kDa in size. Analysis of the sequence indicated that the chitinase contained a catalytic domain belonging to family 18 of glycosyl hydrolases. Three putative binding domains, a chitin binding domain, a novel polycystic kidney disease (PKD) domain, and a fibronectin type III domain, were also identified within the sequence. Pairwise comparisons of each domain to the most closely related sequences found in database searches clearly demonstrated variation in gene sources and the species from which related sequences originated. A 51-kDa protein with chitinolytic activity was purified from culture filtrates of S. maltophilia strain 34S1 by hydrophobic interaction chromatography. Although the protein was significantly smaller than the size predicted from the sequence, the N-terminal sequence verified that the first 15 amino acids were identical to the deduced sequence of the mature protein encoded by chiA. Marker exchange mutagenesis of chiA resulted in mutant strain C5, which was devoid of chitinolytic activity and lacked the 51-kDa protein in culture filtrates. Strain C5 was also reduced in the ability to suppress summer patch disease on Kentucky bluegrass, supporting a role for the enzyme in the biocontrol activity of S. maltophilia.     

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.     

Identification of two group A chitinase genes in Botrytis cinerea which are differentially induced by exogenous chitin

Chitin-degrading enzymes represent potential targets for pesticides in the control of plant pathogenic fungi. Here we describe the cloning, molecular characterization, and expression analysis of two putative chitinases of Botrytis cinerea, a pathogenic fungus infecting a wide range of plants. On the basis of conserved motifs from family 18 of the glycosyl hydrolases and group A of the fungal chitinases, two fragments (BcchiA and BcchiB) were cloned and sequenced. Expression of BcchiA and BcchiB chitinase genes upon growth under different conditions was analysed using RT-PCR. We observed that BcchiA expression was suppressed by glucose, whereas it was strongly stimulated in the presence of chitin or chitin degradation products. Conversely, BcchiB expression was not suppressed by glucose and was not stimulated by chitin or chitin degradation products. The difference in expression regulation is indicative of a functional divergence between the two chitinase paralogous genes.     

Biochemical and genetic characterization of ChiA,the major enzyme component for the solubilization of chitin by<Emphasis Type="Italic"> Cellulomonas uda</Emphasis>

Cellulomonas uda efficiently solubilized chitinous substrates with a simple chitinase system composed of an endochitinase, designated ChiA, which hydrolyzed insoluble substrates into long-chain chitooligosaccharides, and an as yet uncharacterized exochitinase activity. ChiA, isolated from culture supernatant fluids, was found to be a glycosylated endochitinase with an apparent molecular mass of approximately 70 kDa and pI of 8.5. The gene encoding ChiA was cloned in Escherichia coli and sequenced, revealing an open reading frame of 1,716 bp encoding a 571-amino-acid protein with a predicted molecular mass of 59.2 kDa. The region upstream of chiA included a conserved –35 hexamer flanked by two direct repeats analogous to those found in many Streptomyces chitinase promoters, and thought to function as binding sequences for regulatory proteins. Analysis of the deduced amino acid sequence showed a modular protein consisting of a signal peptide at its N terminus, a family 2 carbohydrate-binding module (CBM2) that was closely related to the substrate-binding domains of glycosyl hydrolases from distantly related bacteria, and a family 18 glycosyl hydrolase catalytic module related to Streptomyces chitinases. In contrast to the fibronectin type III domains of Streptomyces chitinases, the linker region between modules in ChiA consisted of a long proline- and threonine-rich module, thought to contribute to the glycosylation and flexibility of the mature protein.Abbreviations CBM Carbohydrate-binding module - P-T Proline- and threonine-rich domain - Fn3 Type III repetitive sequences of fibronectin domain - PKD Polycystic kidney disease I domain     

Serratia marcescens chitinase: One-step purification and use for the determination of chitin

The extracellular chitinase produced by Serratia marcescens was obtained in highly purified form by adsorption-digestion on chitin. After gel electrophoresis in a nondenaturing system, the purified preparation exhibited two major protein bands that coincided with enzymatic activity. A study of the enzyme properties showed its suitability for the analysis of chitin. Thus, the chitinase exhibited excellent stability, a wide pH optimum, and linear kinetics over a much greater range than similar enzymes from other sources. The major product of chitin hydrolysis was chitobiose, which was slowly converted into free N-acetylglucosamine by traces of β-N-acetylglucosaminidase present in the purified preparation. The preparation was free from other polysaccharide hydrolases. Experiments with radiolabeled yeast cell walls showed that the chitinase was able to degrade wall chitin completely and specifically.     

A bacterial GH6 cellobiohydrolase with a novel modular structure

Cel6D from Paenibacillus barcinonensis is a modular cellobiohydrolase with a novel molecular architecture among glycosyl hydrolases of family 6. It contains an N-terminal catalytic domain (family 6 of glycosyl hydrolases (GH6)), followed by a fibronectin III-like domain repeat (Fn3_1,2) and a C-terminal family 3b cellulose-binding domain (CBM3b). The enzyme has been identified and purified showing catalytic activity on cellulosic substrates and cellodextrins, with a marked preference for phosphoric acid swollen cellulose (PASC). Analysis of mode of action of Cel6D shows that it releases cellobiose as the only hydrolysis product from cellulose. Kinetic parameters were determined on PASC showing a K _m of 68.73 mg/ml and a V _max of 1.73 U/mg. A series of truncated derivatives of Cel6D have been constructed and characterized. Deletion of CBM3b caused a notable reduction in hydrolytic activity, while deletion of the Fn3 domain abolished activity, as the isolated GH6 domain was not active on any of the substrates tested. Mutant enzymes Cel6D-D146A and Cel6D-D97A were constructed in the residues corresponding to the putative acid catalyst and to the network for the nucleophilic attack. The lack of activity of the mutant enzymes indicates the important role of these residues in catalysis. Analysis of cooperative activity of Cel6D with cellulases from the same producing P. barcinonensis strain reveals high synergistic activity with processive endoglucanase Cel9B on hydrolysis of crystalline substrates. The characterized cellobiohydrolase can be a good contribution for depolymerization of cellulosic substrates and for the deconstruction of native cellulose.

     

Cloning, expression, and characterization of a chitinase from the chitinolytic bacterium Aeromonas hydrophila strain SUWA-9

The chitinolytic bacterium Aeromonas hydrophila strain SUWA-9, which was isolated from freshwater in Lake Suwa (Nagano Prefecture, Japan), produced several kinds of chitin-degrading enzymes. A gene coding for an endo-type chitinase (chiA) was isolated from SUWA-9. The chiA ORF encodes a polypeptide of 865 amino acid residues with a molecular mass of 91.6 kDa. The deduced amino acid sequence showed high similarity to those of bacterial chitinases classified into family 18 of glycosyl hydrolases. chiA was expressed in Escherichia coli and the recombinant chitinase (ChiA) was purified and examined. The enzyme hydrolyzed N-acetylchitooligomers from trimer to pentamer and produced monomer and dimer as a final product. It also reacted toward colloidal chitin and chitosan with a low degree of deacetylation. When cells of SUWA-9 were grown in the presence of colloidal chitin, a 60 kDa-truncated form of ChiA that had lost the C-terminal chitin-binding domain was secreted.     

X-Ray Crystal Structure of the Full Length Human Chitotriosidase (CHIT1) Reveals Features of Its Chitin Binding Domain

Chitinases are enzymes that catalyze the hydrolysis of chitin. Human chitotriosidase (CHIT1) is one of the two active human chitinases, involved in the innate immune response and highly expressed in a variety of diseases. CHIT1 is composed of a catalytic domain linked by a hinge to its chitin binding domain (ChBD). This latter domain belongs to the carbohydrate-binding module family 14 (CBM14 family) and facilitates binding to chitin. So far, the available crystal structures of the human chitinase CHIT1 and the Acidic Mammalian Chitinase (AMCase) comprise only their catalytic domain. Here, we report a crystallization strategy combining cross-seeding and micro-seeding cycles which allowed us to obtain the first crystal structure of the full length CHIT1 (CHIT1-FL) at 1.95 Å resolution. The CHIT1 chitin binding domain (ChBD_CHIT1) structure shows a distorted β-sandwich 3D fold, typical of CBM14 family members. Accordingly, ChBD_CHIT1 presents six conserved cysteine residues forming three disulfide bridges and several exposed aromatic residues that probably are involved in chitin binding, including the highly conserved Trp465 in a surface- exposed conformation. Furthermore, ChBD_CHIT1 presents a positively charged surface which may be involved in electrostatic interactions. Our data highlight the strong structural conservation of CBM14 family members and uncover the structural similarity between the human ChBD_CHIT1, tachycitin and house mite dust allergens. Overall, our new CHIT1-FL structure, determined with an adapted crystallization approach, is one of the few complete bi-modular chitinase structures available and reveals the structural features of a human CBM14 domain.     

Characterization of two novel bacterial type A <Emphasis Type="Italic">exo</Emphasis>-chitobiose hydrolases having C-terminal 5/12-type carbohydrate-binding modules

Type A chitinases (EC 3.2.1.14), GH family 18, attack chitin ((1 → 4)-2-acetamido-2-deoxy-β-d-glucan) and chito-oligosaccharides from the reducing end to catalyze release of chitobiose (N,N′-diacetylchitobiose) via hydrolytic cleavage of N-acetyl-β-d-glucosaminide (1 → 4)-β-linkages and are thus “exo-chitobiose hydrolases.” In this study, the chitinase type A from Serratia marcescens (SmaChiA) was used as a template for identifying two novel exo-chitobiose hydrolase type A enzymes, FbalChi18A and MvarChi18A, originating from the marine organisms Ferrimonas balearica and Microbulbifer variabilis, respectively. Both FbalChi18A and MvarChi18A were recombinantly expressed in Escherichia coli and were confirmed to exert exo-chitobiose hydrolase activity on chito-oligosaccharides, but differed in temperature and pH activity response profiles. Amino acid sequence comparison of the catalytic β/α barrel domain of each of the new enzymes showed individual differences, but ~69% identity of each to that of SmaChiA and highly conserved active site residues. Superposition of a model substrate on 3D structural models of the catalytic domain of the enzymes corroborated exo-chitobiose hydrolase type A activity for FbalChi18A and MvarChi18A, i.e., substrate attack from the reducing end. A main feature of both of the new enzymes was the presence of C-terminal 5/12 type carbohydrate-binding modules (SmaChiA has no C-terminal carbohydrate binding module). These new enzymes may be useful tools for utilization of chitin as an N-acetylglucosamine donor substrate via chitobiose.     

Differences in the chitinolytic activity of mammalian chitinases on soluble and insoluble substrates

Chitin is an abundant polysaccharide used by many organisms for structural rigidity and water repulsion. As such, the insoluble crystalline structure of chitin poses significant challenges for enzymatic degradation. Acidic mammalian chitinase, a processive glycosyl hydrolase, is the primary enzyme involved in the degradation of environmental chitin in mammalian lungs. Mutations to acidic mammalian chitinase have been associated with asthma, and genetic deletion in mice increases morbidity and mortality with age. We initially set out to reverse this phenotype by engineering hyperactive acidic mammalian chitinase variants. Using a screening approach with commercial fluorogenic substrates, we identified mutations with consistent increases in activity. To determine whether the activity increases observed were consistent with more biologically relevant chitin substrates, we developed new assays to quantify chitinase activity with insoluble chitin, and identified a one‐pot fluorogenic assay that is sufficiently sensitive to quantify changes to activity due to the addition or removal of a carbohydrate‐binding domain. We show that the activity increases from our directed evolution screen were lost when insoluble substrates were used. In contrast, naturally occurring gain‐of‐function mutations gave similar results with oligomeric and insoluble substrates. We also show that activity differences between acidic mammalian chitinase and chitotriosidase are reduced with insoluble substrate, suggesting that previously reported activity differences with oligomeric substrates may have been driven by differential substrate specificity. These results highlight the need for assays against physiological substrates when engineering metabolic enzymes, and provide a new one‐pot assay that may prove to be broadly applicable to engineering glycosyl hydrolases.     

Overview of chitin metabolism enzymes in Manduca sexta: Identification,domain organization,phylogenetic analysis and gene expression

Chitin is one of the most abundant biomaterials in nature. The biosynthesis and degradation of chitin in insects are complex and dynamically regulated to cope with insect growth and development. Chitin metabolism in insects is known to involve numerous enzymes, including chitin synthases (synthesis of chitin), chitin deacetylases (modification of chitin by deacetylation) and chitinases (degradation of chitin by hydrolysis). In this study, we conducted a genome-wide search and analysis of genes encoding these chitin metabolism enzymes in Manduca sexta. Our analysis confirmed that only two chitin synthases are present in M. sexta as in most other arthropods. Eleven chitin deacetylases (encoded by nine genes) were identified, with at least one representative in each of the five phylogenetic groups that have been described for chitin deacetylases to date. Eleven genes encoding for family 18 chitinases (GH18) were found in the M. sexta genome. Based on the presence of conserved sequence motifs in the catalytic sequences and phylogenetic relationships, two of the M. sexta chitinases did not cluster with any of the current eight phylogenetic groups of chitinases: two new groups were created (groups IX and X) and their characteristics are described. The result of the analysis of the Lepidoptera-specific chitinase-h (group h) is consistent with its proposed bacterial origin. By analyzing chitinases from fourteen species that belong to seven different phylogenetic groups, we reveal that the chitinase genes appear to have evolved sequentially in the arthropod lineage to achieve the current high level of diversity observed in M. sexta. Based on the sequence conservation of the catalytic domains and on their developmental stage- and tissue-specific expression, we propose putative functions for each group in each category of enzymes.     

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.     

Molecular and biochemical characterization of three GH62 α-l-arabinofuranosidases from the soil deuteromycete Penicillium funiculosum

Penicillium funiculosum is an industrial fungus exploited for its capacity to secrete a wide array of glycosyl hydrolases (GHs) and glycosyl transferases (GTs). These enzymes are part of an enzymatic cocktail that is commercialized under the name RovabioExcel^®, which is used as feed additive in animal nutrition. The genome sequence of this filamentous fungus has revealed a remarkable richness in several accessory enzymes, and notably in α-l-arabinofuranosidases (α-l-AFases) that participate in the hydrolysis of arabinoxylans (AX) in corn/wheat fibers used in poultry feed. Here, we report on the molecular and biochemical characterization of three GH62 family α-l-AFases encoding genes in this filamentous fungus. Amino acids sequences showed strong similarities (>65%) between them, as well with GH62 enzymes from other filamentous fungi. Interestingly, one of the three PfABF62, namely PfABF62c is unique in bearing at its N-terminus a canonical family 1 carbohydrate-binding module (CBM1) of 37 amino acids length, which was shown to help the protein to bind to microcrystalline cellulose. Also, this PfABF62c showed optimal pH and temperature of 2.8 and 50 °C, respectively, whereas optimal activity for PfABF62a and PfABF62b were measured at 40 °C and at pH ranging between 2.6 and 4.5. Arabinan and arabinoxylan, but no other sugars or polymers were found to augment the thermal transition of the three enzymes by 3–5 °C as measured by differential scanning fluorimetry. Finally, enzymatic hydrolysis fingerprints of heteroxylans allowed concluding that the mode of action of the GH62 enzymes from this fungal species was to remove arabinofuranosyl residues linked in position O-2 and O-3 of substituted xylose units in arabinoxylan chains.