Hideo Kusaoke

Biochemical and genetic properties of Paenibacillus glycosyl hydrolase having chitosanase activity and discoidin domain

Cells of “Paenibacillus fukuinensis” D2 produced chitosanase into surrounding medium, in the presence of colloidal chitosan or glucosamine. The gene of this enzyme was cloned, sequenced, and subjected to site-directed mutation and deletion analyses. The nucleotide sequence indicated that the chitosanase was composed of 797 amino acids and its molecular weight was 85,610. Unlike conventional family 46 chitosanases, the enzyme has family 8 glycosyl hydrolase catalytic domain, at the amino-terminal side, and discoidin domain at the carboxyl-terminal region. Expression of the cloned gene in Escherichia coli revealed β-1,4-glucanase function, besides chitosanase activity. Analyses by zymography and immunoblotting suggested that the active enzyme was, after removal of signal peptide, produced from inactive 81-kDa form by proteolysis at the carboxyl-terminal region. Replacements of Glu115 and Asp176, highly conserved residues in the family 8 glycosylase region, with Gln and Asn caused simultaneous loss of chitosanase and glucanase activities, suggesting that these residues formed part of the catalytic site. Truncation experiments demonstrated indispensability of an amino-terminal region spanning 425 residues adjacent to the signal peptide.

Discoidin Domain of Chitosanase Is Required for Binding to the Fungal Cell Wall

Previously, we reported properties of a glycosylase belonging to GH-8 glycosyl hydrolase (GH) and having both chitosanase and glucanase activities. This enzyme (D2), whose molecular mass (86 kDa) was the largest among the GH-8 group, has its catalytic domain at the N-terminal region, and discoidin domain (DD) at the C-terminal region. Although various chitosanases, chitinases and glucanases have been known, DD is unique to the D2 enzyme. Glucanase and chitinase, but not chitosanase, are known to have functional domain such as carbohydrate-binding module, besides catalytic domain. Accordingly, function of the DD of D2 chitosanase was analyzed, using zygomycete cell wall containing chitosan, glucan and chitin as the basic constituents. The DD specifically and tightly bound to chitosan, but did not participate in affinity for glucan and chitin. Deletion of the DD caused marked reduction in absorbability to cell wall and in hydrolytic activity toward chitosan and glucan. These results suggest that the DD is concerned in binding of the enzyme to cell wall and in effective digestion of the insoluble substrate, through hydrolysis of not only chitosan but also coexisting glucan. Thus, this is the first example of chitosan-binding domain among various carbohydrate-binding modules reported thus far.

Yeast Cell-Surface Expression of Chitosanase from Paenibacillus fukuinensis

To produce chitoorigosaccharides using chitosan, we attempted to construct Paenibacillus fukuinensis chitosanase-displaying yeast cells as a whole-cell biocatalyst through yeast cell-surface engineering. The localization of the chitosanase on the yeast cell surface was confirmed by immunofluorescence labeling of cells. The chitosanase activity of the constructed yeast was investigated by halo assay and the dinitrosalicylic acid method.

The first identification of carbohydrate binding modules specific to chitosan.

Background: Carbohydrate binding modules (CBMs) specific to chitosan have yet to be identified. Results: Two CBMs located at the C terminus of a chitosanase from Paenibacillus sp. IK-5 specifically bound chitosan oligosaccharides. Conclusion: Individual CBMs can accommodate at least two glucosamine units at loops extruded from the core β-sandwich. Significance: The synergistic action of the two CBMs appears to facilitate chitosan hydrolysis. Two carbohydrate binding modules (DD1 and DD2) belonging to CBM32 are located at the C terminus of a chitosanase from Paenibacillus sp. IK-5. We produced three proteins, DD1, DD2, and tandem DD1/DD2 (DD1+DD2), and characterized their binding ability. Transition temperature of thermal unfolding (Tm) of each protein was elevated by the addition of cello-, laminari-, chitin-, or chitosan-hexamer (GlcN)6. The Tm elevation (ΔTm) in DD1 was the highest (10.3 °C) upon the addition of (GlcN)6 and was markedly higher than that in DD2 (1.0 °C). A synergistic effect was observed (ΔTm = 13.6 °C), when (GlcN)6 was added to DD1+DD2. From isothermal titration calorimetry experiments, affinities to DD1 were not clearly dependent upon chain length of (GlcN)n; ΔGr° values were −7.8 (n = 6), −7.6 (n = 5), −7.6 (n = 4), −7.6 (n = 3), and −7.1 (n = 2) kcal/mol, and the value was not obtained for GlcN due to the lowest affinity. DD2 bound (GlcN)n with the lower affinities (ΔGr° = −5.0 (n = 3) ∼ −5.2 (n = 6) kcal/mol). Isothermal titration calorimetry profiles obtained for DD1+DD2 exhibited a better fit when the two-site model was used for analysis and provided greater affinities to (GlcN)6 for individual DD1 and DD2 sites (ΔGr° = −8.6 and −6.4 kcal/mol, respectively). From NMR titration experiments, (GlcN)n (n = 2∼6) were found to bind to loops extruded from the core β-sandwich of individual DD1 and DD2, and the interaction sites were similar to each other. Taken together, DD1+DD2 is specific to chitosan, and individual modules synergistically interact with at least two GlcN units, facilitating chitosan hydrolysis.

Demonstration of catalytic proton acceptor of chitosanase from Paenibacillus fukuinensis by comprehensive analysis of mutant library

Chitosanase from Paenibacillus fukuinensis D2 is an attractive enzyme, and it exhibits both chitosanase and β-1, 4 glucanase activities. In our previous study, we generated P. fukuinensis chitosanase-displaying yeast cells using a yeast cell surface-displaying system. Chitosanase-displaying yeast can be utilized as a chitosanase cluster without many time-consuming purification steps. In this study, using the system, we have investigated whether Glu302, which is supposed as a putative proton acceptor, is an essential amino acid residue for exhibiting chitosanase activity and analyzed the contribution of mutual interaction between Glu302 and Asn312 to the activity. A mutant library in which Glu302 and Asn312 were comprehensively substituted by the other amino acid residues was constructed on the yeast cell surface. From the results of chitosanase and β-1, 4 glucanase activity assays, we demonstrated that Glu302 was a proton acceptor for chitosanase activity, and Asn312 also participated in the hydrolysis of chitosan and cellulose.

Mechanism of chitosan recognition by CBM32 carbohydrate-binding modules from a Paenibacillus sp. IK-5 chitosanase/glucanase

An antifungal chitosanase/glucanase isolated from the soil bacterium Paenibacillus sp. IK-5 has two CBM32 chitosan-binding modules (DD1 and DD2) linked in tandem at the C-terminus. In order to obtain insights into the mechanism of chitosan recognition, the structures of DD1 and DD2 were solved by NMR spectroscopy and crystallography. DD1 and DD2 both adopted a β-sandwich fold with several loops in solution as well as in crystals. On the basis of chemical shift perturbations in(1)H-(15)N-HSQC resonances, the chitosan tetramer (GlcN)4 was found to bind to the loop region extruded from the core β-sandwich of DD1 and DD2. The binding site defined by NMR in solution was consistent with the crystal structure of DD2 in complex with (GlcN)3, in which the bound (GlcN)3 stood upright on its non-reducing end at the binding site. Glu(14)of DD2 appeared to make an electrostatic interaction with the amino group of the non-reducing end GlcN, and Arg(31), Tyr(36)and Glu(61)formed several hydrogen bonds predominantly with the non-reducing end GlcN. No interaction was detected with the reducing end GlcN. Since Tyr(36)of DD2 is replaced by glutamic acid in DD1, the mutation of Tyr(36)to glutamic acid was conducted in DD2 (DD2-Y36E), and the reverse mutation was conducted in DD1 (DD1-E36Y). Ligand-binding experiments using the mutant proteins revealed that this substitution of the 36th amino acid differentiates the binding properties of DD1 and DD2, probably enhancing total affinity of the chitosanase/glucanase toward the fungal cell wall.

Evaluation of chitosan-binding amino acid residues of chitosanase from Paenibacillus fukuinensis.

Chitosan oligosaccharides longer than a hexamer have higher bioactivity than polymer or shorter oligosaccharides, such as the monomer or dimer. In our previous work, we generated Paenibacillus fukuinensis chitosanase-displaying yeast using yeast cell surface displaying system and demonstrated the catalytic base. Here we investigated the specific function of putative four amino acid residues Trp159, Trp228, Tyr311, and Phe406 engaged in substrate binding. Using this system, we generated chitosanase mutants in which the four amino acid residues were substituted with Ala and the chitosanase activity assay and HPLC analysis were performed. Based on these results, we demonstrated that Trp159 and Phe406 were critical for hydrolyzing both polymer and oligosaccharide, and Trp228 and Tyr311 were especially important for binding to oligosaccharide, such as the chitosan-hexamer, not to the chitosan polymer. From the results, we suggested the possibility of the effective strategy for designing useful mutants that produce chitosan oligosaccharides holding higher bioactivity. Graphical Abstract We clarified the difference of importance for binding chitosan oligosaccharides among amino acid residues in substrate binding cleft.

Biomaterials: Chitosan and Collagen for Regenerative Medicine

With contributions from USA, Taiwan, Japan, and Korea, this special issue holds great insight. This special issue offers comprehensive knowledge on chitosan and collagen as biomaterials, especially with respect to their basic biological and chemical properties, as well as clinical applications. Two review articles described the preparation and biological application of chitooligosaccharide and its derivatives and the relevance to clinical dentistry of distinct characteristics of mandibular bone collagen. Original articles reported seven experiments: 3 chitosan topics and 4 collagen topics. The former demonstrated the contributions for a proteomic view of chitosan nanoparticle to hepatic cells, the promotion of D-glucosamine to transfection efficiency, and chitin application as skin substitutes. The latter showed the contributions for hydroxyapatite-gelatin nanocomposite, genipin modification of dentin collagen, dentin phosphophoryn/collagen composite for dental biomaterial, and biological safety of fish collagen.