Characterization of the extracellular domain of sensor histidine kinase NagS from Paenibacillus sp. str. FPU-7: nagS interacts with oligosaccharide binding protein NagB1 in complexes with N, N'-diacetylchitobiose.

We have previously isolated the Gram-positive chitin-degrading bacterium Paenibacillus sp. str. FPU-7. This bacterium traps chitin disaccharide (GlcNAc)2 on its cell surface using two homologous solute-binding proteins, NagB1 and NagB2. Bacteria use histidine kinase (HK) of the two-component regulatory system as an extracellular environment sensor. In this study, we found that nagS, which encodes a HK, is located next to the nagB1 gene. Biochemical experiments revealed that the NagS sensor domain (NagS30-294) interacts with the NagB1-(GlcNAc)2 complex. However, proof of NagS30-294 interacting with NagB1 without (GlcNAc)2 is currently unavailable. In contrast to NagB1, no complex formation was observed between NagS30-294 and NagB2, even in the presence of (GlcNAc)2. The NagS30-294 crystal structure at 1.8 Å resolution suggested that the canonical tandem-Per-Arnt-Sim fold recognizes the NagB1-(GlcNAc)2 complex. This study provides insight into the recognition of chitin oligosaccharides by bacteria.

Induction of plant disease resistance by mixed oligosaccharide elicitors prepared from plant cell wall and crustacean shells.

Basal plant immune responses are activated by the recognition of conserved microbe-associated molecular patterns (MAMPs), or breakdown molecules released from the plants after damage by pathogen penetration, so-called damage-associated molecular patterns (DAMPs). While chitin-oligosaccharide (CHOS), a primary component of fungal cell walls, is most known as MAMP, plant cell wall-derived oligosaccharides, cello-oligosaccharides (COS) from cellulose, and xylo-oligosaccharide (XOS) from hemicellulose are representative DAMPs. In this study, elicitor activities of COS prepared from cotton linters, XOS prepared from corn cobs, and chitin-oligosaccharide (CHOS) from crustacean shells were comparatively investigated. In Arabidopsis, COS, XOS, or CHOS treatment triggered typical defense responses such as reactive oxygen species (ROS) production, phosphorylation of MAP kinases, callose deposition, and activation of the defense-related transcription factor WRKY33 promoter. When COS, XOS, and CHOS were used at concentrations with similar activity in inducing ROS production and callose depositions, CHOS was particularly potent in activating the MAPK kinases and WRKY33 promoters. Among the COS and XOS with different degrees of polymerization, cellotriose and xylotetraose showed the highest activity for the activation of WRKY33 promoter. Gene ontology enrichment analysis of RNAseq data revealed that simultaneous treatment of COS, XOS, and CHOS (oligo-mix) effectively activates plant disease resistance. In practice, treatment with the oligo-mix enhanced the resistance of tomato to powdery mildew, but plant growth was not inhibited but rather tended to be promoted, providing evidence that treatment with the oligo-mix has beneficial effects on improving disease resistance in plants, making them a promising class of compounds for practical application.

Low-Molecular-Weight ß-1,3-1,6-Glucan Derived from Aureobasidium pullulans Exhibits Anticancer Activity by Inducing Apoptosis in Colorectal Cancer Cells.

ß-glucan, a plant polysaccharide, mainly exists in plant cell walls of oats, barley, and wheat. It is attracting attention due to its high potential for use as functional foods and pharmaceuticals. We have previously reported that low-molecular-weight Aureobasidium pullulans-fermented ß-D-glucan (LMW-AP-FBG) could inhibit inflammatory responses by inhibiting mitogen-activated protein kinases and nuclear factor-κB signaling pathways. Bases on previous results, the objective of the present study was to investigate the therapeutic potential of LMW-AP-FBG in BALB/c mice intracutaneously transplanted with CT-26 colon cancer cells onto their backs. Daily intraperitoneal injections of LMW-AP-FBG (5 mg/kg) for two weeks significantly suppressed tumor growth in mice bearing CT-26 tumors by reducing tumor proliferation and inducing apoptosis as compared to phosphate buffer-treated control mice. In addition, LMW-AP-FBG treatment reduced the viability of CT-26 cells in a dose-dependent manner by inducing apoptosis with loss of mitochondrial transmembrane potential and increased activated caspases. Taken together, LMW-AP-FBG exhibits anticancer properties both in vivo and in vitro.

Anti-inflammatory effects of ß-1,3-1,6-glucan derived from black yeast Aureobasidium pullulans in RAW264.7 cells.

ß-glucan derived from the black yeast Aureobasidium pullulans (A. pullulans) is one of the natural products attracting attention due to its high potential for application as a functional food and pharmaceutical. Our team of researchers obtained a highly soluble, low-molecular-weight ß-glucan from the fermentation culture medium of A. pullulans via mechanochemical ball milling method, that is, the low-molecular-weight A. pullulans-fermented ß-D-glucan (LMW-AP-FBG). We investigated the anti-inflammatory effect of LMW-AP-FBG using lipopolysaccharide (LPS)-stimulated murine macrophages (RAW264.7 cells) in the current study. LMW-AP-FBG altered LPS-stimulated inflammatory responses by reducing the release of inflammatory mediators such as nitric oxide (NO), interleukin (IL)-1ß, IL-6 and tumor necrosis factor-α. As well, the mitogen-activated protein kinases (MAPKs) and nuclear factor-κB (NF-κB) signaling pathways were downregulated by LMW-AP-FBG. Furthermore, LMW-AP-FBG markedly reduced LPS-induced expression of cell surface molecules, CD14, CD86, and MHC class II, which mediate macrophage activation. These findings suggest that LMW-AP-FBG can be used as an effective immune modulator to attenuate the progression of inflammatory disease.

Structural characterization of two solute-binding proteins for N,N'-diacetylchitobiose/N,N',N''-triacetylchitotoriose of the gram-positive bacterium, Paenibacillus sp. str. FPU-7.

The chitinolytic bacterium Paenibacillus sp. str. FPU-7 efficiently degrades chitin into oligosaccharides such as N-acetyl-D-glucosamine (GlcNAc) and disaccharides (GlcNAc)2 through multiple secretory chitinases. Transport of these oligosaccharides by P. str. FPU-7 has not yet been clarified. In this study, we identified nagB1, predicted to encode a sugar solute-binding protein (SBP), which is a component of the ABC transport system. However, the genes next to nagB1 were predicted to encode two-component regulatory system proteins rather than transmembrane domains (TMDs). We also identified nagB2, which is highly homologous to nagB1. Adjacent to nagB2, two genes were predicted to encode TMDs. Binding experiments of the recombinant NagB1 and NagB2 to several oligosaccharides using differential scanning fluorimetry and surface plasmon resonance confirmed that both proteins are SBPs of (GlcNAc)2 and (GlcNAc)3. We determined their crystal structures complexed with and without chitin oligosaccharides at a resolution of 1.2 to 2.0 Å. The structures shared typical SBP structural folds and were classified as subcluster D-I. Large domain motions were observed in the structures, suggesting that they were induced by ligand binding via the "Venus flytrap" mechanism. These structures also revealed chitin oligosaccharide recognition mechanisms. In conclusion, our study provides insight into the recognition and transport of chitin oligosaccharides in bacteria.

Structural and biochemical characterisation of a novel alginate lyase from Paenibacillus sp. str. FPU-7.

A novel alginate lyase, PsAly, with a molecular mass of 33 kDa and whose amino acid sequence shares no significant similarity to other known proteins, was biochemically and structurally characterised from Paenibacillus sp. str. FPU-7. The maximum PsAly activity was obtained at 65 °C, with an optimum pH of pH 7-7.5. The activity was enhanced by divalent cations, such as Mg2+, Mn2+, or Co2+, and inhibited by a metal chelator, ethylenediaminetetraacetic acid. The reaction products indicated that PsAly is an endolytic enzyme with a preference for polymannuronate. Herein, we report a detailed crystal structure of PsAly at a resolution of 0.89 Å, which possesses a ß-helix fold that creates a long cleft. The catalytic site was different from that of other polysaccharide lyases. Site-directed mutational analysis of conserved residues predicted Tyr184 and Lys221 as catalytic residues, abstracting from the C5 proton and providing a proton to the glycoside bond, respectively. One cation was found to bind to the bottom of the cleft and neutralise the carboxy group of the substrate, decreasing the pKa of the C5 proton to promote catalysis. Our study provides an insight into the structural basis for the catalysis of alginate lyases and ß-helix polysaccharide lyases.

Structural and functional characterization of a glycoside hydrolase family 3 ß-N-acetylglucosaminidase from Paenibacillus sp. str. FPU-7.

Chitin, a ß-1,4-linked homopolysaccharide of N-acetyl-d-glucosamine (GlcNAc), is one of the most abundant biopolymers on Earth. Paenibacillus sp. str. FPU-7 produces several different chitinases and converts chitin into N,N'-diacetylchitobiose ((GlcNAc)2) in the culture medium. However, the mechanism by which the Paenibacillus species imports (GlcNAc)2 into the cytoplasm and divides it into the monomer GlcNAc remains unclear. The gene encoding Paenibacillus ß-N-acetyl-d-glucosaminidase (PsNagA) was identified in the Paenibacillus sp. str. FPU-7 genome using an expression cloning system. The deduced amino acid sequence of PsNagA suggests that the enzyme is a part of the glycoside hydrolase family 3 (GH3). Recombinant PsNagA was successfully overexpressed in Escherichia coli and purified to homogeneity. As assessed by gel permeation chromatography, the enzyme exists as a 57-kDa monomer. PsNagA specifically hydrolyses chitin oligosaccharides, (GlcNAc)2-4, 4-nitrophenyl N-acetyl ß-d-glucosamine (pNP-GlcNAc) and pNP-(GlcNAc)2-6, but has no detectable activity against 4-nitrophenyl ß-d-glucose, 4-nitrophenyl ß-d-galactosamine and colloidal chitin. In this study, we present a 1.9 Å crystal structure of PsNagA bound to GlcNAc. The crystal structure reveals structural features related to substrate recognition and the catalytic mechanism of PsNagA. This is the first study on the structural and functional characterization of a GH3 ß-N-acetyl-d-glucosaminidase from Paenibacillus sp.

Bacterial Chitinase System as a Model of Chitin Biodegradation.

Chitin, a structural polysaccharide of ß-1,4-linked N-acetyl-D-glucosamine residues, is the second most abundant natural biopolymer after cellulose. The metabolism of chitin affects the global carbon and nitrogen cycles, which are maintained by marine and soil-dwelling bacteria. The degradation products of chitin metabolism serve as important nutrient sources for the chitinolytic bacteria. Chitinolytic bacteria have elaborate enzymatic systems for the degradation of the recalcitrant chitin biopolymer. This chapter introduces chitin degradation and utilization systems of the chitinolytic bacteria. These bacteria secrete many chitin-degrading enzymes, including processive chitinases, endo-acting non-processive chitinases, lytic polysaccharide monooxygenases, and N-acetyl-hexosaminidases. Bacterial chitinases play a fundamental role in the degradation of chitin. Enzymatic properties, catalytic mechanisms, and three-dimensional structures of chitinases have been extensively studied by many scientists. These enzymes can be exploited to produce a range of chitin-derived products, e.g., biocontrol agents against many plant pathogenic fungi and insects. We introduce bacterial chitinases in terms of their reaction modes and structural features.

Biochemical and biotechnological trends in chitin, chitosan, and related enzymes produced by Paenibacillus IK-5 Strain.

We review studies on biochemical characterization of the structures and functions of chitinase, chitosanase, and chitobiase produced by cells of the bacterium, Paenibacillus sp. IK-5. The IK-5 chitinases comprise two GH18 chitinases (ChiA and ChiB), an auxiliary activity family 10 (AA10) chitin oxydehydrolase (ChiC), and a GH19 chitinase (ChiD). The IK-5 chitosanase (ChiE) has a glycosyl hydrolase family 8 (GH8) catalytic domain at the amino-terminus and two discoidin domains (DD) at the carboxyl-terminus. The IK-5 cells also produce chitobiase, containing carbohydrate hydrolase H-20 and S-layer homology domains. Together, these ChiA∼ChiE proteins form a huge complex, designated the "chitinasome". The DD domains bind specifically and tightly to chitosan, suggesting that they are chitosan-specific carbohydrate-binding modules (CBM32); indeed, CBM32 modules have been confirmed to bind to chitosan oligosaccharides (GlcN)2-6. A high-yield secretion system for Ik-5 chitosanase has been constructed using plasmid pNY301 expressed in Bacillus brevis. We also review biotechnological research using chitin, chitosan, crab shell, and IK-5 chitinase and chitosanase. Chitosan has been shown to be useful for efficient gene transfer into microbial and animal cells. IK-5 cell culture and crab shells were effective for the growth of plants and seaweeds.

Solubility-based Separation and Purification of Long-Chain Chitin Oligosaccharides with an Organic-Water Mixed Solvent.

A simple and rapid method for separation and purification of chitin oligosaccharides, (GlcNAc)n, with n ≥ 5 is presented. A commercially available chitin oligosaccharides sample, consisting of (GlcNAc)n with n = 1 - 7, was used as the starting material. Ten milligrams of the material was mixed with 100 µL of the 1 mol/L HCl. All the (GlcNAc)n species were dissolved in the aqueous medium. The aqueous solution was mixed with 900 µL of EtOH; the mixture was centrifuged, and the supernatant was removed to obtain a precipitate. The precipitate was found to consist mainly of (GlcNAc)n with n ≥ 5, indicating the significant difference in solubility between the short-chain (GlcNAc)n species with n ≤ 3 and the longer ones. By the repetition of the operations, a high purity long-chain (GlcNAc)n sample with n ≥ 5 could be prepared successfully. Since the long-chain (GlcNAc)n species are known to have excellent elicitor activity, this sample would be useful in the study of plant pathology, as well as chitin and chitosan chemistry.

Crystal Structure of Chitinase ChiW from Paenibacillus sp. str. FPU-7 Reveals a Novel Type of Bacterial Cell-Surface-Expressed Multi-Modular Enzyme Machinery.

The Gram-positive bacterium Paenibacillus sp. str. FPU-7 effectively hydrolyzes chitin by using a number of chitinases. A unique chitinase with two catalytic domains, ChiW, is expressed on the cell surface of this bacterium and has high activity towards various chitins, even crystalline chitin. Here, the crystal structure of ChiW at 2.1 Å resolution is presented and describes how the enzyme degrades chitin on the bacterial cell surface. The crystal structure revealed a unique multi-modular architecture composed of six domains to function efficiently on the cell surface: a right-handed ß-helix domain (carbohydrate-binding module family 54, CBM-54), a Gly-Ser-rich loop, 1st immunoglobulin-like (Ig-like) fold domain, 1st ß/α-barrel catalytic domain (glycoside hydrolase family 18, GH-18), 2nd Ig-like fold domain and 2nd ß/α-barrel catalytic domain (GH-18). The structure of the CBM-54, flexibly linked to the catalytic region of ChiW, is described here for the first time. It is similar to those of carbohydrate lyases but displayed no detectable carbohydrate degradation activities. The CBM-54 of ChiW bound to cell wall polysaccharides, such as chin, chitosan, ß-1,3-glucan, xylan and cellulose. The structural and biochemical data obtained here also indicated that the enzyme has deep and short active site clefts with endo-acting character. The affinity of CBM-54 towards cell wall polysaccharides and the degradation pattern of the catalytic domains may help to efficiently decompose the cell wall chitin through the contact surface. Furthermore, we clarify that other Gram-positive bacteria possess similar cell-surface-expressed multi-modular enzymes for cell wall polysaccharide degradation.

Determination of Chitin Based on the Colorimetric Assay of Glucosamine in Acidic Hydrolysate.

A colorimetric method for the glucosamine (GlcN) assay was applied for the determination of chitin, which can be hydrolyzed to produce GlcN. A 10-mg sample was mixed with 10 mL of a 5 mol/L HCl aqueous solution, and the mixture was kept at 100°C for 12 h. Under these conditions, chitin was completely depolymerized and deacetylated to produce GlcN, even when the sample was a crab shell. A 20-µL aliquot of the hydrolysate was mixed with 20 µL of a 5 mol/L NaOH aqueous solution and 200 µL of a 50 mmol/L Na2SiO3, 600 mmol/L Na2MoO4, 1.5 mol/L CH3COOH and 30% (v/v) dimethyl sulfoxide solution. The mixture was kept at 70°C for 30 min. In the mixture, GlcN reduced the Mo(VI) species to form a blue molybdosilicate anion, which gave an absorbance maximum at around 750 nm. Since N-acetylglucosamine and chitin oligosaccharides could not render the reaction mixture blue, GlcN in the hydrolysate could be assayed colorimetrically with high selectivity. When a standard chitin sample was examined, the GlcN concentration in the hydrolysate was determined to be 0.97 ± 0.02 g/L (as hydrochloride salt), indicating that the sample contained 10.0 ± 0.2 mg chitin (as an N-acetylglucosamine homopolymer). Calcium cation, amino acids, and proteins did not interfere with the GlcN assay. Thus, the proposed method was successfully applied to determine chitin in a crab shell sample.

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 ß-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 ß-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.

Overexpression, purification, and characterization of Paenibacillus cell surface-expressed chitinase ChiW with two catalytic domains.

Paenibacillus sp. strain FPU-7 produces several different chitinases and effectively hydrolyzes robust chitin. Among the P. FPU-7 chitinases, ChiW, a novel monomeric chitinase with a molecular mass of 150 kDa, is expressed as a cell surface molecule. Here, we report that active ChiW lacking the anchoring domains in the N-terminus was successfully overproduced in Escherichia coli and purified to homogeneity. The two catalytic domains at the C-terminal region were classified as typical glycoside hydrolase family 18 chitinases, whereas the N-terminal region showed no sequence similarity to other known proteins. The vacuum-ultraviolet circular dichroism spectrum of the enzyme strongly suggested the presence of a ß-stranded-rich structure in the N-terminus. Its biochemical properties were also characterized. Various insoluble chitins were hydrolyzed to N,N'-diacetyl-D-chitobiose as the final product. Based on amino acid sequence similarities and site-directed mutagenesis, Glu691 and Glu1177 in the two GH-18 domains were identified as catalytic residues.

Crystallization and preliminary X-ray analysis of the catalytic domains of Paenibacillus sp. strain FPU-7 cell-surface-expressed chitinase ChiW.

The polysaccharide chitin is effectively hydrolyzed and utilized as a carbon and nitrogen source by the Gram-positive bacterium Paenibacillus sp. strain FPU-7. ChiW is a unique cell-surface-expressed chitinase among the Paenibacillus sp. strain FPU-7-secreted chitinases. An N-terminally truncated ChiW protein, primarily comprised of the two catalytic domains of the full-length protein, was successfully overexpressed in Escherichia coli, purified as a functional recombinant protein with a molecular mass of approximately 98 kDa and crystallized. Preliminary X-ray analysis showed that the crystal diffracted to 1.93 Šresolution and belonged to the orthorhombic space group P212121, with unit-cell parameters a = 112.1, b = 128.2, c = 162.6 Å, suggesting the presence of two molecules in an asymmetric unit.

Colorimetric determination of glucosamine and glucose based on the formation of blue molybdosilicate anion and its application to the assay of saccharifying enzyme.

A simple and convenient method of reducing monosaccharide assay is proposed. A Si(IV)-Mo(VI) solution (pH 4.9) was yellow due to the formation of the 11- and/or 12-molybdosilicate(VI) anions. By the addition of a reducing saccharide, glucosamine, the mixture turned to blue gradually, indicating that the Mo(VI) species was reduced by the saccharide to form a blue molybdosilicate anion. The molybdenum blue formation occurred more quickly when a water-miscible organic solvent, dimethyl sulfoxide, was added to the Si(IV)-Mo(VI) solution. Thus, 0.01% level glucosamine can be determined colorimetrically with microtiter plate. Oligochitosan would not interfere with the determination of the glucosamine at the same concentration. Also, a remarkable blue color development of the Si(VI)-Mo(VI) solution was observed by the addition of glucose. On the other hand, maltose, cellobiose, and water-soluble starch at the same concentration level gave no significant coloration of the reaction mixture. Thus, the present monosaccharide assay can be applied advantageously to evaluate the saccharification to produce glucosamine and glucose.

Cooperative degradation of chitin by extracellular and cell surface-expressed chitinases from Paenibacillus sp. strain FPU-7.

Chitin, a major component of fungal cell walls and invertebrate cuticles, is an exceedingly abundant polysaccharide, ranking next to cellulose. Industrial demand for chitin and its degradation products as raw materials for fine chemical products is increasing. A bacterium with high chitin-decomposing activity, Paenibacillus sp. strain FPU-7, was isolated from soil by using a screening medium containing α-chitin powder. Although FPU-7 secreted several extracellular chitinases and thoroughly digested the powder, the extracellular fluid alone broke them down incompletely. Based on expression cloning and phylogenetic analysis, at least seven family 18 chitinase genes were found in the FPU-7 genome. Interestingly, the product of only one gene (chiW) was identified as possessing three S-layer homology (SLH) domains and two glycosyl hydrolase family 18 catalytic domains. Since SLH domains are known to function as anchors to the Gram-positive bacterial cell surface, ChiW was suggested to be a novel multimodular surface-expressed enzyme and to play an important role in the complete degradation of chitin. Indeed, the ChiW protein was localized on the cell surface. Each of the seven chitinase genes (chiA to chiF and chiW) was cloned and expressed in Escherichia coli cells for biochemical characterization of their products. In particular, ChiE and ChiW showed high activity for insoluble chitin. The high chitinolytic activity of strain FPU-7 and the chitinases may be useful for environmentally friendly processing of chitin in the manufacture of food and/or medicine.

The first identification of carbohydrate binding modules specific to chitosan.

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 ß-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.

Properties of the inulinase gene levH1 of Lactobacillus casei IAM 1045; cloning, mutational and biochemical characterization.

Though some genetic features of lactobacillar fructan hydrolases were elucidated, information about their enzymology or mutational analyses were scarce. Lactobacillus casei IAM1045 exhibits extracellular activity degrading inulin. After partial purification of the inulin-degrading protein from the spent culture medium, several fragments were obtained by protease digestion. Based on their partial amino-acid sequences, oligonucleotide primers were designed, and its structural gene (levH1) was determined using the gene library constructed in the E. coli system. The levH1 gene encoded a protein (designated as LevH1), of which calculated molecular mass and pI were 138.8-kDa and 4.66, respectively. LevH1 (1296 amino-acids long) was predicted to have a four-domain structure, containing (i) an N-terminal secretion signal of 40 amino-acids, (ii) variable domain of about 140 residues whose function is unclear, (iii) a catalytic domain of about 630 residues with glycoside-hydrolase activity consisting of two modules, a five-blade ß-propeller module linked to a ß-sandwich module, (iv) a C-terminal domain of about 490 residues comprising five nearly perfect repeat sequences of 80 residues homologous to equivalents of other hypothetical cell surface proteins, followed by 37-residues rich in Ser/Thr/Pro/Gly, a pentad LPQAG (the LPXTG homologue). When overproduced in E. coli, the putative variable-catalytic domain region of about 770 residues exhibited exo-inulinase activity. Deletion analyses demonstrated that the variable-catalytic domain region containing two modules is important for enzymatic activity. Presence of eight conserved motifs (I-VIII) was suggested in the catalytic domain by comparative analysis, among which motif VIII was newly identified in the ß-sandwich module in this study. Site-directed mutagenesis of conserved amino-acids in these motifs revealed that D198, R388, D389 and E440, were crucial for inulinase activity. Moreover, mutations of D502A and D683A in motif VI and VIII respectively caused significant decrease in the activity. These results suggested that the variable domain and ß-sandwich module, besides the ß-propeller module, are important for inulin-degrading activity of LevH1.