Probing the Molecular Structure and Orientation of the Leaf Surface of Brassica oleracea L. by Polarization Modulation-Infrared Reflection-Absorption Spectroscopy.

The surface of most aerial plant organs is covered with the cuticle, a membrane consisting of a variety of organic compounds, including waxes, cutin (a polyester) and polysaccharides. The cuticle serves as the multifunctional interface between the plant and the environment, and plays a major role in protecting plants against various environmental stress factors. Characterization of the molecular arrangements in the intact cuticle is critical for the fundamental understanding of its physicochemical properties; however, this analysis remains technically challenging. Here, we describe the nondestructive characterization of the intact cuticle of Brassica oleracea L. leaves using polarization modulation-infrared (IR) reflection-absorption spectroscopy (PM-IRRAS). PM-IRRAS has a probing depth of less than several hundreds of nanometers, and reveals the crystalline structure of the wax covering the cuticle surface (epicuticular wax) and the nonhydrogen-bonding character of cutin. Combined analysis using attenuated total reflection-IR spectra suggested that hemicelluloses xylan and xyloglucan are present in the outer cuticle region close to the epicuticular wax, whereas pectins are dominant in the inner cuticle region (depth of ≤2 µm). PM-IRRAS can also determine the average orientation of the cuticular molecules, as indicated by the positive and negative spectral peaks. This unique advantage reveals the orientational order in the intact cuticle; the hydrocarbon chains of the epicuticular wax and cutin and the backbones of hemicelluloses are oriented perpendicular to the leaf surface. PM-IRRAS is a versatile, informative and easy-to-use technique for studying plant cuticles because it is nondestructive and does not require sample pretreatment and background measurements.

Expression of cold-adapted ß-1,3-xylanase as a fusion protein with a ProS2 tag and purification using immobilized metal affinity chromatography with a high concentration of ArgHCl.

Cold-adapted ß-1,3-xylanase (P.t.Xyn26A) from the psychrotrophic bacterium, Psychroflexus torquis, was expressed as a fusion protein with tandem repeats of the N-terminal domain of Protein S from Myxocuccus xanthus (ProS2) in Escherichia coli. After cell lysis in phosphate buffer, most of the ProS2-P.t.Xyn26A was located in the insoluble fraction and aggregated during purification. Arginine hydrochloride (ArgHCl) efficiently solubilized the ProS2-P.t.Xyn26A. The solubilized ProS2-P.t.Xyn26A was purified using immobilized metal affinity chromatography (IMAC) with 500 mM ArgHCl. After cleavage of ProS2-P.t.Xyn26A by human rhinovirus 3C protease, we confirmed that recombinant P.t.Xyn26A maintained its native fold. This is the first report of the expression of a cold-adapted enzyme fused with a ProS2 tag under IMAC purification using a high concentration of ArgHCl. These insights into the expression and purification should be useful during the handling of cold-adapted enzymes.

Biochemical characterization of a thermostable ß-1,3-xylanase from the hyperthermophilic eubacterium, Thermotoga neapolitana strain DSM 4359.

The biochemical properties of a putative ß-1,3-xylanase from the hyperthermophilic eubacterium Thermotoga neapolitana DSM 4359 were determined from a recombinant protein (TnXyn26A) expressed in Escherichia coli. This enzyme showed specific hydrolytic activity against ß-1,3-xylan and released ß-1,3-xylobiose and ß-1,3-xylotriose as main products. It displayed maximum activity at 85 °C during a 10-min incubation, and its activity half-life was 23.9 h at 85 °C. Enzyme activity was stable in the pH range 3-10, with pH 6.5 being optimal. Enzyme activity was significantly inhibited by the presence of N-bromosuccinimide (NBS). The insoluble ß-1,3-xylan K m value was 10.35 mg/ml and the k cat value was 588.24 s(-1). The observed high thermostability and catalytic efficiency of TnXyn26A is both industrially desirable and also aids an understanding of the chemistry of its hydrolytic reaction.

Mechanistic insights into the 1,3-xylanases: useful enzymes for manipulation of algal biomass.

Xylanases capable of degrading the crystalline microfibrils of 1,3-xylan that reinforce the cell walls of some red and siphonous green algae have not been well studied, yet they could prove to be of great utility in algaculture for the production of food and renewable chemical feedstocks. To gain a better mechanistic understanding of these enzymes, a suite of reagents was synthesized and evaluated as substrates and inhibitors of an endo-1,3-xylanase. With these reagents, a retaining mechanism was confirmed for the xylanase, its catalytic nucleophile identified, and the existence of -3 to +2 substrate-binding subsites demonstrated. Protein crystal X-ray diffraction methods provided a high resolution structure of a trapped covalent glycosyl-enzyme intermediate, indicating that the 1,3-xylanases likely utilize the (1)S(3) → (4)H(3) → (4)C(1) conformational itinerary to effect catalysis.

Fusion of the OsmC domain from esterase EstO confers thermolability to the cold-active xylanase Xyn8 from Pseudoalteromonas arctica.

The OsmC-region (osmotically induced protein family) of the two-domain esterase EstO from the psychrotolerant bacterium Pseudoalteromonas arctica has been shown to increase thermolability. In an attempt to test if these properties can be conferred to another enzyme, we genetically fused osmC to the 3'-region of the family 8 xylanase encoding gene xyn8 from P. arctica. The chimeric open reading frame xyn8-OsmC was cloned and the chimeric protein was purified after heterologous expression in Escherichia coli. Xyn8 and Xyn8-OsmC showed cold-adapted properties (more than 60% activity at 0°C) using birchwood xylan as the preferred substrate. Maximal catalytic activity is slightly shifted from 15°C (Xyn8) to 20°C for Xyn8-OsmC. Thermostability of Xyn8-OsmC is significantly changed in comparison to wild-type Xyn8. The OsmC-fusion variant showed an apparent decrease in thermostability between 40 and 45°C, while both proteins are highly instable at 50°C.

Expression, crystallization and preliminary X-ray diffraction studies of thermostable ß-1,3-xylanase from Thermotoga neapolitana strain DSM†4359.

Crystals of ß-1,3-xylanase (1,3-ß-D-xylan xylanohydrolase; EC 3.2.1.32) from Thermotoga neapolitana strain DSM 4359 with maximum dimensions of 0.2×0.1×0.02 mm were grown using the sitting-drop vapour-diffusion method at 293 K over 24 h. The crystals diffracted to a resolution of 1.82 Å, allowing structure determination. The crystals belonged to space group P2(1), with unit-cell parameters a=39.061, b=75.828, c=52.140 Å; each asymmetric unit cell contained a single molecule.

Identification and Characterization of a Novel Thermostable and Salt-Tolerant ß-1,3 Xylanase from Flammeovirga pacifica Strain WPAGA1.

ß-1,3 xylanase is an important enzyme in the biorefinery process for some algae. The discovery and characterization of new ß-1,3 xylanase is a hot research topic. In this paper, a novel ß-1,3 xylanase (Xyl88) is revealed from the annotated genome of Flammeovirga pacifica strain WPAGA1. Bioinformatic analysis shows that Xyl88 belongs to the glycoside hydrolase 26 (GH26) with a suspected CBM (carbohydrate-binding module) sequence. The activity of rXyl88 is 75% of the highest enzyme activity (1.5 mol/L NaCl) in 3 mol/L NaCl buffer, which suggests good salt tolerance of rXy188. The optimum reaction temperature in the buffer without NaCl and with 1.5 mol/L NaCl is 45 °C and 55 °C, respectively. Notably, the catalytic efficiency of rXyl88 (kcat/Km) is approximately 20 higher than that of the thermophilic ß-1,3 xylanase that has the highest catalytic efficiency. Xyl88 in this study becomes the most efficient enzyme ever found, and it is also the first reported moderately thermophilic and salt-tolerant ß-1,3 xylanase. Results of molecular dynamics simulation further prove the excellent thermal stability of Xyl88. Moreover, according to the predicted 3D structure of the Xyl88, the surface of the enzyme is distributed with more negative charges, which is related to its salt tolerance, and significantly more hydrogen bonds and Van der Waals force between the intramolecular residues, which is related to its thermal stability.

Cloning of a novel gene encoding beta-1,3-xylosidase from a marine bacterium, Vibrio sp. strain XY-214, and characterization of the gene product.

The beta-1,3-xylosidase gene (xloA) of Vibrio sp. strain XY-214 was cloned and expressed in Escherichia coli. The xloA gene consisted of a 1,608-bp nucleotide sequence encoding a protein of 535 amino acids with a predicted molecular weight of 60,835. The recombinant beta-1,3-xylosidase hydrolyzed beta-1,3-xylooligosaccharides to D-xylose as a final product.

Shifting pH optimum of Bacillus circulans xylanase based on molecular modeling.

Although hydrolases are used in several industrial processes, its industrial applications have some limitations in specific cases since some industrial processes are carried out at pH value which is different from optimum pH of the enzyme. Alkaline side optimum pH of hydrolases is always desirable, and it is proved difficult to achieve that by mutation only. Hence, molecular modeling was applied to select the promising mutants. The changes in electrostatic potential, which was calculated using Delphi, were compared to the changes in pH optimum of four hydolases and their mutants. The results showed that the change in electrostatic potential can be used as an indicator for selecting relevant candidates of mutation. Bacillus circulans xylanase was selected as a model system, and the promising mutants were picked up by the molecular modeling. Q167M and R73V, had a higher pH optimum than the wild type, while K175Q had a similar pH-activity profile of the wild type.

Characterization of a novel polyphenol-specific oligoxyloside transfer reaction by a family 11 xylanase from Bacillus sp. KT12.

A culture filtrate of Bacillus sp. KT12 was used to prepare polyphenyl beta-oligoxylosides from xylan and polyphenols in a one-step reaction. One oligoxyloside transfer enzyme was purified from multiple xylanolytic enzymes in the culture filtrate. N-terminal amino acid sequence determination classified the enzyme as a glycosyl hydrolase family 11 (endo-xylanase). The xylanolytic enzyme activities could be markedly altered; its hydrolytic activity was almost entirely inhibited at acidic pH, whereas near constant transxylosylation activity was observed at pH 4-11. Further, metal ions activated transxylosylation and almost completely inhibited hydrolysis. The enzyme specifically induced a beta-xylosyl transfer reaction to acceptor molecules, such as divalent and trivalent phenolic hydroxyl groups, and displayed no activity toward alcoholic compounds. The Bacillus sp. KT12 xylanolytic enzyme was a suitable enzyme for the synthesis of polyphenyl beta-oligoxylosides.

Identification and characterization of the first ß-1,3-d-xylosidase from a gram-positive bacterium, Streptomyces sp. SWU10.

In previous reports, we characterized four endo-xylanases produced by Streptomyces sp. strain SWU10 that degrade xylans to several xylooligosaccharides. To obtain a set of enzymes to achieve complete xylan degradation, a ß-d-xylosidase gene was cloned and expressed in Escherichia coli, and the recombinant protein, named rSWU43A, was characterized. SWU43A is composed of 522 amino acids and does not contain a signal peptide, indicating that the enzyme is an intracellular protein. SWU43A was revealed to contain a Glyco_hydro_43 domain and possess the three conserved amino acid residues of the glycoside hydrolase family 43 proteins. The molecular mass of rSWU43A purified by Ni-affinity column chromatography was estimated to be 60kDa. The optimum reaction conditions of rSWU43A were pH 6.5 and 40°C. The enzyme was stable up to 40°C over a wide pH range (3.1-8.9). rSWU43A activity was enhanced by Fe2+ and Mn2+ and inhibited by various metals (Ag+, Cd2+, Co2+, Cu2+, Hg2+, Ni2+, and Zn2+), d-xylose, and l-arabinose. rSWU43A showed activity on p-nitrophenyl-ß-d-xylopyranoside and p-nitrophenyl-α-l-arabinofuranoside substrates, with specific activities of 0.09 and 0.06U/mg, respectively, but not on any xylosidic or arabinosidic polymers. rSWU43A efficiently degraded ß-1,3-xylooligosaccharides to produce xylose but showed little activity towards ß-1,4-xylobiose, with specific activities of 1.33 and 0.003U/mg, respectively. These results demonstrate that SWU43A is a ß-1,3-d-xylosidase (EC 3.2.1.72), which to date has only been described in the marine bacterium Vibrio sp. Therefore, rSWU43A of Streptomyces sp. is the first ß-1,3-xylosidase found in gram-positive bacteria. SWU43A could be useful as a specific tool for the structural elucidation and production of xylose from ß-1,3-xylan in seaweed cell walls.

Biochemical characterization of a novel beta-1--3, 1--4 glucan 4-glucanohydrolase from Thermomonospora sp. having a single active site for lichenan and xylan.

A bifunctional high molecular weight (Mr, 64,500 Da) beta-1-3, 1-4 glucan 4-glucanohydrolase was purified to homogeneity from Thermomonospora sp., exhibiting activity towards lichenan and xylan. A kinetic method was used to analyze the active site that hydrolyzes lichenan and xylan. The experimental data was in agreement with the theoretical values calculated for a single active site. Probing the conformation and microenvironment at active site of the enzyme by fluorescent chemo-affinity label, OPTA resulted in the formation of an isoindole derivative with complete inactivation of the enzyme to hydrolyse both lichenan and xylan confirmed the results of kinetic method. OPTA forms an isoindole derivative by cross-linking the proximal thiol and amino groups. The modification of cysteine and lysine residues by DTNB and TNBS respectively abolished the ability of the enzyme to form an isoindole derivative with OPTA, indicating the participation of cysteine and lysine in the formation of isoindole complex.

Kinetic and thermodynamic study of a chemically modified highly active xylanase from Scopulariopsis sp: existence of an essential amino group.

The amino groups of purified least acidic xylanase (LAX) isomer and carboxyl groups of purified highly acidic xylanase (HAX) isomer from Scopulariopsis sp. were chemically modified, resulting in charge neutralization and reversal. Modification of the second amino group was accompanied by the complete loss of enzyme activity in both the absence and presence of xylose. Multiple alignments of family 10 and 11 xylanases revealed that there is a pair of fully conserved Lys residues only in family 10 members. Xylanase structures from family 10 members showed that one of the conserved Lys residues is found near the active-site cleft that makes an H-bond with the substrate. The LAX and HAX isoenzymes in which one amino and three to four carboxyl groups were modified were subjected to kinetic and thermodynamic characterization. There were no differences in pH optima between the native and modified HAX, but there was a broadening of pH optimum toward the alkaline range for charge-neutralized LAX and a double pH optimum for charge-reversed LAX. TheV max/K m of both modified LAX and HAX decreased relative to the native species. The thermodynamics of xylan hydrolysis showed that the decrease in the catalytic activity of modified LAX enzymes was entropically driven. When compared with native enzyme, the thermostabilities of modified LAX enzymes increased in the presence and decreased in the absence of substrate. The thermodynamics of kinetic stability for modified LAX enzymes revealed that this increase in thermolability was owing to the decrease in DeltaH# with a concomitant increase in DeltaS# compared with native LAX. The thermostabilities of all the modified HAX species decreased except that of charge-neutralized HAX, whose half-life significantly increased in 50% (v/v) aqueous dioxan. These results suggest that the altered properties of the modified enzymes were a result of the conformational changes brought about by chemical modification.

Structure of beta-1,3-xylooligosaccharides generated from Caulerpa racemosa var. laete-virens beta-1,3-xylan by the action of beta-1,3-xylanase.

Recently we reported the molecular cloning and characterization of a novel beta-1,3-xylanase from the marine bacterium Vibrio sp. AX-4 [Kiyohara et al. (2005) Biochem. J. 388, 949-957]. We report here the structural analysis of oligosaccharides generated from beta-1,3-xylan of a siphonous green alga, Caulerpa racemosa var. laete-virens, by the action of beta-1,3-xylanase. The enzyme degraded the polysaccharide producing oligosaccharides with different R(f)s on TLC (EX2-EX5). Sugar component, linkage, and MALDI-TOF-MS analyses revealed that EX2 and EX3 were Xyl-1,3-Xyl and Xyl-1,3-Xyl-1,3-Xyl, respectively. On the other hand, EX4 was a mixture of Glc-1,3-Xyl-1,3-Xyl, Xyl-1,4-Xyl-1,3-Xyl and Xyl-1,3-Xyl-1,4-Xyl, while EX5 was a mixture of tetra-saccharides containing 3-substitued Glc in addition to the same components of EX4. Branching was not likely present in EXOs prepared from the polysaccharide by the enzyme. These results strongly suggest that the C. racemosa beta-1,3-xylan is a linear heteropolysaccharide containing 1,3-Glc and 1,4-Xyl both of which are thought to be located within a beta-1,3-Xyl chain and linked via covalent bonds. This report indicates the usefulness of the enzyme for the structural analysis of beta-1,3-xylan.

Molecular cloning and characterization of a novel beta-1,3-xylanase possessing two putative carbohydrate-binding modules from a marine bacterium Vibrio sp. strain AX-4.

We cloned a novel beta-1,3-xylanase gene, consisting of a 1728-bp open reading frame encoding 576 amino acid residues, from a marine bacterium, Vibrio sp. strain AX-4. Sequence analysis revealed that the beta-1,3-xylanase is a modular enzyme composed of a putative catalytic module belonging to glycoside hydrolase family 26 and two putative carbohydrate-binding modules belonging to family 31. The recombinant enzyme hydrolysed beta-1,3-xylan to yield xylo-oligosaccharides with different numbers of xylose units, mainly xylobiose, xylotriose and xylotetraose. However, the enzyme did not hydrolyse beta-1,4-xylan, beta-1,4-mannan, beta-1,4-glucan, beta-1,3-xylobiose or p-nitrophenyl-beta-xyloside. When beta-1,3-xylo-oligosaccharides were used as the substrate, the kcat value of the enzyme for xylopentaose was found to be 40 times higher than that for xylotetraose, and xylotriose was extremely resistant to hydrolysis by the enzyme. A PSI-BLAST search revealed two possible catalytic Glu residues (Glu-138 as an acid/base catalyst and Glu-234 as a nucleophile), both of which are generally conserved in glycoside hydrolase superfamily A. Replacement of these two conserved Glu residues with Asp and Gln resulted in a significant decrease and complete loss of enzyme activity respectively, without a change in their CD spectra, suggesting that these Glu residues are the catalytic residues of beta-1,3-xylanase. The present study also clearly shows that the non-catalytic putative carbohydrate-binding modules play an important role in the hydrolysis of insoluble beta-1,3-xylan, but not that of soluble glycol-beta-1,3-xylan. Furthermore, repeating a putative carbohydrate-binding module strongly enhanced the hydrolysis of the insoluble substrate.

The first crystal structure of a family 31 carbohydrate-binding module with affinity to beta-1,3-xylan.

Here, we present the crystal structure of the family 31 carbohydrate-binding module (CBM) of beta-1,3-xylanase from Alcaligenes sp. strain XY-234 (AlcCBM31) determined at a resolution of 1.25A. The AlcCBM31 shows affinity with only beta-1,3-xylan. The AlcCBM31 molecule makes a beta-sandwich structure composed of eight beta-strands with a typical immunoglobulin fold and contains two intra-molecular disulfide bonds. The folding topology of AlcCBM31 differs from that of the large majority of other CBMs, in which eight beta-strands comprise a beta-sandwich structure with a typical jelly-roll fold. AlcCBM31 shows structural similarity with CBM structures of family 34 and family 9, which also adopt structures based on immunoglobulin folds.

The modular xylanase Xyn10A from Rhodothermus marinus is cell-attached, and its C-terminal domain has several putative homologues among cell-attached proteins within the phylum Bacteroidetes.

Until recently, the function of the fifth domain of the thermostable modular xylanase Xyn10A from Rhodothermus marinus was unresolved. A putative homologue to this domain was however identified in a mannanase (Man26A) from the same microorganism which raised questions regarding a common function. An extensive search of all accessible data-bases as well as the partially sequenced genomes of R. marinus and Cytophaga hutchinsonii showed that homologues of this domain were encoded by multiple genes in microorganisms in the phylum Bacteroidetes. Moreover, the domain occurred invariably at the C-termini of proteins that were predominantly extra-cellular/cell attached. A primary structure motif of three conserved regions including structurally important glycines and a proline was also identified suggesting a conserved 3D fold. This bioinformatic evidence suggested a possible role of this domain in mediating cell attachment. To confirm this theory, R. marinus was grown, and activity assays showed that the major part of the xylanase activity was connected to whole cells. Moreover, immunocytochemical detection using a Xyn10A-specific antibody proved presence of Xyn10A on the R. marinus cell surface. In the light of this, a revision of experimental data present on both Xyn10A and Man26A was performed, and the results all indicate a cell-anchoring role of the domain, suggesting that this domain represents a novel type of module that mediates cell attachment in proteins originating from members of the phylum Bacteroidetes.

Effect of tributylphenyltetraethoxylate on enzyme production of Pleurotus ostreatus.

The effect of the addition of the nonionic surfactant tributylphenyltetraethoxylate to culture media on pH and extracellular protein content, and on production of beta-glucosidase, xylanase, laccase, and manganese-dependent and -independent peroxidases by the edible fungus Pleurotus ostreatus was determined. The relationship between fermentation parameters and concentration of surfactant was assessed by multiple linear regression analysis, and the similarities and differences among the fermentation parameters were elucidated by principal component analysis. Calculations proved that except for xylanase all other cultivation parameters were significantly influenced by the surfactant, with the effect higher at higher surfactant concentrations. Surfactant increased the production of beta-glucosidase and inhibited laccase and manganese-dependent and -independent peroxidase activities.

Co-immobilization mechanism of cellulase and xylanase on a reversibly soluble polymer.

Cellulase and xylanase from Trichoderma reesei were immobilized simultaneously on Eudragit L-100, a reversibly soluble polymer. The effects of polymer concentration and polymer precipitation pH on enzyme activity recovery were investigated at an enzyme complex concentration of 1%. The immobilization mechanism of cellulase and xylanase on the polymer was discussed. An activity recovery of 75% and 59% was obtained for the cellulase and the xylanase, respectively, under the condition of a polymer concentration at 2% and a polymer precipitation pH at 4.0. Most zymoproteins might be connected to the polymer by electrostatic attraction in a medium of pH 4.8. In addition, the covalent coupling between the zymoproteins and the polymer was demonstrated by the infrared spectrograms. It was suggested that dehydration-condensation reaction occurred between the zymoproteins and the polymer during the immobilization.

Inactive fluorescently labeled xylanase as a novel probe for microscopic analysis of arabinoxylan containing cereal cell walls.

A new technique to visualize cereal cell walls by fluorescence microscopy was developed. The novel staining technique is based on an inactive fluorescently labeled xylanase binding to arabinoxylan (AX), an important polysaccharide in grain cell walls in terms of the technological and physiological functionalities of grain. The xylanase probe could stain AX in the seed coat, nucellar epidermis, aleurone layer, and starchy endosperm, but not the highly substituted AX of the pericarp layer. The advantage of this new staining technique over the existing immunolabeling techniques is that the staining procedure is clearly faster and less laborious, and uses a smaller probe that can easily be produced by marking a well characterized enzyme with a fluorescent label. In the future, the here proposed technology can be used to develop probes having specificity also for cell wall components other than AX and thus to study plant cell walls further through fluorescence microscopy.