Enzymatic degradation of algal 1,3-xylan: from synergism of lytic polysaccharide monooxygenases with ß-1,3-xylanases to their intelligent immobilization on biomimetic silica nanoparticles.

Lytic polysaccharide monooxygenases (LPMOs) with synergistic effect on polysaccharide hydrolase represent a revolution in biotechnology, which may accelerate the conversion of biomass to the second-generation biofuels. Discovering more hydrolases that have synergism with LPMOs will considerably expand the knowledge and application of biomass degradation. The LPMOs named CgAA9 were verified to exhibit 1.52-fold synergism when incubated with ß-1,3-xylanase at a molar ratio of 3:1. The ion chromatography results proved that CgAA9 did not alter the endogenous hydrolysis mode of ß-1,3-xylanase. Meanwhile, to decrease the operational cost of enzymes, a novel strategy for immobilizing LPMOs and ß-1,3-xylanases based on the biomimetic silica nanoparticles was developed. It enabled preparation of immobilized enzymes directly from the cell lysate. The immobilization efficiency and activity recovery reached 84.6 and 81.4%. They showed excellent reusability for 12 cycles by retaining 68% of initial activity. The optimum temperature for both free and immobilized biocatalyst were 40 and 37 °C, indicating they were ideal candidates for typical simultaneous saccharification and fermentation (SSF) in ethanol production from algea biomass. This was the first report on the synergy between LPMOs and ß-1,3-xylanase, and the strategy for enzyme self-immobilization was simple, timesaving, and efficient, which might have great potentials in algae biomass hydrolysis. KEY POINTS: • The lytic polysaccharide monooxygenases (LPMOs) from Chaetomium globosum were firstly verified to boost the hydrolysis of ß-1,3-xylanases for ß-1,3-xylan. • A novel strategy for simple preparation of SpyCather-modifed silica nanopartilcles and intelligent immobilization of target enzymes from the cell lysate was proposed. • The immobilized LPMOs and ß-1,3-xylanases could be reasonable alternatives for typical simultaneous saccharification and fermentation (SSF) in manipulation of algae biomass.

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.

The effect of different dietary levels of hybrid rye and xylanase addition on the performance and egg quality in laying hens.

1. In this study, 240 ISA Brown hens were fed diets containing different levels of hybrid rye, and the influence of xylanase addition on laying performance and egg quality was evaluated. 2. Birds were allocated to 10 treatment groups with 12 replicates (cages) of two hens and were fed, from week 26 to 50, isocaloric and isonitrogenous experimental diets. A 5 × 2 experimental arrangement was applied, using diets with increasing level of rye (0%, 10%, 15%, 20% or 25%) with or without xylanase supplementation (200 mg/kg of feed; Ronozyme WX (CT) with minimum xylanase activity of 1,000 FXU/g). 3. Increasing dietary level of rye did not affect daily mass of eggs, mean egg weight or feed conversion ratio (P > 0.05). Laying rate decreased in all groups fed with rye. Egg and eggshell quality indices were unaffected by dietary rye grain (P > 0.05); however, rye inclusion significantly decreased yolk colour on the DSM scale (P < 0.05). In comparison with the control group, high dietary levels of rye (25%) significantly increased viscosity of small intestine content (P < 0.05). Diet supplementation with xylanase had no significant effect on egg production indices and egg quality (except for yolk colour) but decreased the viscosity of intestinal content in laying hens fed high levels of rye (P < 0.05). 4. The results of this experiment suggest that rye may be incorporated to a level of 25% in the diet of laying hens without any strong negative effect on egg performance, while xylanase added to high-rye grain reduced the viscosity of intestinal content; however, it did not positively affect the laying performance or egg quality.

Identification of two transcription factors activating the expression of OsXIP in rice defence response.

BACKGROUND: Xylanase inhibitors have been confirmed to be involved in plant defence. OsXIP is a XIP-type rice xylanase inhibitor, yet its transcriptional regulation remains unknown. RESULTS: Herbivore infestation, wounding and methyl jasmonate (MeJA) treatment enhanced mRNA levels and protein levels of OsXIP. By analyzing different 5' deletion mutants of OsXIP promoter exposed to rice brown planthopper Nilaparvata lugens stress, a 562 bp region (-1451 - -889) was finally identified as the key sequence for the herbivores stress response. Using yeast one-hybrid screening, coupled with chromatin immunoprecipitation analysis, a basic helix-loop-helix protein (OsbHLH59) and an APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF) transcription factor OsERF71 directly binding to the 562 bp key sequence to activate the expression of OsXIP were identified, which is further supported by transient expression assay. Moreover, transcriptional analysis revealed that mechanical wounding and treatment with MeJA resulted in an obvious increase in transcript levels of OsbHLH59 and OsERF71 in root and shoot tissues. CONCLUSIONS: Our data shows that two proteins as direct transcriptional activators of OsXIP responding to stress were identified. These results reveal a coordinated regulatory mechanism of OsXIP, which may probably be involved in defence responses via a JA-mediated signaling pathway.

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.

Heterologous expression of a gene for thermostable xylanase from Chaetomium thermophilum in Pichia pastoris GS115.

We studied heterologous expression of xylanase 11A gene of Chaetomium thermophilum in Pichia pastoris and characterized the thermostable nature of the purified gene product. For this purpose, the xylanase 11A gene of C. thermophilum was cloned in P. pastoris GS115 under the control of AOX1 promoter. The maximum extracellular activity of recombinant xylanase (xyn698: gene with intron) was 15.6 U ml(-1) while that of recombinant without intron (xyn669) was 1.26 U ml(-1) after 96 h growth. The gene product was purified apparently to homogeneity level. The optimum temperature of pure recombinant xylanase activity was 70°C and the enzyme retained its 40.57% activity after incubation at 80°C for 10 min. It exhibited quite lower demand of activation energy, enthalpy, Gibbs free energy, entropy, and xylan binding energy during substrate hydrolysis than that required by that of the donor, thus indicating its thermostable nature. pH-dependent catalysis showed that it was quite stable in a pH range of 5.5-8.5. This revealed that gene was successfully processed in P. pastoris and remained heat stable and may qualify for its potential use in paper and pulp and animal feed applications.

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.

Optimization of xylanase production from Aspergillus niger for biobleaching of eucalyptus pulp.

A crude endo-xylanase produced by Aspergillus niger BCC14405 was investigated for its potential in pre-bleaching of chemical pulp from eucalyptus. The optimal fermentation conditions on the basis of optimization using response surface methodology included cultivation in a complex medium comprising wheat bran, rice bran, and soybean meal supplemented with yeast extract, glucose, peptone, and lactose with a starting pH of 6.0 for 7 d. This resulted in production of 89.5 IU/mL of xylanase with minor cellulase activity. Proteomic analysis using LC/MS/MS revealed that the crude enzyme was a composite of hemicellulolytic enzymes, including endo-ß-1,4-xylanase and other hemicellulolytic enzymes attacking arabinoxylan and mannan. Pretreatment of the pulp at a xylanase dosage of 10 IU/g increased the brightness ceiling after the C-Eop-H bleaching step up to 3.0% using a chlorine charge with a C-factor of 0.16-0.20. Xylanase treatment also led to reduction in chlorine charge of at least 20%, with an acceptable brightness level. The enzyme pretreatment resulted in a slight increase in pulp viscosity, suggesting an increase in relative cellulose content. The crude enzyme was potent in the enzyme-aided bleaching of chemical pulp in an environmentally friendly pulping process.

Optimization of cellulase-free xylanase production by thermophilic Streptomyces thermovulgaris TISTR1948 through Plackett-Burman and response surface methodological approaches.

Cellulase-free xylanase production by thermophilic Streptomyces thermovulgaris TISTR1948 was cultivated in a basal medium with rice straw as sole source of carbon and as an inducible substrate. Variable medium components were selected in accordance with the Plackett-Burman experimental design. The optimization conditions of physical factors (pH and temperature levels) were then combined in further studies through the response surface methodology approach. Only two significant components, rice straw and yeast extract, were chosen for the optimization studies. A second-order quadratic model was constructed by central composite design (CCD). The model revealed that both pH and temperature levels were significant, and were dependent on xylanase production. Under these experimental designs, the xylanase yield increased from 51.11 to 274.49 U/mL (3,400 to 10,000 U/g of rice straw) or about 537% higher than an unoptimized basal medium. The optimum conditions to achieve maximum yield of xylanase were 27.45 g/L of rice straw and 5.42 g/L of yeast extract under relatively neutral conditions of pH 7.11, 50.03 °C, and a incubation period.

[Creation of a heterologous gene expression system on the basis of Aspergillus awamori recombinant strain].

A heterologous gene expression system was created in a domestic Aspergillus awamori Co-6804 strain, which is a producer of the glucoamylase gene. Vector pGa was prepared using promoter and terminator areas of the glucoamylase gene, and A. niger phytase, Trichoderma reesei endoglucanase, and Penicillium canescens xylanase genes were then cloned into pGa vector. Separation of enzyme samples using FPLC showed the amount of the recombinant proteins to be within the 0.6-14% range of total protein.

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.

An endoxylanase rapidly hydrolyzes xylan into major product xylobiose via transglycosylation of xylose to xylotriose or xylotetraose.

Here, we proposed an effective strategy to enhance a novel endoxylanase (Taxy11) activity and elucidated an efficient catalysis mechanism to produce xylooligosaccharides (XOSs). Codon optimization and recruitment of natural propeptide in Pichia pastoris resulted in achievement of Taxy11 activity to 1405.65 ±â€¯51.24 U/mL. Analysis of action mode reveals that Taxy11 requires at least three xylose (xylotriose) residues for hydrolysis to yield xylobiose. Results of site-directed mutagenesis indicate that residues Glu119, Glu210, and Asp53 of Taxy11 are key catalytic sites, while Asp203 plays an auxiliary role. The novel mechanism whereby Taxy11 catalyzes conversion of xylan or XOSs into major product xylobiose involves transglycosylation of xylose to xylotriose or xylotetraose as substrate, to form xylotetraose or xylopentaose intermediate, respectively. Taxy11 displayed highly hydrolytic activity toward corncob xylan, producing 50.44 % of xylobiose within 0.5 h. This work provides a cost-effective and sustainable way to produce value-added biomolecules XOSs (xylobiose-enriched) from agricultural waste.

Expression of endo-1, 4-beta-xylanase from Trichoderma reesei in Pichia pastoris and functional characterization of the produced enzyme.

BACKGROUND: In recent years, xylanases have attracted considerable research interest because of their potential in various industrial applications. The yeast Pichia pastoris can neither utilize nor degrade xylan, but it possesses many attributes that render it an attractive host for the expression and production of industrial enzymes. RESULTS: The Xyn2 gene, which encodes the main Trichoderma reesei Rut C-30 endo-beta-1, 4-xylanase was cloned into the pPICZalphaA vector and expressed in Pichia pastoris. The selected P. pastoris strains produced as 4,350 nkat/ml beta-xylanase under the control of the methanol inducible alcohol oxidase 1 (AOX1) promoter. The secreted recombinant Xyn2 was estimated by SDS-PAGE to be 21 kDa. The activity of the recombinant Xyn2 was highest at 60 degrees C and it was active over a broad range of pH (3.0-8.0) with maximal activity at pH 6.0. The enzyme was quite stable at 50 degrees C and retained more than 94% of its activity after 30 mins incubation at this temperature. Using Birchwood xylan, the determined apparent Km and kcat values were 2.1 mg/ml and 219.2 S-1, respectively. The enzyme was highly specific towards xylan and analysis of xylan hydrolysis products confirmed as expected that the enzyme functions as endo-xylanase with xylotriose as the main hydrolysis products. The produced xylanase was practically free of cellulolytic activity. CONCLUSION: The P. pastoris expression system allows a high level expression of xylanases. Xylanase was the main protein species in the culture supernatant, and the functional tests indicated that even the non-purified enzyme shows highly specific xylanase activity that is free of cellulolytic side acitivities. Therefore, P pastoris is a very useful expression system when the goal is highly specific and large scale production of glycosyl hydrolases.

Gene cloning, expression and characterization of a new cold-active and salt-tolerant endo-beta-1,4-xylanase from marine Glaciecola mesophila KMM 241.

Although a lot of xylanases are studied, only a few xylanases from marine microorganisms have been reported. A new xylanase gene, xynA, was cloned from marine bacterium Glaciecola mesophila KMM 241. Gene xynA contains 1,272 bp and encodes a 423-amino acid xylanase precursor. The recombinant xylanase, XynA, expressed in Escherichia coli BL21 is a monomer with a molecular mass of 43 kDa. Among the characterized xylanases, XynA shares the highest identity (46%) to the xylanase from Flavobacterium sp. strain MSY2. The optimum pH and temperature for XynA is 7.0 and 30 degrees C. XynA retains 23% activity and 27% catalytic efficiency at 4 degrees C. XynA has low thermostability, remaining 20% activity after 60-min incubation at 30 degrees C. Its apparent melting temperature (T (m)) is 44.5 degrees C. These results indicate that XynA is a cold-active xylanase. XynA shows a high level of salt-tolerance, with the highest activity at 0.5 M NaCl and retaining 90% activity in 2.5 M NaCl. It may be the first salt-tolerant xylanase reported. XynA is a strict endo-beta-1,4-xylanase with a demand of at least four sugar moieties for effective cleavage. It efficiently hydrolyzes xylo-oligosaccharides and xylan into xylobiose and xylotriose without producing xylose, suggesting its potential in xylo-oligosaccharides production.

Enzyme complex containing carbohydrases and phytase improves growth performance and bone mineralization of broilers fed reduced nutrient corn-soybean-based diets.

One experiment was conducted to investigate the benefits of a multi-enzyme complex, containing carbohydrases (from Penicillium funiculosum) and phytase (bacterial 6-phytase) activities, on the performance and bone mineralization of broiler chickens fed corn-soybean meal diets. A total of 2,268 male broilers were allocated to 9 treatments, replicated 6 times, in a randomized complete block design from 1 to 43 d. A positive control (PC) diet formulated to be adequate in nutrients and 4 reduced nutrient diets (NC1 to NC4), with gradual decrease on AME, CP, and digestible amino acids (CP-dAA) and available P (avP) and Ca contents, with or without enzyme supplementation, were tested. The nutrient reductions applied were NC1 (-65 kcal/kg, -1.5% CP-dAA) and NC2 (-85 kcal/kg, -3.0% CP-dAA) both with -0.15 percent point avP and -0.12 percent point Ca and NC3 (-65 kcal/kg, -1.5% CP-dAA) and NC4 (-85 kcal/kg, -3.0% CP-dAA) both with -0.20 percent point avP and -0.16 percent point Ca. Supplementation of the NC diets with the enzyme complex increased ADFI (P<0.001), ADG (P<0.001), and reduced feed:gain (P<0.01). The magnitude of the enzyme effect in increasing feed intake and weight gain was greater for the diets with greatest reductions in avP and Ca. Enzyme supplementation increased (P<0.001) feed intake of birds fed on NC diets close to the level of feed consumption of the PC. Enzyme supplementation to NC diets resulted in all cases in lower (P<0.05) feed:gain than the PC. Enzyme supplementation to NC1 and NC3 diets restored bone mineralization to that of the PC, whereas ash and Ca with NC2 and NC4 diets and P with NC4 diet remained lower (P<0.05). These results suggest that the dietary supplementation with a multi-enzyme complex containing nonstarch polysaccharide enzymes and phytase is efficient in reducing the P, energy, protein, and amino acid specifications of corn-soybean meal diets.

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.