Comparison of bovine rumen liquor and bovine faeces as inoculum for an in vitro gas production technique for evaluating forages

Two experiments were undertaken using the in vitro gas production technique of Theodorou et al. [Anim. Feed Sci. Technol. 48 (1994) 185] to compare rumen liquor (RL) and faeces (FA) as inocula for fermenting gramminaceous forages over 96 h periods. Experiment 1 used 12 forages of differing in vivo digestibility (ammonia treated wheat straw, field-cured hay (Lolium perenne) and 10 artificially dried grasses (L. perenne) harvested at different maturities). Experiment 2 used seven maize-silage based forages (whole plant, stover, leaf, lower stem, middle stem, upper stem and husk). In both experiments, rumen liquor and faeces were obtained from two cows in early lactation, each fed daily with 9.4 kg DM of grass silage and 9.0 kg DM of concentrate. Rumen contents were sampled through the fistula, before morning feeding; faeces were sampled from the rectum, immediately afterwards. Rumen liquor (250 ml) was prepared by straining contents through two layers of muslin, adding the solids after blending with 250 ml of buffer and re-straining. Faeces were prepared by mixing (300 ml) with 150 ml of buffer and straining through two layers of muslin and adding a homogenate of the solids and 150 ml of buffer after straining. Data were fitted to the model of France et al. [J. Theor. Biol. 163 (1993) 99]. All model parameters showed FA to have a poorer fermentation capacity than RL. In both experiments, potential gas production volumes (A) were lower (on average 52.9 ml (18.5%)) and lag times longer (on average 2.9 h) for FA compared to RL. Fractional rate of fermentation at half asymptote (T/2) was generally greater for RL than FA (overall means, 0.042, 0.028) and the time required to T/2 being less (overall means, 21.9, 35.4 h). However, potential gas production (A) was highly correlated between RL and FA: Experiment 1 (r²=0.94, 11 forages, excluding ammonia treated straw) and Experiment 2 (r²=0.83, six forages, excluding middle stem). In Experiment 1, organic matter digestibility in vivo (OMD_IV) was also highly correlated with both OMD_FA (r²=0.77, 11 forages) and OMD_RL (r²=0.89, 11 forages); OMD_RL and OMD_FA were also highly correlated (r²=0.81). Similar correlations occurred in Experiment 2. It is concluded that faeces have potential as an alternative inoculum to rumen liquor for in vitro gas production techniques, but methods of overcoming the longer lag phase with faeces require further research.     

Characterization of a recombinant thermostable β-glucosidase from Putranjiva roxburghii expressed in Saccharomyces cerevisiae and its use for efficient biomass conversion

A β-glucosidase gene from Putranjiva roxburghii (PRGH1) was heterologously expressed in Saccharomyces cerevisiae to enable growth on cellobiose. The recombinant enzyme was secreted to the culture medium, purified and biochemically characterized. The enzyme is a glycoprotein with a molecular weight of ∼68 kDa and exhibited enzymatic activity with β‐linked aryl substrates like pNP-Fuc, pNP-Glc, pNP-Gal and pNP-Cel with catalytic efficiency in that order. Significant enzyme activity was observed for cellobiose, however the enzyme activity was decreased with increase in chain length of glycan substrates. Using cellobiose as substrate, the enzyme showed optimal activity at pH 5.0 and 65 °C. The enzyme was thermostable up to 75 °C for 60 min. The enzyme showed significant resistance towards both glucose and ethanol induced inhibition. The recombinant S. cerevisiae strain showed advantages in cell growth, glucose and bio-ethanol production over the native strain with cellobiose as sole carbon source. In simultaneous saccharification and fermentation (SSF) experiments, the recombinant strain was used for bio-ethanol production from two different cellulosic biomass sources. At the end of the SSF, we obtained 9.47 g L⁻¹ and 14.32 g L⁻¹ of bio-ethanol by using carboxymethyl cellulose and pre-treated rice straw respectively. This is first report where a β-glucosidase gene from plant origin has been expressed in S. cerevisiae and used in SSF.     

Fermentative production of succinic acid from straw hydrolysate by Actinobacillus succinogenes

In this work, straw hydrolysates were used to produce succinic acid by Actinobacillus succinogenes CGMCC1593 for the first time. Results indicated that both glucose and xylose in the straw hydrolysates were utilized in succinic acid production, and the hydrolysates of corn straw was better than that of rice or wheat straw in anaerobic fermentation of succinic acid. However, cell growth and succinic acid production were inhibited when the initial concentration of sugar, which was from corn straw hydrolysate (CSH), was higher than 60 g l^?1. In batch fermentation, 45.5 g l^?1 succinic acid concentration and 80.7% yield were attained after 48 h incubation with 58 g l^?1 of initial sugar from corn straw hydrolysate in a 5-l stirred bioreactor. While in fed-batch fermentation, concentration of succinic acid achieved 53.2 g l^?1 at a rate of 1.21 g l^?1 h^?1 after 44 h of fermentation. Our work suggested that corn straw could be utilized for the economical production of succinic acid by A. succinogenes.     

Functional consortium for hydrogen production from cellobiose: Concentration-to-extinction approach

A functional bacterial consortium that can effectively hydrolyze cellobiose and produce bio-hydrogen was isolated by a concentration-to-extinction approach. The sludge from a cattle feedlot manure composting plant was incubated with 2.5–20 g l^?1 cellobiose at 35 °C and pH 6.0. The microbial diversity of serially concentrated suspensions significantly decreased following increasing cellobiose concentration, finally leaving only two viable strains, Clostridium butyricum strain W4 and Enterococcus saccharolyticus strain. This consortium has a maximum specific hydrogen production rate of 2.19 mol H₂ mol hexose^?1 at 5 g l^?1 cellobiose. The metabolic pathways shifted from ethanol-type to acetate-butyrate type as cellobiose concentration increased from 2.5 to >7 g l^?1. The concentration-to-extinction approach is effective for isolating functional consortium from natural microflora. In this case the functional strains of interest are more tolerant to the increased loadings of substrates than the non-functional strains.     

Foliage type specific susceptibility to ozone in Picea abies,Pinus cembra and Larix decidua at treeline: A synthesis

Cumulative ozone uptake (COU, mmol m⁻²) and O₃ flux (FO₃, nmol m⁻² s⁻¹) were related to physiological, morphological and biochemical characteristics of field-grown mature evergreen Norway spruce [Picea abies (L.) Karst.], Cembran pine [Pinus cembra L.], and deciduous European larch [Larix decidua Mill.] trees at treeline. The threshold COU causing a statistically significant decline in photosynthetic capacity (A_max) ranged between 19.6 mmol m⁻² in current-year needles of evergreen conifers and 22.0 6 mmol m⁻² in short-shoot needles of deciduous L. decidua subjected to exposure periods of ≥84 and ≥43 days, respectively. The higher O₃ sensitivity of deciduous L. decidua than of evergreen P abies and P. cembra was associated with differences in FO₃ and specific leaf area (SLA), both being significantly higher in L. decidua. FO₃ was 5.9 nmol m⁻² s⁻¹ in L. decidua and 2.7 nmol m⁻² s⁻¹ in evergreen conifers. Species-dependent differences were also related to detoxification capacity expressed through total surface area based concentrations of reduced ascorbate and α-tocopherol that both increased with SLA. Findings suggest that differences in O₃ sensitivity between evergreen and deciduous conifers can be attributed to foliage type specific differences in SLA, the latter determining physiological and biochemical characteristics of the treeline conifers.     

Enzymatic hydrolysis of corncob and ethanol production from cellulosic hydrolysate

 Enzymatic hydrolysis of corncob and ethanol fermentation from cellulosic hydrolysate were investigated. After corncob was pretreated by 1% H₂SO₄ at 108 °C for 3 h, the cellulosic residue was hydrolyzed by cellulase from Trichoderma reesei ZU-02 and the hydrolysis yield was 67.5%. Poor cellobiase activity in T. reesei cellulase restricted the conversion of cellobiose to glucose, and the accumulation of cellobiose caused severe feedback inhibition to the activities of β-1,4-endoglucanase and β-1,4-exoglucanase in cellulase system. Supplementing cellobiase from Aspergillus niger ZU-07 greatly reduced the inhibitory effect caused by cellobiose, and the hydrolysis yield was improved to 83.9% with enhanced cellobiase activity of 6.5 CBU g⁻¹ substrate. Fed-batch hydrolysis process was started with a batch hydrolysis containing 100 g l⁻¹ substrate, with cellulosic residue added at 6 and 12 h twice to get a final substrate concentration of 200 g l⁻¹. After 60 h of reaction, the reducing sugar concentration reached 116.3 g l⁻¹ with a hydrolysis yield of 79.5%. Further fermentation of cellulosic hydrolysate containing 95.3 g l⁻¹ glucose was performed using Saccharomyces cerevisiae 316, and 45.7 g l⁻¹ ethanol was obtained within 18 h. The research results are meaningful in fuel ethanol production from agricultural residue instead of grain starch.     

Solution for promoting egl3 gene of Trichoderma reesei high-efficiency secretory expression in Escherichia coli and Lactococcus lactis

The objective of this study was to develop a solution for promoting egl3 gene of Trichoderma reesei (coding β-1,4-endoglucanase, EGIII) high-efficiency secretory expression in Escherichia coli and Lactococcus lactis and to investigate the effect of the best recombinant on degrading paper and wheat straw. The coding sequence of the egl3 gene fused with a gene fragment of Usp45 (usp45) of L. lactis was cloned to pMG36e and was expressed in E. coli DH 5α (DH 5α) and L. lactis subsp. lactis MG1363 (MG1363). The maximal productivity in recombinant DH 5α was 226 mU mL⁻¹ for extracellular EGIII and 535 mU mL⁻¹ for intracellular EGIII. The maximal productivity in recombinant MG1363 was 1118 mU mL⁻¹ for extracellular EGIII and 761 mU mL⁻¹ for intracellular EGIII. The plasmid stability in recombinant MG1363 was higher than 85% at 60 generations. Recombinant MG1363 vigorously degraded paper and wheat straw and produced sufficient acids. This study provided EGIII transgenic lactic acid bacteria for processing agricultural byproducts.     

Characterization of inducible cold-active β-glucosidases from the psychrotolerant bacterium Shewanella sp. G5 isolated from a sub-Antarctic ecosystem

The psychrotolerant bacterium Shewanella sp. G5 was used to study differential protein expression on glucose and cellobiose as carbon sources in cold-adapted conditions. This strain was able to growth at 4 °C, but reached the maximal specific growth rate at 37 °C, exhibiting similar growing rates values with glucose (μ: 0.4 h⁻¹) and cellobiose (μ: 0.48 h⁻¹). However, it grew at 15 °C approximately in 30 h, with specific growing rates of 0.25 and 0.19 h⁻¹ for cellobiose and glucose, respectively. Thus, this temperature was used to provide conditions related to the environment where the organism was originally isolated, the intestinal content of Munida subrrugosa in the Beagle Channel, Fire Land, Argentina. Cellobiose was reported as a carbon source more frequently available in marine environments close to shore, and its degradation requires the enzyme β-glucosidase. Therefore, this enzymatic activity was used as a marker of cellobiose catabolism. Zymogram analysis showed the presence of cold-adapted β-glucosidase activity bands in the cell wall as well as in the cytoplasm cell fractions. Two-dimensional gel electrophoresis of the whole protein pattern of Shewanella sp. G5 revealed 59 and 55 different spots induced by cellobiose and glucose, respectively. Identification of the quantitatively more relevant proteins suggested that different master regulation schemes are involved in response to glucose and cellobiose carbon sources. Both, physiological and proteomic analyses could show that Shewanella sp. G5 re-organizes its metabolism in response to low temperature (15 °C) with significant differences in the presence of these two carbon sources.     

Hemicellulose sugar recovery from steam-exploded wheat straw for microbial oil production

There are currently few successful examples of using straw hemicellulose as a carbon source in the fermentation industry. In this paper, hemicellulose hydrolysates were recovered from steam-exploded wheat straw (SEWS) and used to produce microbial oil. The effects of the steam explosion treatment conditions, the elution temperature and the ratio of elution water to SEWS on sugar recovery were examined. A broth with 3.8 g l^?1 of reducing sugar and 22.3 g l^?1 of total soluble sugars was obtained with a 10-fold excess (w/w) of water at 40 °C to wash the SEWS treated under steam explosion conditions at 200 °C for 5 min. This broth was used to produce microbial oil by the oleaginous fungus Microsphaeropsis sp., which was able to secrete xylanase to degrade oligosaccharides from straw hemicellulose and accumulate microbial oil. Under optimized conditions, the oil concentration was 2.6 g l^?1. The yield of oil from sugar consumed was 0.14 g g^?1. The microbial oil produced by this research could be used as feedstock for biodiesel production because the microbial oil was primarily composed of neutral lipids. This research establishes a novel protocol for microbial oil production from straw hemicellulose.     

Nutrient composition,digestion and rumen fermentation in sheep of wheat straw treated with calcium oxide,sodium hydroxide and alkaline hydrogen peroxide

This study tested the effect of calcium oxide (CaO), sodium hydroxide (NaOH) and NaOH plus hydrogen peroxide (H₂O₂; AHP) on cell wall composition, digestion and fermentation of wheat straw (straw) in sheep. Treated straws were prepared by mixing straw either with water followed by dusting with CaO at 160 g kg⁻¹ DM or with a NaOH solution alone at 3 l kg⁻¹ DM to supply 80 g NaOH kg⁻¹ DM (Na) or pre-soaked with Na exactly as in the previous treatment for 27 h followed by mixing with 130 g H₂O₂ kg⁻¹ DM (AHP) for 6 h. After 14 days of storage, the treated straws and an untreated straw (U) were fed automatically every 2 h to four individually housed sheep together with a supplement in a 4×4 latin square experiment. Each kilogram supplement DM contained 422 g CP and 10.8 MJ ME. NDF (p<0.001) and hemicellulose (p<0.01) contents were significantly reduced whereas cellulose was increased (p<0.001) in treated compared to untreated straw. ADL was reduced in Ca (p<0.05) but increased (p<0.05) in Na and AHP compared with U. The rumen and total tract digestibility were significantly (p<0.001) greater in sheep fed treated compared with untreated straw. Significant differences (p<0.05) between treatments for pH, NH₃ and VFA were also observed. All treatments improved the nutritive value of straws compared with untreated through modification of cell wall with a subsequent increase in digestibility by sheep. Although the digestibility for Ca was lower than that for Na despite reduction in cell wall, its use to treat straws may be more safe and cost effective than Na. AHP was the most effective and could also improve the energy value of other low quality forages for ruminants. However, the need of AHP for a large amount of NaOH to achieve highly alkaline pH limits its farm scale application. Therefore, further studies should either consider reducing the amount of NaOH or finding alternative alkalis that are cost effective and user-friendly.     

Microbial processes and bacterial populations associated to anaerobic treatment of sulfate-rich wastewater

A pilot-scale (1.2 m³) anaerobic sequencing batch biofilm reactor (ASBBR) containing mineral coal for biomass attachment was fed with sulfate-rich wastewater at increasing sulfate concentrations. Ethanol was used as the main organic source. Tested COD/sulfate ratios were of 1.8 and 1.5 for sulfate loading rates of 0.65–1.90 kgSO₄²⁻/cycle (48 h-cycle) or of 1.0 in the trial with 3.0 gSO₄²⁻ l⁻¹. Sulfate removal efficiencies observed in all trials were as high as 99%. Molecular inventories indicated a shift on the microbial composition and a decrease on species diversity with the increase of sulfate concentration. Beta-proteobacteria species affiliated with Aminomonas spp. and Thermanaerovibrio spp. predominated at 1.0 gSO₄²⁻ l⁻¹. At higher sulfate concentrations the predominant bacterial group was Delta-proteobacteria mainly Desulfovibrio spp. and Desulfomicrobium spp. at 2.0 gSO₄²⁻ l⁻¹, whereas Desulfurella spp. and Coprothermobacter spp. predominated at 3.0 gSO₄²⁻ l⁻¹. These organisms have been commonly associated with sulfate reduction producing acetate, sulfide and sulfur. Methanogenic archaea (Methanosaeta spp.) was found at 1.0 and 2.0 gSO₄²⁻ l⁻¹. Additionally, a simplified mathematical model was used to infer on metabolic pathways of the biomass involved in sulfate reduction.     

Purification and characterization of cyanogenic β-glucosidase from wild apricot (Prunus armeniaca L.)

β-Glucosidase catalyzes the sequential breakdown of cyanogenic glycosides in cyanogenic plants. The β-glucosidase from Prunus armeniaca L. was purified to 8-fold, and 20% yield was obtained, with a specific activity of 281 U/mg protein. The enzyme showed maximum activity in 0.15 M sodium citrate buffer, pH 6, at 35 °C with p-nitrophenylglucopyranoside as substrate. The β-glucosidase from wild apricot was used successfully for the saccharification of cellobiose into D-glucose. This enzyme has a V_max of 131.6 μmol min⁻¹ mg⁻¹ protein, K_m of 0.158 mM, K_cat of 144.8 s⁻¹, K_cat/K_m of 917.4 mM⁻¹ s⁻¹, and K_m/V_max of 0.0012 mM min mg μmole⁻¹, using cellobiose as substrate. The half-life, deactivation rate coefficient, and activation energy of this β-glucosidase were 12.76 h, 1.509 × 10⁻⁵ s⁻¹, and 37.55 kJ/mol, respectively. These results showed that P. armeniaca is a potential source of β-glucosidase, with high affinity and catalytic capability for the saccharification of cellulosic material.     

Characterizing the ensiling properties of sugarbeets with dry feedstuffs

A study was conducted to evaluate the ensiling characteristics of chopped sugarbeets with dry feedstuffs and the corresponding change in the nutritive composition of the silages with the addition of dry substrates. Pre-calculated amounts of each feedstuff were weighed individually to achieve desired proportions of each silage product and thoroughly mixed for 5 min. After mixing, the silage was distributed evenly into three 19-L buckets and sealed to provide an anaerobic environment. The treatments for this study were arranged in a 4 × 4 + 1 factorial design to determine the effects of DM level and source of dry feedstuff on the ensiling properties of sugarbeets following a 42-d fermentation period. Treatments were ensiled sugarbeets alone (250 g/kg) or based on (1) formulated silage DM concentrations of 275, 350, 425, and 500 g/kg and (2) the inclusion of dry feedstuffs (alfalfa hay, dry-rolled corn, wheat middlings, and wheat straw). Fermentation and nutritive characteristics of ensiled sugarbeets were influenced with the addition of dry substrates. A linear increase (P<0.001) in silage pH was observed with the addition of alfalfa, dry-rolled corn, wheat middlings, and wheat straw to ensiled sugarbeets. Lactic acid increased (P<0.001) with the addition of wheat middlings. Alfalfa addition to sugarbeet silage did not alter (P<0.001) lactate concentration. Concentration of lactate decreased (P=0.01) when corn was added, while wheat straw addition did not influence (P=0.37) lactate. A contrast was used to compare ensiling characteristics of sugarbeets alone (250 g/kg DM) to 350 g/kg DM (sugarbeets with dry substrates). Results indicated fermentative parameters were altered; pH increased (P<0.001) for all dry substrates while lactate was lower (P=0.003) for the sugarbeets ensiled with dry-rolled corn compared with sugarbeets ensiled alone. Alfalfa, wheat straw, and wheat middlings decreased (P<0.001) while dry-rolled corn did not affect (P=0.54) in vitro DM digestion. These results indicate the inclusion of dry feedstuffs with sugarbeets altered fermentation and with the exception of corn, decreased in vitro DM digestion. Nutrient composition and DM content of ensiled sugarbeets was altered with the addition of dry substrates.     

Optimization of integrated alkaline–extrusion pretreatment of barley straw for sugar production by enzymatic hydrolysis

In this work, an integrated one-step alkaline–extrusion process was tested as pretreatment for sugar production from barley straw (BS) biomass. The influence of extrusion temperature (T) and the ratio NaOH/BS dry matter (w/w) (R) into the extruder on pretreatment effectiveness was investigated in a twin-screw extruder at bench scale. A 2³ factorial design of experiments was used to analyze the effect of process conditions [T: 50–100 °C; R: 2.5–7.5% (w/w)] on composition and enzymatic digestibility of pretreated substrate (extrudate). The optimum conditions for a maximum glucan to glucose conversion were determined to be R = 6% and T = 68 °C. At these conditions, glucan yield reached close to 90% of theoretical, while xylan conversion was 71% of theoretical. These values are 5 and 9 times higher than that of the untreated material, which supports the great potential of this one-step combined pre-treatment technology for sugar production from lignocellulosic substrates. The absence of sugar degradation products is a relevant advantage over other traditional methods for a biomass to ethanol production process since inhibitory effect of such product on sugar fermentation would be prevented.     

Effect of inoculum and pepsin–pancreatin hydrolysis on fibre fermentation measured by the gas production technique in pigs

Two experiments were undertaken to adapt the in vitro gas production technique in syringes, used for ruminants, to fibre fermentation studies in the large intestine of pigs.In a first experiment, two inocula (faeces and large intestine content) were compared at four dilution levels in a buffer solution (0.025, 0.05, 0.1 and 0.2 g ml⁻¹) with two substrates: wheat bran and sugar–beet pulp. The accumulated gas produced over 72 h was modelled and the kinetics parameters compared. The time to half asymptote was lower for the intestinal inoculum (5.5 versus 8.0 h, P<0.02), but the 2 inocula yielded similar fractional rates of degradation (0.16 h⁻¹) and gave equal final gas production (252 ml g⁻¹ substrate). No interaction (P>0.05) was observed between inocula and substrates. The dilution of the samples in the buffer solution increased (P<0.001) the lag time (from 0.9 to 2.1 h for dilution rates ranging from 0.2 to 0.025 g ml⁻¹, respectively) and decreased (P<0.001) the rates of substrate degradation (from 0.18 to 0.13 h⁻¹).A second experiment aimed to study the effect of an in vitro pepsin–pancreatin hydrolysis of the sample prior to the gas test. Six substrates were tested: maize, wheat bran, sugar–beet pulp, lupins, peas and soybean meal. The enzymatic hydrolysis affected (P<0.001) the kinetics parameters and the ranking order of the fermented substrates. The lag times also increased for all ingredients. The rate of degradation decreased when peas, lupins, maize and wheat bran were hydrolysed (P<0.001) but it increased with soybean meal (P=0.014) and sugar–beet pulp (P<0.001). Final gas production increased with peas and soybean meal (P<0.001), remained unchanged for lupins and decreased for the other substrates (P<0.001).In conclusion, the method using faeces as a source of microbial inoculum is reliable to characterise the fermentation kinetics of ingredients in the large intestine of pigs. However, it is important to hydrolyse the substrates with pepsin and pancreatin before the gas tests.     

Biochemical properties of a β-mannanase and a β-xylanase produced by Ceriporiopsis subvermispora during biopulping conditions

One mannanase and one of the three xylanases produced by Ceriporiopsis subvermispora grown on Pinus taeda wood chips were characterized. A combination of ion exchange chromatography and SDS-PAGE data revealed the existence of a high-molecular-weight mannanase of 150 kDa that was active against galactoglucomannan and xylan. Its activity was optimal at pH 4.5. The K_m value with galactoglucomannan as substrate was 0.50 mg ml^?1. One xylanase with molecular mass of 79 kDa was also purified and characterized. Its activity was optimal at 60 °C and pH 8.0. Its K_m value with birchwood xylan as substrate was 1.65 mg ml^?1. Both the mannanase and the 79 kDa xylanase displayed relatively high activity on carboxymethyl cellulose. The sensitivity of the xylanase and mannanase to various salts was evaluated. None of the tested salts inhibited the xylanase, but Mn⁺², Fe⁺³, and Cu⁺² were strong inhibitors for the mannanase.     

Biochemical characterization of xylanase produced from Streptomyces sp. CS624 using an agro residue substrate

An extracellular and cellulase-free xylanase (EX624) was produced by Streptomyces sp. CS624 using an agricultural residue (wheat bran) as a growth substrate. EX624 was purified from culture supernatant using ammonium sulfate precipitation, ion exchange and gel filtration chromatography. The SDS-PAGE and the zymogram analysis of the purified xylanase indicated molecular mass of 40 kDa. Biochemical characterization of the purified EX624 revealed its highest activity at a temperature of 60 °C and pH 6.0. The xylanase was adequately stable in the pH range 4.5–10.0 and at temperatures ≤50 °C. EX624 displayed enhanced activity in the presence of several metal ions including Fe²⁺, Co²⁺ and Ca²⁺. HPLC results showed that EX624 was not only able to hydrolyze commercially available pure beechwood xylan to xylose, xylobiose and xylotriose, but also abundantly available lignocellulosic agricultural residues in nature such as wheat bran to xylooligosaccharides.     

Paenibacillus curdlanolyticus B-6 xylanase Xyn10C capable of producing a doubly arabinose-substituted xylose, α-l-Araf-(1 → 2)-[α-l-Araf-(1 → 3)]-d-Xylp,from rye arabinoxylan

Paenibacillus curdlanolyticus B-6 Xyn10C is a single module xylanase consisting of a glycoside hydrolase family-10 catalytic module. The recombinant enzyme, rXyn10C, was produced by Escherichia coli and characterized. rXyn10C was highly active toward soluble xylans derived from rye, birchwood, and oat spelt, and slightly active toward insoluble wheat arabinoxylan. It hydrolyzed xylooligosaccharides larger than xylotetraose to produce xylotriose, xylobiose, and xylose. When rye arabinoxylan and oat spelt xylan were treated with the enzyme and the hydrolysis products were analyzed by thin layer chromatography (TLC), two unknown hydrolysis products, U1 and U2, were detected in the upper position of xylose on a TLC plate. Electrospray ionization mass spectrometry and enzymatic analysis using Bacillus licheniformis α-l-arabinofuranosidase Axh43A indicated that U1 was α-l-Araf-(1 → 2)-[α-l-Araf-(1 → 3)]-d-Xylp and U2 was α-l-Araf-(1 → 2)-d-Xylp, suggesting that rXyn10C had strong activity toward a xylosidic linkage before and after a doubly arabinose-substituted xylose residue and was able to accommodate an α-1,2- and α-1,3-linked arabinose-substituted xylose unit in both the −1 and +1 subsites. A molecular docking study suggested that rXyn10C could accommodate a doubly arabinose-substituted xylose residue in its catalytic site, at subsite −1. This is the first report of a xylanase capable of producing α-l-Araf-(1 → 2)-[α-l-Araf-(1 → 3)]-d-Xylp from highly arabinosylated xylan.     

Utilization of agro-industrial waste for xylanase production by Aspergillus foetidus MTCC 4898 under solid state fermentation and its application in saccharification

Xylanase production by Aspergillus foetidus MTCC 4898 was carried out under solid state fermentation using wheat bran and anaerobically treated distillery spent wash. Response surface methodology involving Box–Behnken design was employed for optimizing xylanase production. The interactions among various fermentation parameters viz. moisture to substrate ratio, inoculum size, initial pH, effluent concentration and incubation time were investigated and modeled. The predicted xylanase activity under optimized parameters was 8200–8400 U/g and validated xylanase activity was 8450 U/g with very poor cellulase activity. Crude xylanase was used for enzymatic saccharification of agroresidues like wheat straw, rice straw and corncobs. Dilute NaOH and ammonia pretreatments were found to be beneficial for the efficient enzymatic hydrolysis of all the three substrates. Dilute NaOH pretreated wheat straw, rice straw and corncobs yielded 4, 4.2, 4.6 g/l reducing sugars, respectively whereas ammonia treated wheat straw, rice straw and corncobs yielded 4.9, 4.7, 4.6 g/l reducing sugars, respectively. The hydrolyzates were analysed by HPTLC. Xylose was found to be the major end product with traces of glucose in the enzymatic hydrolyzates of all the substrates.