Production of cellulases and xylanase by Aspergillus fumigatus SK1 using untreated oil palm trunk through solid state fermentation

Direct utilization of untreated oil palm trunk (OPT) for cellulases and xylanase production by Aspergillus fumigatus SK1 was conducted under solid-state fermentation (SSF). The highest activities of extracellular cellulases and xylanases were produced at 80% moisture level, initial pH 5.0, 1 × 10⁸ spore/g (inoculum) with 125 μm of OPT as sole carbon source. The cellulases and xylanase activities obtained were 54.27, 3.36, 4.54 and 418.70 U/g substrates for endoglucanase (CMCase), exoglucanase (FPase), β-glucosidase and xylanase respectively. The crude cellulases and xylanase required acidic condition to retain their optimum activities (pH 4.0). Crude cellulases and xylanase were more stable at 40 °C compared to their optimum activities conditions (60 °C for FPase and 70 °C for CMCase, β-glucosidase and xylanase). SDS-PAGE and zymogram analysis showed that Aspergillus fumigatus SK1 could secrete cellulases (endoglucanase, exoglucanase and β-glucosidase), xylanase and protease. Enzymatic degradation of alkaline treated OPT with concentrated crude cellulases and xylanases resulted in producing polyoses.     

Assessment of the efficacy of varying experimental exogenous fibrolytic enzymes using in vitro fermentation characteristics

This study evaluated a series of recombinant, single activity experimental enzyme products including 13 endoglucanases (END) and 10 xylanases (XY), for their potential to improve in vitro ruminal degradation of alfalfa hay in two experiments. Based on the endoglucanase or xylanase enzymatic activities measured using complex substrates at the optimal conditions (pH 5.4, 37 °C) for the enzymes, a dose level (1 unit/g dry matter [DM]) was chosen for addition of enzymes to substrate. Enzyme products, re-suspended with water, were added to alfalfa hay (0.5 or 1.0 g DM) in culture vials in six replications. Anaerobic buffer medium (20 or 40 ml) adjusted to pH 6.0 and strained ruminal fluid (5 or 10 ml) were sequentially added to the vials and incubated for 18 h. Headspace gas production (GP) was measured throughout the incubation, and degradability of organic matter (OMD) and fibre and volatile fatty acid (VFA) concentrations were determined after 18 h of incubation. The enzyme products had a wide range of added endoglucanase or xylanase activities when determined using pure substrates and physiological conditions typical of the rumen (pH 6.0, 39 °C). In experiment 1, many END, and some XY, products increased GP and OMD. The correlation between added endoglucanase activity determined at ruminal conditions and OMD improvement was high (r = 0.71; P<0.01), whereas added activity of xylanase was not associated with OMD improvement. Two END and two XY products selected from experiment 1 were further assessed because they substantially improved GP and OMD. In experiment 2, all enzyme treatments, alone or in combination, increased total GP and DM and fibre degradabilities (P<0.05). However, the combinations of END and XY did not increase degradation of alfalfa beyond that of the component enzymes. Total VFA production was not affected by enzyme treatments although some products changed the acetate to propionate ratio. Experimental exogenous enzyme products with either endoglucanase or xylanase activity substantially improved in vitro ruminal degradation of alfalfa hay, but further improvement by combining these activities did not occur.     

Purification and characterisation of a xylanase from Thermomyces lanuginosus and its functional expression by Pichia pastoris

A xylanase produced by Thermomyces lanuginosus 195 by solid state fermentation (SSF) was purified 9.3-fold from a crude koji extract, with a 7.6% final yield. The purified xylanase (with an estimated mass of 22 kDa by SDS-PAGE) retained 18% relative activity when treated for 10 min at 100 °C and approximately 90% relative activity when incubated at pH values ranging from 6 to 10. Xylanase activity in the purified preparation was significantly enhanced following treatment with manganese and potassium chlorides (p < 0.05) but significantly reduced by calcium, cobalt and iron (p < 0.05). The purified enzyme was also shown to be exclusively xylanolytic. The gene encoding xylanase activity from T. lanuginosus 195 was functionally expressed by Pichia pastoris. MALDI-ToF mass spectrometry and zymography were employed to confirm functional recombinant expression. Maximum xylanase titres were achieved following 120 h induction of the recombinant culture, yielding 26.8 U/mL. Achieving functional protein expression facilitates future efforts to optimise the cultivation conditions for heterologous xylanase production.     

A highly-active endo-1,3-1,4-β-glucanase from thermophilic Talaromyces emersonii CBS394.64 with application potential in the brewing and feed industries

A 1245-bp endoglucanase gene of glycoside hydrolase (GH) family 7, egl7A, was cloned from the acidothermophilic fungus Talaromyces emersonii CBS394.64 and successfully expressed in Pichia pastoris. Sequence alignments indicated that Egl7A had highest identity of 62.7% at the amino acid level with the functionally characterized endoglucanase from Aspergillus terreus NIH2624. Purified recombinant Egl7A exhibited the maximum activity at pH 4.5 and 70 °C, retained stable over the pH range of 2.0–12.0 and at 65 °C, and was strongly resistant to acidic and neutral proteases, most metal ions and SDS. The enzyme exhibited the highest specific activity reported so far (11,299 U mg⁻¹) when using barley β-glucan as the substrate. Egl7A exhibited broad substrate specificity, including barley β-glucan, lichenin, CMC-Na, and xylan and had capacity to cleave cellopentaose and cellohexaose into smaller units rapidly. Under simulated mashing conditions, addition of Egl7A reduced the mash viscosity by 12.40%; when combined with a GH10 xylanase, more viscosity reduction (27.75%) was observed, which is significantly higher than that of the commercial enzyme Ultraflo XL (17.91%). All these properties make Egl7A attractive for potential applications in the feed and brewing industries.     

Isolation and identification of a novel Rhodococcus sp. ML-0004 producing epoxide hydrolase and optimization of enzyme production

Rhodococcus sp. ML-0004, a novel strain for producing epoxide hydrolase, was isolated from soil in this study. The epoxide hydrolase can catalyze the stereo-specific hydrolysis of cis-epoxysuccinic acid to generate l(+)-tartaric acid. By examining physiological, biochemical characteristics and comparing its 16S rDNA gene sequence, it was identified as Rhodococcus opacus, and named R. opacus ML-0004. The optimal conditions for epoxide hydrolase production from R. opacus ML-0004 were also investigated. Propanediol and (NH₄)₂SO₄ were selected as carbon source and nitrogen source, respectively, for the production of R. opacus ML-0004 epoxide hydrolase. The optimal conditions for epoxide hydrolase production were fermentation temperature = 28 °C, pH 7.0, and cultivation time = 26 h. Under these conditions, the maximum epoxide hydrolase activity reached 10.5 U mL⁻¹.     

Multi-functional glycoside hydrolase: Blon_0625 from Bifidobacterium longum subsp. infantis ATCC 15697

We here describe a unique β-D-glucosidase (BGL; Blon_0625) derived from Bifidobacterium longum subsp. infantis ATCC 15697. The Blon_0625 gene was expressed by recombinant Escherichia coli. Purified recombinant Blon_0625 retains hydrolyzing activity against both p-nitrophenyl-β-D-glucopyranoside (pNPG; 17.3 ± 0.24 U mg⁻¹) and p-nitrophenyl-β-D-xylopyranoside (pNPX; 16.7 ± 0.32 U mg⁻¹) at pH 6.0, 30 °C. To best of our knowledge, no previously described BGL retains the same level of both pNPGase and pNPXase activity. Furthermore, Blon_0625 also retains the activity against 4-nitrophenyl-α-l-arabinofranoside (pNPAf; 5.6 ± 0.09 U mg⁻¹). In addition, the results of the degradation of phosphoric acid swollen cellulose (PASC) or xylan using endoglucanase from Thermobifida fusca YX (Tfu_0901) or xylanase from Kitasatospora setae KM-6054 (KSE_59480) show that Blon_0625 acts as a BGL and as a β-D-xylosidase (XYL) for hydrolyzing oligosaccharides. These results clearly indicate that Blon_0625 is a multi-functional glycoside hydrolase which retains the activity of BGL, XYL, and also α-l-arabinofuranosidase. Therefore, the utilization of multi-functional Blon_0625 may contribute to facilitating the efficient degradation of lignocellulosic materials and help enhance bioconversion processes.     

Improvement in natamycin production by Streptomyces natalensis with the addition of short-chain carboxylic acids

Natamycin is an important tetraene (polyene) antibiotic produced in submerged culture by different strains of Streptomyces sp. In the present work, the effects of the addition of short-chain carboxylic acids (acetic, propionic and butyric) on cell growth and the kinetics of natamycin production were investigated during submerged cultivation of Streptomyces natalensis. The addition of acetic and propionic acids showed stimulatory effects on natamycin production when added to the fermentation medium at concentrations below 2 g L^?1 at the beginning of cultivation. In addition, when acetic and propionic acids were added in a mixture (7:1) at a total concentration of 2 g L^?1, antibiotic production increased significantly, reaching 3.0 g L^?1 (approximately 223% and 250% increases in volumetric and specific antibiotic production, respectively, compared with the control culture). Moreover, the addition of carboxylic acids not only increased the antibiotic yield but also decreased the production time from 96 h to only 84 h in shake-flask cultures. A further enhancement in natamycin production was achieved by cultivation in a 2-L stirred-tank bioreactor under controlled pH conditions. The maximum volumetric production of 3.98 g L^?1 was achieved after 84 h in carboxylic acid-supplemented culture (acetate and propionate in a ratio of 7:1).     

Identification of a novel cellulose-binding domain within the endo-β-1,4-xylanase KRICT PX-3 from Paenibacillus terrae HPL-003

The model 3-D structure of xylanase KRICT PX3 (JF320814) identified by DNA sequence analysis revealed a catalytic domain and CBM4-9 which functions as a xylan binding domain (XBD). To identify its role in xylan hydrolysis, six expression plasmids were constructed encoding the N-terminal CBM plus the catalytic domain or different glycosyl hydrolases, and the biochemical properties of the recombinant enzymes were compared to the original structure of PX3 xylanase. All six of the recombinant xylanases with the addition of CBM in the pIVEX-GST expression vector showed no improved PX3 hydrolytic activity. However, the absence of the CBM domain resulted in a decrement of 40% in thermostability, movement of the optimal temperature from 55 °C to 45 °C, alteration of the optimal pH range from 5⿿10 to 6⿿8, and reduction of the enzymatic activity to one-second under the same condition, respectively. The putative XBD in PX3 comprises a new N-terminal domain homologous to the catalytic thermostabilizing domains from other xylanases. Analysis of the main products released from xylan indicate that the recombinant enzymes act as endo-1,4-β-xylanases but differ in their hydrolysis of xylan from beech wood, birch wood, and oat spelt.     

Functional and structural analyses of a 1,4-β-endoglucanase from Ganoderma lucidum

Ganoderma lucidum is a saprotrophic white-rot fungus which contains a rich set of cellulolytic enzymes. Here, we screened an array of potential 1,4-β-endoglucanases from G. lucidum based on the gene annotation library and found that one candidate gene, GlCel5A, exhibits CMC-hydrolyzing activity. The recombinant GlCel5A protein expressed in Pichia pastoris is able to hydrolyze CMC and β-glucan but not xylan and mannan. The enzyme exhibits optimal activity at 60 °C and pH 3–4, and retained 50% activity at 80 and 90 °C for at least 15 and 10 min. The crystal structure of GlCel5A and its complex with cellobiose, solved at 2.7 and 2.86 Å resolution, shows a classical (β/α)₈ TIM-barrel fold as seen in other members of glycoside hydrolase family 5. The complex structure contains a cellobiose molecule in the +1 and +2 subsites, and reveals the interactions with the positive sites of the enzyme. Collectively, the present work provides the first comprehensive characterization of an endoglucanase from G. lucidum that possesses properties for industrial applications, and strongly encourages further studying in the cellulolytic enzyme system of G. lucidum.     

Molecular cloning and characterization of a novel thermostable xylanase from Paenibacillus campinasensis BL11

An open reading frame (XylX) with 1131 nucleotides from Paenibacillus campinasensis BL11 was cloned and expressed in E. coli. It encodes a family 11 endoxylanase, designated as XylX, of 41 kDa. The homology of the amino acid sequence deduced from XylX is only 73% identical to the next closest sequence. XylX contains a family 11 catalytic domain of the glycoside hydrolase and a family 6 cellulose-binding module. The recombinant xylanase was fused to a His-tag for affinity purification. The XylX activity was 2392 IU/mg, with a K_m of 6.78 mg/ml and a V_max of 4953 mol/min/mg under optimal conditions (pH 7, 60 °C). At pH 11, 60 °C, the activity was still as high as 517 IU/mg. Xylanase activities at 60 °C under pH 5 to pH 9 remained at more than 69.4% of the initial activity level for 8 h. The addition of Hg²⁺ at 5 mM almost completely inhibited xylanase activity, whereas the addition of tris-(2-carboxyethyl)-phosphine (TCEP) and 2-mercaptoethanol stimulated xylanase activity. No relative activities for Avicel, CMC and d-(+)-cellobiose were found. Xylotriose constitutes the majority of the hydrolyzed products from oat spelt and birchwood xylan. Broad pH and temperature stability shows its application potentials for biomass conversion, food and pulp/paper industries.     

Production of yeast chitin–glucan complex from biodiesel industry byproduct

Crude glycerol from the biodiesel industry was used as carbon source for high cell density fed-batch cultivation of Pichia pastoris aiming at producing a chitin–glucan complex (CGC). More than 100 g L^?1 biomass was obtained in less than 48 h. The yield of biomass on a glycerol basis was 0.55 g g^?1 during the batch phase and 0.63 g g^?1 during the fed-batch phase. The chitin–glucan complex was recovered from the yeast cell wall by hot alkaline extraction. CGC content in the cell wall was found to be relatively constant throughout the cultivation (18–26%) with a volumetric productivity of 1.28 g L^?1 h^?1 at the end of the fed-batch phase. The molar ratio of chitin:β-glucan in the extracted biopolymer was 16:84, close to other CGC extracted from Aspergillus biomass. The extracted polymer was characterized by Differential Scanning Calorimetry (DCS) and solid-state Nuclear Magnetic Resonance (NMR) spectroscopy and compared with commercial biopolymers, namely, crab shell chitin and/or chitosan, algal β-glucan (laminarin) and fungal chitin–glucan complex (kiOsmetine).     

Cloning and characterization of a recombinant family 5 endoglucanase from Bacillus licheniformis strain B-41361

The gene encoding a family 5 endoglucanase, cel5A, was cloned from the moderate thermophile Bacillus licheniformis strain B-41361. The primary structure of the translated cel5A gene predicts a 49 amino acid putative secretion signal and a 485 residue endoglucanase consisting of an N-terminal family 5 catalytic domain and C-terminal family 3 cellulose binding domain. The endoglucanase portion of the gene was expressed in Escherichia coli, but soluble activity in cell lysates was due to a truncated enzyme with an apparent mass of 42 kDa, the equivalent of the predicted catalytic domain. Insoluble protein renatured from inclusion bodies was protected against truncation, yielding an active holoenzyme (rCel5A) with apparent mass of 62 kDa. The recombinant rCel5A was optimally active at 65 °C and pH 6.0, but retained only 10% activity after 1 h incubation at this temperature. At 55 °C, rCel5A had a broad pH range for activity and stability, with greater than 75% relative activity from pH 4.5–7.0, and retaining greater than 80% relativity activity across the range pH 4.5–8.0 following 1 h incubation at 55 °C. It readily hydrolyzed pNPC, carboxymethylcellulose, barley β-glucan, and lichenan, but despite binding to cellulose, had only weak activity against avicel. Hydrolysis products from soluble polysaccharides included glucose, cellobiose, cellotriose, and cellotetraose. The catalytic properties, broad pH range and thermostability of the recombinant B. licheniformis endoglucanase may prove suitable for industrial applications.     

Manipulation of B. megaterium growth for efficient 2-KLG production by K. vulgare

In the two-step Vitamin C fermentative production, its precursor 2-keto-l-gulonic acid (2-KLG) was synthesized by Ketogulonicigenium vulgare through co-culture with Bacillus megaterium. The rates of K. vulgare cell growth and 2-KLG production were closely related with B. megaterium concentration in the co-culture system. To enhance the 2-KLG production efficiency, a strategy of manipulating B. megaterium growth in the co-culture system and properly releasing its intracellular components was introduced. Lysozyme was used specifically to damage B. megaterium cell wall structure and subsequently inhibit its cell growth. When 10,000 U mL⁻¹ lysozyme was fed to the co-culture system at 12 h, the growth rate of K. vulgare, sorbose consumption rate, and 2-KLG productivity could increase 27.4%, 37.1%, and 28.2%, respectively.     

Improved capsular polysaccharide production by Streptococcus pneumoniae serotype 14 using continuous cultivation

The capsular polysaccharide (PS) is the most important pneumococcal virulence factor and is currently used as antigen in all pneumococcal vaccines. Despite its physiological and epidemiological importance, meager studies have been devoted to improve PS production and understand its relationship with pneumococcal central metabolism. In this study, kinetics of growth and production of PS by Streptococcus pneumoniae serotype 14 (PS14) in batch and continuous cultivation were investigated. Strong cell lysis was observed in batch cultivation, while accumulation of organic acids and autolysis was avoided in continuous cultivation. In the continuous cultivation was possible to achieve higher concentration of biomass and PS14. Calculation of kinetic parameters demonstrated that PS14 is a cell-associated product. The coefficients for growth-associated stoichiometric true yield and maintenance were determined as 0.25 g_glucose g_biomass⁻¹ and 1.24 g_glucose (g_biomass h)⁻¹, respectively. The maximum productivity of PS14 released in the supernatant (PS14_R) and cell-bound PS14 (PS14_C) were obtained at a dilution rate of 0.8 h⁻¹, respectively, 85 and 122 mg  (g_biomass h)⁻¹. Compared to batch fermentation, both PS14_R and PS14_C productivities were increased by about 300% in the continuous process. These findings demonstrate that continuous cultivation is a promising strategy for PS production to be used in pneumococcal vaccines.     

Improved β-carotene production of Rhodotorula glutinis in fermented radish brine by continuous cultivation

Fermentation kinetics of growth and β-carotene production by Rhodotorula glutinis DM28 in batch and continuous cultures using fermented radish brine, a waste generated from fermented vegetable industry, as a cultivation medium were investigated. The suitable brine concentration for β-carotene production by R. glutinis DM28 was 30 g l^?1. Its growth and β-carotene production obtained by batch culture in shake flasks were 2.2 g l^?1 and 87 μg l^?1, respectively, while, in a bioreactor were 2.6 g l^?1 and 186 μg l^?1, respectively. Furthermore, its maximum growth rate and β-carotene productivity in continuous culture obtained at the dilution rate of 0.24 h^?1 were 0.3 g l^?1 h^?1 and 19 μg l^?1 h^?1, respectively, which were significantly higher than those in the batch. Therefore, improved growth rate and β-carotene productivity of R. glutinis in fermented radish brine could be accomplished by continuous cultivation.     

Cloning,expression and characterization of a novel acidic xylanase,XYL11B,from the acidophilic fungus Bispora sp. MEY-1

A xylanase gene (xyl11B) was cloned from Bispora sp. MEY-1 and expressed in Pichia pastoris. xyl11B, with a 66-bp intron, encodes a mature protein of 219 residues with highest identity (57.1%) to the Trichoderma reesei xylanase of glycoside hydrolase family 11. The purified recombinant XYL11B was acidophilic, exhibiting maximum activity at pH 2.6 and 65 °C. The enzyme was also thermostable, pH stable, and was highly resistant to both pepsin and trypsin, suggesting good performance in the digestive tract as a feed supplement to improve animal nutrition. The activity of XYL11B was enhanced by most metal ions but was inhibited weakly by Hg²⁺, Pb²⁺and Cu²⁺, which strongly inhibit many other xylanases. The specific activity of XYL11B for oat spelt xylan substrate was 2049 U mg^?1. The main hydrolysis products of xylan were xylose and xylobiose.     

Fed-batch fermentation of recombinant Citrobacter freundii with expression of a violacein-synthesizing gene cluster for efficient violacein production from glycerol

The extensive prospects of violacein in the pharmaceutical industry have attracted increasing interest. However, the fermentation levels of violacein are currently inadequate to meet the demands of industrial production. This study was undertaken to develop an efficient process for the production of violacein by recombinant Citrobacter freundii. The effects of dissolved oxygen (DO) and pH on cell growth and violacein production in batch cultures were investigated first. When the DO and pH of the medium were controlled at around 25% and 7.0, respectively, the biomass and concentration of violacein were maximized. Based on the consumption of nutrients in the medium observed during batch culture, a fed-batch fermentation strategy with controlled DO and pH was implemented. By continuously feeding glycerol, NH₄Cl, and l-tryptophan at a constant feeding rate of 16 mL h⁻¹, the final concentration of violacein reached 4.13 g L⁻¹, which was 4.09-fold higher than the corresponding batch culture, and the maximal dry cell weight (DCW) and average violacein productivity obtained for the fed-batch culture were 3.34 g DCW L⁻¹ and 82.6 mg L⁻¹ h⁻¹, respectively. To date, this is the first report on the efficient production of violacein by genetically engineered strains in a fermentor.     

Continuous 2-keto-gluconic acid (2KGA) production from corn starch hydrolysate by Pseudomonas fluorescens AR4

A continuous fermentation process for 2-keto-gluconic acid (2KGA) production from cheap raw material corn starch hydrolysate was developed using the strain Pseudomonas fluorescens AR4. The dilution rate and feeding glucose concentration had a significant effect on the cell concentrations, glucose utilization and 2KGA production performance. The optimal operating factors were obtained as: 0.065 h⁻¹ of dilution rate, 180 g/L of feeding glucose concentration, and 16 h of batch fermentation time as the starting point. Under these conditions, the steady state had the 135.92 g/L of produced 2KGA concentration, 8.83 g/L.h of average volumetric productivity, and 0.9510 g/g of yield. In conclusion, the proposed efficient and stable continuous fermentation process for 2KGA production by the strain P. fluorescens AR4 is potentially competitive for industrial production from corn starch hydrolysate in terms of 2KGA productivity and yield.     

Recombinant expression,characterization, and pulp prebleaching property of a Phanerochaete chrysosporium endo-β-1,4-mannanase

A Phanerochaete chrysosporium cDNA predicted to encode endo-1,4-β-d-mannanase, man5D, was cloned and expressed in Aspergillus niger. The coding region of the gene man5D was predicted to contain, in order from the N-terminal: a secretory signal peptide, cellulose-binding domain, linker region, and glycosyl hydrolase family 5 catalytic site. The enzyme was purified from culture filtrate of A. niger transformants that carried the recombinant man5D. Recombinant Man5D had an apparent molecular size of about 65 kDa by SDS-PAGE, and optimal activity at pH 4.0–6.0 and 60 °C. It was stable from pH 4.0 to 8.0 and up to 60 °C. The enzyme showed affinity for Avicel cellulose, suggesting that the predicted cellulose-binding domain is biologically functional. The specific activities of Man5D on mannan, galactomannan, and glucomannan at pH 5 and 60 °C ranged from 160 to 460 μmol/(min mg), with apparent K_m values from 0.54 to 2.3 mg/mL. Product analysis results indicated that Man5D catalyzes endo-cleavage, and appears to have substantial transglycosylase activity. When used to treat softwood kraft pulp, Man5D hydrolyzed mainly glucomannan and exhibited a positive effect as a prebleaching agent. Compared to a commercial prebleaching with xylanase, the prebleaching effect of Man5D was weaker but with reduced loss of fibre yield as determined by the release of solubilized sugars.     

Novel thermophilic hemicellulases for the conversion of lignocellulose for second generation biorefineries

The biotransformation of lignocellulose biomasses into fermentable sugars is a very complex procedure including, as one of the most critical steps, the (hemi) cellulose hydrolysis by specific enzymatic cocktails. We explored here, the potential of stable glycoside hydrolases from thermophilic organisms, so far not used in commercial enzymatic preparations, for the conversion of glucuronoxylan, the major hemicellulose of several energy crops. Searches in the genomes of thermophilic bacteria led to the identification, efficient production, and detailed characterization of novel xylanase and α-glucuronidase from Alicyclobacillus acidocaldarius (GH10-XA and GH67-GA, respectively) and a α-glucuronidase from Caldicellulosiruptor saccharolyticus (GH67-GC). Remarkably, GH10-XA, if compared to other thermophilic xylanases from this family, coupled good specificity on beechwood xylan and the best stability at 65 °C (3.5 days). In addition, GH67-GC was the most stable α-glucuronidases from this family and the first able to hydrolyse both aldouronic acid and aryl-α-glucuronic acid substrates. These enzymes, led to the very efficient hydrolysis of beechwood xylan by using 7- to 9-fold less protein (concentrations <0.3 μM) and in much less reaction time (2 h vs 12 h) if compared to other known biotransformations catalyzed by thermophilic enzymes. In addition, remarkably, together with a thermophilic β-xylosidase, they catalyzed the production of xylose from the smart cooking pre-treated biomass of one of the most promising energy crops for second generation biorefineries. We demonstrated that search by the CAZy Data Bank of currently available genomes and detailed enzymatic characterization of recombinant enzymes allow the identification of glycoside hydrolases with novel and interesting properties and applications.