Regioselectivity-driven evolution of CYP102D1 for improved synthesis of 3′-ortho-dihydroxyisoflavone

Daidzein is a major component of isoflavones, and its hydroxylated forms are valuable phytochemicals with anti-cancer and anti-oxidant activity. Due to the limitations of chemical synthesis of these hydroxylated structures, alternative enzymatic synthesis has been attempted. Previously, several protein-engineering approaches using CYP102D1 were investigated; these produced mutants with daidzein hydroxylation activity and regioselectivity through rational design (F96V/M246I) and saturation mutagenesis (A273H/G274E/T277G). However, the generated mutants have low regioselectivity (F96V/M246I) or low hydroxylation activity (A273H/G274E/T277G). Here, we characterized mutants capable of catalyzing C3′-specific daidzein hydroxylation with enhanced hydroxylation activity and regioselectivity. In order to obtain regioselectivity toward the daidzein C3′-position, site-saturation mutagenesis on the substrate-binding region of CYP102D1 F96V/M246I was investigated. A high-throughput screening assay was then performed, based on O-dealkylation activity against the daidzein analog substrate 4′-O-methyl-daidzein. This resulted in a mutant with more than 23-fold improved hydroxylation activity (55.6 ± 17.9 μM⁻¹ min⁻¹, or 48.4 mg/L titer) and regioselectivity over the 3′/6-position that was increased by three-fold (from 0.9 to 2.6) compared with the F96V/M246I template enzyme. Furthermore, we carried out docking simulation studies that could partially explain the effects of these mutations on C3′-specific hydroxylation activity.     

Ala258Phe substitution in Bacillus sp. YX-1 glucose dehydrogenase improves its substrate preference for xylose

Bacillus sp. YX-1 glucose dehydrogenase (BsGDH) with good solvent resistance catalyzes the oxidation of β-d-glucose to d-glucono-1,5-lactone. Xylose is a recyclable resource from hemicellulase hydrolysis. In this work, to improve the preference of BsGDH for xylose, we designed seven mutants inside or adjacent to the substrate binding pocket using site-directed mutagenesis. Among all mutants, Ala258Phe mutant displayed the highest activity of 7.59 U mg⁻¹ and nearly 8-folds higher k_cat/K_m value towards xylose than wild-type BsGDH. The kinetic constants indicated that the A258F mutation effectively altered the transition state. By analysis of modeled protein structure, Ala258Phe created a space to facilitate the reactivity towards xylose. A258F mutant retained good solvent resistance in glycol, ethyl caprylate, octane, decane, cyclohexane, nonane, etc. as with BsGDH. This work provides a protein engineering approach to modify the substrate stereo-preference of alcohol dehydrogenase and a promising enzyme for cofactor regeneration in chiral catalysis.     

LaaA,a novel high-active alkalophilic alpha-amylase from deep-sea bacterium Luteimonas abyssi XH031T

Alpha-amylase is a kind of broadly used industrial enzymes, most of which have been exploited from terrestrial organism. Comparatively, alpha-amylase from marine environment was largely undeveloped. In this study, a novel alkalophilic alpha-amylase with high activity, Luteimonas abyssi alpha-amylase (LaaA), was cloned from deep-sea bacterium L. abyssi XH031^T and expressed in Escherichia coli BL21. The gene has a length of 1428 bp and encodes 475 amino acids with a 35-residue signal peptide. The specific activity of LaaA reached 8881 U/mg at the optimum pH 9.0, which is obvious higher than other reported alpha-amylase. This enzyme can remain active at pH levels ranging from 6.0 to 11.0 and temperatures below 45 °C, retaining high activity even at low temperatures (almost 38% residual activity at 10 °C). In addition, 1 mM Na⁺, K⁺, and Mn²⁺ enhanced the activity of LaaA. To investigate the function of potential active sites, R227G, D229K, E256Q/H, H327V and D328V mutants were generated, and the results suggested that Arg227, Asp229, Glu256 and Asp328 were total conserved and essential for the activity of alpha-amylase LaaA. This study shows that the alpha-amylase LaaA is an alkali-tolerant and high-active amylase with strong potential for use in detergent industry.     

Rapid ethanol production at elevated temperatures by engineered thermotolerant Kluyveromyces marxianus via the NADP(H)-preferring xylose reductase-xylitol dehydrogenase pathway

Conversion of xylose to ethanol by yeasts is a challenge because of the redox imbalances under oxygen-limited conditions. The thermotolerant yeast Kluyveromyces marxianus grows well with xylose as a carbon source at elevated temperatures, but its xylose fermentation ability is weak. In this study, a combination of the NADPH-preferring xylose reductase (XR) from Neurospora crassa and the NADP⁺-preferring xylitol dehydrogenase (XDH) mutant from Scheffersomyces stipitis (Pichia stipitis) was constructed. The xylose fermentation ability and redox balance of the recombinant strains were improved significantly by over-expression of several downstream genes. The intracellular concentrations of coenzymes and the reduced coenzyme/oxidized coenzyme ratio increased significantly in these metabolic strains. The byproducts, such as glycerol and acetic acid, were significantly reduced by the disruption of glycerol-3-phosphate dehydrogenase (GPD1). The resulting engineered K. marxianus YZJ088 strain produced 44.95 g/L ethanol from 118.39 g/L xylose with a productivity of 2.49 g/L/h at 42 °C. Additionally, YZJ088 realized glucose and xylose co-fermentation and produced 51.43 g/L ethanol from a mixture of 103.97 g/L xylose and 40.96 g/L glucose with a productivity of 2.14 g/L/h at 42 °C. These promising results validate the YZJ088 strain as an excellent producer of ethanol from xylose through the synthetic xylose assimilation pathway.     

Purification and biochemical characterization of a mycelial glucose- and xylose-stimulated β-glucosidase from the thermophilic fungus Humicola insolens

A mycelial β-glucosidase from the thermophilic mold Humicola insolens was purified and biochemically characterized. The enzyme showed carbohydrate content of 21% and apparent molecular mass of 94 kDa, as estimated by gel filtration. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis showed a single polypeptide band of 55 kDa, suggesting that the native enzyme was a homodimer. Mass spectrometry analysis showed amino acid sequence similarity with a β-glucosidase from Humicola grisea var. thermoidea, with about 22% coverage. Optima of temperature and pH were 60 °C and 6.0–6.5, respectively. The enzyme was stable up to 1 h at 50 °C and showed a half-life of approximately 44 min at 55 °C. The β-glucosidase hydrolyzed cellobiose, lactose, p-nitrophenyl-β-d-glucopyranoside, p-nitrophenyl-β-d-fucopyranoside, p-nitrophenyl-β-d-xylopyranoside, p-nitrophenyl-β-d-galactopyranoside, o-nitrophenyl-β-d-galactopyranoside, and salicin. Kinetic studies showed that p-nitrophenyl-β-d-fucopyranoside and cellobiose were the best enzyme substrates. Enzyme activity was stimulated by glucose or xylose at concentrations up to 400 mM, with maximal stimulatory effect (about 2-fold) around 40 mM. The high catalytic efficiency for the natural substrate, good thermal stability, strong stimulation by glucose or xylose, and tolerance to elevated concentrations of these monosaccharides qualify this enzyme for application in the hydrolysis of cellulosic materials.     

Complete genome sequence,metabolic model construction and phenotypic characterization of Geobacillus LC300, an extremely thermophilic,fast growing,xylose-utilizing bacterium

We have isolated a new extremely thermophilic fast-growing Geobacillus strain that can efficiently utilize xylose, glucose, mannose and galactose for cell growth. When grown aerobically at 72 °C, Geobacillus LC300 has a growth rate of 2.15 h⁻¹ on glucose and 1.52 h⁻¹ on xylose (doubling time less than 30 min). The corresponding specific glucose and xylose utilization rates are 5.55 g/g/h and 5.24 g/g/h, respectively. As such, Geobacillus LC300 grows 3-times faster than E. coli on glucose and xylose, and has a specific xylose utilization rate that is 3-times higher than the best metabolically engineered organism to date. To gain more insight into the metabolism of Geobacillus LC300 its genome was sequenced using PacBio׳s RS II single-molecule real-time (SMRT) sequencing platform and annotated using the RAST server. Based on the genome annotation and the measured biomass composition a core metabolic network model was constructed. To further demonstrate the biotechnological potential of this organism, Geobacillus LC300 was grown to high cell-densities in a fed-batch culture, where cells maintained a high xylose utilization rate under low dissolved oxygen concentrations. All of these characteristics make Geobacillus LC300 an attractive host for future metabolic engineering and biotechnology applications.     

Elimination of substrate inhibition of a β-N-acetyl-d-hexosaminidase by single site mutation

Substrate inhibition hinders chitinolytic β-N-acetyl-d-hexosaminidases in producing N-acetyl-d-glucosamine (GlcNAc), the valuable chemical widely applied in medical and food industries. Here we focused on a promising chitinolytic enzyme, OfHex1 from the insect, Ostrinia furnacalis. By structural analysis of OfHex1, five residues nearby the active pocket including V327, E328, Y471, V484 and W490 were chosen and nine mutants including V327G, E328Q, E328A, Y471V, V484R, W490A, W490H, V327G/V484R/W490A and V327G/Y471V/W490H were constructed and recombinantly expressed in Pichia pastoris. The best-performing mutant, W490A, obtained by a higher yield of 5 mg/L, did not show substrate inhibition even when 5 mM of the substrates, (GlcNAc)_2–4, were applied. The k_cat/K_m values for (GlcNAc)_2–4 are 239.8, 111.3 and 79.8 s^?1 mM^?1, respectively. Besides, the pH stability of the mutant ranges from pH 4 to 11 and the thermal stability is up to 50 °C. This work suggests the W490A mutant might be an ideal biocatalyst for GlcNAc production from chitin.     

Rational design of xylose dehydrogenase for improved thermostability and its application in development of efficient enzymatic biofuel cell

In this paper, the construction of 3D model structure of xylose dehydrogenase (XDH) by using homology modeling to guide the rational design of the enzyme for improving thermostability was reported. Three XDH mutants of NA-1 (+249L), NA-2 (G149P) and NA-3 (+249L/G149P) were designed and displayed on the surface of bacteria. Among them, bacteria displaying NA-1 (NA-1-bacteria) exhibited superior thermostability without compromising its activity and substrate specificity in comparison with its wild-type counterpart. NA-1-bacteria retained its original activity after incubation at room temperature for one-month with the half-life of 9.8 days at 40 °C. Finally, the NA-1-bacteria were applied to construct xylose/O₂ based biofuel cell with good performance including enhanced operational stability. Thus, the approach described here could be explored for engineering of other enzymes for improving certain characters without three-dimensional structure identified by experimental methods.     

A novel thermophilic xylanase from Achaetomium sp. Xz-8 with high catalytic efficiency and application potentials in the brewing and other industries

Thermophilic xylanases are of great interest for their wide industrial application prospects. Here we identified a thermophilic xylanase (XynC01) of glycoside hydrolase (GH) family 10 in a thermophilic fungal strain Achaetomium sp. Xz-8. The deduced amino acids of XynC01 showed the highest identity of ≤52% to experimentally verified xylanases. XynC01 was functionally expressed in Pichia pastoris, showed optimal activity at pH 5.5 and 75 °C with stability over a broad pH range (pH 4.0–10.0) and at temperatures of 55 °C and below. XynC01 had the highest catalytic efficiency (k_cat/K_m, 3710 mL/s/mg) ever reported for all GH 10 xylanases, and was resistant to all tested metal ions and chemical reagents. Its hydrolysis products of various xylans were simple, mainly consisting of xylobiose and xylose. Under simulated mashing conditions, XynC01 alone had a comparable effect on filtration improvement with Ultraflo from Novozymes (20.24% vs. 20.71%), and showed better performance when combined with a commercial β-glucanase (38.50%). Combining all excellent properties described above, XynC01 may find diverse applications in industrial fields, especially in the brewing industry.     

Enhanced catalysis of l-asparaginase from Bacillus licheniformis by a rational redesign

l-Asparaginase (3.5.1.1) being antineoplastic in nature are used in the treatment of acute lymphoblastic leukemia (ALL). However glutaminase activity is the cause of various side effects when used as a drug against acute lymphoblastic leukemia (ALL). Therefore, there is a need of a novel l-asparaginase (L-ASNase) with low or no glutaminase activity. Such a property has been observed with L-ASNase from B. licheniformis (BliA). The enzyme being glutaminase free in nature paved the way for its improvement to achieve properties similar to or near to the commercially available L-ASNases. Rational enzyme engineering approach resulted in four mutants: G238N, E232A, D103V and Q112H. Among these the mutant enzyme, D103V, had a specific activity of 597.7 IU/mg, which is higher than native (rBliA) (407.65 IU/mg). Moreover, when the optimum temperature and in vitro half life were studied and compared with native BliA, D103V mutant BliA was better, showing tolerance to higher temperatures and a 3 fold higher half life. Kinetic studies revealed that the mutant D103V L-ASNase has increased substrate affinity, with K_m value of 0.42 mM and V_max of 2778.9 μmol min⁻¹.     

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.     

Characterization and comparison of thermostability of purified β-glucosidases from a mesophilic Aureobasidium pullulans and a thermophilic Thermoascus aurantiacus

The thermophilic fungus Thermoascus aurantiacus 179-5 and the mesophilic Aureobasidium pullulans ER-16 were cultivated in corn-cob by solid state fermentation for β-glucosidase production. After fermentation both enzymes were purified. The β-glucosidases produced by the strains A. pullulans and T. aurantiacus were most active at pH 4.0–4.5 and 4.5, with apparent optimum temperatures at 80 and 75 °C, respectively. Surprisingly, the enzyme produced by the mesophilic A. pullulans was stable over a wider range of pH (4.5–9.5 against 4.5–6.5) and more thermostable (98% after 1 h at 75 °C against 98% after 1 h at 70 °C) than the enzyme from the thermophilic T. aurantiacus. The t_(1/2) at 80 °C were 90 and 30 min for A. pullulans and T. aurantiacus, respectively. β-Glucosidase thermoinactivation followed first-order kinetics and the energies of denaturation were 414 and 537 kJ mol⁻¹ for T. aurantiacus and A. pullulans, respectively. The result showed that β-glucosidase obtained from the mesophilic A. pullulans is more stable than that obtained from the thermophilic T. aurantiacus.     

Hepatitis B virus precore G1896A mutation in chronic liver disease patients with HBeAg negative serology from North India

Hepatitis B with precore stop codon mutation is related with severe liver damage in HBeAg negative patients. It is of utmost importance to screen the G1896A precore mutation. The study was designed to assess the impact of G1986A mutations in patients with different clinical spectra of the liver disease by PCR–LCR. 210 HBV positive patients with HBeAg negative serology of different kind of liver diseases (AVH = 72, FH = 21, CH = 79, Cirrhosis = 20 and HCC = 18) were screened. Patients were screened for the presence or absence of precore G1896A mutation by PCR–LCR. Direct nucleotide sequencing was done to confirm the results of LCR. Precore mutant in HCC was 94.4% (17/18), 85.7% (18/21) in FH, 60% (12/20) in liver cirrhosis, 48.1% (38/79) in chronic hepatitis and 27.7% (20/72) in AVH cases. The serum ALT level was statistically significant between HBeAg negative WT and G1896A mutants in chronic hepatitis cases. ALT level and HBV DNA level was slightly raised in the pre core mutant but and was not significant. Genotype D had a higher prevalence (79.5%) as compared to genotype A (20.5%). The mutations detected by PCR–LCR were in 100% concordance with direct sequencing. The exceptionally high prevalence of G1896A in FH and HCC demonstrates that the precore mutants are strongly associated with the progression of liver diseases in patients with HBeAg negative serology. The findings are also suggestive of screening HBV precore G1896A mutation particularly in HBeAg negative cases. The precore G1896A mutation increases proportionately in severe form of liver diseases. LCR can be a suitable tool for screening of G1896A mutations.     

Efficiencies of designed enzyme combinations in releasing arabinose and xylose from wheat arabinoxylan in an industrial ethanol fermentation residue

The generation of a fermentable hydrolysate from arabinoxylan is an important prerequisite for utilization of wheat hemicellulose in production of ethanol or other value added products. This study examined the individual and combined efficiencies of four selected, commercial, multicomponent enzyme preparations Celluclast 1.5 L (from Trichoderma reesei), Finizym (from Aspergillus niger), Ultraflo L (from Humicola insolens), and Viscozyme L (from Aspergillus aculeatus) in catalyzing arabinose and xylose release from water-soluble wheat arabinoxylan in an industrial fermentation residue (still bottoms) in lab scale experiments. Different reaction conditions, i.e. enzyme dosage, reaction time, pH, and temperature, were evaluated in response surface and ternary mixture designs. Ultraflo L treatment gave optimal arabinose release: treatment (6 h, 60 °C, pH 6) with this enzyme preparation liberated up to 46% by weight (wt.%) of the theoretically maximal arabinose yield from the substrate. Celluclast 1.5 L was superior to the other enzyme preparations in releasing xylose and catalyzed release of up to 25 wt.% of the theoretical maximum xylose yield (6 h, 60 °C, pH 4). Prolonged treatment for 24 h with a 50:50 mixture of Celluclast 1.5 L and Ultraflo L at 50 °C, pH 5 exhibited a synergistic effect in xylose release and 62 wt.% of the theoretically maximal xylose yield was achieved. Addition of pure β-xylosidase from T. reesei to the Ultraflo L preparation released the same amounts of xylose from the substrate as the 50:50 mixture of Celluclast 1.5 L and Ultraflo L. The data thus signified that the synergistic effect in xylose release between Celluclast 1.5 L and Ultraflo L is the result of a three-step interaction mechanism involving α-l-arabinofuranosidase and different xylan degrading enzyme activities in the two enzyme preparations.     

Microbial communities involved in biogas production from wheat straw as the sole substrate within a two-phase solid-state anaerobic digestion

Microbial communities involved in biogas production from wheat straw as the sole substrate were investigated. Anaerobic digestion was carried out within an up-flow anaerobic solid-state (UASS) reactor connected to an anaerobic filter (AF) by liquor recirculation. Two lab-scale reactor systems were operated simultaneously at 37 °C and 55 °C. The UASS reactors were fed at a fixed organic loading rate of 2.5 g L⁻¹ d⁻¹, based on volatile solids. Molecular genetic analyses of the bacterial and archaeal communities within the UASS reactors (digestate and effluent liquor) and the AFs (biofilm carrier and effluent liquor) were conducted under steady-state conditions. The thermophilic UASS reactor had a considerably higher biogas and methane yield in comparison to the mesophilic UASS, while the mesophilic AF was slightly more productive than the thermophilic AF. When the thermophilic and mesophilic community structures were compared, the thermophilic system was characterized by a higher Firmicutes to Bacteroidetes ratio, as revealed by 16S rRNA gene (rrs) sequence analysis. The composition of the archaeal communities was phase-separated under thermophilic conditions, but rather stage-specific under mesophilic conditions. Family- and order-specific real-time PCR of methanogenic Archaea supported the taxonomic distribution obtained by rrs sequence analysis. The higher anaerobic digestion efficiency of the thermophilic compared to the mesophilic UASS reactor was accompanied by a high abundance of Firmicutes and Methanosarcina sp. in the thermophilic UASS biofilm.     

Atrial fibrillation-associated Connexin40 mutants make hemichannels and synergistically form gap junction channels with novel properties

Mutations of Cx40 (GJA5) have been identified in people with lone chronic atrial fibrillation including G38D and M163V which were found in the same patient. We used dual whole cell patch clamp procedures to examine the transjunctional voltage (V_j) gating and channel conductance properties of these two rare mutants. Each mutant exhibited slight alterations of V_j gating properties and increased the gap junction channel conductance (γ_j) by 20–30 pS. While co-expression of the two mutations had similar effects on V_j gating, it synergistically increased γ_j by 50%. Unlike WTCx40 or M163V, G38D induced activity of a dominant 271 pS hemichannel.     

Metabolic engineering of Thermoanaerobacterium saccharolyticum for n-butanol production

The thermophilic anaerobe Thermoanaerobacterium saccharolyticum JW/SL-YS485 was investigated as a host for n-butanol production. A systematic approach was taken to demonstrate functionality of heterologous components of the clostridial n-butanol pathway via gene expression and enzymatic activity assays in this organism. Subsequently, integration of the entire pathway in the wild-type strain resulted in n-butanol production of 0.85 g/L from 10 g/L xylose, corresponding to 21% of the theoretical maximum yield. We were unable to integrate the n-butanol pathway in strains lacking the ability to produce acetate, despite the theoretical overall redox neutrality of n-butanol formation. However, integration of the n-butanol pathway in lactate deficient strains resulted in n-butanol production of 1.05 g/L from 10 g/L xylose, corresponding to 26% of the theoretical maximum.     

Molecular characterization of Rifr mutations in Pseudomonas aeruginosa and Pseudomonas putida

The rpoB gene encoding for β subunit of RNA polymerase is a target of mutations leading to rifampicin resistant (Rif^r) phenotype of bacteria. Here we have characterized rpoB/Rif^r system in Pseudomonas aeruginosa and Pseudomonas putida as a test system for studying mutational processes. We found that in addition to the appearance of large colonies which were clearly visible on Rif selective plates already after 24 h of plating, small colonies grew up on these plates for 48 h. The time-dependent appearance of the mutant colonies onto selective plates was caused by different levels of Rif resistance of the mutants. The Rif^r clusters of the rpoB gene were sequenced and analyzed for 360 mutants of P. aeruginosa and for 167 mutants of P. putida. The spectrum of Rif^r mutations characterized for P. aeruginosa grown at 37 °C and that characterized for P. putida grown at 30 °C were dissimilar but the differences almost disappeared when the mutants of both strain were isolated at the same temperature, at 30 °C. The strong Rif^r phenotype of P. aeruginosa and P. putida was accompanied only with substitutions of these residues which belong to the putative Rif-binding pocket. Approximately 70% of P. aeruginosa mutants, which were isolated at 37 °C and expressed weak Rif^r phenotype, contained base substitutions in the N-terminal cluster of the rpoB gene. The differences in the spectra of mutations at 30 °C and 37 °C can be explained by temperature-sensitive growth of several mutants in the presence of rifampicin. Thus, our results imply that both the temperature for the growth of bacteria and the time for isolation of Rif^r mutants from selective plates are critical when the rpoB/Rif^r test system is employed for comparative studies of mutagenic processes in Pseudomonas species which are conventionally cultivated at different temperatures.     

Description of a new anaerobic thermophilic bacterium,Thermoanaerobacterium butyriciformans sp. nov.

Strain USBA-019^T, an anaerobic and thermophilic strain, was identified as a new member of the genus Thermoanaerobacterium. USBA-019^T cells are gram-positive, strictly anaerobic, thermophilic, chemoorganotrophic, moderately acidophilic, non-motile, endospore-forming, slightly curved, and rod-shaped. Cells measure 0.4 × 3.0–7.0 μm. Optimal growth occurs at 50–55 °C (35–65 °C). Optimum pH is 5.0–5.5 (4.0–8.5). Thiosulfate, elemental sulfur and nitrate were utilized as electron acceptors. Fermentation of glucose, lactose, cellobiose, galactose, arabinose, xylose, starch and xylan primarily produced acetate and butyrate. Xylan, starch and cellobiose produced ethanol and starch, cellobiose, galactose, arabinose and mannose produced lactic acid. Phylogenetic analyses based on 16S rRNA gene sequence comparison and genomic relatedness indices show the close relation of USBA-019^T to Thermoanaerobacterium thermostercoris and Thermoanaerobacterium aotearoense (similarity value: 99%). Hybridization of USBA-019^T, Th. thermostercoris DSM22141^T and Th. aotearoense DMS10170^T found DNA–DNA relatedness of 33.2% and 18.2%, respectively. Based on phenotypic, chemotaxonomic and phylogenetic evidence, along with low identity at whole genome level, USBA-019^T is a novel species of the genus Thermoanaerobacterium which we propose to name Thermoanaerobacterium butyriciformans sp. nov. The type strain is USBA-019^T (=CMPUJ U-019^T = DSM 101588^T).     

Expression,characterization and mutagenesis of an FAD-dependent glucose dehydrogenase from Aspergillus terreus

An FAD-dependent glucose dehydrogenase (FAD-GDH) from Aspergillus terreus NIH2624 was expressed in Escherichia coli with a yield of 228 ± 16 U/L of culture. Co-expression with chaperones DnaK/DnaJ/GrpE and osmotic stress induced by simple carbon sources enhanced productivity significantly, improving the yield to 23883 ± 563 U/L after optimization. FAD-GDH was purified in two steps with the specific activity of 604 U/mg. Using d-glucose as substrate, the optimal pH and temperature for FAD-GDH were determined to be 7.5 and 50 °C, respectively. Activity was stable across the pH range 3.5–9.0, and the half-life was 52 min at 42 °C. K_m and V_max were calculated as 86.7 ± 5.3 mM and 928 ± 35 U/mg, and the molecular weight was approximately 65.6 kDa based on size exclusion chromatography, indicating a monomeric structure. The 3D structure of FAD-GDH was simulated by homology modelling using the structure of A. niger glucose oxidase (GOD) as template. From the model, His551, His508, Asn506 and Arg504 were identified as key residues, and their importance was verified by site-directed mutagenesis. Furthermore, three additional mutants (Arg84Ala, Tyr340Phe and Tyr406Phe) were generated and all exhibited a higher degree of substrate specificity than the native enzyme. These results extend our understanding of the structure and function of FAD-GDH, and could assist potential commercial applications.