A study on the fatty acid composition of lipids in truffles selected from Europe and Africa.

The gas chromatography of hexane extracts from ascocarps of Tuber maculatum (mostly the first report), T. aestivum/unicantum, T. borchii, T. melanosporum and Tirmania nivea dominantly showed palmitic, stearic, oleic and linoleic acids followed by traces of polyunsaturated fatty acids. The fatty acid content varied from ca. 8-61 mg g- 1, dry-weight-basis with species with highest for T. maculatum. Polyunsaturated fatty acids contributions varied from ca. 42-59%. The dominant fatty acid varied with the species. A comparison with existing reports on same species cultivated in different regions showed differences in contributions by saturated, monounsaturated and polyunsaturated fatty acids as well as dominant fatty acids detected. Lesser explored species such as T. borchii, T. maculatum call for further research. This is a preliminary study that indicates fatty acid composition as a potential tool for distinction like aroma between truffle species and geographies of cultivation. This forms the basis for further studies in different species and regions.

The effect of direct and counter-current flow-through delignification on enzymatic hydrolysis of wheat straw, and flow limits due to compressibility.

This article compares the processes for wheat straw lignocellulose fractionation by percolation, counter-current progressing batch percolation and batch reaction at low NaOH-loadings (3-6% of DM). The flow-through processes were found to improve delignification and subsequent enzymatic saccharification, reduce NaOH-consumption and allow reduction of thermal severity, whereas hemicellulose dissolution was unaffected. However, contrary to previous expectations, a counter-current process did not provide additional benefits to regular percolation. The compressibility and flow properties of a straw bed were determined and used for simulation of the packing density profile and dynamic pressure in an industrial scale column. After dissolution of 30% of the straw DM by delignification, a pressure drop above 100 kPa m-1 led to clogging of the flow due to compaction of straw. Accordingly, the maximum applicable feed pressure and volumetric straw throughput was determined as a function of column height, indicating that a 10 m column can be operated at a maximum feed pressure of 530 kPa, corresponding to an operation time of 50 min and a throughput of 163 kg m-3 h-1 . Biotechnol. Bioeng. 2016;113: 2605-2613. © 2016 Wiley Periodicals, Inc.

Enhancement of acetyl xylan esterase activity on cellulose acetate through fusion to a family 3 cellulose binding module.

The current study investigates the potential to increase the activity of a family 1 carbohydrate esterase on cellulose acetate through fusion to a family 3 carbohydrate binding module (CBM). Specifically, CtCBM3 from Clostridium thermocellum was fused to the carboxyl terminus of the acetyl xylan esterase (AnAXE) from Aspergillus nidulans, and active forms of both AnAXE and AnAXE-CtCBM3 were produced in Pichia pastoris. CtCBM3 fusion had negligible impact on the thermostability or regioselectivity of AnAXE; activities towards acetylated corncob xylan, 4-methylumbelliferyl acetate, p-nitrophenyl acetate, and cellobiose octaacetate were also unchanged. By contrast, the activity of AnAXE-CtCBM3 on cellulose acetate increased by two to four times over 24 h, with greater differences observed at earlier time points. Binding studies using microcrystalline cellulose (Avicel) and a commercial source of cellulose acetate confirmed functional production of the CtCBM3 domain; affinity gel electrophoresis using acetylated xylan also verified the selectivity of CtCBM3 binding to cellulose. Notably, gains in enzyme activity on cellulose acetate appeared to exceed gains in substrate binding, suggesting that fusion to CtCBM3 increases functional associations between the enzyme and insoluble, high molecular weight cellulosic substrates.

Determination of surface-accessible acidic hydroxyls and surface area of lignin by cationic dye adsorption.

A new colorimetric method for determining the surface-accessible acidic lignin hydroxyl groups in lignocellulose solid fractions was developed. The method is based on selective adsorption of Azure B, a basic dye, onto acidic hydroxyl groups of lignin. Selectivity of adsorption of Azure B on lignin was demonstrated using lignin and cellulose materials as adsorbents. Adsorption isotherms of Azure B on wheat straw (WS), sugarcane bagasse (SGB), oat husk, and isolated lignin materials were determined. The maximum adsorption capacities predicted by the Langmuir isotherms were used to calculate the amounts of surface-accessible acidic hydroxyl groups. WS contained 1.7-times more acidic hydroxyls (0.21 mmol/g) and higher surface area of lignin (84 m(2)/g) than SGB or oat husk materials. Equations for determining the amount of surface-accessible acidic hydroxyls in solid fractions of the three plant materials by a single point measurement were developed. A method for high-throughput characterization of lignocellulosic materials is now available.

Enzymatic saccharification of pretreated wheat straw: comparison of solids-recycling, sequential hydrolysis and batch hydrolysis.

In the enzymatic hydrolysis of lignocellulose materials, the recycling of the solid residue has previously been considered within the context of enzyme recycling. In this study, a steady state investigation of a solids-recycling process was made with pretreated wheat straw and compared to sequential and batch hydrolysis at constant reaction times, substrate feed and liquid and enzyme consumption. Compared to batch hydrolysis, the recycling and sequential processes showed roughly equal hydrolysis yields, while the volumetric productivity was significantly increased. In the 72h process the improvement was 90% due to an increased reaction consistency, while the solids feed was 16% of the total process constituents. The improvement resulted primarily from product removal, which was equally efficient in solids-recycling and sequential hydrolysis processes. No evidence of accumulation of enzymes beyond the accumulation of the substrate was found in recycling. A mathematical model of solids-recycling was constructed, based on a geometrical series.

Production of L-xylose from L-xylulose using Escherichia coli L-fucose isomerase.

L-Xylulose was used as a raw material for the production of L-xylose with a recombinantly produced Escherichia coli L-fucose isomerase as the catalyst. The enzyme had a very alkaline pH optimum (over 10.5) and displayed Michaelis-Menten kinetics for L-xylulose with a K(m) of 41 mM and a V(max) of 0.23 µmol/(mg min). The half-lives determined for the enzyme at 35 °C and at 45 °C were 6h 50 min and 1h 31 min, respectively. The reaction equilibrium between L-xylulose and L-xylose was 15:85 at 35 °C and thus favored the formation of L-xylose. Contrary to the L-rhamnose isomerase catalyzed reaction described previously [14]L-lyxose was not detected in the reaction mixture with L-fucose isomerase. Although xylitol acted as an inhibitor of the reaction, even at a high ratio of xylitol to L-xylulose the inhibition did not reach 50%.

Increased water resistance of CTMP fibers by oat (Avena sativa L.) husk lignin.

The insertion of oat husk lignin onto chemithermomechanical pulp (CTMP) fibers was studied to increase fiber hydrophobicity. The pretreated pulp samples were subsequently used for preparation of handsheets for characterization. Treatment of CTMP with laccase in the presence of oat husk lignin resulted in a significant increase in hydrophobicity of the handsheet surface, as indicated by dynamic contact angle analysis. Water absorption time of 8 s was obtained with initial contact angle of 118°. Although the handsheet's brightness was reduced by 33%, tensile index was only subtly decreased. Neither laccase nor oat husk lignin alone gave much improved water absorption times. Therefore, handsheets made of laccase-treated pulp with and without oat husk lignin were further examined by XPS, which suggested that both laccase and oat husk lignin were inserted onto CTMP fibers. The oat husk lignin was distributed as heterogeneous aggregates on the handsheet surface whereas laccase was uniformly distributed. Evidence was obtained that the adsorbed laccase layer formed a noncovalent base for the insertion of oat husk lignin onto fiber surfaces.

Stochastic boundary molecular dynamics simulation of L-ribose in the active site of Actinoplanes missouriensis xylose isomerase and its Val135Asn mutant with improved reaction rate.

We used molecular dynamics simulations to study how a non-natural substrate, L-ribose, interacts with the active site of Actinoplanes missouriensis xylose isomerase. The simulations showed that L-ribose does not stay liganded in the active site in the same way as D-xylose, in which the oxygens O2 and O4 are liganded to the metal M1. The oxygen O4 of L-ribose moved away from the metal M1 to an upside down position. Furthermore, the distances of the carbons C1 and C2 of L-ribose to the catalytic metal M2 were higher than in the case of D-xylose. These findings explain the extremely low reaction rate of xylose isomerase with L-ribose. The mutation V135N close to the C5-OH of the substrate increased the reaction efficiency 2- to 4-fold with L-ribose. V135N did not affect the reaction with D-xylose and L-arabinose, whereas the reaction with D-glucose was impaired, probably due to a hydrogen bond between Asn-135 and the substrate. When L-ribose was the substrate, Asn-135 formed a hydrogen bond to Glu-181. As a consequence, O4 of L-ribose stayed liganded to the metal M1 in the V135N mutant in molecular dynamics simulations. This explains the decreased K(m) of the V135N mutant with L-ribose.

Xylanase production by Trichoderma reesei Rut C-30 grown on L-arabinose-rich plant hydrolysates.

The suitability of L-arabinose-rich plant hydrolysates as carbon sources and inducers of xylanase production in Trichoderma reesei Rut C-30 was tested. Significantly higher xylanase activities were obtained in cultures on oat husk and sugar beet pulp hydrolysates than on lactose. In batch culture with oat husk hydrolysate and lactose, the xylanase activity was about 9 times higher ( approximately 510 IU/ml) than in lactose ( approximately 60 IU/ml). Even higher xylanase activity ( approximately 630 IU/ml) was obtained when the batch cultivations were done on sugar beet pulp hydrolysate and lactose. In a fed-batch culture using oat husk hydrolysate-lactose the xylanase activity was as high as 1350 IU/ml in 4 days. The cellulase production clearly decreased when T. reesei was cultured on both hydrolysates compared to the cultivation on lactose. Moreover, the relative amounts of the xylanases I-III were similar regardless the used carbon source.

Engineering the substrate specificity of xylose isomerase.

Xylose isomerase (XI) catalyzes the isomerization and epimerization of hexoses, pentoses and tetroses. In order to clarify the reasons for the low reaction efficiency of a pentose sugar, L-arabinose, we determined the crystal structure of Streptomyces rubiginosus XI complexed with L-arabinose. The crystal structure revealed that, when compared with D-xylose and D-glucose, L-arabinose binds to the active site in a partially different position, in which the ligand has difficulties in binding the catalytic metal M2. Lys183 has been thought to stabilize the open substrate conformation by hydrogen bonding to oxygen O1. Our results with L-arabinose showed that the substrate stays in a linear form even without a hydrogen bond between Lys183 and oxygen O1. We engineered mutations to the active site of Actinoplanes missouriensis XI to improve the reaction efficiency with L-arabinose. The mutation F26W was intended to shift the position of oxygen O1 of L-arabinose closer to the catalytic metal M2. This effect of F26W was modeled by free energy perturbation simulations. In line with this, F26W increased 2-fold the catalytic efficiency of XI with L-arabinose; the increase was seen mainly in kcat. The mutation Q256D was outside the sphere of the catalytic residues and probably modified the electrostatic properties of the active site. It improved 3-fold the catalytic efficiency of XI with L-arabinose; this increase was seen in both Km and kcat. This study showed that it is possible to engineer the substrate specificity of XI.