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A hyperthermostable xylanase XYN10B from Thermotoga maritima (PDB code 1VBR, GenBank accession number KR078269) was subjected to site-directed and error-prone PCR mutagenesis. From the selected five mutants, the two site-directed mutants (F806H and F806V) showed a 3.3-3.5-fold improved enzyme half-life at 100 °C. The mutant XYNA generated by error-prone PCR showed slightly improved stability at 100 °C and a lower K

The biochemical conversion route in the utilization of biomass has required intensive development of processing methods for reducing recalcitrance of cellulose to enzymatic hydrolysis. Since discov...

The pulp and paper industry produces a diverse range of side-streams from multi-stage processes, but these remain underutilized despite their high potential for use as biofuels. This study investigated acetone-butanol-ethanol (ABE) fermentation by Clostridium acetobutylicum DSM 1731 from the side-streams of three different stages of the pulp and paper manufacturing process (PI, PII, and PIII). Biomass specimens with and without water washing were pretreated with 0.2% H2SO4 at 180 °C for 10 min, followed by enzymatic hydrolysis, to obtain fermentable sugars. The results showed that the produced ABE solvent concentrations were 12.8 g/L, 5.2 g/L, and 6.3 g/L from PI, PII, and PIII, respectively. The butanol yields of PI, PII, and PIII were 0.25, 0.18, and 0.19 g/g sugars, respectively. Among the tested side-streams, PI was shown to have potential as a feedstock for butanol production without prewashing prior to dilute acid pretreatment, enzymatic hydrolysis, and microbial fermentation.

The use of enzymes in aqueous solutions of ionic liquids (ILs) could be useful for the enzymatic treatment of lignocellulose. Hydrophilic ILs that dissolve lignocellulose are harmful to enzymes. The toleration limits and enzyme-friendly superbase IL combinations were investigated for the hyperthermophilic Thermopolyspora flexuosa GH10 xylanase (endo-1,4-beta-xylanase EC 3.2.1.8) TfXYN10A and Dictyoglomus thermophilum GH11 xylanase DtXYN11B. TfXYN10A was more tolerant than DtXYN11B to acetate or propionate-based ILs. However, when the anion of the ILs was bigger (guaiacolate), GH11 xylanase showed higher tolerance to ILs. 1-Ethyl-3-methylimidazolium acetate ([EMIM]OAc), followed by 1,1,3,3-tetramethylguanidine acetate ([TMGH]OAc), were the most enzyme-friendly ILs for TfXYN10A and [TMGH](+)-based ILs were tolerated best by DtXYN11B. Double-ring cations and a large size anion were associated with the strongest enzyme inhibition. Competitive inhibition appears to be a general factor in the reduction of enzyme activity. However, with guaiacolate ILs, the denaturation of proteins may also contribute to the reduction in enzyme activity. Molecular docking with IL cations and anions indicated that the binding mode and shape of the active site affect competitive inhibition, and the cobinding of cations and anions to separate active site positions caused the strongest enzyme inhibition.

Formate dehydrogenases (FDHs) are NAD(P)H-dependent enzymes that catalyse the reversible oxidation of formate to CO2. The main goal was to use directed evolution to obtain variants of the FDH from Chaetomium thermophilum (CtFDH) with enhanced reduction activity in the conversion of CO2 into formic acid. Four libraries were constructed targeting five residues in the active site. We identified two variants (G93H/I94Y and R259C) with enhanced reduction activity which were characterised in the presence of both aqueous CO2(g) and HCO3−. The A1 variant (G93H/I94Y) showed a 5.4-fold increase in catalytic efficiency (kcat/KM) compared to that of the wild-type for HCO3− reduction. The improved biocatalysts were also applied as a coupled cofactor recycling system in the enantioselective oxidation of 4-phenyl-2-propanol catalysed by the alcohol dehydrogenase from Streptomyces coelicolor A3 (ScADH). Conversions in these reactions increased from 56 to 91% when the A1 variant was used instead of wild-type CtFDH. Two variants presenting up to five-fold increase in catalytic efficiency and kcat were obtained and characterised. They constitute a promising enzymatic alternative for CO2 utilization and will serve as scaffolds to be further developed in order to meet industrial requirements.

Objectives Ionic liquids (ILs) that dissolve biomass are harmful to the enzymes that degrade lignocellulose. Enzyme hyperthermostability promotes a tolerance to ILs. Therefore, the limits of hyperthemophilic Pyrococcus horikoschii endoglucanase (PhEG) to tolerate 11 superbase ILs were explored. Results PhEG was found to be most tolerant to 1-ethyl-3-methylimidazolium acetate ([EMIM]OAc) in soluble 1% carboxymethylcellulose (CMC) and insoluble 1% Avicel substrates. At 35% concentration, this IL caused an increase in enzyme activity (up to 1.5-fold) with CMC. Several ILs were more enzyme inhibiting with insoluble Avicel than with soluble CMC. Km increased greatly in the presence ILs, indicating significant competitive inhibition. Increased hydrophobicity of the IL cation or anion was associated with the strongest enzyme inhibition and activation. Surprisingly, PhEG activity was increased 2.0–2.5-fold by several ILs in 4% substrate. Cations exerted the main role in competitive inhibition of the enzyme as revealed by their greater binding energy to the active site. Conclusions These results reveal new ways to design a beneficial combination of ILs and enzymes for the hydrolysis of lignocellulose, and the strong potential of PhEG in industrial, high substrate concentrations in aqueous IL solutions.

Background. Sahara is one of the largest deserts in the world. The hard conditions especially high temperature and aridity lead to unique adaptation of organisms that could be a potential source of new metabolites. In this respect, two Saharan soils from El Oued Souf and Beni Abbes in Algeria were collected. The bacterial isolates were selected by screening the antibacterial, antifungal, and enzymatic activities. The whole genomes of the two native Saharan strains were sequenced to study the desert Streptomyces microbiology and ecology from genomic perspective. Results. The strains Babs14 (from Beni Abbes, Algeria) and Osf17 (from El Oued Souf, Algeria) were initially identified by 16S rRNA sequence as belonging to the Streptomyces genus. The whole genome sequencing of the two strains was performed by using Pacific Biosciences Sequel II technology (PacBio) and it showed that Babs14 and Osf17 have a linear chromosome of 8.00 Mb and 7.97 Mb, respectively. The number of identified proteins is 6910 in Babs14 and 6894 in Osf17. No plasmids were found in Babs14, whereas three plasmids were detected in Osf17 and no genome duplications were detected in the two strains. Although the strains have different phenotypes and are from different regions, they are slightly different from each other in protein sequence level but, differ more from their earlier published relatives. 29 biosynthetic gene clusters (BGCs) coding for secondary metabolites were predicted in each strain indicating antifungal, antibiotic, and other activities of Babs14 and Osf17. Moreover, many other BGCs were related to biosynthesis of factors needed in response to environmental stress in temperature, UV light and osmolarity. Conclusion. The genome sequencing of Saharan Streptomyces strains revealed factors that are related to their adaptation to an extreme environment and stress conditions. The genome information gives tools to study the ecological adaptation in desert environment and to explore the bioactive compounds of these microorganisms. The two whole genome sequences are among the first ones for the Streptomyces genus of Algerian Sahara. The present research was undertaken as a first step to explore more profoundly the desert microbiome.

BACKGROUNDThis study elaborates the possibility to apply combined ionic liquid (IL) and enzyme treatments for pulp fibre modification. The approach involves swelling of fibre surfaces with IL followed by enzymatic modification of the disrupted fibre surface using carbohydrate active enzymes. RESULTSThe capacity of seven cellulose-dissolving or cellulose-swelling ionic liquids to swell pulp fibres was compared. In addition, thirteen cellulases and five xylanases were screened for their IL tolerance, which determines their applicability in combined or sequential IL-enzyme treatments of fibres. Among the studied ionic liquids, 1-ethyl-3-methylimidazolium dimethylphosphate ([EMIM]DMP) and 1,3-dimethylimidazolium dimethylphosphate ([DMIM]DMP) had the strongest effect on fibre swelling. These solvents were also found to be the least inactivating for the studied enzymes. CONCLUSIONEnzyme compatibility and cellulose-dissolving capability are not two conflicting properties of an ionic liquid. (c) 2017 Society of Chemical Industry

Formate dehydrogenases (FDHs) are NAD(P)H-dependent enzymes that catalyse the reversible oxidation of formate to COFour libraries were constructed targeting five residues in the active site. We identified two variants (G93H/I94Y and R259C) with enhanced reduction activity which were characterised in the presence of both aqueous COTwo variants presenting up to five-fold increase in catalytic efficiency and k

The gene of Thermotoga maritima GH10 xylanase (TmXYN10B) was synthesised to study the extreme limits of this hyperthermostable enzyme at high temperatures in the presence of biomass-dissolving hydrophilic ionic liquids (ILs). TmXYN10B expressed from Pichia pastoris showed maximal activity at 100 °C and retained 92 % of maximal activity at 105 °C in a 30-min assay. Although the temperature optimum of activity was lowered by 1-ethyl-3-methylimidazolium acetate ([EMIM]OAc), TmXYN10B retained partial activity in 15–35 % hydrophilic ILs, even at 75–90 °C. TmXYN10B retained over 80 % of its activity at 90 °C in 15 % [EMIM]OAc and 15–25 % 1-ethyl-3-methylimidazolium dimethylphosphate ([EMIM]DMP) during 22-h reactions. [EMIM]OAc may rigidify the enzyme and lower Vmax. However, only minor changes in kinetic parameter Km showed that competitive inhibition by [EMIM]OAc of TmXYN10B is minimal. In conclusion, when extended enzymatic reactions under extreme conditions are required, TmXYN10B shows extraordinary potential. Electronic supplementary material The online version of this article (doi:10.1007/s00792-016-0841-y) contains supplementary material, which is available to authorized users.

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.

Desert truffles have traditionally been used as food in Libya. Desert truffle grows and gives fruit sporadically when adequate and properly distributed rainfall occurs with existence of suitable soil and mycorrhizal host plant. The present study aimed to identify and characterize two kinds of wild desert truffles from ecological and nutritional points that were collected from the studied area. The truffle samples were identified as Terfezia (known as red or black truffle) and Tirmania (known as white truffle). The nutritional values (protein, lipid and carbohydrate) of both Libyan wild truffle (Terfezia and Tirmania) were determined on a dry weight basis and result showed that Tirmania and Terfezia contained 16.3 and 18.5% protein, 6.2 and 5.9% lipid, 67.2 and 65% carbohydrate, respectively, in ascocarp biomass. The soil pH of the upper and lower regions of the Hamada Al-Hamra ranged between 8.2 and 8.5 giving suitable conditions for fructification. The plants, Helianthemum kahiricum and Helianthemum lippii were the dominant plants in Hamada Al-Hamra region found to form a mycorrhiza with desert truffles. The phylogenetic analysis of the genomic rDNA ITS region showed that, out of five collections three represented Tirmania pinoyi (Maire) Malencon, one Tirmania nivea (Desf.) Trappe, and one Terfezia boudieri Chatin.

Xylan-debranching enzymes facilitate the complete hydrolysis of xylan and can be used to alter xylan chemistry. Here, the family GH62 α- l -arabinofuranosidase from Streptomyces thermoviolaceus (SthAbf62A) was shown to have a half-life of 60 min at 60°C and the ability to cleave α-1,3 l -arabinofuranose ( l -Ara f ) from singly substituted xylopyranosyl (Xyl p ) backbone residues in wheat arabinoxylan; low levels of activity on arabinan as well as 4-nitrophenyl α- l -arabinofuranoside were also detected. After selective removal of α-1,3 l -Ara f substituents from disubstituted Xyl p residues present in wheat arabinoxylan, SthAbf62A could also cleave the remaining α-1,2 l -Ara f substituents, confirming the ability of SthAbf62A to remove α- l -Ara f residues that are (1→2) and (1→3) linked to monosubstituted β- d -Xyl p sugars. Three-dimensional structures of SthAbf62A and its complex with xylotetraose and l -arabinose confirmed a five-bladed β-propeller fold and revealed a molecular Velcro in blade V between the β1 and β21 strands, a disulfide bond between Cys27 and Cys297, and a calcium ion coordinated in the central channel of the fold. The enzyme-arabinose complex structure further revealed a narrow and seemingly rigid l -arabinose binding pocket situated at the center of one side of the β propeller, which stabilized the arabinofuranosyl substituent through several hydrogen-bonding and hydrophobic interactions. The predicted catalytic amino acids were oriented toward this binding pocket, and the catalytic essentiality of Asp53 and Glu213 was confirmed by site-specific mutagenesis. Complex structures with xylotetraose revealed a shallow cleft for xylan backbone binding that is open at both ends and comprises multiple binding subsites above and flanking the l -arabinose binding pocket.

In the present study, an extremophilic GH11 xylanase was stabilized by an engineered N-terminal disulphide bridge. The effect of the stabilization was then tested against high temperatures and in the presence of a biomass-dissolving ionic liquid, 1-ethyl-3-methylimidazolium acetate ([emim]OAc). The N-terminal disulfide bridge increased the half-life of a GH11 xylanase (XYNB) from the hyperthermophilic bacterium Dictyoglomus thermophilum by 10-fold at 100 °C. The apparent temperature optimum increased only by ∼5 °C, which is less than the corresponding increase in mesophilic (∼15 °C) and moderately thermophilic (∼10 °C) xylanases. The performance of the enzyme was increased significantly at 100–110 °C. The increasing concentration of [emim]OAc almost linearly increased the inactivation level of the enzyme activity and 25% [emim]OAc inactivated the enzyme almost fully. On the contrary, the apparent temperature optimum did not decrease to a similar extent, and the degree of denaturation of the enzyme was also much lower according to the residual activity assays. Also, 5% [emim]OAc largely counteracted the benefit obtained by the stabilizing disulfide bridge in the temperature-dependent activity assays, but not in the stability assays. K m was increased in the presence of [emim]OAc, indicating that [emim]OAc interfered the substrate–enzyme interactions. These results indicate that the effect of [emim]OAc is targeted more to the functioning of the enzyme than the basic stability of the hyperthermophilic GH11 xylanase.

While formate dehydrogenases (FDHs) have been used for cofactor recycling in chemoenzymatic synthesis, the ability of FDH to reduce CO

A fairly recent whole-genome duplication (WGD) event in yeast enables the effects of gene duplication and subsequent functional divergence to be characterized. We examined 15 ohnolog pairs (i.e. paralogs from a WGD) out of c. 500 Saccharomyces cerevisiae ohnolog pairs that have persisted over an estimated 100 million years of evolution. These 15 pairs were chosen for their high levels of asymmetry, i.e. within the pair, one ohnolog had evolved much faster than the other. Sequence comparisons of the 15 pairs revealed that the faster evolving duplicated genes typically appear to have experienced partially – but not fully – relaxed negative selection as evidenced by an average nonsynonymous/synonymous substitution ratio (dN/dSavg=0.44) that is higher than the slow-evolving genes' ratio (dN/dSavg=0.14) but still

While formate dehydrogenases (FDHs) have been used for cofactor recycling in chemoenzymatic synthesis, the ability of FDH to reduce CO2 could also be utilized in the conversion of CO2 to useful products via formate (HCOO-). In this study, we investigated the reduction of CO2 in the form of hydrogen carbonate (HCO3-) to formate by FDHs from Candida methylica (CmFDH) and Chaetomium thermophilum (CtFDH) in a NADH-dependent reaction. The catalytic performance with HCO3- as a substrate was evaluated by measuring the kinetic rates and conducting productivity assays. CtFDH showed a higher efficiency in converting HCO3- to formate than CmFDH, whereas CmFDH was better in the oxidation of formate. The pH optimum of the reduction was at pH 7-8. However, the high concentrations of HCO3- reduced the reaction rate. CtFDH was modeled in the presence of HCO3- showing that it fits to the active site. The active site setting for hydride transfer in CO2 reduction was modeled. The hydride donated by NADH would form a favorable contact to the carbon atom of HCO3-, resulting in a surplus of electrons within the molecule. This would cause the complex formed by hydrogen carbonate and the hydride to break into formate and hydroxide ions.

This work describes the synthesis of temperature-responsive polystyrene-block-poly(N-isopropylacrylamide)-block-polystyrene triblock copolymers, i.e., PS-b-PNIPAM-b-PS, their self-assembly and phase behavior in bulk, and demonstration of aqueous thermoresponsive membranes. A series of PS-b-PNIPAM-b-PS triblock copolymers were synthesized using reversible addition-fragmentation chain transfer (RAFT) polymerization. The hydrophobic PS end blocks were selected to form the minority component, whereas the temperature-responsive PNIPAM midblock accounted for the majority component. The self-assembly and phase behavior in bulk of PS-b-PNIPAM-b-PS as well as selected blends with low molecular weight PNIPAM homopolymers were studied using transmission electron microscopy (TEM). Classical lamellar, cylindrical, spherical, and bicontinuous double gyroid morphologies were observed in the dried state. In aqueous solutions, the glassy PS domains act as physical cross-links, and hydrogels were therefore formed. The bulk block copolymer morphology had a strong effect on the degree of swelling in aqueous solutions upon cooling below the coil-globule transition temperature of the PNIPAM midblock. Bulk compositions with spherical PS domains and PNIPAM continuous phase swelled in water up to 58 times by weight, whereas composition having cylindrical PS domains or bicontinous gyroid structure in bulk swelled 20 or 10 times by weight, respectively. Finally, lamellar compositions did not show any swelling. Composite membranes for separation studies were prepared by spin-coating thin films of PS-b-PNIPAM-b-PS on top of meso/macroporous polyacrylonitrile (PAN) support membrane. The permeability was measured as a function of temperature using aqueous mixture of poly(ethylene glycol) (PEG) with several well-defined molecular weights. The permeability showed a temperature switchable on/off behavior, where higher permeability is obtained below transition temperature of PNIPAM, and the molecular cutoff limits for the PEG molecules are surprisingly low-between 108 and 660 g/mol. The results encourage to further develop and optimize these materials for responsive nanofiltration applications.

The extraordinary properties of natural proteins demonstrate that life-like protein engineering is both achievable and valuable. Rapid progress and impressive results have been made towards this goal using rational design and random techniques or a combination of both. However, we still do not have a general theory on how to specify a structure that is suited to a target function nor can we specify a sequence that folds to a target structure. There is also overreliance on the Darwinian blind search to obtain practical results. In the long run, random methods cannot replace insight in constructing life-like proteins. For the near future, however, in enzyme development, we need to rely on a combination of both.

Intramolecular mobility and conformational changes of flexible loops have important roles in the structural and functional integrity of proteins. The Achaetomium sp. Xz8 endo-polygalacturonase (PG8fn) of glycoside hydrolase (GH) family 28 is distinguished for its high catalytic activity (28,000 U/mg). Structure modeling indicated that PG8fn has a flexible T3 loop that folds partly above the substrate in the active site, and forms a hydrogen bond to the substrate by a highly conserved residue Asn94 in the active site cleft. Our research investigates the catalytic roles of Asn94 in T3 loop which is located above the catalytic residues on one side of the substrate. Molecular dynamics simulation performed on the mutant N94A revealed the loss of the hydrogen bond formed by the hydroxyl group at O34 of pentagalacturonic acid and the crucial ND2 of Asn94 and the consequent detachment and rotation of the substrate away from the active site, and that on N94Q caused the substrate to drift away from its place due to thelonger side chain. In line with the simulations, site-directed mutagenesis at this site showed that this position is very sensitive to amino acid substitutions. Except for the altered Km values from 0.32 (wild type PG8fn) to 0.75–4.74 mg/ml, all mutants displayed remarkably lowered kcat (~3–20,000 fold) and kcat/Km (~8–187,500 fold) values and significantly increased △(△G) values (5.92–33.47 kJ/mol). Taken together, Asn94 in the GH28 T3 loop has a critical role in positioning the substrate in a correct way close to the catalytic residues.

Xylanase was produced by growing Thermomyces lanuginosus DSM 10635 in different carbohydrate containing media. The xylanase was purified to electrophoretic homogeneity and some of its enzymatic properties were studied. The molecular mass of this xylanase estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was 25.5 kDa, and the p I was approximately 3.7. Applying mass spectrometric analysis, the average molecular mass of the xylanase was determined to be 21295.17 Da, which is practically the same as the theoretical molecular mass of the T. lanuginosus DSM 5826 xylanase (crystal structure 1yna). Accordingly, the thermostability and enzymatic properties of DSM 10635 xylanase were similar to DSM 5826 xylanase. The inactivation of DSM 10635 xylanase during the elevation of temperature proceeded slowly at pH 6.5 and 8.0, whereas at pH 5.0, the inactivation profile was steep, indicating that changes in the electrostatic interactions decrease the stability at lower pH. The thermostabilizing effect of the substrate on DSM 10635 xylanase was studied and it was observed to be significant only under acidic conditions. The half-life in the presence of 1% birchwood xylan (w/v) was 7-fold, 4-fold and 1.5-fold higher than without the substrate at pH 4.0, 5.0 and 6.5, respectively. No increase in the stability by the presence of substrate was observed at pH 9.0.

We have successfully engineered a disulphide bridge into the N-terminal region of Trichoderma reesei endo-1,4-beta-xylanase II (XYNII) by substituting Thr-2 and Thr-28 with cysteine. The T2C:T28C mutational changes increased the half-life in thermal inactivation of this mesophilic enzyme from approximately 40 s to approximately 20 min at 65 degrees C, and from less than 10 s to approximately 6 min at 70 degrees C. Therefore, the N-terminal disulphide bridge enables the use of XYNII at substantially higher temperatures than permitted by its native mesophilic counterpart. Altogether, thermostability increased by about 15 degrees C. The kinetic properties of the mutant XYNII were maintained at the level of the wild type enzyme. Our findings demonstrated that a properly designed disulphide bridge, here within the N-terminal region of XYNII, can be very effective in resisting thermal inactivation.

Trichoderma reesei Rut C-30 was grown on eight different natural or rare aldopentoses as the main carbon source and on mixtures of an aldopentose with D-glucose or lactose. The fungal cells consumed all aldopentoses tested, except L-xylose and L-ribose. The highest total xylanase and cellulase activities were achieved when cells were grown on L-arabinose as the main carbon source. The total xylanase activity produced by cells grown on L-arabinose was even higher than that produced by cells grown on an equal amount of lactose. In co-metabolism of D-glucose (15 g l(-1)) and L-arabinose (5 g l(-1)), the total volumetric and specific xylanase productivities were improved (derepressed) approximately 23- and 18-fold, respectively, compared to a cultivation on only D-glucose (20 g l(-1)). In a similar experiment, in which cells were grown on a mixture of lactose and L-arabinose, the xylanase productivity was approximately doubled, compared to a cultivation on only lactose. The cellulase productivities, however, were not improved by the addition of L-arabinose. Compared with a typical industrial fungal enzyme production process with lactose as the main carbon source, better volumetric and specific xylanase productivities were achieved both on a lactose/arabinose mixture and on a glucose/arabinose mixture.

The crystal structures of thermophilic xylanases from Chaetomium thermophilum and Nonomuraea flexuosa were determined at 1.75 and 2.1 A resolution, respectively. Both enzymes have the overall fold typical to family 11 xylanases with two highly twisted beta-sheets forming a large cleft. The comparison of 12 crystal structures of family 11 xylanases from both mesophilic and thermophilic organisms showed that the structures of different xylanases are very similar. The sequence identity differences correlated well with the structural differences. Several minor modifications appeared to be responsible for the increased thermal stability of family 11 xylanases: (a) higher Thr : Ser ratio (b) increased number of charged residues, especially Arg, resulting in enhanced polar interactions, and (c) improved stabilization of secondary structures involved the higher number of residues in the beta-strands and stabilization of the alpha-helix region. Some members of family 11 xylanases have a unique strategy to improve their stability, such as a higher number of ion pairs or aromatic residues on protein surface, a more compact structure, a tighter packing, and insertions at some regions resulting in enhanced interactions.

The functional properties of extremophilic Dictyoglomus thermophilum xylanase (XYNB) and the N-terminal disulphide-bridge mutant (XYNB-DS) were studied at high pressure and temperature. The enzymes were quite stable even at the pressure of 500MPa at 80°C. The half-life of inactivation in these conditions was over 30h. The inactivation at 80°C in atmospheric pressure was only 3-times slower. The increase of pressure up to 500MPa at 80°C decreased only slightly the enzyme's stability, whereas in 500MPa the increase of temperature from 22 to 80°C decreased significantly more the enzyme's stability. While the high temperature (80-100°C) decreased the enzyme reaction with short xylooligosaccharides (xylotetraose and xylotriose), the high pressure (100-300MPa) had an opposite effect. The temperature of 100°C strongly increased the Km but did not affect the kcat to the same extent, thus indicating that the interaction of the substrate with the active site suffers before the catalytic reaction begins to decrease as the temperature rises. Circular dichroism spectroscopy showed the high structural stability of XYNB and XYNB-DS at 93°C.

Thermal stability and other functional properties of Trichoderma reesei endo-1,4-beta-xylanase II (XYNII; family 11) were studied by designed mutations. Mutations at three positions were introduced to the XYNII mutant containing a disulfide bridge (S110C-N154C) in the alpha-helix. The disulfide bridge increased the half-life of XYNII from less than 1 min to 14 min at 65 degrees C. An additional mutation at the C-terminus of the alpha-helix (Q162H or Q162Y) increased the half-life to 63 min. Mutations Q162H and Q162Y alone had a stabilizing effect at 55 degrees C but not at 65 degrees C. The mutations N11D and N38E increased the half-life to about 100 min. Due to the stabilizing mutations the pH stability increased in a wide pH range, but at the same time the activity decreased both in acidic and neutral-alkaline pH, the pH optimum being at pH region 5-6. There was no essential difference between the specific activities of the mutants and the wild-type XYNII.

Endo-1,4-β-xylanase II (XYNII) from Trichoderma reesei is a 21kDa enzyme that catalyses the hydrolysis of xylan, the major plant hemicellulose. It has various applications in the paper, food and feed industries. Previous thermostability studies have revealed a significant decrease in enzymic activity of the protein at elevated temperatures in citrate buffer [Tenkanen, Puls and Poutanen (1992) Enzyme Microb. Technol. 14, 566–574]. Here, thermostability of XYNII was investigated using both conventional and nanoelectrospray ionization Fourier-transform ion cyclotron resonance MS and hydrogen/deuterium (H/D)-exchange reactions. In addition, dynamic light scattering (DLS) was used as a comparative method to observe possible changes in both tertiary and quaternary structures of the protein. We observed a significant irreversible conformational change and dimerization when the protein was exposed to heat. H/D exchange revealed two distinct monomeric protein populations in a narrow transition temperature region. The conformational change in both the water and buffered solutions occurred in the same temperature region where enzymic-activity loss had previously been observed. Approx. 10–30% of the protein was specifically dimerized when exposed to the heat treatment. However, adding methanol to the solution markedly lowered the transition temperature of conformational change as well as increased the dimerization up to 90%. DLS studies in water confirmed the change in conformation observed by electrospray ionization MS. We propose that the conformational change is responsible for the loss of enzymic activity at temperatures over 50°C and that the functioning of the active site in the enzyme is unfeasible in a new, more labile solution conformation.

MUC1, a transmembrane member of the mucin family, is believed to have anti-adhesive properties because of its highly sialylated, extended, and rigid rod-like conformation. The ERM proteins (ezrin, radixin, and moesin) function as membrane–cytoskeletal linkers. MUC1 and ezrin are enriched in microvilli in MCF-7az breast carcinoma cells. Similar localization was also found in peripheral membrane areas and in filopodium-like protrusions. Whereas ezrin was consistently detected in the cell–cell contact region, MUC1 was less frequently found there. MUC1 was distinctly expressed in long filopodial protrusions and was highly concentrated at their tips, which also contained ezrin, whereas F-actin was found along the stalk. This localization of MUC1 suggests a role for MUC1 in transient cell structures of migrating cells and transient cell adhesion. No direct association has yet been found between MUC1 and ezrin. However, both MUC1 and ezrin had a similar overall distribution pattern in microvilli and filopodium-like protrusions in immunoelectron tomography. In addition, MUC1 and ezrin showed spatial association, because several 10-nm gold particles used to decorate ezrin were seen in the vicinity close to the clusters of 5-nm gold particles decorating MUC1. Therefore, MUC1 appears to be associated with ezrin, but the nature of this association requires further study.

An extracellular beta-galactosidase from Trichoderma reesei was crystallized from sodium cacodylate buffer using polyethylene glycol (PEG) as a precipant. Crystals grown by homogenous streak-seeding belonged to space group P1, with unit-cell parameters a = 67.3, b = 69.1, c = 81.5 A, alpha = 109.1, beta = 97.3, gamma = 114.5 degrees . The crystals diffracted to 1.8 A resolution using a rotating-anode generator and to 1.2 A resolution using a synchrotron source. On the basis of the Matthews coefficient (V(M) = 3.16 A(3) Da(-1)), one molecule is estimated to be present in the asymmetric unit. The aim of the determination of the crystal structure is to increase the understanding of this industrially significant enzyme.

Protein engineering is commonly used to improve the robustness of enzymes for activity and stability at high temperatures. In this study, we identified four residues expected to affect the thermostability of Streptomyces sp. strain S9 xylanase XynAS9 through multiple-sequence analysis (MSA) and molecular dynamic simulations (MDS). Site-directed mutagenesis was employed to construct five mutants by replacing these residues with proline or glutamic acid (V81P, G82E, V81P/G82E, D185P/S186E, and V81P/G82E/D185P/S186E), and the mutant and wild-type enzymes were expressed in Pichia pastoris . Compared to the wild-type XynAS9, all five mutant enzymes showed improved thermal properties. The activity and stability assays, including circular dichroism and differential scanning calorimetry, showed that the mutations at positions 81 and 82 increased the thermal performance more than the mutations at positions 185 and 186. The mutants with combined substitutions (V81P/G82E and V81P/G82E/D185P/S186E) showed the most pronounced shifts in temperature optima, about 17°C upward, and their half-lives for thermal inactivation at 70°C and melting temperatures were increased by >9 times and approximately 7.0°C, respectively. The mutation combination of V81P and G82E in adjacent positions more than doubled the effect of single mutations. Both mutation regions were at the end of long secondary-structure elements and probably rigidified the local structure. MDS indicated that a long loop region after positions 81 and 82 located at the end of the inner β-barrel was prone to unfold. The rigidified main chain and filling of a groove by the mutations on the bottom of the active site canyon may stabilize the mutants and thus improve their thermostability.

Very little is known about the receptors and target molecules involved in natural killer (NK) cell activity. Here we present a model system in which interleukin-2-activated killing by NK cells depends on the intercellular adhesion molecule ICAM-2 and is regulated by the distribution of ICAM-2. The level of ICAM-2 expression in NK-sensitive and resistant cells is similar, but in sensitive cells ICAM-2 is concentrated into bud-like cellular projections known as uropods, whereas in resistant cells it is evenly distributed. The cytoskeletal-membrane linker protein ezrin is also localized in uropods. Transfection of human ezrin into NK-resistant cells induces uropods formation, redistribution of ICAM-2 and ezrin, and sensitizes target cells to interleukin-2-activated killing. These results reveal a new mechanism of target-cell recognition: cytotoxic cells recognize adhesion molecules that are already present on normal cells, but in diseased cells are concentrated into a biologically active cell-surface region by cytoskeletal reorganization. The results also highlight the importance of cytoskeletal interactions in the regulation of ICAM-2-mediated adhesive phenomena.

We studied the pressure stability of disulphide bridge mutants of Trichoderma reesei XYNII at 500-5000 bar. The inactivation of XYNII and its mutants was strongest above 4000 bar. The pressure stability correlated with the thermostability order of the XYNII mutants, indicating that the stabilising mutations in protein regions important for thermostability also protect the enzyme at high pressure. In combination with high pressure, a mild heating had already inactivated the wild-type enzyme; the thermostabilising mutations largely counteracted this effect. At a low temperature, the mutations did not have any remarkable pressure stabilisation effect. Thus, thermal inactivation appeared to dominate over pressure inactivation at higher temperatures. Kinetic calculations indicated that pressure compressibility correlated with the thermostability of xylanase mutants.

Ezrin, previously also known as cytovillin, p81, and 80K, is a cytoplasmic protein enriched in microvilli and other cell surface structures. Ezrin is postulated to have a membrane-cytoskeleton linker role. Recent findings have also revealed that the NH2-terminal domain of ezrin is associated with the plasma membrane and the COOH-terminal domain with the cytoskeleton (Algrain, M., O. Turunen, A. Vaheri, D. Louvard, and M. Arpin. 1993. J. Cell Biol. 120: 129-139). Using bacterially expressed fragments of ezrin we now demonstrate that ezrin has an actin-binding capability. We used glutathione-S-transferase fusion proteins of truncated ezrin in affinity chromatography to bind actin from the cell extract or purified rabbit muscle actin. We detected a binding site for filamentous actin that was localized to the COOH-terminal 34 amino acids of ezrin. No binding of monomeric actin was detected in the assay. The region corresponding to the COOH-terminal actin-binding site in ezrin is highly conserved in moesin, actin-capping protein radixin and EM10 protein of E. multilocularis, but not in merlin/schwannomin. Consequently, this site is a potential actin-binding site also in the other members of the protein family. Furthermore, the actin-binding site in ezrin shows sequence homology to the actin-binding site in the COOH terminus of the beta subunit of the actin-capping protein CapZ and one of the potential actin-binding sites in myosin heavy chain. The actin-binding capability of ezrin supports its proposed role as a membrane-cytoskeleton linker.

l-Xylulose was used as a raw material for the production of l-xylose with a recombinantly produced Escherichia colil-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 Km of 41 mM and a Vmax of 0.23 μmol/(mg min). The half-lives determined for the enzyme at 35 °C and at 45 °C were 6 h 50 min and 1 h 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%.

Ezrin, a widespread protein present in actin-containing cell-surface structures, is a substrate of some protein tyrosine kinases. Based on its primary and secondary structure similarities with talin and band 4.1 it has been suggested that this protein could play a role in linking the cytoskeleton to the plasma membrane (Gould, K.L., A. Bretscher, F.S. Esch, and T. Hunter. 1989. EMBO (Eur. Mol. Biol. Organ.), J. 8:4133-4142; Turunen, O., R. Winqvist, R. Pakkanen, K.-H. Grzeschik, T. Wahlström, and A. Vaheri. 1989. J. Biol. Chem. 264:16727-16732). To test this hypothesis, we transiently expressed the complete human ezrin cDNA, or truncated cDNAs encoding the amino- and carboxy-terminal domains of the protein, in CV-1 cells. Protein epitope tagging was used to unambiguously determine the subcellular distribution of the protein encoded by the transfected cDNA. We show that this protein is concentrated underneath the dorsal plasma membrane in all actin-containing structures and is partially detergent insoluble. The amino-terminal domain displays the same localization but is readily extractable by nonionic detergent. The carboxy-terminal domain colocalizes with microvillar actin filaments as well as with stress fibers and remains associated with actin filaments after detergent extraction, and with disorganized actin structures after cytochalasin D treatment. Our results clearly demonstrate that ezrin interacts with membrane-associated components via its amino-terminal domain, and with the cytoskeleton via its carboxy-terminal domain. The amino-terminal domain could include the main determinant that restricts the entire protein to the cortical cytoskeleton in contact with the dorsal plasma membrane and its specialized microdomains such as microvilli, microspikes and lamellipodia.

The effects of different structural features on the thermostability of Thermopolyspora flexuosa xylanase XYN10A were investigated. A C-terminal carbohydrate binding module had only a slight effect, whereas a polyhistidine tag increased the thermostability of XYN10A xylanase. In contrast, glycosylation at Asn26, located in an exposed loop, decreased the thermostability of the xylanase. The presence of a substrate increased stability mainly at low pH.

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.

We studied the effects of increase in the number of surface arginines on the enzyme activity and stability of Trichoderma reesei endo-1,4-beta-xylanase II. The number of arginines was increased in two mutant series. The first set contained six arginines on different sides of the protein surface. These arginines had no significant effect on the thermostability. However, the optimal pH region became narrower. Another series of five arginines was engineered into the 'Ser/Thr surface', formed of part of the double-layered beta-sheet located on one side of the 'right-hand-like' xylanase. These mutations shifted the activity profile to the alkaline region by approximately 0.5-1.0 pH units. In addition, the arginines on the Ser/Thr surface increased the enzyme activity at high temperature, although the enzyme stability in the absence of substrate decreased significantly at 50-55 degrees C. In the presence of the substrate, the thermostability increased 4-5-fold at 60-65 degrees C. Thus, the substrate neutralized the destabilizing effect of Ser/Thr surface arginines and revealed a stabilizing effect of the same mutations. The stabilizing effect of arginines at high temperatures was seen clearly only when five arginines were introduced into the Ser/Thr surface.

The ERM proteins, ezrin, radixin, and moesin, act as linkers between the plasma membrane and actin cytoskeleton. They are involved in a variety of cellular functions, such as cell adhesion, migration, and the organization of cell surface structures, and are highly homologous, both in protein sequence and in functional activity, with merlin/schwannomin, a neurofibromatosis-2-associated tumor-suppressor protein. We report here the genomic structure and intron junction sequences of the human ezrin gene. Ezrin consists of 13 exons and spans approximately 24 kb genomic DNA. The coding parts of the exons range in size from 12 bp to 275 bp and the introns from 182 bp to 7 kb. The genomic structures of ezrin and moesin are highly conserved, suggesting their recent divergence. Radiation hybrid mapping has refined the location of ezrin to the interval between D6S442 and D6S281.

Aalto University, School of Chemical Technology, Department of Biotechnology and Chemical Technology, P.O. Box 16100, 00076 Aalto, Finland South-Central University for Nationalities, College of Life Science, Engineering Research Centre of Bioresources in Southern China, Wuhan 430074, China Aalto University, School of Chemical Technology, Department of Forest Products Technology, P.O. Box 16300, 00076 Aalto, Finland

Neurofibromatosis 2 (NF2) protein (merlin; schwannomin) is a tumor suppressor involved in tumorigenesis of NF2-associated and sporadic schwannomas and meningiomas. The protein shares the domain structure of three homologous proteins: ezrin, radixin and moesin (ERM). ERM proteins function as membrane organizers and may act as linkers between plasma membrane molecules, such as CD44 and ICAM-2, and the cytoskeleton. We analyzed the distribution and effects of transfected NF2 protein in COS-1, CHO and 293 cells, and endogenous NF2 protein in U251 glioma cells. The distribution was compared to ezrin, CD44 and F-actin. Both transfected and endogenous NF2 protein localized underneath the plasma membrane in a pattern typical of an ERM protein. In COS-1 transfectants, NF2 protein typically codistributed with ezrin but, in cells with poorly developed actin cytoskeleton, it replaced ezrin in filopodia and ruffling edges. NF2 protein colocalized with CD44, which in transfected cells accumulated into restructured cell membrane protrusions. The association of CD44 and NF2 protein was further suggested by binding of CD44 from cellular lysates to recombinant NF2 protein. Interaction between NF2 protein and the actin-containing cytoskeleton was indicated by partial colocalization, by cytochalasin B-induced coclustering, and by retention of NF2 protein in the detergent-insoluble fraction. Transfected NF2 protein induced morphogenic changes. The cells contained restructured membrane extensions and blebs, and CHO cells expressing NF2 protein were more elongated than control transfectants. In conclusion, NF2 protein possesses functional properties of an ERM family member.

We have previously purified from cultured JEG-3 choriocarcinoma cells an Mr 75,000 protein, originally detected using antibodies to a retrovirus-related synthetic peptide. Using polyclonal antibodies, we have now localized this protein immunocytochemically in JEG-3 cells at both light and electron microscopic levels. In immunofluorescence microscopy of saponin-permeabilized cells, the antigen appeared as dots and short strands at the apical cell surface. In pre-embedding immunoperoxidase electron microscopy, the Mr 75,000 protein was specifically localized to microvilli on the apical cell surface. Immunoferritin electron microscopy was used to assess more quantitatively the antigen distribution in the plane of the plasma membrane, and to define the position of the antigenic site(s) with respect to the membrane. The immunoferritin results confirmed the microvillus specificity of the Mr 75,000 protein and showed that the antigenic portion of the protein is within a few nanometers from, and on the cytoplasmic side of, the lipid bilayer. In detergent extraction experiments, the Mr 75,000 antigen was highly enriched in the soluble fractions. These results demonstrate that the Mr 75,000 protein is a membrane protein highly specific for microvilli.