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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

L-asparaginases (L-ASNase, EC 3.5.1.1) are amidohydrolases catalysing the conversion of L-Asn to L-Asp and ammonia and to a lower extent L-Gln to L-Glu (L-glutaminase activity, L-GLNase). This enzyme is exploited as an anti-neoplastic agent to treat acute lymphoblastic leukaemia. Currently, Escherichia coli and Erwinia chrysanthemi L-ASNases are used as oncological drugs. However, harmful side-effects and hypersensitivity reactions are the main limitations of these therapeutics. A link has been proposed between L-GLNase activity and harmful side effects. Therefore, finding of new L-ASNases with low glutaminase activity, thus potentially with lower side effect risk, is important for medical applications. A detailed biochemical characterization of a wide range of L-ASNases linked with in silico approaches could contribute for discovering better oncologic ASNase drug candidates. In this study, ten bacterial and yeast L-ASNase genes belonging to type I and II class of L-ASNase families, were synthesized and cloned into T7 bacterial expression vector for production in E. coli BL21 cells. The optimum pH and temperature of the expressed L-ASNases were at pH 7.0-9.0 and 35°C-50°C range, respectively. While the optimum temperature and pH of the ten L-ASNases were similar as those previously reported for type I and II L-ASNases, none of them displayed detectable glutaminase activity. Structural comparisons of these ten ASNases with quite much differing kinetic properties showed that the residues with a catalytic role are conserved and some differences at position 59 close to the substrate may affect the kinetic parameters. The type I L-ASNase from Lachancea thermotolerans yeast ( LtASNase) exhibited the highest specific activity of 313.82 U/mg and catalytic efficiency for L-Asn. Therefore, LtASNase is a promising enzyme for further investigation in pharmaceutical applications.

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.

NAD-dependent formate dehydrogenase (FDH) enzymes are frequently used in industrial and scientific applications. FDH is a reversible enzyme that reduces the NAD molecule to NADH and produces CO2 by oxidation of the formate ion, whereas it causes CO2 reduction in the reverse reaction. Some transition metal elements - Fe3+, Mo6+ and W6 + - can be found in the FDH structure of anaerobic and archaeal microorganisms, and these enzymes require cations and other redox-active cofactors for their FDH activity. While NAD-dependent FDHs do not necessarily require any metal cations, the presence of various metal cations can still affect FDH activities. To study the effect of 11 different metal ions, NAD-dependent FDH enzymes from ten different microorganisms were tested: Ancylobacter aquaticus (AaFDH), Candida boidinii (CboFDH), Candida methylica (CmFDH), Ceriporiopsis subvermispora (CsFDH), Chaetomium thermophilum (CtFDH), Moraxella sp. (MsFDH), Myceliophthora thermophila (MtFDH), Paracoccus sp. (PsFDH), Saccharomyces cerevisiae (ScFDH) and Thiobacillus sp. (TsFDH). It was found that metal ions (mainly Cu2+ and Zn2+) could have quite strong inhibition effects on several enzymes in the forward reaction, whereas several cations (Li+, Mg2+, Mn2+, Fe3+ and W6+) could increase the forward reaction of two FDHs. The highest activity increase (1.97 fold) was caused by Fe3+ in AaFDH. The effect on the reverse reaction was minimal. The modelled structures of ten FDHs showed that the active site is formed by 15 highly conserved amino acid residues spatially settling around the formate binding site in a conserved way. However, the residue differences at some of the sites close to the substrate do not explain the activity differences. The active site space is very tight, excluding water molecules, as observed in earlier studies. Structural examination indicated that smaller metal ions might be spaced close to the active site to affect the reaction. Metal ion size showed partial correlation to the effect on inhibition or activation. Affinity of the substrate may also affect the sensitivity to the metal's effect. In addition, amino acid differences on the protein surface may also be important for the metal ion effect. WOS:000576745100001 33043425 Q4

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.

In recent years, CO

In recent years, CO2 reduction and utilization have been proposed as an innovative solution for global warming and the ever-growing energy and raw material demands. In contrast to various classical methods, including chemical, electrochemical, and photochemical methods, enzymatic methods offer a green and sustainable option for CO2 conversion. In addition, enzymatic hydrogenation of CO2 into platform chemicals could be used to produce economically useful hydrogen storage materials, making it a win-win strategy. The thermodynamic and kinetic stability of the CO2 molecule makes its utilization a challenging task. However, Nicotine adenine dinucleotide (NAD(+))-dependent formate dehydrogenases (FDHs), which have high selectivity and specificity, are attractive catalysts to overcome this issue and convert CO2 into fuels and renewable chemicals. It is necessary to improve the stability, cofactor necessity, and CO2 conversion efficiency of these enzymes, such as by combining them with appropriate hybrid systems. However, metal-independent, NAD(+)-dependent FDHs, and their CO2 reduction activity have received limited attention to date. This review outlines the CO2 reduction ability of these enzymes as well as their properties, reaction mechanisms, immobilization strategies, and integration with electrochemical and photochemical systems for the production of formic acid or formate. The biotechnological applications of FDH, future perspectives, barriers to CO2 reduction with FDH, and aspects that must be further developed are briefly summarized. We propose that constructing hybrid systems that include NAD(+)-dependent FDHs is a promising approach to convert CO2 and strengthen the sustainable carbon bio-economy.

A novel versatile glycoside hydrolase Cel5M was identified from Pectobacterium carotovorum and used in ramie degumming. The full-length Cel5M (GenBank: MH544226) containing the catalytic domain Cel5MD and its carbohydrate-binding module (CBM3) were expressed in E. coli. Both Cel5M and Cel5MD showed a broad substrate spectrum cleaving konjac glucomannan (KGM), carboxymethylcellulose (CMC) and oat spelt xylan. The optimal temperature of Cel5MD was 55oC with CMC and KGM, whereas the optimum temperature of Cel5M was 65oC with KGM and 60oC with CMC. The improved thermal activity with KGM was probably reflected also in the much lower K m with KGM than with CMC, indicating stronger binding of the full-length enzyme with KGM. Both enzymes were highly stable over a broad pH range from pH 5.0 to 9.0 with KGM. The use of the Cel5M enzyme together with the wild-type P. carotovorum HG-49 strain showed significant improvement when compared to using the mere strain HG-49 for application in ramie degumming. Moreover, the whole P. carotovorum Cel5M gene was over-expressed in P. carotovorum HG-49 itself to improve the ramie-degumming capacity. Altogether, this study indicates that in situ production of processing enzymes could be utilized in the textile industry.

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

A codon optimized synthetic alkaline thermostable Thermobifida fusca β-mannanase ManB ( KJ806638 ) was expressed in Pichia pastoris and used in ramie degumming. To improve the expression level, a multi-copy secretion expression vector pAOhr was constructed to introduce the ManB gene into Pichia pastoris GS115. The highest secretion yield was obtained from a transformant strain containing six copies of ManB gene. The size of ManB protein was 34 kDa in SDS-PAGE and the secreted protein was the main protein in the culture broth. The optimal activity region of ManB was at pH 7–9 and the enzyme was quite stable at pH 6–10. At pH 9, the specific activity of ManB was 493.8 IU/mg and the optimum temperature was 70–75 °C. ManB appeared to be inhibited by Tris buffer. Molecular docking showed that Tris molecule can bind to the enzyme active site. ManB exhibited high activity for locust bean gum, whereas it showed in practice no activity for CMC-Na. Ramie degumming was performed with combined treatment by ManB and Bacillus sp. HG-28 expressing pectinase and xylanase. The obtained results demonstrated that the combination treatment with additional mannanase enzyme was more efficient in removing the gums than the treatment merely by the bacterial strain.

Maize ( Zea mays L.) is one of the main crops, which is easily susceptable to Aspergillus flavus infection resulting in huge losses worldwide. This study was carried out to investigate the effect of combining heat and irradiation treatments in controlling the fungal growth in maize grains. Surface disinfected maize grains were artificially contaminated with spores of Aspergillus flavus Link NRRL 5906, and then exposed to gamma radiation with doses of 3.0, 4.0 and 5.0 kGy. The samples were additionally heat treated at 60 °C for 30 min. The heat and irradiation treatments showed a synergistic effect on controlling Aspergillus flavus growth. The heat treatment reduced the required radiation dose of about 0.5–1.0 kGy when 4.0 kGy or 5.0 kGy irradiation was used. The combined heat and irradiation treatment of moisture reduced the average CFU by 8 log cycles when 4 kGy or 5 kGy irradiation was used and by 7 log cycles when 3 kGy irradiation was used. The heat treatment of moisture alone reduced the average CFU by only by 0.8 log cycles. Combining irradiation with heat treatment to reduce the required radiation dose is very useful especially when there is a concern over biological side effects of irradiation.

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

Interaction of truffle mycelium with the host plant involves the excretion of extracellular enzymes. The ability of Tuber maculatum mycelium to produce an extracellular cellulase during submerged fermentation was demonstrated for the first time. T. maculatum mycelia were isolated and tested for extracellular cellulase production at variable pH on solid agar medium, and the highest activity was observed at pH 7.0. Furthermore, T. maculatum was subjected to submerged fermentation in basal salt medium for cellulase production. Under optimized conditions using sodium carboxymethyl cellulose (0.5 % w/v) as carbon source and an initial pH of 7.0, the enzyme production yielded 1.70 U/mL of cellulase in the cell-free supernatant after 7 days of incubation time. The optimum of the obtained cellulase’s activity was at pH 5.0 and a temperature of 50 °C. The enzyme showed good thermostability at 50 °C by retaining 99 % of its maximal activity over an incubation time of 100 min. The cellulase activity was inhibited by Fe2+ and slightly activated by Mn2+ and Cu2+ at 1 mM concentration. The results indicated that truffle mycelium is utilizing cellulosic energy source in the root system, and the optimal conditions are those existing in the acidic Finnish soil.

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 present study investigated the ability of pressurized microwave pretreatment to convert softwood lignocellulose to fermentable monosaccharides. Norway spruce lignocellulose was subjected to microwave pretreatment (600 and 1200 W) under high pressure at different temperatures. Microwave pretreatment at mild acid concentrations (0.05–0.1 % H2SO4), temperatures of 170 and 200 °C, and a very short incubation time (5 min) released 84–100 % of hemicellulosic monosaccharides (mannose, galactose, and xylose). In addition, minimal amounts of degradation products (5-(hydroxymethyl)-2-furaldehyde, levulinic acid) were formed. The highest yield of fermentable sugars was 75 %, after both the pressurized microwave pretreatment with conditions 0.05 % H2SO4/600 W/200 °C/5 min and enzymatic hydrolysis with 20 FPU Celluclast 1.5 L, 400 nkat of Novozyme 188, and polyethyleneglycol (PEG) 4000 (0.3 g/g of pretreated material). Results showed that already 0.05 % H2SO4 used in microwave pretreatment could effectively liberate hemicellulose monosaccharides without serious monosaccharide degradation and form a basis for enzymatic hydrolysis.

BACKGROUND High-temperature enzymatic hydrolysis of cellulose has been studied recently with promising results. High temperatures can improve reaction rates and mass transfer, and lower viscosity. Tests were made to determine how short high-temperature prehydrolysis steps affect the final hydrolysis with commercial cellulase mixture. RESULTS Continuous mixing improved the hydrolysis of 3% Avicel by Pyrococcus horikoshii endoglucanase (PhEG) at 70°C but not at 100°C. Differences in the temperature effect between 90°C and ∼103°C were much higher than between 70°C and 90°C. It was essential that the dosed enzyme activity was the same at each temperature, meaning lower volumetric amount of enzyme at higher temperature. PhEG prehydrolysis (2 h) of steam pretreated spruce before final hydrolysis by commercial cellulase mixture was more efficient at 100°C than at 80°C, improving the total enzymatic hydrolysis of cellulose by 15–30%. Xylanase and mannase prehydrolysis at 80°C (2 h) improved the cellulase hydrolysis by 20–33%, while all three enzymes together improved the final enzymatic yield by 50–70%. CONCLUSION A temperature of 100–105°C provides clear process advantages for enzymatic treatments when compared with 70–90°C. Enzymatic high-temperature prehydrolysis could be applicable to circumvent the problems of high viscosity and to reduce the required amount of cellulases in the subsequent hydrolysis. © 2015 Society of Chemical Industry

Sulfolobus acidocaldarius β-glycosidase (BGAL_SULAC) was used as an extremophilic enzyme model to study the effect of mutations close to the catalytic residues on the enzyme activity and the pH-activity profile. We report here the results for three mutations (N211D, V212D and V212T) changing the polarity close to the putative acid/base catalyst E209. N211D was outside the H-bonding distance from E209, whereas V212D and V212T were in H-bonding distance from E209. V212D and V212T shifted the pH-activity profile towards acidic pH with both lactose and cellobiose as substrates. N211D and V212D decreased clearly the activity. Although V212T increased 6-fold the K m value with cellobiose, the mutant showed higher specific activity in high substrate concentrations. The reason was greatly reduced production of trisaccharide by V212T from cellobiose by transglycosylation. Threonine differs by the terminal oxygen from valine, indicating that additional hydrogen bonding to substrate or reaction products may affect the reaction behavior of the enzyme. Although the mutations in the active site are often harmful, the mutation V212T showed biotechnologically promising properties.

Thermopolyspora flexuosa GH11 xylanase (XYN11A) shows optimal activity at pH 6-7 and 75-80 °C. We studied how mutation to aspartic acid (N46D and V48D) in the vicinity of the catalytic acid/base affects the pH activity of highly thermophilic GH11 xylanase. Both mutations shifted the pH activity profile toward acidic pH. In general, the Km values were lower at pH 4-5 than at pH 6, and in line with this, the rate of hydrolysis of xylotetraose was slightly faster at pH 4 than at pH 6. The N46D mutation and also lower pH in XYN11A increased the hydrolysis of xylotriose. The Km value increased remarkably (from 2.5 to 11.6 mg/mL) because of V48D, which indicates the weakening of binding affinity of the substrate to the active site. Xylotetraose functioned well as a substrate for other enzymes, but with lowered reaction rate for V48D. Both N46D and V48D increased the enzyme inactivation by ionic liquid [emim]OAc. In conclusion, the pH activity profile could be shifted to acidic pH due to an effect from two different directions, but the tightly packed GH11 active site can cause steric problems for the mutations.

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.

GH10 xylanase from Thermoascus aurantiacus strain SL16W (TasXyn10A) showed high stability and activity up to 70–75 °C. The enzyme’s half-lives were 101 h, 65 h, 63 min and 6 min at 60, 70, 75 and 80 °C, respectively. The melting point (T m), as measured by DSC, was 78.5 °C, which is in line with a strong activity decrease at 75–80 °C. The biomass-dissolving ionic liquid 1-ethyl-3-methylimidazolium acetate ([emim]OAc) in 30 % concentration had a small effect on the stability of TasXyn10A; T m decreased by only 5 °C. It was also observed that [emim]OAc inhibited much less GH10 xylanase (TasXyn10A) than the studied GH11 xylanases. The K m of TasXyn10A increased 3.5-fold in 15 % [emim]OAc with xylan as the substrate, whereas the approximate level of V max was not altered. The inhibition of enzyme activity by [emim]OAc was lesser at higher substrate concentrations. Therefore, high solid concentrations in industrial conditions may mitigate the inhibition of enzyme activity by ionic liquids. Molecular docking experiments indicated that the [emim] cation has major binding sites near the catalytic residues but in lower amounts in GH10 than in GH11 xylanases. Therefore, [emim] cation likely competes with the substrate when binding to the active site. The docking results indicated why the effect is lower in GH10.

Large numbers of Finnish lakes accumulate biomass sediments due to large nutrient inputs from forest areas, agricultural and industrial activities. The theoretical biofuel production potential was studied for lake bottom biomass from mesotrophic (ML) and eutrophic lakes (EL) in eastern Finland. Methane potentials of the EL and ML biomass were 136.6 ml g−1 volatile solid and 38.9 ml g−1 volatile solid, respectively. The estimated ethanol yields of the EL and ML biomass from fermentation were 137 l tonne−1, and 40 l tonne−1, whereas the yields from mixed alcohol synthesis after gasification were 244.5 l tonne−1 and 57.1 l tonne−1, respectively. The ash from both lake bottom biomass contained harmful elements below the detectable limit, and therefore, could be used as fertilizer. Techno-economic estimation showed that bioethanol production from the fermentation of EL biomass is the most profitable process and is a potential non-food lignocellulosic biomass source for the biorefining industry.

Thermophilic Thermopolyspora flexuosa GH10 xylanase (TfXYN10A) was studied in the presence of biomass-dissolving hydrophilic ionic liquids (ILs) [EMIM]OAc, [EMIM]DMP and [DBNH]OAc. The temperature optimum of TfXYN10A with insoluble xylan in the pulp was at 65–70 °C, with solubilised 1 % xylan at 70–75 °C and with 3 % xylan at 75–80 °C. Therefore, the amount of soluble substrate affects the enzyme activity at high temperatures. The experiments with ILs were done with 1 % substrate. TfXYN10A can partially hydrolyse soluble xylan even in the presence of 40 % (v/v) ILs. Although ILs decrease the apparent temperature optimum, a surprising finding was that at the inactivating temperatures (80–90 °C), especially [EMIM]OAc increases the stability of TfXYN10A indicating that the binding of IL molecules strengthens the protein structure. Earlier kinetic studies showed an increased K m with ILs, indicating that ILs function as competitive inhibitors. TfXYN10A showed low increase of K m, which was 2-, 3- and 4-fold with 15 % [EMIM]OAc, [DBNH]OAc and [EMIM]DMP, respectively. One reason for the low competitive inhibition could be the high affinity to the substrate (low K m). Xylanases with low K m (~1 mg/mL) appear to show higher tolerance to ILs than xylanases with higher K m (~2 mg/mL). Capillary electrophoresis showed that TfXYN10A hydrolyses xylan to the end-products in 15–35 % ILs practically as completely as without IL, also indicating good binding of the short substrate molecules by TfXYN10A despite of major apparent IL binding sites above the catalytic residues. Substrate binding interactions in the active site appear to explain the high tolerance of TfXYN10A to ILs.

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

In order to increase the stability of thermophilic Thermomyces lanuginosus GH11 xylanase, TLX, a disulfide bridge Q1C-Q24C was introduced into the N-terminal region of the enzyme. The apparent temperature optimum shifted upwards at pH 6.5 by about 10°C to 75°C. The resistance to thermal inactivation also increased by about 10°C. The melting temperature measured by CD spectroscopy increased from 66 to 74°C. Therefore the N-terminal disulfide bridge increased both kinetic and thermodynamic stability almost equally. At pH 8 and 70°C, the disulfide bridge increased the enzyme half-life 20-fold in the presence of substrate. In contrast to the situation in acidic-neutral pH, the substrate decreased the thermostability of xylanases in alkaline pH. The upper limit for the performance of the disulfide bridge mutant at pH 9 was 75°C. This study showed that N-terminal disulfide bridges can stabilize even thermostable family GH11 xylanases.

The structure of softwoods, which confers resistance to degradation through hydrolysis and decay, currently limits their use for the production of biofuels. However, since wood is very heterogeneous, it is possible that differences in wood properties within and between trees could differentially affect its processability. In this research, heartwood (inner) and sapwood (outer) from Norway spruce (Picea abies (L.) Karst.) clones were enzymatically hydrolyzed by Trichoderma viride cellulases after concentrated acid pretreatment. Wood sections with two particle sizes were compared based on their susceptibility to enzymatic hydrolysis, evaluated by assaying the formation of hydrolysis products and measured as reducing sugar yield (RSY). We also studied the relationship between RSY and the susceptibility to Heterobasidion parviporum wood decay and whether these traits are reflected in wood density and yield. Wood from the outer section produced more RSY with higher glucan but lower lignin content than wood from the inner section. Furthermore, susceptibility to enzymatic hydrolysis was positively correlated with H. parviporum wood decay, while both processes were negatively correlated with wood density. Our results revealed the importance of clonal trials for identifying suitable lignocellulosic biomass when considering wood properties and indicate that potential genotypes for the production of biofuels are not necessarily the most productive.

In this work, carbon nanotubes (CNTs) functionalized by acidic amino acids were used as a framework, which aims to form a mimetic structure of an active site of the glycoside hydrolases. It was demonstrated that the glycosidic bonds of the disaccharides were cleaved by the fabricated biofunctionalized CNTs. It was implied that the number of carboxyl groups and their individual pKa values in the amino acids, and the distance between the NH2 and the side chain carboxyl groups of the amino acid are predominant factors for determining the reaction efficiency and the optimum pH. It was suggested that glutamic acid functionalized CNTs framework showed the highest efficiency in the cleavage of glycosidic bond of cellobiose than other acidic biomolecules. It was also suggested that the glutamic acid functionalized CNT framework showed preference to the types of glycosidic bonds in the following order: β-1,2-glycoside > β-1,4-glycoside > α-1,4-glycoside [Formula: see text] α-1,1-glycoside bond.

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

Kinetic as well as energetic aspects of the thermal denaturation of Trichoderma reesei endo-1,4-β-xylanase II (TRX II) and its three thermostable disulfide mutants were characterized by means of differential scanning calorimetry (DSC) in different solution conditions. The calorimetric transitions were strongly scan-rate dependent, characteristic for an irreversible, kinetically controlled protein denaturation. The DSC-determined T *-values (the temperature at which the denaturation rate constant equals 1 min −1 ), and the activation free energies for the transitions are consistent with the apparent transition temperatures of TRX II determined earlier by mass spectrometry. Protein aggregation, connected with the irreversibility of the transitions, was present in all cases but was less pronounced with the mutants as well as highly dependent on experimental conditions.

In our recent studies, it was demonstrated that the IONCELL-P process selectively dissolves hemicelluloses from bleached birch kraft pulp in a mixture of 1-ethyl-3-methylimidazolium acetate ([emim][OAc]) and water as a solvent system. The IONCELL-P method refines paper-grade pulp to dissolving pulp with

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.