Exploring the potential of a new thermotolerant xylanase from Rasamsonia composticola (XylRc): production using agro-residues, biochemical studies, and application to sugarcane bagasse saccharification.

Xylanases from thermophilic fungi have a wide range of commercial applications in the bioconversion of lignocellulosic materials and biobleaching in the pulp and paper industry. In this study, an endoxylanase from the thermophilic fungus Rasamsonia composticola (XylRc) was produced using waste wheat bran and pretreated sugarcane bagasse (PSB) in solid-state fermentation. The enzyme was purified, biochemically characterized, and used for the saccharification of sugarcane bagasse. XylRc was purified 30.6-fold with a 22% yield. The analysis using sodium dodecyl sulphate-polyacrylamide gel electrophoresis revealed a molecular weight of 53 kDa, with optimal temperature and pH values of 80 °C and 5.5, respectively. Thin-layer chromatography suggests that the enzyme is an endoxylanase and belongs to the glycoside hydrolase 10 family. The enzyme was stimulated by the presence of K+, Ca2+, Mg2+, and Co2+ and remained stable in the presence of the surfactant Triton X-100. XylRc was also stimulated by organic solvents butanol (113%), ethanol (175%), isopropanol (176%), and acetone (185%). The Km and Vmax values for oat spelt and birchwood xylan were 6.7 ± 0.7 mg/mL, 2.3 ± 0.59 mg/mL, 446.7 ± 12.7 µmol/min/mg, and 173.7 ± 6.5 µmol/min/mg, respectively. XylRc was unaffected by different phenolic compounds: ferulic, tannic, cinnamic, benzoic, and coumaric acids at concentrations of 2.5-10 mg/mL. The results of saccharification of PSB showed that supplementation of a commercial enzymatic cocktail (Cellic® CTec2) with XylRc (1:1 w/v) led to an increase in the degree of synergism (DS) in total reducing sugar (1.28) and glucose released (1.05) compared to the control (Cellic® HTec2). In summary, XylRc demonstrated significant potential for applications in lignocellulosic biomass hydrolysis, making it an attractive alternative for producing xylooligosaccharides and xylose, which can serve as precursors for biofuel production.

N-acetylation of toxic aromatic amines by fungi: Strain screening, cytotoxicity and genotoxicity evaluation, and application in bioremediation of 3,4-dichloroaniline.

Aromatic amines (AA) are one of the most commonly used classes of compounds in industry and the most common pollutants found in both soil and water. 3,4-Dichloaniline (3,4-DCA) is a persistent residue of the phenylurea herbicide in the environment. In this study, we used a colorimetric method as a new approach to screen 12 filamentous fungal strains of the genera Aspergillus, Chaetomium, Cladosporium, and Mucor to assess their capacity to perform AA N-acetylation since it is considered a potential tool in environmental bioremediation. Subsequently, the selected strains were biotransformed with different AA substrates to evaluate the product yield. The strains Aspergillus niveus 43, Aspergillus terreus 31, and Cladosporium cladosporioides showed higher efficiencies in the biotransformation of 3,4-DCA at 500 µM into its N-acetylated product. These fungal strains also showed great potential to reduce the phytotoxicity of 3,4-DCA in experiments using Lactuca sativa seeds. Furthermore, N-acetylation was shown to be effective in reducing the cytotoxic and genotoxic effects of 3,4-DCA and other AA in the immortalized human keratinocyte (HaCaT) cell line. The isolated products after biotransformation showed that fungi of the genera Aspergillus and Cladosporium appeared to have N-acetylation as the first and main AA detoxification mechanism. Finally, A. terreus 31 showed the highest 3,4-DCA bioremediation potential, and future research can be carried out on the application of this strain to form microbial consortia with great potential for the elimination of toxic AA from the environment.

New derivatives of the iridoid specioside from fungal biotransformation.

Iridoids are widely found from species of Bignoniaceae family and exhibit several biological activities, such as anti-inflammatory, antimicrobial, antioxidant, and antitumor. Specioside is an iridoid found from Tabebuia species, mainly in Tabebuia aurea. Thus, here fungus-mediated biotransformation of the iridoid specioside was investigated by seven fungi. The fungus-mediated biotransformation reactions resulted in a total of nineteen different analogs by fungus Aspergillus niger, Aspergillus flavus, Aspergillus japonicus, Aspergillus terreus, Aspergillus niveus, Penicillium crustosum, and Thermoascus aurantiacus. Non-glycosylated specioside was the main metabolite observed. The other analogs were yielded from ester hydrolysis, hydroxylation, methylation, and hydrogenation reactions. The non-glycosylated specioside and coumaric acid were yielded by all fungi-mediated biotransformation. Thus, fungus applied in this study showed the ability to perform hydroxylation and glycosidic, as well as ester hydrolysis reactions from glycosylated iridoid. KEY POINTS: • The biotransformation of specioside by seven fungi yielded nineteen analogs. • The non-glycosylated specioside was the main analog obtained. • Ester hydrolysis, hydroxylation, methylation, and hydrogenation reactions were observe.

Electrostatic interaction optimization improves catalytic rates and thermotolerance on xylanases.

Understanding the aspects that contribute to improving proteins' biochemical properties is of high relevance for protein engineering. Properties such as the catalytic rate, thermal stability, and thermal resistance are crucial for applying enzymes in the industry. Different interactions can influence those biochemical properties of an enzyme. Among them, the surface charge-charge interactions have been a target of particular attention. In this study, we employ the Tanford-Kirkwood solvent accessibility model using the Monte Carlo algorithm (TKSA-MC) to predict possible interactions that could improve stability and catalytic rate of a WT xylanase (XynAWT) and its M6 xylanase (XynAM6) mutant. The modeling prediction indicates that mutating from a lysine in position 99 to a glutamic acid (K99E) favors the native state stabilization in both xylanases. Our lab results showed that mutated xylanases had their thermotolerance and catalytic rate increased, which conferred higher processivity of delignified sugarcane bagasse. The TKSA-MC approach employed here is presented as an efficient computational-based design strategy that can be applied to improve the thermal resistance of enzymes with industrial and biotechnological applications.

High stabilization and hyperactivation of a Recombinant ß-Xylosidase through Immobilization Strategies.

Attainment of a stable and highly active ß-xylosidase is of major importance for the efficient and cost-competitive hydrolysis of hemicellulose xylan, as well as for its industrial conversion into biofuels and biochemicals. Here, a recombinant ß-xylosidase of the glycoside hydrolase family (GH43) from Bacillus subtilis was produced in Escherichia coli culture, purified, and subsequently immobilized on agarose and chitosan. Glutaraldehyde and glyoxyl groups were evaluated as activating agents to select the most efficient derivative. Multi-point immobilization on agarose led to an extraordinary thermal stability (half-lives 3604 and 164-fold higher than the free enzyme, at 50° and 35 °C, respectively). Even for chitosan activated with glutaraldehyde, a low-cost support, thermal stability of the immobilized enzyme was 326 and 12-fold higher than the free enzyme at 50° and 35°C, respectively. Immobilized enzymes showed no release of any subunit for the agarose-glyoxyl derivative, and only a few ones for the support activated with glutaraldehyde. Most remarkably, the enzyme kinetic behavior after immobilization increased up to 4-fold in relation to the free one. ß-xylosidase, a tetrameric enzyme with four identical subunits, exists in equilibrium between the monomeric and oligomeric forms in solution. Depending on the pH of immobilization, the enzyme oligomerization can be favored, thus explaining the hyperactivation phenomenon. Both glyoxyl-agarose and chitosan-glutaraldehyde derivatives were used to catalyze corncob xylan hydrolysis, reaching 72 % conversion, representing a xylose productivity of around 20 g L-1 h-1. After ten 4h-cycles (pH 6.0, 35 °C), the xylan-to-xylose conversion remained approximately unchanged. Therefore, the immobilized ß-xylosidases prepared in this work can be of great interest as biocatalysts in a biorefinery context.

Identification of a cold-adapted and metal-stimulated ß-1,4-glucanase with potential use in the extraction of bioactive compounds from plants.

Cold-adapted endo-ß-1,4-glucanases hold great potential for industrial processes requiring high activity at mild temperatures such as in food processing and extraction of bioactive compounds from plants. Here, we identified and explored the specificity, mode of action, kinetic behavior, molecular structure and biotechnological application of a novel endo-ß-1,4-glucanase (XacCel8) from the phytopathogen Xanthomonas citri subsp. citri. This enzyme belongs to an uncharacterized phylogenetic branch of the glycoside hydrolase family 8 (GH8) and specifically cleaves internal ß-1,4-linkages of cellulose and mixed-linkage ß-glucans releasing short cello-oligosaccharides ranging from cellobiose to cellohexaose. XacCel8 acts in near-neutral pHs and in a broad temperature range (10-50 °C), which are distinguishing features from conventional thermophilic ß-1,4-glucanases. Interestingly, XacCel8 was greatly stimulated by cobalt ions, which conferred higher conformational stability and boosted the enzyme turnover number. The potential application of XacCel8 was demonstrated in the caffeine extraction from guarana seeds, which improved the yield by 2.5 g/kg compared to the traditional hydroethanolic method (HEM), indicating to be an effective additive in this industrial process. Therefore, XacCel8 is a metal-stimulated and cold-adapted endo-ß-1,4-glucanase that could be applied in a diverse range of biotechnological processes under mild conditions such as caffeine extraction from guarana seeds.

High stabilization and hyperactivation of a recombinant β-Xylosidase through immobilization strategies

Attainment of a stable and highly active β-xylosidase is of major importance for the efficient and cost-competitive hydrolysis of hemicellulose xylan, as well as for its industrial conversion into biofuels and biochemicals. Here, a recombinant β-xylosidase of the glycoside hydrolase family (GH43) from Bacillus subtilis was produced in Escherichia coli culture, purified, and subsequently immobilized on agarose and chitosan. Glutaraldehyde and glyoxyl groups were evaluated as activating agents to select the most efficient derivative. Multi-point immobilization on agarose led to an extraordinary thermal stability (half-lives 3604 and 164-fold higher than the free enzyme, at 50° and 35 °C, respectively). Even for chitosan activated with glutaraldehyde, a low-cost support, thermal stability of the immobilized enzyme was 326 and 12-fold higher than the free enzyme at 50° and 35°C, respectively. Immobilized enzymes showed no release of any subunit for the agarose-glyoxyl derivative, and only a few ones for the support activated with glutaraldehyde. Most remarkably, the enzyme kinetic behavior after immobilization increased up to 4-fold in relation to the free one. β-xylosidase, a tetrameric enzyme with four identical subunits, exists in equilibrium between the monomeric and oligomeric forms in solution. Depending on the pH of immobilization, the enzyme oligomerization can be favored, thus explaining the hyperactivation phenomenon. Both glyoxyl-agarose and chitosan-glutaraldehyde derivatives were used to catalyze corncob xylan hydrolysis, reaching 72 % conversion, representing a xylose productivity of around 20 g L−1 h−1. After ten 4h-cycles (pH 6.0, 35 °C), the xylan-to-xylose conversion remained approximately unchanged. Therefore, the immobilized β-xylosidases prepared in this work can be of great interest as biocatalysts in a biorefinery context.

Designing a cocktail containing redox enzymes to improve hemicellulosic hydrolysate fermentability by microorganisms.

Bioproducts production using monomeric sugars derived from lignocellulosic biomass presents several challenges, such as to require a physicochemical pretreatment to improve its conversion yields. Hydrothermal lignocellulose pretreatment has several advantages and results in solid and liquid streams. The former is called hemicellulosic hydrolysate (HH), which contains inhibitory phenolic compounds and sugar degradation products that hinder microbial fermentation products from pentose sugars. Here, we developed and applied a novel enzyme process to detoxify HH. Initially, the design of experiments with different redox activities enzymes was carried out. The enzyme mixture containing the peroxidase (from Armoracia rusticana) together with superoxide dismutase (from Coptotermes gestroi) are the most effective to detoxify HH derived from sugarcane bagasse. Butanol fermentation by the bacteria Clostridium saccharoperbutylacetonicum and ethanol production by the yeast Scheffersomyces stipitis increased by 24.0× and 2.4×, respectively, relative to the untreated hemicellulosic hydrolysates. Detoxified HH was analyzed by chromatographic and spectrometric methods elucidating the mechanisms of phenolic compound modifications by enzymatic treatment. The enzyme mixture degraded and reduced the hydroxyphenyl- and feruloyl-derived units and polymerized the lignin fragments. This strategy uses biocatalysts under environmentally friendly conditions and could be applied in the fuel, food, and chemical industries.

Crystal structure of a novel xylose isomerase from Streptomyces sp. F-1 revealed the presence of unique features that differ from conventional classes.

BACKGROUND: Enzymatic isomerization is a promising strategy to solve the problem of xylose fermentation and, consequently, to leverage the production of advanced biofuels and biochemicals. In a previous work, our research group discovered a new strain of Streptomyces with great biotechnological potential due to its ability to produce a broad arsenal of enzymes related to lignocellulose degradation. METHODS: We applied a multidisciplinary approach involving enzyme kinetics, biophysical methods, small angle X-ray scattering and X-ray crystallography to investigate two novel xylose isomerases, XylA1F1 and XylA2F1, from this strain. RESULTS: We showed that while XylA1F1 prefers to act at lower temperatures and relatively lower pH, XylA2F1 is extremely stable at higher temperatures and presents a higher turnover number. Structural analysis revealed that XylA1F1 exhibits unique properties in the active site not observed in classical XylAs from classes I and II nor in its ortholog XylA2F1. It encompasses the natural substitutions, M86A and T93K, that create an extra room for substrate accommodation and narrow the active-site entrance, respectively. Such modifications may contribute to the functional differentiation of these enzymes. CONCLUSIONS: We have characterized two novel xylose isomerases that display distinct functional behavior and harbor unprecedented amino-acid substitutions in the catalytic interface. GENERAL SIGNIFICANCE: Our findings contribute to a better understanding of the functional and structural aspects of xylose isomerases, which might be instrumental for the valorization of the hemicellulosic fraction of vegetal biomass.

A Collagenolytic Aspartic Protease from Thermomucor indicae-seudaticae Expressed in Escherichia coli and Pichia pastoris.

Proteases are produced by the most diverse microorganisms and have a wide spectrum of applications. However, the use of wild microorganisms, mainly fungi, for enzyme production has some drawbacks. They are subject to physiological instability due to metabolic adaptations, causing complications and impairments in the production process. Thus, the objective of this work was to promote the heterologous expression of a collagenolytic aspartic protease (ProTiN31) from Thermomucor indicae seudaticae in Escherichia coli and Pichia pastoris. The pET_28a (+) and pPICZαA vectors were synthesized containing the gene of the enzyme and transformed into E. coli and P. pastoris, respectively. The recombinant enzymes produced by E. coli and P. pastoris showed maximum activity at pH 5.0 and 50 °C, and pH 5.0 and 60 °C, respectively. The enzyme produced by P. pastoris showed better thermostability when compared to that produced by E. coli. Both enzymes were stable at pH 6.0 and 6.5 for 24 h at 4 °C, and sensitive to pepstatin A, ß-mercaptoethanol, and Hg2+. Comparing the commercial collagen hydrolysate (Artrogen duo/Brazil) and gelatin degradation using protease from P. pastoris, they showed similar peptide profiles. There are its potential applications in a wide array of industrial sectors that use collagenolytic enzymes.

Structure-guided design combined with evolutionary diversity led to the discovery of the xylose-releasing exo-xylanase activity in the glycoside hydrolase family 43.

Rational design is an important tool for sculpting functional and stability properties of proteins and its potential can be much magnified when combined with in vitro and natural evolutionary diversity. Herein, we report the structure-guided design of a xylose-releasing exo-ß-1,4-xylanase from an inactive member of glycoside hydrolase family 43 (GH43). Structural analysis revealed a nonconserved substitution (Lys247 ) that results in the disruption of the hydrogen bond network that supports catalysis. The mutation of this residue to a conserved serine restored the catalytic activity and crystal structure elucidation of the mutant confirmed the recovery of the proper orientation of the catalytically relevant histidine. Interestingly, the tailored enzyme can cleave both xylooligosaccharides and xylan, releasing xylose as the main product, being the first xylose-releasing exo-ß-1,4-xylanase reported in the GH43 family. This enzyme presents a unique active-site topology when compared with closely related ß-xylosidases, which is the absence of a hydrophobic barrier at the positive-subsite region, allowing the accommodation of long substrates. Therefore, the combination of rational design for catalytic activation along with naturally occurring differences in the substrate binding interface led to the discovery of a novel activity within the GH43 family. In addition, these results demonstrate the importance of solvation of the ß-propeller hollow for GH43 catalytic function and expand our mechanistic understanding about the diverse modes of action of GH43 members, a key and polyspecific carbohydrate-active enzyme family abundant in most plant cell-wall-degrading microorganisms.

A novel ß-glucosidase isolated from the microbial metagenome of Lake Poraquê (Amazon, Brazil).

The Amazon region holds most of the biological richness of Brazil. Despite their ecological and biotechnological importance, studies related to microorganisms from this region are limited. Metagenomics leads to exciting discoveries, mainly regarding non-cultivable microorganisms. Herein, we report the discovery of a novel ß-glucosidase (glycoside hydrolase family 1) gene from a metagenome from Lake Poraquê in the Amazon region. The gene encodes a protein of 52.9 kDa, named AmBgl-LP, which was recombinantly expressed in Escherichia coli and biochemically and structurally characterized. Although AmBgl-LP hydrolyzed the synthetic substrate p-nitrophenyl-ß-d-glucopyranoside (pNPßG) and the natural substrate cellobiose, it showed higher specificity for pNPßG (kcat/Km = 6 s-1·mM-1) than cellobiose (kcat/Km = 0.6 s-1·mM-1). AmBgl-LP showed maximum activity at 40 °C and pH 6.0 when pNPßG was used as the substrate. Glucose is a competitive inhibitor of AmBgl-LP, presenting a Ki of 14 mM. X-ray crystallography and Small Angle X-ray Scattering were used to determine the AmBgl-LP three-dimensional structure and its oligomeric state. Interestingly, despite sharing similar active site architecture with other structurally characterized GH1 family members which are monomeric, AmBgl-LP forms stable dimers in solution. The identification of new GH1 members by metagenomics might extend our understanding of the molecular mechanisms and diversity of these enzymes, besides enabling us to survey their industrial applications.

Draft genome sequence of Streptomyces sp. strain F1, a potential source for glycoside hydrolases isolated from Brazilian soil

ABSTRACT Here, we show the draft genome sequence of Streptomyces sp. F1, a strain isolated from soil with great potential for secretion of hydrolytic enzymes used to deconstruct cellulosic biomass. The draft genome assembly of Streptomyces sp. strain F1 has 69 contigs with a total genome size of 8,142,296 bp and G + C 72.65%. Preliminary genome analysis identified 175 proteins as Carbohydrate-Active Enzymes, being 85 glycoside hydrolases organized in 33 distinct families. This draft genome information provides new insights on the key genes encoding hydrolytic enzymes involved in biomass deconstruction employed by soil bacteria.

New Heterofunctional Supports Based on Glutaraldehyde-Activation: A Tool for Enzyme Immobilization at Neutral pH.

Immobilization is an exciting alternative to improve the stability of enzymatic processes. However, part of the applied covalent strategies for immobilization uses specific conditions, generally alkaline pH, where some enzymes are not stable. Here, a new generation of heterofunctional supports with application at neutral pH conditions was proposed. New supports were developed with different bifunctional groups (i.e., hydrophobic or carboxylic/metal) capable of adsorbing biocatalysts at different regions (hydrophobic or histidine richest place), together with a glutaraldehyde group that promotes an irreversible immobilization at neutral conditions. To verify these supports, a multi-protein model system (E. coli extract) and four enzymes (Candidarugosa lipase, metagenomic lipase, ß-galactosidase and ß-glucosidase) were used. The immobilization mechanism was tested and indicated that moderate ionic strength should be applied to avoid possible unspecific adsorption. The use of different supports allowed the immobilization of most of the proteins contained in a crude protein extract. In addition, different supports yielded catalysts of the tested enzymes with different catalytic properties. At neutral pH, the new supports were able to adsorb and covalently immobilize the four enzymes tested with different recovered activity values. Notably, the use of these supports proved to be an efficient alternative tool for enzyme immobilization at neutral pH.

Draft genome sequence of Streptomyces sp. strain F1, a potential source for glycoside hydrolases isolated from Brazilian soil.

Here, we show the draft genome sequence of Streptomyces sp. F1, a strain isolated from soil with great potential for secretion of hydrolytic enzymes used to deconstruct cellulosic biomass. The draft genome assembly of Streptomyces sp. strain F1 has 69 contigs with a total genome size of 8,142,296bp and G+C 72.65%. Preliminary genome analysis identified 175 proteins as Carbohydrate-Active Enzymes, being 85 glycoside hydrolases organized in 33 distinct families. This draft genome information provides new insights on the key genes encoding hydrolytic enzymes involved in biomass deconstruction employed by soil bacteria.

The Coptotermes gestroi aldo-keto reductase: a multipurpose enzyme for biorefinery applications.

BACKGROUND: In nature, termites can be considered as a model biological system for biofuel research based on their remarkable efficiency for lignocellulosic biomass conversion. Redox enzymes are of interest in second-generation ethanol production because they promote synergic enzymatic activity with classical hydrolases for lignocellulose saccharification and inactivate fermentation inhibitory compounds produced after lignocellulose pretreatment steps. RESULTS: In the present study, the biochemical and structural characteristics of the Coptotermes gestroi aldo-keto reductase (CgAKR-1) were comprehensively investigated. CgAKR-1 displayed major structural differences compared with others AKRs, including the differences in the amino acid composition of the substrate-binding site, providing basis for classification as a founding member of a new AKR subfamily (family AKR1 I). Immunolocalization assays with anti-CgAKR-1 antibodies resulted in strong fluorescence in the salivary gland, proventriculus, and foregut. CgAKR-1 supplementation caused a 32% reduction in phenolic aldehydes, such as furfural, which act as fermentation inhibitors of hemicellulosic hydrolysates, and improved ethanol fermentation by the xylose-fermenting yeast Scheffersomyces stipitis by 45%. We observed synergistic enzymatic interactions between CgAKR-1 and commercial cellulosic cocktail for sugarcane bagasse saccharification, with a maximum synergism degree of 2.17 for sugar release. Our data indicated that additive enzymatic activity could be mediated by reactive oxygen species because CgAKR-1 could produce hydrogen peroxide. CONCLUSION: In summary, we identified the founding member of an AKRI subfamily with a potential role in the termite digestome. CgAKR-1 was found to be a multipurpose enzyme with potential biotechnological applications. The present work provided a basis for the development and application of integrative and multipurpose enzymes in the bioethanol production chain.

A Novel Carbohydrate-binding Module from Sugar Cane Soil Metagenome Featuring Unique Structural and Carbohydrate Affinity Properties.

Carbohydrate-binding modules (CBMs) are appended to glycoside hydrolases and can contribute to the degradation of complex recalcitrant substrates such as the plant cell wall. For application in bioethanol production, novel enzymes with high catalytic activity against recalcitrant lignocellulosic material are being explored and developed. In this work, we report the functional and structural study of CBM_E1, which was discovered through a metagenomics approach and is the founding member of a novel CBM family, CBM81. CBM_E1, which is linked to an endoglucanase, displayed affinity for mixed linked ß1,3-ß1,4-glucans, xyloglucan, Avicel, and cellooligosaccharides. The crystal structure of CBM_E1 in complex with cellopentaose displayed a canonical ß-sandwich fold comprising two ß-sheets. The planar ligand binding site, observed in a parallel orientation with the ß-strands, is a typical feature of type A CBMs, although the expected affinity for bacterial crystalline cellulose was not detected. Conversely, the binding to soluble glucans was enthalpically driven, which is typical of type B modules. These unique properties of CBM_E1 are at the interface between type A and type B CBMs.

Engineering increased thermostability in the GH-10 endo-1,4-ß-xylanase from Thermoascus aurantiacus CBMAI 756.

The GH10 endo-xylanase from Thermoascus aurantiacus CBMAI 756 (XynA) is industrially attractive due to its considerable thermostability and high specific activity. Considering the possibility of a further improvement in thermostability, eleven mutants were created in the present study via site-directed mutagenesis using XynA as a template. XynA and its mutants were successfully overexpressed in Escherichia coli Rosetta-gami DE3 and purified, exhibiting maximum xylanolytic activity at pH 5 and 65°C. Three of the eleven mutants, Q158R, H209N, and N257D, demonstrated increased thermostability relative to the wild type at 70°C and 75°C.Q158R and N257D were stable in the pH range 5.0-10.0, while WT and H209N were stable from pH 8-10. CD analysis demonstrated that the WT and the three mutant enzymes were expressed in a folded form. H209N was the most thermostable mutant, showing a Tm of 71.3°C. Molecular dynamics modeling analyses suggest that the increase in H209N thermostability may beattributed to a higher number of short helices and salt bridges, which displayed a positive charge in the catalytic core, stabilizing its tertiary structure.

Bioethanol production by recycled Scheffersomyces stipitis in sequential batch fermentations with high cell density using xylose and glucose mixture.

Here, it is shown three-step investigative procedures aiming to improve pentose-rich fermentations performance, involving a simple system for elevated mass production by Scheffersomyces stipitis (I), cellular recycle batch fermentations (CRBFs) at high cell density using two temperature strategies (fixed at 30°C; decreasing from 30 to 26°C) (II), and a short-term adaptation action seeking to acclimatize the microorganism in xylose rich-media (III). Cellular propagation provided 0.52gdrycellweightgRS(-1), resulting in an expressive value of 45.9gdrycellweightL(-1). The yeast robustness in CRBF was proven by effective ethanol production, reaching high xylose consumption (81%) and EtOH productivity (1.53gL(-1)h(-1)). Regarding the short-term adaptation, S. stipitis strengthened its robustness, as shown by a 6-fold increase in xylose reductase (XR) activity. The short fermentation time (20h for each batch) and the fermentation kinetics for ethanol production from xylose are quite promising.

Oligomerization as a strategy for cold adaptation: Structure and dynamics of the GH1 ß-glucosidase from Exiguobacterium antarcticum B7.

Psychrophilic enzymes evolved from a plethora of structural scaffolds via multiple molecular pathways. Elucidating their adaptive strategies is instrumental to understand how life can thrive in cold ecosystems and to tailor enzymes for biotechnological applications at low temperatures. In this work, we used X-ray crystallography, in solution studies and molecular dynamics simulations to reveal the structural basis for cold adaptation of the GH1 ß-glucosidase from Exiguobacterium antarcticum B7. We discovered that the selective pressure of low temperatures favored mutations that redesigned the protein surface, reduced the number of salt bridges, exposed more hydrophobic regions to the solvent and gave rise to a tetrameric arrangement not found in mesophilic and thermophilic homologues. As a result, some solvent-exposed regions became more flexible in the cold-adapted tetramer, likely contributing to enhance enzymatic activity at cold environments. The tetramer stabilizes the native conformation of the enzyme, leading to a 10-fold higher activity compared to the disassembled monomers. According to phylogenetic analysis, diverse adaptive strategies to cold environments emerged in the GH1 family, being tetramerization an alternative, not a rule. These findings reveal a novel strategy for enzyme cold adaptation and provide a framework for the semi-rational engineering of ß-glucosidases aiming at cold industrial processes.