Novel Ethanol- and 5-Hydroxymethyl Furfural-Stimulated β-Glucosidase Retrieved From a Brazilian Secondary Atlantic Forest Soil Metagenome

Beta-glucosidases are key enzymes involved in lignocellulosic biomass degradation for bioethanol production, which complete the final step during cellulose hydrolysis by converting cellobiose into glucose. Currently, industry requires enzymes with improved catalytic performance or tolerance to process-specific parameters. In this sense, metagenomics has become a powerful tool for accessing and exploring the biochemical biodiversity present in different natural environments. Here, we report the identification of a novel β-glucosidase from metagenomic DNA isolated from soil samples enriched with decaying plant matter from a Secondary Atlantic Forest region. For this, we employed a functional screening approach using an optimized and synthetic broad host-range vector for library production. The novel β-glucosidase – named Lfa2 – displays three GH3-family conserved domains and conserved catalytic amino acids D283 and E487. The purified enzyme was most active in pH 5.5 and at 50°C, and showed hydrolytic activity toward several pNP synthetic substrates containing β-glucose, β-galactose, β-xylose, β-fucose, and α-arabinopyranose, as well as toward cellobiose. Lfa2 showed considerable glucose tolerance, exhibiting an IC50 of 300 mM glucose and 30% of remaining activity in 600 mM glucose. In addition, Lfa2 retained full or slightly enhanced activity in the presence of several metal ions. Further, β-glucosidase activity was increased by 1.7-fold in the presence of 10% (v/v) ethanol, a concentration that can be reached in conventional fermentation processes. Similarly, Lfa2 showed 1.7-fold enhanced activity at high concentrations of 5-hydroxymethyl furfural, one of the most important cellulase inhibitors in pretreated sugarcane bagasse hydrolysates. Moreover, the synergistic effect of Lfa2 on Bacillus subtilis GH5-CBM3 endoglucanase activity was demonstrated by the increased production of glucose (1.6-fold). Together, these results indicate that β-glucosidase Lfa2 is a promissory enzyme candidate for utilization in diverse industrial applications, such as cellulosic biomass degradation or flavor enhancement in winemaking and grape processing

Enzymatically and/or thermally treated Macroalgae biomass as feedstock for fermentative H2 production

Due to its high carbohydrate content, algae biomass can be employed as feedstock to produce hydrogen (H2)by fermentation. However, to make the carbohydrates entrapped within the cell wall more bioavailable, algaebiomass must be treated before fermentation. We submitted Kappaphyccus alvarezzi macroalgae biomass toautoclave (at 120 °C and 1 atm for 6 h) treatment and/or enzymatic (Celluclast® and/or a recombinant β-glucosidase) hydrolysis, to break down complex carbohydrates into available sugars that were used to produceH2 by fermentation. Macroalgae biomass treated with Celluclast®+β-glucosidase and with combinedthermal treatment and enzymatic hydrolysis reached very similar TRS productivities, 0.24 and 0.22 g ofTRS/L.h, respectively. The enzymatically treated biomass was employed as feedstock to produce H2 byClostridium beijerinckii Br21, which afforded high yield: 21.3 mmol of H2/g of dry algae biomass. Hence,treatment with Celluclast® and recombinant β-glucosidase provided macroalgae biomass for enhanced bioconversionto H2 by C. beijerinckii Br21.Keywords: Kappaphyccus alvarezzi, Clostridium beijerinckii, Biohydrogen, Cellulase, β-glucosidas

Structure-function relationship of a glucose-xylose-stimulated -glucosidase from thermophilic fungus Humicola insolens: studies of directed evolution

Um dos pré-requisitos para a produção economicamente viável de etanol a partir da biomassa lignocelulósica é o desenvolvimento de processos eficientes e baratos de hidrólise enzimática de celulose e hemicelulose. As enzimas respondem por altas percentagens dos custos de hidrólise, pois é necessário utilizar altas cargas enzimáticas para obter rendimentos aceitáveis devido à inibição das enzimas lignocelulolíticas pelos produtos, que se intensifica com o uso de altas concentrações iniciais de biomassa. Uma das estratégias para melhorar a eficiência e diminuir os custos da hidrólise é a identificação de enzimas mais eficientes, com grande atenção para aquelas tolerantes e/ou estimuladas pelos produtos de reação. Nesse contexto, a engenharia de proteínas é uma poderosa ferramenta para o melhoramento e o entendimento da relação estrutura-função destas enzimas. O presente trabalho visou avaliar o efeito da glicosilação sobre as características bioquímicas de uma - glucosidase estimulada por glicose e xilose de Humicola insolens, comparando a enzima nativa e as enzimas recombinantes, expressas em Escherichia coli (Bglhi) e Pichia pastoris (BglhiPp), além de estudar a relação estrutura-função por meio de técnicas de evolução dirigida objetivando o entendimento dos mecanismos envolvidos na estimulação da enzima pelos monossacarídeos. Com relação à glicosilação, a expressão e caracterização da BglhiPp permitiu avaliar que as principais características influenciadas por diferentes conteúdos de carboidratos na enzima foram a temperatura ótima e a termoestabilidade. Já o estudo de evolução dirigida culminou na geração de 4 mutantes com padrão de estimulação por glicose e xilose diferentes da Bglhi (utilizada como controle). Todos os mutantes contêm uma das duas substituições (D237V e N235S) agrupadas ao redor dos subsítios de ligação da aglicona (+1 e +2). Os dados cinéticos e de transglicosilação permitiram sugerir que o mecanismo de estimulação destas enzimas envolve interações alostéricas, modulação das rotas de hidrólise e transglicosilação e competição entre substrato e monossacarídeos pela ligação aos subsítios do sítio ativo. A mutação D237V (presente nos mutantes 4-12D e 5-7H) favoreceu a rota de hidrólise em detrimento à de transglicosilação e a atividade pNP-glucosidásica, mas não a celobiásica, foi estimulada por xilose. A substituição N235S (presente nos mutantes 1-6D e 5-7C) aboliu a preferência por hidrólise ou transglicosilação e a atividade celobiásica, mas não a pNP-glucosidásica, foi fortemente inibida por xilose. Além disso, ambas as mutações diminuíram a tolerância das enzimas pelos monossacarídeos. Estes resultados mostraram que a modulação fina da atividade da Bglhi e das enzimas dos mutantes por glicose e/ou xilose é regulada pelas afinidades relativas dos subsítios da glicona e da aglicona pelos substratos e pelos monossacarídeos livres. As mudanças na topologia e nas propriedades físico-químicas dos subsítios +1 e +2 da aglicona foi proposta por racionalizar os dados cinéticos e de transglicosilação.One of the prerequisites for the economically viable production of ethanol from the lignocellulosic biomass is the development of efficient and inexpensive processes of enzymatic hydrolysis of cellulose and hemicellulose. The enzymes are responsible for high percentages of hydrolysis costs, since it is necessary to use high enzymatic loads for acceptable yields due to inhibition of lignocellulolitic enzymes by products, which is intensified by the use of high initial concentrations of biomass. One of the strategies to improve efficiency and decrease the costs of hydrolysis is the identification of more efficient enzymes with great attention to those that are tolerant and/or stimulated by the reaction products. In this context, protein engineering is a powerful tool for the improvement and understanding of the structure-function relationship of these enzymes. The present work aimed to evaluate the effect of glycosylation on the biochemical characteristics of glucose and xylose-stimulated -glucosidase from Humicola insolens, comparing the native enzyme and the recombinant enzymes expressed in Escherichia coli (Bglhi) and Pichia pastoris (BglhiPp) and study the structure-function relationship through directed evolution techniques aiming the understanding of the mechanisms involved in the stimulation of the enzyme by the monosaccharides. With regard to glycosylation, the expression and characterization of BglhiPp allowed to evaluate that the main characteristics influenced by different carbohydrate contents in the enzyme were optimum temperature and thermostability. The study of directed evolution culminated in the generation of 4 mutants with pattern of stimulation by glucose and xylose different from Bglhi (used as control). All mutants contain one of the two substitutions (D237V and N235S) grouped around the aglycone binding sites (+1 and +2). The kinetic and transglycosylation data allowed us to suggest that the mechanism of stimulation of these enzymes involves allosteric interactions, modulation of the hydrolysis and transglycosylation routes, and competition between substrate and monosaccharides by binding to the subsites in active site. The mutation D237V (present in mutants 4-12D and 5-7H) favored the hydrolysis route over that of transglycosylation and pNP-glucosidase activity, but not cellobiase activity, was stimulated by xylose. The substitution N235S (present in mutants 1-6D and 5-7C) abolished the preference for hydrolysis or transglycosylation and cellobiase activity, but not pNP-glucosidase activity, was strongly inhibited by xylose. In addition, both mutations decreased the tolerance of the enzymes by the monosaccharides. These results showed that fine modulation of Bglhi and mutant enzymes activities by glucose and/or xylose is regulated by the relative affinities of the glycone and aglycone subsites for the substrates and the free monosaccharides. The changes in the topology and physicochemical properties of the +1/+2 aglycone sites of the mutants have been proposed to rationalize the kinetic and transglycosylation data

Engineering the GH1 β-glucosidase from Humicola insolens: Insights on the stimulation of activity by glucose and xylose.

The activity of the GH1 β-glucosidase from Humicola insolens (Bglhi) against p-nitrophenyl-β-D-glucopyranoside (pNP-Glc) and cellobiose is enhanced 2-fold by glucose and/or xylose. Kinetic and transglycosylation data showed that hydrolysis is preferred in the absence of monosaccharides. Stimulation involves allosteric interactions, increased transglycosylation and competition of the substrate and monosaccharides for the -1 glycone and the +1/+2 aglycone binding sites. Protein directed evolution has been used to generate 6 mutants of Bglhi with altered stimulation patterns. All mutants contain one of three substitutions (N235S, D237V or H307Y) clustered around the +1/+2 aglycone binding sites. Two mutants with the H307Y substitution preferentially followed the transglycosylation route in the absence of xylose or glucose. The strong stimulation of their pNP-glucosidase and cellobiase activities was accompanied by increased transglycosylation and higher monosaccharide tolerance. The D237V mutation favoured hydrolysis over transglycosylation and the pNP-glucosidase activity, but not the cellobiase activity, was stimulated by xylose. The substitution N235S abolished the preference for hydrolysis or transglycosylation; the cellobiase, but not the pNP-glucosidase activity of the mutants was strongly inhibited by xylose. Both the D237V and N235S mutations lowered tolerance to the monosaccharides. These results provide evidence that the fine modulation of the activity of Bglhi and mutants by glucose and/or xylose is regulated by the relative affinities of the glycone and aglycone binding sites for the substrate and the free monosaccharides

Kinetic parameters for the stimulation of the cellobiase activity of Bglhi and mutants by cellobiose in the presence or absence of fixed concentrations of xylose.

<p>Kinetic parameters for the stimulation of the cellobiase activity of Bglhi and mutants by cellobiose in the presence or absence of fixed concentrations of xylose.</p

Time-course analysis of the reaction products formed by Bglhi and mutants against cellobiose.

<p>(a) Bglhi, (b) N89Y/H307Y, (c) H307Y, (d) D237V/P389H/E395G/K475R, (e) D237V, (f) A141T/N235S, (g) N235S. The reactions were performed at 90–95% saturating concentrations of cellobiose (see legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0188254#pone.0188254.g004" target="_blank">Fig 4</a>) in the absence (indicated as “control”) or presence of glucose (indicated as “Plus G₁”) or xylose (indicated as “Plus X₁”). The final concentrations of each monosaccharide were equal to MC_max (Bglhi, N89Y/H307Y and H307Y) or MT (D237V/P389H/E395G/K475R, D237V, A141T/N235S and N235S). The reaction times were 0 (lanes t₀), 5 min (lanes t₁), 10 min (lanes t₂) and 24 h (lanes t₃). Standards: G₁, glucose; G₂, cellobiose; G₃, cellotriose; G₄, cellotetraose; Ge, gentibiose; X₁, xylose; X₂, xylobiose; SG₂, equimolar mixture of sophorose and cellobiose. The volumes of each aliquot applied to the TLC plates were adjusted aiming the best visualization of the products.</p

Catalytic reaction mechanism of the retaining β-glycosidases.

<p>After the formation of the glucosyl-enzyme intermediate (step 1), the entry of a water molecule leads to hydrolysis (step 2) and the entry of a sugar leads to transglycosylation (step 3).</p

Kinetic parameters for the stimulation of the <i>p</i>NP-glucosidase activity of Bglhi and mutants against <i>p</i>NP-Glc in the absence or presence of fixed concentrations of glucose or xylose.

<p>Kinetic parameters for the stimulation of the <i>p</i>NP-glucosidase activity of Bglhi and mutants against <i>p</i>NP-Glc in the absence or presence of fixed concentrations of glucose or xylose.</p

Engineering the GH1 β-glucosidase from <i>Humicola insolens</i>: Insights on the stimulation of activity by glucose and xylose - Fig 6

<p><b>Tandem mass spectrometry analysis of glucopyranosyl-xylose (A), cellotriose (B) and cellotetraose (C).</b> The sodium adducts of glucopyranosyl-xylose (<i>m/z</i> 335), cellotriose (<i>m/z</i> 527) and cellotetraose (<i>m/z</i> 689) were analyzed by MS/MS and the proposed interpretation of mass spectra are indicated on the structures.</p