RESUMO
This study reported that surfactants could facilitate the organosolv pretreatment of lignocellulosic biomass (LCB) to produce fermentable sugars and highly active lignin. Under the optimized conditions, the surfactant-assisted glycerol organosolv (saGO) pretreatment achieved 80.7% delignification with a retention of 93.4% cellulose and 83.0% hemicellulose. The saGO pretreated substrate exhibited an excellent enzymatic hydrolyzability, achieving 93% of glucose yield from the enzymatic hydrolysis at 48 h. Structural analysis showed that the saGO lignin contained rich ß-O-4 bondings with less repolymerization and lower phenolic hydroxyl groups, thus forming highly reactive lignin fragments. The analysis evidenced that the surfactant graft the lignin by structural modification, which was responsible for the excellent substrate hydrolyzability. The co-production of fermentable sugars and organosolv lignin almost recovered a gross energy (87.2%) from LCB. Overall, the saGO pretreatment holds a lot of promise for launching a novel pathway towards lignocellulosic fractionation and lignin valorization.
Assuntos
Glicerol , Lignina , Açúcares , Tensoativos , Biomassa , HidróliseRESUMO
Enzymatic hydrolysis is a critical process for the cellulase-mediated lignocellulosic biorefinery to produce sugar syrups that can be converted into a whole range of biofuels and biochemicals. Such a process operating at high-solid loadings (i.e., scarcely any free water or roughly ≥ 15% solids, w/w) is considered more economically feasible, as it can generate a high sugar concentration at low operation and capital costs. However, this approach remains restricted and incurs "high-solid effects", ultimately causing the lower hydrolysis yields with increasing solid loadings. The lack of available water leads to a highly viscous system with impaired mixing that exhibits strong transfer resistance and reaction limitation imposed on enzyme action. Evidently, high-solid enzymatic hydrolysis involves multi-scale mass transfer and multi-phase enzyme reaction, and thus requires a synergistic perspective of transfer and biotransformation to assess the interactions among water, biomass components, and cellulase enzymes. Porous particle characteristics of biomass and its interface properties determine the water form and distribution state surrounding the particles, which are summarized in this review aiming to identify the water-driven multi-scale/multi-phase bioprocesses. Further aided by the cognition of rheological behavior of biomass slurry, solute transfer theories, and enzyme kinetics, the coupling effects of flow-transfer-reaction are revealed under high-solid conditions. Based on the above basic features, this review lucidly explains the causes of high-solid hydrolysis hindrances, highlights the mismatched issues between transfer and reaction, and more importantly, presents the advanced strategies for transfer and reaction enhancements from the viewpoint of process optimization, reactor design, as well as enzyme/auxiliary additive customization.
Assuntos
Celulase , Lignina , Hidrólise , Lignina/química , Celulase/química , Glucose/metabolismo , Água , BiomassaRESUMO
The valorization of organosolv pretreatment (OP) is a required approach to the industrialization of the current enzyme-mediated lignocellulosic biorefinery. Recent literature has demonstrated that the solvolysis happening in the OP can modify the soluble components into value-added active compounds, namely organosolv modified lignin (OML) and organosolv modified sugars (OMSs), in addition to protecting them against excessive degradation. Among them, the OML is coincidental with the "lignin-first" strategy that should render a highly reactive lignin enriched with ß-O-4 linkages and less condensed structure by organosolv grafting, which is desirable for the transformation into phenolic compounds. The OMSs are valuable glycosidic compounds mainly synthesized by trans-glycosylation, which can find potential applications in cosmetics, foods, and healthcare. Therefore, a state-of-the-art OP holds a big promise of lowering the process cost by the valorization of these active compounds. Recent advances in organosolv modified components are reviewed, and perspectives are made for addressing future challenges.
Assuntos
Alimentos , Lignina , Biomassa , GlicosilaçãoRESUMO
N, N-dimethylformamide is frequently present in industrial wastewater and is environmentally detrimental. The current study aims to assess the utilization and biodegradation of N, N-dimethylformamide-containing wastewater to lessen the associated environmental load. Results show that addition of wastewater containing N, N-dimethylformamide to Trichoderma reesei fermentation media enhances cellulase production and facilitates cellulose hydrolysis. However, N, N-dimethylformamide is a cellulase enhancer that is not degraded during cellulase production in T. reesei fermentation and is retained in the N, N-dimethylformamide-enhanced cellulase solution. Indeed, the cellulosic sugar solution generated via lignocellulose hydrolysis with N, N-dimethylformamide-enhanced cellulase retains N, N-dimethylformamide. We further identified three core enzyme modulesâN, N-dimethylformamidase, dimethylamine dehydrogenase, and methylamine dehydrogenase enzymeâwhich were inserted into Escherichia coli to develop metabolically engineered strains. These strains degraded N, N-dimethylformamide and produced succinate using N, N-dimethylformamide-enhanced cellulosic sugar as the substrate. The platform described here can be applied to effectively convert waste into valuable bioproducts.
Assuntos
Celulase , Trichoderma , Dimetilformamida/metabolismo , Trichoderma/metabolismo , Águas Residuárias , Engenharia Metabólica , Celulase/metabolismo , Celulose/metabolismo , Hidrólise , Fermentação , Carboidratos , Ácido Succínico/metabolismo , Açúcares/metabolismoRESUMO
Fermentable sugar production from lignocellulosic biomass has received considerable attention and has been dramatic progress recently. However, due to low enzymatic hydrolysis (EH) yields and rates, a high dosage of the costly enzyme is required, which is a bottleneck for commercial applications. Over the last decades, various strategies have been developed to reduce cellulase enzyme costs. The progress of the non-catalytic additive proteins in mitigating inhibition in EH is discussed in detail in this review. The low efficiency of EH is mostly due to soluble lignin compounds, insoluble lignin, and harsh thermal and mechanical conditions of the EH process. Adding non-catalytic proteins into the EH is considered a simple and efficient approach to boost hydrolysis yield. This review discussed the multiple mechanical steps involved in the EH process. The effect of physicochemical properties of modified lignin on EH and its interaction with cellulase and cellulose are identified and discussed, which include hydrogen bonding, hydrophobic, electrostatic, and cation-π interactions, as well as physical barriers. Moreover, the effects of different conditions of EH that lead to cellulase deactivation by thermal and mechanical mechanisms are also explained. Finally, recent advances in the development, potential mechanisms, and economic feasibility of non-catalytic proteins on EH are evaluated and perspectives are presented.
Assuntos
Celulase , Lignina , Biomassa , Celulase/química , Celulase/metabolismo , Celulose/química , Celulose/metabolismo , Hidrólise , Lignina/química , Estudos Prospectivos , AçúcaresRESUMO
BACKGROUND: Lignin plays an important role in biochemical conversion of biomass to biofuels. A significant amount of lignin is precipitated on the surface of pretreated substrates after organosolv pretreatment. The effect of this residual lignin on enzymatic hydrolysis has been well understood, however, their effect on subsequent ABE fermentation is still unknown. RESULTS: To determine the effect of residual extractable lignin on acetone-butanol-ethanol (ABE) fermentation in separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) processes, we compared ABE production from ethanol-washed and unwashed substrates. The ethanol organosolv pretreated loblolly pine (OPLP) was used as the substrate. It was observed that butanol production from OPLP-UW (unwashed) and OPLP-W (washed) reached 8.16 and 1.69 g/L, respectively, in SHF. The results showed that ABE production in SHF from OPLP-UW prevents an "acid crash" as compared the OPLP-W. In SSF process, the "acid crash" occurred for both OPLP-W and OPLP-UW. The inhibitory extractable lignin intensified the "acid crash" for OPLP-UW and resulted in less ABE production than OPLP-W. The addition of detoxified prehydrolysates in SSF processes shortened the fermentation time and could potentially prevent the "acid crash". CONCLUSIONS: The results suggested that the residual extractable lignin in high sugar concentration could help ABE production by lowering the metabolic rate and preventing "acid crash" in SHF processes. However, it became unfavorable in SSF due to its inhibition of both enzymatic hydrolysis and ABE fermentation with low initial sugar concentration. It is essential to remove extractable lignin of substrates for ABE production in SSF processes. Also, a higher initial sugar concentration is needed to prevent the "acid crash" in SSF processes.
RESUMO
The development of sustainable bioethanol fuel production from food waste has increasingly become an attractive topic. Food waste is recognized as the most available and costless feedstock. Therefore, ethanol production has been adopted as cost-efficient and an ecological way for FW disposal. This paper reviewed the microorganisms utilized for ethanol fermentation, the effect of enzymatic hydrolysis on ethanol concentration, optimization of accurate process parameters, and recycling of huge volumes of stillage for ethanol production towards reducing any incurred environmental burdens and minimizing the cost. The statistical tools which may enhance the process efficiency had been presented. Also, the perspective and the future development were introduced. All these aimed to fully utilize the food waste and also reduce the cost for side-product in this process; proper operation conditions and the control methods for stillage recycling were considered as the methods to improve ethanol fermentation from food waste.
Assuntos
Biocombustíveis/análise , Etanol/análise , Fermentação , Alimentos , Reciclagem/métodos , Resíduos/análise , Hidrólise , Reciclagem/economiaRESUMO
BACKGROUND: Biomass prehydrolysates from dilute acid pretreatment contain a considerable amount of fermentable sugars for biofuels production. However, carbonyl degradation compounds present severe toxicity to fermentation microbes. Furans (such as furfural and hydroxymethylfurfural), aliphatic acids (such as acetic acid, formic acid and levulinic acid) and phenolic compounds (such as vanillin and syringaldehyde) have been suggested to be the main inhibitors in biomass prehydrolysates. However, no single compound has been determined as the dominant toxic inhibitor. The effects of various detoxification methods on inhibitors removal have not been fully understood. RESULTS: The effects of overliming and activated carbon (AC) detoxification on the removal of inhibitors and butanol fermentation of the poplar prehydrolysates were investigated. Gas chromatography-mass spectrometry (GC/MS) was used to identify and quantify 46 carbonyl compounds as potential inhibitors. It was observed that overliming and AC treatment alone did not make the prehydrolysates fermentable with Clostridium saccharobutylicum. The sequential overliming and AC resulted in a remarkable fermentability and a high butanol yield at 0.22 g g-1 sugar. The inhibitor removal in the prehydrolysates treated by overliming and AC was also examined by GC/MS. Overliming removed 75.6% of furan derivatives and 68.1% of aromatic monomers. In comparison, AC (5.0% w/v) removed 77.9% of furan derivatives and 98.6% of aromatic monomers. In addition, overliming removed much more 2,5-furandicarboxyaldehyde, 5-ethylfuran-2-carbaldehyde and 2,5-hexanedione than AC did. On the contrary, AC could remove considerably more phenolic acids than overliming. In the sequential detoxification, both dialdehydes/diketones and phenolic acids were extensively removed. This could be the main reason why the sequential detoxification enabled a remarkable ABE fermentation for the prehydrolysates. CONCLUSIONS: This study indicated that the effect of overliming and AC treatment on inhibitors removal was related to their chemical structures. Overliming removed more dialdehydes and diketones than AC treatment, while AC removed more phenolic acids than overliming. Sequential overliming and AC treatment were required to make the prehydrolysates fermentable with C. saccharobutylicum. The study also suggested different detoxification method was needed for ABE fermentation of the prehydrolysate as compared to ethanol fermentation.
RESUMO
Bioconversion of lignocellulose to biofuels suffers from the degradation compounds formed during pretreatment and acid hydrolysis. In order to achieve an efficient biomass to biofuel conversion, detoxification is often required before enzymatic hydrolysis and microbial fermentation. Prehydrolysates from ethanol organosolv-pretreated pine wood were used as substrates in butanol fermentation in this study. Six detoxification approaches were studied and compared, including overliming, anion exchange resin, nonionic resin, laccase, activated carbon, and cysteine. It was observed that detoxification by anion exchange resin was the most effective method. The final butanol yield after anion exchange resin treatment was comparable to the control group, but the fermentation was delayed for 72 h. The addition of Ca(OH)2 was found to alleviate this delay and improve the fermentation efficiency. The combination of Ca(OH)2 and anion exchange resin resulted in completion of fermentation within 72 h and acetone-butanol-ethanol (ABE) production of 11.11 g/L, corresponding to a yield of 0.21 g/g sugar. The cysteine detoxification also resulted in good detoxification performance, but promoted fermentation towards acid production (8.90 g/L). The effect of salt on ABE fermentation was assessed and the possible role of Ca(OH)2 was to remove the salts in the prehydrolysates by precipitation.
Assuntos
Resinas de Troca Aniônica/metabolismo , Butanóis/metabolismo , Cisteína/metabolismo , Fermentação , Pinus/metabolismo , Biocombustíveis , Biomassa , Clostridium acetobutylicum/metabolismo , Hidrólise , Inativação Metabólica , Sulfatos/químicaRESUMO
A single-chamber microbial fuel cell (MFC) was used in this study to treat recycled stillage obtained from food waste ethanol fermentation. Corresponding substrates inside the system were evaluated by fluorescence spectra, and microbial communities were also investigated. Results demonstrated that output voltage and current, respectively, reached 0.29 V and 1.4 mA with an external resistance of 200 Ω. Corresponding total organic carbon and chemical oxygen demand removal efficiency reached more than 50% and 70%, respectively. Results of fluorescence spectra demonstrated that tryptophan-like aromatic, soluble microbial by-product-like and humic acid-like substances accumulated and were not easily degraded. Microbial community analysis by high-throughput sequence indicated that Advenella and Moheibacter occupied the highest proportion among all genera at the anode instead of Geobacter. These results may be due to complicated accumulated stillage, and potential tetracyclines possibly influenced microbial communities. Details on how stillage affects MFC operation should be further studied, and a solution on relieving effects should be established.
RESUMO
The effect of ethanol organosolv lignin (EOL) on enzymatic hydrolysis was examined at pH 4.8-6.0. The addition of EOL prepared from sweetgum enhanced the enzymatic hydrolysis of organosolv-pretreated loblolly pine (OPLP) by 38.8% and 88.0% at pH 4.8 and 5.6, respectively. The addition of EOL prepared from loblolly pine inhibited the enzymatic hydrolysis of OPLP at pH 4.8 but improved it by 43.0% at pH 5.6. This suggests that the addition of EOL and increase in pH act synergistically to improve the enzymatic hydrolysis of OPLP. The effect of EOL addition on cellulase adsorption onto residual lignins was examined. The results revealed that increasing the pH intensified the suppression of non-productive binding between enzymes and residual lignins by EOL. The potential stabilization effects of EOL on enzymes can contribute to the improvement of enzymatic hydrolysis with EOL at higher pH.
RESUMO
BACKGROUND: Lignin typically inhibits enzymatic hydrolysis of cellulosic biomass, but certain organosolv lignins or lignosulfonates enhance enzymatic hydrolysis. The hydrophobic and electrostatic interactions between lignin and cellulases play critical roles in the enzymatic hydrolysis process. However, how to incorporate these two interactions into the consideration of lignin effects has not been investigated. RESULTS: We examined the physicochemical properties and the structures of ethanol organosolv lignins (EOL) from hardwood and softwood and ascertained the association between lignin properties and their inhibitory and stimulatory effects on enzymatic hydrolysis. The zeta potential and hydrophobicity of EOL lignin samples, isolated from organosolv pretreatment of cottonwood (CW), black willow (BW), aspen (AS), eucalyptus (EH), and loblolly pine (LP), were determined and correlated with their effects on enzymatic hydrolysis of Avicel. EOLs from CW, BW, and AS improved the 72 h hydrolysis yield by 8-12%, while EOLs from EH and LP decreased the 72 h hydrolysis yield by 6 and 16%, respectively. The results showed a strong correlation between the 72 h hydrolysis yield with hydrophobicity and zeta potential. The correlation indicated that the hydrophobicity of EOL had a negative effect and the negative zeta potential of EOL had a positive effect. HSQC NMR spectra showed that ß-O-4 linkages in lignin react with ethanol to form an α-ethoxylated ß-O-4' substructure (A') during organosolv pretreatment. Considerable amounts of C2,6-H2,6 correlation in p-hydroxybenzoate (PB) units were observed for EOL-CW, EOL-BW, and EOL-AS, but not for EOL-EH and EOL-LP. CONCLUSIONS: This study revealed that the effect of lignin on enzymatic hydrolysis is a function of both hydrophobic interactions and electrostatic repulsions. The lignin inhibition is controlled by lignin hydrophobicity and the lignin stimulation is governed by the negative zeta potential. The net effect of lignin depends on the combined influence of hydrophobicity and zeta potential. This study has potential implications in biomass pretreatment for the reduction of lignin inhibition by increasing lignin negative zeta potential and decreasing hydrophobicity.
RESUMO
The Cel6A deficiency has become one of the limiting factors for cellulose saccharification in biochemical conversion of cellulosic biomass to fuels and chemicals. The work attempted to use codon optimization to enhance Trichoderma reesei Cel6A expression in Pichia pastoris. Two recombinants P. pastoris GS115 containing AOX1 and GAP promotors were successfully constructed, respectively. The optimal temperatures and pHs of the expressed Cel6A from two recombinants were consistent with each other, were also in the extremely similar range to that reported on the native Cel6A from T. reesei. Based on the shake flask fermentation, AOX1 promotor enabled the recombinant to produce 265U/L and 300mg/L of the Cel6A enzyme, and the GAP promotor resulted in 145U/L and 200mg/L. High cell density fed batch (HCDFB) fermentation significantly improved the enzyme titer (1100U/L) and protein yield (2.0g/L) for the recombinant with AOX1 promotor. Results have showed that the AOX1 promotor is more suitable than the GAP for the Cel6A expression in P. pastoris. And the HCDFB cultivation is a favorable way to express the Cel6A highly in the methanol inducible yeast.
Assuntos
Celulose 1,4-beta-Celobiosidase/genética , Celulose 1,4-beta-Celobiosidase/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Pichia/enzimologia , Pichia/genética , Trichoderma/enzimologia , Trichoderma/genética , Sequência de Aminoácidos , Sequência de Bases , Celulose 1,4-beta-Celobiosidase/química , Códon/genética , Fermentação , Proteínas Fúngicas/química , Expressão Gênica , Genes Fúngicos , Modelos Moleculares , Conformação de Ácido Nucleico , Regiões Promotoras Genéticas , RNA Fúngico/química , RNA Fúngico/genética , RNA Mensageiro/química , RNA Mensageiro/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Paper mill sludge (PS), a solid waste from pulp and paper industry, was investigated as a feedstock for acetone-butanol-ethanol (ABE) production by simultaneous saccharification and fermentation (SSF). ABE fermentation of paper sludge by Clostridium acetobutylicum required partial removal of ash in PS to enhance its enzymatic digestibility. Enzymatic hydrolysis was found to be a rate-limiting step in the SSF. A total of 16.4-18.0g/L of ABE solvents were produced in the SSF of de-ashed PS with solid loading of 6.3-7.4% and enzyme loading of 10-15FPU/g-glucan, and the final solvent yield reached 0.27g/g sugars. No pretreatment and pH control were needed in ABE fermentation of paper sludge, which makes it an attractive feedstock for butanol production. The results suggested utilization of paper sludge should not only consider the benefits of buffering effect of CaCO3 in fermentation, but also take into account its inhibitory effect on enzymatic hydrolysis.
Assuntos
Acetona/metabolismo , Butanóis/metabolismo , Metabolismo dos Carboidratos , Etanol/metabolismo , Fermentação , Papel , Esgotos/química , Álcalis/farmacologia , Metabolismo dos Carboidratos/efeitos dos fármacos , Celulose/metabolismo , Clostridium acetobutylicum/efeitos dos fármacos , Clostridium acetobutylicum/metabolismo , Fermentação/efeitos dos fármacos , Glucose/metabolismo , Hidrólise , Panicum/efeitos dos fármacos , Xilose/metabolismoRESUMO
Carbonyl compounds generated in biomass pretreatment hinder the biochemical conversion of biomass hydrolysates to biofuels. A novel approach of detoxifying hydrolysates with amino acids for ethanol production was developed. Among the 20 amino acids assessed for their detoxification efficiency and nucleophilicity, cysteine was the most effective one. It increased both ethanol productivity and final yield of biomass hydrolysates from 0.18 (untreated) to 1.77 g/L/h and from 0.02 to 0.42 g/g, respectively. Detoxification efficiency was followed by histidine and it increased the final yield to 0.42 g/g, then by lysine, tryptophan and asparagine. It was observed all five effective amino acids contained reactive side-chain functional groups, which played important roles in the amino acid detoxification reaction. The study further showed cysteine and glycine detoxifications were temperature and pH dependent. The mechanistic study using mass spectrometry revealed thiazolidine carboxylic acid, a Schiff base, was formed by condensation of aldehyde and cysteine.
Assuntos
Aminoácidos/metabolismo , Etanol/metabolismo , Fermentação/fisiologia , Biocombustíveis/microbiologia , Biomassa , Concentração de Íons de Hidrogênio , Hidrólise , TemperaturaRESUMO
Biomass degradation compounds significantly inhibit biochemical conversion of biomass prehydrolysates to biofuels and chemicals, such as lactic acid. To characterize the structure-activity relationship of carbonyl inhibition on lactic acid fermentation, we examined effects of eight carbonyl compounds (furfural, 5-hydroxymethyl furfural, vanillin, syringaldehyde, 4-hydroxybenzaldehyde, phthalaldehyde, benzoic acid, and pyrogallol aldehyde) and creosol on lactic acid production by Lactobacillus delbrueckii. Pyrogallol aldehyde reduced the cell growth rate by 35 % at 1.0 mM and inhibited lactic acid production completely at 2.0 mM. By correlating the molecular descriptors to the inhibition constants in lactic acid fermentation, we found a good relationship between the hydrophobicity (Log P) of aldehydes and their inhibition constants in fermentation. The inhibitory effect of carbonyl inhibitors appeared to correlate with their thiol reactivity as well. In addition, we found that H2O2 detoxified pyrogallol aldehyde and phthalaldehyde inhibitory activity. H2O2 detoxification was applied to real biomass prehydrolysates in lactic acid fermentation.
Assuntos
Biomassa , Ácido Láctico/metabolismo , Lactobacillus delbrueckii/química , Biocombustíveis , Etanol/química , Fermentação , Peróxido de Hidrogênio/química , Hidrólise , Ácido Láctico/química , Lactobacillus delbrueckii/metabolismo , Relação Estrutura-AtividadeRESUMO
Identifying an appropriate parameter to elucidate effects of lignin on enzymatic hydrolysis is essential to understand the interactions between enzymes and lignin. Contrasting effects of hardwood organosolv lignin (EOL-SG) and softwood organosolv lignin (EOL-LP) on enzymatic hydrolysis were observed. The addition of EOL-SG (8 g/L) significantly improved the 72 h hydrolysis yields of organosolv pretreated sweetgum (OPSG) and loblolly pine (OPLP) from 49.3% to 68.6% and from 41.2% to 60.8%, respectively. In contrast, the addition of EOL-LP decreased the 72 h hydrolysis yields of OPSG and OPLP to 42.0% and 38.1%, respectively. A strong correlation between the distribution coefficients of cellulase enzymes on lignins and the changes of hydrolysis yields indicated that the inhibitory or stimulatory effects of organosolv lignins on enzymatic hydrolysis were governed by the distribution coefficients (R). The different R values probably were related to the electrostatic interactions, hydrophobic interactions and hydrogen bondings between enzymes and lignin.
Assuntos
Celulase/metabolismo , Lignina/metabolismo , Madeira , Hidrólise , Interações Hidrofóbicas e Hidrofílicas , Espectroscopia de Prótons por Ressonância MagnéticaRESUMO
Low solvent concentration effect on substrate digestibility of ethanol organosolv pretreated sweetgum was examined. Surprisingly, lower ethanol concentration in organosolv pretreatments resulted in faster initial rates and higher 72h hydrolysis yields in pretreated substrates. A strong correlation (r(2)=0.96) between pretreatment combined severity factor and residual xylan/glucan ratio was observed. The residual xylan/glucan ratio was associated with the initial hydrolysis rate closely. Furthermore, it was found that preserving extractable lignin in the pretreated substrates could improve enzymatic hydrolysis yield by 33%. This has an important implication in reducing the pretreatment and enzyme cost, because the typical solvent washing after pretreatment could be eliminated and preserving extractable lignin could reduce enzyme loading. Finally, we observed that xylan removal by xylanase could improve the initial rate by 53% and increase the 72h hydrolysis yield by 21%. The extractable lignin precipitation on pretreated substrates increased the 72h hydrolysis yield by 10%.
Assuntos
Biotecnologia/métodos , Celulase/metabolismo , Lignina/isolamento & purificação , Solventes/farmacologia , Madeira/efeitos dos fármacos , Precipitação Química , Etanol/metabolismo , Glucanos/metabolismo , Hidrólise/efeitos dos fármacos , Liquidambar/efeitos dos fármacos , Xilanos/metabolismoRESUMO
The 2,367-bp ORF of TtAFase from Thermotoga thermarum DSM 5069 encodes a calculated 90-kDa α-L-arabinofuranosidase (TtAFase), which does not belonging to any reported glycosyl hydrolase families α-L-arabinofuranosidases in the database and represents a novel one of glycosyl hydrolase family 2. The purified recombinant TtAFase produced in Escherichia coli BL21 (DE3) had optimum activity at pH 5.5 and at 80 °C. It was stable up to 80 °C and from pH 4.5-8.5. Kinetic experiments at 80 °C with p-nitrophenyl α-L-arabinofuranoside as a substrate gave a K m of 0.77 mM, V max of 2.3 µmol mg(-1) min(-1) and k cat of 4.5 s(-1). The enzyme had no apparent requirement of metal ions for activity, and its activity was significantly inhibited by Cu(2+) or Zn(2+).