RESUMO
OBJECTIVE: A correlation among different volumetric oxygen transfer coefficients (kLa) and the oxireduction potential (ORP) in batch fermentations using Scheffersomyces stipitis was evaluated. Experiments were performed using a mixture of xylose and glucose as the substrates. RESULTS: Microaerophilic condition (kLa = 4.9 h-1) have shown to be suitable when compared to complete anaerobiosis (kLa = 0), providing an ethanol yield and a productivity after 48 h of 64.3% and 0.45 g ethanol L-1 h-1, respectively; the maximum ethanol titer obtained was 21.50 g ethanol L-1. Values of ORP varying from - 270 to - 330 mV resulted in high ethanol production from xylose by S. stipitis. CONCLUSIONS: Different ORP values were found in anaerobiosis and kLa 4.9 h-1, suggesting that for ethanol production by S. stipitis, values from - 270 to - 330 mV are favorable under the studied circumstances. In this ORP range, the greatest rates of xylose consumption and ethanol production were registered. ORP monitoring was demonstrated to be a suitable option for online control throughout the fermentation processes, which may provide a more efficient bioprocess operation with a very low O2 concentration.
Assuntos
Meios de Cultura/química , Etanol/metabolismo , Fermentação , Glucose/metabolismo , Oxirredução , Saccharomycetales/metabolismo , Xilose/metabolismo , Aerobiose , Anaerobiose , Biotransformação , Saccharomycetales/crescimento & desenvolvimentoRESUMO
Xylanases catalyze the hydrolysis of ß-1,4-linked xylosyl moieties from xylan chains, one of the most abundant hemicellulosic polysaccharides found in plant cell walls. These enzymes can exist either as single catalytic domains or as modular proteins composed of one or more carbohydrate-binding modules (CBMs) appended to the catalytic core. However, the molecular mechanisms governing the synergistic effects between catalytic domains and their CBMs are not fully understood. Thus, the goal of this study was to evaluate the functional effects of the fusion of a CBM belonging to family 6, which exhibits high affinity to xylan, with the GH11 xylanase from Bacillus subtilis, which does not have a CBM in its wild-type form. The wild-type enzyme (BsXyl11) and the chimeric protein (BsXyl11-CBM6) were heterologously produced in Escherichia coli and purified to homogeneity for biochemical characterization. The molecular fusion did not alter the pH and temperature dependence, but kinetic data revealed an increase of 65% in the catalytic efficiency of the chimeric enzyme. Furthermore, the BsXyl11-CBM6 chimera was used to supplement the commercial cocktail Accellerase® 1500 and improved the reducing sugar release by 17% from pretreated sugarcane bagasse. These results indicate that CBM6 can be used as a molecular tool to enhance the catalytic performance of endo-xylanases (GH11) and provide a new strategy for the development of optimized biocatalysts for biotechnological applications.
Assuntos
Endo-1,4-beta-Xilanases/química , Endo-1,4-beta-Xilanases/metabolismo , Xilanos/metabolismo , Bacillus subtilis/enzimologia , Bacillus subtilis/genética , Biotecnologia , Catálise , Domínio Catalítico , Celulose , Endo-1,4-beta-Xilanases/genética , Hidrólise , Cinética , Domínios Proteicos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Saccharum , Especificidade por Substrato , Xilanos/químicaRESUMO
Xylan is an abundant plant cell wall polysaccharide and its reduction to xylose units for subsequent biotechnological applications requires a combination of distinct hemicellulases and auxiliary enzymes, mainly endo-xylanases and ß-xylosidases. In the present work, a bifunctional enzyme consisting of a GH11 endo-1,4-ß-xylanase fused to a GH43 ß-xylosidase, both from Bacillus subtilis, was designed taking into account the quaternary arrangement and accessibility to the substrate. The parental enzymes and the resulting chimera were successfully expressed in Escherichia coli, purified and characterized. Interestingly, the substrate cleavage rate was altered by the molecular fusion improving at least 3-fold the xylose production using specific substrates as beechwood xylan and hemicelluloses from pretreated biomass. Moreover, the chimeric enzyme showed higher thermotolerance with a positive shift of the optimum temperature from 35 to 50 °C for xylosidase activity. This improvement in the thermal stability was also observed by circular dichroism unfolding studies, which seems to be related to a gain of stability of the ß-xylosidase domain. These results demonstrate the superior functional and stability properties of the chimeric enzyme in comparison to individual parental domains, suggesting the molecular fusion as a promising strategy for enhancing enzyme cocktails aiming at lignocellulose hydrolysis.
Assuntos
Glicosídeo Hidrolases/metabolismo , Xilose/biossíntese , Bacillus subtilis/enzimologia , Bacillus subtilis/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biomassa , Biotecnologia , Dicroísmo Circular , Endo-1,4-beta-Xilanases/química , Endo-1,4-beta-Xilanases/genética , Endo-1,4-beta-Xilanases/metabolismo , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/genética , Hidrólise , Lignina/metabolismo , Modelos Moleculares , Polissacarídeos/metabolismo , Estrutura Quaternária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Especificidade por Substrato , Temperatura , Xilanos/metabolismo , Xilosidases/química , Xilosidases/genética , Xilosidases/metabolismoRESUMO
Beta-glucosidases (BGLs) are enzymes of great potential for several industrial processes, since they catalyze the cleavage of glucosidic bonds in cellobiose and other short cellooligosaccharides. However, features such as good stability to temperature, pH, ions and chemicals are required characteristics for industrial applications. This work aimed to provide a comparative biochemical analysis of three thermostable BGLs from Pyrococcus furiosus and Thermotoga petrophila. The genes PfBgl1 (GH1 from P. furiosus), TpBgl1 (GH1 from T. petrophila) and TpBgl3 (GH3 from T. petrophila) were cloned and proteins were expressed in Escherichia coli. The purified enzymes are hyperthermophilic, showing highest activity at temperatures above 80°C at acidic (TpBgl3 and PfBgl1) and neutral (TpBgl1) pHs. The BGLs showed greatest stability to temperature mainly at pH 6.0. Activities using a set of different substrates suggested that TpBgl3 (GH3) is more specific than GH1 family members. In addition, the influence of six monosaccharides on BGL catalysis was assayed. While PfBgl1 and TpBgl3 seemed to be weakly inhibited by monosaccharides, TpBgl1 was activated, with xylose showing the strongest activation. Under the conditions tested, TpBgl1 showed the highest inhibition constant (Ki=1100.00mM) when compared with several BGLs previously characterized. The BGLs studied have potential for industrial use, specifically the enzymes belonging to the GH1 family, due to its broad substrate specificity and weak inhibition by glucose and other saccharides.
Assuntos
Indústrias , beta-Glucosidase/metabolismo , Análise de Variância , Biocatálise/efeitos dos fármacos , Dissacarídeos/metabolismo , Ácido Edético/farmacologia , Concentração de Íons de Hidrogênio , Hidrólise/efeitos dos fármacos , Cinética , Polissacarídeos/metabolismo , Pyrococcus furiosus/enzimologia , Especificidade por Substrato/efeitos dos fármacos , TemperaturaRESUMO
Multifunctional enzyme engineering can improve enzyme cocktails for emerging biofuel technology. Molecular dynamics through structure-based models (SB) is an effective tool for assessing the tridimensional arrangement of chimeric enzymes as well as for inferring the functional practicability before experimental validation. This study describes the computational design of a bifunctional xylanase-lichenase chimera (XylLich) using the xynA and bglS genes from Bacillus subtilis. In silico analysis of the average solvent accessible surface area (SAS) and the root mean square fluctuation (RMSF) predicted a fully functional chimera, with minor fluctuations and variations along the polypeptide chains. Afterwards, the chimeric enzyme was built by fusing the xynA and bglS genes. XylLich was evaluated through small-angle X-ray scattering (SAXS) experiments, resulting in scattering curves with a very accurate fit to the theoretical protein model. The chimera preserved the biochemical characteristics of the parental enzymes, with the exception of a slight variation in the temperature of operation and the catalytic efficiency (kcat/Km). The absence of substantial shifts in the catalytic mode of operation was also verified. Furthermore, the production of chimeric enzymes could be more profitable than producing a single enzyme separately, based on comparing the recombinant protein production yield and the hydrolytic activity achieved for XylLich with that of the parental enzymes.
Assuntos
Bacillus subtilis/enzimologia , Endo-1,4-beta-Xilanases/química , Glicosídeo Hidrolases/química , Simulação de Dinâmica Molecular , Proteínas Recombinantes de Fusão/química , Simulação por Computador , Endo-1,4-beta-Xilanases/genética , Endo-1,4-beta-Xilanases/metabolismo , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Modelos Moleculares , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Espalhamento a Baixo ÂnguloRESUMO
Cellulases participate in a number of biological events, such as plant cell wall remodelling, nematode parasitism and microbial carbon uptake. Their ability to depolymerize crystalline cellulose is of great biotechnological interest for environmentally compatible production of fuels from lignocellulosic biomass. However, industrial use of cellulases is somewhat limited by both their low catalytic efficiency and stability. In the present study, we conducted a detailed functional and structural characterization of the thermostable BsCel5A (Bacillus subtilis cellulase 5A), which consists of a GH5 (glycoside hydrolase 5) catalytic domain fused to a CBM3 (family 3 carbohydrate-binding module). NMR structural analysis revealed that the Bacillus CBM3 represents a new subfamily, which lacks the classical calcium-binding motif, and variations in NMR frequencies in the presence of cellopentaose showed the importance of polar residues in the carbohydrate interaction. Together with the catalytic domain, the CBM3 forms a large planar surface for cellulose recognition, which conducts the substrate in a proper conformation to the active site and increases enzymatic efficiency. Notably, the manganese ion was demonstrated to have a hyper-stabilizing effect on BsCel5A, and by using deletion constructs and X-ray crystallography we determined that this effect maps to a negatively charged motif located at the opposite face of the catalytic site.