Expression and characterization of a novel ß-1,4-endoglucanase from Bacillus subtilis strain isolated from a pulp and paper mill wastewater.

The production of fermentable sugars from lignocellulosic biomass is achieved by the synergistic action of a group of enzymes called cellulases. Cellulose is a long chain of chemically linked glucoses by ß-1,4 bonds. The enzyme ß-1,4-endoglucanase is the first cellulase involved in the degradation, breaking the bond of the amorphous regions. A ß-1,4-endoglucanase enzyme with high activity was obtained from a Bacillus subtilis strain isolated from wastewater of a pulp and paper mill. Sequencing and bioinformatic analysis showed that the gene amplified by PCR consisting of 1407 nucleotides and coding for a ß-1,4-endoglucanase enzyme of approximately 55 kDa. The open reading frame (ORF) encoding the mature endoglucanase (eglS) was successfully inserted in a modified cloning plasmid (pITD03) and into the pYD1 plasmid used for its expression in yeast. Carboxymethylcellulose (CMC) plate assay, SDS-PAGE, and zymogram confirmed the production and secretion by the transformed E. coli BL21-SI strain of a 39 kDa ß-1,4-endoglucanase consistent with the catalytic domain without the cellulose-binding module (CBM). The results showed that the truncated ß-1,4-endoglucanase had higher activity and stability.

Gradually supply of isoamyl alcohol increases the isoamyl acetate production in solid-state fermentation.

Nowadays, isoamyl acetate production is carried out by chemical synthesis with a recent interest in developing biological producing processes, mainly based on microorganisms in submerged fermentation. This work assayed producing isoamyl acetate through solid-state fermentation (SSF), feeding the precursor in the gas phase. Polyurethane foam functioned as the inert support to contain 20 ml of a solution of molasses (10% w/v, pH 5.0). The yeast Pichia fermentans was inoculated at 3 × 107 cells per gram of initial dry weight. The airstream to supply oxygen also served to supply the precursor. Slow supply was obtained using an isoamyl alcohol solution of 5 g l-1 in the bubbling columns and an air stream of 50 ml min-1. For fast supply, fermentations were aerated using 10 g l-1 and 100 ml min-1 for isoamyl alcohol solution and air stream, respectively. It demonstrated the feasibility of isoamyl acetate production in SSF. Moreover, the slow supply of the precursor increased isoamyl acetate production up to 390 mg l-1, which is 12.5 times higher than that obtained without precursor (32 mg l-1). On the other hand, fast supply caused an evident inhibition of the growth and production capacity of the yeast.

Improvement in the Thermostability of a Recombinant ß-Glucosidase Immobilized in Zeolite under Different Conditions.

ß-Glucosidase is part of the cellulases and is responsible for degrading cellobiose into glucose, a compound that can be used to produce biofuels. However, the use of the free enzyme makes the process more expensive. Enzyme immobilization improves catalytic characteristics and supports, such as zeolites, which have physical-chemical characteristics and ion exchange capacity that have a promising application in the biotechnological industry. This research aimed to immobilize by adsorption a recombinant ß-glucosidase from Trichoderma reesei, obtained in Escherichia coli BL21 (DE3), in a commercial zeolite. A Box Behnken statistical design was applied to find the optimal immobilization parameters, the stability against pH and temperature was determined, and the immobilized enzyme was characterized by SEM. The highest enzymatic activity was determined with 100 mg of zeolite at 35 °C and 175 min. Compared to the free enzyme, the immobilized recombinant ß-glucosidase presented greater activity from pH 2 to 4 and greater thermostability. The kinetic parameters were calculated, and a lower KM value was obtained for the immobilized enzyme compared to the free enzyme. The obtained immobilization parameters by a simple adsorption method and the significant operational stability indicate promising applications in different fields.

Expression of a ß-glucosidase from Trichoderma reesei in Escherichia coli using a synthetic optimized gene and stability improvements by immobilization using magnetite nano-support.

The enzymatic conversion of lignocellulosic biomass to fermentable sugars is determined by the enzymatic activity of cellulases; consequently, improving enzymatic activity has attracted great interest in the scientific community. Cocktails of commercial cellulase often have low ß-glucosidase content, leading to the accumulation of cellobiose. This accumulation inhibits the activity of the cellulolytic complex and can be used to determine the enzymatic efficiency of commercial cellulase cocktails. Here, a novel codon optimized ß-glucosidase gene (B-glusy) from Trichoderma reesei QM6a was cloned and expressed in three strains of Escherichia coli (E. coli). The synthetic sequence containing an open reading frame (ORF) of 1491 bp was used to encode a polypeptide of 497 amino acid residues. The ß-glucosidase recombinant protein that was expressed (57 kDa of molecular weight) was purified by Ni agarose affinity chromatography and visualized by SDS-PAGE. The recombinant protein was better expressed in E. coli BL21 (DE3), and its enzymatic activity was higher at neutral pH and 30 °C (22.4 U/mg). Subsequently, the ß-glucosidase was immobilized using magnetite nano-support, after which it maintained >65% of its enzymatic activity from pH 6 to 10, and was more stable than the free enzyme above 40 °C. The maximum immobilization yield had enzyme activity of 97.2%. In conclusion, ß-glucosidase is efficiently expressed in the microbial strain E. coli BL21 (DE3) grown in a simplified culture medium.

Study of the Enzymatic Capacity of Kluyveromyces marxianus for the Synthesis of Esters.

Recently, biotechnological opportunities have been found in non-Saccharomyces yeasts because they possess metabolic characteristics that lead to the production of compounds of interest. It has been observed that Kluyveromyces marxianus has a great potential in the production of esters, which are aromatic compounds of industrial importance. The genetic bases that govern the synthesis of esters include a large group of enzymes, among which the most important are alcohol acetyl transferases (AATases) and esterases (AEATases), and it is known that some are present in K. marxianus, because it has genetic characteristics like S. cerevisiae. It also has a physiology suitable for biotechnological use since it is the eukaryotic microorganism with the fastest growth rate and has a wide range of thermotolerance with respect to other yeasts. In this work, the enzymatic background of K. marxianus involved in the synthesis of esters is analyzed, based on the sequences reported in the NCBI database.

Stabilization of dimeric ß-glucosidase from Aspergillus niger via glutaraldehyde immobilization under different conditions.

The dimeric enzyme ß-glucosidase from Aspergillus niger has been immobilized on different amino-agarose beads at pH 5 and 7, exploiting the versatility of glutaraldehyde. The stability of the free enzyme depended on enzyme concentration. Immobilization via ion exchange improved enzyme stability/activity, depending on the immobilization pH. However, the enzyme was desorbed in 75 mM NaCl at pH 7 and some stability/enzyme concentration dependence still existed. TREATMENT: of these biocatalysts with glutaraldehyde increased enzyme stability (e.g. at pH 5, after incubation under conditions where the enzyme just ionically exchanged was fully inactivated, the activity of the glutaraldehyde treated enzyme remained unaltered). Immobilization on glutaraldehyde pre-activated supports yielded a higher increase in enzyme activity, but the stabilization was lower. While when measuring the enzyme activity at pH 4 there were no changes after immobilization, all immobilized enzymes were more active than the free enzyme at pH 6 and 7 (2-3 times). The Ki/Km ratio did not significantly decrease in any immobilized biocatalysts, and in some cases it worsened in a significant way (by a 9 fold factor using preactivated supports). The new biocatalysts are significantly more stable and avoid enzyme subunit desorption, being the immobilization pH a key point in their design.

Comparison of five pretreatments for the production of fermentable sugars obtained from Pinus pseudostrobus L. wood.

To benefit from the use of a waste product such as pine sawdust from a sawmill in Michoacán, Mexico, five different pretreatments for the production of reducing sugars by enzymatic hydrolysis were evaluated (sodium hydroxide, sulfuric acid, steam explosion, organosolv and combined method nitric acid / sodium hydroxide). The main finding of the study was that the pretreatment with 6 % HNO3 and 1 % NaOH led to better yields than those obtained with sodium hydroxide, dilute sulfuric acid, steam explosion, and organosolv pretreatments. Also, HNO3 yields were maximized by the factorial method. With those results the maxima concentration of reducing sugar found was 97.83 ± 1.59, obtained after pretreatment with 7.5 % HNO3 at 120 °C for 30 minutes; followed by 1 % of NaOH at 90 °C for 30 minutes at pH 4.5 for 168 hours with a load enzyme of 25 FPU/g of total carbohydrates. Comparing the results obtained by the authors with those reported in the literature, the combined method was found to be suitable for use in the exploitation of sawdust.

Study of the rheological properties of a fermentation broth of the fungus Beauveria bassiana in a bioreactor under different hydrodynamic conditions.

Fermentation with filamentous fungi in a bioreactor is a complex dynamic process that is affected by flow conditions and the evolution of the rheological properties of the medium. These properties are mainly affected by the biomass concentration and the morphology of the fungus. In this work, the rheological properties of a fermentation with the fungus Beauveria bassiana under different hydrodynamic conditions were studied and the rheological behavior of this broth was simulated through a mixture of carboxymethyl cellulose sodium and cellulose fibers (CMCNa-SF). The bioreactor was a 10 L CSTR tank operated at different stir velocities. Rheological results were similar at 100 and 300 rpm for both systems. However, there was a significant increase in the viscosity accompanied by a change in the consistence index, calculated according to the power law model, for both systems at 800 rpm. The systems exhibited shear-thinning behavior at all stir velocities, which was determined with the power law model. The mixing time was observed to increase as the cellulose content in the system increased and, consequently, the efficiency of mixing diminished. These results are thought to be due to the rheological and morphological similarities of the two fungal systems. These results will help in the optimization of scale-up production of these fungi.

Effect of solid substrate fermentation on the nutritional quality of agro-industrial residues

El objetivo de este trabajo fue evaluar la composición química y la producción de gas in vitro de cinco residuos agro-industriales, antes y después de aplicar un proceso de fermentaci¢n en medio sólido. Paja de avena, paja de haba, rastrojo de maíz, bagaso de manzana y bagaso de agave fueron tratados usando dos hongos filamentosos, Trichoderma harzianum y Phanaerochaete chrysosporium. Los análisis químicos realizados fueron: determinación de proteína cruda, fibra detergente neutro, fibra detergente ácido, lignina, celulosa y hemicelulosa. Para la determinación de la producción de gas in vitro se utilizaron jeringuas de cristal calibradas, mientras que el perfil de ácidos grasos volátiles (AGV's) fue obtenido por cromatografía de gases. Los datos de producción de gas in vitro fueron ajustados usando las ecuaciones p=a+b(1-e-ct) propuesta por Orskov y McDonald y G=A/(1+[B/t]) propuesta por France et al (2000). Se observaron aumentos en el contenido de proteína cruda (P<0,05) al tratar paja de avena (20 por ciento), paja de haba (83 por ciento) y rastrojo de maíz (16 por ciento) con P. chrysosporium. El contenido de fibra detergente neutro fue reducido un 9,5 por ciento en paja de avena por los tratamientos (P<0,05). Con respecto a los parámetros de la producción del gas, los valores de b y c en bagaso de agave fueron mayores (57,4 por ciento y 7,5 por ciento, respectivamente) en el substrato tratado con P. chrysosporium en relación con otros tratamientos. De manera similar, valores más altos para el parámetro de A fueron observados en rastrojo de maíz (+22 por ciento) y bagaso de agave (+13 por ciento) tratados con P. chrysosporium. A pesar de estas diferencias aisladas, el análisis realizado demostró que la composición química, principalmente carbohidratos estructurales, no fue modificada bajo las condiciones de este estudio.