Cloning and expression of an endoglucanase gene from the thermotolerant fungus Aspergillus fumigatus DBiNU-1 in Kluyveromyces lactis

Abstract An intronless endoglucanase from thermotolerant Aspergillus fumigatus DBINU-1 was cloned, characterized and expressed in the yeast Kluyveromyces lactis. The full-length open reading frame of the endoglucanase gene from A. fumigatus DBiNU-1, designated Cel7, was 1383 nucleotides in length and encoded a protein of 460 amino acid residues. The predicted molecular weight and the isoelectric point of the A. fumigatus Cel7 gene product were 48.19 kDa and 5.03, respectively. A catalytic domain in the N-terminal region and a fungal type cellulose-binding domain/module in the C-terminal region were detected in the predicted polypeptide sequences. Furthermore, a signal peptide with 20 amino acid residues at the N-terminus was also detected in the deduced amino acid sequences of the endoglucanase from A. fumigatus DBiNU-1. The endoglucanase from A. fumigatus DBiNU-1 was successfully expressed in K. lactis, and the purified recombinant enzyme exhibited its maximum activity at pH 5.0 and 60 °C. The enzyme was very stable in a pH range from 4.0 to 8.0 and a temperature range from 30 to 60 °C. These features make it suitable for application in the paper, biofuel, and other chemical production industries that use cellulosic materials.

Asymmetric bioreduction of ketoesters derivatives by Kluyveromyces marxianus: influence of molecular structure on the conversion and enantiomeric excess

ABSTRACT This study presents the bioreduction of six β-ketoesters by whole cells of Kluyveromyces marxianus and molecular investigation of a series of 13 β-ketoesters by hologram quantitative structure-activity relationship (HQSAR) in order to relate with conversion and enantiomeric excess of β-stereogenic-hydroxyesters obtained by the same methodology. Four of these were obtained as (R)-configuration and two (S)-configuration, among them four compounds exhibited >99% enantiomeric excess. The β-ketoesters series LUMO maps showed that the β-carbon of the ketoester scaffold are exposed to undergo nucleophilic attack, suggesting a more favorable β-carbon side to enzymatic reduction based on adopted molecular conformation at the reaction moment. The HQSAR method was performed on the β-ketoesters derivatives separating them into those provided predominantly (R)- or (S)-β-hydroxyesters. The HQSAR models for both (R)- and (S)-configuration showed high predictive capacity. The HQSAR contribution maps suggest the importance of β-ketoesters scaffold as well as the substituents attached therein to asymmetric reduction, showing a possible influence of the ester group carbonyl position on the molecular conformation in the enzyme catalytic site, exposing a β-carbon side to the bioconversion to (S)- and (R)-enantiomers.

Volatile metabolites produced from agro-industrial wastes by Na-alginate entrapped Kluyveromyces marxianus

Abstract The aim of this study was to evaluate the effects of alginate entrapment on fermentation metabolites of Kluyveromyces marxianus grown in agrowastes that served as the liquid culture media. K. marxianus cells entrapped in Na-alginate were prepared using the traditional liquid-droplet-forming method. Whey and pomaces from processed tomatoes, peppers, and grapes were used as the culture media. The changes in the concentrations of sugar, alcohol, organic acids, and flavor compounds were analyzed using gas chromatography-mass spectrometry (GC-MS) and high pressure liquid chromatography (HPLC). Both free and entrapped, K. marxianus were used individually to metabolize sugars, organic acids, alcohols, and flavor compounds in the tomato, pepper, grape, and acid whey based media. Marked changes in the fermentation behaviors of entrapped and free K. marxianus were observed in each culture. A 1.45-log increase was observed in the cell numbers of free K. marxianus during fermentation. On the contrary, the cell numbers of entrapped K. marxianus remained the same. Both free and entrapped K. marxianus brought about the fermentation of sugars such as glucose, fructose, and lactose in the agrowaste cultures. The highest volume of ethanol was produced by K. marxianus in the whey based media. The concentrations of flavor compounds such as ethyl acetate, isoamyl alcohol, isoamyl acetate, 2-phenylethyl isobutyrate, phenylethyl acetate, and phenylethyl alcohol were higher in fermented agrowaste based media compared to the control.

Thermotolerant and mesophylic fungi from sugarcane bagasse and their prospection for biomass-degrading enzyme production

Nineteen fungi and seven yeast strains were isolated from sugarcane bagasse piles from an alcohol plant located at Brazilian Cerrado and identified up to species level on the basis of the gene sequencing of 5.8S-ITS and 26S ribosomal DNA regions. Four species were identified: Kluyveromyces marxianus, Aspergillus niger, Aspergillus sydowii and Aspergillus fumigatus, and the isolates were screened for the production of key enzymes in the saccharification of lignocellulosic material. Among them, three strains were selected as good producers of hemicellulolitic enzymes: A. niger (SBCM3), A. sydowii (SBCM7) and A. fumigatus (SBC4). The best β-xylosidase producer was A. niger SBCM3 strain. This crude enzyme presented optimal activity at pH 3.5 and 55 °C (141 U/g). For β-glucosidase and xylanase the best producer was A. fumigatus SBC4 strain, whose enzymes presented maximum activity at 60 °C and pH 3.5 (54 U/g) and 4.0 (573 U/g), respectively. All these crude enzymes presented stability around pH 3.0–8.0 and up to 60 °C, which can be very useful in industrial processes that work at high temperatures and low pHs. These enzymes also exhibited moderate tolerance to ethanol and the sugars glucose and xylose. These similar characteristics among these fungal crude enzymes suggest that they can be used synergistically in cocktails in future studies of biomass conversion with potential application in several biotechnological sectors.

Hypocholesterolemic effects of Kluyveromyces marxianus M3 isolated from Tibetan mushrooms on diet-induced hypercholesterolemia in rat

To investigate the effects of Kluyveromyces marxianus M3 isolated from Tibetan mushrooms on diet-induced hypercholesterolemia in rats, female Wistar rats were fed a high-cholesterol diet (HCD) for 28 d to generate hyperlipidemic models. Hyperlipidemic rats were assigned to four groups, which were individually treated with three different dosages of K. marxianus M3+HCD or physiological saline+HCD via oral gavage for 28 d. The total cholesterol (TC), triglycerides (TG), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) levels in the serum and liver of the rats were measured using commercially available enzyme kits. In addition, the liver morphology was also examined using hematoxylin and eosin staining and optical microscopy. According to our results, the serum and liver TC, TG, LDL-C levels and atherogenic index (AI) were significantly decreased in rats orally administered K. marxianus M3 (p <0.01), and the HDL-C levels and anti atherogenic index (AAI) were significantly increased (p <0.01) compared to the control group. Moreover, K. marxianus M3 treatment also reduced the build-up of lipid droplets in the liver and exhibited normal hepatocytes, suggesting a protective effect of K. marxianus M3 in hyperlipidemic rats.

Feasibility of biohydrogen production from industrial wastes using defined microbial co-culture

BACKGROUND: The development of clean or novel alternative energy has become a global trend that will shape the future of energy. In the present study, 3 microbial strains with different oxygen requirements, including Clostridium acetobutylicum ATCC 824, Enterobacter cloacae ATCC 13047 and Kluyveromyces marxianus 15D, were used to construct a hydrogen production system that was composed of a mixed aerobic-facultative anaerobic-anaerobic consortium. The effects of metal ions, organic acids and carbohydrate substrates on this system were analyzed and compared using electrochemical and kinetic assays. It was then tested using small-scale experiments to evaluate its ability to convert starch in 5 L of organic wastewater into hydrogen. For the one-step biohydrogen production experiment, H1 medium (nutrient broth and potato dextrose broth) was mixed directly with GAM broth to generate H2 medium (H1 medium and GAM broth). Finally, Clostridium acetobutylicum ATCC 824, Enterobacter cloacae ATCC 13047 and Kluyveromyces marxianus 15D of three species microbial co-culture to produce hydrogen under anaerobic conditions. For the two-step biohydrogen production experiment, the H1 medium, after cultured the microbial strains Enterobacter cloacae ATCC 13047 and Kluyveromyces marxianus 15D, was centrifuged to remove the microbial cells and then mixed with GAM broth (H2 medium). Afterward, the bacterial strain Clostridium acetobutylicum ATCC 824 was inoculated into the H2 medium to produce hydrogen by anaerobic fermentation. RESULTS: The experimental results demonstrated that the optimum conditions for the small-scale fermentative hydrogen production system were at pH 7.0, 35°C, a mixed medium, including H1 medium and H2 medium with 0.50 mol/L ferrous chloride, 0.50 mol/L magnesium sulfate, 0.50 mol/L potassium chloride, 1% w/v citric acid, 5% w/v fructose and 5% w/v glucose. The overall hydrogen production efficiency in the shake flask fermentation group was 33.7 mL/h-1.L-1, and those the two-step and the one-step processes of the small-scale fermentative hydrogen production system were 41.2 mLVh-1.L-1 and 35.1 mL/h-1.L-1, respectively. CONCLUSION: Therefore, the results indicate that the hydrogen production efficiency of the two-step process is higher than that of the one-step process.

Whole cells in enantioselective reduction of benzyl acetoacetate

The β-ketoester benzyl acetoacetate was enantioselectively reduced to benzyl (S)-3-hydroxybutanoate by seven microorganism species. The best result using free cells was obtained with the yeast Hansenula sp., which furnished 97% ee and 85% of conversion within 24 h. After immobilization in calcium alginate spheres, K.marxianus showed to be more stable after 2 cycles of reaction.

Improvement of SCP production and BOD removal of whey with mixed yeast culture

This research emphasizes on single cell protein (SCP) production and Biochemical Oxygen Demand (BOD) removal from whey with mixed yeast culture. For this purpose, 11 yeast strains were isolated from dairy products (M1-M11) and the strains were identified by morphological and physiological properties. These yeast strains were tested for their ability to reduce the BOD and to produce SCP from whey. Among these strains, K. lactis (M2) had the most SCP production from whey with the yield of 11.79 g/l. Ammonium sulphate as nitrogen source had an increasing effect on biomass yield. The mixed culture of the isolated yeast strains with Saccharomyces cerevisiae was used in order to increase the biomass yield and BOD removal. The highest biomass yield (22.38 g/l) and reduction of initial BOD from 30000 to 3450 mg/l were obtained with the mixed culture of K. lactis (M2) and S. cerevisiae.

Inulin hydrolysis by kluyveromyces marxianus inulase produced on some agricultural wastes

High fructose syrup is produced by the action of inulase on inulin. Many yeast strains were used in this study for inulase production on some agricultural wastes. Results showed that Kluyveromyces marxianus was the most potential strain in inulase production among sixteen yeast strains. K. lactis and K. fragilis were found in the second order. 15 min. was chosen as the best reaction time. PH 5.5 and 6.0 was found as the optimum pH for K. lactis and K. fragilis and K. marxianus, respectively. Enzyme activity was maximal at 35C. K. marxianus inulase was maximized after 3 days of incubation and pH 7.5 at 30C. Biological wastes played an important role in K. marxianus inulase production. Dahlia tubers were the best carbon source for inulase synthesis. Also, chicory roots, orange peel and beet pulp gave high amount of inulase compared to other carbon sources. Ammonium nitrate induced inulase production by K. marxianus which was found as the best nitrogen source between all inorganic and organic ones. The degree of inulin hydrolysis reached 76.50% after 2 hr with 40 units of enzyme in 5% inulin solution. This revealed the importance of this enzyme in the production of fructose syrup from inulin; that industry is very important from healthy and economic viewpoints

Producción de biomasa de Kluyveromices fragilis en suero de leche desproteinizado / Production of Kluyveromices fragilis biomass in deproteinized milk whey

The milk whey from a mature cheese factory deproteinised by acid thermic coagulation (pH 4.5 and 90 degrees C), provides a good culture media for the production of Kluyveromices fragilis biomass. The optimal experimental conditions for the maximal production of biomass were established by using fermenters with different capacity and design. For lactose concentration of 15 g/l, pH 4.5, 30 degrees C and aireation between 0.25 and 1 VVM, the duplication time was below two hours and 98 per cent of the lactose was consumed. The obtained yield in dried weight was between 36 and 49 per cent (g biomass/g lactose). The biomass (without broken cell) contain 46 per cent protein on dry base and showed an "in vitro" digestibility of 65 per cent. The organic mass decreased 80 per cent after 12 hour of fermentation. This process eliminates a polluting agent and simutaneously, produces a biomass that could have industrial use as a protein complement in feeds.

Actividad de beta-galactosidasa de cepas de Kluyveromyces spp. y produccíon de etanol a partir de lactosa / Beta-galactosidase activity of strains of Kluyveromyces spp. and production of ethanol from lactose

Se realizaron estudios sobre levaduras del género Kluyveromyces (K. fragilis 507, K. lactis 29 y K. lactis 10), que crecen en lactosa como única fuente de carbono, ya que poseen el sistema enzimático para la utilización de este azucar. Se determinó la actividad de ß-galactosidasa en medios con glucosa y con lactosa, en cultivos de estas tres cepas en fase log. Al agregar entre ) y 12% v/v de etanol a células tratadas con tolueno, no se observó inhibición enzimática para la cepa K. lactis 10 que tenía la mayor actividad enzimática (704,4 Unidades). Como existe la posibilidad de utilizar industrialmente suero concentrado 4 veces como sustrato que contenga lactosa, se realizaron ensayos de fermentación a 30 C las tres cepas, en medios que contenían inicialmente 16,5 y 24,5% de lactosa. Al cabo de 48 h la lactosa residual fue prácticamente cero, lográndose concentraciones de etanol directo entre 7,60 y 10,10% v/v. Es de esperar que la velocidad de fermentación de un disacárido como lactosa, esté relacionada con la velocidad de hidrólisis del mismo azúcar, por lo que cepas con una mayor velocidad enzimática hidrolítica debieran poseer mayor velocidad de fermentación. En este caso no se observó tal comportamiento, por cuanto cepas con actividad enzimática tan distinta como K. lactis 10 (704,4 U) y K lactis 29(189,7 U) no presentaron mayores diferencias en la producción de etanol a partir de lactosa

Optimización de condiciones para la producción discontínua de proteina unicelular utilizando suero de leche / Optimizing conditions for the batch production of single-cell protein from cheese whey

El suero de quesería es uno de los efluentes de mayor importancia que se desechan en nuestra zona. Por estas razones, se procedió a estudiar la optimización de condiciones referentes al aprovechamiento de este desecho con el objeto de obtener biomasas proteicas en un fermentador de tipo discontinuo. Para ello se propagó una cepa de levadura (Kluyveromyces marxianus var. lactis) en un medio a base de suero de quesería, optimizándose las siguientes condiciones de desarrollo celular: temperatura, pH y concentración inicial de lactosa. Se propuso uma metodología basada en la realización de un conjunto de ensayos ordenados de acuerdo a una estructura de cuadrados latinos, que permite estudiar tres factores simultáneamente con un número reducido de experiencias. Las mismas se realizaron en un fermentador coluniforme de vidrio, lográndose un máximo rendimiento celular (4,78 g/l en peso seco) al trabajar con una concentración inicial de lactosa del 4,8%. Para todos los niveles de temperatura (27-C, 30-C y 35-C) y de pH (3,4 y 5) ensayados, se logró un aumento en el peso seco a medida que se aumentó la concentración inicial de lactosa, desde el 2% al 4,8%. Se aconseja trabajar a una temperatura de 27-C y pH 4, condiciones éstas que minimizan la contaminación bacteriana y con una concentración de lactosa de 4,8% que es la que posee el suero residual de quesería. De este modo se obtiene un rendimiento óptimo en biomasa proteica; lográndose un buen aprovechamiento de este enfluente y disminuyendo a la vez su Demanda Biológica de Oxígeno inicial de 60000 a 15000 ppm