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.

Enzyme activity of ß-galactosidase from Kluyveromyces lactis and Aspergillus oryzae on simulated conditions of human gastrointestinal system / Atividade de ß-galactosidase de Kluyveromyces lactis e Aspergillus oryzae, em condições simuladas do sistema gastrintestinal humano

An alternative to relieve the symptoms of lactose intolerance is the intake of the enzyme ?-galactosidase in pharmaceutical dosage forms. The ability of ?-galactosidase produced by Kluyveromyces lactis and Aspergillus oryzae to hydrolyze lactose in simulated conditions of the human gastrointestinal tract was investigated. The experiment was carried out in the optimum temperature for each enzyme activity, 40 and 55°C, respectively, and at the normal human body temperature (37°C) at concentrations of 1.5, 3.0, and 5.0 g/L (enzyme from A. oryzae) or mL/L (enzyme from K. lactis). Both enzymes were completely inactivated under simulated gastric conditions (pH 2). When the enzymes were subjected to simulated small intestine conditions (pH 7.4), lactose hydrolysis has occurred, but at 37°C the percentage was lower than that under the optimal temperatures. At concentrations of 1.5, 3.0, and 5.0 mL/L the enzyme from K. lactis hydrolyzed 76.63%, 88.91% and 94.80% of lactose at 40°C, and 55.99%, 80.91% and 81.53% at 37°C, respectively. In contrast, the enzyme from A. oryzae hydrolyzed 7.11%, 16.18% and 21.29% at 55°C, and 8.4%, 11.85% and 16.43% at 37°C. It was observed that under simulated intestinal conditions, the enzyme from K. lactis was more effective on lactose hydrolysis as compared to the enzyme from A. oryzae. Considering the findings of this study, it is extremely necessary to use an enteric coating on ?-galactosidase capsules so that this enzyme is released only in the small intestine, which is its site of action, thus not suffering the action of the stomach pH.(AU)

Uma das alternativas para amenizar os sintomas da intolerância à lactose é a ingestão de ?-galactosidase em formas farmacêuticas. Neste trabalho avaliou-se a capacidade de hidrólise de ?-galactosidase produzida por Kluyveromyces lactis e Aspergillus oryzae simulando as condições do trato gastrintestinal humano. O teste foi realizado nas temperaturas ótimas de ação para cada enzima, 40 e 55°C, respectivamente, e na temperatura corpórea humana (37°C), nas concentrações de 1,5; 3,0 e 5,0 g/L para a enzima de Aspergillus oryzae ou mL/L para a de Kluyveromyces lactis. Na simulação da condição estomacal humana (pH 2), ambas enzimas foram totalmente inativadas. Quando as enzimas foram submetidas às condições simuladas do intestino delgado (pH 7,4), observou-se hidrólise da lactose, porém, a 37°C, a porcentagem foi menor do que a observada nas temperaturas ótimas de cada enzima. A enzima de K. lactis nas concentrações de 1,5; 3,0 e 5,0 mL/L apresentou hidrólise de 76,63%, 88,91% e 94,80% a 40°C e 55,99%, 80,91% e 81,53%, a 37°C, respectivamente. Nas concentrações 1,5; 3,0 e 5,0 g/L, a porcentagem de hidrólise pela enzima de A. oryzae a 55°C foi de 7,11%, 16,18% e 21,29%. Para esta enzima, nessas concentrações, a hidrólise obtida a 37°C foi 8,4%, 11,85% e de 16,43%. Sob condições intestinais simuladas, a enzima de K. lactis apresentou maior eficiência na hidrólise da lactose quando comparada à enzima de A. oryzae. Considerando-se as etapas avaliadas neste estudo, observa-se que é extremamente necessário o uso de um revestimento entérico em cápsulas de ?-galactosidase, para que esta enzima seja liberada somente no intestino delgado, seu local de ação, não sofrendo, portanto, a ação do pH estomacal.(AU)

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.

Producción y caracterización parcial de beta-galactosidasa de Kluyveromyces lactis propagada en suero de leche desproteinizado / Production and partial characterization of beta-galactosidase from Kluyveromyces lactis grown in deproteinized whey

The purpose of this work was to optimize the beta-galactosidase production by Kluyveromyces lactis, applying the Surface Response Methodology (SRM) and using deproteinized whey as fermentation medium. An Orthogonal Central Compound Design (OCCD) was used without repetition, with four factors: temperature, pH, agitation speed and fermentation time. Then, enzyme activity (U/ml) as response variable was used. Thirty trials in twenty-five treatments, with six repetitions at the central point, were carried out, in a New Brunswick Bioflo 2000 fermentor with a volume of 2 liters. The deproteinized whey obtained by thermocoagulation was chemically analyzed. The results were: moisture 93.83 per cent, total solids 6.17 per cent, protein 0.44 per cent, lactose 4.85 per cent, acidity 0.43 per cent and pH 4.58. The best conditions in the enzyme production were: temperature 30.3 degrees C, pH 4.68, agitation speed 191 r.p.m. and fermentation time 18.5 h. with an enzyme production of 8.3 U/ml. The degree of purification obtained was 7.4 times and the yield was 50.8 per cent. The purified enzyme had an optimum temperature of 60 degrees C and a pH of 6.2. This work shows that the yeast Kluyveromyces lactis grown in deproteinized whey is able to produce the enzyme beta-galactosidase and SRM can be used in the fermentology processes, specifically in determining the best suitable operation conditions.

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

UDP-galactose 4-epimerase from Kluyveromyces fragilis: equilibrium unfolding studies.

UDP-galactose 4-epimerase from yeast (Kluyveromyces fragilis) is a homodimer of total molecular mass 150 kDa having possibly one mole of NAD/dimer acting as a cofactor. The molecule could be dissociated and denatured by 8 M urea at pH 7.0 and could be functionally reconstituted after dilution with buffer having extraneous NAD. The unfolded and refolded equilibrium intermediates of the enzyme between 0-8 M urea have been characterized in terms of catalytic activity, NADH like characteristic coenzyme fluorescence, interaction with extrinsic fluorescence probe 1-anilino 8-naphthelene sulphonic acid (ANS), far UV circular dichroism spectra, fluorescence emission spectra of aromatic residues and subunit dissociation. While denaturation monitored by parameters associated with active site region e.g. inactivation and coenzyme fluorescence, were found to be cooperative having delta G between -8.8 to -4.4 kcals/mole, the overall denaturation process in terms of secondary and tertiary structure was however continuous without having a transition point. At 3 M urea a stable dimeric apoenzyme was formed having 65% of native secondary structure which was dissociated to monomer at 6 M urea with 12% of the said structure. The unfolding and refolding pathways involved identical structures except near the final stage of refolding where catalytic activity reappeared.

Purification of microbial (beta)-galactosidase from Kluyveromyces fragilis by bioaffinity partitioning

This work investigated the partitioning of (beta)-galactosidase from "Kluyveromyces fragilis" in aqueous two-phase systems (ATPS) by bioaffinity. PEG 4000 was chemically activated with thresyl chloride, and the biospecific ligand p-aminophenyl 1-thio-(beta)-D-galactopyranoside (APGP) was attached to the activated PEG 4000. A new two-step method for extraction and purification of the enzyme (beta)-galactosidase from "Kluyveromyces fragilis" was developed. In the first step, a system composed of 6(per cent) PEG 4000-APGP and 8(per cent) dextran 505 was used, where (beta)-galactosidase was strongly partitioned to the top phase (K = 2.330). In the second step, a system formed of 13(per cent) Peg-APGP and 9(per cent) phosphate salt was used to revert the value of the partition coefficient of (beta)-galactosidase (K = 2.0E-5) in order to provide the purification and recovery of 39(per cent) of the enzyme in the bottom salt-rich phase

Microenvironment at the substrate binding subsite of the active site of UDPglucose 4-epimerase from Kluyveromyces fragilis using a fluorescent analog of UMP.

A chromophorics and fluorescent analog of uridine 5'-monophosphate (UMP), a known competitive inhibitor of UDPglucose 4-epimerase was synthesised. This analog, namely 2',3'-O-(2,4,6-trinitrocyclohexadienylidene) uridine 5'-monophosphate, was found to be a powerful reversible inhibitor of UDPglucose 4-epimerase indicating its interaction with the substrate binding site of the enzyme. The extreme sensitivity of the fluorescence emission spectrum of this analog to solvent polarity makes it an excellent probe for the study of the environment at the active site of the enzyme. We report here the effective use of this UMP analog to demonstrate that the hydroxyl groups of the ribose moiety of UMP and presumably the substrates (UDPgalactose and UDPglucose) do not reside in a hydrophobic milieu.

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

Treatments for B-glucosidase release from yeast cells

Among various treatments tested for releasing active beta-glucosidase in a good yield from Kfragilis NRRL Y-1137 cells, sonication was the most efficient method followed in descending order by organic solvents, homogenization breakage and freezing and thawingOptimum activity of the crude beta-glucosidase was obtained at 50C for 10 min at pH 6.2In addition, 0.05 Mof potassium phosphate was the most favorable buffer for beta-glucosidase activity