Browsing by Keyword "Applied Microbiology and Biotechnology"
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Item Efficient production of acetoin by fermentation using the newly isolated mutant strain Lactococcus lactis subsp. lactis CML B4(2017-07) Roncal, Tomás; Caballero, Susana; Díaz de Guereñu, María del Mar; Rincón, Inés; Prieto-Fernández, Soraya; Ochoa-Gómez, Jose R.; Tecnalia Research & Innovation; BIOECONOMÍA Y CO2; TECNOLOGÍA DE MEMBRANAS E INTENSIFICACIÓN DE PROCESOS; TECNOLOGÍAS DE HIDRÓGENO; GENERALWith the aim of applying biotechnology to produce acetoin, a chemical that can be used as an aroma and as a building block for other compounds, several putative mutants with reduced lactic acid synthesis were obtained from a wild-type homolactic strain of L. lactis subjected to chemical mutagenesis. Among these mutants, a strain was isolated, CML B4, that showed reduced lactate dehydrogenase (LDH) and increased NADH oxidase (NOX) activities. Shaken flask cultures of this mutant strain mainly produced acetoin, increasing the levels produced compared to the wild-type strain by 15-fold. A point mutation detected in the ldh gene encoding LDH was probably the genetic defect responsible for this phenotype. In pH-controlled aerobic batch fermentation, the CML B4 strain produced more than 40 gL−1 acetoin, which was increased by up to 59 gL−1 in fed-batch fermentations, with yields close to 88 and 74%, respectively, and productivities exceeding 2 gL−1 h−1. These results indicate that this strain could be used industrially as a cell factory for the production of acetoin from bioresources.Item Hydrolysis of animal fats by immobilized Candida rugosa lipase(1994-01) Virto, María D.; Agud, Isabel; Montero, Sol; Blanco, Alicia; Solozabal, Rodolfo; Lascaray, JoséM M.; Llama, María J.; Serra, Juan L.; Landeta, L. Carlos; de Renobales, Mertxe; Centros PRE-FUSION TECNALIA - (FORMER); Tecnalia Research & InnovationLipase (triacylglycerol ester hydrolase, EC 3.1.1.3) from Candida rugosa was immobilized by adsorption on a commercially available microporous polypropylene support of 200- to 400-μm particle size. A contact period of 90 min allowed the highest degrees of hydrolysis to be achieved, particularly in the second and third hydrolysis reactions. The optimal hydrolysis conditions were 0.10 kg enzyme per kilogram fat, 50% (w/v) fat, and 40°C for 24 h. The immobilized enzyme can be repeatedly used and hydrolysis degrees of 90% or higher can be achieved. Of the three animal fats studied, edible pork lard consistently yielded the highest degrees of hydrolysis (95%) in the first hydrolysis reaction and inedible beef tallow the lowest (65%). The immobilized enzyme lost its activity above 45°C. The support could be easily recovered and reused up to 5 times.Item Hydrolysis of animall fats by lipase at temperatures below their melting points(1992-08) de Renobales, Mertxe; Agud, I.; Lascaray, J. M.; Múgica, J. C.; Landeta, L. C.; Solozábal, R.; Centros PRE-FUSION TECNALIA - (FORMER); Tecnalia Research & InnovationWe have studied the hydrolysis of high melting animal fats by the lipase from Candidarugosa at temperatures between 20°C and 37°C without the addition of surfactants or organic solvents. To establish the practical applications of this process we investigated the optimal conditions of the reaction at high substrate concentrations (50% fat w/v) to achieve 95% hydrolysis (or better) in 24 hours. Experiments were conducted in solid emulsions without constant stirring (500 ml total reaction volume). Under all conditions tested, edible pork lard was a better substrate than inedible beef tallow yielding up to 96% hydrolysis with as low as 0.3 g lipase/Kg fat or 98% hydrolysis with 0.5 g lipase/Kg fat. The optimum temperature for the hydrolysis of edible pork lard was around 30°C. Inedible beef tallow and pork lard did not exhibit a clear optimum temperature. Inedible lard gave results intermediate between those of edible lard and inedible beef tallow.Item Kinetic properties of soluble and immobilized Candida rugosa lipase(1995-02) Virto, Mar'ia D.; Agud, ISabel; Montero, Sol; Blanco, Alicia; Solozabal, Rodolfo; Lascaray, Jos'e M.; Llama, Mar'ia J.; Serra, Juan L.; Landeta, L. Carlos; De Renobales, Mertxe; Centros PRE-FUSION TECNALIA - (FORMER); Tecnalia Research & InnovationImmobilized lipase (triacylglycerol ester hydrolase, EC 3.1.1.3) from Candida rugosa has been immobilized on commercially available microporous polypropylene and used for the batch hydrolysis of different animal fats. The effect of the reaction products at concentrations similar to those obtained at 90% hydrolysis, both on soluble and immobilized lipase, was studied. Glycerol showed low inhibitory effect but oleic acid caused 50% inhibition. A mixture of free fatty acids present in the complete hydrolysis of beef tallow inhibited lipase activity more than 70%. The stability of the enzyme (both soluble and immobilized) was highest in the presence of 20% isooctane. The apparent Michaelis constant for each substrate for the soluble enzyme did not change on immobilization.Item Protein hydrolysate from organic fraction of municipal solid waste compost as nitrogen source to produce lactic acid by Lactobacillus fermentum ATCC 9338 and Lactobacillus plantarum NCIMB 8826(2020-01) Izaguirre, Jon Kepa; Dietrich, Thomas; Villarán, M. Carmen; Castañón, Sonia; Alimentación SostenibleIn this work a strategy for obtaining free amino-acids concentrate from an organic fraction of municipal solid waste compost and its use as a nitrogen source for lactic acid production, a compound widely used in different industries, using L. fermentum ATCC 9338 and L. plantarum NCIMB 8826 strains is described. Enzymatic digestion is based on the combined action of endoprotease Alcalase 1.5 MG and exoprotease Flavourzyme 500 MG. The highest degree of hydrolysis obtained under the optimal conditions was 41%. The use of glucanase Viscozyme L prior to protein hydrolysis helped to reduce the viscosity of the solution and promote the action of proteases, increasing its hydrolysis degree by 76%. The hydrolysate contained all 21 amino-acids, making it ideal for lactic acid bacteria growth. During shake flask cultivations the culture media was complemented with glucose as carbon source. Finally, with the hydrolysate, a maximum lactic acid concentration of 9.0 ± 0.2 g·L−1 and 11.1 ± 0.1 g·L−1 for L. fermentum ATCC 9338 and L. plantarum NCIMB 8826 respectively was obtained after 27 h. The innovation of the approach lies in exploiting the overproduction of compost for the production of lactic acid.Item Two-step oxidation of glycerol to glyceric acid catalyzed by the Phanerochaete chrysosporium glyoxal oxidase(2012-02-10) Roncal, Tomás; Muñoz, Carmen; Lorenzo, Leire; Maestro, Belén; Díaz de Guereñu, María del Mar; BIOECONOMÍA Y CO2; TECNOLOGÍA DE MEMBRANAS E INTENSIFICACIÓN DE PROCESOSGlyoxal oxidase of P. chrysosporium is a radical copper oxidase that catalyzes oxidation of aldehydes to carboxylic acids coupled to dioxygen reduction to H 2O 2. In addition to known substrates, glycerol is also found to be a substrate for glyoxal oxidase. During enzyme turnover, glyoxal oxidase undergoes a reversible inactivation, probably caused by loss of the active site free radical, resulting in short-lasting enzyme activities and undetectable substrate conversions. Enzyme activity could be extended by including two additional enzymes, horseradish peroxidase and catalase, in addition to a redox chemical activator, such as Mn(III) (or Mn(II)+H 2O 2) or hexachloroiridate. Using this three-enzyme system glycerol was converted in glyceric acid in a two-step reaction, with glyceraldehyde as intermediate. A possible operation mechanism is proposed in which the three enzymes would work coordinately allowing to maintain a sustained glyoxal oxidase activity. In the course of its catalytic cycle, glyoxal oxidase alternates between two functional and interconvertible reduced and oxidized forms resulting from a two-electron transfer process. However, glyoxal oxidase can also undergo an one-electron reduction to a catalytically inactive form lacking the active site free radical. Horseradish peroxidase could use glyoxal oxidase-generated H 2O 2 to oxidize Mn(II) to Mn(III) which, in turn, would reoxidize and reactivate the inactive form of glyoxal oxidase. Catalase would remove the excess of H 2O 2 generated during the reaction. In spite of the improvement achieved using the three-enzyme system, glyoxal oxidase inactivation still occurred, which resulted in low substrate conversions. Possible causes of inactivation, including end-product inhibition, are discussed.