Jacinto Catalán

Molecular cloning of TvDAO1, a gene encoding a D-amino acid oxidase from Trigonopsis variabilis and its expression in Saccharomyces cerevisiae and Kluyveromyces lactis

The DAO1 gene of Trigonopsis variabilis encoding a D‐amino acid oxidase (EC 1.4.3.3) was isolated from genomic clones selected for their specific hybridization to synthetic oligodeoxyribonucleotide probes based on regions of the enzyme that have been conserved through evolution. The nucleotide sequence of the gene predicts a protein with similarities to human, pig, rabbit, mouse and Fusarium solani D‐amino acid oxidases. The open reading frame of the T. variabilis DAO1 gene was interrupted by an intron. The Dao1p sequence displays two regions, one in the N‐terminal section—the FAD binding site—and the other near the C‐terminal region that contains conserved signatures found in all the D‐amino acid oxidases. The three C‐terminal amino acids suggest that the enzyme may be located in peroxisomes. Northern blot experiments showed that no transcriptional activation occurred in the presence of D‐methionine. The cDNA encoding Dao1p was expressed in Saccharomyces cerevisiae and Kluyveromyces lactis. Both yeast species are able to synthesize a functional enzyme under the control of the GAL1 promoter. In K. lactis, up to six times more enzyme units per gram of dry weight are produced with a multicopy plasmid in comparison with the wild‐type strain of T. variabilis. The yeast expression system we describe may constitute an alternative source for the production of D‐amino acid oxidases at industrial level. The sequence presented here has been submitted to the EMBL data library under Accession Number Z50019.

Prediction of kLa in yeast broths

Abstract Oxygen transfer rate, can play an important role in the scale-up and economy of many microbial processes and values of the volumetric oxygen transfer coefficient (kLa) for specific fermentation culture media need to be evaluated and correlated to obtain the appropriate design tools. The purpose of this work was to determinate the values of kLa in yeast broths (Trigonopsis variabilis) in a mechanically-stirred, sparger-aerated and baffled reactor, the most common fermenter type, over a wide range of superficial air velocities impeller rotational speeds and geometric parameters. Three different mixing vessels (2, 5 and 15 litres) were used in order to consider the effect of the fermenter scale-up on kLa. Once kLa data are obtained, several empirical and theoretical correlations were used to fit the experimental data. A new correlation is proposed k L a=3.2·10 −3 P V 0.35 U sg 0.41 based on the power input per unit volume of liquid (P/V) and the superficial gas velocity (Usg). The correlation improves the prediction of kLa values in culture media with respect to other generic correlations, mainly because early correlations were developed for strong coalescent and non-coalescent fluids, whereas the medium used in this work and in most the yeast broths behaves as a typical Newtonian, slightly non-coalescence fluid, due to the moderate presence of mineral salts.

Kinetics and heat-inactivation mechanisms of D-amino acid oxidase

Abstract A study was made of the stability of crude hog kidney d -amino acid oxidase ( d -AAO) under different experimental conditions of temperature, enzyme concentration, buffer, and in the presence of flavine adenine dinucleotide (FAD)_ and glycerol. A deactivation mechanism is proposed. The kinetic deactivation studies were performed in a buffer at pH over a temperature range of 277-327K (4–54°C). The activity for d -AAO at 1 U/ml at 277 K in potassium phosphate and potassium pyrophosphate buffer remains almost constant for 20 days. For d -AAO at 0·02 U/ml at 303 K (30°C) in phosphate buffer, the presence of FAD and glycerol causes the deactivation constant to decrease, while the initial activity and the half-life time increase, the latter doubling on passing from buffer alone to buffer with FAD and 10% glycerol. In the case of d -AAO at 0·2 U/ml, the stability increases considerably with respect to 0·02 U/ml. Both FAD and glycerol enhance this stabilization. For temperatures above 313 K (40°C), the deactivation of d -AAO at 0·2 U/ml showed values of the thermodynamic variables for the overall deactivation constant and for the deactivation constant due to the loss of FAD that indicate that the dissociation of FAD is the main deactivation mechanism. Finally, deactivation due to the loss of FAD is higher for d -AAO at 0·1 U/ml as compared with a concentration of 0·2 U/ml.

Inhibition ofd-amino acid oxidase by α-keto acids analogs of amino acids

The inhibition of D-amino acid oxidase by certain alpha-keto acids products of the reaction with D-amino acids, in particular alpha-keto acids that are analogs of the amino acids alanine, valine, leucine, phenylanaline, phenylglycine, tyrosine and tryptophan, is reported. All the alpha-keto acids assayed behaved as substrate competitive inhibitors of the enzyme. The relationship between the degree of inhibition and the structure of the inhibitor is discussed.

Oxygen Kinetic and Metabolic Parameters for the Yeast Trigonopsis variabilis

The metabolic and kinetic parameters of oxygen consumption by the yeast Trigonopsis variabilis under conditions of induced and non-induced D-amino acid oxidase are described for the first time. These parameters can be calculated from the results of a single batch fermentation running at an oxygen-limiting condition. The values obtained for the parameters may be used as a basis for the calculation of the aeration and stirring requirements and optimal oxygen conditions for the growth of T. variabilis in batch, fed-batch or continuous fermentations.

Inhibition of D-amino Acid Oxidase by α-Keto-γ-Methiolbutyric Acid, product of the reaction with D-Methionine

SummaryThe inhibition of D-Amino Acid Oxidase by α-Keto Acid, product of the reaction with D-Amino Acid is described for the first time. Inhibition of the enzyme by α-Keto-γ-Methiolbutyric Acid (4-methylthio-2-oxobutanoic acid), product of the reaction with D-Methionine, was studied. From the results obtained it is deduced that inhibition is competitive with the substrate, the value of the inhibition constant being 1.85 10−3 M.

PERMEABILIZATION OF TRIGONOPSIS VARIABILIS FOR ENHANCED D-AMINO ACID OXIDASE ACTIVITY

The permeabilization of T. variabilis for use as a whole-cell biocatalyst has been little studied even though it is one of the most important microbial sources of D-Amino Acid Oxidase (DAO) and is useful for the separation of racemic amino acid mixtures, and the production of α-keto acids and 7-amino cephalosporanic acid. In the present work, for the first time a comparative study has been carried out on the efficiency of the permeabilization of the yeast T. variabilis (CBS 4095) grown in a culture medium with D-methionine as an inducer of the synthesis of DAO using different detergents, organic solvents, and freeze-thaw cycles. The best results were obtained with treatment with cetyltrimethylammonium bromide (CTAB), 2% of wet weight of cells, at 45°C for 30 min. The efficiency of the permeabilization procedure was seen to depend on the detergent/cell ratio, being independent of the concentration of detergent in the reaction medium over a broad range: 0.016–0.8% (w/v). The main advantages of the CTAB procedure are the high activity and reproducibility obtained, together with the fact that it can be used with either fresh or frozen cells with almost identical results, unlike the other detergents studied (CHAPS, Triton X-100, Sarcosyl), and especially treatment with toluene-ethanol at 0°C, where a previous sample-freezing step is determinant in the cell permeabilization process. The permeabilization procedure reported here can be used for both accurate in vivo quantification of DAO activity in T. variabilis and to prepare a whole-cell biocatalyst with a high level of activity and stability.

A GENETIC STRUCTURED MODEL IN RELATION TO D-AMINO ACID OXIDASE PRODUCTION IN TRIGONOPSIS VARIABILIS

ABSTRACT In this article, a genetic structured model regarding the synthesis of D-amino acid oxidase (DAO) by the yeast Trigonopsis variabilis growing under batch conditions is proposed for the first time. The basis of the model is a system of coupled ordinary differential equations (ODEs) accounting for the rate of messenger-ribonucleic acid (mRNA) transcription, the rate of DAO translation, and the rates of deactivation of both mRNA and DAO. The present model quantitatively represents the influence of methionine and ammonium on the synthesis of the enzyme. The values of the calculated parameters point to a repression in the transcription phase as responsible for the low levels of DAO observed in cells growing in culture media containing ammonium. The model reasonably explains the experimental data from this work in both the exponential and stationary phases of the cell growth.

A Metabolic Model Describing Growth and Substrate Uptake of Trigonopsis variabilis

Abstract As a part of the study on the production of the enzyme D -Amino acid oxidase (D-AAO) by the yeast Trigonopsis variabilis, a chemically structured model capable of explaining cellular growth and substrate uptake of T. variabilis is presented. The model makes use of a simple sequence of biochemical reactions produced inside the cell and is capable of simulating a range of metabolic responses obtained from the T. variabilis growth in discontinuous culture. The model can serve to understand the behavior of the yeast under different growth conditions and could be used as a basis for the design of an industrial fermentor for D-AAO production. Since some modifications have been introduced to the basic metabolic model to provide even more generality, this new model can be applied to any yeast growing under discontinuous conditions.