Optimization of media composition for D-amino acid oxidase production by Trigonopsis variabilis using biostatistical analysis.

D-amino acid oxidase (DAAO) is biotechnologically relevant enzyme that is used in various food and pharmaceutical industries. DAAO from the yeast Trigonopsis variabilis is an important agent for use in commercial applications because of its high activity with cephalosporin C and is reasonable resistant to the oxidants O2 and H2O2 byproducts of reaction. In this study, response surface methodology (RSM) in shake flask culture was used to enhance the production of DAAO from T. variabilis by optimization of fermentation media composition. The effects of six factors (DL-alanine, glucose, pH, ZnSO4, (NH4)2SO4 and temperature) were evaluated on DAAO production. Results of Placket-Burman design showed that DL-alanine, pH, glucose and ZnSO4 were significant factors for DAAO production (P<0.05). The optimum values of media components as predicted by the central composite design were inducer (DL-alanine) concentration 3 g/L, pH 7.7, glucose 17 g/L and ZnSO4 34 mg/L. At these optimum values of media composition, maximum production of DAAO was 153 U/g yeast dry weight. Two-fold increase in DAAO production was achieved after optimization of the physical parameters by RSM.

Fusion expression of D-amino acid oxidase from Trignoposis variabilis with maltose binding protein and Vitreoscilla hemoglobin / 生物工程学报

D-amino acid oxidase (DAAO) is one of important industrial enzymes. To increase the solubility and activity of the TvDAAO from Trignoposis variabilis expressed in recombinant Escherichia coli (E. coli), a maltose binding protein (MBP) and Vitreoscilla hemoglobin (VHb) was introduced to fuse with N-terminal of the TvDAAO, respectively. Fusion protein of MBP-TvDAAO was constitutively expressed in JM105/pMKC-DAAO and inductively expressed in JM105/pMKL-DAAO. With respect to the control strain of BL21 (DE3)/pET-DAAO without MBP fusion, the constitutive fusion expression obtained 28% of soluble protein with 3.7 folds of solubility improvement. As for the inductive fusion expression, corresponding results changed to 17% and 1.8 folds, respectively. However, the DAAO activity significantly decreased in the MBP-fusing expression. Fusion protein of VHb-TvDAAO was constructed and inductively expressed in BL21 (DE3)/pET-VDAAO. Its DAAO activity highly reached 3.24 u/mL in flask culture, about 90% increase in contrast to the control without VHb.

Effect of peroxisomal proliferators on rat's liver peroxisomal enzymes

Treatment of rodents with peroxisome proliferators as nafinopin [NAF], perflurooctanoic acid [PFOA], 2, 4- Dichlorophenoxyacetic acid [2, 4-D] and 2, 4, 5 trichlorophenoxyacetic acid [2, 4, 5-T] is suggested to be associated with a remarkable increase in peroxisomal beta-oxidation of fatty acids with subsequent marked production of acyl CoA oxidase and other peroxisomal enzymes e.g. amino acid oxidase and glycolate oxidase. The substantial role of these enzymes in increasing H[2]O[2] concentration in cellular steady state could lead to the generation of chemically reactive oxygen species e.g. superoxide anion, singlet oxygen as well as hydroxyl radical, therefore, initiating radical mediated deleterious effects. The present study aimed at estimation of the effect of mentioned peroxisome proliferators on these enzymes activity.Lipid peroxidation was assessed by measuring malondialdehyde liver tissue level. This study was conducted on 80 male Wistar rats classified into five groups, minimum of 15 rats per group. Group I animals received only basal diet [BD] [laboratory rodent diet, a constant nutrition TM formulation]. Groups from II to V, have recieved NAF 0.1%, PFOA 0.02%, 2,4-D 0.05% and 2, 4, 5-T 0.05% which were admixed to BD respectively. Fourteen weeks later animals were sacrificed and their livers were used for biochemical analysis and measurement of enzymes activity. The activity of acyl CoA oxidase was significantly increased by about 50-, 20-, 3- and 2- fold in groups II, III, V and IV respectively. The activity of D-amino acid oxidase showed a significant increase [p < 0.01] only in group V, while the activity of glycolate oxidase was not significantly changed. Malondialdehyde was measured as a predictor of lipid peroxidation and it showed a significant increase [p < 0.05] in groups II and III. Results of this study confirmed the remarkable increase in peroxisomal beta-oxidation of fatty acids as a result of treatment by peroxisomal proliferators

Compared D-amino acid oxidase expression in different Pichia pastoris host strains / 生物工程学报

To compare the DAAO expression level in different Pichia pastoris host strains, the gene encoding DAAO from Trigonopsis variabilis was cloned into plasmid pPIC3.5k and then transformed into P. pastoris GS115 and KM71 respectively. The positive transformants PDK13 (MutS) and PD27 (Mut+) were obtained by PCR analysis. Their optimal and different expression conditions were investigated. To compare with PD27, PDK13 was determined to poss a slower consumption of methanol, a longer induction time, a lower oxygen request and apparently higher expression of DAAO. The highest expression levels were reached up to 2700, 2500 IU/L in shaking flask and 10140, 8463.5 IU/L in fermentor respectively. The over-expression of DAAO can meet its large demand for production of 7-ACA, alpha-keto acid and L-amino acid. In addition, the phenylpyruvate and L-phenylalanine were obtained by crude DAAO reacting with DL-phenylalanine at 37 degrees C for 3h.

Study on in vitro killing activity of DAAO/D-Ala system to K562e cells / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the in vitro killing efficiency of D-amino acid oxidase (DAAO)/D-alanine (D-Ala) system on K562e cells.</p><p><b>METHODS</b>K(DfGC) cell line stably expressing DAAO was obtained by retrovirus transfection technique. The integration and expression of DAAO gene were identified by PCR and in situ hybridization. The killing activities of D-Ala to DAAO(+) cells alone or the mixtures of DAAO(+) and DAAO(-) cells in different ratios were observed. H(2)O(2) production by K(DfGC) cell was measured by phenol red oxidation assay.</p><p><b>RESULTS</b>PCR and in situ hybridization analysis confirmed the integration of DAAO gene in positive clone and its mRNA expression. There was no significant difference in cell proliferation between the two kinds of K562 cells. Ninety percent of K(DfGC) cells was killed by 12.5 mmol/L D-Ala after 24 hour-treatment and the H(2)O(2) levels were in accord with the killing activities of D-Ala. When K(DfGC) was mixed with K562e at different ratio, no significant bystander effect could be found after treating with 15.0 mmol/L D-Ala for 24 hours.</p><p><b>CONCLUSION</b>The leukemia cell line K562e was sensitive to DAAO/D-Ala system and there was no significant bystander effects observed within this cells.</p>

Expression of d-amino acid oxidase gene and green fluorescence protein gene transferred into k562e cells by retroviral vector containing internal ribosome entry site sequence / 中国实验血液学杂志

The internal ribosome entry site (IRES) sequence was derived from encephalomyocarditis virus. It allows to translate two open reading frames at one mRNA, so two genes conjoined by IRES have the same expression rate. K(DfGC) and K(DfGd) cell lines, stably expressing D-amino acid oxidase (DAAO) gene and green fluorescence protein (GFP) genes, were obtained by transfection of K562e cells with retroviral vector pLDfG containing IRES sequence, DAAO cDNA and GFP gene. Fluorescence positive rate and fluorescence intensity of the two cell lines were measured with flow cytometry. H(2)O(2) production by K(DfGC) and K(DfGd) cells treated with D-alanine was measured by the phenol red oxidation assay. The fluorescence positive rate and fluorescence intensity in K(DfGC) and K(DfGd) cell were 94.64% and 96.31% and 202 units and 174 units per 2 x 10(4) cells, respectively. There was exponential correlation between fluorescence intensity and H(2)O(2) level. The above-mentioned results demonstrate that DAAO gene and GFP gene were simultaneously expressed in K562e cell line by the regulation of IRES sequence, and DAAO level was correlated with fluorescence intensity of GFP.

D-amino acid oxidase: its potential in the production of 7-aminocephalosporanic acid.

D-Amino acid oxidase (DAAO) used in the preparation of alpha-keto acids, in the determination of D-amino acids and in the resolution of racemic mixture of amino acids is produced by a wide range of microorganisms. In the recent past this enzyme is being recognized for its potential in the commercial production of 7-aminocephalosporanic acid (7-ACA), a starting material for various semisynthetic cephalosporins. Though this enzyme is widespread among microorganisms, very few microbial species have been explored for the production of 7-ACA; this is because cephalosporin C is quantitatively deaminated by limited microbial DAAOs. Comparison of physico-chemical properties of enzyme preparations indicate wide variations, however in general DAAOs are specific for D-configuration of amino acids. Both immobilized enzyme and cell preparations are developed for its various applications. The advantages of DAAO in the production of 7-ACA are discussed.

Stability of D-amino acid oxidase: denaturation by guanidine hydrochloride and urea.

The guanidine hydrochloride-induced and urea-induced denaturations of swine D-amino acid oxidase (EC 1.4.3.3) dimer were studied by the measurements of the difference absorption spectra in the near-ultra violet region and specific activity. Spectral measurements were made at different pH values (8.3 and 3.2) and temperatures (27, 37 and 47 degrees C). It has been observed that (1), enzyme looses all its activity in concentrated solutions of the denaturants, and (2), the product of urea and guanidine hydrochloride denaturation is only partially unfolded as judged by the measurements of the intrinsic viscosity and exposures of the tyrosyl, tryptophyl and sulphhydryl residues.