Genetic polymorphisms of glutathione S-transferases and cytochrome P450 enzymes as susceptibility factors to systemic lupus erythematosus in southern Brazilian patients

Systemic lupus erythematosus (SLE) is an autoimmune chronic inflammatory disease that presents several clinical manifestations, affecting multiple organs and systems. Immunological, environmental, hormonal and genetic factors may contribute to disease. Genes and proteins involved in metabolism and detoxification of xenobiotics are often used as susceptibility markers to diseases with environmental risk factors. Cytochrome P450 (CYP) enzymes activate the xenobiotic making it more reactive, while the Glutathione S-transferases (GST) enzymes conjugate the reduced glutathione with electrophilic compounds, facilitating the toxic products excretion. CYP and GST polymorphisms can alter the expression and catalytic activity of enzymes. This study aimed to investigate the role of genetic variants of CYP and GST in susceptibility and clinical expression of SLE, through the analysis of GSTM1 null, GSTT1 null, GSTP1*Ile105Val, CYP1A1*2C and CYP2E1*5B polymorphisms. 371 SLE patients from Hospital de Clínicas de Porto Alegre and 522 healthy blood donors from southern Brazil were evaluated. GSTP1 and CYP variants were genotyped using PCR–RFLP and GSTT1 and GSTM1 variants were analyzed by multiplex PCR. Among European-derived individuals, a lower frequency of GSTP1*Val heterozygous genotypes was found in SLE patients when compared to controls (p = 0.005). In African-derived SLE patients, the CYP2E1*5B allelic frequency was higher in relation to controls (p = 0.054). We did not observe any clinical implication of the CYP and GST polymorphisms in patients with SLE. Our data suggest a protective role of the GSTP1*Ile/Val heterozygous genotype against the SLE in European-derived and a possible influence of the CYP2E1*5B allele in SLE susceptibility among African-derived individuals.     

Characterization and expression of glucosamine-6-phosphate synthase from Saccharomyces cerevisiae in Pichia pastoris

Glucosamine-6-phosphate (GlcN-6-P) synthase from Saccharomyces cerevisiae was expressed in Pichia pastoris SMD1168 GIVING maximum activity of 96 U ml^?1 for the enzyme in the culture medium. By SDS-PAGE, the enzyme, a glycosylated protein, had an apparent molecular mass of 90 kDa. The enzyme was purified by gel exclusion chromatography to near homogeneity, with a 90 % yield and its properties were characterized. Optimal activities were at pH 5.5 and 55 °C, respectively, at which the highest specific activity was 6.8 U mg protein ^?1. The enzyme was stable from pH 4.5 to 5.5 and from 45 to 60 °C. The K_m and V_max of the GlcN-6-P synthase towards d-fructose 6-phosphate were 2.8 mM and 6.9 μmol min^?1 mg^?1, respectively.     

A-ring ortho-specific monohydroxylation of daidzein by cytochrome P450s of Nocardia farcinica IFM10152

The bioconversion of the isoflavonoid daidzein using whole cell Nocardia farcinica IFM10152 showed two kinds of major metabolic modifications, i.e. mono-hydroxylation and subsequent O-methylation. The major hydroxylated products of daidzein prior to the O-methylation reaction were 3',4',7-trihydroxyisoflavone (3'-ODI), 4',6,7-trihydroxyisoflavone (6-ODI) and 4',7,8-trihydroxyisoflavone (8-ODI), which are mono-hydroxylated at the ortho position of each hydroxyl group of daidzein. To identify monooxygenases playing a key role in the monohydroxylation of the A-ring of daidzein, all genes of 27 cytochrome P450s from N. farcinica IFM10152 were cloned and transformed into a E. coli BL21 (DE3) host system. By this enzymatic reaction using the mutants and the genome sequence analysis of N. farcinica IFM10152, it was revealed that nfa12130 and nfa33880 P450 genes clustered with their own ferredoxins and ferredoxin reductases (nfa12140+nfa12150 and nfa338870+nfa33860, respectively) are responsible for the hydroxylation of the A-ring of daidzein, and their major reaction products were 6-ODI and 8-ODI, respectively.     

Purification of an amide hydrolase DamH from Delftia sp. T3-6 and its gene cloning,expression, and biochemical characterization

A highly active amide hydrolase (DamH) was purified from Delftia sp. T3-6 using ammonium sulfate precipitation, diethylaminoethyl anion exchange, hydrophobic interaction chromatography, and Sephadex G-200 gel filtration. The molecular mass of the purified enzyme was estimated to be 32 kDa by sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis. The sequence of the N-terminal 15 amino acid residues was determined to be Gly-Thr-Ser-Pro-Gln-Ser-Asp-Phe-Leu-Arg-Ala-Leu-Phe-Gln-Ser. Based on the N-terminal sequence and results of peptide mass fingerprints, the gene (damH) was cloned by PCR amplification and expressed in Escherichia coli BL21(DE3). DamH was a bifunctional hydrolase showing activity to amide and ester bonds. The specific activities of recombinant DamH were 5,036 U/mg for 2′-methyl-6′-ethyl-2- chloroacetanilide (CMEPA) (amide hydrolase function) and 612 U/mg for 4-nitrophenyl acetate (esterase function). The optimum substrate of DamH was CMEPA, with K _m and k _cat values of 0.197 mM and 2,804.32 s^?1, respectively. DamH could also hydrolyze esters such as 4-nitrophenyl acetate, glycerol tributyrate, and caprolactone. The optimal pH and temperature for recombinant DamH were 6.5 and 35 °C, respectively; the enzyme was activated by Mn²⁺ and inhibited by Cu²⁺, Zn²⁺, Ni²⁺, and Fe²⁺. DamH was inhibited strongly by phenylmethylsulfonyl and SDS and weakly by ethylenediaminetetraacetic acid and dimethyl sulfoxide.     

Anti-apoptosis effect of polysaccharide isolated from the seeds of Cuscuta chinensis Lam on cardiomyocytes in aging rats

To investigate the mechanism of apoptosis in myocardial cells of aging rats induced by d-galactose and to study the effect of the Polysaccharide isolated from the seeds of Cuscuta chinensis Lam (PCCL) on apoptosis of cardiomyocytes and its corresponding machinasim in aging rat model. Fifty male SD rats were randomly divided into 5 groups. Normal control group (NC). d-galactose (100 mg·kg^?1d^?1 for 56 day) indued aging group (MC), d-galactose plus 100 mg kg^?1 d^?1 PCCL group (ML), d-galactose plus 200 mg kg^?1 d^?1 PCCL group (MM), and d-galactose plus 400 mg kg^?1 d^?1 PCCL group (MH). Same volume of solution (water, or PCCL aqueous solution) was given by gavage for 56 days. Then the hearts were collected and apoptosis parameters were evaluated. Caspase-3 and Cyt c were determined by fluorescence spectrometer, the apoptosis rate was assessed by AnnexinV-FITC method by Flow-Cytometry, [Ca²⁺]i and [Ca²⁺]i overloaded by KCL were observed by laser scanning confocal microscopy (LSCM); Bcl-2 and Bax were examined by immunohistochemistry. The content of Cyt C, [Ca²⁺]i of cardiomyocytes, the activity of Caspase-3, Bax expression level in d-galactose induced aging group were higher than NC (p < 0.05). The ratio of Bcl-2/Bax was decreased in d-galactose induced aging group compared to NC. On the other hand, the content of Cyt C, [Ca²⁺]i of cardiomyocytes, the activity of Caspase-3 and apoptosis rate, as well as Bax expression level in all three PCCL groups were decreased compared to galactose induced group (p < 0.05). Bcl-2/Bax ratio was increased in all PCCL groups compared to galactose induced aging group. PCCL could decrease the apoptosis of cardiomyocytes by the mitochondria apoptosis pathway.     

Identification of methylation quantitative trait loci (mQTLs) influencing promoter DNA methylation of alcohol dependence risk genes

Interaction of DNA methylation and sequence variants that are methylation quantitative trait loci (mQTLs) may influence susceptibility to diseases such as alcohol dependence (AD). We used genome-wide genotype data from 268 African Americans (AAs: 129 AD cases and 139 controls) and 143 European Americans (EAs: 129 AD cases and 14 controls) to identify mQTLs that were associated with promoter CpGs in 82 AD risk genes. 282 significant mQTL–CpG pairs (9.9 × 10^?100 ≤ P _nominal ≤ 7.7 × 10^?8) in AAs and 313 significant mQTL–CpG pairs (2.7 × 10^?53 ≤ P _nominal ≤ 9.9 × 10^?8) in EAs were identified [i.e., mQTL–CpG associations survived multiple-testing correction, q values (false discovery rate) ≤ 0.05]. The most significant mQTL was rs1800759, which was strongly associated with CpG cg12011299 in both AAs (P _nominal = 9.9 × 10^?100; q = 6.7 × 10^?91) and EAs (P _nominal = 2.7 × 10^?53; q = 1.4 × 10^?44). Rs1800759 (previously known to be associated to AD) and CpG cg12011299 (distance: 37 bp) are both located in alcohol dehydrogenase (ADH) 4 gene (ADH4) promoter region. In general, the strength of association between mQTLs and CpGs was inversely correlated with the distance between them. Association was also influenced by race and AD. Additionally, 48.3 % of the mQTLs identified in AAs and 65.6 % of the mQTLs identified in EAs were predicted to be expression QTLs. Three mQTLs (rs2173201, rs4147542, and rs4147541 in ADH1B-AHD1C gene cluster region) found in AAs were previously identified by our genome-wide association studies as being significantly associated with AD in AAs. Thus, DNA methylation, which can be influenced by sequence variants and is implicated in gene expression regulation, appears to at least partially underlie the association of genetic variation with AD.     

Bacterial community analysis in chlorpyrifos enrichment cultures via DGGE and use of bacterial consortium for CP biodegradation

The organophosphate pesticide chlorpyrifos (CP) has been used extensively since the 1960s for insect control. However, its toxic effects on mammals and persistence in environment necessitate its removal from contaminated sites, biodegradation studies of CP-degrading microbes are therefore of immense importance. Samples from a Pakistani agricultural soil with an extensive history of CP application were used to prepare enrichment cultures using CP as sole carbon source for bacterial community analysis and isolation of CP metabolizing bacteria. Bacterial community analysis (denaturing gradient gel electrophoresis) revealed that the dominant genera enriched under these conditions were Pseudomonas, Acinetobacter and Stenotrophomonas, along with lower numbers of Sphingomonas, Agrobacterium and Burkholderia. Furthermore, it revealed that members of Bacteroidetes, Firmicutes, α- and γ-Proteobacteria and Actinobacteria were present at initial steps of enrichment whereas β-Proteobacteria appeared in later steps and only Proteobacteria were selected by enrichment culturing. However, when CP-degrading strains were isolated from this enrichment culture, the most active organisms were strains of Acinetobacter calcoaceticus, Pseudomonas mendocina and Pseudomonas aeruginosa. These strains degraded 6–7.4 mg L^?1 day^?1 of CP when cultivated in mineral medium, while the consortium of all four strains degraded 9.2 mg L^?1 day^?1 of CP (100 mg L^?1). Addition of glucose as an additional C source increased the degradation capacity by 8–14 %. After inoculation of contaminated soil with CP (200 mg kg^?1) disappearance rates were 3.83–4.30 mg kg^?1 day^?1 for individual strains and 4.76 mg kg^?1 day^?1 for the consortium. These results indicate that these organisms are involved in the degradation of CP in soil and represent valuable candidates for in situ bioremediation of contaminated soils and waters.     

Clostridium butyricum reduce lipogenesis through bacterial wall components and butyrate

Intervention strategies for obesity are global issues that require immediate attention. The objective of this study was to assess the possibility that Clostridium butyricum and its potential components could reduce lipogenesis. Co-culture experiments of Caco-2 cells and 1?×?10⁶, 1?×?10⁷, and 1?×?10⁸ CFU/ml of C. butyricum were set up to monitor the cytotoxicity of C. butyricum and the changes of angiopoietin-like protein 4 (ANGPTL4) mRNA expression. It was found that cell viability was not affected by C. butyricum, and ANGPTL4 mRNA expression in Caco-2 cells was highly induced by 1?×?10⁷ CFU/ml of C. butyricum. Co-culture experiment of Caco-2 cells and potential components of C. butyricum were set up to monitor any ensuing alteration in ANGPTL4. It was observed that bacterial wall components and potentially secreted factors from C. butyricum could induce ANGPTL4 mRNA expression and protein secretion. To determine whether butyrate could affect the ANGPTL4 production in Caco-2 cells, the role of monocarboxylate transporter 1 (MCT1) in mediating potentially secreted factors from C. butyricum-induced ANGPTL4 production in Caco-2 cells and the effect of 0.1 mM of butyrate on ANGPTL4 production in Caco-2 cells were investigated. It is confirmed that butyrate was the factor secreted by C. butyricum to stimulate ANGPTL4 production. Besides, the soluble factors secreted by live C. butyricum-Caco-2 cells interaction, bacterial wall components-Caco-2 cells interaction, and the main metabolites butyrate-Caco-2 cells interaction reduced lipogenic gene expression in HepG2 cells. In conclusion, 1?×?10⁷ CFU/ml of C. butyricum could reduce lipogenesis through the bacterial wall components and the metabolites such as butyrate.     

Inter-animal radiation as potential heat stressor in lying animals

A model for predicting inter-animal radiant heat exchange in shaded animals is presented, with emphasis on mature cattle. When a cow’s surface temperature is 35 °C, as is common in warmer climates, it loses ～510 Watt m^?2 as radiant heat. Net radiant heat balance depends on radiation coming from bodies in the vicinity. In the 30 °C radiant temperature shaded environment typical of warm climates, net radiant loss from a lactating cow is ～60 Watt m^?2, i.e., 30 % of its ～173 Watt m^?2 heat production. Cows rest for 8–14 h day^?1. The heat exchange of a lying cow differs from that of a standing one: the body center is low and 20–30 % of its surface contacts a surface of relatively low heat conductance. Lying reduces the impact of the surrounding shaded area on heat exchange but increases that of heat radiating from neighboring cows. When a cow rests adjacent to other cows, with 1.25 m between body centers when in stalls, it occupies about 140° of the horizontal plane of view. Heat emitted from the animal’s surface reduces the net radiant heat loss of a resting cow by ～30 Watt m^?2. In contrast, the presence of cows at 5 and 10 m distance, e.g., cows resting on straw in loose yard housing, reduces the net radiant heat loss of the resting cow by 9 and 5 Watt m^?2, respectively. Radiant heat input increases with animal density, which is beneficial in cooler climates, but acts as a stressor in warm climates.     

Estimates of heavy metal tolerance and chromium(VI) reducing ability of Pseudomonas aeruginosa CCTCC AB93066: chromium(VI) toxicity and environmental parameters optimization

The potential role of parameters in the reduction of hexavalent chromium [Cr(VI)] by Pseudomonas aeruginosa is not well documented. In this study, laboratory batch studies were conducted to assess the effect of a variety of factors, e.g., carbon sources, salinity, initial Cr(VI) concentrations, co-existing ions and a metabolic inhibitor, on microbial Cr(VI) reduction to Cr(III) by P. aeruginosa AB93066. Strain AB93066 tolerated up to 400 mg/L of Cr(VI) in nutrient broth medium compared to only 150 mg/L of Cr(VI) in nutrient agar. This bacteria exhibited different levels of resistance against Pb(II) (200 mg/L), Cd(II) (100 mg/L), Ni(II) (100 mg/L), Cu(II) (100 mg/L), Co(II) (50 mg/L) and Hg(II) (5 mg/L). Cr(VI) reduction was significantly promoted by the addition of glucose and glycerine but was strongly inhibited by the presence of methanol and phenol. The rate of Cr(VI) reduction increased with increasing concentrations of Cr(VI) and then decreased at higher concentrations. The presence of Ni(II) stimulated Cr(VI) reduction, while Pb(II), Co(II) and Cd(II) had adverse impact on reduction ability of this strain. Cr(VI) reduction was also inhibited by high levels of NaCl, various concentrations of sodium azide and 20 mM of SO₄ ^2?, MoO₄ ^2?, NO₃ ^?, PO₄ ^3?. No significant relationship was observed between Cr(VI) reduction and redox potential of the culture medium. Scanning electron microscopy showed visible morphological changes in the cells due to chromate stress. Fourier transform infrared spectroscopy analysis revealed chromium species was likely to form complexes with certain functional groups such as carboxyl and amino groups on the surface of P. aeruginosa AB93066. Overall, above results are beneficial to the bioremediation of chromate-polluted industrial wastewaters.     

Effects of metabolic pathway precursors and polydimethylsiloxane (PDMS) on poly-(gamma)-glutamic acid production by Bacillus subtilis BL53

The aims of this study were to evaluate the effects of the addition of metabolic precursors and polydimethylsiloxane (PDMS) as an oxygen carrier to cultures of Bacillus subtilis BL53 during the production of γ-PGA. Kinetics analyses of cultivations of different media showed that B. subtilis BL53 is an exogenous glutamic acid-dependent strain. When the metabolic pathway precursors of γ-PGA synthesis, l-glutamine and a-ketoglutaric acid, were added to the culture medium, production of the biopolymer was increased by 20 % considering the medium without these precursors. The addition of 10 % of the oxygen carrier PDMS to cultures caused a two-fold increase in the volumetric oxygen mass transfer coefficient (k_La), improving γ-PGA production and productivity. Finally, bioreactor cultures of B. subtilis BL53 adopting the combination of optimized medium E, added of glutamine, α-ketoglutaric acid, and PDMS, showed a productivity of 1 g L^?1 h^?1 of g-PGA after only 24 h of cultivation. Results of this study suggest that the use of metabolic pathway precursors glutamine and a-ketolgutaric acid, combined with the addition of PDMS as an oxygen carrier in bioreactors, can improve γ-PGA production and productivity by Bacillus strains .     

Effects of inorganic nanoparticles on viability and catabolic activities of Agrobacterium sp. PH-08 during biodegradation of dibenzofuran

This study investigated the cytotoxicity, genotoxicity, and growth inhibition effects of four different inorganic nanoparticles (NPs) such as aluminum (nAl), iron (nFe), nickel (nNi), and zinc (nZn) on a dibenzofuran (DF) degrading bacterium Agrobacterium sp. PH-08. NP (0–1,000 mg L^?1) -treated bacterial cells were assessed for cytotoxicity, genotoxicity, growth and biodegradation activities at biochemical and molecular levels. In an aqueous system, the bacterial cells treated with nAl, nZn and nNi at 500 mg L^?1 showed significant reduction in cell viability (30–93.6 %, p < 0.05), while nFe had no significant inhibition on bacterial cell viability. In the presence of nAl, nZn and nNi, the cells exhibited elevated levels of reactive oxygen species (ROS), DNA damage and cell death. Furthermore, NP exposure showed significant (p < 0.05) impairment in DF and catechol biodegradation activities. The reduction in DF biodegradation was ranged about 71.7–91.6 % with single NPs treatments while reached up to 96.3 % with a mixture of NPs. Molecular and biochemical investigations also clearly revealed that NP exposure drastically affected the catechol-2,3-dioxygenase activities and its gene (c23o) expression. However, no significant inhibition was observed in nFe treatment. The bacterial extracellular polymeric materials and by-products from DF degradation can be assumed as key factors in diminishing the toxic effects of NPs, especially for nFe. This study clearly demonstrates the impact of single and mixed NPs on the microbial catabolism of xenobiotic-degrading bacteria at biochemical and molecular levels. This is the first study on estimating the impact of mixed NPs on microbial biodegradation.     

Correlation between oxidative stress and G6PD activity in neonatal jaundice

Fetal distress represents a pathophysiological condition in which oxygen is not available to the fetus in sufficient quantities. In cases of glucose 6-phosphate dehydrogenase (G6PD) deficiency, under conditions of oxidative stress, the residual G6PD and complimentary antioxidant mechanisms may become insufficient to neutralize the large amounts of ROS and to prevent severe hemolysis. Alteration in the oxidant–antioxidant profile is also known to occur in neonatal jaundice. The study group included 22 neonates presented with fetal distress during labor and 24 neonates with no evidence of fetal distress (control group). Umbilical cord blood samples were taken immediately after delivery, and the following blood tests were carried out after birth and at discharge from the hospital: erythrocyte count, total bilirubin, G6PD activity, and parameters presenting oxidative status [thiobarbituric acid reactive substances (TBARS), NO, O₂ ^?, H₂O₂, SOD, CAT, O₂ ^?/SOD, and H₂O₂/CAT]. There were no significant differences in TBARS and NO values among neonates with or without fetal distress. However, the values of O₂ ^?, H₂O₂, SOD, O₂ ^?/SOD, and H₂O₂/CAT among neonates born after fetal distress were significantly higher than in neonates without fetal distress (p < 0.01). In neonates with fetal distress, the total number of RBCs at delivery was significantly lower, accompanied with higher bilirubin content. Also neonates with fetal distress had lower activity of G6PD and lower CAT activity. Higher values of oxidative stress parameters in newborns delivered after fetal distress do not indicate strictly what occurred first—oxidative stress or basic lower G6PD activity.     

Effects of Long-Term Treatment with Quercetin on Cognition and Mitochondrial Function in a Mouse Model of Alzheimer’s Disease

Amyloid-β (Aβ)-induced mitochondrial dysfunction has been recognized as a prominent, early event in Alzheimer’s disease (AD). Therefore, therapeutics targeted to improve mitochondrial function could be beneficial. Quercetin, a bioflavanoid, has been reported to have potent neuro-protective effects, but its preventive effects on Aβ-induced mitochondrial dysfunction and cognitive impairment have not been well characterised. Three-month-old APPswe/PS1dE9 transgenic mice were randomly assigned to a vehicle group, two quercetin (either 20 or 40 mg kg^?1 day^?1) groups, or an Aricept (2 mg kg^?1 day^?1) group. After 16 weeks of treatment, we observed beneficial effects of quercetin (40 mg kg^?1 day^?1), including lessening learning and memory deficits, reducing scattered senile plaques, and ameliorating mitochondrial dysfunction, as evidenced by restoration of mitochondrial membrane potential, reactive oxygen species and ATP levels in mitochondria isolated from the hippocampus compared to control. Furthermore, the AMP-activated protein kinase (AMPK) activity significantly increased in the quercetin-treated (40 mg kg^?1 day^?1) group. These findings suggest that a reduction in plaque burden and mitochondrial dysfunction through the activation of AMPK may be one of the mechanisms by which quercetin improves cognitive functioning in the APPswe/PS1dE9 transgenic mouse model of AD.     

Loss of EphB6 protein expression in human colorectal cancer correlates with poor prognosis

Erythropoietin-producing hepatocyte (Eph) receptor family constitutes the largest family of tyrosine kinase receptors in the human genome. Loss of EphB6, a kinase-deficient receptor, correlated with a negative outcome in several carcinomas. This study aimed to investigate the expression of EphB6 protein and mRNA levels in colorectal cancers (CRCs) and possible correlations with clinicopathological variables and prognosis. To assess protein expression level, 124 CRCs and 57 colorectal adenomas samples were examined by immunostaining, the mRNA level of 43 paired CRC and the adjacent normal tissues were detected by using SYBR Green real-time PCR method. Decreased expression of EphB6 protein was found in CRC as compared with adenoma and normal tissues (χ² = 10.146, P = 0.001 and χ² = 45.333, P < 0.001, respectively). Low EphB6 mRNA expression was detected in 83.8 % of cancers with negative or low EphB6 protein expression. The loss of EphB6 protein in CRC was positively associated with poorly differentiation (P < 0.001), lymph node metastasis (P = 0.006), Dukes stage (P = 0.002) and depth of invasion (P = 0.016). The patients with lymph node metastasis had a worse prognosis independently of gender, age, tumor site, stage and differentiation (RR = 0.404, CI 0.267–0.213, P < 0.001). Low levels of EphB6 protein expression are associated with a shorter mean duration of survival in colorectal cancer. Our results demonstrated that EphB6 may represent a novel, useful tissue biomarker for the prediction of survival rate in CRC.     

Expression of the NAD-dependent FDH1 β-subunit from Methylobacterium extorquens AM1 in Escherichia coli and its characterization

The efficient regeneration of nicotinamide cofactors is an important process for industrial applications because of their high cost and stoichiometric requirements. In this study, the FDH1 β-subunit of NAD-dependent formate dehydrogenase from Methylobacterium extorquens AM1 was heterologously expressed in Escherichia coli. It showed water-forming NADH oxidase (NOX-2) activity in the absence of its α-subunit. The β-subunit oxidized NADH and generated NAD⁺. The enzyme showed a low NADH oxidation activity (0.28 U/mg enzyme). To accelerate electron transfer from the enzyme to oxygen, four electron mediators were tested; flavin mononucleotide, flavin adenine dinucleotide, benzyl viologen (BV), and methyl viologen. All tested electron mediators increased enzyme activity; addition of 250 μM BV resulted in the largest increase in enzyme activity (9.98 U/mg enzyme; a 35.6-fold increase compared with that in the absence of an electron mediator). Without the aid of an electron mediator, the enzyme had a substrate-binding affinity for NADH (K _m) of 5.87 μM, a turnover rate (k _cat) of 0.24/sec, and a catalytic efficiency (k _cat/K _m) of 41.31/mM/sec. The addition of 50 μM BV resulted in a 22.75-fold higher turnover rate (k _cat, 5.46/sec) and a 2.64-fold higher catalytic efficiency (k _cat/K _m, 107.75/mM/sec).     

Regioselective hydroxylation of daidzein using P450 (CYP105D7) from Streptomyces avermitilis MA4680

Regiospecific 3′‐hydroxylation reaction of daidzein was performed with CYP105D7 from Streptomyces avermitilis MA4680 expressed in Escherichia coli. The apparent K_m and k_cat values of CYP105D7 for daidzein were 21.83 ± 6.3 µM and 15.01 ± 0.6 min^?1 in the presence of 1 µM of CYP105D7, putidaredoxin (CamB) and putidaredoxin reductase (CamA), respectively. When CYP105D7 was expressed in S. avermitilis MA4680, its cytochrome P450 activity was confirmed by the CO‐difference spectra at 450 nm using the whole cell extract. When the whole‐cell reaction for the 3′‐hydroxylation reaction of daidzein was carried out with 100 µM of daidzein in 100 mM of phosphate buffer (pH 7.5), the recombinant S. avermitilis grown in R2YE media overexpressing CYP105D7 and ferredoxin FdxH (SAV7470) showed a 3.6‐fold higher conversion yield (24%) than the corresponding wild type cell (6.7%). In a 7 L (working volume 3 L) jar fermentor, the recombinants S. avermitilis grown in R2YE media produced 112.5 mg of 7,3′,4′‐trihydroxyisoflavone (i.e., 29.5% conversion yield) from 381 mg of daidzein in 15 h. Biotechnol. Bioeng. 2010. 105: 697–704. © 2009 Wiley Periodicals.