Study on the decrease of renal d-amino acid oxidase activity in the rat after renal ischemia by chiral ligand exchange capillary electrophoresis

d-Amino acid oxidase (DAAO) in mammal kidney regulates the renal reactive oxygen species (ROS) levels directly and plays a leading role in the development of ROS-mediated renal pathologic damages based on its crucial role in the oxidative deamination of d-amino acids and the consequent generation of H₂O₂. Quantitative measurement of DAAO activity in the process of renal ischemia, which could help to understand the molecular mechanisms of this gripping acute renal disease, was conducted through the determination of chiral substrate by capillary electrophoresis (CE) in our study. In this study, a chiral ligand exchange CE method was explored with Zn(II)-l-alaninamide complex as the chiral selector to investigate DAAO activity by determining the decreased concentration of the chiral substrate of DAAO-mediated enzymatic reaction. Then, the change of DAAO activity following 60-min acute renal ischemia in rats was observed with the proposed method. The study showed that the operation of renal ischemia resulted in a 45.49 ± 8.30% (n = 8) decrease in the DAAO-induced consumption of substrate, indicating a sharp decrease in renal DAAO activity following this acute renal injury. This phenomenon, with the possible reason of metabolic acidosis, could pave a new way for the study of oxidative stress in the development of renal ischemia injury.     

Development of an aminotransferase-driven biocatalytic cascade for deracemization of d,l-phosphinothricin

2-oxo-4-[(hydroxy)(methyl)phosphinoyl]butyric acid (PPO) is the essential precursor keto acid for the asymmetric biosynthesis of herbicide l -phosphinothricin (l -PPT). Developing a biocatalytic cascade for PPO production with high efficiency and low cost is highly desired. Herein, a d -amino acid aminotransferase from Bacillus sp. YM-1 (Ym DAAT) with high activity (48.95 U/mg) and affinity (K_m = 27.49 mM) toward d -PPT was evaluated. To circumvent the inhibition of by-product d -glutamate (d -Glu), an amino acceptor (α-ketoglutarate) regeneration cascade was constructed as a recombinant Escherichia coli (E. coli D), by coupling Ym d -AAT, d -aspartate oxidase from Thermomyces dupontii (TdDDO) and catalase from Geobacillus sp. CHB1. Moreover, the regulation of the ribosome binding site was employed to overcome the limiting step of expression toxic protein TdDDO in E. coli BL21(DE3). The aminotransferase-driven whole-cell biocatalytic cascade (E. coli D) showed superior catalytic efficiency for the synthesis of PPO from d ,l -phosphinothricin (d ,l -PPT). It revealed the production of PPO exhibited high space–time yield (2.59 g L⁻¹ h⁻¹) with complete conversion of d -PPT to PPO at high substrate concentration (600 mM d ,l -PPT) in 1.5 L reaction system. This study first provides the synthesis of PPO from d ,l -PPT employing an aminotransferase-driven biocatalytic cascade.     

Triple fusion of d-amino acid oxidase from Trigonopsis variabilis with polyhistidine and Vitreoscilla hemoglobin

Fusion proteins of d-amino acid oxidase from Trigonopsis variabilis (TvDAAO) with Vitreoscilla Hemoglobin (VHb) and (His)₆-tag were constructed and expressed in recombinant Escherichia coli. A fusing-position effect was revealed that (His)₆-tag’s N-terminal fusion with TvDAAO (HDAAO) reduced the specific activity by ~29%, while the C-terminal fusion (DAAOH) remained unreduced. The N-terminal fusion of VHb with TvDAAO and DAAOH significantly improved their activity. As in a 5 l fermentor, the activity of the triple fusion VHb-TvDAAO-(His)₆ (VDAAOH) reached 2.53 U/mg dry cell at 9 h, ~58% increase than that of DAAOH together with ~40% biomass increase, confirming the positive effect of VHb expression on cell level. After purification, the UV–visible and fluorescence spectrum of DAAOH and VDAAOH were characterized. Enzyme kinetics studies further indicated that VDAAOH behaved a higher K _cat, but a weaker substrate affinity of K _m relative to DAAOH, revealing two distinct impacts of VHb-coupling with TvDAAO on protein level.     

Continuous colorimetric screening assay for detection of d-amino acid aminotransferase mutants displaying altered substrate specificity

d-Amino acid aminotransferase (DAAT) catalyzes the synthesis of numerous d-amino acids, making it an attractive biocatalyst for the production of enantiopure d-amino acids. To bolster its biocatalytic applicability, improved variants displaying increased activity toward non-native substrates are desired. Here, we report the development of a high-throughput, colorimetric, continuous coupled enzyme assay for the screening of DAAT mutant libraries that is based on the use of d-amino acid oxidase (DAAO). In this assay, the d-amino acid product of DAAT is oxidized by DAAO with concomitant release of hydrogen peroxide, which is detected colorimetrically by the addition of horseradish peroxidase and o-dianisidine. Using this assay, we measured apparent K_M and k_cat values for DAAT and identified mutants displaying altered substrate specificity via the screening of cell lysates in 96-well plates. The DAAO coupled assay is sensitive in that it allowed the detection of a DAAT mutant displaying an approximately 2000-fold decrease in k_cat/K_M relative to wild type. In addition, the DAAO assay enabled the identification of two DAAT mutants (V33Y and V33G) that are more efficient than wild type at transaminating the non-native acceptor phenylpyruvate. We expect that this assay will be useful for the engineering of additional mutants displaying increased activity toward non-native substrates.     

Kinetic Resolution of 3-Fluoroalanine Using a Fusion Protein of D-Amino Acid Oxidase with Vitroscilla Hemoglobin

In this study, a fusion protein (VHb-DAAO) of D-amino acid oxidase (DAAO) with Vitreoscilla hemoglobin (VHb) was functionally expressed in Escherichia coli and purified. The k _cat value VHb-DAAO (47.1 s^?1) towards rac-3-flouroalanine was about 2-fold higher than that of DAAO (21.9 s^?1). rac-3-Flouroalanine (500 mM) was kinetically resolved into (R)-3-fluoroalanine with high enatiomeric excess (>99%) by VHb-DAAO with about 52% conversion.     

Low modularity of aminoacyl-tRNA substrates in polymerization by the ribosome

Aminoacyl-transfer RNAs contain four standardized units: amino acids, an invariant 3′-terminal CCA, trinucleotide anticodons and tRNA bodies. The degree of interchangeability of the three variable modules is poorly understood, despite its role in evolution and the engineering of translation to incorporate unnatural amino acids. Here, a purified translation system is used to investigate effects of various module swaps on the efficiency of multiple ribosomal incorporations of unnatural aminoacyl-tRNA substrates per peptide product. The yields of products containing three to five adjacent l-amino acids with unnatural side chains are low and cannot be improved by optimization or explained simply by any single factor tested. Though combinations of modules that allow quantitative single unnatural incorporations are found readily, finding combinations that enable efficient synthesis of products containing multiple unnatural amino acids is challenging. This implies that assaying multiple, as opposed to single, incorporations per product is a more stringent assay of substrate activity. The unpredictability of most results illustrates the multifactorial nature of substrate recognition and the value of synthetic biology for testing our understanding of translation. Data indicate that the degree of interchangeability of the modules of aminoacyl-tRNAs is low.     

Properties and applications of microbial D-amino acid oxidases: current state and perspectives

D-Amino acid oxidase (DAAO) is a biotechnologically relevant enzyme that is used in a variety of applications. DAAO is a flavine adenine dinucleotide-containing flavoenzyme that catalyzes the oxidative deamination of D-isomer of uncharged aliphatic, aromatic, and polar amino acids yielding the corresponding imino acid (which hydrolyzes spontaneously to the α-keto acid and ammonia) and hydrogen peroxide. This enzymatic activity is produced by few bacteria and by most eukaryotic organisms. In the past few years, DAAO from mammals has been the subject of a large number of investigations, becoming a model for the dehydrogenase-oxidase class of flavoproteins. However, DAAO from microorganisms show properties that render them more suitable for the biotechnological applications, such as a high level of protein expression (as native and recombinant protein), a high turnover number, and a tight binding of the coenzyme. Some important DAAO-producing microorganisms include Trigonopsis variabilis, Rhodotorula gracilis, and Fusarium solani. The aim of this paper is to provide an overview of the main biotechnological applications of DAAO (ranging from biocatalysis to convert cephalosporin C into 7-amino cephalosporanic acid to gene therapy for tumor treatment) and to illustrate the advantages of using the microbial DAAOs, employing both the native and the improved DAAO variants obtained by enzyme engineering.      

Optimization of culture condition for the production of D-amino acid oxidase in a recombinant Escherichia coli

The gene encoding D-amino acid oxidase (DAAO) from Trigonopsis variabilis CBS 4095 has been cloned and expressed in Escherichia coli BL21 (DE3). Unfortunately, it was observed that the host cell was negatively affected by the expressed DAAO, resulting in a remarkable decrease in cell growth. To overcome this problem, we investigated several factors that affect cell growth rate and DAAO production such as addition time of inducer and dissolved oxygen (DO) concentration. The addition time of lactose, which was used as an inducer, and DO concentration appeared to be critical for the cell growth of E. coli BL21 (DE3)/pET-DAAO. A two-stage DO control strategy was developed, in which the DO concentration was controlled above 50% until specific stage of bacterial growth (OD₆₀₀ 30–40) and then downshifted to 30% by changing the agitation speed and aeration rate, and they remained at these rates until the end of fermentation. With this strategy, the maximum DAAO activity and cell growth reached 18.5 U/mL and OD₆₀₀ 81, respectively. By reproducing these optimized conditions in a 12-m³ fermentor, we were able to produce DAAO at a productivity of 19 U/mL with a cell growth of OD₆₀₀ 80.     

Purification and characterization of an -amino acid deaminase used to prepare unnatural amino acids

-Amino acid deaminase ( -AAD) from Proteus myxofaciens was cloned and over-expressed in Escherichia coli K12. This enzyme has a broad substrate specificity, working on both natural and unnatural -amino acids. Of the 20 naturally occurring -amino acids, -AAD prefers amino acid substrates that have aliphatic, aromatic or sulfur-containing side chains; those with charged side chains (–CO₂⁻ or –NH₃⁺) are poor or non-substrates. Enzyme activity was monitored using a microtiter-plate-based assay, which measures the formation of phenylpyruvic acid from -phenylalanine. The reaction has an absolute requirement for O₂, releases NH₃ and does not produce H₂O₂. Substrate comparisons were carried out by using an O₂ electrode to measure the O₂ utilization rates. Studies on partially purified enzyme show a pH optimum of 7.5 with a subunit molecular weight of approximately 51 kDa. Additional purification and characterization strategies will be presented. The use of whole cells containing -AAD will be discussed to prepare chiral pharmaceutical intermediates.     

Role of the active site residues arginine-216 and arginine-237 in the substrate specificity of mammalian <Emphasis Type="SmallCaps">d</Emphasis>-aspartate oxidase

d-Aspartate oxidase (DDO) and d-amino acid oxidase (DAO) are flavin adenine dinucleotide-containing flavoproteins that catalyze the oxidative deamination of d-amino acids. Unlike DAO, which acts on several neutral and basic d-amino acids, DDO is highly specific for acidic d-amino acids. Based on molecular modeling and simulated annealing docking analyses, a recombinant mouse DDO carrying two substitutions (Arg-216 to Leu and Arg-237 to Tyr) was generated (R216L-R237Y variant). This variant and two previously constructed single-point mutants of mouse DDO (R216L and R237Y variants) were characterized to investigate the role of Arg-216 and Arg-237 in the substrate specificity of mouse DDO. The R216L-R237Y and R216L variants acquired a broad specificity for several neutral and basic d-amino acids, and showed a considerable decrease in activity against acidic d-amino acids. The R237Y variant, however, did not show any additional specificity for neutral or basic d-amino acids and its activity against acidic d-amino acids was greatly reduced. The kinetic properties of these variants indicated that the Arg-216 residue is important for the catalytic activity and substrate specificity of mouse DDO. However, Arg-237 is, apparently, only marginally involved in substrate recognition, but is important for catalytic activity. Notably, the substrate specificity of the R216L-R237Y variant differed significantly from that of the R216L variant, suggesting that Arg-237 has subsidiary effects on substrate specificity. Additional experiments using several DDO and DAO inhibitors also suggested the involvement of Arg-216 in the substrate specificity and catalytic activity of mouse DDO and that Arg-237 is possibly involved in substrate recognition by this enzyme. Collectively, these results indicate that Arg-216 and Arg-237 play crucial and subsidiary role(s), respectively, in the substrate specificity of mouse DDO.     

N-Carbamyl-L-Amino Acid Amidohydrolase of Pseudomonas sp. Strain NS671: Purification and Some Properties of the Enzyme Expressed in Escherichia coli

An N-carbamyl-L-amino acid amidohydrolase was purified from cells of Escherichia coli in which the gene for N-carbamyl-L-amino acid amidohydrolase of Pseudomonas sp. strain NS671 was expressed. The purified enzyme was homogeneous by the criterion of SDS–polyacrvlamide gel electrophoresis. The results of gel filtration chromatography and SDS–polyacrylamide gel electrophoresis suggested that the enzyme was a dimeric protein with 45-kDa identical subunits. The enzyme required Mn²⁺ ion (above 1 mM) for the activity. The optimal pH and temperature were 7.5 and around 40°C, respectively, with N-carbamyl-L-methionine as the substrate. The enzyme activity was inhibited by ATP and was iost completely with p-chloromercuribenzoate (1 mM). The enzyme was strictly L-specific and showed a broad substrate specificity for N-carbamyl-L-α-amino acids.     

In vitro study of the metabolic effects of D-amino acids

While the L -configuration of amino acids predominates in all known living systems, D -enantiomers of amino acids have been detected with highly sensitive chromatographic techniques in human physiological fluids. In the present study, the survival of Chinese hamster ovary cells (CHO) and HeLa cells was inhibited by exposure to high concentrations of some D - or L -amino acids. Inhibition of colony formation, though, was not necessarily observed to be chiral-dependent. Some L -amino acids (L AAS) were found to be toxic while other D -amino acids (D AAS) were innocuous in both cultures. This is contradictory to the previous observations that D AAS were generally considered to be harmful. Frequently it was implied, although not experimentally proven, that the L AAS were not toxic. One of the metabolites produced by oxidative deamination of D - or L AAS is hydrogen peroxide (H₂O₂), a reactive oxygen species (ROS) that is decomposed by catalase. Increased intracellular H₂O₂ can result in peroxidation of lipids. We measured catalase activity and the lipid peroxide levels (LPO) after incubating cells in either D - or L AAS. The amino acids (AAS) that were found to inhibit colony formation were found to be associated with higher levels of catalase activity and LPO. Therefore, we hypothesize that enhanced ROS generation may be, in part, responsible for the observed toxicity of some amino acids. © 1996 Wiley-Liss, Inc.     

Purification and Biochemical Characterization of Indole-3-acetyl-aspartic Acid Synthetase from Immature Seeds of Pea (Pisum sativum)

Indole-3-acetic acid (IAA) amidosynthetases catalyzing the ATP-dependent conjugation of IAA and amino acids play an important role in the maintenance of auxin homeostasis in plant cells. A new amidosynthetase, indole-3-acetic acid:l-aspartic acid ligase (IAA-Asp synthetase) involved in IAA-amino acid biosynthesis, was isolated via a biochemical approach from immature seeds of the pea (Pisum sativum L). The enzyme was purified to homogeneity by a three-step procedure, involving PEG 6000 fractionation, DEAE-Sephacel anion-exchange chromatography, and preparative PAGE, and characterized as a 70-kDa monomeric protein by analytical gel filtration and SDS-PAGE. Rabbit antiserum against recombinant AtGH3.5 cross-reacted with the pea IAA-Asp synthetase, and a single immunoreactive polypeptide band was observed at 70 kDa. The purified enzyme had an apparent isoelectric point at pH 4.7, the highest activity at pH 8.2, preferred Mg²⁺ as a cofactor, and was strongly activated by reducing agents. Similar to known recombinant GH3 enzymes, an IAA-Asp synthetase from pea catalyzes the conjugation of phytohormone acyl substrates to amino acids. The enzyme had the highest synthesizing activity on IAA, followed by 1-NAA, SA, 2,4-D, and IBA, whereas activities on l-Trp, IPA, PAA, (±)JA, and 2-NAA were not significant or not detected. Of 14 amino acids tested, the enzyme had the highest activity on Asp and lower activity on Ala and Lys. Glutamate was found to be a very poor substrate and no conjugating activity was observed on the rest of the amino acids. Steady-state kinetic analysis indicated that IAA and aspartate were preferred substrates for the pea IAA-Asp synthetase. The enzyme exhibited both higher affinities for IAA and Asp (K _m = 0.2 and 2.5 mM, respectively) and catalytic efficiencies (k _cat/K _m = 682,608.7 and 5080 s⁻¹ M⁻¹, respectively) compared with other auxins and amino acids examined. This study describes the first amidosynthetase isolated and purified from plant tissue and provides the foundation for future genetic approaches to explain the role of IAA-Asp in Pisum sativum physiology.     

The cytosolic isoenzyme of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase in Spinacia oleracea and other higher plants: extreme substrate ambiguity and other properties

The cytosolic isoenzyme of 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase (DS-Co: EC 4.1.2.15) in Spinacia oleracea, Solanum tubersosum and many other higher plants was found to use a diversity of substrates. Diose (glycolaldehyde), triose (D-glyceraldehyde, L-glyceraldehyde and DL-glyceraldehyde 3-phosphate), tetrose (D-erythrose, L-erythrose, D-erythrose 4-phosphate, D-threose and L-threose), and pentose (D-ribose 5-phosphate and D-arabinose 5-phosphate) were utilized in combination with phosphoenolpyruvate (PEP) to make the corresponding 2-keto-3-deoxy sugar acids. Glyoxylate was also utilized by DS-Co. Glycoladehyde exhibited the highest reaction velocity when substrates were tested at 3 mM concentrations. Pentoses were poor substrates except when phsophorylated, an effect which is probably due to an increased fraction of the molecules being in the open-chain form. Little stereoselective discrimination exists since comparable velocities were demonstrated with the D and L isomers of glyceraldehyde, erythrose or threose. The enzyme is not a reversible aldolase since pyruvate failed to substitute for PEP. The use of D-erythrose 4-phsophate or glycolaldehyde resulted in K_m values of 1.95 mM and 8.60 mM, respectively. However, glycolaldehyde exhibited the largest V_maxK_m ratio, suggesting a greater catalytic efficiency for this substrate. Glycolaldehyde is an ideal substrate for inexpensive assays of DS-Co that are absolutely selective in the presence of two other plant enzymes which also utilize erythrose 4-phosphate and PEP. The spinach DS-Co enzymes required divalent metals for activity. The presence of 20 mM Mg²⁺, 1 mM Co²⁺ and 1 mM Mn²⁺ yielded relative activities of 100, 70 and 15, respectively. The pH optimum was 9.5 and temperature optimum for activity was 49°C. The molecular masses of DS-Co from spinach, tobacco and pea were all in the range of 400 kDa. The possible roles of DS-Co in biosynthesis of α-ketoglutarate and aromatic amino acids, in biosynthesis of components of cell wall and phytotoxin, and in acting as a sink for 2-and 3-carbon sugars are discussed.     

Investigating the role of active site residues of Rhodotorula gracilis D-amino acid oxidase on its substrate specificity

D-amino acid oxidase (DAAO) is a flavoprotein that catalyzes stereospecifically the oxidative deamination of D-amino acids. The wild-type DAAO is mainly active on neutral D-amino acids, while basic D-amino acids are poor substrates and the acidic ones are virtually not oxidized. To present a comprehensive picture of how the active site residues can modulate the substrate specificity a number of mutants at position M213, Y223, Y238, R285, S335, and Q339 were prepared in the enzyme from the yeast Rhodotorula gracilis. All DAAO mutants have spectral properties similar to those of the wild-type enzyme and are catalytically active, thus excluding an essential role in catalysis; a lower activity on neutral and basic amino acids was observed. Interestingly, an increase in activity and (k(cat)/K(m))(app) ratio on D-aspartate was observed for all the mutants containing an additional charged residue in the active site. The active site of yeast DAAO appears to be a highly evolved scaffold built up through evolution to optimize the oxidative deamination of neutral D-amino acids without limiting its substrate specificity. It is noteworthy, that introduction of a sole, additional, positively charged residue in the active site is sufficient to optimize the reactivity on acidic D-amino acids, giving rise to kinetic properties similar to those of D-aspartate oxidase.     

Evaluation of substrate promiscuity of an L‐carbamoyl amino acid amidohydrolase from Geobacillus stearothermophilus CECT43

N‐carbamoyl‐amino‐acid amidohydrolase (also known as N‐carbamoylase) is the stereospecific enzyme responsible for the chirality of the D ‐ or L ‐amino acid obtained in the “Hydantoinase Process.” This process is based on the dynamic kinetic resolution of D ,L ‐5‐monosubstituted hydantoins. In this work, we have demonstrated the capability of a recombinant L ‐N‐carbamoylase from the thermophilic bacterium Geobacillus stearothermophilus CECT43 (BsLcar) to hydrolyze N‐acetyl and N‐formyl‐L ‐amino acids as well as the known N‐carbamoyl‐L ‐amino acids, thus proving its substrate promiscuity. BsLcar showed faster hydrolysis for N‐formyl‐L ‐amino acids than for N‐carbamoyl and N‐acetyl‐L ‐derivatives, with a catalytic efficiency (k_cat/K_m) of 8.58 × 10⁵, 1.83 × 10⁴, and 1.78 × 10³ (s^?1 M^?1), respectively, for the three precursors of L ‐methionine. Optimum reaction conditions for BsLcar, using the three N‐substituted‐L ‐methionine substrates, were 65°C and pH 7.5. In all three cases, the metal ions Co²⁺, Mn²⁺, and Ni²⁺ greatly enhanced BsLcar activity, whereas metal‐chelating agents inhibited it, showing that BsLcar is a metalloenzyme. The Co²⁺‐dependent activity profile of the enzyme showed no detectable inhibition at high metal ion concentrations. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

An influence of methane concentration on the methanotrophic activity of a model landfill cover

Evaluation of kinetic parameters of methane oxidation under various conditions, on the basis of an analysis of the literature and the authors’ own laboratory research, is presented. Variation in methanotrophic activity in the profile of a simulated landfill cover was observed. The greatest activity was found at a depth of 60 cm. A low affinity (1/K_M) and high potential activity (V_max) were observed. V_max values ranged from 0.11 × 10⁻³ to 0.86 × 10⁻³ units. The values of K_M ranged from 0.6 to 2.9% of CH₄ (v/v).     

Probing active-site residues of pyranose 2-oxidase from Trametes multicolor by semi-rational protein design

D -Tagatose is a sweetener with low caloric and non-glycemic characteristics. It can be produced by an enzymatic oxidation of D -galactose specifically at C2 followed by chemical hydrogenation. Pyranose 2-oxidase (P2Ox) from Trametes multicolor catalyzes the oxidation of many aldopyranoses to their corresponding 2-keto derivatives. Since D -galactose is not the preferred substrate of P2Ox, semi-rational design was employed to improve the catalytic efficiency with this poor substrate. Saturation mutagenesis was applied on all positions in the active site of the enzyme, resulting in a library of mutants, which were screened for improved activity in a 96-well microtiter plate format. Mutants with higher activity than wild-type P2Ox were chosen for further kinetic investigations. Variant V546C was found to show a 2.5-fold increase of k_cat with both D -glucose and D -galactose when oxygen was used as electron acceptor. Because of weak substrate binding, however, k_cat/K_M is lower for both sugar substrates compared to wild-type TmP2Ox. Furthermore, variants at position T169, i.e., T169S and T169N, showed an improvement of the catalytic characteristics of P2Ox with D -galactose. Batch conversion experiments of D -galactose to 2-keto-D -galactose were performed with wild-type TmP2O as well as with variants T169S, T169N, V546C and V546C/T169N to corroborate the kinetic properties determined by Michaelis-Menten kinetics.     

Amino acid uptake in skeletal muscle of rats bearing the Yoshida AH-130 ascites hepatoma

Rats bearing the Yoshida AH-130 ascites hepatoma show decreased activity of neutral amino acid transport in skeletal muscle measuredin vivo as the tissue accumulation of the analogue -amino [1-¹⁴C]isobutyrate (AIB). The decreased accumulation of AIB observed is not merely a consequence of the hypoinsulinaemia present in these animals (as a result of tumour burden) sincein vitro experiments carried out using incubations of isolated soleus muscles also showed a decreased uptake of neutral amino acids. In these preparations the addition of insulin results in similar increases in uptake both in the pair-fed controls and the tumour-bearing animals, thus suggesting similar insulin sensitivities. The decrease in amino acid uptake in soleus muscle is associated with a decrease in the activity of system A, while systems L and ASC show no particular changes as a result of the tumour growth. The kinetic characterisation of system A in the Yoshida-bearing rats shows a decrease in V_max together with a decrease in K_M in relation with the pair-fed animals.     

Single-pot conversion of cephalosporin C to 7-aminocephalosporanic acid in the absence of hydrogen peroxide

In this study, d-amino acid oxidase (DAAO) and catalase (CAT) in the permeabilized recombinant Pichia pastori cells were well investigated. It appeared that their thermal stability was negatively correlated with the apparent enzymatic activities. The frozen-melted cells presented the best stability and the lowest apparent activities of DAAO and CAT, whereas the cetyltrimethylammonium bromide (CTAB) permeabilized cells displayed the weakest stability and the highest apparent activities of the two enzymes. Simultaneous action of DAAO and CAT in the CTAB-permeabilized cells and glutaryl-7-aminocephalosporanic acid acylase (GA) immobilized on carrier contributed to the conversion of cephalosporin C (CPC) to 7-aminocephalosporanic acid (7-ACA) with a yield of 76.2%. During such a reaction cycle, no visible activity loss occurred at the immobilized GA, whereas the loss rates of DAAO and CAT activities were about 0.029 and 1.13 U min⁻¹, respectively. Nevertheless, this problem could be easily solved by continuous feeding of the new permeabilized cell suspension at the rate of 6 ml h⁻¹ to the reactor. Following such a fed-batch strategy, these permeabilized cells and the immobilized GA could be efficiently reused for 6 and 15 reaction cycles, respectively, yielding around 76% 7-ACA at each reaction cycle.