Conversion of ammonia or urea into essential amino acids, L-leucine, L-valine, and L-isoleucine, using artificial cells containing an immobilized multienzyme system and dextran-NAD+. 2. Yeast alcohol dehydrogenase for coenzyme recycling.

Semipermeable nylon-polyethylenimine artificial cells containing leucine dehydrogenase (EC 1.4.1.9), alcohol dehydrogenase (EC 1.1.1.1), urease (EC 3.5.1.5), and dextran-NAD+ were prepared. Artificial cells could convert ammonia or urea into L-leucine, L-valine, and L-isoleucine. For batch conversion in 20.0 mM of ammonium acetate substrate solutions, in 2 h 0.2 ml of artificial cells could produce 4.48 mumol of L-leucine, 9.98 mumol of L-valine, or 5.96 mumol of L-isoleucine. The corresponding conversion ratios were 22.4, 49.9, and 29.8%. In 20.0 mM of urea substrate solutions, 13.71 mumol of L-leucine, 16.12 mumol of L-valine, or 13.44 mumol of L-isoleucine was produced and the conversion ratios were 68.6, 80.6, and 67.2%. The substrate specificity of leucine dehydrogenase for the reductive amination was determined. Of the three branched-chain amino acids produced, the production rates of L-valine were the highest. The apparent Km values were as follows: 0.32 mM for alpha-ketoisocaproate, 1.63 mM for alpha-ketoisovalerate, and 0.73 mM for Dl-alpha-keto-beta-methyl-n-valerate. The leucine dehydrogenase multienzyme system had a good storage stability. It retained 72.0% of the original activity with artificial cells were stored at 4 degrees C for 6 weeks. The optimum conversion pH and temperature were 8.5-9.0 and 35-40 degrees C. The effects of urea and ammonium salts on conversion rate were also studied. The relative activities in ammonium salts solutions were 45.1-75.9% of those in urea solutions.     

Regulation of lysine and threonine synthesis in carrot cell suspension cultures and whole carrot roots

Aspartokinase (EC 2.7.2.4), homoserine-dehydrogenase (EC 1.1.1.3) and dihydrodipicolinic-acid-synthase (EC 4.2.1.52) activities were examined in extracts from 1-year-old and 11-year-old cell suspension cultures and whole roots of garden carrot (Daucus carota L.). Aspartokinase activity from suspension cultures was inhibited 85% by 10 mM L-lysine and 15% by 10mM L-threonine. In contrast, aspartokinase activity from whole roots was inhibited 45% by 10 mM lysine and 55% by 10 mM threonine. This difference may be based upon alterations in the ratios of the two forms (lysine-and threonine-sensitive) of aspartokinase, since the activity is consistently inhibited 100% by lysine+threonine. Only one form each of homoserine dehydrogenase and of dihydrodipicolinic acid synthase was found in extracts from cell suspension cultures and whole roots. The regulatory properties of either enzyme were identical from the two sources. In both the direction of homoserine formation and aspartic--semialdehyde formation, homoserine dehydrogenase activities were inhibited by 10mM threonine and 10 mM L-cysteine in the presence of NADH or NADPH. KCl increased homoserine dehydrogenase activity to 185% of control values and increased the inhibitory effect of threonine. Dihydrodipicolinic acid synthase activities from both sources were inhibited over 80% by 0.5 mM lysine. Aspartokinase was less sensitive to inhibition by low concentrations of lysine and threonine than were dihydrodipicolinic acid synthase and homoserine dehydrogenase to inhibition by the respective inhibitors.     

Production of essential L-branched-chain amino acids in bioreactors containing artificial cells immobilized multienzyme systems and dextran-NAD+

We prepared artificial cells each containing leucine dehydrogenase (EC 1.4.1.9), urease (EC 3.5.1.5), soluble dextran-NAD(+), and one of the following coenzyme regenerating dehydrogenases: glucose dehydrogenase (EC 1.1.1.47); yeast alcohol dehydrogenase (EC 1.1.1.1); malate dehydrogenase (EC 1.1.1.37); or lactate dehydrogenase (EC 1.1.1.27). Artificial cells were packed in small columns. L-Leucine, L-valine, and L-isoleucine were continuously produced with simultaneous dextran-NADH regeneration. The maximum production ratios depended on the coenzyme regenerating systems used: 83-93% for D-glucose and glucose dehydrogenase system; 90% for ethanol and yeast alcohol dehydrogenase system; 45-55% for L-malate and malate dehydrogenase system; and 64-78% for L-lactate and lactate dehydrogenase system. Kinetic experiments were also carried out. The apparent K(m) values are as follows: 0.33 mM for alpha-ketoisocaproate (KIC); 0.51 mM for alpha-ketoisovalerate (KIV); 0.58 mM for DL-alpha-keto-beta-methyl-n-valerate (KMV); 3.52 mM for urea; 27.82 mM for D-glucose; 3.89 mM for ethanol; 3.02 mM for L-malate; and 16.67 mM for L-lactate. Kinetic analysis showed that KIC, KIV, and KMV were all competitive inhibitors in the reactions catalyzed by leucine dehydrogenase. Their inhibitor constants were the corresponding K(m) values.     

酶法转化L-谷氨酸生产a-酮戊二酸

利用L-谷氨酸氧化酶(LGOX),对酶法转化L-谷氨酸生产α-酮戊二酸(α-KG)的工艺条件进行了研究。首先对野生菌链霉菌Streptomyces sp.FMME066进行亚硝基胍诱变,获得一株遗传性状稳定的突变株Streptomyces sp.FMME067;突变株在最优培养基(g/L):果糖10,蛋白胨7.5,KH2PO4 1,CaCl2 0.05条件下,LGOX酶活为0.14 U/mL。LGOX的生化特征为最适pH 8.5、温度35℃,Mn2+是激活剂。对LGOX转化L-谷氨酸生产α-KG的条件进行优化,在最优条件下转化24 h,α-KG产量为38.1 g/L,转化率为81.4%。研究结果为开发LGOX酶法转化生产α-KG的工业化奠定了坚实的基础。     

Activity of enzymes of carbon metabolism during the induction of Crassulacean acid metabolism in Mesembryanthemum crystallinum L.

The maximum extractable activities of twenty-one photosynthetic and glycolytic enzymes were measured in mature leaves of Mesembryanthemum crystallinum plants, grown under a 12 h light 12 h dark photoperiod, exhibiting photosynthetic characteristics of either a C₃ or a Crassulacean acid metabolism (CAM) plant. Following the change from C₃ photosynthesis to CAM in response to an increase in the salinity of in the rooting medium from 100 mM to 400 mM NaCl, the activity of phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31) increased about 45-fold and the activities of NADP malic enzyme (EC 1.1.1.40) and NAD malic enzyme (EC 1.1.1.38) increased about 4- to 10-fold. Pyruvate, Pi dikinase (EC 2.7.9.1) was not detected in the non-CAM tissue but was present in the CAM tissue; PEP carboxykinase (EC 4.1.1.32) was detected in neither tissue. The induction of CAM was also accompanied by large increases in the activities of the glycolytic enzymes enolase (EC 4.2.1.11), phosphoglyceromutase (EC 2.7.5.3), phosphoglycerate kinase (EC 2.7.2.3), NAD glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12), and glucosephosphate isomerase (EC 2.6.1.2). There were 1.5- to 2-fold increases in the activities of NAD malate dehydrogenase (EC 1.1.1.37), alanine and aspartate aminotransferases (EC 2.6.1.2 and 2.6.1.1 respectively) and NADP glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.13). The activities of ribulose-1,5-bisphosphate (RuBP) carboxylase (EC 4.1.1.39), fructose-1,6-bisphosphatase (EC 3.1.3.11), phosphofructokinase (EC 2.7.1.11), hexokinase (EC 2.7.1.2) and glucose-6-phosphate dehydrogenase (EC 1.1.1.49) remained relatively constant. NADP malate dehydrogenase (EC 1.1.1.82) activity exhibited two pH optima in the non-CAM tissue, one at pH 6.0 and a second at pH 8.0. The activity at pH 8.0 increased as CAM was induced. With the exceptions of hexokinase and glucose-6-phosphate dehydrogenase, the activities of all enzymes examined in extracts from M. crystallinum exhibiting CAM were equal to, or greater than, those required to sustain the maximum rates of carbon flow during acidification and deacidification observed in vivo. There was no day-night variation in the maximum extractable activities of phosphoenolpyruvate carboxylase, NADP malic enzyme, NAD malic enzyme, fructose-1,6-bisphosphatase and NADP malate dehydrogenase in leaves of M. crystallinum undergoing CAM.Abbreviations CAM Crassulacean acid metabolism - PEP phosphoenolpyruvate - RuBP ribulose-1,5-bisphosphate     

Red-cell amino acid transport. Evidence for the presence of system ASC in mature human red blood cells.

The properties of Na+-dependent L-alanine transport in human erythrocytes were investigated using K+ as the Na+ substitute. Initial rates of Na+-dependent L-alanine uptake (0.2 mM extracellular amino acid) for erythrocytes from 22 donors ranged from 40 to 180 mumol/litre of cells per h at 37 degrees C. Amino acid uptake over the concentration range 0.1-8 mM was consistent with a single saturable component of Na+-dependent L-alanine transport. Apparent Km and Vmax. values at 37 and 5 degrees C measured in erythrocytes from the same donor were 0.27 and 0.085 mM respectively, and 270 and 8.5 mumol/litre of cells per h respectively. The transporter responsible for this uptake was identified as system ASC on the basis of cross-inhibition studies with a series of 42 amino acids and amino acid analogues. Apparent Ki values for glycine, L-alpha-amino-n-butyrate, L-serine and L-leucine as inhibitors of Na+-dependent L-alanine uptake at 37 degrees C were 4.2, 0.12, 0.16 and 0.70 mM respectively. Reticulocytes from a patient with inherited pyruvate kinase deficiency were found to have a 10-fold elevated activity of Na+-dependent L-alanine uptake compared with erythrocytes from normal donors. Separation of erythrocytes according to cell density (cell age) established that even the oldest mature erythrocytes retained significant Na+-dependent L-alanine transport activity. Amino acid transport was, however, a more sensitive indicator of cell age than acetylcholinesterase activity. Erythrocytes were found to accumulate L-alanine against its concentration gradient (distribution ratio approx. 1.5 after 4 h incubation), an effect that was abolished in Na+-free media. Na+-dependent L-alanine uptake was shown to be associated with L-alanine-dependent Na+ influx, the measured coupling ratio being 1:1.     

Developmental changes in rat blood choline concentration.

1. Serum choline concentration in the newborn rat is extremely high and declines as the rat matures until adult values are attained at 20 days of age. 2. Rat milk is a rich source of choline, and rat pups denied access to milk had significantly lower serum choline concentrations than did fed littermates. We conclude that dietary intake of choline contributes to the maintenance of high serum choline concentrations in the neonatal rat. 3. In vivo, choline disappears with a half-life of 70 min. It is converted into betaine, phosphocholine and phosphatidylcholine. The rate of phosphocholine formation is identical in 3- and 10-day-old rats (3.3 mumol/h), whereas the rate of betaine formation is slower in younger animals (0.15 mumol/h at 3 days versus 0.69 mumol/h at 10 days). In vitro, choline oxidase activity [choline dehydrogenase (EC 1.1.99.1) and betaine aldehyde dehydrogenase (EC 1.2.1.8)] increased between birth and 40 days of age. The age-related acceleration in choline's conversion into betaine probably tends to diminish unesterified choline concentration in the rat.     

Bioconversion of benzonitrile to benzoic acid using free and agar entrapped cells of Nocardia globerula NHB-2

The free and agar immobilized cells of Nocardia globerula NHB-2 having nitrilase (EC 3.5.5.1) activity were used to catalyse the transformation of benzonitrile to benzoic acid. The whole cells of N. globerula NHB-2 were immobilized in agar which exhibited maximum conversion of benzonitrile to benzoic acid in 0.1 M potassium phosphate buffer pH 7.5 (free cells) 8.0 (immobilized cells), temperature 40 degrees C, cells 2 mg dcm ml(-1) reaction mixture and benzonitrile (4% v/v) in 4 h (free cells). The effect of temperature on the stability of nitrilase was studied and cells retained 100% activity at 30 degrees C and lost 50% activity at 40 degrees C. In a fed batch mode of reaction 108 and 84 gl(-1) benzoic acid was produced using free and agar entrapped cells (2 g dcm). The agar immobilized cells were recycled up to three times and 80, 62, 20 gl(-1) benzoic acid was again produced respectively in each of three cycles and a total 244 g benzoic acid was produced by recycling the same mass of immobilized biocatalyst.     

Rat liver mitochondrial malate dehydrogenase: purification, kinetic properties, and role in ethanol metabolism

Malate dehydrogenase was purified from the mitochondrial fraction of rat liver by ion-exchange chromatography with affinity elution. The kinetic parameters for the enzyme were determined at pH 7.4 and 37 degrees C, yielding the following values (microM): Ka, 72; Kia, 11; Kb, 110; Kp, 1600; Kip, 7100; Kq, 170; Kiq, 1100, where a = NADH, b = oxalacetate, p = malate, and q = NAD+. Kib was estimated to be about 100 microM. The maximum velocities for mitochondrial malate dehydrogenase in rat liver homogenates, at pH 7.4 and 37 degrees C, were 380 +/- 40 mumol/min per gram of liver, wet weight, for oxalacetate reduction and 39 +/- 3 mumol/min per gram of liver, wet weight, for malate oxidation. Rates of the reaction catalyzed by mitochondrial malate dehydrogenase under conditions similar to those in vivo were calculated using these kinetic parameters and were much lower than the maximum velocity of the enzyme. Since mitochondrial malate dehydrogenase is not saturated with malate at physiological concentrations, its kinetic parameters are probably important in the regulation of mitochondrial malate concentration during ethanol metabolism. For the mitochondrial enzyme to operate at a rate comparable to the flux through cytosolic malate dehydrogenase during ethanol metabolism (about 4 mumol min-1 per gram liver), the mitochondrial [malate] would need to be about 2 mM and the mitochondrial [oxalacetate] would need to be less than 1 microM.     

NADP+ production using thermostable NAD+ kinase of corynebacterium flaccumfaciens AHU-1622

A sonicate of Corynebacterium flaccumfaciens AHU-1622 had the highest NAD+ kinase activity (1.22 mU/mL culture broth) of the strains of bacteria we investigated. This enzyme was thermostable, with activity maintained at 50 degrees C for 1 h. This treatment inactivated phosphatase activity. Resting cells of the bacterium also had NAD+ kinase activity when treated at 60 degrees C for 30 min with 0.2% Triton X-100. NADP+ production was achieved using 8 mumol NAD+, 8 mumol ATP, 16 mumol MgCl2, 1.6 mumol NaN3, and 12 mU NAD+ kinase (0.1 g of permeabilized wet cells) in 2 mL of 0.1 M phosphate buffer, pH 7.5. The conversion ratio of NADP+ from NAD+ was 75% after 10 h of incubation at 50 degrees C, and the amount of accumulated NADP+ was 3 mumol/mL of reaction mixture. The NAD+ kinase activity of the permeabilized cells was stable and did not decrease after repeated use.     

Sensitivity of canine oocytes to low temperature

This study compared the viability of canine oocytes after storage for 5 h at 4 or 38 degrees C. The ovaries were collected after ovariohysterectomy of bitches and transported to the laboratory within 5 h at 4 or 38 degrees C. The collected oocytes were matured in DMEM supplemented with 10% FBS, 0.6 mM/mL cysteine, 0.2 mM pyruvic acid, 20 ng/mL E2 and 1 microg/mL rbST, and incubated for 0, 24 and 48 h, at 38 degrees C and in 95% air with 5% CO2. The viability of the oocytes after 0 h did not differ significantly between 4 and 38 degrees C group (79.6% versus 83.9%), but after 24 and 48 h, significant differences were apparent (13.2% versus 77.8% after 24 h and 0.0% versus 72.9% after 48 h; P < 0.05). Therefore, canine oocytes were remarkably sensitive to low temperatures.     

NAD-dependent formate dehydrogenase from methylotrophic bacterium, strain 1. Purification and characterization.

1. NAD-dependent formate dehydrogenase was isolated from gram-negative methylotrophic bacteria, strain 1, grown on methanol. The purification procedure involved ammonium sulfate fractionation, ion-exchange chromatography and preparative isotachophoresis or gel filtration; it resulted in a yield of 40%. 2. The final enzyme preparations were homogeneous as judged by sedimentation in an ultracentrifuge. Formate dehydrogenase purified in the presence of EDTA reveals two bands on electrophoresis in polyacrylamide gel both after protein and activity staining. Two components are transformed into a single one after prolonged storage in the presence of 2-mercaptoethanol. 3. Formate dehydrogenase is a dimer composed of identical or very similar subunits. The molecular weight of the enzyme is about 80 000. 4. Amino acid composition and some other physico-chemical properties of the enzyme were studied. 5. Formate dehydrogenase is specific for formate and NAD as electron acceptor. The Michaelis constant was 0.11 mM for NAD and 15 mM for formate (pH 7.0, 37 degrees C). 6. Formate dehydrogenase was rapidly inactivated in the absence of -SH compounds. The enzyme retained full activity upon storage at ambient temperature in solution for half a year in the presence of 2-mercaptoethanol or EDTA.     

Effect of antioxidants on preservation of motility,viability and acrosomal integrity of equine spermatozoa during storage at 5 degrees C

Preservation of liquid semen at 5 degrees C is an important technique in the breeding management of horses. Oxidative damage to spermatozoa during storage is a potential cause of the decline in motility and fertility during hypothermic storage of liquid semen. The objective of this study was to evaluate the use of water-soluble and lipid-soluble antioxidants to improve the maintenance of motility of equine spermatozoa at 5 degrees C during storage for 72 to 96 h. In Experiment 1, the effect of addition of catalase on the maintenance of motility, viability and acrosomal integrity was determined. Semen was collected, and these treatments were applied: catalase (0, 100 or 200 U/mL) in nonfat, dried skim milk extender (NFDSM; with or without seminal plasma) or 10% seminal plasma + NFDSM. Motility was determined by computerized semen analysis (CASA) at 0, 24, 48 and 72 h. Viability and acrosomal integrity were determined at 72 h of storage. There was no significant treatment effect on the maintenance of sperm motility during 72 h storage. In Experiment 2, the effect of adding lipid-soluble antioxidants on maintenance of motility was evaluated. Semen was diluted to a final concentration of 25 x 10(6) sperm/mL in NFDSM containing butylated hydroxytoluene (BHT; 2.0, 1.0, or 0.5 mM), Vitamin E (4.0, 2.0, 1.0 mM), or Tempo (2.0, 1.0, or 0.5 mM). Although the addition of BHT significantly reduced (P < 0.05) progressive motility during storage compared to the control, there were no positive treatment effects of either Vitamin E or Tempo on maintenance of motility. In Experiment 3, the effect of adding water-soluble antioxidants on maintenance of motility was evaluated. Semen was diluted in NFDSM containing these treatments: Trolox (2.0 mM), Tempo (1.0 mM), Vitamin C (0.45 mg/mL), BSA (3% w/v), combinations of these antioxidants, or control. Adding these water-soluble antioxidants did not significantly improve the maintenance of motility during cooled storage at 5 degrees C. In conclusion, adding the enzyme scavenger, catalase, or a variety of lipid- and water-soluble antioxidants did not significantly improve the maintenance of motility during liquid semen storage at 5 degrees C.     

Production of extracellular exoinulinase from Kluyveromyces marxianus YS-1 using root tubers of Asparagus officinalis

Root tubers of Asparagus officinalis were used as a source of raw inulin for the production of exoinulinase (EC 3.2.1.7) from Kluyveromyces marxianus YS-1. Root extract prepared at 10kg/cm2 pressure for 10min showed maximum inulinase production. Medium components and process parameters were standardized to improve the enzyme production. Inulinase yield of 40.2IU/mL in a medium containing raw inulin (3.5%), beef extract (2%), SDS (0.001%), Mn2+ (2.0mM), Mg2+ (1.5mM), Co2+ (2mM) and pH 6.5 has been obtained under agitation (150rpm) after 60h of incubation at 30 degrees C at shake flask level. After optimization, the enzyme production was 4.8 times more than the basal medium. To test the feasibility of raw inulin from A. officinalis for the production of inulinase, trials were also made in a bioreactor (1.5L). Inulinase activity of 50.2IU/mL was obtained from raw inulin (4.0%) under agitation (200rpm) and aeration (0.75vvm) at 30 degrees C after 60h of fermentation. Inulinase yield in bioreactor was almost six times higher than the basal medium used initially in shake flask.     

Uptake of L-lysine by a double mutant ofSaccharomyces cerevisiae

A gap1 can1 mutant of Saccharomyces cerevisiae with a single lysine transport system remaining was used to study detailed kinetics of this transport. Its half-saturation constant was 78 mumol per litre, its maximum rate of transport was 0.29 mumol L-lysine per g dry matter per minute, both parameters being lower by more than an order of magnitude in comparison with the GAP system. The pH optimum lay at very acid values of about 3, the temperature dependence without any transition point showed an activation energy of 48 kJ/mol. The transport was inhibited by common metabolic inhibitors (3'-chlorophenylhydrazonomalononitrile, antimycin, 2-deoxy-D-glucose, sodium arsenate) as well as by a membrane-active one (uranyl nitrate). The specificity of the system was extremely high, none of the natural amino acids acting as competitor to L-lysine. The maximum accumulation ratio attained (at about 5 mg dry matter per mL) was 100: 1-120: 1, in agreement with the measured protonmotive force under the assumption of 1 H+ ion being transported with 1 lysine molecule. The ratio decreased with increasing external concentration of lysine to as little as 4: 1 at 1 mmol lysine per litre. It also decreased with increasing suspension density and it was at extremely low suspension densities (0.2 mg dry matter per mL) that ratios of as much as 500: 1 were reached. Application of group-specific inhibitors showed that the active site of the carrier contains an essential histidine residue.     

Purification and properties of the bifunctional proline dehydrogenase/1-pyrroline-5-carboxylate dehydrogenase from Pseudomonas aeruginosa

Proline dehydrogenase/1-pyrroline-5-carboxylate dehydrogenase (Pro/P5C dehydrogenase), a bifunctional enzyme catalyzing the two consecutive reactions of the oxidation of proline to glutamic acid, was purified from Pseudomonas aeruginosa strain PAO1. Pro/P5C dehydrogenase oxidized L-proline in an FAD-dependent reaction to L-delta 1-pyrroline-5-carboxylic acid and converted this intermediate with NAD or NADP as cosubstrates to L-glutamic acid. The purification procedure involved DEAE-cellulose chromatography, affinity chromatography on Matrex gel red A and gel filtration on Sephadex G-200. It resulted, after 40-fold purification with 11% yield, in a homogeneous preparation (greater than 98% pure). The molecular weight of the single subunit was determined as 119,000. Gel filtration of purified Pro/P5C dehydrogenase yielded a molecular weight of 242,000 while polyacrylamide gel electrophoresis under native conditions led to the appearance of two catalytically active forms of the enzyme with molecular weights of 241,000 and 470,000. Manual Edman degradation revealed proline, alanine and aspartic acid as the N-terminal amino acid sequence. Pro/P5C dehydrogenase was highly specific for the L-forms of proline and delta 1-pyrroline-5-carboxylic acid. Its apparent Km values were 45 mM for L-proline, 0.03 mM for NAD and 0.17 mM for NADP. The saturation function for delta 1-pyrroline-5-carboxylic acid was non-hyperbolic.     

Repeated batch production of L-phenylalanine from phenylpyruvate and NH(4)Cl by immobilized cells of Nocardia opaca under hydrogen high pressure

Among various microbial cells examined under screening conditions, Nocardia opaca showed the highest activity for production of phenylalanine from phenylpyruvate. Here NH(4)Cl as well as amino acids were used as an amino donor for phenylalanine production. The phenylalanine production rate increased with increasing hydrogen pressure. The specific activity of phenylalanine dehydrogenase was increased by culturing N. opaca cells in nutrient broth containing 0.3% phenylalanine. As a result, the phenylalanine production rate increased from 0.69 to 4.4 mumol/min g dry cells. Immobilized cells were activated in nutrient broth containing ZnCl(2) before phenylalanine production. Phenylalanine dehydrogenase activity and cell number in the gel increased with increasing incubation time, and the maximum phenylalanine dehydrogenase activity was obtained at 36 h incubation. Then, phenylalanine was produced from phenylpyruvate, NH(4)Cl, and 100 atm H(2) with the activated immobilized cells. The rate of phenylalanine production was 0.24 mumol/min cm(3) gel. The conversion of phenylpyruvate to phenylalanine was 82%. Immobilized cells retained 76% of the initial phenylalanine production rate after 10 h reactions were repeated 11 times with two intervening reactivations.     

A thermostable shikimate 5-dehydrogenase from the archaeon Archaeoglobus fulgidus

Shikimate 5-dehydrogenase (SKDH; EC 1.1.1.25) catalyzes the reversible reduction of 3-dehydroshikimate to shikimate and is a key enzyme in the aromatic amino acid biosynthesis pathway. The shikimate 5-dehydrogenase gene, aroE, from Archaeoglobus fulgidus was cloned and overexpressed in Escherichia coli. The recombinant enzyme purified as a homodimer and yielded a maximum specific activity of 732 U/mg at 87 degrees C (with NADP+ as coenzyme). Apparent Km values for shikimate, NADP+, and NAD+ were estimated at 0.17+/-0.03 mM, 0.19+/-0.01 mM, and 11.4+/-0.4 mM, respectively. The half-life of the A. fulgidus SKDH is 2 h at the assay temperature (87 degrees C) and 17 days at 60 degrees C. Addition of 1 M NaCl or KCl stabilized the enzyme's half-life to approximately 70 h at 87 degrees C and approximately 50 days at 60 degrees C. This work presents the first kinetic analysis of an archaeal SKDH.     

Activities of branched-chain 2-oxo acid dehydrogenase and its components in skin fibroblasts from normal and classical-maple-syrup-urine-disease subjects.

1. Comparisons of the activity and kinetics of the branched-chain 2-oxo acid dehydrogenase in cultured skin fibroblasts from normal and classical maple-syrup-urine-disease (MSUD) subjects provide a kinetic explanation for the enzyme defect. 2. In the intact cell assays, normal fibroblasts demonstrated hyperbolic kinetics with 3-methyl-2-oxo[1-14C]butyrate as a substrate. Intact fibroblasts from four classical MSUD patients showed no decarboxylation over a substrate concentration range of 0.25 to 5.0 mM, and thiamin (4 mM) was without effect. 3. The overall reaction of the multienzyme complex was efficiently reconstituted by using a disrupted-cell system. Normals again showed typical hyperbolic kinetics at the 2-oxo acid concentrations of 0.1 to 5 mM. The Vmax. and apparent Km values were 0.10 +/- 0.02 m-unit/mg of protein and 0.05-0.1 mM respectively, with 3-methyl-2-oxobutyrate. In contrast, classical MSUD patients exhibited sigmoidal kinetics (Hill coefficient, 2.5) with activity approaching 40-60% of the normal value at 5 mM substrate. The K0.5 values from the Hill plots for MSUD patients were 4-7 mM. 4. The E1 (branched-chain 2-oxo acid decarboxylase) component of the multienzyme complex was measured in disrupted-particulate preparations. Normals again showed hyperbolic kinetics with the 2-oxo acid, whereas MSUD preparations exhibited sigmoidal kinetics with the activity of E1 strictly dependent on substrate concentration. Apparent Km or K0.5 were 0.1 and 1.0 mM for normal and MSUD subjects respectively. 5. Measurements of E2 (dihydrolipoyl transacylase) and E3 (dihydrolipoyl dehydrogenase) in MSUD preparations showed them to be in the normal range. 6. The above data suggest a defect in the E1 step of branched-chain 2-oxo acid dehydrogenase in classical MSUD patients.     

Purification and characterization of two alpha-L-arabinofuranosidases from Streptomyces diastaticus.

A nonsporulating strain of Streptomyces diastaticus producing alpha-L-arabinofuranosidase activity (EC 3.2-1.55) was isolated from soil. Two alpha-L-arabinosidases were purified by ion-exchange chromatography and chromatofocusing. The enzymes had molecular weights of 38,000 (C1) and 60,000 (C2) and pIs of 8.8 and 8.3, respectively. The optimum pH range of activity for both enzymes was between 4 and 7. The apparent Km values with p-nitrophenyl arabinofuranoside as the substrate were 10 mM (C1) and 12.5 mM (C2). C1 retained 50% of its activity after 8 h of incubation at 25 degrees C, while C2 retained 80% activity. After 3 h of incubation at 50 degrees C, C1 lost 90% of its initial activity while C2 lost only 40%. The purified enzymes hydrolyzed p-nitrophenyl alpha-L-arabinofuranoside and liberated arabinose from arabinoxylan and from a debranched beta-1,5-arabinan.