Production of l-Phenylalanine from Starch by Analog-Resistant Mutants of Bacillus polymyxa

p-Fluorophenylalanine-resistant mutants of starch-degrading Bacillus polymyxa ATCC 842, generated by ethyl methanesulfonate mutagenesis followed by incubation with caffeine, overproduced small amounts of l-phenylalanine (l-phe) from starch. A beta-2-thienylalanine-resistant mutant (BT-7) derived from p-fluorophenylalanine mutant (C-4000 FP-4) and resistant to both p-fluorophenylalanine and beta-2-thienylalanine produced 0.5 g of l-phe and 0.15 g of l-tyrosine per liter from 10 g of starch per liter when growing in a minimal medium. trans-Cinnamic acid (CA) was also excreted by both mutants, indicating the possibility of l-phenylalanine ammonia-lyase-induced deamination of l-phe to CA. The amount of l-phe-derived CA detected in BT-7 was less compared with mutant C-4000 FP-4. CA production was induced in the parent only when l-phe was used as a sole nitrogen source. Time of CA production in the two mutants could be delayed by addition of other nitrogen sources, an indication of possible l-phenylalanine ammonia-lyase inhibition or repression. The presence of l-phenylalanine ammonia-lyase in B. polymyxa mutant C-4000 FP-4 was confirmed by assays of cell-free extracts from cells grown in starch minimal medium containing l-phe as the sole nitrogen source. Preliminary studies of the regulation of deoxy-d-arabino-heptulosonate-7-phosphate synthase and prephenate dehydratase in the wild-type strain showed that deoxy-d-arabino-heptulosonate-7-phosphate synthase was subject to feedback inhibition by l-phe, l-tyrosine, and l-tryptophan. Inhibition by each amino acid was to a similar extent singly or in combination at a 0.5 mM level of each amino acid. Prephenate dehydratase was feedback inhibited by l-phe, but not by l-tyrosine or l-tryptophan or both. In the double analog-resistant mutant BT-7, deoxy-d-arabino-heptulosonate-7-phosphate synthase had specific activity similar to that in the wild type, and the enzyme was still subject to feedback inhibition. However, prephenate dehydratase had increased specific activity and it was also insensitive to feedback inhibition by l-phe. The overproduction of aromatic amino acids by BT-7 was thought to be due, at least in part, to deregulation of feedback inhibition of prephenate dehydratase. Chorismate mutase was not subject to feedback inhibition in the wild type and was unaffected in the mutant.     

Purification and general properties of δ-aminolaevulate dehydratase from Nicotiana tabacum L

1. delta-Aminolaevulate dehydratase (EC 4.2.1.24) was purified 80-fold from tobacco leaves and its properties were studied. 2. The enzyme had optimum pH7.4 in potassium phosphate buffer, K(m)6.25x10(-4)m at 37 degrees and pH7.4, optimum temperature 45 degrees and an activation energy of 11100 cal./mole. 3. The enzyme lost activity when prepared in the absence of cysteine, and this activity was only partly restored by the later addition of thiols. Reagents for thiol groups inactivated the enzyme. 4. Mg(2+) was essential for activity, and EDTA and Fe(2+) were inhibitory; Mn(2+) was an activator or an inhibitor depending on the concentration.     

High-performance liquid chromatographic method for the determination of a novel thymidylate synthase inhibitor, AG 331, in human serum

AG 331 is a novel thymidylate synthase inhibitor currently in Phase I clinical trial. To determine the pharmacokinetic parameters of AG 331 in human subjects, a suitable analytical method was developed using high-performance liquid chromatography. Serum and urine samples were prepared using both solid-phase extraction and solvent extraction. Either 4,4′-diaminodiphenyl sulfone or benz[cd]indole-2(1H)-one were used as internal standards for the method. A reversed-phase C₁₈ analytical column completely resolved the drug and internal standard peaks from non-specific substances present in biological matrix. The method was validated for precision, accuracy, and reproducibility in serum and was linear over a concentration range of 50–2000 ng/ml, with a limit of detection of 20.0 ng/ml and a quantifiable limit of 50 ng/ml.     

Production and Purification of Extracellular D-Xylose Isomerase from an Alkaliphilic, Thermophilic Bacillus sp.

An alkaliphilic, thermophilic Bacillus sp. (NCIM 59) produced extracellular xylose isomerase at pH 10 and 50°C by using xylose or wheat bran as the carbon source. The distribution of xylose isomerase as a function of growth in comparison with distributions of extra- and intracellular marker enzymes such as xylanase and β-galactosidase revealed that xylose isomerase was truly secreted as an extracellular enzyme and was not released because of sporulation or lysis. The enzyme was purified to homogeneity by ammonium sulfate precipitation followed by gel filtration, preparative polyacrylamide gel electrophoresis, and ion-exchange chromatography. The molecular weight of xylose isomerase was estimated to be 160,000 by gel filtration and 50,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating the presence of three subunits. The enzyme is most active at pH 8.0 and with incubation at 85°C for 20 min. Divalent metal ions Mg²⁺, Co²⁺, and Mn²⁺ were required for maximum activity of the enzyme. The K_m values for D-xylose and D-glucose at 80°C and pH 7.5 were 6.66 and 142 mM, respectively, while K_cat values were 2.3 × 10² s^-1 and 0.5 × 10² s^-1, respectively.     

Antioxidant and Antidiabetic Properties of Extracts from Three Underutilized Food Plants of North East India

Three underutilized leafy vegetables Sarcochlamys pulcherrima (Roxb.) Gaudich (SP), Ipomoea aquatica Forssk. (IA) and Zanthoxylum rhetsa (Roxb.) DC (ZR) were extracted with different solvents viz. 95 % ethyl alcohol, methanol and hot water. The extracts were evaluated for their antioxidant potential via DPPH, ABTS and FRAP assay along with electroanalytical studies using cyclic voltammetry. The antidiabetic potential was determined by recording their α-amylase and α-glucosidase inhibitory assay. The total phenolic content (TPC), total flavonoid content (TFC) and the liquid chromatography-mass spectrometry (LC/MS) based phytochemical profiles of the extracts were also determined. All three extracts of SP exhibited significant antioxidant capacity. The antidiabetic potential of the IA and ZR extracts was found to be higher than or at par with that of standard acarbose. LC/MS studies reveal the presence of hitherto reported antioxidant and antidiabetic compounds like gamma-aminobutyric acid, cinnamic acid, caffeic acid, α-viniferin, piperlonguminine, niacin, kaempferol, etc., in the extracts.     

Bimodal oxygen uptake in juveniles and adults amphibious fish,Channa (= Ophiocephalus) marulius

Oxygen uptake of Channa marulius was studied under water with and without access to air. There was a significant increase in the oxygen uptake through the gills when access to air was prevented. However, this value (0.863 ± 0.058 mlO₂/indiv./h) was quite low in comparison to the total bimodal oxygen uptake (2.04 ± 0.14 mlO₂/indiv./h) in juveniles. In adult fish the oxygen uptake per unit time increased appreciably (4.673 ± 0.404 mlO₂/indiv./h). In juveniles as well as in adults the air breathing dominated over aquatic breathing. This fish showed a definite circadian rhythm in the bimodal oxygen uptake during different hours of the day.This work was performed in the Ichthyology Laboratory, P. G. Dept. of Zoology, Bhagalpur University, and was supported by a research grant from Bhagalpur University     

Role of catecholamines in hypotensive response to dieting.

The effect of dieting on blood pressure and catecholamine metabolism was assessed in 11 normotensive obese women by providing first a weight-maintenance regimen high in carbohydrate and then a low-energy diet. All dietary constituents other than carbohydrate were maintained constant throughout the 18-day study. The low-carbohydrate diet led within 48 hours to a 41% fall in the urinary output of 4-hydroxy-3-methoxy mandelate and a significant fall in systolic and diastolic blood pressure. Plasma noradrenaline concentrations also fell and the hypotensive effect of the diet continued despite a maintained total body sodium. Thus the fall in blood pressure appeared to be mediated by changes in catecholamine metabolism independent of sodium intake. This may explain both the usefulness of weight reduction in hypertensive patients and the fainting that occurs in some normotensive obese subjects taking slimming regimens low in carbohydrate.