P.Madhusudanan Nair

Stabilization of phenylalanine ammonia lyase containing Rhodotorula glutinis cells for the continuous synthesis of L-phenylalanine methyl ester/96/

A procedure for the direct one-step synthesis of l-phenylalanine methyl ester, a precursor of the artificial sweetener, aspartame, by using phenylalanine ammonia lyase (PAL)-containing Rhodotorula glutinis cells in a 2:1 heptane: 0.1 m Tris-sulfate buffer pH 9.0 biphasic system was reported earlier; however, the yeast cells lost PAL activity rapidly during the reaction and were rendered unsuitable for repeated use. Stabilization of the enzyme during the biotransformation for the continuous use is described. Immobilization of PAL containing Rhodotorula glutinis cells by different conventional methods could not prevent the inactivation of the enzyme. The addition of a low concentration of Mg2+ to the incubation mixtures resulted in significant stabilization of PAL which was further enhanced by the presence of glycerol. At 4 mm MgSO4 and 10% glycerol concentrations, the system could produce l-phenylalanine methyl ester for nine cycles of repeated use while the controls lost activity by the fourth cycle. The total yield of l-phenylalanine methyl ester in nine cycles was 92 g l−1.

Purification of phenylalanine ammonia lyase from Rhodotorula glutinis

Abstract A simple and rapid method employing four steps; sonication, salt precipitation, gel filtration on Sephacryl-S-400 followed by hydrophobic interaction chromatography on Phenyl Sepharose CL-4B for the purification of phenylalanine ammonia lyase from the yeast Rhodotorula glutinis is described. The preparation gave an overall yield of 33%, 195-fold purification and a high specific activity of 4.69 U mg −1 protein. The final preparation was homogeneous as judged from native polyacrylamide gel electrophoresis.

δ-Aminolevulinic acid synthetase from cold-stored potatoes

Abstract δ-Aminolevulinic acid synthetase has been obtained in a cell-free state from greening potatoes, which are stored in low temperature (0–4°C) and dim light (30–40 lux). The maximum enzyme activity is developed within 3 weeks of storage. Higher temperatures or light intensities do not favour the formation of this enzyme. The pH optima for δ-aminolevulinic acid synthetase is 7.0. Supplementation of the reaction mixture with pyridoxal phosphate, Mg 2+ , ATP. CoA and succinyl-CoA synthetase gives maximum δ-Aminolevulinic acid synthesis. Chlorophyllide a at 10 μg level in the reaction mixture inhibits the enzyme activity while chlorophyll a and pheophytin a are ineffective.

Potato tuber succinate semialdehyde dehydrogenase: purification and characterization.

Succinate semialdehyde dehydrogenase (SSADH) has been purified from potato tubers with 39% yield, 832-fold purification, and a specific activity of 6.5 units/mg protein. The final preparation was homogeneous as judged from native and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Gel filtration on Sepharose 6B gave a relative molecular mass (Mr) of 145,000 for the native enzyme. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis gave a single polypeptide band of Mr 35,000. Thus the enzyme appears to be a tetramer of identical subunits. Chromatofocusing of the enzyme gave a pI of 8.7. The enzyme was maximally active at pH 9.0 in 100 mM sodium pyrophosphate buffer. In 100 mM Tris-HCl buffer, pH 9.0, the enzyme gave only 20% of the activity found in pyrophosphate buffer and had a shorter linear rate. The enzyme was specific for succinate semialdehyde (SSA) as substrate and could not utilize acetaldehyde, glyceraldehyde 3-phosphate, malonaldehyde, lactate, or ethanol as substrates. The enzyme was also specific for NAD+ as cofactor and NADP+ and 3-acetylpyridine adenine dinucleotide could not serve as cofactors. Potato SSADH had a Km of 4.6 microM for SSA when assayed in pyrophosphate buffer and was inhibited by that substrate at concentrations greater than 120 microM. The Km for NAD+ was found to be 31 microM. The enzyme required exogenous addition of a thiol compound for maximal activity and was inhibited by the thiol-directed reagents p-hydroxymercuribenzoate, dithionitrobenzoate, and N-ethyl-maleimide, by heavy metal ions Hg2+, Cu2+, Cd2+, and Zn2+, and by arsenite. These results indicate a requirement of a SH group for catalytic activity.

Novel direct synthesis of l-phenylalanine methyl ester by using Rhodotorula glutinis phenylalanine ammonia lyase in an organic-aqueous biphasic system

A procedure for the direct, one-step enzymatic conversion of trans-cinnamyl methyl ester to L-phenylalanine methyl ester is described. The reverse reaction of phenylalanine ammonia lyase from Rhodotorula glutinis was utilized for this conversion. Insolubility of substrate trans-cinnamyl methyl ester in aqueous buffer solution was overcome by employing an organic-aqueous biphasic system, heptane:0.1 m Tris-sulfate buffer, pH 9.0 (2:1). Different conditions were optimized for the maximal conversion such as time (16–18 h), temperature (30°C), pH (9.0), concentration of substrates, 0.1 m trans-cinnamyl methyl ester and 1 m (NH4)2 SO4, and nature of the organic solvent (heptane); about 70% conversion of substrate to product was obtained under these conditions. Formation of the product, l-phenylalanine methyl ester, was identified by paper chromatography and was further confirmed by autoradiography and NMR spectral analyses.

Alterations in carbohydrate metabolism of γ-irradiated cavendish banana

Abstract γ-Irradiation of preclimacteric banana resulted in a gradual increase in fructose content, which reached a maximum in 6 days. Although the catabolism of glucose-U-14C was less in irradiated banana, incorporation of label into fructose was high. Initial fructose accumulation in irradiated banana may be due to a shift in glucose utilization from the glycolytic to the pentose phosphate pathway. The ratio of resporatory CO2 from glucose-6-14C and glucose-1-14C was halved in irradiated bananas indicating predominance of the pentose phosphate pathway. The radioactivity of fructose derived from glucose-6-14C was almost twice that from glucose-1-14C in irradiated bananas, whilst in control both fruit the labelled precursors yielded equal amounts. Studies on individual enzymes in these two pathways showed an increase in phosphorylase, phosphoglucomutase, glucose-6-phosphate dehydrogenase and fructose-6-phosphatase and a decrease in hexokinase in irradiated banana.

Stimulation of the glyoxylate shunt in gamma-irradiated banana

Abstract Succinic dehydrogenase was the most susceptible among the TCA cycle enzymes to gamma irradiation in preclimacteric banana. Maximum inhibition occurred at the 3rd day after irradiation. Impairment of this activity did not affect operation of the TCA cycle, assessed from the incorporation pattern of acetate [2- 14 C] into the organic acids such as citric, malic and succinic. Nevertheless, incorporation into keto acids like glyoxylate, α-keto-glutarate and oxaloacetate showed a difference. The rate of labelling into α-ketoglutarate and oxaloacetate was reduced on the 3rd day while incorporation into glyoxylate was increased indicating the operation of glyoxylate shunt pathway. Studies on the individual enzymes of this pathway, isocitrate lyase and malate synthetase confirmed its operation. The reduction in oxalo-acetate has been attributed to the increased gluconeogenesis.

Asparagine synthetase from γ-irradiated potatoes

Abstract γ-Irradiation of potatoes at sprout-inhibiting dose was found to enhance the asparagine synthetase activity. This activation was dose-dependent and maximum activation was obtained between 5 and 25 krads; 10 krad being the optimum dose. Asparagine synthetase was purified to about 50-fold. l -Asparagine was identified as the product of the reaction. The reaction was specific for l -aspartic acid and devoid of transferase activity. Hydroxylamine at very high concentration could replace NH4 + for substrate. The reaction showed a definite requirement for NIH4 + , ATP, and Mg 2+ . The mechanism of enzyme activity resembled that reported for other higher plants, where l -asparagine, ADP, and Pi are the products of the reaction. No AMP was detected as the product of the enzyme reaction. There was strict stoichiometry between asparagine and ADP formed and Pi liberated during the reaction. Mn 2+ ions could replace Mg 2+ for activity. But the pH optima shifted from 7.2 to 6.0 when Mg 2+ ions were supplemented in the reaction.

Properties of acidic and alkaline fructose-1,6-diphosphatase from γ-irradiated banana

Abstract Irradiation of preclimacteric Dwarf Cavendish bananas at 35 krad caused a gradual activation of fructose-1,6-diphosphatase, which reached a maximal 5-fold increase in activity in 3 days. Two enzymic components showing activity in acid (pH 6·6) and alkali (pH 8·8) were separated by calcium phosphate gel adsorption and elution. Both the enzymes required Mg 2+ ions for activity. The catalytic properties of acidic and alkaline FDPase were quite different. The alkaline enzyme was more sensitive towards the concentration of substrate, and allosteric inhibitor AMP. Activation by disulfide reagent and EDTA was observed only in the case of alkaline enzyme.

Studies on determination of active site amino acid residues in glyoxylate synthetase from potato tuber chloroplasts

Abstract A homogeneous preparation of glyoxylate synthetase from greening potato tubers was used to study the functional role of disulphide groups, lysine and tryptophan residues in enzyme catalysis. The formation of a thioisoindole derivative was demonstrated by spectral analysis of the reduced and o -phthalaldehyde-treated enzymes. o -Phthalaldehyde modification resulted in about a 25 % loss of tryptophan emission at 336 nm and the appearance of a 410-nm emission peak characteristic of a thioisoindole. Ferrous iron was capable of generating thiol groups and addition of substrate resulted in a faster disappearance of these thiols. The optimal time for maximum glyoxylate synthesis by glyoxylate synthetase paralleled the disappearance of these thiols. Involvement of lysine and tryptophan residues in the enzyme reaction was demonstrated by the inhibition of activity by pyridoxal 5′-phosphate and dimethyl(2-hydroxy 5-nitrobenzyl) sulphonium bromide (DMHNB), respectively. Pyridoxal phosphate strongly and reversibly inhibited glyoxylate synthetase, and substrate and metal ion provided significant protection against inhibition. The results suggest that the lysine residue may be at or near the active binding site. The lysyl residue formed a Schiff base with pyridoxal phosphate which was stabilised by NaBH 4 . Glyoxylate synthetase was also irreversibly inactivated by a tryptophan selective reagent, DMHNB, while substrate provided substantial protection against inactivation. Kinetic analysis and correlation of the spectral data at 410 nm indicated that complete inactivation by DMHNB resulted from the modification of 5 tryptophan residues/subunit, of which one was essential for activity. The available evidence suggests a possible concerted action of enzyme disulphides, ferrous iron, lysine and aromatic amino acid residues in the synthesis of glyoxylate by this enzyme.