Effects of solvent, water activity and temperature on lipase and hydroxynitrile lyase enantioselectivity

The influence of the reaction conditions on the enantioselectivity of reactions catalysed by lipases or hydroxynitrile lyases (HNLs) in organic solvents was investigated. The lipases catalysed kinetic resolution of chiral secondary alcohols or chiral carboxylic acids and the HNLs catalysed asymmetric addition of hydrogen cyanide to aldehydes.

The temperature effects on enantioselectivity were studied in detail. From measurements of the enantiomeric ratio (E) at different temperatures the activation parameters ΔΔH^# and ΔΔS^# were determined. In the lipase-catalysed reactions the enthalpic and entropic effects on E always counteracted, while in a few of the HNL-catalysed reactions, ΔΔH^# and ΔΔS^# had opposite signs and therefore the effects cooperated to give high E values (−RTlnE = ΔΔG^# = ΔΔH^# − TΔΔS^#). In all the HNL-catalysed reactions and most of the lipase-catalysed ones, the enantioselectivity increased with decreasing reaction temperature. However, in one of the lipase-catalysed reactions, the enantioselectivity decreased with decreasing temperature. The theoretical background of these observations was discussed.

In the HNL-catalysed reactions, the enantioselectivity increased with increasing water content up to water saturation, while in the lipase-catalysed reactions the opposite trend was found in one case and in the others no significant effect was observed. Solvent mixtures of diisopropylether and hexane were used to obtain solvents with different log P values. The log P value of the solvent did not influence the enantioselectivity in the HNL-catalysed reactions, while the enantioselectivity increased with increasing log P value in two of the lipase-catalysed reactions. The reaction temperature was shown to be a very useful way to influence enzyme selectivity and the effects obtained could be rationalised. The influence of the reaction medium (solvent and water activity) is much more difficult to rationalise and predict.     

Expression of the Kidney Bean Phenylalanine-Ammonia Lyase Gene in the Hairy Roots of Astragalus sinicus

Using Agrobacterium rhizogenes, Astragalus sinicus plants were transformed with the kidney bean pal5 gene coding for phenylalanine-ammonia lyase (PAL). The hairy root culture thus obtained manifested enhanced PAL activity and lignin content in the cell walls; in addition, the transformed cells differed from the wild-type ones in several electrophysiological indices. In particular, the diffusion component of the total membrane potential of plasmalemma increased in the pal-transformed roots. The authors presume that the volume density of the protein-related negative charge of the cytoplasm increases in the transformed root cells along with changes in the cytoplasmic pH and pCa²⁺, the extent of coupling of these two indices, and the hydraulic conductivity of plasmodesmata.     

Pectin lyase production by Penicillium griseoroseum grown in sugar cane juice in repeated batch cultures

Penicillium griseoroseum cultured in the presence of sucrose and yeast extract produces pectin lyase (EC 4.2.2.10) (PL) in the absence of its natural inducer pectin. This fungus was cultured in a fermenter at an aeration rate of 0.5 l/min for the first 25 h and 1.0 l/min for the remainder of the culture period, and at a stirring rate of 200 rev/min for the entire culture period. Fungal spores were inoculated directly into the fermenter at a final concentration of 5 × 10⁴ spores/ml. The fungus was cultured in minimal medium supplemented with powdered dehydrated sugar cane juice, producing PL without added yeast extract. Maximum PL activity (0.067 IU/ml) was obtained after 65 h in batch culture. Pellet morphology of the mycelia made it possible to carry out three cycles of repeated batch culture. The same medium was used for renewal as for the single batch culture. The initial cycle was 53 h, after which approximately 0.103 IU/ml of PL was obtained. After this period, the medium was renewed and fermentation continued for two more cycles, which lasted approximately 20 h. Activity of PL obtained in the second cycle was approximately 0.118 IU/ml and in the third, approximately 0.109 IU/ml.     

The nucleotide sequence of the yeast ARG4 gene

The complete nucleotide sequence of a 2296-bp DNA fragment containing the yeast (Saccharomyces cerevisiae) ARG4 gene has been determined. This gene specifies the synthesis of the arginine biosynthetic enzyme, argininosuccinate lyase (EC 4.3.2.1). The sequence contains one major open reading frame of 463 codons, giving a calculated M_r of 52010 for the protein, in good agreement with the experimentally determined value of 53 000. The sequence upstream from the ARG4 gene shares structural features in common with other yeast genes subject to general amino acid control.     

C-C bond formation and cleavage in radical enzymes, a theoretical perspective

Quantum chemical methods are today a viable tool in the study of enzyme catalysis. The development of new density functional techniques and the enormous advancement in computer power have made it possible to accurately describe active sites of enzymes. This review gives a brief account of the methods and models used in this field. Three specific enzymes are discussed: pyruvate-formate lyase (PFL), spore photoproduct lyase (SPL), and benzylsuccinate synthase (BSS). What these enzymes have in common is that they use radical chemistry to catalyze C-C bond formation or cleavage reactions.     

Regulatory mutations affecting the synthesis of pectate lyase in Xanthomonas campestris

Four classes of Xanthomonas campestris mutants were identified with respect to pectate lyase. Pectate lyase production in the wild-type and classes I and IIb mutants was partially dependent on the growth-phase whereas in classes IIa and III it was totally dependent. Enzyme activity in some of the mutants was constitutive and resistant to catabolite repression.     

Intracellular location of ATP citrate lyase in leaves of Pisum sativum L.

The aim of this work was to discover if there is enough ATP citrate lyase (EC 4.1.3.8) in the cytosol of the leaves of Pisum sativum L. to catalyse the synthesis of the acetyl CoA needed for terpenoid synthesis. Estimates of the maximum catalytic activity of the enzyme in leaves of 7-d-old peas gave values of 113 nmol min^-1 g^-1 fresh weight. The rate of carotenoid accumulation in these leaves corresponded to a requirement for acetyl CoA of 0.7 nmol min^-1 g^-1 fresh weight. The distribution of marker enzymes during fractionation of homogenates of leaves from 7 to 10-d-old peas showed that differential centrifugation led to the isolation in reasonable yields of chloroplasts, mitochondria, peroxisomes and the endomembrane system. None of the above components of the leaf contained appreciable detectable activity of ATP citrate lyase, the distribution of which closely paralleled that of the cytosolic marker. It was concluded that in young leaves of pea most of the ATP citrate lyase is in the cytosol.     

Purification and Characterization of a Cold-adapted Isocitrate Lyase and a Malate Synthase from Colwellia maris,a Psychrophilic Bacterium

Isocitrate lyase (ICL) and malate synthase (MS) of a psychrophilic marine bacterium, Colwellia maris, were purified to electrophoretically homogeneous state. The molecular mass of the ICL was found to be 240 kDa, composed of four identical subunits of 64.7 kDa. MS was a dimeric enzyme composed of 76.3 kDa subunits. N-Terminal amino acid sequences of the ICL and MS were analyzed. Purified ICL had its maximum activity at 20°C and was rapidly inactivated at the temperatures above 30°C, but the optimum temperature for the activity of MS was 45°C. NaCl was found to protect ICL from heat inactivation above 30°C, but the salt did not stabilize MS. Effects of temperatures on the kinetic parameters of both the enzymes were examined. The K_m for the substrate (isocitrate) of ICL was decreased with decreasing temperature. On the other hand, the K_m for the substrate (glyoxylate) of MS was increased with decreasing temperature. The calculated value of free energy of activation of ICL was on the same level as that of MS.     

Review: Organophosphonates: occurrence, synthesis and biodegradation by microorganisms

The organophosphonates are biogenic and xenobiotic compounds characterized by the presence of a stable carbon to phosphorus (C-P) bond. The C-P bond imparts upon these molecules a relative resistance to (bio)degradation and fears have been expressed over their environmental recalcitrance and possible ecotoxicity, as more than 20×103 tonnes of these compounds enter the environment annually in the U.S.A. and western Europe alone (Egli, 1988). Biodegradation of organophosphonates is generally accepted to be dependent upon the phosphate status of the cell, with biodegradation occurring only under conditions of phosphate limitation. In recent years, however, several novel bacteria capable of completely mineralizing both natural and man-made organophosphonates have been isolated. These organisms represent a departure, both at a physiological and genetic level, from the accepted consensus that organophosphonates are utilized only phosphorus sources. This review covers all aspects of our knowledge of organophosphonate metabolism over the last 50 years, concentrating on the advances made in the last 10 years.