Phenylalanine ammonia lyase (PAL; E.C.4.3.1.5), which catalyses the biotransformation of L-phenylalanine to trans-cinnamic acid and ammonia, was first described in 1961 by Koukol and Conn. Since its discovery, much knowledge has been gathered with reference to the enzyme's catabolic role in microorganisms and its importance in the phenyl propanoid pathway of plants. The 3-dimensional structure of the enzyme has been characterized using X-ray crystallography. This has led to a greater understanding of the mechanism of PAL-catalyzed reactions, including the discovery of a recently described cofactor, 3,5-dihydro-5-methyldiene-4H-imidazol-4-one. In the past 3 decades, PAL has gained considerable significance in several clinical, industrial, and biotechnological applications. The reversal of the normal physiological reaction can be effectively employed in the production of optically pure L-phenylalanine, which is a precursor of the noncalorific sweetener aspartame (L-phenylalanyl-L-aspartyl methyl ester). The enzyme's natural ability to break down L-phenylalanine makes PAL a reliable treatment for the genetic condition phenylketonuria. In this mini-review, we discuss prominent details relating to the physiological role of PAL, the mechanism of catalysis, methods of determination and purification, enzyme kinetics, and enzyme activity in nonaqueous media. Two topics of current study on PAL, molecular biology and crystal structure, are also discussed.

Peroxidase (EC 1.11.1.7) from horseradish ( Armoracia rusticana ) roots was purified using a simple, rapid, three-step procedure: ultrasonication, ammonium sulfate salt precipitation, and hydrophobic interaction chromatography on phenyl Sepharose CL-4B. The preparation gave an overall yield of 71%, 291-fold purification, and a high specific activity of 772 U mg(-1) protein. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the purified enzyme was homogeneous and had a molecular weight of approximately 40 kDa. The isolated enzyme had an isoelectric point of 8.8 and a Reinheitszahl value of 3.39 and was stable when stored in the presence of glycerol at -20 degrees C, with >95% retention of original enzyme activity for at least 6 months. Maximal activity of purified horseradish peroxidase (HRP) was obtained under different optimized conditions: substrate (guaiacol and H(2)O(2)) concentrations (0.5 and 0.3 mM, respectively), type of buffer (50 mM phosphate buffer), pH (7.0), time (1.0 min), and temperature of incubation (30 degrees C). In addition, the effect of HRP and H(2)O(2) in a neutral-buffered aqueous solution for the oxidation of phenol and 2-chlorophenol substrates was also studied. Different conditions including concentrations of phenol/2-chlorophenol, H(2)O(2), and enzyme, time, pH, and temperature were standardized for the maximal activity of HRP with these substrates; under these optimal conditions 89.6 and 91.4% oxidations of phenol and 2-chlorophenol were obtained, respectively. The data generated from this work could have direct implications in studies on the commercial production of this biotechnologically important enzyme and its stability in different media.

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[lignin-14C]Lignocellulose was prepared from wheat seedlings, injected with L-[U-' 4C]-phenylalanine, by a procedure that selectively removed radiolabelled low molecular weight compounds and protein. 4C was mainly localized in the acid-insoluble fraction ('Klason lignin') of the lignocellulose and the polymeric nature of the radiolabelled material was confirmed by gel permeation chromatography. Ferulic and p-coumaric acids were found to be esterified to both carbohydrate and lignin fractions of the wheat lignocellulose, but contained only a small proportion of the total 14C. The white-rot fungi Phanerochaete chrysosporium, Sporotrichum pulverulentum and Coriolus versicolor degraded the radiolabelled substrate; the S . pulverulentum culture was the most active, releasing almost 50% of the 14C as 14C02 in 15 d at 37 "C. In all three species I4CO2 evolution commenced after a lag period during which growth occurred, and was greatly enhanced by reducing the nitrogen concentration in the medium. This indicated that grass lignin degradation by these fungi is subject to the same nutrient nitrogen regulation as the degradation of wood and synthetic lignins. In a culture of P . chrysosporium grown on the radiolabelled substrate for 50d at 37"C, I4CO2 was the main product. Low molecular weight degradation products present in the culture medium were acid-soluble and accounted for a much smaller proportion of the original [ 14C]lignin.

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