Lyases assay

A modification of the acylneuraminate pyruvate-lyase assay using pyruvate oxidase to generate H2O2, and measurement of this with peroxidase and p-chlorophenol-4-aminoantipyrine has been described (Sugahara et al. 1980). The measurement of the chromophore is at 505 nm and a molar extinction coefficient of 1.14 X 104 was found at this wavelength. This test has been used in conjunction with sialidase for estimation of serum sialic acid (Sugahara et al. 1980), but remains less sensitive than the method coupled with lactate dehydrogenase. 

Hydroperoxide lyase assay. Hydroperoxide lyase was determined by measurement of the formation of hexanal from 13-hydroperoxlde of linoleic acid at pH 6.3.(1 ) The substrate (6 pmol) dissolved In diethyl ether was pipetted Into a 50-ml flask and the solvent was evaporated 1 vacuo. Then, 10 ml of chloroplast suspension or leaf homogenate was added. The mixture was sealed In the flask with a rubber stopper and Incubated at 35°C for 10 mLn. The Cg-aldehydes formed were measured by the headspace method with GLC. 

Pectin lyase and pectate lyase activities were assayed by measuring the increasing of the absorbancy at 235 nm by the method of Albersheim and Killias (12, 13) when pectin or sodium pectate was used as the substrate, repectively. 

Pectin lyase (PNL) activity was measured spectrophotometrically by the increase in absorbance at 235 nm of the 4,5-unsaturated reaction products. Reaction mixtures containing 0.25 ml of culture filtrate, 0.25 ml of distilled water and 2.0 ml of 0.24% pectin from apple (Fluka) in 0.05M tris-HCl buffer (pH 8.0) with ImM CaCl2, were incubated at 37 C for 10 minutes. One unit of enzyme is defined as the amount of enzyme which forms Ipmol of 4,5-unsaturated product per minute under the conditions of the assay. The molar extinction coefficients of the unsaturated products is 5550 M cm [25]. Also viscosity measurements were made using Cannon-Fenske viscometers or Ostwald micro-viscosimeter, at 37°C. Reaction mixtures consisted of enzyme solution and 0.75% pectin in 0.05 M tris-HCl buffer (pH 8.0) with 0.5 mM CaCl2. One unit is defined as the amount of enzyme required to change the inverse specific viscosity by 0.001 min under the conditions of reaction. Specific viscosity (n p) is (t/to)-l, where t is the flow time (sec) of the reaction mixture and t is the flow time of the buffer. The inverse pecific viscosity (n p ) is proportional to the incubation time and the amount of enzyme used [26]. Units of enzyme activity were determined for 10 min of reaction. 

Figure 2. CM-cellulose chromatography of pectolytic enzymes. The activity peaks of the flow-through of a DEAE-cellulose chromatography was applied to a CM-cellulose column. The column was eluted with a NaCl (0-0.5M) continuous gradient at a flow rate of 34 ml/h. 10 ml fractions were collected and assayed for pectolytic activities Symbols (0) pectate lyase ( ) polygalacturonase (reducing sugar-releasing activity) (x) protein. Other details in Methods.

Andexer, J., Guterl, J.-K., Pohl, M. and Eggert, T. (2006) A high-throughput screening assay for hydroxynitrile lyase activity. Chemical Communications, 4201 4203. 

Krammer, B., Rumbold, K., Tschemmernegg, M. et al. (2007) A novel screening assay for hydroxynitrile lyases suitable for high-throughput screening. Journal of Biotechnology, 129, 151-161. 

In addition to the foregoing methods, all of the methods reported in Section V,4 can be used for the assay of lyase activity. 

ENZ enzyme assays, SC structural composition, MM molecular methods, IL isotopic labeling, IF isotopic fractionation, INH inhibition studies, UNK unknown, LOX lipoxogenase, EPSP synthase 5-enolpyruvylshikimate-3-phosphate, SDH shikimate dehydrogenase, PAL phenylalanine ammonium lyase, PKS polyketide synthase, NRPS nonribosomal peptide synthase 1 Gerwick 1999 2 Liu et al. 1994 3 Boonprab et al. 2003 4 Cvejic and Rohmer 1999 5 Disch et al. 1998 6 Chikaraishi et al. 2006 7 Schwender et al. 2001 8 Schwender et al. 1997 9 Mayes et al. 1993 10 Shick et al. 1999 11 Richards et al. 2006 12 Bouarab et al. 2004 13 Pelletreau et al., unpublished data 14 Dittman and Weigand 2006 15 Rein and Barrone 1999 Empty columns imply no direct evidence of these enzymes from these systems... 

A new hydroxynitrile lyase (HNL) was isolated from the seed of Japanese apricot Prunus mume). It accepts benzaldehyde and a large number of unnatural substrates for the addition of HCN to produce the corresponding (7 )-cyanohydrins in excellent optical and chemical yields. A new high-performance liquid chromatography (HPLC)-based enantioselective assay technique was developed for the enzyme, which promotes the addition of KCN to benzaldehyde in a buffered solution (pH 4.0). Asymmetric synthesis of (7 )-cyanohydrins by a new HNL is described (Figure 8.4). ... 

Figure 6. HPLC kinetics of polygalacturonic acid depolymerization by extracellular pectate lyases from crude supernatants of Erwinia chrysanthemi and Lachnospira multiparus cultures. A panels are full scale representations of products found over the reaction time sequence. B panels have expanded ordinates to better demonstrate the kinetics of minor products. Area unit refers to integration from HPLC tracings of product absorbance at 235 nm. Numbers in the panels refer to the degree of polymerization for individual products. Conditions for enzyme assay and product detection were the same as described for Figure 5.

Figure 7. HPLC kinetic profiles of reaction products of pectate lyases obtained from chromatofocused fractions of Erwinia chrysanthemi culture supernatants. Chromatofocused enzymes, eluted at pH 8.6 (A), pH 8.3 (B), pH 6.0 (C), and < pH 6.0 (D), were assayed under conditions similar to those described in Figure 5.

Schols and Voragen, 2002). Other substrates, such as citrus pectin, may be used in some cases, but overall PGase activities will probably be lower on the esterified pectins, and results from assays using the esterified pectins are more likely to be influenced by non-PGase enzymes, particularly pectin lyase and pectin methyl esterase (see Commentary). 

LVE region, see Linear viscoelastic region Lyase, pectic characterized, 342 PGase assay, interference in,... 

Reliable enzymatic assays for SeMet are not available as specific SeMet metabolizing enzymes have not been identified and enzymes such as glutamine transaminase react with Met equally as well as with SeMet (Blazon et al., 1994). However, with some enzymes reaction rates for SeMet and Met differ sufficiently to be of some use in SeMet analysis. For example, SeMet is a better substrate than Met for the a,y-elimination by i.-methionine y-lyase of Pseudomonas putida (Esaki et al., 1979). The adenosyl methionine transferase from rat liver reacts with L-SeMet at 51% of the rate with L-Met, and with the corresponding D-isomers at only 13 and 10% of the rate of L-Met (Pan and Tarver, 1967). Other adenosyl methionine transferases, such as that from yeast, react with SeMet more rapidly and with higher stereoselectivity than with Met, providing an indirect means for SeMet determination (Mudd and Cantoni, 1957 Sliwkowski, 1984 Uzar and Michaelis, 1994). 

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