Biotechnological applications of bioluminescence and chemiluminescence

Biotechnology applications of horseradish peroxidase (HRP) would benefit from access to tailor-made variants with greater specific activity, lower K(m) for peroxide, and higher thermostability. Starting with a mutant that is functionally expressed in Saccharomyces cerevisiae, we used random mutagenesis, recombination, and screening to identify HRP-C mutants that are more active and stable to incubation in hydrogen peroxide at 50 degrees C. A single mutation (N175S) in the HRP active site was found to improve thermal stability. Introducing this mutation into an HRP variant evolved for higher activity yielded HRP 13A7-N175S, whose half-life at 60 degrees C and pH 7.0 is three times that of wild-type (recombinant) HRP and a commercially available HRP preparation from Sigma (St. Louis, MO). The variant is also more stable in the presence of H(2)O(2), SDS, salts (NaCl and urea), and at different pH values. Furthermore, this variant is more active towards a variety of small organic substrates frequently used in diagnostic applications. Site-directed mutagenesis to replace each of the four methionine residues in HRP (M83, M181, M281, M284) with isoleucine revealed no mutation that significantly increased the enzyme's stability to hydrogen peroxide.

Functional expression and stabilization of horseradish peroxidase by directed evolution in Saccharomyces cerevisiae

Luminol-type chemiluminescence derivatization reagents for liquid chromatography and capillary electrophoresis.

Firefly luciferase is a member of the acyl-adenylate/thioester-forming superfamily of enzymes and catalyzes the oxidation of firefly luciferin with molecular oxygen to emit light. Knowledge of the luminescence mechanism catalyzed by firefly luciferase has been gathered, leading to the discovery of a novel catalytic function of luciferase. Recently, we demonstrated that firefly luciferase has a catalytic function of fatty acyl-CoA synthesis from fatty acids in the presence of ATP, Mg2+ and coenzyme A. Based on identification of fatty acyl-CoA genes in firefly, Drosophila, and non-luminous click beetles, we then proposed that the evolutionary origin of firefly luciferase is a fatty acyl-CoA synthetase in insects. Further, we succeeded in converting the fatty acyl-CoA synthetase of non-luminous insects into functional luciferase showing luminescence activity by site-directed mutagenesis.

Firefly luciferase: an adenylate-forming enzyme for multicatalytic functions.

The chemistry of pyrimidines and their benzo derivatives, including quinazolines, perimidines and benzo[gh]perimidines is reviewed in this chapter, with particular attention paid to work published between 1996 and 2007, which covers the period since the publication of the second edition of Comprehensive Heterocyclic Chemistry. Fully aromatic, and both partially and fully reduced species are included, and oxo derivatives, including nucleosides and nucleotides are also covered. New routes to the synthesis of many of these heterocycles, as well as modern synthetic techniques, including microwave assisted procedures, are described. Both ring synthesis and substituent modification procedures are covered. The relative reactivity of different substituent groups and/or substituent positions is also discussed, including several examples of selective metal catalyzed cross-coupling reactions. Metal catalyzed amination and amidation reactions are also described. The importance of pyrimidine and quinazoline heterocyclic compounds in biological systems is also emphasized, since two of the four DNA bases are pyrimidine derivatives, and many modern pharmaceutical agents contain either pyrimidine or quinazoline fragments. Several examples of biologically important pyrimidine and quinazoline derivatives are identified, and in this context the signal transduction inhibitor imatinib (gleevec) is highlighted as one of the most significant new pyrimidine derivatives to have been identified in the last few years.

Pyrimidines and their Benzo Derivatives

A bifunctional luminogenic substrate for two luminescent enzymes: firefly luciferase and horseradish peroxidase.

The effects of three opiate analgesics, isolated from opium, on the firefly luciferase enzyme have been studied Morphine (MN), 6-acetylmorphine (MAM), and diacetylmorphine (DAM) inhibited the enzyme activity at different levels At lower concentrations, MN and MAM enhanced enzyme activity, effecting inhibition at higher concentrations However, DAM inhibited the enzyme activity at all concentrations investigated The stimulating activity of MN and MAM is attributed to the hydrophilic interaction of the proton donor-acceptor type with the polar regions of the luciferase located outside the binding pocket of the active site The inhibition at higher concentrations of MN and MAM and at all concentrations of DAM is found to be competitive in nature, with the analgesics competing for the binding of the enzyme's natural substrate luciferin The binding site of the luciferase could accommodate only one analgesic molecule Binding constants determined from bioluminescence studies showed that the inhibitor binding site is hydrophobic in nature The inhibition constants of analgesics are in the order MN > MAM > DAM The greater binding of DAM to luciferase is attributed to its ability to form a ground state complex with ATP and greater hydrophobicity At higher concentrations of ATP, the binding constants increased The results obtained are explained assuming that the firefly luciferase acts as a subtype mu-opioid receptor model

T. Sudhaharan

Papers

A bifunctional luminogenic substrate for two luminescent enzymes: firefly luciferase and horseradish peroxidase.

Opiate analgesics' dual role in firefly luciferase activity.

Dye induced quenching of firefly luciferase-luciferin bioluminescence.