The coupled oxidation of protoheme IX at the active sites of heme proteins exposed to ascorbate in the presence of dioxygen has been known for over 65 years and is related to the catalytic conversion of protoheme IX to biliverdin by the enzyme heme oxygenase. The present report demonstrates that replacement of two residues present in the distal heme pocket of horse heart myoglobin (Mb), Val67 and Val68, with alanine and serine, respectively, increases the efficiency of coupled oxidation in the active site of myoglobin. HPLC analysis of the heme oxidation product demonstrates that the specificity of wild-type myoglobin for opening the heme ring at the R-meso carbon is retained by the variant. The infrared spectrum of the carbonyl derivative of the variant exhibits ν CO bands of comparable intensity at 1948 and 1958 cm -1 , which are consistent with the presence of the polar serine residue in close proximity to the bound ligand. The high-resolution crystal structure of the Val67Ala/Val68Ser metMb variant establishes that a hydrogen bond forms between the hydroxyl group of Ser68 and the coordinated water molecule. The increased rate of coupled oxidation exhibited by the variant is attributed to the increased polarity of the distal pocket created by the amino acid substitutions. These results are discussed in light of recent work concerning the active site of heme oxygenase.
Avermectins produced by Streptomyces avermitilis are potent against a broad spectrum of nematode and arthropod parasites with low-level side effects on the host organisms. This study was designed to investigate a high-throughput screening strategy for the efficient identification of avermectin high-yield strains. The production protocol was miniaturized in 96 deep-well microplates. UV absorbance at 245 nm was used to monitor avermectin production. A good correlation between fermentation results in both 96 deep-well microplates and conventional Erlenmeyer flasks was observed. With this protocol, the production of avermectins was determined in less than 10 min for a full plate without compromising accuracy. The high-yield strain selected through this protocol was also tested in 360 m(3) batch fermentation with 1.6-fold improved outcome. Thus, the development of this protocol is expected to accelerate the selection of superior avermectin-producing strains.
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