Setting the stage for evolution of a new enzyme

The evolution of novel enzymes has fueled the diversification of life on earth for billions of years. Insights into events that set the stage for the evolution of a new enzyme can be obtained from ancestral reconstruction and laboratory evolution. Ancestral reconstruction can reveal the emergence of a promiscuous activity in a pre-existing protein and the impact of subsequent mutations that enhance a new activity. Laboratory evolution provides a more holistic view by revealing mutations elsewhere in the genome that indirectly enhance the level of a newly important enzymatic activity. This review will highlight recent studies that probe the early stages of the evolution of a new enzyme from these complementary points of view.     

Characterization of glucose oxidase-negative mutants of a lignin degrading basidiomycete Phanerochaete chrysosporium

The isolation and characterization of glucose oxidase-negative (gox ^-) mutants of Phanerochaete chrysosporium, is described. These mutants are deficient not only in their ability to produce hydrogen peroxide (H₂O₂) but also in lignin degradation (2-¹⁴C-synthetic lignin¹⁴CO₂), ligninase and peroxidase activities, decolorization of the dye poly-R 481, and production of ethylene from -oxo--methylthiobutyric acid (KTBA). The gox ^- mutants retained, albeit at a lower level, the capacity to produce veratryl alcohol, a typical secondary metabolite, and produced conidia at a level comparable to that of the wild type. The addition of ligninase and/or glucose oxidase to a gox ^- mutant (GOX-10) did not enhance its capacity to degrade lignin. The Gox⁺ revertant strains regained glucose oxidase activity, the ability to degrade lignin, as well as the other characteristics that were missing in the gox ^- mutants. The results suggest that the genetic lesion in these mutants affects the regulation of a set of secondary metabolic characteristics.Abbreviations Gox glucose oxidase - KTBA -oxo--methylthiobutyric acid Journal article no. 11740 from the Michigan Agricultural Experiment Station     

Influence of some surfactants and related compoundson ligninolytic activity of Phanerochaete chrysosporium

Abstract Several surfactants were tested as possible stimulators of ligninase production in agitated culture of Phanerochaete chrysosporium . In addition to Tween 80 and Tween 20, surfactants already known to have a stimulatory effect, polyoxyethylene oleate (15EO)C_18:1 and the hydrophilic fraction of Tween 80, were also found to enhance ligninase activity over 200-fold, indicating that surfactants as such may not be the true active substances. Cultures with increased ligninase activities exhibited preferential enrichment of total and polar lipids in palmitoleic acid and an increased unsaturation index.     

Development of a stirred tank reactor system for the production of lignin peroxidases (ligninases) byPhanerochaete chrysosporium BKM-F-1767

Summary Lignin peroxidases produced byPhanerochaete chrysosporium have several important potential industrial applications based on their ability to degrade lignin and lignin-like compounds. A stirred tank reactor system for the production of lignin peroxidases is described here. Included in this study is an examination of the mechanics of pellet biocatalyst formation and the optimization of an acetate buffered medium. Higher levels of lignin peroxidase were obtained with acetate buffer compared to the other buffer systems tested. Concentrations of 0.05% (w/v) Tween 80 and 0.4 mM veratryl alcohol gave optimal lignin peroxidase activity in acetate buffered medium. In shake flask cultures, mycelial fragments in the inoculum aggregated into pellets during the first eight hours of incubation and thereafter increased in size through the eighth day. The agitation rate in shake flask cultures affected pellet size, the number of pellets formed, and lignin peroxidase activity. Transfer of fungal pellets from shake flask culture to a continuously oxygenated baffled stirred tank reactor (STR) resulted in production of high lignin peroxidase titres comparable to those of shake flask cultures when the agitation rate, oxygen dispersion and foaming were closely controlled.     

Modification of malachite green by Fomes sclerodermeus and reduction of toxicity to Phanerochaete chrysosporium

Malachite green (MG) is a triphenylmethane dye used as a fungicide but also possesses a high toxicity to mammalian cells. The toxicity of MG to Fomes sclerodermeus and Phanerochaete chrysosporium was assessed. P. chrysosporium was highly sensitive to the dye and it was unable to grow on solid media containing 64 microM of MG, lower concentrations caused a delay in growth. The radial growth of F. sclerodermeus was not affected at this concentration and up to 128 microM. In liquid media both fungi were more sensitive. F. sclerodermeus not only was able to grow in the presence of high concentrations of MG, but also it was able to decolorize and detoxify the dye. MG treated with supernatants containing high laccase activity in the presence or absence of 1-hydroxybenzotriazole (1-HBT) gave a colorless product (DMG) that was not toxic to P. chrysosporium and other white rot fungi tested. On the basis of the data of maximal absorbance, it is probable that the mechanism involved in the modification of the dye was different if 1-HBT was added to the reaction.     

Bioconversion of lignocellulose and simultaneous production of cellulase,ligninase and bioflocculants by Alcaligenes faecalis-X3

Lignocelluloses have been used as carbon sources for bioflocculant production. However, the low bioconversion efficiency of lignocellulose to bioflocculants is a major challenge. In this study, a lignocellulolytic strain of Alcaligenes faecalis-X3 was cultivated in ramie bio-degumming wastewater. Optimal production of ligninase, cellulase and bioflocculants (MBF-X3) was evaluated. The highest activity of MBF-X3 under the optimal conditions of pH 6.0 at 48 h of fermentation was 95.44%, with the maximum production of ligninase and cellulase (0.27 and 0.12 U/mL, respectively). The crude ligninase and cellulase had optimum activities at pH 5.0 and 40 °C and pH 6.0 and 50 °C, respectively. The cellulase activity was increased by Mn²⁺, Ca²⁺, Zn²⁺, and Mg²⁺ at 1 mM. The ligninase activity was significantly enhanced in the presence of Zn²⁺ at 10 mM. The flocculating activity of MBF-X3 was not changed by the addition of any metal cation. The results demonstrated that A. faecalis possesses an excellent enzyme system for the efficient bioconversion of lignocellulose into MBF-X3. Additionally, MBF-X3 has a high flocculating efficiency of Disperse Blue-2BLN (85.7%) at a dose of 1.0 g/L.