Characterization and purification of coal depolymerizing enzymes and use of RAPD methods for developing genus and strain-specific Streptomyces probes.

摘要

Actinomycetes, in particular Streptomyces, have been isolated and cultured for commercial production of hundreds of bioactive compounds. Isolation, screening, environmentally tracking and taxonomic identification of those strains that produce similar products is important.; The first manuscript used a Randomly Amplified Polymorphic DNA (RAPD) method for generating strain- and taxon-specific DNA probes for environmental tracking of Streptomyces. A Streptomyces-specific and two DNA fragments unique for Streptomyces lydicus WYEC108 were found and analyzed for specificity. Environmental extraction of DNA and subsequent probing of the indigenous soil/rhizosphere DNA showed that RAPD can target and track specific bacterial strains.; Lignites are characterized by low calorific value due to their low carbon and high oxygen contents thus they are unsuitable for power production. It is therefore important to seek economically feasible methods to utilize this resource.; Pseudomonas cepacia DLC-62 produces an extracellular product or products that non-oxidatively depolymerize lignite coal. This transformation operates at ambient pressures and temperatures while decreasing the average molecular weight of the parent coal. The last two manuscripts describe potential enzymatic processes that may have a role in coal microbial biotransformation. The enzymes present in active extracellular culture filtrates of DLC-62 were characterized and found to be composed of multiple esterases and peroxidases. Partially purified or purified esterases from hydrophobic interaction and anion exchange chromatography were incubated with coal polymer. Under these conditions, they exhibited no depolymerizing activity. However, crude ultra-filtered culture filtrates containing both esterases and peroxidases were active. In the latter case, however, coal-depolymerizing activity was independent of exogenously added H{dollar}sb2{dollar}O{dollar}sb2{dollar}. The esterases of strain DLC-62 have been purified by hydrophobic interaction chromatography (HIC) and Fast Protein Liquid Chromatography (FPLC).; A novel coal-ligand affinity chromatography procedure was developed for separation of enzymes based on their affinity for coal. The chromatography procedure was used to isolate and characterize the affinity of the three esterase isoforms for coal.