Hans-Peter Schär

Biodegradation of chemical waste by specialized methylotrophs, an alternative to physical methods of waste disposal

Abstract Microbial waste treatment systems were established for the disposal of chemical wastes by using specialized methylotrophs in pure, mixed or enriched cultures. Three typical mother liquors originating from the production of large-scale chemicals were selected as model wastes: a monomethyl sulfate containing mother liquor from a methylation process, a trimethylethylammonium chloride containing mother liquor from a dealkylation process and a N,N-dimethyl-formamide containing mother liquor. The microorganisms enriched and isolated for the biodegradation (mineralization) of the three model wastes were characterized and found to be bacteria of the genera Pseudomonas (trimethylethylammonium chloride utilizers and N,N-dimethylformamide utilizers) and Hyphomicrobium (monomethyl sulfate utilizers). Degradation pathways for monomethyl sulfate, trimethylethylammonium chloride and N,N-dimethylformamide are proposed. Some of the postulated novel enzymes were detected in cell-free extracts. The substrate spectra of our new species of specialized methylotrophs were investigated in order to evaluate their potential use for the biodegradation of selected organic solvents (Cl- and C2-compounds) currently used in synthesis processes (acetamide, acetonitrile, ethanol etc.). Pilot fermentations with pure substrates and model wastes are described and technical aspects and requirements of specialized microbial waste treatment systems are discussed.

Enzymatic Determination of Dimethylformamide in Waste Water Using Flow Injection Analysis

Abstract The novel enzyme N,N-dimethylformamidase has been immobilized on controlled pore glass beads and introduced into a Flow Injection Analysis manifold. Using this system it was possible to determine the dimethylformamide content of industrial waste water and process effluents in 8 of 10 examined samples. Concentrations up to 15mM could be determined with a detection limit of 0.05mM. The immobilized enzyme is stable for months and has an operational lifetime of several days. The analytical system can be used for a permanent control of the dimethylformamide concentration in industrial waste water and process effluents.

Purification and properties of an acetyl specific carboxylesterase from Nocardia mediterranei

SummaryAn acetyl specific carboxylesterase has been purified from Nocardia mediterranei. The purified enzyme is homogeneous as shown by SDS polyacrylamide gel electrophoresis. The esterase has a molecular weight of 68,000 and is composed of two identical subunits. The enzyme exhibits optimal activity at pH 7.5 and at 35°C and is stable below 40°C. The enzyme activity is inhibited by several sulfhydryl reagents. The esterase hydrolyzes preferentially acetyl esters. Propionyl esters are cleaved very slowly whereas butyryl esters are no substrates at all. In addition, the esterase shows a pronounced regiospecificity. On the other hand the enantiospecificity is rather low as demonstrated by the hydrolysis of prochiral and racemic substrates.

Applications of Microbes and Microbial Enzymes in Environmental Control and Organic Synthesis

Specialized methylotrophs, originally isolated for the specific biodegradation of chemical wastes, were found to be an excellent source of novel microbial enzymes, such as methylsulfatase, N,N-dimethylformamidase (DMFase), esterases, methanol dehydrogenase etc. The properties and possible applications of these microbes and enzymes are discussed. Mutant strains of Nocardia mediterranei (rifamycin producer) were successfully applied for the microbial production of D(−)-ribulose and shikimic acid.

d-Ribulokinase from Klebsiella pneumoniae for continuous production of d-(−)-ribulose-5-phosphate

SummaryThe production of d-ribulose-5-phosphate in an enzyme membrane reactor was examined. Phosphoryl transfer from ATP to d-ribulose was catalysed by d-ribulokinase isolated from Klebsiella pneumoniae. For production of d-ribulose-5-phosphate the phosphoryl donor ATP was used either in stoichiometric or in catalytic amounts. Using catalytic amounts of ATP requires a second enzyme, e.g. pyruvate kinase, to regenerate ATP. The kinetic parameters for d-ribulokinase and pyruvate kinase were determined to calculate the performance of an enzyme membrane reactor for continuous production of d-ribulose-5-phosphate. Both processes operated for more than 200 h. Regardless of whether ATP was used in catalytic or stoichiometric amounts, about the same production parameters were determined. In continuous production space/time yields of 117 g (with ATP regeneration) and 103 g (without ATP regeneration) of d-ribulose-5-phosphate 1−1 per day were reached.