Production of a polyester degrading extracellular hydrolase from Thermomonospora fusca.
The production of a polyester-degrading hydrolase from the thermophilic actinomycete Thermomonospora fusca was investigated with regard to its potential technical application. Only in the presence of a polyester (random aliphatic-aromatic copolyester from 1,4-butanediol, terephthalic acid, and adipic acid with around 40-50 mol % terephthalic acid in the acid component), the excretion of the extracellular enzyme could be achieved with an optimized synthetic medium using pectin and NH(4)Cl as nitrogen source. Compared to complex media, a significantly higher specific activity at comparable volumetric yields could be obtained, thus reducing the expenditure for purification. The activity profile in the medium is controlled by a complex process involving (1) induction of enzyme excretion, (2) enzyme adsorption on the hydrophobic polyester surface, (3) inhibition of enzyme generation by monomers produced by polyester cleavage, and (4) enzyme denaturation. Diafiltration with cellulose acetate membranes as the sole downstream processing step led to a product of high purity and with sufficient yield (60% of total activity). Scaling-up from shaking flasks to a fermentor scale of 100 L revealed no specific problems. However, the excretion of the hydrolase by the actinomycete turned out to be inhibited by the degradation products (monomers) of the aliphatic-aromatic copolyester used as inductor for the enzyme production. The crude enzyme exhibited generally similar properties (temperature and pH optimum) as the highly purified hydrolase described previously; however, the storage capability and thermal stability is improved when the crude enzyme solution is diafiltrated.
D000192:Actinomycetales / Q000145:classification / Q000201:enzymology*; D001673:Biodegradation, Environmental; D019149:Bioreactors; D002208:Caproates / Q000378:metabolism*; D002460:Cell Line; D004789:Enzyme Activation; D004795:Enzyme Stability; D005285:Fermentation; D006863:Hydrogen-Ion Concentration; D006867:Hydrolases / Q000302:isolation & purification* / Q000378:metabolism*; D006868:Hydrolysis; D007783:Lactones / Q000378:metabolism*; D008049:Lipase / Q000302:isolation & purification / Q000378:metabolism; D010865:Pilot Projects; D011091:Polyesters / Q000378:metabolism* / Q000494:pharmacology; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013045:Species Specificity; D013379:Substrate Specificity; D013696:Temperature; D014462:Ultrafiltration
D002208:Caproates; D007783:Lactones; D011091:Polyesters; C472388:ecoflex; C121056:caprolactone; D006867:Hydrolases; D008049:Lipase