Production of a Polyester Degrading Extracellular Hydrolase from Thermomonospora fusca

Abstract

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.

Degradation of poly(ε-caprolactone) by a thermophilic bacterium Ralstonia sp. strain MRL-TL isolated from hot spring

“…SEM indicates changes in surface morphology such as roughness, groves, pits and cracks formation as a result of microbial growth at its surface (Das and Kumar, 2013). The film degradation in our experiment is in accord with those previously reported, and indicates that the prominent surface structure changes occurs due to the microbial degradability of polymers (Gouda et al, 2002;Li et al, 2012). However, the factors such as crystallinity of polymer film and increased secretion of PCL degrading enzyme in the culture should also be considered.…”

A Practical Fluorescence-Based Screening Protocol for Polyethylene Terephthalate Degrading Microorganisms

“…A comparable result was observed by Gouda et al 29 when a polyester-degrading hydrolase was only produced in the presence of a BTA copolyester (1,4-butanediol, terephthalic acid and adipic acid; around 40-50 mol% terephthalic acid in the acid component). A previous similar approach to induce PET degrading enzymes resulted in 34% increase in lipase activity when BHET was used as the inducer, but was lower (4%) when a PET short fiber was used.…”

Enzymatic Degradation of Poly(ethylene terephthalate): Rapid Hydrolyse using a Hydrolase from T. fusca

“…[9] rTfH is a recombinant hydrolase (gene of TfH from Thermobifida fusca transferred into E. Coli), which was purified by chromatography to gel-chromatographic purity. [8] The lipase from Pseudomonas sp.…”