Laccase catalyzed elimination of morphine from aqueous systems.

Pharmaceuticals contaminate the environment for several reasons, including metabolic excretion after intake, industrial waste and improper disposal. The narcotic drug morphine is commonly utilized for chronic pain management, and the distribution of morphine in aqueous systems and in waste waters is of high concern. Here, the removal of morphine by a laccase from Myceliophthora thermophila both in its free form as well as immobilized on Accurel MP1000 beads was investigated. Complete morphine elimination was achieved within 30 min for the free and the immobilized enzyme (70% bound protein) for concentrations between 1 and 1,000 mg L-1 according to LC-TOF mass spectrometry analysis. Higher morphine concentrations up to 60 g L-1 were also tested and total elimination was achieved within 6 h. Therefore, laccases are ideal candidates for removing morphine from aqueous systems.

Data on synthesis of oligomeric and polymeric poly(butylene adipate-co-butylene terephthalate) model substrates for the investigation of enzymatic hydrolysis.

The aliphatic-aromatic copolyester poly(butylene adipate-co-butylene terephthalate) (PBAT), also known as ecoflex, contains adipic acid, 1,4-butanediol and terephthalic acid and is proven to be compostable [1], [2], [3]). We describe here data for the synthesis and analysis of poly(butylene adipate-co-butylene terephthalate variants with different adipic acid:terephatalic acid ratios and 6 oligomeric PBAT model substrates. Data for the synthesis of the following oligomeric model substrates are described: mono(4-hydroxybutyl) terephthalate (BTa), bis(4-(hexanoyloxy)butyl) terephthalate (HaBTaBHa), bis(4-(decanoyloxy)butyl) terephthalate (DaBTaBDa), bis(4-(tetradecanoyloxy)butyl) terephthalate (TdaBTaBTda), bis(4-hydroxyhexyl) terephthalate (HTaH) and bis(4-(benzoyloxy)butyl) terephthalate (BaBTaBBa). Polymeric PBAT variants were synthesized with adipic acid:terephatalic acid ratios of 100:0, 90:10, 80:20, 70:30, 60:40 and 50:50. These polymeric and oligomeric substances were used as ecoflex model substrates in enzymatic hydrolysis experiments in the article "Substrate specificities of cutinases on aliphatic-aromatic polyesters and on their model substrates" [4].

An Esterase from Anaerobic Clostridium hathewayi Can Hydrolyze Aliphatic-Aromatic Polyesters.

Recently, a variety of biodegradable polymers have been developed as alternatives to recalcitrant materials. Although many studies on polyester biodegradability have focused on aerobic environments, there is much less known on biodegradation of polyesters in natural and artificial anaerobic habitats. Consequently, the potential of anaerobic biogas sludge to hydrolyze the synthetic compostable polyester PBAT (poly(butylene adipate-co-butylene terephthalate) was evaluated in this study. On the basis of reverse-phase high-performance liquid chromatography (RP-HPLC) analysis, accumulation of terephthalic acid (Ta) was observed in all anaerobic batches within the first 14 days. Thereafter, a decline of Ta was observed, which occurred presumably due to consumption by the microbial population. The esterase Chath_Est1 from the anaerobic risk 1 strain Clostridium hathewayi DSM-13479 was found to hydrolyze PBAT. Detailed characterization of this esterase including elucidation of the crystal structure was performed. The crystal structure indicates that Chath_Est1 belongs to the α/ß-hydrolases family. This study gives a clear hint that also micro-organisms in anaerobic habitats can degrade manmade PBAT.

Substrate specificities of cutinases on aliphatic-aromatic polyesters and on their model substrates.

The enzymatic hydrolysis of the biodegradable polyester ecoflex and of a variety of oligomeric and polymeric ecoflex model substrates was investigated. For this purpose, substrate specificities of two enzymes of typical compost inhabitants, namely a fungal cutinase from Humicola insolens (HiC) and a bacterial cutinase from Thermobifida cellulosilytica (Thc_Cut1) were compared. Model substrates were systematically designed with variations of the chain length of the alcohol and the acid as well as with varying content of the aromatic constituent terephthalic acid (Ta). HPLC/MS identification and quantification of the hydrolysis products terephthalic acid (Ta), benzoic acid (Ba), adipic acid (Ada), mono(4-hydroxybutyl) terephthalate (BTa), mono-(2-hydroxyethyl) terephthalate (ETa), mono-(6-hydroxyhexyl) terephthalate (HTa) and bis(4-hydroxybutyl) terephthalate (BTaB) indicated that these enzymes indeed hydrolyze the tested esters. Shorter terminal chain length acids but longer chain length alcohols in oligomeric model substrates were generally hydrolyzed more efficiently. Thc_Cut1 hydrolyzed aromatic ester bonds more efficiently than HiC resulting in up to 3-fold higher concentrations of the monomeric hydrolysis product Ta. Nevertheless, HiC exhibited a higher overall hydrolytic activity on the tested polyesters, resulting in 2-fold higher concentration of released molecules. Thermogravimetry and differential scanning calorimetry (TG-DSC) of the polymeric model substrates revealed a general trend that a lower difference between melting temperature (Tm) and the temperature at which the enzymatic degradation takes place resulted in higher susceptibility to enzymatic hydrolysis.

Hydrolysis of synthetic polyesters by Clostridium botulinum esterases.

Two novel esterases from the anaerobe Clostridium botulinum ATCC 3502 (Cbotu_EstA and Cbotu_EstB) were expressed in Escherichia coli BL21-Gold(DE3) and were found to hydrolyze the polyester poly(butylene adipate-co-butylene terephthalate) (PBAT). The active site residues (triad Ser, Asp, His) are present in both enzymes at the same location only with some amino acid variations near the active site at the surrounding of aspartate. Yet, Cbotu_EstA showed higher kcat values on para-nitrophenyl butyrate and para-nitrophenyl acetate and was considerably more active (sixfold) on PBAT. The entrance to the active site of the modeled Cbotu_EstB appears more narrowed compared to the crystal structure of Cbotu_EstA and the N-terminus is shorter which could explain its lower activity on PBAT. The Cbotu_EstA crystal structure consists of two regions that may act as movable cap domains and a zinc metal binding site.