Anita Eberl

Enzymatic surface hydrolysis of poly(ethylene terephthalate) and bis(benzoyloxyethyl) terephthalate by lipase and cutinase in the presence of surface active molecules

A lipase from Thermomyces lanuginosus and cutinases from Thermobifida fusca and Fusarium solani hydrolysed poly(ethylene terephthalate) (PET) fabrics and films and bis(benzoyloxyethyl) terephthalate (3PET) endo-wise as shown by MALDI-Tof-MS, LC-UVD/MS, cationic dyeing and XPS analysis. Due to interfacial activation of the lipase in the presence of Triton X-100, a seven-fold increase of hydrolysis products released from 3PET was measured. In the presence of the plasticizer N,N-diethyl-2-phenylacetamide (DEPA), increased hydrolysis rates of semi-crystalline PET films and fabrics were measured both for lipase and cutinase. The formation of novel polar groups resulted in enhanced dye ability with additional increase in colour depth by 130% and 300% for cutinase and lipase, respectively, in the presence of plasticizer.

Enzymatic hydrolysis of PTT polymers and oligomers

Oligomers and polymers (film, fabrics) of the linear aromatic polyester poly(trimethylene terephthalate) (PTT) were treated with polyesterases from Thermomyces lanuginosus, Penicillium citrinum, Thermobifida fusca and Fusarium solani pisi. The cutinase from T. fusca was found to release the highest amounts of hydrolysis products from PTT materials and was able to open and hydrolyse a cyclic PTT dimer according to RP-HPLC-UV detection. In contrast, the lipase from T. lanuginosus also showed activity on the PTT fibres and on bis(3-hydroxypropyl) terephthalate (BHPT) but was not able to hydrolyse the polymer film, mono(3-hydroxypropyl) terephthalate (MHPT) nor the cyclic dimer of PTT. As control enzymes inhibited with mercury chloride were used. Surface hydrophilicity changes were investigated with contact angle measurements and the degree of crystallinity changes were determined with DSC.

Hydrolysis of PET and bis-(benzoyloxyethyl) terephthalate with a new polyesterase from Penicillium citrinum

A polyethylene terephthalate (PET) model substrate, bis-(benzoyloxyethyl)terephthalate (3PET), was used to screen for micro-organisms producing enzymes hydrolyzing PET. From this screen, a strain growing on 3PET was isolated and identified as Penicillium citrinum. The polyesterase responsible for 3PET and PET hydrolysis was purified to electrophoretic homogeneity. The polyesterase had a molecular weight of 14.1 kDa, and the Km and Kcat values on 4-nitrophenyl butyrate were 0.57 mM and 0.21 s−1, respectively. Highest enzyme activities were obtained when P. citrinum was grown on a medium containing cutin, which was hydrolyzed by the polyesterase. Surface hydrolysis of PET with the enzyme lead to an increase in hydrophilicity based on rising height (+5.1 cm) and drop dissipation measurements (55 s). Both from PET and 3PET bis-(2-hydroxyethyl)terephthalate and mono-(2-hydroxyethyl)terephthalate were released, while only low amounts of terephthalic acid were liberated.

A Novel Aryl Acylamidase From Nocardia farcinica Hydrolyses Polyamide

An alkali stable polyamidase was isolated from a new strain of Nocardia farcinica. The enzyme consists of four subunits with a total molecular weight of 190 kDa. The polyamidase cleaved amide and ester bonds of water insoluble model substrates like adipic acid bishexylamide and bis(benzoyloxyethyl)terephthalate and hydrolyzed different soluble amides to the corresponding acid. Treatment of polyamide 6 with this amidase led to an increased hydrophilicity based on rising height and tensiometry measurements and evidence of surface hydrolysis of polyamide 6 is shown. In addition to amidase activity, the enzyme showed activity on p‐nitrophenylbutyrate. On hexanoamide the amidase exhibited a Km value of 5.5 mM compared to 0.07 mM for p‐nitroacetanilide. The polyamidase belongs to the amidase signature family and is closely related to aryl acylamidases from different strains/species of Nocardia and to the 6‐aminohexanoate‐cyclic dimer hydrolase (EI) from Arthrobacter sp. KI72. Biotechnol. Bioeng. 2009;102: 1003–1011.

Enzymatic surface hydrolysis of PET enhances bonding in PVC coating

The effect of polyesterase preparations from Thermomyces lanuginosus and Beauveria brongniartii on the hydrophilicity of PET materials was assessed. As a result of polyesterase treatment the hydrophilicity of PET fabrics was increased by up to 8 cm in terms of rising height with increases in surface tension from 6.2 mNm (heat-inactivated control samples) to above 8 mNm. Both enzymes were able to increase the amount of hydroxyl groups on PET from 90 to a maximum of 182 mmol kg−1, while only the B. brongniartii polyesterase released significant amounts of terephthalic acid from PET. Enzymatic surface hydrolysis of PET increased the bonding strength in PVC coating to 13.40 daN 5cm−1 using 0.5% adhesive compared to 11.5 daN 5cm−1 obtained without enzyme pretreatment and 6% of adhesive.

Surface hydrolysis of polyamide with a new polyamidase from Beauveria brongniartii

Twelve fungi were screened for the potential of their extracellular enzymes to increase the hydrophilicity of polyamide (PA) materials. The most pronounced increase in hydrophilicity was found for enzymes from Beauveria brongniartii and B. bassiana. The 55 kDa polyamidase from B. brongniartii was purified using ultrafiltration, anion exchange chromatography and size exclusion chromatography. This polyamidase was able to hydrolyse adipic acid bishexylamide and various typical amidase substrates, but did not show protease activity. In contrast, the 27 kDa protease from B. brongniartii did not show activity on PA. The improvement of hydrophilicity due to hydrolysis with the 55 kDa polyamidase from B. brongniartii based on rising height was 11 cm for PA 6 Perlon fibres and 5 cm for PA 6.6 Nylon. The drop dissipation measurement corroborated the improvement of the hydrophilicity giving 7 s and less than 1 s for the two enzyme treated materials, respectively. The surface tension s of Perlon increased from 46.1 to 67.4 mNm after enzyme treatment.

Hydroxylation of polypropylene using the monooxygenase mutant 139-3 from Bacillus megaterium BM3

Enzymatic hydroxylation of polypropylene (PP) was investigated in order to increase hydrophilicity. A mutant (139-3) of the P450monooxygenase from Bacillus megaterium expressed in E. coli DH5α was purified using anion exchange chromatography. Hydroxylation of PP fabrics led to a dramatic increase of hydrophilicity as indicated by a water drop dissipation time of below 1 s compared to the hydrophobic reference material. Likewise, a 4.9 cm increase of rising height was measured which remained consistent after 144 h of storage. Similarly, enzymatic hydroxylation of PP films lead to a decrease of the WCA from 104.6° to 77.3° with no major change after exposure to air for 6 days. Using X-ray photoelectron spectroscopy, an increase in normalized atomic concentrations of oxygen from 1.40 to 4.98% for the CO-inhibited and enzyme treated sample, respectively, was measured confirming enzymatic hydroxylation.