A two step enzymatic process for grafting phenolics onto polyamides (PAs) was developed in order to impart special functionalities to inert PA. Therefore, a polyamidase (NfpolyA) from Nocardia farcinica was overexpressed in Escherichia coli BL21-Gold(DE3) and purified in a single step. With p-nitroacetanilide as a substrate, NfpolyA revealed a specific activity of 20Umg−1 compared to 1.5Umg−1 for the wild-type enzyme. NfpolyA showed a KM value of 0.12±0.01mM and a kcat of 19.1s−1 which were both higher than measured for the wild-type enzyme (kcat=3.5s−1; KM=0.06mM). A laccase from Trametes hirsuta was used to oxidize ferulic acid, used as a phenol model substrate and study the covalent grafting of n-butylamine, as model substrate for PA. According to LC–MS, up to three equivalents of n-butylamine were bound to ferulic acid after laccase oxidation of ferulic acid. Both enzymes were used sequentially in a two step process. In a first step the polyamidase is used to partially hydrolyze the amide bond, leading to a surface with amine and carboxylic acids. In a second step, by using a laccase from T. hirsuta ferulic acid was grafted onto the surface of PA as confirmed with FTIR-ATR analysis.

From a screening on agar plates with bis(benzoyloxyethyl) terephthalate (3PET), a Bacillus subtilis p ‐nitrobenzylesterase (BsEstB) was isolated and demonstrated to hydrolyze polyethyleneterephthalate (PET). PET‐hydrolase active strains produced clearing zones and led to the release of the 3PET hydrolysis products terephthalic acid (TA), benzoic acid (BA), 2‐hydroxyethyl benzoate (HEB), and mono‐(2‐hydroxyethyl) terephthalate (MHET) in 3PET supplemented liquid cultures. The 3PET‐hydrolase was isolated from non‐denaturating polyacrylamide gels using fluorescein diacetate (FDA) and identified as BsEstB by LC‐MS/MS analysis. BsEstB was expressed in Escherichia coli with C‐terminally fused StrepTag II for purification. The tagged enzyme had a molecular mass of 55.2 kDa and a specific activity of 77 U/mg on p ‐nitrophenyl acetate and 108 U/mg on p ‐nitrophenyl butyrate. BsEstB was most active at 40°C and pH 7.0 and stable for several days at pH 7.0 and 37°C while the half‐life times decreased to 3 days at 40°C and only 6 h at 45°C. From 3PET, BsEstB released TA, MHET, and BA, but neither bis(2‐hydroxyethyl) terephthalate (BHET) nor hydroxyethylbenzoate (HEB). The k cat values decreased with increasing complexity of the substrate from 6 and 8 (s−1) for p ‐nitrophenyl‐acetate (4NPA) and p ‐nitrophenyl‐butyrate (4NPB), respectively, to 0.14 (s−1) for bis(2‐hydroxyethyl) terephthalate (BHET). The enzyme hydrolyzed PET films releasing TA and MHET with a concomitant decrease of the water‐contact angle (WCA) from 68.2° ± 1.7° to 62.6° ± 1.1° due to formation of novel hydroxyl and carboxyl groups. These data correlated with a fluorescence emission intensity increase seen for the enzyme treated sample after derivatization with 2‐(bromomethyl)naphthalene. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011

Functionalisation of synthetic polymers by using enzymes has been recently demonstrated. The major advantage of enzymes over chemical processes lies in their surface specific and endo-wise mode of action. Surface hydrophilisation of PET with lipases and cutinases leads to a dramatic increase of the surfacial acid and hydroxyl group content while conventional chemical treatment does not cause any change. However, this PET-hydrolysing activity by enzymes from distinct classes has not yet been correlated to activity on natural polyesters. Here, we show that lipases, cutinases and a PHA-depolymerase are all capable of hydrolysing PET, while only lipases and cutinases also hydrolysed cutin to various degrees. Lipases showed a higher specificity for terminal fatty acids while the cutinases preferred hydroxy fatty acids during cutin hydrolysis.

Macromolecular SymposiaVolume 296, Issue 1 p. v-xix ContentsFree Access Macromol. Symp. 296 First published: 29 November 2010 https://doi.org/10.1002/masy.201090028AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Volume296, Issue1Special Issue: Modern Trends in Polymer Science – EPF'09 –October 2010Pages v-xix RelatedInformation

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 K m 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. © 2008 Wiley Periodicals, Inc.

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.

Alkaline and enzymatic hydrolyzes of poly(ethylene terephthalate) fabrics (PET) were mechanistically compared based on released degradation products (HPLC-UV-RI) and changes in surface properties [hydrophilicity, cationic dyeing, Xray photoelectron spectroscopy (XPS)]. Enzymatic hydrolysis led to an increase in the amount of hydroxyl and carboxyl groups on the surface resulting in an enhanced water absorption and dyeability. Enzymes partially adsorbed to PET fabrics during hydrolysis were completely removed by subsequent extraction according to XPS analysis. In contrast to the enzyme treatment, alkaline hydrolysis did not lead to an increase of hydroxyl and acid groups according to XPS while both treatments caused a substantial increase in hydrophilicity and were more effective on amorphous fibers. Alkaline hydrolysis led to a greater increase in the K/S value after cationic dyeing due to enlarged surface area. Consequently, ESEM-images demonstrated that alkaline treatment drastically affected the surface morphology of the polymer resulting in crater-like structures of the fibers, whereas after enzymatic treatment the morphology of the fibers remained unchanged. To reach similar benefits in hydrophilicity, drastically higher amounts of degradation products were released during alkaline hydrolysis as also indicated by >6% weight loss compared to <1% after enzyme treatment. (C) 2008 Wiley Periodicals, Inc.

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 new Micrococcus luteus strain BST20 was isolated with ability to metabolize PAN polymers as sole carbon source. Out of seven synthetized PAN copolymers containing different moieties of acrylic acid and/or vinyl acetate the polymer with lowest crystallinity (PAN with 5% vinyl acetate) was most easily metabolized. (13)C labelled PAN was completely converted to the acrylic acid by this strain. M. luteus BST20 produced membrane-bound nitrile hydrolysing enzymes able to convert nitrile groups on PAN powder surface to the corresponding acids. Similarly, nitrile groups on PAN fabrics were transformed to the corresponding acid as indicated by an K/S increased after dying with Methylene blue and the released ammonia. On small soluble substrates the enzyme system showed a preference for aliphatic and aromatic substituted aliphatic nitriles.

A new strain of Agrobacterium tumefaciens (BST05) was found to grow on polyacrylonitrile (PAN; C-13 labelled) converting the polymer to polyacrylic acid as shown by solid state NMR. When cultivated in a medium containing acetonitrile the bacterium produced nitrile hydratase and amidase activity. Activity recovery after lyophilisation and enzyme stability was significantly enhanced in the presence of 5% sorbitol leading to half life times of 12, 72 and 154 days at 25 degrees C, 4 degrees C and -20 degrees C. The enzymes were able to convert 1.1% of the nitrile groups of PAN-powder to the corresponding acids. PAN fabrics were mainly converted to the amides as shown by an 80% increase of the O/C ratio in ESCA analysis. These data were confirmed by cationic dyeing and FTIR-ATR analysis.

Recently the potential of enzymes for surface hydrophilisation and/or functionalisation of polyethyleneterephthalate (PET) and polyamide (PA) has been discovered. However, there was no correlation between enzyme class/activity (e.g. esterase, lipase, cutinase) and surface hydrolysis of these polymers and consequently no simple assay to estimate this capability. Enzymes active on the model substrates bis (benzoyloxyethyl) terephthalate and adipic acid bishexyl-amide, were also capable of increasing the hydrophilicity of PET and PA. When dosed at the identical activity on 4-nitrophenyl butyrate, only enzymes from Thermobifida fusca, Aspergillus sp., Beauveria sp. and commercial enzymes (TEXAZYME PES sp5 and Lipase PS) increased the hydrophilicity of PET fibres while other esterases and lipases did not show any effect. Activity on PET correlated with the activity on the model substrate. Hydrophilicity of fibres was greatly improved based on increases in rising height of up to 4.3 cm and the relative decrease of water absorption time between control and sample of the water was up to 76%. Similarly, enzymes increasing the hydrophilicity of PA fibres such as from Nocardia sp., Beauveria sp. and F. solani hydrolysed the model substrate; however, there was no common enzyme activity (e.g. protease, esterase, amidase) which could be attributed to all these enzymes.