Journal
BIOCATALYSIS AND BIOTRANSFORMATION
Volume 30, Issue 1, Pages 2-9Publisher
INFORMA HEALTHCARE
DOI: 10.3109/10242422.2012.644435
Keywords
Thermobifida alba; PET; 3PET; cutinase
Funding
- Federal Ministry of Economy, Family and Youth (BMWFJ)
- Federal Ministry of Traffic, Innovation and Technology (bmvit)
- Styrian Business Promotion Agency SFG
- Standortagentur Tirol and ZIT - Technology Agency of the City of Vienna
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A new cutinase from Thermobifi da alba (Tha_Cut1) was cloned and characterized for polyethylene terephthalate (PET) hydrolysis. Tha_Cut1 showed a high degree of identity to a T. cellulolysitica cutinase with only four amino acid differences outside the active site area, according to modeling data. Yet, Tha_Cut1 was more active in terms of PET surface hydrolysis leading to considerable improvement in hydrophilicity quantified based on a decrease of the water contact angle from 87.7 degrees to 45.0 degrees. The introduction of new carboxyl groups was confirmed and measured after esterification with the fluorescent reagent alkyl bromide, 2-(bromomethyl) naphthalene (BrNP), resulting in a fluorescence emission intensity increase from 980 to 1420 a.u. On the soluble model substrates p-nitrophenyl acetate (PNPA) and p-nitrophenyl butyrate (PNPB), the cutinase showed K m values of 213 and 1933 mu M and k(cat) values of 2.72 and 6.03 s(-1) respectively. The substrate specificity was investigated with bis(benzoyloxyethyl) terephthalate (3PET) and Tha_Cut1 was shown to release primarily 2-hydroxyethyl benzoate. This contrasts with the well-studied Humicula insolens cutinase which preferentially liberates terminal benzoic acid from 3PET.
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