Recombinant polyesterase (Est119) from Thermobifida alba AHK119 was purified by two chromatography steps. The final protein was observed as a single band in SDS-PAGE, and the specific activity of Est119 for p-nitrophenyl butyrate was 2.30 u/mg. Purified Est119 was active with aliphatic and aliphatic-co-aromatic polyesters. Kinetic data indicated that p-nitrophenyl butyrate (pNPB) or hexanoate was the best substrate for Est119 among p-nitrophenyl acyl esters. Calcium was required for full activity and thermostability of Est119, which was stable at 50 °C for 16 h. Three-dimensional modeling and biochemical characterization showed that Est119 is a typical cutinase-type enzyme that has the compact ternary structure of an α/β-hydrolase. Random and site-directed mutagenesis of wild-type Est119 resulted in improved activity with increased hydrophobic interaction between the antiparallel first and second β-sheets (A68V had the greatest effect). Introduction of a proline residue (S219P) in a predicted substrate-docking loop increased the thermostability. The specific activity of the A68V/S219P mutant on pNPB was increased by more than 50-fold over the wild type. The mutant was further activated by 2.6-fold (299 u/mg) with 300 mM Ca(2+) and was stable up to 60 °C with 150 mM Ca(2+). Another identical gene was located in tandem in the upstream of est119.
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http://dx.doi.org/10.1007/s00253-011-3781-6 | DOI Listing |
J Environ Manage
September 2024
National Energy R&D Center for Biorefinery, Beijing Key Laboratory of Bioprocess, College of Life Science and Technology, Beijing University of Chemical Technology, Beijing, 100029, China. Electronic address:
Bioresour Technol
March 2021
College of Engineering, Jiangxi Agricultural University, Nanchang 330045, PR China; Jiangxi Engineering Research Center of Animal Husbandry Facility Technology Exploitation, Nanchang 330045, PR China.
The aim of this work was to explore the ability of cutinase in the decolorization of molasses wastewater. Thermophilic cutinase from Thermobifida alba eliminated 76.1-78.
View Article and Find Full Text PDFJ Biotechnol
November 2020
Laboratory of Sustainable Biotechnology (LIBioS), National University of Quilmes, Roque Sáenz Peña 352, Bernal, B1876BXD, Argentina; National Scientific and Technical Research Council (CONICET), Godoy Cruz 2290, CABA, C1425FQB, Argentina. Electronic address:
Cladribine (2-chloro-2'-deoxy-β-d-adenosine) is a 2'-deoxyadenosine analogue, approved by the FDA for the treatment of hairy cell leukemia and more recently has been proved for therapeutic against many autoimmune diseases as multiple sclerosis. The biosynthesis of this compound using Thermomonospora alba CECT 3324 as biocatalyst is herein reported. This thermophilic microorganism was successfully entrapped in polyacrylamide gel supplemented with nanoclays such as bentonite.
View Article and Find Full Text PDFFEBS J
June 2019
Graduate School of Comprehensive Rehabilitation, College of Health and Human Sciences, Osaka Prefecture University, Habikino, Japan.
Cutinases are enzymes known to degrade polyester-type plastics. Est119, a plastic-degrading type of cutinase from Thermobifida alba AHK119 (herein called Ta_cut), shows a broad substrate specificity toward polyesters, and can degrade substrates including polylactic acid (PLA). However, the PLA-degrading mechanism of cutinases is still poorly understood.
View Article and Find Full Text PDFJ Biosci Bioeng
May 2019
Center for Fiber and Textile Science, Kyoto Institute of Technology, Kyoto 606-8181, Japan.
Thermobifida alba AHK119 exhibits sufficient filter paper-degradation activity in its culture supernatant. AHK119-bMs (1365 bp) and AHK119-E5 (1425 bp), which encode novel GH5 family endoglucanases, were cloned from the genomic DNA of T. alba AHK119.
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