Thermostabilization of the uronate dehydrogenase from Agrobacterium tumefaciens by semi-rational design
Abstract Aldaric acids represent biobased ‘top value-added chemicals’ that have the potential to substitute petroleum-derived chemicals. Until today they are mostly produced from corresponding aldoses using strong chemical oxidizing agents. An environmentally friendly and more selective process coul...
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doaj-48010ea617b94e33a3621658d0e3057b2020-11-25T01:25:45ZengSpringerOpenAMB Express2191-08552017-05-01711910.1186/s13568-017-0405-2Thermostabilization of the uronate dehydrogenase from Agrobacterium tumefaciens by semi-rational designTeresa Roth0Barbara Beer1André Pick2Volker Sieber3Chair of Chemistry of Biogenic Resources, Straubing Centre of Science, Technical University of MunichChair of Chemistry of Biogenic Resources, Straubing Centre of Science, Technical University of MunichChair of Chemistry of Biogenic Resources, Straubing Centre of Science, Technical University of MunichChair of Chemistry of Biogenic Resources, Straubing Centre of Science, Technical University of MunichAbstract Aldaric acids represent biobased ‘top value-added chemicals’ that have the potential to substitute petroleum-derived chemicals. Until today they are mostly produced from corresponding aldoses using strong chemical oxidizing agents. An environmentally friendly and more selective process could be achieved by using natural resources such as seaweed or pectin as raw material. These contain large amounts of uronic acids as major constituents such as glucuronic acid and galacturonic acid which can be converted into the corresponding aldaric acids via an enzyme-based oxidation using uronate dehydrogenase (Udh). The Udh from Agrobacterium tumefaciens (UdhAt) features the highest catalytic efficiency of all characterized Udhs using glucuronic acid as substrate (829 s−1 mM−1). Unfortunately, it suffers from poor thermostability. To overcome this limitation, we created more thermostable variants using semi-rational design. The amino acids for substitution were chosen according to the B factor in combination with four additional knowledge-based criteria. The triple variant A41P/H101Y/H236K showed higher kinetic and thermodynamic stability with a T 50 15 value of 62.2 °C (3.2 °C improvement) and a ∆∆GU of 2.3 kJ/mol compared to wild type. Interestingly, it was only obtained when including a neutral mutation in the combination.http://link.springer.com/article/10.1186/s13568-017-0405-2Uronate dehydrogenaseGlucuronic acidAgrobacterium tumefaciensThermostabilityB factorNeutral drift |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Teresa Roth Barbara Beer André Pick Volker Sieber |
spellingShingle |
Teresa Roth Barbara Beer André Pick Volker Sieber Thermostabilization of the uronate dehydrogenase from Agrobacterium tumefaciens by semi-rational design AMB Express Uronate dehydrogenase Glucuronic acid Agrobacterium tumefaciens Thermostability B factor Neutral drift |
author_facet |
Teresa Roth Barbara Beer André Pick Volker Sieber |
author_sort |
Teresa Roth |
title |
Thermostabilization of the uronate dehydrogenase from Agrobacterium tumefaciens by semi-rational design |
title_short |
Thermostabilization of the uronate dehydrogenase from Agrobacterium tumefaciens by semi-rational design |
title_full |
Thermostabilization of the uronate dehydrogenase from Agrobacterium tumefaciens by semi-rational design |
title_fullStr |
Thermostabilization of the uronate dehydrogenase from Agrobacterium tumefaciens by semi-rational design |
title_full_unstemmed |
Thermostabilization of the uronate dehydrogenase from Agrobacterium tumefaciens by semi-rational design |
title_sort |
thermostabilization of the uronate dehydrogenase from agrobacterium tumefaciens by semi-rational design |
publisher |
SpringerOpen |
series |
AMB Express |
issn |
2191-0855 |
publishDate |
2017-05-01 |
description |
Abstract Aldaric acids represent biobased ‘top value-added chemicals’ that have the potential to substitute petroleum-derived chemicals. Until today they are mostly produced from corresponding aldoses using strong chemical oxidizing agents. An environmentally friendly and more selective process could be achieved by using natural resources such as seaweed or pectin as raw material. These contain large amounts of uronic acids as major constituents such as glucuronic acid and galacturonic acid which can be converted into the corresponding aldaric acids via an enzyme-based oxidation using uronate dehydrogenase (Udh). The Udh from Agrobacterium tumefaciens (UdhAt) features the highest catalytic efficiency of all characterized Udhs using glucuronic acid as substrate (829 s−1 mM−1). Unfortunately, it suffers from poor thermostability. To overcome this limitation, we created more thermostable variants using semi-rational design. The amino acids for substitution were chosen according to the B factor in combination with four additional knowledge-based criteria. The triple variant A41P/H101Y/H236K showed higher kinetic and thermodynamic stability with a T 50 15 value of 62.2 °C (3.2 °C improvement) and a ∆∆GU of 2.3 kJ/mol compared to wild type. Interestingly, it was only obtained when including a neutral mutation in the combination. |
topic |
Uronate dehydrogenase Glucuronic acid Agrobacterium tumefaciens Thermostability B factor Neutral drift |
url |
http://link.springer.com/article/10.1186/s13568-017-0405-2 |
work_keys_str_mv |
AT teresaroth thermostabilizationoftheuronatedehydrogenasefromagrobacteriumtumefaciensbysemirationaldesign AT barbarabeer thermostabilizationoftheuronatedehydrogenasefromagrobacteriumtumefaciensbysemirationaldesign AT andrepick thermostabilizationoftheuronatedehydrogenasefromagrobacteriumtumefaciensbysemirationaldesign AT volkersieber thermostabilizationoftheuronatedehydrogenasefromagrobacteriumtumefaciensbysemirationaldesign |
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