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Pregled bibliografske jedinice broj: 1118042

Thermostability engineering of a class II pyruvate aldolase from Escherichia coli by in vivo folding interference


(Carbazymes) Bosch, Sandra; Sanchez-Freire, E.; del Pozo, M. L.; Česnik, Morana; Quesada, J.; Mate, D. M.; Hernández, Karel; Qi, Yun; Clapés, Pere; Vasić- Rački, Đurđa et al.
Thermostability engineering of a class II pyruvate aldolase from Escherichia coli by in vivo folding interference // ACS Sustainable Chemistry & Engineering, 9 (2021), 15; 5430-5436 doi:10.1021/acssuschemeng.1c00699 (međunarodna recenzija, članak, znanstveni)


CROSBI ID: 1118042 Za ispravke kontaktirajte CROSBI podršku putem web obrasca

Naslov
Thermostability engineering of a class II pyruvate aldolase from Escherichia coli by in vivo folding interference

Autori
Bosch, Sandra ; Sanchez-Freire, E. ; del Pozo, M. L. ; Česnik, Morana ; Quesada, J. ; Mate, D. M. ; Hernández, Karel ; Qi, Yun ; Clapés, Pere ; Vasić- Rački, Đurđa ; Findrik Blažević, Zvjezdana ; Berenguer Jesus, Hidalgo, Aurelio

Kolaboracija
Carbazymes

Izvornik
ACS Sustainable Chemistry & Engineering (2168-0485) 9 (2021), 15; 5430-5436

Vrsta, podvrsta i kategorija rada
Radovi u časopisima, članak, znanstveni

Ključne riječi
pyruvate aldolase, thermostabilization

Sažetak
The use of enzymes in industrial processes is often limited by the unavailability of biocatalysts with prolonged stability. Thermostable enzymes allow increased process temperature and thus higher substrate and product solubility, reuse of expensive biocatalysts, resistance against organic solvents and better “evolvability” of enzymes. In this work, we have used an activity-independent method for the selection of thermostable variants of any protein in Thermus thermophilus through folding interference at high temperature of a thermostable antibiotic reporter protein at the C-terminus of a fusion protein. To generate a monomeric folding reporter, we have increased the thermostability of the moderately thermostable Hph5 variant of the hygromycin B phosphotransferase from Escherichia coli to meet the method requirements. The final Hph17 variant showed 1.5 ºC higher melting temperature (Tm) and 3-fold longer half-life at 65 ºC compared to parental Hph5, with no changes in the steady- state kinetic parameters. Additionally, we demonstrate the validity of the reporter by stabilizing the 2-oxoacid utilizing 2-keto-3- deoxy-l-rhamnonate aldolase from E. coli (YfaU). The most thermostable multiple-mutated variants thus obtained, YfaU99 and YfaU103, showed increases of 2 and 2.9 ºC in Tm compared to the wild-type enzyme, but a severely lower retro- aldol activity (150-fold and 120-fold, respectively). After segregation of the mutations, the most thermostable single variant, Q107R, showed a Tm 8.9 °C higher, a 16-fold improvement in half-life at 60 ºC and higher operational stability than the wild-type, without substantial modification of the kinetic parameters.

Izvorni jezik
Engleski

Znanstvena područja
Kemijsko inženjerstvo, Biotehnologija



POVEZANOST RADA


Projekti:
EK-H2020-635595 - Sustainable industrial processes based on a C-C bond-forming enzyme platform (CARBAZYMES) (Findrik Blažević, Zvjezdana, EK - H2020-LEIT-BIO-2014-1) ( POIROT)

Ustanove:
Fakultet kemijskog inženjerstva i tehnologije, Zagreb

Citiraj ovu publikaciju

(Carbazymes) Bosch, Sandra; Sanchez-Freire, E.; del Pozo, M. L.; Česnik, Morana; Quesada, J.; Mate, D. M.; Hernández, Karel; Qi, Yun; Clapés, Pere; Vasić- Rački, Đurđa et al.
Thermostability engineering of a class II pyruvate aldolase from Escherichia coli by in vivo folding interference // ACS Sustainable Chemistry & Engineering, 9 (2021), 15; 5430-5436 doi:10.1021/acssuschemeng.1c00699 (međunarodna recenzija, članak, znanstveni)
(Carbazymes) (Carbazymes) Bosch, S., Sanchez-Freire, E., del Pozo, M., Česnik, M., Quesada, J., Mate, D., Hernández, K., Qi, Y., Clapés, P. & Vasić- Rački, Đ. (2021) Thermostability engineering of a class II pyruvate aldolase from Escherichia coli by in vivo folding interference. ACS Sustainable Chemistry & Engineering, 9 (15), 5430-5436 doi:10.1021/acssuschemeng.1c00699.
@article{article, year = {2021}, pages = {5430-5436}, DOI = {10.1021/acssuschemeng.1c00699}, keywords = {pyruvate aldolase, thermostabilization}, journal = {ACS Sustainable Chemistry and Engineering}, doi = {10.1021/acssuschemeng.1c00699}, volume = {9}, number = {15}, issn = {2168-0485}, title = {Thermostability engineering of a class II pyruvate aldolase from Escherichia coli by in vivo folding interference}, keyword = {pyruvate aldolase, thermostabilization} }
@article{article, year = {2021}, pages = {5430-5436}, DOI = {10.1021/acssuschemeng.1c00699}, keywords = {pyruvate aldolase, thermostabilization}, journal = {ACS Sustainable Chemistry and Engineering}, doi = {10.1021/acssuschemeng.1c00699}, volume = {9}, number = {15}, issn = {2168-0485}, title = {Thermostability engineering of a class II pyruvate aldolase from Escherichia coli by in vivo folding interference}, keyword = {pyruvate aldolase, thermostabilization} }

Časopis indeksira:


  • Current Contents Connect (CCC)
  • Web of Science Core Collection (WoSCC)
    • Science Citation Index Expanded (SCI-EXP)
    • SCI-EXP, SSCI i/ili A&HCI
  • Scopus


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