Enhancing the stability of a novel D-allulose 3-epimerase from Ruminococcus sp. CAG55 by interface interaction engineering and terminally attached a self-assembling peptide.
Int J Biol Macromol
; 269(Pt 1): 131986, 2024 Jun.
Article
en En
| MEDLINE
| ID: mdl-38697423
ABSTRACT
D-allulose, a highly desirable sugar substitute, is primarily produced using the D-allulose 3-epimerase (DAE). However, the availability of usable DAE enzymes is limited. In this study, we discovered and engineered a novel DAE Rum55, derived from a human gut bacterium Ruminococcus sp. CAG55. The activity of Rum55 was strictly dependent on the presence of Co2+, and it exhibited an equilibrium conversion rate of 30.6 % and a half-life of 4.5 h at 50 °C. To enhance its performance, we engineered the interface interaction of Rum55 to stabilize its tetramer structure, and the best variant E268R was then attached with a self-assembling peptide to form active enzyme aggregates as carrier-free immobilization. The half-life of the best variant E268R-EKL16 at 50 °C was dramatically increased 30-fold to 135.3 h, and it maintained 90 % of its activity after 13 consecutive reaction cycles. Additionally, we identified that metal ions played a key role in stabilizing the tetramer structure of Rum55, and the dependence on metal ions for E268R-EKL16 was significantly reduced. This study provides a useful route for improving the thermostability of DAEs, opening up new possibilities for the industrial production of D-allulose.
Palabras clave
Texto completo:
1
Colección:
01-internacional
Asunto principal:
Estabilidad de Enzimas
/
Ingeniería de Proteínas
/
Ruminococcus
Idioma:
En
Revista:
Int J Biol Macromol
Año:
2024
Tipo del documento:
Article
País de afiliación:
China