Engineering a thermostable chondroitinase for production of specifically distributed low-molecular-weight chondroitin sulfate.
Biotechnol J
; 16(5): e2000321, 2021 May.
Article
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| MEDLINE
| ID: mdl-33350041
ABSTRACT
Chondroitinase ABC I (csABC I) has attracted intensive attention because of its great potential in heparin refining and the enzymatic preparation of low-molecular-weight chondroitin sulfate (LMW-CS). However, low thermal resistance (<30â) restricts its applications. Herein, structure-guided and sequence-assisted combinatorial engineering approaches were applied to improve the thermal resistance of Proteus vulgaris csABC I. By integrating the deletion of the flexible fragment R166-L170 at the N-terminal domain and the mutation of E694P at the C-terminal domain, variant NΔ5/E694P exhibited 247-fold improvement of its half-life at 37â and a 2.3-fold increase in the specific activity. Through batch fermentation in a 3-L fermenter, the expression of variant NΔ5/E694P in an Escherichia coli host reached 1.7 g L-1 with the activity of 1.0 × 105 U L-1 . Finally, the enzymatic approach for the preparation of LMW-CS was established. By modulating enzyme concentration and controlling depolymerization time, specifically distributed LMW-CS (7000, 3400, and 1900 Da) with low polydispersity was produced, demonstrating the applicability of these processes for the industrial production of LMW-CS in a more environmentally friendly way.
Palabras clave
Texto completo:
1
Base de datos:
MEDLINE
Asunto principal:
Sulfatos de Condroitina
/
Condroitina ABC Liasa
Idioma:
En
Revista:
Biotechnol J
Asunto de la revista:
BIOTECNOLOGIA
Año:
2021
Tipo del documento:
Article
País de afiliación:
China