Structural and functional analyses of GH51 alpha-L-arabinofuranosidase of Geobacillus vulcani GS90 reveal crucial residues for catalytic activity and thermostability
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Dosyalar
Tarih
2022
Yazarlar
Dergi Başlığı
Dergi ISSN
Cilt Başlığı
Yayıncı
John Wiley and Sons Inc
Erişim Hakkı
info:eu-repo/semantics/closedAccess
Özet
Alpha-L-arabinofuranosidase (Abf) is of big interest in various industrial areas. Directed evolution is a powerful strategy to identify significant residues underlying Abf properties. Here, six active variants from GH51 Abf of Geobacillus vulcani GS90 (GvAbf) by directed evolution were overproduced, extracted, and analyzed at biochemical and structural levels. According to the activity and thermostability results, the most-active and the least-active variants were found as GvAbf51 and GvAbf52, respectively. GvAbf63 variant was more active than parent GvAbf by 20% and less active than GvAbf51. Also, the highest thermostability belonged to GvAbf52 with 80% residual activity after 1 h. Comparative sequence and structure analyses revealed that GvAbf51 possessed L307S displacement. Thus, this study suggested that L307 residue may be critical for GvAbf activity. GvAbf63 had H30D, Q90H, and L307S displacements, and H30 was covalently bound to E29 catalytic residue. Thus, H30D may decrease the positive effect of L307S on GvAbf63 activity, preventing E29 action. Besides, GvAbf52 possessed S215N, L307S, H473P, and G476C substitutions and S215 was close to E175 (acid–base residue). S215N may partially disrupt E175 action. Overall effect of all substitutions in GvAbf52 may result in the formation of the C–C bond between C171 and C213 by becoming closer to each other. © 2022 International Union of Biochemistry and Molecular Biology, Inc.
Açıklama
Anahtar Kelimeler
directed evolution, error-prone PCR (epPCR), Geobacillus vulcani, GH51 family, ?-L-arabinofuranosidase, Stability, Arabinofuranosidase, Biochemical level, Directed evolution, Error prone PCR, Error-prone PCR, Geobacillus vulcani, Gh51 family, Industrial area, Property, ?-L-arabinofuranosidase, Catalyst activity
Kaynak
Biotechnology and Applied Biochemistry
WoS Q Değeri
Scopus Q Değeri
Q3
