Functional and biophysical characterization of a hyperthermostable GH51 α-L-arabinofuranosidase from Thermotoga petrophila.
A hyperthermostable glycoside hydrolase family 51 (GH51) α-L-arabinofuranosidase from Thermotoga petrophila RKU-1 (TpAraF) was cloned, overexpressed, purified and characterized. The recombinant enzyme had optimum activity at pH 6.0 and 70°C with linear α-1,5-linked arabinoheptaose as substrate. The substrate cleavage pattern monitored by capillary zone electrophoresis showed that TpAraF is a classical exo-acting enzyme producing arabinose as its end-product. Far-UV circular dichroism analysis displayed a typical spectrum of α/β barrel proteins analogously observed for other GH51 α-L-arabinofuranosidases. Moreover, TpAraF was crystallized in two crystalline forms, which can be used to determine its crystallographic structure.
dos Santos
Camila Ramos
CR
Laboratório Nacional de Biociências (LNBio), Centro Nacional de Pesquisa em Energia e Materiais, Rua giuseppe maximo scolfaro, 10000, Campinas, SP, 13083-970, Brazil.
Squina
Fábio Márcio
FM
Navarro
Andréia Meza
AM
Oldiges
Daiane Patrícia
DP
Paes Leme
Adriana Franco
AF
Ruller
Roberto
R
Mort
Andrew John
AJ
Prade
Rolf
R
Murakami
Mário Tyago
MT
D001089:Arabinose / Q000378:metabolism; D001419:Bacteria / Q000201:enzymology*; D002942:Circular Dichroism; D003001:Cloning, Molecular; D003460:Crystallization; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004795:Enzyme Stability; D015870:Gene Expression; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity
D004269:DNA, Bacterial; D011994:Recombinant Proteins; D001089:Arabinose; D006026:Glycoside Hydrolases; C019713:alpha-N-arabinofuranosidase