title: Enzymatic fine-tuning for 2-(6-hydroxynaphthyl) β-D-xylopyranoside synthesis catalyzed by the recombinant β-xylosidase BxTW1 from Talaromyces amestolkiae.
abstract: BACKGROUND
  Glycosides are compounds displaying crucial biological roles and plenty of applications. Traditionally, these molecules have been chemically obtained, but its efficient production is limited by the lack of regio- and stereo-selectivity of the chemical synthesis. As an interesting alternative, glycosidases are able to catalyze the formation of glycosides in a process considered green and highly selective. In this study, we report the expression and characterization of a fungal β-xylosidase in Pichia pastoris. The transglycosylation potential of the enzyme was evaluated and its applicability in the synthesis of a selective anti-proliferative compound demonstrated.
  
  
  RESULTS
  The β-xylosidase BxTW1 from the ascomycete fungus Talaromyces amestolkiae was cloned and expressed in Pichia pastoris GS115. The yeast secreted 8 U/mL of β-xylosidase that was purified by a single step of cation-exchange chromatography. rBxTW1 in its active form is an N-glycosylated dimer of about 200 kDa. The enzyme was biochemically characterized displaying a Kmand kcatagainst p-nitrophenyl-β-D-xylopyranoside of 0.20 mM and 69.3 s-1respectively, and its maximal activity was achieved at pH 3 and 60 °C. The glycan component of rBxTW1 was also analyzed in order to interpret the observed loss of stability and maximum velocity when compared with the native enzyme. A rapid screening of aglycone specificity was performed, revealing a remarkable high number of potential transxylosylation acceptors for rBxTW1. Based on this analysis, the enzyme was successfully tested in the synthesis of 2-(6-hydroxynaphthyl) β-D-xylopyranoside, a well-known selective anti-proliferative compound, enzymatically obtained for the first time. The application of response surface methodology, following a Box-Behnken design, enhanced this production by eightfold, fitting the reaction conditions into a multiparametric model. The naphthyl derivative was purified and its identity confirmed by NMR.
  
  
  CONCLUSIONS
  A β-xylosidase from T. amestolkiae was produced in P. pastoris and purified. The final yields were much higher than those attained for the native protein, although some loss of stability and maximum velocity was observed. rBxTW1 displayed remarkable acceptor versatility in transxylosylation, catalyzing the synthesis of a selective antiproliferative compound, 2-(6-hydroxynaphthyl) β-D-xylopyranoside. These results evidence the interest of rBxTW1 for transxylosylation of relevant products with biotechnological interest.
authors.affiliation: Department of Environmental Biology, Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, 28040, Madrid, Spain.
authors.forename: Manuel
authors.initials: M
authors.lastname: Nieto-Domínguez
authors.affiliation: Department of Environmental Biology, Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, 28040, Madrid, Spain.
authors.forename: Alicia
authors.initials: A
authors.lastname: Prieto
authors.affiliation: Department of Chemical and Physical Biology, Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, 28040, Madrid, Spain.
authors.forename: Beatriz
authors.initials: B
authors.lastname: Fernández de Toro
authors.affiliation: Department of Chemical and Physical Biology, Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, 28040, Madrid, Spain.
authors.forename: Francisco Javier
authors.initials: FJ
authors.lastname: Cañada
authors.affiliation: Department of Environmental Biology, Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, 28040, Madrid, Spain.
authors.forename: Jorge
authors.initials: J
authors.lastname: Barriuso
authors.affiliation: Department of Chemistry, Centre for High-Throughput Biology, University of British Columbia, Vancouver, Canada.
authors.forename: Zach
authors.initials: Z
authors.lastname: Armstrong
authors.affiliation: Department of Chemistry, Centre for High-Throughput Biology, University of British Columbia, Vancouver, Canada.
authors.forename: Stephen G
authors.initials: SG
authors.lastname: Withers
authors.affiliation: Department of Environmental Biology, Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, 28040, Madrid, Spain. lidem@cib.csic.es.
authors.forename: Laura I
authors.initials: LI
authors.lastname: de Eugenio
authors.affiliation: Department of Environmental Biology, Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, 28040, Madrid, Spain. mjmartinez@cib.csic.es.
authors.forename: María Jesús
authors.initials: MJ
authors.lastname: Martínez
mesh_terms: D000595:Amino Acid Sequence; D055162:Biocatalysis; D006027:Glycosides / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism; D006031:Glycosylation; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D009284:Naphthols / Q000737:chemistry / Q000378:metabolism; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D013379:Substrate Specificity; D032901:Talaromyces / Q000201:enzymology* / Q000235:genetics; D014994:Xylose / Q000378:metabolism; D014995:Xylosidases / Q000235:genetics* / Q000378:metabolism*
chemical_list: C111149:2-(6-hydroxynaphthyl)-O-beta-D-xylopyranoside; D006027:Glycosides; D009284:Naphthols; D014994:Xylose; D014995:Xylosidases; C026579:exo-1,4-beta-D-xylosidase
keywords: 2-(6-hydroxynaphthyl) β-D-xylopyranoside; Box-Behnken design; N-Glycosylation; Pichia pastoris; Response surface methodology; Transxylosylation; β-Xylosidase