title: A thermostable exo-β-fructosidase immobilised through rational design.
abstract: Thermotoga maritima exo-β-fructosidase (BfrA) secreted by a recombinant Pichia pastoris strain was optimally immobilised on Glyoxyl-Sepharose CL 4B using the Rational Design of Immobilised Derivatives (RDID) strategy. Covalent attachment of the N-glycosylated BfrA onto the activated support at pH 10 allowed total recovery of the loaded enzyme and its activity. The immobilisation process caused no variation in the catalytic properties of the enzyme and allowed further enhancement of the thermal stability. Complete inversion of cane sugar (2.04 M) in a batch stirred tank reactor at 60 °C was achieved with a productivity of 22.2 g of substrate hydrolysed/gram of biocatalyst/hour. Half-life of the immobilised enzyme of 5 days at 60 °C was determined in a continuously operated fixed-bed column reactor. Our results promote the applicability of the BfrA-immobilised biocatalyst for the complete hydrolysis of concentrated sucrose solutions under industrial conditions, especially at a high reaction temperature.
authors.affiliation: Fermentation Laboratory, Center for Genetic Engineering and Biotechnology Sancti Spíritus (CIGBSS), Circunvalante Norte S/N, Olivos 3, Apartado Postal 83, Sancti Spíritus 60200, Cuba. Electronic address: duniesky.martinez@cigb.edu.cu.
authors.forename: Duniesky
authors.initials: D
authors.lastname: Martínez
authors.forename: Bessy
authors.initials: B
authors.lastname: Cutiño-Avila
authors.forename: Enrique Rosendo
authors.initials: ER
authors.lastname: Pérez
authors.forename: Carmen
authors.initials: C
authors.lastname: Menéndez
authors.forename: Lázaro
authors.initials: L
authors.lastname: Hernández
authors.forename: Alberto
authors.initials: A
authors.lastname: Del Monte-Martínez
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D055162:Biocatalysis; D002847:Chromatography, Agarose; D019295:Computational Biology / Q000379:methods; D019422:Dietary Sucrose / Q000378:metabolism*; D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005103:Expert Systems; D005511:Food Handling; D005632:Fructose / Q000378:metabolism*; D005947:Glucose / Q000378:metabolism*; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006031:Glycosylation; D006038:Glyoxylates / Q000737:chemistry; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008958:Models, Molecular; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012685:Sepharose / Q000031:analogs & derivatives / Q000737:chemistry; D020124:Thermotoga maritima / Q000201:enzymology
chemical_list: D001426:Bacterial Proteins; D019422:Dietary Sucrose; D004800:Enzymes, Immobilized; D006038:Glyoxylates; D011994:Recombinant Proteins; C070399:glyoxyl agarose; D005632:Fructose; C035771:Sepharose CL 4B; D012685:Sepharose; D006026:Glycoside Hydrolases; C044118:fructan beta-fructosidase; C008133:invert sugar; D005947:Glucose
keywords: Covalent immobilisation; Invert syrup; Invertase; Rational design