title: Complete sucrose hydrolysis by heat-killed recombinant Pichia pastoris cells entrapped in calcium alginate.
abstract: BACKGROUND
  An ideal immobilized biocatalyst for the industrial-scale production of invert sugar should stably operate at elevated temperatures (60-70°C) and high sucrose concentrations (above 60%, w/v). Commercial invertase from the yeast Saccharomyces cerevisiae is thermolabile and suffers from substrate inhibition. Thermotoga maritima β-fructosidase (BfrA) is the most thermoactive and thermostable sucrose-hydrolysing enzyme so far identified and allows complete inversion of the substrate in highly concentrated solutions.
  
  
  RESULTS
  In this study, heat-killed Pichia pastoris cells bearing N-glycosylated BfrA in the periplasmic space were entrapped in calcium alginate beads. The immobilized recombinant yeast showed maximal sucrose hydrolysis at pH 5-7 and 90°C. BfrA was 65% active at 60°C and had no activity loss after incubation without the substrate at this temperature for 15 h. Complete inversion of cane sugar (2.04 M) at 60°C was achieved in batchwise and continuous operation with respective productivities of 4.37 and 0.88 gram of substrate hydrolysed per gram of dry beads per hour. The half-life values of the biocatalyst were 14 and 20 days when operated at 60°C in the stirred tank and the fixed-bed column, respectively. The reaction with non-viable cells prevented the occurrence of sucrose fermentation and the formation of by-products. Six-month storage of the biocatalyst in 1.46 M sucrose (pH 5.5) at 4°C caused no reduction of the invertase activity.
  
  
  CONCLUSIONS
  The features of the novel thermostable biocatalyst developed in this study are more attractive than those of immobilized S. cerevisiae cells for application in the enzymatic manufacture of inverted sugar syrup in batch and fixed-bed reactors.
authors.forename: Duniesky
authors.initials: D
authors.lastname: Martínez
authors.forename: Carmen
authors.initials: C
authors.lastname: Menéndez
authors.forename: Félix M
authors.initials: FM
authors.lastname: Echemendia
authors.forename: Enrique R
authors.initials: ER
authors.lastname: Pérez
authors.affiliation: Plant-Microbe Interactions Laboratory, Center for Genetic Engineering and Biotechnology (CIGB), Ave 31 entre 158 y 190, Apartado Postal 6162, Habana 10600, Cuba. luis.trujillo@cigb.edu.cu.
authors.forename: Luis E
authors.initials: LE
authors.lastname: Trujillo
authors.forename: Alina
authors.initials: A
authors.lastname: Sobrino
authors.forename: Ricardo
authors.initials: R
authors.lastname: Ramírez
authors.forename: Yamira
authors.initials: Y
authors.lastname: Quintero
authors.forename: Lázaro
authors.initials: L
authors.lastname: Hernández
mesh_terms: D000464:Alginates / Q000737:chemistry*; D001426:Bacterial Proteins / Q000378:metabolism; D061249:Batch Cell Culture Techniques; D055162:Biocatalysis; D018533:Biomass; D018914:Cells, Immobilized; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D020723:Glucuronic Acid / Q000737:chemistry; D006207:Half-Life; D006603:Hexuronic Acids / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D010843:Pichia / Q000254:growth & development / Q000378:metabolism*; D013395:Sucrose / Q000378:metabolism*; D013696:Temperature; D020124:Thermotoga maritima / Q000201:enzymology; D043324:beta-Fructofuranosidase / Q000235:genetics / Q000378:metabolism*
chemical_list: D000464:Alginates; D001426:Bacterial Proteins; D005656:Fungal Proteins; D006603:Hexuronic Acids; D013395:Sucrose; D020723:Glucuronic Acid; D043324:beta-Fructofuranosidase