Development of soluble and immobilized biocatalysts based on a recombinant thermostable ß-fructosidase enabling complete sucrose inversion at pasteurization temperatures

Biocatalysts for the industrial production of invert sugar are preferred to stably operate at high sucrose concentrations and pasteurization temperatures. Thermotoga maritima ß-fructosidase (BfrA) is more thermostable and less susceptible to substrate inhibition than the current commercial invertase from Saccharomyces cerevisiae. In this research, the non-saccharolytic host Pichia pastoris was engineered for BfrA production. Fed-batch fermentation of the recombinant yeast for 72 h using cane sugar as a non-expensive energy source yielded cultures of cell densities over 100 g/L (dry biomass) with invertase activity exceeding 300 U/mL. BfrA was secreted to the cell periplasmic space and the culture medium as a fully active glycoprotein with unaltered thermostability. The extracellularly-released BfrA representing 85 % of the total proteins in the culture supernatant was either dried into powder to generate a soluble free enzyme biocatalyst (specific activity 15 000 U per gram of powder) or covalently immobilized on Glyoxyl-Sepharose CL 4B to generate an insoluble enzyme biocatalyst (specific activity 9249 U per gram of dry support) for reuse. As a third approach, the biomass bearing the periplasmic BfrA was submitted to a killing heat treatment and entrapped in calcium alginate beads to generate a reusable non-viable cell biocatalyst (specific activity 103 U per gram of dry beads). The three biocatalysts completely hydrolyzed cane sugar (70 %, w/v) in batchwise or continuous operation at 60 ºC, offering alternative cost-effective options for the industrial manufacture of food-grade inverted sugar syrup. This research granted the 2013 Award of the Cuban National Academy of Sciences.