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

Abstract

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.

Keywords

• invertase
• invert syrup
• immobilization
• Thermotoga maritima
• Pichia pastoris