BackgroundAn 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.ResultsIn 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.ConclusionsThe 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.
Sucrose hydrolysis was carried out in a constant-volume batch reactor, using recombinant Pichia pastoris BfrA4X whole cells expressing Thermotoga maritima invertase, entrapped in calcium alginate beads. The kinetics of the enzymatic hydrolysis of sucrose by the biocatalyst was examined at substrate concentrations ranging between 0.03 M and 2.04 M. The reaction rate increases until 0.31 M after which the reaction velocity was constant until 1.16 M, above this concentration, the reaction rate decreases with increasing sucrose concentration. The experimental data obtained with two weight of the biocatalyst were incorporated into two kinetic models to predict the reaction time needed for sucrose hydrolysis. One model was applied for sucrose concentrations bellow 1.16 M while a second one could be used at inhibitory range between 1.46 and 2.04 M with a k value as function of initial sucrose concentration and biocatalyst weight.
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