Evaluation of different glycerol fed-batch strategies in a lab-scale bioreactor for the improved production of a novel engineered ß-fructofuranosidase enzyme in Pichia pastoris.

The ß-fructofuranosidase enzyme from Aspergillus niger has been extensively used to commercially produce fructooligosaccharides from sucrose. In this study, the native and an engineered version of the ß-fructofuranosidase enzyme were expressed in Pichia pastoris under control of the glyceraldehyde-3-phosphate dehydrogenase promoter, and production was evaluated in bioreactors using either dissolved oxygen (DO-stat) or constant feed fed-batch feeding strategies. The DO-stat cultivations produced lower biomass concentrations but this resulted in higher volumetric activity for both strains. The native enzyme produced the highest volumetric enzyme activity for both feeding strategies (20.8% and 13.5% higher than that achieved by the engineered enzyme, for DO-stat and constant feed, respectively). However, the constant feed cultivations produced higher biomass concentrations and higher volumetric productivity for both the native as well as engineered enzymes due to shorter process time requirements (59 h for constant feed and 155 h for DO-stat feed). Despite the DO-stat feeding strategy achieving a higher maximum enzyme activity, the constant feed strategy would be preferred for production of the ß-fructofuranosidase enzyme using glycerol due to the many industrial advantages related to its enhanced volumetric enzyme productivity.

Influence of codon optimization, promoter, and strain selection on the heterologous production of a ß-fructofuranosidase from Aspergillus fijiensis ATCC 20611 in Pichia pastoris.

Fructooligosaccharides (FOS) are compounds possessing various health properties and are added to functional foods as prebiotics. The commercial production of FOS is done through the enzymatic transfructolysation of sucrose by ß-fructofuranosidases which is found in various organisms of which Aureobasidium pullulans and Aspergillus niger are the most well known. This study overexpressed two differently codon-optimized variations of the Aspergillus fijiensis ß-fructofuranosidase-encoding gene (fopA) under the transcriptional control of either the alcohol oxidase (AOX1) or glyceraldehyde-3-phosphate dehydrogenase (GAP) promoters. When cultivated in shake flasks, the two codon-optimized variants displayed similar volumetric enzyme activities when expressed under control of the same promoter with the GAP strains producing 11.7 U/ml and 12.7 U/ml, respectively, and the AOX1 strains 95.8 U/ml and 98.6 U/ml, respectively. However, the highest production levels were achieved for both codon-optimized genes when expressed under control of the AOX1 promoter. The AOX1 promoter was superior to the GAP promoter in bioreactor cultivations for both codon-optimized genes with 13,702 U/ml and 2718 U/ml for the AOX1 promoter for ATUM and GeneArt®, respectively, and 6057 U/ml and 1790 U/ml for the GAP promoter for ATUM and GeneArt®, respectively. The ATUM-optimized gene produced higher enzyme activities when compared to the one from GeneArt®, under the control of both promoters.

The effect of growth rate on the production and vitality of non-Saccharomyces wine yeast in aerobic fed-batch culture.

Non-Saccharomyces wine yeasts are of increasing importance due to their influence on the organoleptic properties of wine and thus the factors influencing the biomass production of these yeasts, as starter cultures, are of commercial value. Therefore, the effects of growth rates on the biomass yield (Yx/s) and fermentation performance of non-Saccharomyces yeasts at bench and pilot scale were examined. The fermentative performance and (Yx/s) were optimised, in aerobic fed-batch cultivations, to produce commercial wine seed cultures of Lachancea thermotolerans Y1240, Issatchenkia orientalis Y1161 and Metschnikowia pulcherrima Y1337. Saccharomyces cerevisiae (Lalvin EC1118) was used as a benchmark. A Crabtree positive response was shown by L. thermotolerans in a molasses-based industrial medium, at growth rates exceeding 0.21 h-1 (µcrit), resulting in a Yx/s of 0.76 g/g at 0.21 h-1 (46% of µmax) in the aerobic bioreactor-grown fed-batch culture at bench scale. At pilot scale and 0.133 h-1 (36% of µmax), this yeast exhibited ethanol concentrations reaching 10.61 g/l, as a possible result of substrate gradients. Crabtree negative responses were observed for I. orientalis and M. pulcherrima resulting in Yx/s of 0.83 g/g and 0.68 g/g, respectively, below 32% of µmax. The Yx/s of M. pulcherrima, I. orientalis and L. thermotolerans was maximised at growth rates between 0.10 and 0.12 h-1 and the fermentative capacity of these yeasts was maximised at these lower growth rates.

Amberlite IRA 900 versus calcium alginate in immobilization of a novel, engineered ß-fructofuranosidase for short-chain fructooligosaccharide synthesis from sucrose.

The immobilization of ß-fructofuranosidase for short-chain fructooligosaccharide (scFOS) synthesis holds the potential for a more efficient use of the biocatalyst. However, the choice of carrier and immobilization technique is a key to achieving that efficiency. In this study, calcium alginate (CA), Amberlite IRA 900 (AI900) and Dowex Marathon MSA (DMM) were tested as supports for immobilizing a novel engineered ß-fructofuranosidase from Aspergillus japonicus for scFOS synthesis. Several immobilization parameters were estimated to ascertain the effectiveness of the carriers in immobilizing the enzyme. The performance of the immobilized biocatalysts are compared in terms of the yield of scFOS produced and reusability. The selection of carriers and reagents was motivated by the need to ensure safety of application in the production of food-grade products. The CA and AI900 both recorded impressive immobilization yields of 82 and 62%, respectively, while the DMM recorded 47%. Enzyme immobilizations on CA, AI900 and DMM showed activity recoveries of 23, 27, and 17%, respectively. The CA, AI900 immobilized and the free enzymes recorded their highest scFOS yields of 59, 53, and 61%, respectively. The AI900 immobilized enzyme produced a consistent scFOS yield and composition for 12 batch cycles but for the CA immobilized enzyme, only 6 batch cycles gave a consistent scFOS yield. In its first record of application in scFOS production, the AI900 anion exchange resin exhibited potential as an adequate carrier for industrial application with possible savings on cost of immobilization and reduced technical difficulty.