Direct Isomaltulose Synthesis From Beet Molasses by Immobilized Sucrose Isomerase

Isomaltulose is becoming a focus as a functional sweetener for sucrose substitutes; however, isomaltulose production using sucrose as the substrate is not economical. Low-cost feedstocks are needed for their production. In this study, beet molasses (BM) was introduced as the substrate to produce isomaltulose for the first time. Immobilized sucrose isomerase (SIase) was proved as the most efficient biocatalyst for isomaltulose synthesis from sulfuric acid (H2SO4) pretreated BM followed by centrifugation for the removal of insoluble matters and reducing viscosity. The effect of different factors on isomaltulose production is investigated. The isomaltulose still achieved a high concentration of 446.4 ± 5.5 g/L (purity of 85.8%) with a yield of 0.94 ± 0.02 g/g under the best conditions (800 g/L pretreated BM, 15 U immobilized SIase/g dosage, 40°C, pH of 5.5, and 10 h) in the eighth batch. Immobilized SIase used in repeated batch reaction showed good reusability to convert pretreated BM into isomaltulose since the sucrose conversion rate remained 97.5% in the same batch and even above 94% after 11 batches. Significant cost reduction of feedstock costs was also confirmed by economic analysis. The findings indicated that this two-step process to produce isomaltulose using low-cost BM and immobilized SIase is feasible. This process has the potential to be effective and promising for industrial production and application of isomaltulose as a functional sweetener for sucrose substitute.

Isolation of various hyperactive mutants of phenylalanine ammonia-lyase containing yeasts

Various strains of phenylalanine ammonia-lyase (PAL) containing yeasts were mutagenised using ultraviolet (UV) irradiation. Two of the yeast, SPA 1 and SPA 17, produced UV kill curves with a distinct shoulder reflecting their diploid nature. Of the analogues used, p-fluoro-D,L-phenylalanine and β-2-thienyl-D,L-alanine selected the greatest frequency of mutants with the highest PAL activities: one such mutant, FP10M6, exhibited five times the PAL activity of the parent SPA 10. Mutants constitutive for PAL synthesis could not be isolated using any selection regime, including resistance to relatively high concentrations of 2-deoxyglucose. Three of the hyperactive PAL mutants examined were not impaired for growth and their PAL-induction profiles were not different from the respective parents. Inactivation of PAL shortly after peak synthesis was found with all mutants examined, although this was not extensively investigated. Of the 21 strains mutagenised, mutants from three of the wild-type yeasts were found to exhibit moderate growth at 37 °C, while growth of the parent strains was greatly impaired. The latter temperature-resistant mutants exhibited a twofold increase in PAL activity as compared with the parent strains at either 30 or 37 °C. Under biotransformation reaction conditions, several of the mutants were capable of producing more than 15 g L-phenylalanine∙L−1. Although the stability of the PAL catalyst varied markedly among different mutants, some of the most productive mutants did exhibit good reuseability under sequentially repeated batch reaction conditions.