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- Recent advances in β-galactosidase and fructosyltransferase immobilization technologyPublication . Ureta, Maria Micaela; Martins, Gonçalo Nuno; Figueira, Onofre; Pires, Pedro Filipe; Castilho, Paula Cristina; Gomez-Zavaglia, AndreaThe highly demanding conditions of industrial processes may lower the stability and affect the activity of enzymes used as biocatalysts. Enzyme immobilization emerged as an approach to pro mote stabilization and easy removal of enzymes for their reusability. The aim of this review is to go through the principal immobilization strategies addressed to achieve optimal industrial proc esses with special care on those reported for two types of enzymes: b-galactosidases and fructo syltransferases. The main methods used to immobilize these two enzymes are adsorption, entrapment, covalent coupling and cross-linking or aggregation (no support is used), all of them having pros and cons. Regarding the support, it should be cost-effective, assure the reusability and an easy recovery of the enzyme, increasing its stability and durability. The discussion provided showed that the type of enzyme, its origin, its purity, together with the type of immobilization method and the support will affect the performance during the enzymatic synthesis. Enzymes’ immobilization involves interdisciplinary knowledge including enzymology, nanotechnology, molecular dynamics, cellular physiology and process design. The increasing availability of facilities has opened a variety of possibilities to define strategies to optimize the activity and re-usability of b-galactosidases and fructosyltransferases, but there is still great place for innovative developments.
- Profile analysis of Oligosaccharides in Yacon (Smallanthus sonchifolius) roots: extraction optimization and inulin hydrolysisPublication . Figueira, Onofre Agapito da Silva; Castilho, Paula Cristina Machado FerreiraFructo-oligosaccharides (FOS) and inulin are prebiotic fructose-based saccharides. They are structurally similar differing mainly in their molecular length: while FOS are short-chain linear molecules, inulin is a polysaccharide, with chains up to 60 or more fructose units. In the industry, FOS are synthetically produced through transfructosylation reactions. However, these reactions require difficult conditions to maintain the transfructosylation activity without hydrolytic activity. The use of natural matrices, such as yacon (Smallanthus sonchifolius) roots, is proposed for the extraction of FOS and inulin to overcome the disadvantages of synthetic production. This can be achieved through the extraction and purification of endogenous FOS, and by their production through the hydrolysis of inulin using endo-inulinase. In the present work, three different yacon extracts were prepared and analyzed by HPLC-RI, TLC and MALDI-TOF. The concentration of FOS obtained ranged from 57.1 mg/g to 150.9 mg/g. The DP of these FOS were determined to be over 7 by TLC. Inulin was precipitated using anti-solvents and analyzed by MALDI-TOF revealing inulin with maximum DP up to 20. Several purification methods were tested on an extract containing 74.5 mg/g of saccharides using Saccharomyces cerevisiae, activated charcoal and a combination of activated charcoal and Celite®. The best results were observed with activated charcoal with the recovery of 31.5% of inulin and 21.3% of FOS. Endoinulinase was successfully immobilized in calcium alginate beads, with 97% immobilization yield, and used to hydrolyze the inulin in the yacon extract. The method proved effective, with total consumption of inulin and the consequent production of FOS, with no enzymatic leakage detected by the Bradford method. A TLC-densitometry method for the quantification of FOS in the hydrolysis was performed and proved to be a good, cheap and reliable method to follow the hydrolysis process, before advancing to more expensive techniques.