The laccase from the ascomycete Myceliophthora thermophila was covalently immobilized on polymethacrylate-based polymers (Sepabeads EC-EP3 and Dilbeads NK) activated with epoxy groups. The enzyme immobilized on Sepabeads EC-EP3 exhibited notable activity (203 U/g) along with remarkably improved stability towards pH, temperature and storage time, but no increased resistance to organic solvents. In addition, the biocatalyst also showed good operational stability, maintaining 84% of its initial activity after 17 cycles of oxidation of ABTS. The immobilized laccase was applied to the decolorization of six synthetic dyes (Reactive Black 5, Acid Blue 25, Methyl Orange, Remazol Brilliant Blue B, Methyl Green and Acid Green 27) with or without the redox mediator 1-hydroxybenzotriazole. The laccase immobilized in Sepabeads EC-EP3 retained 41% activity in the decolorization of Methyl Green in a fixed-bed reactor after five cycles. The features of these biocatalysts are very attractive for their application on the decolorization of dyes in the textile industry in batch and continuous fixed-bed bioreactors. #
A fructosyltransferase present in Pectinex Ultra SP-L, a commercial enzyme preparation from Aspergillus aculeatus, was purified to 107-fold and further characterised. The enzyme was a dimeric glycoprotein (20% (w/w) carbohydrate content) with a molecular mass of around 135 kDa for the dimer. Optimal activity/stability was found in the pH range 5.0-7.0 and at 60 degrees C. It was stable or slightly activated (upto 1.4-fold) in the presence of reducing agents, such as dithiothreitol and 2-mercaptoethanol, and detergents, such as sodium dodecylsulphate and Tween 80. The enzyme was able to transfer fructosyl groups from sucrose as donor producing the corresponding series of fructooligosaccharides: 1-kestose, nystose and fructosylnystose. Using sucrose as substrate, the k(cat) and K(m) values for transfructosylating activity were 1.62+/-0.09 x 10(4)s(-1) and 0.53+/-0.05 M, whereas for hydrolytic activity the corresponding values were 775+/-25s(-1) and 27+/-3 mM. At elevated sucrose concentrations, the fructosyltransferase from A. aculeatus showed a high transferase/hydrolase ratio that confers it a great potential for the industrial production of prebiotic fructooligosaccharides.
The transfructosylating activity present in two commercial pectinase preparations (Pectinex Ultra SP-L, from Aspergillus aculeatus, and Rapidase TF, from Aspergillus niger) was studied. Pectinex Ultra SP-L, which has a high transferase/hydrolase ratio, was covalently immobilised on a polymethacrylate-based polymer (Sepabeads EC) activated with epoxy groups. The influence of pore volume and average pore size on biocatalyst performance was studied for two of these carriers (Sepabeads EC-EP3 and EC-EP5). Several parameters that affect immobilisation such as buffer concentration, pH and amount (mg) of protein added per gram of support (varied over the range 30:1 to 200:1) were analysed. We found that Pectinex Ultra SP-L can be efficiently immobilised on these supports without adding any external salt or buffer. Using Sepabeads EC-EP5-whose pore volume (1.67 cm 3 /g) and pore size (800 nm) are higher than those corresponding to Sepabeads EC-EP3-the activity towards sucrose reached 25.9 U/g biocatalyst. The immobilised fructosyltransferase was applied to the batch synthesis of fructo-oligosaccharides (FOS) using 630 g/l sucrose to shift activity towards transfructosylation in detriment of hydrolysis. The FOS concentration reached a maximum value of 387 g/l after 36 h (240 g/l 1-kestose, 144 g/l nystose and 3 g/l 1 Ffructofuranosyl-nystose), which corresponds to 61.5% (w/w) of the total carbohydrates in the mixture. The features of these immobilised biocatalysts are very attractive for their application in batch and fixed-bed bioreactors.
Unnatural selection: A fungal laccase was tailored by directed evolution to be active at neutral/alkaline pH. After five generations, the final mutant showed a broader pH profile while retaining 50 to 80 % of its activity at neutral pH.
The transglycosylation activity of a novel a-glucosidase from the basidiomycetous yeast Xanthophyllomyces dendrorhous (formerly Phaffia rhodozyma) was studied using maltose as glucosyl donor. The enzyme synthesized oligosaccharides with a-(1 ! 2), a-(1 ! 4) and a-(1 ! 6) bonds. Using 200 g/l maltose, the yield of oligosaccharides was 53.8 g/l, with prebiotic oligosaccharides containing at least one a-(1 ! 6) linkage (panose, 6-O-a-glucosyl-maltotriose and 6-O-a-isomaltosyl-maltose) being the major products (47.1 g/l). The transglycosylatying yield was 3.6 times higher than the observed with the a-glucosidase from Saccharomyces cerevisiae (53.8 vs. 14.7 g/l). Moreover, when increasing the maltose concentration up to 525 g/l, the maximum production of tri-and tetrasaccharides reached 167.1 g/l, without altering the percentage of oligosaccharides in the mixture. Compared with other microbial a-glucosidases in which the main transglycosylation product is a disaccharide, the enzyme from X. dendrorhous yields a final product enriched in trisaccharides and tetrasaccharides. #
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