Engineering CGTase to improve synthesis of alkyl glycosides.

Alkyl glycoside surfactants with elongated carbohydrate chains are useful in different applications due to their improved biocompatibility. Cyclodextrin glucanotransferases can catalyze the elongation process through the coupling reaction. However, due to the presence of a hydrophobic tail, the interaction between an alkyl glycoside acceptor and the active site residues is weaker than the interaction with maltooligosaccharides at the corresponding site. Here we report the mutations of F197, G263 and E266 near the acceptor subsites in the CGTase CspCGT13 from Carboxydocella sp. The results showed that substitutions of both F197 and G263 were important for the binding of acceptor substrate dodecyl maltoside during coupling reaction. The double mutant F197Y/G263A showed enhanced coupling activity and displayed a 2-fold increase of the primary coupling product using γ-cyclodextrin as donor when compared to wildtype CspCGT13. Disproportionation activity was also reduced, which was also the case for another double mutant (F197Y/E266A) that however not showed the corresponding increase in coupling. A triple mutant F197Y/G263A/E266A maintained the increase in primary coupling product (1.8-fold increase) using dodecyl maltoside as acceptor, but disproportionation was approximately at the same level as in the double mutants. In addition, hydrolysis of starch was slightly increased by the F197Y and G263A substitutions, indicating that interactions at both positions influenced the selectivity between glycosyl and alkyl moieties.

Agarose-coated anion exchanger prevents cell-adsorbent interactions.

A common problem during recovery of bioproducts by adsorption from particulate broths is fouling of the adsorbent material as a result of the interaction of cells and cell debris, which present negative charges, with the positively charged anion exchangers commonly used in bioprocesses. The effect of shielding an adsorbent with a layer of agarose on reducing the binding of cells while still allowing the low-molecular-mass bioproducts to be adsorbed was studied. Coating the anion-exchange resin Amberlite IRA-400 with agarose followed by cross-linking the agarose layer effectively prevented the binding of Escherichia coli, Saccharomyces cerevisiae, and Lactobacillus casei cells but allowed binding of lactic acid to the adsorbent. The cross-linked agarose layer was stable during recycling of the adsorbent.

Structure and biocompatibility of poly(vinyl alcohol)-based and agarose-based monolithic composites with embedded divinylbenzene-styrene polymeric particles.

Macroporous monolithic composites with embedded divinylbenzene-styrene (DVB-ST) polymeric particles were prepared by cryogelation techniques using poly(vinyl alcohol) or agarose solutions. Scanning electron microscopy images showed multiple interconnected pores with an average diameter in the range of 4 to 180 µm and quite homogeneous distribution of DVB-ST particles in the composites. Biocompatibility of the composites was assessed by estimation of the C5a fragment of complement in the blood serum and concentration of fibrinogen in the blood plasma which contacted the composites. A time-dependent generation of C5a fragment indicated weak activation of the complement system. At the same time, the difference in fibrinogen concentration, one of the most important proteins in the coagulation system of the blood, between the pristine blood plasma and the plasma, circulated through the monolithic columns, was insignificant.