The galactosidase from Aspergillus oryzae is a monomeric enz

The β-galactosidase from Aspergillus oryzae is a monomeric enzyme of 113 kDa [58] that has been widely used in GOS synthesis [49,53,[59], [60], [61]], exhibiting a high activity of lactose transgalactosylation [62]. Different immobilization methods have been used for the synthesis of lactose-derived oligosaccharides with β-galactosidases immobilized in chitosan [[63], [64], [65], [66], [67], [68], [69], [70]]. For the immobilization of A. oryzae β-galactosidase, chitosan functionalized with glutaraldehyde has been used as support in most of the cases and the enzyme has been covalently linked to the aldehyde groups of the support [[63], [64], [65], [66]]. The aim of this work was to evaluate the use of chitosan heterofunctionality for the covalent immobilization of A. oryzae β-galactosidase in a two-step process. In order to analyze if the activity and stability of the final biocatalyst was modified as a consequence of such immobilization, the covalent immobilization in chitosan by a one-step and a two-step process were compared. Additionally, the impact of the carrier was analyzed, comparing and optimizing the covalent immobilization of the enzyme by a two-step process in chitosan and amino-glyoxyl agarose, since in both cases the enzyme should be immobilized through the surface region having the highest net negative charge [38]. The optimized biocatalysts were applied in GOS synthesis, comparing them in terms of their specific activity, thermal stability, and performance in GOS synthesis under sequential batch operation.
Materials and methods
Results and discussion
Conclusions An effective methodology of immobilization in chitosan by a two-step process was developed using the heterofunctionality of the Parthenolide carrying amino and aldehyde groups. The immobilization of A. oryzae β-galactosidase in C-E by this strategy offered the best compromise between specific activity and thermal stability, overpassing the performance of the enzyme immobilized only through the aldehyde groups. The modification of C-E aldehyde density allowed the improvement of the biocatalyst specific activity, showing the impact of the carrier properties on the biocatalyst performance. The comparison of the biocatalysts immobilized in C-ETS-200 and A-G-An under optimized conditions, showed that the use of this new methodology of immobilization in chitosan and the correct design of the carrier allowed obtaining a biocatalyst competitive with its agarose counterpart, showing the high versatility of this low-cost carrier. The biocatalysts immobilized in C-ETS-200 and A-G-An under optimized conditions were successfully applied to GOS synthesis in sequential batch reactor operation, obtaining a better performance with the former due to its higher specific activity. Even though lower GOS yields were obtained with immobilized biocatalysts than with the soluble enzyme, only three sequential batches with the immobilized enzymes were enough to obtain a cumulative specific productivity higher than obtained with the soluble enzyme.
Acknowledgements Work financed by Chilean Fondecyt Grant 1100050. Doctoral fellowship to Ms. Urrutia from Conicyt-Chile is acknowledged.
Introduction Cyclodextrins (CDs) are nontoxic macrocyclic oligosaccharides, consisting of (α-1, 4)-linked α-l-glucopyranose units. CDs are very attractive ingredients for making artificial enzymes and other biomimetic materials (Tong, 2001). The most significant characteristic of CDs is the certain size of stereoscopic chiral cavity with hydrophobic central cavity and hydrophilic outer surface (Del Valle, 2004). According to the number of glucose units, the degrees of polymerization are 6, 7 and 8 for α-, β- and γ-CD, respectively. CDs have the ability to form inclusion or non-inclusion complexes with different compounds, such as organic molecules, rare gases and inorganic compounds (Hedges, 1998, Szejtli, 1998). This fact allows CDs to slow the release of some target compounds, improving the solubilization and the antioxidant potential of some components as well as masking some negative properties of some products (eg. bad smell). For all the above mentioned reasons, over the last years, people have become more and more interested in the study of CDs and the research regarding the modification of cyclodextrin is nowadays widely used in food, medicine, chemistry, agriculture, environmental protection and other fields.