Advantages of Supports Activated with Divinyl Sulfone in Enzyme Coimmobilization: Possibility of Multipoint Covalent Immobilization of the Most Stable Enzyme and Immobilization via Ion Exchange of the Least Stable Enzyme

Abstract

Vinyl sulfone (VS)–agarose beads were used to develop a new strategy to coimmobilize enzymes with very different stabilities, enabling the reuse of the most stable immobilized enzyme. Two model combi-biocatalysts were prepared. First, trypsin and chymotrypsin were multipoint covalently coimmobilized on VS–agarose, the support was blocked with ethylenediamine, and then β-galactosidase from Aspergillus oryzae was coimmobilized via anion exchange. Both immobilized proteases were more stable than the immobilized lactase, which could be released from the triple combi-biocatalyst by incubation in 400 mM ammonium sulfate at pH 7.0. Four cycles of combi-biocatalyst incubation at high temperature, β-galactosidase partial inactivation, and release and reimmobilization of the β-galactosidase in the combi-protease biocatalysts were performed, maintaining 80% of the activity of immobilized trypsin and 90% of immobilized chymotrypsin. The second model system was the coimmobilization of trypsin and ficin. Ficin was inactivated when immobilized on VS–agarose supports, but it could be immobilized on this support blocked with aspartic acid. Trypsin was covalently immobilized on the support blocked with aspartic acid and ficin was coimmobilized via cation exchange. As the covalently immobilized enzyme was more stable than ionically exchanged ficin, the combi-biocatalyst was submitted to four cycles as described above with similar results.

Is enzyme immobilization a mature discipline? Some critical considerations to capitalize on the benefits of immobilization

“…120 Here, the development of new enzyme immobilization strategies to solve the problem of poorly matched enzyme stability plays a special role and is an area of particular growth. [136][137][138][139][140] Enzyme immobilization may also be coupled to other techniques to improve enzyme features, such as site-directed mutagenesis, directed evolution or chemical modification. 10,109,[141][142][143][144] In fact, of special interest are those strategies where site-directed mutagenesis or chemical modification has been developed to improve enzyme immobilization itself.…”

“…For example, it has been shown that this blocking step may be used to perform the co-immobilization of several enzymes, enabling the reuse of the most stable enzyme after the inactivation of the least stable. 137,139 4.10. Immobilization protocols are not finished when all the enzyme activity is incorporated in the support Capitalizing on enzyme immobilization to the full, it is not only necessary to utilize a suitable support, an adequate degree of activation of the support and a convenient reactive group on the support.…”

“…In some instances, the enzyme is readily inactivated when immobilized in an activated support. 139 Only a proper investigation will explain if the problem is the active group or it is the nature of the support itself. For this reason, we recommend characterizing a new immobilization protocol for a specific enzyme using an inert and hydrophilic support (such as agarose), 164 and then try to reproduce the protocol with the ''target'' support.…”

“…For example, it has been shown that this blocking step may be used to perform the co-immobilization of several enzymes, enabling the reuse of the most stable enzyme after the inactivation of the least stable. 137,139…”

Application of magnetic field (MF) as an effective method to improve the activity of immobilized Candida antarctica lipase B (CALB)

“…Enzymatic processes have been widely used due to the advantages of fast speed, strong specificity, green environmental protection, and saving reagents and energy. [1][2][3][4][5][6] The designed enzyme-catalyzed chemical reactions have high selectivity and efficiency in saving energy. As a green catalyst, such as other enzymes, Candida antarctica lipase B (CALB, 3.1.1.3) has advantages when used as a catalyst, such as mild reaction conditions, chemo-and enantioselectivity, and high catalytic activity under mild reaction conditions.…”