Improvement of Enzyme Properties with a Two-Step Immobilizaton Process on Novel Heterofunctional Supports

• Published in: Biomacromolecules 2010-11, Vol.11 (11), p.3112-3117
• Main Authors: Mateo, Cesar, Bolivar, Juan Manuel, Godoy, Cesar A, Rocha-Martin, Javier, Pessela, Benevides C, Curiel, Jose Antonio, Muñoz, Rosario, Guisan, Jose M, Fernández-Lorente, Gloria
• Format: Article
• Language: English
• Subjects: Adsorption; Animals; Applied sciences; Biological and medical sciences; Biotechnology; Carboxylic Ester Hydrolases - chemistry; Carboxylic Ester Hydrolases - metabolism; Chymotrypsin - chemistry; Chymotrypsin - metabolism; Enzyme Stability; Enzymes, Immobilized - chemistry; Enzymes, Immobilized - metabolism; Exact sciences and technology; Fundamental and applied biological sciences. Psychology; Geobacillus - enzymology; Glyoxylates - chemistry; Hydrogen-Ion Concentration; Immobilization of enzymes and other molecules; Immobilization techniques; Lactobacillus plantarum - enzymology; Lipase - chemistry; Lipase - metabolism; Methods. Procedures. Technologies; Natural polymers; Pancreas - enzymology; Physicochemistry of polymers; Sepharose - chemistry; Starch and polysaccharides; Surface Properties; Swine
• ISSN: 1525-7797; 1526-4602
• DOI: 10.1021/bm100916r

Novel heterofunctional glyoxyl-agarose supports were prepared. These supports contain a high concentration of groups (such as quaternary ammonium groups, carboxyl groups, and metal chelates) that are capable of adsorbing proteins, physically or chemically, at neutral pH as well as a high concentration of glyoxyl groups that are unable to immobilize covalently proteins at neutral pH. By using these supports, a two-step immobilization protocol was developed. In the first step, enzymes were adsorbed at pH 7.0 through adsorption of surface regions, which are complementary to the adsorbing groups on the support, and in the second step, the immobilized derivatives were incubated under alkaline conditions to promote an intramolecular multipoint covalent attachment between the glyoxyl groups on the support and the amino groups on the enzyme surface. These new derivatives were compared with those obtained on a monofunctional glyoxyl support at pH 10, in which the region with the greatest number of lysine residues participates in the first immobilization step. In some cases, multipoint immobilization on heterofunctional supports was much more efficient than what was achieved on the monofunctional support. For example, derivatives of tannase from Lactobacillus plantarum on an amino-glyoxyl heterofunctional support were 20-fold more stable than the best derivative on a monofunctional glyoxyl support. Derivatives of lipase from Geobacillus thermocatenulatus (BTL2) on the amino-glyoxyl supports were two times more active and four times more enantioselective than the corresponding monofunctional glyoxyl support derivative.