Covalent immobilization of cyclodextrin glucosyltransferase (CGTase) in activated silica and Sepharose.
Cyclodextrin glucanotransferase is a non-Leloir glycosyltransferase that directly employs the free energy of cleavage of starch to produce cyclodextrins. In presence of appropriate acceptors, this enzyme synthesizes oligosaccharides containing alpha(1-->4) bonds. We have investigated the covalent immobilization of CGTase onto different activated supports. Silica was aminated and further activated with glutaraldehyde. The maximum amount of bound protein was about 4 mg CGTase per gram of support; however, the catalytic efficiency of the immobilized enzyme was lower than 6%. Sepharose 4B activated with cyanogen bromide (CNBr-activated Sepharose) and Sepharose 4B with a spacer arm of 1,6-diaminohexane (EAH Sepharose) were also assayed. These gels react with the amino and carboxylic groups of CGTase, respectively. With CNBr-activated Sepharose, a low percentage of enzyme was bound to the support but with a significant catalytic efficiency (29%). A higher recovery of protein was obtained with EAH Sepharose (62%), but only 2.4% of the initial activity was present in the immobilized biocatalyst. The results were discussed in terms of CGTase structure and mechanism. In addition, the solvent accessibility of amino or carboxylic groups, calculated using the NACCESS software, was considered.
Departamento de Biocatálisis, Instituto de Catálisis, CSIC, Campus UAM Cantoblanco, 28049 Madrid, Spain.
M Teresa
MT
Martín
Miguel
M
Alcalde
Francisco J
FJ
Plou
Antonio
A
Ballesteros
D000818:Animals; D020134:Catalytic Domain; D003505:Cyclodextrins / Q000737:chemistry; D003959:Diamines / Q000737:chemistry; D004798:Enzymes / Q000737:chemistry; D004800:Enzymes, Immobilized / Q000737:chemistry*; D005964:Glucosyltransferases / Q000737:chemistry*; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry; D008956:Models, Chemical; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry; D012685:Sepharose / Q000737:chemistry*; D012822:Silicon Dioxide / Q000737:chemistry*; D012984:Software; D046968:Thermoanaerobacter / Q000378:metabolism; D013997:Time Factors
D003505:Cyclodextrins; D003959:Diamines; D004798:Enzymes; D004800:Enzymes, Immobilized; D007074:Immunoglobulin G; D011506:Proteins; D012822:Silicon Dioxide; D012685:Sepharose; D005964:Glucosyltransferases; C019963:cyclomaltodextrin glucanotransferase; C007297:1,6-diaminohexane