Novel support for enzyme immobilization prepared by chemical activation with cysteine and glutaraldehyde

• Published in: Journal of molecular catalysis. B, Enzymatic Vol. 102; pp. 218 - 224
• Main Authors: Bezbradica, Dejan I., Mateo, Cesar, Guisan, Jose M.
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 01-04-2014; Elsevier
• Subjects: Agarose; Bioconversions. Hemisynthesis; Biological and medical sciences; Biotechnology; Cysteine; Enzyme immobilization; Fundamental and applied biological sciences. Psychology; Glutaraldehyde; Methods. Procedures. Technologies; Stability; Cysteine; Enzyme immobilization; Glutaraldehyde; Stability; Agarose; Support; Enzyme; Immobilization; Activation; Glutaral
• ISSN: 1381-1177; 1873-3158
• DOI: 10.1016/j.molcatb.2014.02.021

•Novel glutaraldehyde-activated support without a net charge is presented.•Protein immobilization is performed without previous adsorption.•Linkages protein-support were firstly reversible being converted into irreversible with the time or after reduction.•Reactive groups are quite stable allowing the incubation at alkaline pH.•Incubated derivatives were more stable than that just immobilized at pH 7.
Immobilization of enzymes on glutaraldehyde-activated supports has been largely used on supports previously activated with amine groups. Therefore, the supports are positively charged hence usually the immobilization is promoted through a two step mechanism: in a first step the enzyme is adsorbed on the support via an anionic exchange mechanism and then, the covalent immobilization occurs. In this paper a new glutaraldehyde activated support without a net charge is presented and characterized in immobilizations of trypsin, penicillin acylase G, lipase and E. coli BL21 cell extract. Immobilization mechanism was studied and this was produced without an adsorption step. This support promoted initially a reversible immobilization, converting into irreversible after incubation of the enzyme-support for several days or after a reduction step. In addition the stability of glutaraldehyde groups was studied retaining around 50 and 25% of its immobilization capacity for 24h at pH 7 and 10 respectively. This fact allows the incubation of the enzyme with the support even at alkaline pH promoting an extra stabilization factor for trypsin on this support.