Authors: Braham, SA; Morellon-Sterling, R; de Andrades, D; Rodrigues, RC; Siar, EH; Aksas, A; Pedroche, J; Millan, MD; Fernandez-Lafuente, R

Article; Early Access.
Appl. Biochem. Biotechnol.. vol: . page: 0273-2289.
Date: . .
Doi: 10.1007/s12010-021-03570-4.

Tris is an extensively used buffer that presents a primary amine group on its structure. In the present work trypsin, chymotrypsin and penicillin G acylase (PGA) were immobilized/stabilized on glyoxyl agarose in presence of different concentrations of Tris (from 0 to 20 mM). The effects of the presence of Tris during immobilization were studied analyzing the thermal stability of the obtained immobilized biocatalysts. The results indicate a reduction of the enzyme stability when immobilized in the presence of Tris. This effect can be observed in inactivations carried out at pH 5, 7, and 9 with all the enzymes assayed. The reduction of enzyme stability increased with the Tris concentration. Another interesting result is that the stability reduction was more noticeable for immobilized PGA than in the other immobilized enzymes, the biocatalysts prepared in presence of 20 mM Tris lost totally the activity at pH 7 just after 1 h of inactivation, while the reference at this time still kept around 61 % of the residual activity. These differences are most likely due to the homogeneous distribution of the Lys groups in PGA compared to trypsin and chymotrypsin (where almost 50% of Lys group are in a small percentage of the protein surface). The results suggest that Tris could be affecting the multipoint covalent immobilization in two different ways, on one hand, reducing the number of available glyoxyl groups of the support during immobilization, and on the other hand, generating some steric hindrances that difficult the formation of covalent bonds..