Sol–gel immobilization of Alcalase from Bacillus licheniformis for application in the synthesis of C-terminal peptide amides

Abstract

Precusor mixtures from organically modified silanes, having general formula (R 1 ,R 2 )Si(OCH 3 ) n ( n = 2 or 3), and tetramethoxysilane (at 1:1 molar ratio) have been used for sol–gel entrapment of Alcalase, revealing immobilization yields around 80%, while the highest activity in amidation of Z-Ala-Phe-OMe dipeptide ester was showed by the biocatalyst obtained from the precursor with two methyl nonhydrolizable groups. ► Commercial Alcalase was successfully entrapped in sol–gel matrices derived from different silane precursors. ► Encapsulated protease had good storage stability at room temperature and against heat treatment. ► Immobilized biocatalyst could be reused in repeated batch biotransformation runs, showing a good operational and mechanical stability. Alcalase 2.4L FG, a commercial preparation of Subtilisin A, was physically entrapped in glass sol–gel matrices using alkoxysilanes of different types mixed with tetramethoxysilane (TMOS). The materials were used for catalyzing C-terminal amidation of Z-Ala-Phe-OMe in a mixture of tert -butanol/DMF. From the screening of silane monomers in the sol–gel coating process, it was concluded that dimethyldimethoxysilane (DMDMOS) gave the best performance, and Alcalase immobilized therein exhibited the highest activity in the ammonolysis of Z-Ala-Phe-OMe. The percentage of protein immobilization was in the range of 68–98%, and total amidation activity of the immobilized Alcalase was up to 1.76 μmol/h/mg gel. We investigated the immobilization efficiency for a protein mass range of 2.8–9.7 mg per mmol total silanes, to determine the immobilization capacity of the biosilica support. The optimum enzyme loading capacity in the silica matrix was 115 mg/g dry silica xerogel (11.5%, w/w). The amount of the DMDMOS silicate was optimized by adjusting the molar ratio of silane mixture (DMDMOS and TMOS at 1:1). Biocatalyst sol–gel particles prepared at optimum immobilization conditions retained 100% of the original activity even after 14 cycles of repeated use. Reproducibility of the immobilization technique was also investigated by evaluating the catalytic efficiency of the obtained preparations. The thermal stability of the protease at 70 °C increased threefold upon entrapment in sol–gel materials, and twofold under storage for 50 days at ambient temperature.