Biotechnology, Bioprocess, and Biocatalysis Group, Food Science and Technology Institute, Federal University of Rio Grande do Sul, Av. Bento Gonçalves 9500, PO Box 15090, ZC 91501-970 Porto Alegre, RS, Brazil.
Center of Medical Sciences and Pharmaceutical, Western Paraná State University, Rua Universitária 2069, ZC 85819-110 Cascavel, PR, Brazil.
Int J Biol Macromol. 2019 May 15;129:672-678. doi: 10.1016/j.ijbiomac.2019.02.057. Epub 2019 Feb 14.
Three β-glucosidases (Pectinex Ultra SP-L, Pectinex Ultra Clear and homemade preparation from Aspergillus niger) were immobilized using different strategies: ionic adsorption on aminated (MANAE)-agarose beads at pH 5, 7, and 9, followed by biocatalysts modification with glutaraldehyde, or on glutaraldehyde pre-activated supports. The pH of the immobilization was altered to allow different enzyme molecule orientations on the support surface. The biocatalysts from Pectinex Ultra SP-L showed the highest thermal and operational stabilities when immobilized on MANAE-agarose-glutaraldehyde at pH 7. The β-glucosidase from Pectinex Ultra Clear and from A. niger produced best results when immobilized on MANAE-agarose beads at pH 5 and 7, respectively, which was later treated with glutaraldehyde. The best immobilization results using pre-activated supports were observed for the enzyme present in Pectinex Ultra SP-L, to which the highest thermal stabilities were obtained. Remarkably, the enzyme from A. niger, immobilized on MANAE-agarose at pH 9 and subsequently treated with glutaraldehyde, produced the highest stabilization (approximately 560 times more stable than soluble enzyme at 60 °C). Results showed that optimal protocol for β-glucosidases immobilizations using the glutaraldehyde chemistry must be individually tested and tailored to each type of enzyme.
三种β-葡萄糖苷酶（Pectinex Ultra SP-L、Pectinex Ultra Clear 和来自黑曲霉的自制制剂）采用不同策略进行固定化：在 pH 5、7 和 9 下通过离子吸附到氨甲基琼脂糖珠上，然后用戊二醛修饰生物催化剂，或在戊二醛预激活的载体上进行固定化。固定化的 pH 值发生变化，以允许支持物表面上不同的酶分子取向。当在 pH 7 的氨甲基琼脂糖-戊二醛上固定化时，来自 Pectinex Ultra SP-L 的β-葡萄糖苷酶显示出最高的热稳定性和操作稳定性。来自 Pectinex Ultra Clear 和黑曲霉的β-葡萄糖苷酶在 pH 5 和 7 下分别在氨甲基琼脂糖珠上固定化效果最佳，随后用戊二醛处理。使用预激活载体进行最佳固定化的结果是在 Pectinex Ultra SP-L 中存在的酶，获得了最高的热稳定性。值得注意的是，在 pH 9 下固定化在氨甲基琼脂糖上的黑曲霉来源的酶，随后用戊二醛处理，产生了最高的稳定性（在 60°C 下比可溶性酶稳定约 560 倍）。结果表明，使用戊二醛化学固定化β-葡萄糖苷酶的最佳方案必须单独测试，并针对每种类型的酶进行调整。
