Biotechnology, Bioprocess, and Biocatalysis Group, Food Science and Technology Institute, Federal University of Rio Grande do Sul, Porto Alegre, RS, Brazil.
Department of Biocatalysis, ICP-CSIC, Campus UAM-CSIC, Madrid, Spain.
Biotechnol Prog. 2019 Nov;35(6):e2890. doi: 10.1002/btpr.2890. Epub 2019 Aug 19.
β-Glucosidases from two different commercial preparations, Pectinex Ultra SP-L and Celluclast® 1.5L, were immobilized on divinylsulfone (DVS) supports at pH 5.0, 7.0, 9.0, and 10. In addition, the biocatalysts were also immobilized in agarose beads activated by glyoxyl, and epoxide as reagent groups. The best immobilization results were observed using higher pH values on DVS-agarose, and for Celluclast® 1.5L, good results were also obtained using the glyoxil-agarose immobilization. The biocatalyst obtained using Pectinex Ultra SP-L showed the highest thermal stability, at 65°C, and an operational stability of 67% of activity after 10 reuses cycles when immobilized on DVS-agarose immobilized at pH 10 and blocked with ethylenediamine. The β-glucosidase from Celluclast® 1.5L produced best results when immobilized on DVS-agarose immobilized at pH 9 and blocked with glycine, reaching 7.76-fold higher thermal stability compared to its free form and maintaining 76% of its activity after 10 successive cycles. The new biocatalysts obtained by these protocols showed reduction of glucose inhibition of enzymes, demonstrating the influence of immobilization protocols, pH, and blocking agent.
来自两种不同商业制剂的β-葡萄糖苷酶，果胶酶 Ultra SP-L 和 Celluclast®1.5L，在 pH 值为 5.0、7.0、9.0 和 10.条件下通过二乙烯砜（DVS）载体固定化。此外，这些生物催化剂也通过乙二醛和环氧化物作为活化试剂基团的琼脂糖珠固定化。在 DVS-琼脂糖上，使用较高的 pH 值可获得最佳的固定化效果，而对于 Celluclast®1.5L，使用乙二醛-琼脂糖固定化也可获得良好的效果。使用 Pectinex Ultra SP-L 获得的生物催化剂在 65°C 时表现出最高的热稳定性，在 10 次重复使用循环后，当在 pH 值为 10 的 DVS-琼脂糖上固定化并用乙二胺封闭时，其活性保持了 67%。当在 pH 值为 9 的 DVS-琼脂糖上固定化并用甘氨酸封闭时，Celluclast®1.5L 的β-葡萄糖苷酶可获得最佳的固定化效果，与游离形式相比，其热稳定性提高了 7.76 倍，在连续 10 个循环后仍保持 76%的活性。通过这些方案获得的新型生物催化剂显示出酶的葡萄糖抑制作用降低，表明固定化方案、pH 值和封闭剂的影响。
