Departamento de Biocatalisis, ICP-CSIC, Campus UAM-CSIC Cantoblanco, Madrid, Spain; TECNOLÓGICO NACIONAL DE MÉXICO, INSTITUTO TECNOLÓGICO DEDURANGO, Blvd. Felipe Pescador 1130 Ote. Col., Nueva Vizcaya, CP 34080, Durango, Dgo., México.
Departamento de Biocatalisis, ICP-CSIC, Campus UAM-CSIC Cantoblanco, Madrid, Spain.
Enzyme Microb Technol. 2018 Mar;110:38-45. doi: 10.1016/j.enzmictec.2017.12.007. Epub 2017 Dec 22.
The dimeric enzyme β-glucosidase from Aspergillus niger has been immobilized on different amino-agarose beads at pH 5 and 7, exploiting the versatility of glutaraldehyde. The stability of the free enzyme depended on enzyme concentration. Immobilization via ion exchange improved enzyme stability/activity, depending on the immobilization pH. However, the enzyme was desorbed in 75 mM NaCl at pH 7 and some stability/enzyme concentration dependence still existed.
of these biocatalysts with glutaraldehyde increased enzyme stability (e.g. at pH 5, after incubation under conditions where the enzyme just ionically exchanged was fully inactivated, the activity of the glutaraldehyde treated enzyme remained unaltered). Immobilization on glutaraldehyde pre-activated supports yielded a higher increase in enzyme activity, but the stabilization was lower. While when measuring the enzyme activity at pH 4 there were no changes after immobilization, all immobilized enzymes were more active than the free enzyme at pH 6 and 7 (2-3 times). The Ki/Km ratio did not significantly decrease in any immobilized biocatalysts, and in some cases it worsened in a significant way (by a 9 fold factor using preactivated supports). The new biocatalysts are significantly more stable and avoid enzyme subunit desorption, being the immobilization pH a key point in their design.
黑曲霉来源的二聚体酶β-葡萄糖苷酶已在 pH5 和 7 下通过戊二醛的多功能性固定在不同的氨基琼脂糖珠上。游离酶的稳定性取决于酶浓度。通过离子交换进行的固定化提高了酶的稳定性/活性，这取决于固定化 pH 值。然而，在 pH7 下用 75mM NaCl 洗脱时，酶会被洗脱出来，并且仍然存在一些稳定性/酶浓度依赖性。
用戊二醛处理这些生物催化剂可以提高酶的稳定性（例如，在 pH5 下孵育时，仅通过离子交换的酶完全失活，戊二醛处理的酶的活性保持不变）。在戊二醛预激活载体上固定化可使酶活性提高更高，但稳定性较低。虽然在 pH4 下测量酶活性时固定化后没有变化，但所有固定化酶在 pH6 和 7 时都比游离酶更活跃（2-3 倍）。在任何固定化生物催化剂中，Ki/Km 比值均未显著降低，在某些情况下，该比值显著恶化（使用预激活载体时，降低了 9 倍）。新型生物催化剂的稳定性显著提高，避免了酶亚基的洗脱，固定化 pH 值是其设计的关键。
