Gennari Adriano, Mobayed Francielle H, da Silva Rafael Ruan, Rodrigues Rafael C, Sperotto Raul A, Volpato Giandra, Volken de Souza Claucia F
Laboratório de Biotecnologia de Alimentos, Programa de Pós-Graduação em Biotecnologia, Universidade do Vale do Taquari - Univates, Lajeado, RS, Brazil.
Biotechnology, Bioprocess and Biocatalysis Group, Institute of Food Science and Technology, Federal University of Rio Grande do Sul, Porto Alegre, RS, Brazil.
Biotechnol Prog. 2018 Jul;34(4):934-943. doi: 10.1002/btpr.2652. Epub 2018 Jun 4.
We studied the modification of Immobead 150 support by either introducing aldehyde groups using glutaraldehyde (Immobead-Glu) or carboxyl groups through acid solution (Immobead-Ac) for enzyme immobilization by covalent attachment or ion exchange, respectively. These two types of immobilization were compared with the use of epoxy groups that are now provided on a commercial support. We used Aspergillus oryzae β-galactosidase (Gal) as a model protein, immobilizing it on unmodified (epoxy groups, Immobead-Epx) and modified supports. Immobilization yield and efficiency were tested as a function of protein loading (10-500 mg g support). Gal was efficiently immobilized on the Immobeads with an immobilization efficiency higher than 75% for almost all supports and protein loads. Immobilization yields significantly decreased when protein loadings were higher than 100 mg g support. Gal immobilized on Immobead-Glu and Immobead-Ac retained approximately 60% of its initial activity after 90 days of storage at 4°C. The three immobilized Gal derivatives presented higher half-lifes than the soluble enzyme, where the half-lifes were twice higher than the free Gal at 73°C. All the preparations were moderately operationally stable when tested in lactose solution, whey permeate, cheese whey, and skim milk, and retained approximately 50% of their initial activity after 20 cycles of hydrolyzing lactose solution. The modification of the support with glutaraldehyde provided the most stable derivative during cycling in cheese whey hydrolysis. Our results suggest that the Immobead 150 is a promising support for Gal immobilization. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:934-943, 2018.
我们研究了Immobead 150载体的改性方法，即分别使用戊二醛引入醛基（Immobead-Glu）或通过酸溶液引入羧基（Immobead-Ac），以便分别通过共价连接或离子交换固定酶。将这两种固定化方法与现在市售载体上提供的环氧基团的使用进行了比较。我们使用米曲霉β-半乳糖苷酶（Gal）作为模型蛋白，将其固定在未改性的（环氧基团，Immobead-Epx）和改性载体上。固定化产率和效率作为蛋白质负载量（10 - 500 mg g载体）的函数进行了测试。Gal能有效地固定在Immobeads上，几乎所有载体和蛋白质负载量的固定化效率都高于75%。当蛋白质负载量高于100 mg g载体时，固定化产率显著降低。固定在Immobead-Glu和Immobead-Ac上的Gal在4°C储存90天后保留了其初始活性的约60%。三种固定化的Gal衍生物的半衰期比可溶性酶更长，在73°C时半衰期比游离Gal高出两倍。在乳糖溶液、乳清渗透液、干酪乳清和脱脂牛奶中测试时，所有制剂的操作稳定性适中，在水解乳糖溶液20个循环后保留了其初始活性的约50%。用戊二醛对载体进行改性在干酪乳清水解循环过程中提供了最稳定的衍生物。我们的结果表明，Immobead 150是固定Gal的一种有前景的载体。© 2018美国化学工程师学会生物技术进展，34:934 - 943，2018。
