Department of Chemical and Biochemical Engineering, Chair of Chemical Biotechnology, TU Dortmund, Dortmund 44221, Germany.
Molecules. 2011 Jul 18;16(7):5975-88. doi: 10.3390/molecules16075975.
A straightforward one-pot procedure combining enrichment and immobilization of recombinantely expressed FADH₂ dependent styrene monooxygenase (StyA) directly from Escherichia coli cell extracts was investigated. Sepabeads EC-EA and EC-Q1A anion-exchange carriers were employed to non-covalently adsorb StyA from the cell extracts depending on basic parameters such as varying initial protein concentrations and pH. The protein fraction of the cell extract contained around 25% StyA. At low initial protein concentrations (2.5 mg mL⁻¹) and pH 6, the enzyme could be enriched up to 52.4% on Sepabeads EC-EA and up to 46.0% on Sepabeads EC-Q1A, accounting for an almost complete StyA adsorption from the cell extracts. Higher initial protein concentrations were necessary to exploit the high loading capacity of the beads. At 20 mg mL⁻¹, up to 37.6% of the theoretical bead loading capacity could be utilized for StyA binding using Sepabeads EC-EA, and 34.0% using Sepabeads EC-Q1A. For both carriers, protein leakage under reaction conditions could be reduced to less than 2%. During assays, the FADH₂ cofactor necessary for StyA activity was supplied by the NADH-FAD reductase component styrene monooxygenase B (StyB). StyA immobilized on Sepabeads EC-Q1A displayed twice as high styrene epoxidation rates (0.2 U mg(StyA)⁻¹) as compared to Sepabeads EC-EA. This activity could be increased to 0.7 U mg(StyA)⁻¹ by co-immobilizing StyB on Sepabeads EC-Q1A, which corresponds to 33% of the soluble StyA activity.
本研究探索了一种简便的一锅法，可直接从大肠杆菌细胞提取物中富集和固定重组表达的黄素腺嘌呤二核苷酸（FADH₂）依赖性苯乙烯单加氧酶（StyA）。采用 Sepabeads EC-EA 和 EC-Q1A 阴离子交换载体，根据初始蛋白浓度和 pH 等基本参数，非共价吸附细胞提取物中的 StyA。细胞提取物中的蛋白部分含有约 25%的 StyA。在低初始蛋白浓度（2.5 mg mL⁻¹）和 pH 6 条件下，酶可在 Sepabeads EC-EA 上富集至 52.4%，在 Sepabeads EC-Q1A 上富集至 46.0%，几乎可实现从细胞提取物中完全吸附 StyA。需要更高的初始蛋白浓度来利用珠子的高载量。在 20 mg mL⁻¹时，Sepabeads EC-EA 可利用高达 37.6%的理论珠载量结合 StyA，Sepabeads EC-Q1A 可利用 34.0%。对于两种载体，在反应条件下的蛋白泄漏均可降低至 2%以下。在测定中，苯乙烯单加氧酶 B（StyB）的 NADH-FAD 还原酶组分提供了 StyA 活性所需的 FADH₂辅因子。固定在 Sepabeads EC-Q1A 上的 StyA 的苯乙烯环氧化速率（0.2 U mg(StyA)⁻¹）是固定在 Sepabeads EC-EA 上的两倍。通过在 Sepabeads EC-Q1A 上共固定 StyB，可将此活性提高至 0.7 U mg(StyA)⁻¹，相当于可溶性 StyA 活性的 33%。
