Yang Fangfang, Backov Rénal, Blin Jean-Luc, Fáklya Bernadett, Tron Thierry, Mekmouche Yasmina
Aix Marseille Univ, Centrale Marseille, CNRS, iSm2, Marseille, France.
Université de Bordeaux, CNRS, CRPP, UMR5031, 115 Avenue Albert Schweitzer, F-33600, Pessac, France.
Biotechnol Rep (Amst). 2021 Jun 9;31:e00645. doi: 10.1016/j.btre.2021.e00645. eCollection 2021 Sep.
We immobilized a fungal laccase with only two spatially close lysines available for functionalization into macrocellular Si(HIPE) monoliths for the purpose of continuous flow catalysis. Immobilization (30-45 % protein immobilization yields) was obtained using a covalent bond forming reaction between the enzyme and low glutaraldehyde (0.625 % (w/w)) functionalized foams. Testing primarily HBT-mediated RB5 dye decolorization in continuous flow reactors, we show that the activity of the heterogeneous catalyst is comparable to its homogeneous counterpart. More, its operational activity remains as high as 60 % after twelve consecutive decolorization cycles as well as after one-year storage, performances remarkable for such a material. We further immobilized two variants of the laccase containing a unique lysine: one located in the vicinity of the substrate oxidation site (K157) and one at the opposite side of this oxidation site (K71) to study the effect of the proximity of the Si(HIPE) surface on enzyme activity. Comparing activities on different substrates for monoliths with differentially oriented catalysts, we show a twofold discrimination for ABTS relative to ascorbate. This study provides ground for the development of neo-functionalized materials that beyond allowing stability and reusability will become synergic partners in the catalytic process.
我们将一种仅含有两个空间上相邻且可用于功能化的赖氨酸的真菌漆酶固定在大孔硅（HIPE）整体柱中，用于连续流动催化。通过酶与低浓度戊二醛（0.625%（w/w））功能化泡沫之间形成共价键的反应实现固定化（蛋白质固定化产率为30 - 45%）。在连续流动反应器中主要测试HBT介导的RB5染料脱色，我们发现多相催化剂的活性与其均相对应物相当。此外，在连续十二次脱色循环以及一年储存后，其操作活性仍高达60%，对于这样一种材料而言，这些性能非常出色。我们进一步固定了含有一个独特赖氨酸的漆酶的两个变体：一个位于底物氧化位点附近（K157），另一个位于该氧化位点的另一侧（K71），以研究Si（HIPE）表面的接近程度对酶活性的影响。通过比较具有不同取向催化剂的整体柱对不同底物的活性，我们发现相对于抗坏血酸，对ABTS有两倍的区分度。这项研究为新型功能化材料的开发奠定了基础，这种材料不仅具有稳定性和可重复使用性，还将成为催化过程中的协同伙伴。
