Catálisis Heterogénea en Síntesis Orgánicas Selectivas, Instituto de Síntesis Química y Catálisis Homogénea (ISQCH-CSIC), University of Zaragoza, Pedro Cerbuna, 12, 50009 Zaragoza, Spain; Heterogeneous Biocatalysis Laboratory, CICbiomaGUNE Basque Research and Technology Alliance (BRTA), Paseo de Miramón, 182, 20014 Donostia-San Sebastián, Spain.
Laboratorio de Biotecnología, Universidad ORT Uruguay, Cuareim 1441, 11100 Montevideo, Uruguay.
Int J Biol Macromol. 2020 Dec 1;164:4318-4328. doi: 10.1016/j.ijbiomac.2020.09.003. Epub 2020 Sep 6.
Transaminases are a class of enzymes with promising applications for the preparation and resolution of a vast diversity of valued amines. Their poor operational stability has fueled many investigations on its stabilization due to their biotechnological relevance. In this work, we screened the stabilization of the tetrameric ω-transaminase from Pseudomonas fluorescens (PfωTA) through both carrier-bound and carrier-free immobilization techniques. The best heterogeneous biocatalyst was the PfωTA immobilized as cross-linked enzyme aggregates (PfωTA-CLEA) which resulted after studying different parameters as the precipitant, additives and glutaraldehyde concentrations. The best conditions for maximum recovered activity (29 %) and maximum thermostability at 60 ºC and 70 ºC (100 % and 71 % residual activity after 1 h, respectively) were achieved by enzyme precipitation with 90% acetone or ethanol, in presence of BSA (100 mg/mL) and employing glutaraldehyde (100 mM) as cross-linker. Studies on different conditions for PfωTA-CLEA preparation yielded a biocatalyst that exhibited 31 and 4.6 times enhanced thermal stability at 60 °C and 70 °C, respectively, compared to its soluble counterpart. The PfωTA-CLEA was successfully used in the bioamination of 4-hydroxybenzaldehyde to 4-hydroxybenzylamine. To the best of our knowledge, this is the first report describing a transaminase cross-linked enzyme aggregates as immobilization strategy to generate a biocatalyst with outstanding thermostability.
转氨酶是一类具有广阔应用前景的酶，可用于制备和拆分各种有价值的胺类化合物。由于其在生物技术中的重要性，其较差的操作稳定性促使人们对其稳定性进行了大量研究。在这项工作中，我们通过载体结合和无载体固定化技术筛选了荧光假单胞菌（Pseudomonas fluorescens）中的ω-转氨酶（PfωTA）的稳定性。最佳的非均相生物催化剂是 PfωTA 交联酶聚集体（PfωTA-CLEA），通过研究不同的参数如沉淀剂、添加剂和戊二醛浓度得到。在最佳条件下，最大活性回收率（29%）和在 60°C 和 70°C 时的最大热稳定性（分别在 1 小时后保留 100%和 71%的活性）通过 90%丙酮或乙醇沉淀、添加 BSA（100mg/mL）和使用戊二醛（100mM）作为交联剂实现。对 PfωTA-CLEA 制备的不同条件的研究得到了一种生物催化剂，与可溶性酶相比，其在 60°C 和 70°C 时的热稳定性分别提高了 31 倍和 4.6 倍。PfωTA-CLEA 成功地用于 4-羟基苯甲醛的生物胺化反应，生成 4-羟基苯甲胺。据我们所知，这是首次报道将转氨酶交联酶聚集体作为固定化策略，生成具有出色热稳定性的生物催化剂。
