Substrate bio-imprinted CLEAs of type B feruloyl esterase from Aspergillus terreus: A selective heterogeneous biocatalyst towards butyl caffeate

IF 4 3区生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Process Biochemistry Pub Date : 2025-03-01 Epub Date: 2025-01-10 DOI:10.1016/j.procbio.2025.01.004

Daniel A. Grajales-Hernández , Mariana A. Armendáriz-Ruiz , Jorge A. Rodríguez , Susana Velasco-Lozano , Fernando López-Gallego , Juan Carlos Mateos-Díaz

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Abstract

The biological properties of caffeic acid esters, compel the search for selective biocatalysts for its synthesis since caffeic acid is generally mixed with ferulic acid in nature. Here, we employed a type B feruloyl esterase from Aspergillus terreus bio-imprinted and crosslinked (AtFAEB-CLEAs) to increase its selectivity towards the synthesis of butyl caffeate in caffeic/ferulic acid equimolar mixtures. The best AtFAEB-CLEAs were prepared using n-butanol/caffeic acid (50 mM) mixture for enzyme precipitation/bio-imprinting, polyethyleneimine as co-feeder, and glutaraldehyde as a crosslinker. Selected AtFAEB-CLEAs showed an expressed activity 2.6-fold higher (218 U·mg⁻¹) than free enzyme, and a 13.5-fold enhancement in synthesis selectivity on a hexane: n-butanol: water system, inverting the initial preference for ferulic acid towards caffeic acid. Intrinsic protein fluorescence profiles strongly suggest a freeze-up of conformational changes induced by bioimprinting in the AtFAEB. Additionally, immobilization increased the thermal and solvent stability of AtFAEB-CLEAS 7.9 and 5.7-fold, respectively. Final reaction tests (0.1 mg/mL) achieved 88 % conversion in 6 hours and demonstrated a strong preference for butyl caffeate synthesis of 26-fold in the first hour of reaction. Improvement through CLEA fabrication of selectivity and stability of AtFAEB could potentially lead to the valorization of agro-industrial by-products into relevant molecules for the cosmetic and pharmaceutic industries.

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地曲霉B型阿魏酰酯酶的底物生物印迹CLEAs：一种选择性非均相生物催化剂

咖啡酸酯的生物学特性迫使人们寻找选择性的生物催化剂来合成咖啡酸，因为咖啡酸在自然界中通常与阿魏酸混合。在这里，我们使用了一种来自地曲霉生物印迹交联的B型阿魏酰酯酶（AtFAEB-CLEAs），以提高其在咖啡酸/阿魏酸等摩尔混合物中合成咖啡酸丁酯的选择性。以正丁醇/咖啡酸（50 mM）混合酶沉淀/生物印迹，聚乙烯亚胺为共进料剂，戊二醛为交联剂制备最佳的AtFAEB-CLEAs。选择的AtFAEB-CLEAs的表达活性比游离酶高2.6倍（218 U·mg−1），在正己烷：正丁醇：水体系上的合成选择性提高13.5倍，逆转了最初对阿威酸的偏好，而不是咖啡酸。固有蛋白荧光谱强烈提示生物印迹诱导的AtFAEB构象变化冻结。此外，固定化使AtFAEB-CLEAS的热稳定性和溶剂稳定性分别提高了7.9倍和5.7倍。最终的反应测试（0.1 mg/mL）在6 小时内达到88 %的转化率，并显示在反应的第一个小时内强烈倾向于合成26倍的咖啡酸丁酯。通过CLEA制备提高AtFAEB的选择性和稳定性，可能会导致农用工业副产品转化为化妆品和制药工业的相关分子。

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来源期刊

Process Biochemistry 生物-工程：化工

CiteScore

8.30

自引率

4.50%

发文量

374

审稿时长

53 days

期刊介绍： Process Biochemistry is an application-orientated research journal devoted to reporting advances with originality and novelty, in the science and technology of the processes involving bioactive molecules and living organisms. These processes concern the production of useful metabolites or materials, or the removal of toxic compounds using tools and methods of current biology and engineering. Its main areas of interest include novel bioprocesses and enabling technologies (such as nanobiotechnology, tissue engineering, directed evolution, metabolic engineering, systems biology, and synthetic biology) applicable in food (nutraceutical), healthcare (medical, pharmaceutical, cosmetic), energy (biofuels), environmental, and biorefinery industries and their underlying biological and engineering principles.