Substrate specificity modification of paraben hydrolase and tannase from Aspergillus oryzae

IF 3.4 3区生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Enzyme and Microbial Technology Pub Date : 2024-08-07 DOI:10.1016/j.enzmictec.2024.110495

Michiko Hakoda, Tomoe Kato, Chihiro Takahashi, Yoshihito Shiono, Takuya Koseki

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Abstract

Paraben hydrolase and tannase catalyze the hydrolysis of parabens (4-hydroxybenzoic acid esters) and gallic acid (3,4,5-trihydroxybenzoic acid) esters, respectively. Paraben hydrolase (AoPrbA) and tannase (AoTanB) from Aspergillus oryzae belong to the tannase family in the ESTHER database. However, the substrate specificities of AoPrbA and AoTanB are narrow. Based on structural information of Aspergillus niger tannase (PDB code 7k4o), we constructed five single variants of AoPrbA (Thr200Glu, Phe231Gln, Leu232Gln, Ile361Tyr, and Leu428Ser) and four of AoTanB (Glu203Asp, Glu203Thr, His237Ala, and Ser440Leu) to investigate substrate discrimination between AoPrbA and AoTanB. Each variant was expressed in Pichia pastoris and were purified from the culture supernatant. Five purified variants of AoPrbA and four variants of AoTanB showed reduced paraben hydrolase and tannase activities compared with AoPrbA and AoTanB wild types, respectively. Interestingly, the AoPrbA wild type did not hydrolyze gallic acid methyl ester, whereas the Thr200Glu, Leu232Gln, and Leu428Ser variants did, indicating that these three variants acquired tannase activity. In particular, the Leu428Ser variant exhibited considerably greater hydrolysis of gallic acid and protocatechuic acid methyl esters. Meanwhile, the AoTanB wild type, and Glu203Asp, His237Ala and Ser440Leu variants hydrolyzed the protocatechuate methyl and 4-hydroxybenzoate ethyl esters; however, the Glu203Thr variant did not hydrolyze above-mentioned substrates. Additionally, the ratio of paraben hydrolase activity to tannase activity in Ser440Leu was markedly elevated.

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对羟基苯甲酸酯水解酶和黑曲霉单宁酶的底物特异性修饰

对羟基苯甲酸酯水解酶和单宁酸酶分别催化对羟基苯甲酸酯（4-羟基苯甲酸酯）和没食子酸（3,4,5-三羟基苯甲酸）酯的水解。在ESTHER数据库中，来自黑曲霉的对羟基苯甲酸酯水解酶（AoPrbA）和单宁酸酶（AoTanB）属于单宁酸酶家族。然而，AoPrbA 和 AoTanB 的底物特异性较窄。根据黑曲霉单宁酶（PDB 代码 7k4o）的结构信息，我们构建了 AoPrbA 的五个单变体（Thr200Glu、Phe231Gln、Leu232Gln、Ile361Tyr 和 Leu428Ser）和 AoTanB 的四个单变体（Glu203Asp、Glu203Thr、His237Ala 和 Ser440Leu），以研究 AoPrbA 和 AoTanB 的底物特异性。每个变体都在 Pichia pastoris 中表达，并从培养上清液中纯化。与 AoPrbA 和 AoTanB 野生型相比，纯化的 5 个 AoPrbA 变体和 4 个 AoTanB 变体分别显示出较低的对羟基苯甲酸酯水解酶和单宁酶活性。有趣的是，AoPrbA 野生型不能水解没食子酸甲酯，而 Thr200Glu、Leu232Gln 和 Leu428Ser 变体却能，这表明这三个变体获得了单宁酶活性。特别是，Leu428Ser 变体对没食子酸和原儿茶酸甲酯的水解能力要强得多。同时，AoTanB 野生型、Glu203Asp、His237Ala 和 Ser440Leu 变体可水解原儿茶酸甲酯和 4-羟基苯甲酸乙酯；但 Glu203Thr 变体不能水解上述底物。此外，Ser440Leu 变体的对羟基苯甲酸酯水解酶活性与单宁酸酶活性之比明显升高。

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来源期刊

Enzyme and Microbial Technology 生物-生物工程与应用微生物

CiteScore

7.60

自引率

5.90%

发文量

142

审稿时长

38 days

期刊介绍： Enzyme and Microbial Technology is an international, peer-reviewed journal publishing original research and reviews, of biotechnological significance and novelty, on basic and applied aspects of the science and technology of processes involving the use of enzymes, micro-organisms, animal cells and plant cells. We especially encourage submissions on: Biocatalysis and the use of Directed Evolution in Synthetic Biology and Biotechnology Biotechnological Production of New Bioactive Molecules, Biomaterials, Biopharmaceuticals, and Biofuels New Imaging Techniques and Biosensors, especially as applicable to Healthcare and Systems Biology New Biotechnological Approaches in Genomics, Proteomics and Metabolomics Metabolic Engineering, Biomolecular Engineering and Nanobiotechnology Manuscripts which report isolation, purification, immobilization or utilization of organisms or enzymes which are already well-described in the literature are not suitable for publication in EMT, unless their primary purpose is to report significant new findings or approaches which are of broad biotechnological importance. Similarly, manuscripts which report optimization studies on well-established processes are inappropriate. EMT does not accept papers dealing with mathematical modeling unless they report significant, new experimental data.