Institute of Biochemistry, Department of Biotechnology and Enzyme Catalysis, Greifswald University, Felix-Hausdorff-Strasse 4, 17487 Greifswald, Germany.
Appl Microbiol Biotechnol. 2012 Feb;93(3):1119-26. doi: 10.1007/s00253-011-3464-3. Epub 2011 Jul 16.
An esterase from Pseudomonas putida JD1 (PPE) was successfully cloned, actively expressed in Escherichia coli, and characterized. It was discovered that PPE is more active towards short-chain esters, hydrolyzed δ-valerolactone, and ε-caprolactone and was most active at 37°C and pH 8. After purification to homogeneity by Ni-NTA-assisted affinity chromatography, the kinetic parameters K(M) and k(cat) were determined for p-nitrophenyl acetate and butyrate, respectively, showing better catalytic efficiency for hydrolysis of the acetate residue. Investigation of the protein sequence revealed not only the classical catalytic triad for carboxylesterases, additionally the interesting GGG(A)X-motif, which is associated to activity towards tertiary alcohols, was found. Indeed, enzymatic activity was shown for a set of different tertiary alcohols with enantioselectivities up to E = 20, suggesting PPE to be a promising biocatalyst. In addition, PPE also hydrolyzed 4-hydroxyphenyl acetate, the product of a Baeyer-Villiger monooxygenase-catalyzed oxidation of 4-hydroxyacetophenone with a specific activity of 34.36 U/mg suggesting a physiological role in P. putida JD1.
从恶臭假单胞菌 JD1（PPE）中成功克隆、在大肠杆菌中活性表达并鉴定了一种酯酶。研究发现，PPE 对短链酯具有更高的活性，可水解 δ-戊内酯和 ε-己内酯，最适反应温度和 pH 值分别为 37°C 和 8。通过 Ni-NTA 辅助亲和层析进行纯化为均相后，分别测定了对硝基苯乙酸酯和丁酸酯的动力学参数 K(M)和 k(cat)，表明其对乙酸酯残基的水解具有更好的催化效率。对蛋白质序列的研究不仅揭示了经典的羧酸酯酶催化三联体，还发现了有趣的 GGG(A)X 基序，该基序与对叔醇的活性有关。事实上，研究显示 PPE 对一系列不同的叔醇具有酶活性，对映选择性高达 E = 20，表明 PPE 是一种很有前途的生物催化剂。此外，PPE 还可水解 4-羟基苯乙酸酯，这是 4-羟基苯乙酮在 Baeyer-Villiger 单加氧酶催化氧化作用下的产物，其比活性为 34.36 U/mg，表明其在恶臭假单胞菌 JD1 中具有生理作用。
