Perz Veronika, Baumschlager Armin, Bleymaier Klaus, Zitzenbacher Sabine, Hromic Altijana, Steinkellner Georg, Pairitsch Andris, Łyskowski Andrzej, Gruber Karl, Sinkel Carsten, Küper Ulf, Ribitsch Doris, Guebitz Georg M
acib-Austrian Centre of Industrial Biotechnology, Konrad Lorenz Strasse 20, Tulln, 3430, Austria.
acib-Austrian Centre of Industrial Biotechnology, Graz, Austria.
Biotechnol Bioeng. 2016 May;113(5):1024-34. doi: 10.1002/bit.25874. Epub 2015 Nov 20.
Two novel esterases from the anaerobe Clostridium botulinum ATCC 3502 (Cbotu_EstA and Cbotu_EstB) were expressed in Escherichia coli BL21-Gold(DE3) and were found to hydrolyze the polyester poly(butylene adipate-co-butylene terephthalate) (PBAT). The active site residues (triad Ser, Asp, His) are present in both enzymes at the same location only with some amino acid variations near the active site at the surrounding of aspartate. Yet, Cbotu_EstA showed higher kcat values on para-nitrophenyl butyrate and para-nitrophenyl acetate and was considerably more active (sixfold) on PBAT. The entrance to the active site of the modeled Cbotu_EstB appears more narrowed compared to the crystal structure of Cbotu_EstA and the N-terminus is shorter which could explain its lower activity on PBAT. The Cbotu_EstA crystal structure consists of two regions that may act as movable cap domains and a zinc metal binding site.
从肉毒梭菌ATCC 3502中分离出的两种新型酯酶（Cbotu_EstA和Cbotu_EstB）在大肠杆菌BL21-Gold(DE3)中表达，并发现它们能够水解聚对苯二甲酸丁二醇酯-己二酸丁二醇酯共聚物（PBAT）。两种酶的活性位点残基（Ser、Asp、His三联体）位于相同位置，只是在天冬氨酸周围的活性位点附近存在一些氨基酸差异。然而，Cbotu_EstA对丁酸对硝基苯酯和乙酸对硝基苯酯表现出更高的kcat值，并且对PBAT的活性要高得多（六倍）。与Cbotu_EstA的晶体结构相比，模拟的Cbotu_EstB活性位点的入口似乎更窄，并且N端较短，这可以解释其对PBAT的活性较低。Cbotu_EstA的晶体结构由两个可能作为可移动帽结构域的区域和一个锌金属结合位点组成。
