IFP, Institut Français du Pétrole, Rueil-Malmaison, France.
Lett Appl Microbiol. 2009 Dec;49(6):673-8. doi: 10.1111/j.1472-765X.2009.02734.x. Epub 2009 Aug 29.
The objective of the study was to produce and characterize the cinnamoyl esterase EstA from the anaerobic fungus Piromyces equi for potential industrial applications.
The catalytic domain EstA was produced in Trichoderma reesei. Because the two fungi displayed different genome features, including different codon usage and GC content, a synthetic gene was designed and expressed, leading to the production of the corresponding protein at around 33 mg per litre in the T. reesei culture medium. After the recombinant protein was purified, biochemical characterization showed that EstA presents peak activity at pH 6.5 and at 50-60 degrees C. Furthermore, EstA remained stable at pH 6-8 and below 50 degrees C. EstA was compared to cinnamoyl esterases FaeA and FaeB from Aspergillus niger in terms of ferulic acid (FA) release from wheat bran (WB), maize bran (MB) and sugar beet pulp (SBP).
The synthetic gene was successfully cloned and overexpressed in T. reesei. EstA from P. equi was demonstrated to efficiently release FA from various natural substrates.
Recombinant EstA produced in an industrial enzyme producer, T. reesei, was biochemically characterized, and its capacity to release an aromatic compound (FA) for biotechnological applications was demonstrated.
本研究的目的是从厌氧真菌 Piromyces equi 中产生并表征肉桂酰酯酶 EstA，以用于潜在的工业应用。
催化结构域 EstA 在里氏木霉中产生。由于两种真菌显示出不同的基因组特征，包括不同的密码子使用和 GC 含量，因此设计并表达了一个合成基因，导致在里氏木霉培养基中每升约产生 33 毫克的相应蛋白质。在纯化重组蛋白后，生化特性表明 EstA 在 pH6.5 和 50-60°C 时呈现出最高活性。此外，EstA 在 pH6-8 和 50°C 以下保持稳定。EstA 与黑曲霉中的肉桂酰酯酶 FaeA 和 FaeB 进行了比较，比较了它们从麦麸（WB）、玉米麸（MB）和糖甜菜浆（SBP）中释放阿魏酸（FA）的能力。
成功地在里氏木霉中克隆和过表达了合成基因。来自 P. equi 的 EstA 被证明能够有效地从各种天然基质中释放 FA。
在工业酶生产商里氏木霉中产生的重组 EstA 进行了生化特性分析，并证明了其释放芳香族化合物（FA）的能力可用于生物技术应用。
