Abstract:
The enzymatic production of 2,5-furandicarboxylic acid (FDCA) from 5-hydroxymethylfurfural (HMF) has gained interest in recent years, as FDCA is a renewable precursor of poly(ethylene-2,5-furandicarboxylate) (PEF). 5-Hydroxymethylfurfural oxidases (HMFOs) form a flavoenzyme family with genes annotated in a dozen bacterial species but only one enzyme purified and characterized to date (after heterologous expression of a Methylovorus sp. HMFO gene). This oxidase acts on both furfuryl alcohols and aldehydes and, therefore, is able to catalyze the conversion of HMF into FDCA through 2,5-diformylfuran (DFF) and 2,5-formylfurancarboxylic acid (FFCA), with only the need of oxygen as a cosubstrate. To enlarge the repertoire of HMFO enzymes available, genetic databases were screened for putative HMFO genes, followed by heterologous expression in Escherichia coli After unsuccessful trials with other bacterial HMFO genes, HMFOs from two Pseudomonas species were produced as active soluble enzymes, purified, and characterized. The Methylovorus sp. enzyme was also produced and purified in parallel for comparison. Enzyme stability against temperature, pH, and hydrogen peroxide, three key aspects for application, were evaluated (together with optimal conditions for activity), revealing differences between the three HMFOs. Also, the kinetic parameters for HMF, DFF, and FFCA oxidation were determined, the new HMFOs having higher efficiencies for the oxidation of FFCA, which constitutes the bottleneck in the enzymatic route for FDCA production. These results were used to set up the best conditions for FDCA production by each enzyme, attaining a compromise between optimal activity and half-life under different conditions of operation.IMPORTANCE HMFO is the only enzyme described to date that can catalyze by itself the three consecutive oxidation steps to produce FDCA from HMF. Unfortunately, only one HMFO enzyme is currently available for biotechnological application. This availability is enlarged here by the identification, heterologous production, purification, and characterization of two new HMFOs, one from Pseudomonas nitroreducens and one from an unidentified Pseudomonas species. Compared to the previously known Methylovorus HMFO, the new enzyme from P. nitroreducens exhibits better performance for FDCA production in wider pH and temperature ranges, with higher tolerance for the hydrogen peroxide formed, longer half-life during oxidation, and higher yield and total turnover numbers in long-term conversions under optimized conditions. All these features are relevant properties for the industrial production of FDCA. In summary, gene screening and heterologous expression can facilitate the selection and improvement of HMFO enzymes as biocatalysts for the enzymatic synthesis of renewable building blocks in the production of bioplastics.
摘要:
近年来,由5-羟甲基糠醛(HMF)酶法生产2,5-呋喃二甲酸(FDCA)已引起人们的兴趣,FDCA是聚(2,5-呋喃二甲酸乙二醇酯)(PEF)的可再生前体。5-羟甲基糠醛氧化酶(HMFOs)形成了一个黄素酶家族,其基因在十几种细菌中得到了注释,但迄今为止只有一种酶得到了纯化和表征(在异源表达了Methypalvorussp。HMFO基因)。这种氧化酶作用于糠醇和醛,因此,能够通过2,5-二甲酰呋喃(DFF)和2,5-甲酰呋喃甲酸(FFCA)催化HMF转化为FDCA,只需要氧作为共底物。为了扩大可用的HMFO酶库,遗传数据库筛选推定的HMFO基因,在其他细菌HMFO基因试验不成功后,在大肠杆菌中进行异源表达,来自两种假单胞菌的HMFOs作为活性可溶性酶产生,纯化,和特点。甲基vorussp.酶也平行产生和纯化用于比较。酶对温度的稳定性,pH值,和过氧化氢,应用的三个关键方面,进行了评估(以及最佳活动条件),揭示了三种HMFO之间的差异。此外,HMF的动力学参数,DFF,并测定了FFCA氧化,新的HMFO对FFCA的氧化具有更高的效率,这构成了FDCA生产的酶促途径的瓶颈。这些结果用于建立每种酶生产FDCA的最佳条件,在不同的操作条件下实现最佳活性和半衰期之间的折衷。重要性HMFO是迄今为止描述的唯一酶,其自身可以催化三个连续的氧化步骤以从HMF产生FDCA。不幸的是,目前只有一种HMFO酶可用于生物技术应用。这种可用性在这里通过识别得到了扩大,异源生产,净化,以及两个新的HMFOs的表征,一种来自硝基还原假单胞菌,一种来自未鉴定的假单胞菌。与以前已知的甲基HMFO相比,新的酶从P.硝基还原表现出更好的性能FDCA生产在更宽的pH和温度范围,对形成的过氧化氢有更高的耐受性,氧化过程中的半衰期更长,以及在优化条件下的长期转换中更高的收益率和总周转数。所有这些特征都是FDCA工业生产的相关特性。总之,基因筛选和异源表达可以促进HMFO酶作为生物催化剂的选择和改进,用于生物塑料生产中可再生结构单元的酶合成。