Department of Biochemical Engineering, University College London, Bernard Katz Building, Gower Street, London, WC1E 6BT, UK.
Chemical Metrology and Biometry Department, National Institute of Metrology, 3/4-5 Moo 3, Klong 5, Klong Luang, 12120, Pathumthani, Thailand.
Sci Rep. 2021 Dec 8;11(1):23584. doi: 10.1038/s41598-021-03001-8.
Transketolase (TK) has been previously engineered, using semi-rational directed evolution and substrate walking, to accept increasingly aliphatic, cyclic, and then aromatic substrates. This has ultimately led to the poor water solubility of new substrates, as a potential bottleneck to further exploitation of this enzyme in biocatalysis. Here we used a range of biophysical studies to characterise the response of both E. coli apo- and holo-TK activity and structure to a range of polar organic co-solvents: acetonitrile (AcCN), n-butanol (nBuOH), ethyl acetate (EtOAc), isopropanol (iPrOH), and tetrahydrofuran (THF). The mechanism of enzyme deactivation was found to be predominantly via solvent-induced local unfolding. Holo-TK is thermodynamically more stable than apo-TK and yet for four of the five co-solvents it retained less activity than apo-TK after exposure to organic solvents, indicating that solvent tolerance was not simply correlated to global conformational stability. The co-solvent concentrations required for complete enzyme inactivation was inversely proportional to co-solvent log(P), while the unfolding rate was directly proportional, indicating that the solvents interact with and partially unfold the enzyme through hydrophobic contacts. Small amounts of aggregate formed in some cases, but this was not sufficient to explain the enzyme inactivation. TK was found to be tolerant to 15% (v/v) iPrOH, 10% (v/v) AcCN, or 6% (v/v) nBuOH over 3 h. This work indicates that future attempts to engineer the enzyme to better tolerate co-solvents should focus on increasing the stability of the protein to local unfolding, particularly in and around the cofactor-binding loops.
转酮醇酶（TK）之前已经通过半理性定向进化和底物行走进行了工程改造，以接受越来越多的脂肪族、环状，然后是芳香族底物。这最终导致新底物的水溶性较差，成为进一步开发该酶在生物催化中应用的潜在瓶颈。在这里，我们使用一系列生物物理研究来表征大肠杆菌脱辅基和全酶 TK 活性和结构对一系列极性有机共溶剂的响应：乙腈（AcCN）、正丁醇（nBuOH）、乙酸乙酯（EtOAc）、异丙醇（iPrOH）和四氢呋喃（THF）。发现酶失活的机制主要是通过溶剂诱导的局部展开。全酶 TK 比脱辅基 TK 热力学上更稳定，但对于五种共溶剂中的四种，在暴露于有机溶剂后，其保留的活性低于脱辅基 TK，这表明溶剂耐受性与全局构象稳定性并不简单相关。完全失活所需的共溶剂浓度与共溶剂 log(P) 成反比，而展开速率成正比，这表明溶剂通过疏水接触与酶相互作用并部分展开酶。在某些情况下会形成少量聚集体，但这不足以解释酶失活。发现 TK 可耐受 15%（v/v）iPrOH、10%（v/v）AcCN 或 6%（v/v）nBuOH 超过 3 小时。这项工作表明，未来尝试工程改造酶以更好地耐受共溶剂应侧重于提高蛋白质对局部展开的稳定性，特别是在辅因子结合环内和周围。
