Rotilio Laura, Swoboda Alexander, Ebner Katharina, Rinnofner Claudia, Glieder Anton, Kroutil Wolfgang, Mattevi Andrea
Department of Biology and Biotechnology, University of Pavia, via Ferrata 9, 27100 Pavia, Italy.
Austrian Centre of Industrial Biotechnology, c/o Institute of Chemistry, University of Graz, Heinrichstraße 28, 8010 Graz, Austria.
ACS Catal. 2021 Sep 17;11(18):11511-11525. doi: 10.1021/acscatal.1c03065. Epub 2021 Sep 2.
Unspecific peroxygenases (UPO) are glycosylated fungal enzymes that can selectively oxidize C-H bonds. UPOs employ hydrogen peroxide as oxygen donor and reductant. With such an easy-to-handle co-substrate and without the need of a reducing agent, UPOs are emerging as convenient oxidative biocatalysts. Here, an unspecific peroxygenase from (UPO) was identified in an activity-based screen of six putative peroxygenase enzymes that were heterologously expressed in . The enzyme was found to tolerate selected organic solvents such as acetonitrile and acetone. UPO is a versatile catalyst performing various reactions, such as the oxidation of - and -alcohols, epoxidations and hydroxylations. Semi-preparative biotransformations were demonstrated for the non-enantioselective oxidation of racemic 1-phenylethanol (TON = 13000), giving the product with 88% isolated yield, and the oxidation of indole to give indigo (TON = 2800) with 98% isolated yield. UPO features a compact and rigid three-dimensional conformation that wraps around the heme and defines a funnel-shaped tunnel that leads to the heme iron from the protein surface. The tunnel extends along a distance of about 12 Å with a fairly constant diameter in its innermost segment. Its surface comprises both hydrophobic and hydrophilic groups for dealing with small-to-medium size substrates of variable polarities. The structural investigation of several protein-ligand complexes revealed that the active site of UPO is accessible to molecules of varying bulkiness and polarity with minimal or no conformational changes, explaining the relatively broad substrate scope of the enzyme. With its convenient expression system, robust operational properties, relatively small size, well-defined structural features, and diverse reaction scope, UPO is an exploitable candidate for peroxygenase-based biocatalysis.
非特异性过氧酶（UPO）是一种糖基化真菌酶，能够选择性地氧化碳氢键。UPO利用过氧化氢作为氧供体和还原剂。由于这种共底物易于处理且无需还原剂，UPO正成为方便的氧化生物催化剂。在此，通过基于活性的筛选，在六种在[具体宿主]中异源表达的假定过氧酶中鉴定出一种来自[具体来源]的非特异性过氧酶（UPO）。发现该酶能耐受某些有机溶剂，如乙腈和丙酮。UPO是一种多功能催化剂，可进行各种反应，如α-和β-醇的氧化、环氧化和羟基化反应。已证明半制备生物转化可用于外消旋1-苯乙醇的非对映选择性氧化（TON = 13000），产物分离产率为88%，以及吲哚氧化生成靛蓝（TON = 2800），分离产率为98%。UPO具有紧凑且刚性的三维构象，环绕着血红素，并形成一个从蛋白质表面通向血红素铁的漏斗形通道。该通道沿约12埃的距离延伸，其最内段直径相当恒定。其表面包含疏水和亲水基团，可处理不同极性的中小尺寸底物。对几种蛋白质-配体复合物的结构研究表明，UPO的活性位点对不同体积和极性的分子都可及，且构象变化最小或没有变化，这解释了该酶相对较宽的底物范围。凭借其便捷的表达系统、稳健的操作特性、相对较小的尺寸、明确的结构特征以及多样的反应范围，UPO是基于过氧酶的生物催化中一个可开发的候选物。
