Review of research on unspecific peroxygenases (UPOs)

doi:10.12211/2096-8280.2022-028

Abstract

Abstract:

The selective insertion of oxygen species through unactivated C—H bonds is one of the most challenging tasks in organic synthesis. Fungal unspecific peroxygenases (UPOs) are a class of highly glycosylated thioheme enzymes that catalyze reactions including hydroxylation of unactivated C—H bonds in n-alkanes, epoxidation of alkenes and aromatics, oxidation of heteroatom (N, S) compounds, ether cleavage, N-dealkylation, deacylation and one-electron oxidation of phenols. As one of the most promising oxidases in synthetic chemistry,UPOs use H₂O₂ as the oxygen donor and the electron acceptor, and do not require cofactors other than heme. This paper reviews the classification and development process of UPOs, and focuses on the heterologous expression, selectivity engineering and H₂O₂in situ regeneration of UPOs. Since the first discovery of UPOs from Agrocybe aegerita in 2004, UPOs have attracted much attention due to the advantages described above. However, the difficulties of heterologous expression and poor selectivity of UPOs still limit their development. The difficulty of heterologous expression makes it hard to mine new variants of UPOs, and native UPOs are difficult to be characterized and applied in biocatalysis due to the slow growth rate of their hosts. In the past two years, important breakthroughs have been made in the heterologous expression of UPOs through the modification or replacement of signal peptides, revealing the important role of signal peptides in this process. However, the specific role of signal peptides in the secretory expression and three-dimensional structure formation of UPOs remain elusive. With the in-depth research on the mechanism of signal peptides affecting the heterologous expression of UPOs and the development of artificial intelligence (AI) algorithms, the combination of genome mining and signal peptide prediction will be the key for discovering new UPOs. The poor selectivity of UPOs also hinders the development and application of UPOs. This paper reviews different types of reactions that UPOs catalyze, and reveals the problem that UPOs have broad substrate range but poor selectivity. In-depth research on the structure-function relationship of UPOs and the development of protein structure prediction algorithms will help the engineering of UPOs and lay a foundation for solving the problem of poor substrate selectivity. This paper also compares several methods for the in situ regeneration of H₂O₂, and concluded that the multi-enzyme cascade method is the most economical and practical method for the in situ regeneration of H₂O₂.

Key words: unspecific peroxygenase(UPO), unactivated C—H bond, oxidation reaction, heterologous expression, selectivity

摘要：

未活化的C—H键选择性插入活性氧是目前有机合成面临的最具挑战性之一。真菌非特异性过加氧酶（UPO）是一类高度糖基化的硫代血红素酶，催化的反应包括正构烷烃中未活化的C—H键的羟基化、烯烃和芳烃的环氧化、含杂原子（N、S）化合物的氧化、乙醚裂解、N-脱烷基化、脱酰化和酚类的单电子氧化。UPO以H₂O₂为氧供体与电子受体，不需要任何辅因子，是目前最具发展潜力的氧化酶之一。然而，UPO的异源表达困难与选择性差的问题仍限制着UPO的发展。近两年，通过信号肽改造或更换的方法在UPO的异源表达方面取得了重要突破，对UPO结构功能关系的深入研究以及蛋白结构预测算法的发展也将助力UPO的分子改造，为解决UPO选择性差的问题奠定基础。本文聚焦于UPO的异源表达、选择性问题与H₂O₂原位再生，综述了UPO的最新发展以及存在的技术瓶颈，并对解决这些瓶颈问题的方案做出展望。

关键词: 非特异性过加氧酶, 未活化C—H键, 氧化反应, 异源表达, 选择性

CLC Number:

Q814

Mingyuan LAI, Jian WEI, Jianhe XU, Huilei YU. Review of research on unspecific peroxygenases (UPOs)[J]. Synthetic Biology Journal, 2022, 3(6): 1235-1249.

赖铭元, 韦健, 许建和, 郁惠蕾. 非特异性过加氧酶（UPO）的研究综述[J]. 合成生物学, 2022, 3(6): 1235-1249.

Figures/Tables 16

Tab. 1 Classification of oxidoreductases (according to the webpage Enzyme Nomenclature of the NC-IUBMB https://iubmb.qmul.ac.uk/enzyme/EC1/)

名称	编号	辅因子	电子受体	催化作用
氧化酶	EC 1.x.3.x	金属离子(Cu等)	O₂	将电子从底物或辅因子转移到分子氧上
氧化酶	EC 1.x.3.x	黄素	O₂、Fe³⁺、醌^-等	将电子从底物或辅因子转移到分子氧上
过氧化物酶	EC 1.11.1.x	血红素	H₂O₂	催化两个电子从底物转移到H₂O₂
过加氧酶	EC 1.11.2.1	血红素	H₂O₂	催化氧原子从H₂O₂转移到底物上
单加氧酶	EC 1.13.12.x	血红素、黄素等	O₂	催化1个氧原子从O₂转移到底物中
双加氧酶	EC 1.13.11.x EC 1.14.11.x EC 1.14.12.x	黄素、金属离子等	O₂	催化2个氧原子从O₂转移到底物中

Tab. 2 UPO that have been successfully heterologously expressed

UPO	来源	异源表达宿主	方法
MroUPO	M. rotula	E. coli	原序列，自诱导培养基^[26]
DcaUPO	D. caldariorum	E. coli	原序列^[27]
CviUPO	C. virescens	E. coli	原序列^[27]
HinUPO	H. insolens	Aspergillus oryzae	原序列^[34]
CciUPO	C. cinerea	Aspergillus oryzae	原序列^[32]
PviUPO	P.virgatula	Aspergillus oryzae	原序列^[34]
ThyUPO	T.hyrcaniae	Aspergillus oryzae	原序列^[34]
LfuUPO	C. fumago	Aspergillus niger	原序列^[39]
AaeUPO (wild-type)	A. aegerita	无细胞	无细胞蛋白表达系统^[38]
AaeUPO (PADAⅠ)	A. aegerita	S. cerevisiae & P. pastoris	信号肽改造^[30-31]
MroUPO	M. rotula	S. cerevisiae & P. pastoris	更换信号肽^[29]
CglUPO	C. globosum	S. cerevisiae & P. pastoris	更换信号肽^[29]
MthUPO	M. thermophila	S. cerevisiae & P. pastoris	更换信号肽^{[29, 36]}
TteUPO	T. terrestris	S. cerevisiae & P. pastoris	更换信号肽^{[29, 36]}
PabUPO	P. aberdarensis	S. Cerevisiae & P. pastoris	更换信号肽^[37]
MfeUPO	M. fergusii	P. pastoris	更换信号肽^[36]
MhiUPO	M. hinnulea	P. pastoris	更换信号肽^[36]
DcaUPO	D. caldariorum	P. pastoris	更换信号肽^[36]
HspUPO	Hypoxylon sp	P. pastoris	原序列^[40]
AniUPO	Aspergillus niger	P. pastoris	信号肽预测^[41]
CabUPO 1	C. aberdarensis	P. pastoris	信号肽预测^[41]
CabUPO 2	C. aberdarensis	P. pastoris	信号肽预测^[41]

Fig. 1 Mechanism of thioheme-catalyzed oxidation[The process of cytochrome P450 monooxygenase catalyzation consists of six steps and requires the participation of NAD(P)H and O2(Steps 1-6). The catalytic oxidation of UPO is indicated by red lines(Steps a-d). UPO do not catalyze the reduction and activation of molecular oxygen, but directly use hydrogen peroxide to form catalytically active ferric oxide groups, so it does not rely on the expensive NAD(P)H and has a more concise electron transport chain.]

Fig. 2 The general formula for the oxidation of alkanes by UPO

Fig. 3 Oxidation of alkanes and fatty acids by UPO(a)～(d) AaeUPO oxidizes linear (branched) alkanes and cycloalkanes; (e) AaeUPO hydroxylates fatty acids; (f) MroUPO oxidizes fatty acids for decarboxylation; (g) AaeUPO/CciUPO catalyze fatty acid carbon chain shortening (removal of two carbon atom)

Fig. 4 The general formula for the oxidation of olefin by UPO

Fig. 5 Oxidation of Olefins by UPO(a)～(d) AaeUPO oxidizes different olefins, and the difference in olefin structure will affect the ratio of hydroxylation products to epoxidation products

Fig. 6 General formula for the oxidation of aromatic hydrocarbons by UPO

Fig. 7 Oxidation of aromatic hydrocarbons by UPO(a) AaeUPO oxidized benzene[56]; (b) AaeUPO oxidized naphthalene[55]; (c) AaeUPO oxidized flavonoids[57]

Fig. 8 Oxidation of compounds containing S, N or Br by UPO

Fig. 9 Oxidation of aryl alkyl sulfides by AaeUPO[58]

Fig. 10 UPO catalyzes the cleavage reaction(a) AaeUPO catalyzed the ring-opening reaction of tetrahydrofuran; (b) 5-nitro-1,3-benzodioxole (NBD) was dealkylated under the action of UPO to form formic acid and 4-nitrocatechol; (c) AaeUPO selectively catalyzes the N-dealkylation of the N-methylpiperazine ring of the drug sildenafil

Fig. 11 Oxidation of 5-hydroxymethylfurfural (HMF) by AaeUPO to 2,5-furandicarboxylic acid (FDCA)

Fig. 12 Au-TiO2-mediated photocatalytic water oxidation to generate H2O2in situ for driving AaeUPO-catalyzed oxidation reaction

Fig. 13 In situ generation of H2O2 by electrochemical method to drive peroxygenase-catalyzed oxidation

Fig. 14 In situ generation of H2O2 by a formate oxidase (AoFOx)-peroxygenase coupled system

References 84

1	GYGLI G, VAN BERKEL W J H. Oxizymes for biotechnology[J]. Current Biotechnology, 2015, 4(2): 100-110.
2	MARTÍNEZ A T, RUIZ-DUEÑAS F J, CAMARERO S, et al. Oxidoreductases on their way to industrial biotransformations[J]. Biotechnology Advances, 2017, 35(6): 815-831.
3	YAMAGUCHI J, YAMAGUCHI A D, ITAMI K. C—H bond functionalization: emerging synthetic tools for natural products and pharmaceuticals[J]. Angewandte Chemie (International Ed in English), 2012, 51(36): 8960-9009.
4	ZHANG R K, HUANG X Y, ARNOLD F H. Selective C-H bond functionalization with engineered heme proteins: new tools to generate complexity [J]. Current Opinion in Chemical Biology, 2019, 49: 67-75.
5	BATHE U, TISSIER A. Cytochrome P450 enzymes: a driving force of plant diterpene diversity[J]. Phytochemistry, 2019, 161: 149-162.
6	HOLTMANN D, HOLLMANN F. The oxygen dilemma: a severe challenge for the application of monooxygenases? [J]. ChemBioChem, 2016, 17(15): 1391-1398.
7	WANG Y H, LAN D M, DURRANI R, et al. Peroxygenases en route to becoming dream catalysts. What are the opportunities and challenges? [J]. Current Opinion in Chemical Biology, 2017, 37: 1-9.
8	HOFRICHTER M, ULLRICH R. Heme-thiolate haloperoxidases: versatile biocatalysts with biotechnological and environmental significance[J]. Applied Microbiology and Biotechnology, 2006, 71(3): 276-288.
9	KLEBANOFF S J. Myeloperoxidase: friend and foe[J]. Journal of Leukocyte Biology, 2005, 77(5): 598-625.
10	LEE D S, YAMADA A, SUGIMOTO H, et al. Substrate recognition and molecular mechanism of fatty acid hydroxylation by cytochrome P450 from Bacillus subtilis: crystallographic, spectroscopic, and mutational studies[J]. The Journal of Biological Chemistry, 2003, 278(11): 9761-9767.
11	HANANO A, BURCKLEN M, FLENET M, et al. Plant seed peroxygenase is an original heme-oxygenase with an EF-hand calcium binding motif[J]. The Journal of Biological Chemistry, 2006, 281(44): 33140-33151.
12	TANG M C, FU C Y, TANG G L. Characterization of SfmD as a heme peroxidase that catalyzes the regioselective hydroxylation of 3-methyltyrosine to 3-hydroxy-5-methyltyrosine in saframycin A biosynthesis[J]. The Journal of Biological Chemistry, 2012, 287(7): 5112-5121.
13	CONNOR K L, COLABROY K L, GERRATANA B. A heme peroxidase with a functional role as an L-tyrosine hydroxylase in the biosynthesis of anthramycin[J]. Biochemistry, 2011, 50(41): 8926-8936.
14	ULLRICH R, NÜSKE J, SCHEIBNER K, et al. Novel haloperoxidase from the Agrocybe aegerita oxidizes aryl alcohols and aldehydes[J]. Applied & Environmental Microbiology, 2004, 70(8): 4575-4581.
15	MORRIS D R, HAGER L P. Chloroperoxidase. I. Isolation and properties of the crystalline glycoprotein[J]. The Journal of Biological Chemistry, 1966, 241(8): 1763-1768.
16	MANOJ K M, HAGER L P. Chloroperoxidase, a janus enzyme[J]. Biochemistry, 2008, 47(9): 2997-3003.
17	SANFILIPPO C, NICOLOSI G. Catalytic behaviour of chloroperoxidase from Caldariomyces fumago in the oxidation of cyclic conjugated dienes[J]. Cheminform, 2003, 13(17): 1889-1892.
18	KLUGE M G, ULLRICH R, SCHEIBNER K, et al. Spectrophotometric assay for detection of aromatic hydroxylation catalyzed by fungal haloperoxidase-peroxygenase[J]. Applied Microbiology and Biotechnology, 2007, 75(6): 1473-1478.
19	ULLRICH R, HOFRICHTER M. The haloperoxidase of the agaric fungus Agrocybe aegerita hydroxylates toluene and naphthalene[J]. FEBS Letters, 2005, 579(27): 6247-6250.
20	HOFRICHTER M, ULLRICH R. Oxidations catalyzed by fungal peroxygenases[J]. Current Opinion in Chemical Biology, 2014, 19: 116-125.
21	FAIZA M, LAN D M, HUANG S F, et al. UPObase: an online database of unspecific peroxygenases[J]. Database-the Journal of Biological Databases and Curation, 2019, 2019(8): baz122.
22	KELLNER H, LUIS P, PECYNA M J, et al. Widespread occurrence of expressed fungal secretory peroxidases in forest soils[J]. PLoS One, 2014, 9(4): e95557.
23	HOFRICHTER M, KELLNER H, PECYNA M J, et al. Fungal unspecific peroxygenases: heme-thiolate proteins that combine peroxidase and cytochrome p450 properties[J]. Advances in Experimental Medicine and Biology, 2015, 851: 341-368.
24	KENIGSBERG P, FANG G H, HAGER L P. Post-translational modifications of chloroperoxidase from Caldariomyces fumago [J]. Archives of Biochemistry and Biophysics, 1987, 254(2): 409-415.
25	ZONG Q, OSMULSKI P A, HAGER L P. High-pressure-assisted reconstitution of recombinant chloroperoxidase[J]. Biochemistry, 1995, 34(38): 12420-12425.
26	CARRO J, GONZÁLEZ-BENJUMEA A, FERNÁNDEZ-FUEYO E, et al. Modulating fatty acid epoxidation vs hydroxylation in a fungal peroxygenase[J]. ACS Catalysis, 2019, 9(7): 6234-6242.
27	LINDE D, OLMEDO A, GONZÁLEZ-BENJUMEA A, et al. Two new unspecific peroxygenases from heterologous expression of fungal genes in Escherichia coli [J]. Applied and Environmental Microbiology, 2020, 86(7): e02899-e02819.
28	GONZÁLEZ-BENJUMEA A, CARRO J, RENAU-MÍNGUEZ C, et al. Fatty acid epoxidation by Collariella virescensperoxygenase and heme-channel variants[J]. Catalysis Science & Technology, 2020, 10(3): 717-725.
29	PÜLLMANN P, KNORRSCHEIDT A, MÜNCH J, et al. A modular two yeast species secretion system for the production and preparative application of unspecific peroxygenases[J]. Communications Biology, 2021, 4: 562.
30	MOLINA-ESPEJA P, GARCIA-RUIZ E, GONZALEZ-PEREZ D, et al. Directed evolution of unspecific peroxygenase from Agrocybe aegerita [J]. Applied & Environmental Microbiology, 2014, 80(11): 3496-3507.
31	MOLINA-ESPEJA P, MA S, MATE D M, et al. Tandem-yeast expression system for engineering and producing unspecific peroxygenase[J]. Enzyme and Microbial Technology, 2015, 73/74: 29-33.
32	PECYNA M, SCHNORR K M, ULLRICH R, et al. Fungal peroxygenases and methords of application. WO/2008/119780[P]. 2008-09-10.
33	BABOT E D, DEL RÍO J C, KALUM L, et al. Oxyfunctionalization of aliphatic compounds by a recombinant peroxygenase from Coprinopsis cinerea [J]. Biotechnology & Bioengineering, 2013, 110(9): 2323-2332.
34	LUND H, KALUM L, HOFRICHTER M. US Pat.,9908860B2 [P], 2018.
35	ARANDA C, MUNICOY M, GUALLAR V, et al. Selective synthesis of 4-hydroxyisophorone and 4-ketoisophorone by fungal peroxygenases[J]. Catalysis Science & Technology, 2019, 9(6): 1398-1405.
36	PÜLLMANN P, WEISSENBORN M J. Improving the heterologous production of fungal peroxygenases through an episomal pichia pastoris promoter and signal peptide shuffling system[J]. ACS Synthetic Biology, 2021, 10(6): 1360-1372.
37	GOMEZ DE SANTOS P, HOANG M D, KIEBIST J, et al. Functional expression of two unusual acidic peroxygenases from Candolleomyces aberdarensis in yeasts by adopting evolved secretion mutations[J]. Applied and Environmental Microbiology, 2021, 87(19): e0087821.
38	SCHRAMM M, FRIEDRICH S, SCHMIDTKE K U, et al. Cell-free protein synthesis with fungal lysates for the rapid production of unspecific peroxygenases[J]. Antioxidants, 2022, 11(2): 284.
39	CONESA A, VAN DE VELDE F, VAN RANTWIJK F, et al. Expression of the Caldariomyces fumagochloroperoxidase in Aspergillus niger and characterization of the recombinant enzyme[J]. Journal of Biological Chemistry, 2001, 276(21): 17635-17640.
40	ROTILIO L, SWOBODA A, EBNER K, et al. Structural and biochemical studies enlighten the unspecific peroxygenase from Hypoxylon sp. EC38 as an efficient oxidative biocatalyst[J]. ACS Catalysis, 2021, 11(18): 11511-11525.
41	BORMANN S, KELLNER H, HERMES J, et al. Broadening the biocatalytic toolbox-screening and expression of new unspecific peroxygenases[J]. Antioxidants, 2022, 11(2): 223.
42	PIONTEK K, STRITTMATTER E, ULLRICH R, et al. Structural basis of substrate conversion in a new aromatic peroxygenase: Cytochrome P450 functionality with benefits[J]. The Journal of Biological Chemistry, 2013, 288(48): 34767-34776.
43	PECYNA M J, ULLRICH R, BITTNER B, et al. Molecular characterization of aromatic peroxygenase from Agrocybe aegerita [J]. Applied Microbiology and Biotechnology, 2009, 84(5): 885-897.
44	LEWIS D F V, ITO Y. Cytochrome P450 structure and function: an evolutionary perspective[M]//Issues in Toxicology. Cambridge: Royal Society of Chemistry, 2008: 3-45.
45	PIONTEK K, ULLRICH R, LIERS C, et al. Crystallization of a 45 kDa peroxygenase/peroxidase from the mushroom Agrocybe aegerita and structure determination by SAD utilizing only the haem iron[J]. Acta Crystallographica Section F, 2010, 66(6): 693-698.
46	HOFRICHTER M, KELLNER H, HERZOG R, et al. Fungal peroxygenases: a phylogenetically old superfamily of heme enzymes with promiscuity for oxygen transfer reactions[M]. Grand Challenges in Fungal Biotechnology, 2020, 369-403.
47	RAMIREZ-ESCUDERO M, MOLINA-ESPEJA P, GOMEZ DE SANTOS P, et al. Structural insights into the substrate promiscuity of a laboratory-evolved peroxygenase[J]. ACS Chemical Biology, 2018, 13(12): 3259-3268.
48	LECINA D, GILABERT J F, GUALLAR V. Adaptive simulations, towards interactive protein-ligand modeling[J]. Scientific Reports, 2017, 7: 8466.
49	PETER S, KINNE M, WANG X S, et al. Selective hydroxylation of alkanes by an extracellular fungal peroxygenase[J]. The FEBS Journal, 2011, 278(19): 3667-3675.
50	PETER S, KARICH A, ULLRICH R, et al. Enzymatic one-pot conversion of cyclohexane into cyclohexanone: comparison of four fungal peroxygenases[J]. Journal of Molecular Catalysis B: Enzymatic, 2014, 103: 47-51.
51	GUTIÉRREZ A, BABOT E D, ULLRICH R, et al. Regioselective oxygenation of fatty acids, fatty alcohols and other aliphatic compounds by a basidiomycete heme-thiolate peroxidase[J]. Archives of Biochemistry and Biophysics, 2011, 514(1/2): 33-43.
52	OLMEDO A, RÍO J C D, KIEBIST J, et al. Fatty acid chain shortening by a fungal peroxygenase[J]. Chemistry, 2017, 23(67): 16985-16989.
53	OLMEDO A, ULLRICH R, HOFRICHTER M, et al. Novel fatty acid chain-shortening by fungal peroxygenases yielding 2C-shorter dicarboxylic acids[J]. Antioxidants, 2022, 11(4): 744.
54	PETER S, KINNE M, ULLRICH R, et al. Epoxidation of linear, branched and cyclic alkenes catalyzed by unspecific peroxygenase[J]. Enzyme & Microbial Technology, 2013, 52(6/7): 370-376.
55	KLUGE M, ULLRICH R, DOLGE C, et al. Hydroxylation of naphthalene by aromatic peroxygenase from Agrocybe aegerita proceeds via oxygen transfer from H₂O₂ and intermediary epoxidation[J]. Applied Microbiology and Biotechnology, 2009, 81(6): 1071-1076.
56	KARICH A, KLUGE M, ULLRICH R, et al. Benzene oxygenation and oxidation by the peroxygenase of Agrocybe aegerita [J]. AMB Express, 2013, 3(1): 5.
57	BARKOVÁ K, KINNE M, ULLRICH R, et al. Regioselective hydroxylation of diverse flavonoids by an aromatic peroxygenase[J]. Tetrahedron, 2011, 67(26): 4874-4878.
58	BASSANINI I, FERRANDI E E, VANONI M, et al. Peroxygenase-catalyzed enantioselective sulfoxidations[J]. European Journal of Organic Chemistry, 2017, 2017(47): 7186-7189.
59	KINNE M, PORAJ-KOBIELSKA M, RALPH S A, et al. Oxidative cleavage of diverse ethers by an extracellular fungal peroxygenase[J]. Journal of Biological Chemistry, 2009, 284(43): 29343-29349.
60	PORAJ-KOBIELSKA M, KINNE M, ULLRICH R, et al. A spectrophotometric assay for the detection of fungal peroxygenases[J]. Analytical Biochemistry, 2012, 421(1): 327-329.
61	PORAJ-KOBIELSKA M, KINNE M, ULLRICH R, et al. Preparation of human drug metabolites using fungal peroxygenases[J]. Biochemical Pharmacology, 2011, 82(7): 789-796.
62	HYLAND R, ROE E G H, JONES B C, et al. Identification of the cytochrome P450 enzymes involved in the N-demethylation of sildenafil[J]. British Journal of Clinical Pharmacology, 2001, 51(3): 239-248.
63	ARANDA E, KINNE M, KLUGE M, et al. Conversion of dibenzothiophene by the mushrooms Agrocybe aegerita and Coprinellus radians and their extracellular peroxygenases[J]. Applied Microbiology and Biotechnology, 2009, 82(6): 1057-1066.
64	CARRO J, FERREIRA P, RODRÍGUEZ L, et al. 5-hydroxymethylfurfural conversion by fungal aryl-alcohol oxidase and unspecific peroxygenase[J]. The FEBS Journal, 2015, 282(16): 3218-3229.
65	KLUGE M, ULLRICH R, SCHEIBNER K, et al. Stereoselective benzylic hydroxylation of alkylbenzenes and epoxidation of styrene derivatives catalyzed by the peroxygenase of Agrocybe aegerita [J]. Green Chemistry, 2012, 14(2): 440-446.
66	ULLRICH R, HOFRICHTER M. Enzymatic hydroxylation of aromatic compounds[J]. Cellular and Molecular Life Sciences, 2007, 64(3): 271-293.
67	LUCAS F, BABOT E D, CAÑELLAS M, et al. Molecular determinants for selective C25-hydroxylation of vitamins D₂ and D₃ by fungal peroxygenases[J]. Catalysis Science & Technology, 2016, 6(1): 288-295.
68	BORMANN S, GOMEZ BARAIBAR A, NI Y, et al. Specific oxyfunctionalisations catalysed by peroxygenases: Opportunities, challenges and solutions[J]. Catalysis Science & Technology, 2015, 5(4): 2038-2052.
69	ZHANG W Y, FERNÁNDEZ-FUEYO E, NI Y, et al. Selective aerobic oxidation reactions using a combination of photocatalytic water oxidation and enzymatic oxyfunctionalizations[J]. Nature Catalysis, 2018, 1(1): 55-62.
70	WILLOT S J P, FERNÁNDEZ-FUEYO E, TIEVES F, et al. Expanding the spectrum of light-driven peroxygenase reactions[J]. ACS Catalysis, 2019, 9(2): 890-894.
71	YUAN B, MAHOR D, FEI Q, et al. Water-soluble anthraquinone photocatalysts enable methanol-driven enzymatic halogenation and hydroxylation reactions[J]. ACS Catalysis, 2020, 10(15): 8277-8284.
72	WAPSHOTT-STEHLI H L, GRUNDEN A M. In situ H₂O₂ generation methods in the context of enzyme biocatalysis[J]. Enzyme and Microbial Technology, 2021, 145: 109744.
73	HOLTMANN D, KRIEG T, GETREY L, et al. Electroenzymatic process to overcome enzyme instabilities[J]. Catalysis Communications, 2014, 51: 82-85.
74	GUILLET N, ROUÉ L, MARCOTTE S, et al. Electrogeneration of hydrogen peroxide in acid medium using pyrolyzed cobalt-based catalysts: Influence of the cobalt content on the electrode performance[J]. Journal of Applied Electrochemistry, 2006, 36(8): 863-870.
75	SAHA M S, NISHIKI Y, FURUTA T, et al. Electrolytic synthesis of peroxyacetic acid using in situ generated hydrogen peroxide on gas diffusion electrodes[J]. Journal of the Electrochemical Society, 2004, 151(9): D93.
76	CHOI D S, NI Y, FERNÁNDEZ-FUEYO E, et al. Photoelectroenzymatic oxyfunctionalization on flavin-hybridized carbon nanotube electrode platform[J]. ACS Catalysis, 2017, 7(3): 1563-1567.
77	BORMANN S, VAN SCHIE M M C H, DE ALMEIDA T P, et al. H₂O₂ production at low overpotentials for electroenzymatic halogenation reactions[J]. ChemSusChem, 2019, 12(21): 4759-4763.
78	NGUYEN L T, YANG K L. Combined cross-linked enzyme aggregates of horseradish peroxidase and glucose oxidase for catalyzing cascade chemical reactions[J]. Enzyme & Microbial Technology, 2017, 100: 52-59.
79	NI Y, FERNÁNDEZ-FUEYO E, BARAIBAR A G, et al. Peroxygenase-catalyzed oxyfunctionalization reactions promoted by the complete oxidation of methanol[J]. Angewandte Chemie International Edition, 2016, 55(2): 798-801.
80	TIEVES F, WILLOT S J P, VAN SCHIE M M C H, et al. Formate oxidase (FO_x) from Aspergillus oryzae: one catalyst enables diverse H₂O₂-dependent biocatalytic oxidation reactions[J]. Angewandte Chemie International Edition, 2019, 58(23): 7873-7877.
81	VAITHYANATHAN V K, RAVI S, LEDUC R, et al. Utilization of biosolids for glucose oxidase production: a potential bio-fenton reagent for advanced oxidation process for removal of pharmaceutically active compounds[J]. Journal of Environmental Management, 2020, 271: 110995.
82	KARICH A, ULLRICH R, SCHEIBNER K, et al. Fungal unspecific peroxygenases oxidize the majority of organic EPA priority pollutants[J]. Frontiers in Microbiology, 2017, 8: 1463.
83	JUMPER J, EVANS R, PRITZEL A, et al. Highly accurate protein structure prediction with AlphaFold[J]. Nature, 2021, 596(7873): 583-589.
84	卞佳豪, 杨广宇. 人工智能辅助的蛋白质工程[J]. 合成生物学, 2022, 3(3): 429-444.
	BIAN J H, YANG G Y. Artificial intelligence-assisted protein engineering[J]. Synthetic Biology Journal, 2022, 3(3): 429-444.

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