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Nano-Evolution and Protein-Based Enzymatic Evolution Predicts the Novel View Article Online View Journal Nanoscale Advances Accepted Manuscript This article can be cited before page numbers have been issued, to do this please use: G. Nazarbek, A. Kutzhanova, L. Nurtay , C. Mu, B. Kazybay, X. Li, C. Ma, A. Amin and Y. Xie, Nanoscale Adv., 2021, DOI: 10.1039/D1NA00475A. Volume 1 Number 1 This is an Accepted Manuscript, which has been through the January 2018 Pages 1-200 Royal Society of Chemistry peer review process and has been accepted Nanoscale for publication. Advances Accepted Manuscripts are published online shortly after acceptance, rsc.li/nanoscale-advances before technical editing, formatting and proof reading. Using this free service, authors can make their results available to the community, in citable form, before we publish the edited article. We will replace this Accepted Manuscript with the edited and formatted Advance Article as soon as it is available. You can find more information about Accepted Manuscripts in the Information for Authors. Please note that technical editing may introduce minor changes to the text and/or graphics, which may alter content. The journal’s standard ISSN 2516-0230 Terms & Conditions and the Ethical guidelines still apply. In no event shall the Royal Society of Chemistry be held responsible for any errors or omissions in this Accepted Manuscript or any consequences arising from the use of any information it contains. rsc.li/nanoscale-advances Page 1 of 25 Nanoscale Advances 1 Nano-evolution and protein-based enzymatic evolution predicts the novel View Article Online 2 types of natural product nanozyme of traditional Chinese medicine:DOI: 10.1039/D1NA00475A cases of 3 herbzymes of Taishan-Huangjing (Rhizoma polygonati) and Goji (Lycium 4 chinense) 5 Guldan Nazarbek1, Aidana Kutzhanova1 ,Lazzat Nurtay1, Chenglin Mu2, Bexultan 6 Kazybay1, Xugang Li2, Cuiping Ma3, Amr Amin4,5 , Yingqiu Xie1* 7 8 1Biology Department, Nazarbayev University, 010000, Nur-Sultan, Kazakhstan; 9 2 Sino-German Joint Research Center on Agricultural Biology, State Key Laboratory of Crop Biology, 10 College of Life Sciences, Shandong Agricultural University, Tai'an, 271018, China Manuscript 11 3 Shandong Provincial Key Laboratory of Biochemical Engineering, Qingdao Nucleic Acid Rapid 12 Detection Engineering Research Center, College of Marine Science and Biological Engineering, 13 Qingdao University of Science and Technology, Qingdao, 266042, China. 4 14 Creative Commons Attribution-NonCommercial 3.0 Unported Licence. Biology Department, UAE University, Al Ain 15551, UAE 15 5 The College, The University of Chicago, Chicago, IL 60637, USA Accepted 16 17 18 This article is licensed under a * Correspondence. Yingqiu Xie 53 Kabanbay Batyr Ave, Nur-Sultan,010000, Republic of 19 Kazakhstan; Tel: +7 (7172) 694686; Email: [email protected]; 20 [email protected] Open Access Article. Published on 12 August 2021. Downloaded 9/24/2021 6:26:24 PM. 21 All authors Emails: [email protected], [email protected], Advances 22 [email protected] [email protected]; 23 [email protected] [email protected] [email protected] 24 [email protected] 25 Lazzat Nurtay Tel+7(778)6078187; Email: [email protected] 26 Nazarbek Guldan Tel +7(778) 7884887; Email:[email protected] Nanoscale 27 Bexultan Kazybay Tel +7(771) 3378827; Email: [email protected] 28 Words Count for the abstract: 29 A complete manuscript word count: 3897 1 Nanoscale Advances Page 2 of 25 30 Number of references: 26 31 Key Words: Enzyme, Nanozyme, evolution, association study View Article Online DOI: 10.1039/D1NA00475A 32 33 34 35 Abstract 36 Nanozyme and natural product-derived herbzyme have been identified in different enzyme types 37 simulating the natural protein-based enzyme function. How to explore and predict enzyme types 38 of novel nanozyme when synthesized remain elusive. Informed analysis might be useful for the Manuscript 39 prediction. Here we applied a protein-evolution analysis method to predict novel types of enzyme 40 with experimental validation. First, reported nanozymes were analyzed by chemical- Creative Commons Attribution-NonCommercial 3.0 Unported Licence. 41 classification and nano-evolution. We found nanozymes predominantly are classified as protein- 42 based EC1 oxidoreductase. In comparison, protein-based natural enzymes were analyzed for Accepted 43 evolution by phylogenetic tree and the most conserved enzymes were found as peroxidase and 44 This article is licensed under a lyase. From this finding of correlation, the natural products were further predicted to explore and 45 test the potent new types of mimicking natural protein-based enzymes according to the Open Access Article. Published on 12 August 2021. Downloaded 9/24/2021 6:26:24 PM. Advances 46 conserved enzyme types by natural simplicity simulation of enzyme evolution. The experimental 47 validation showed the natural products from total extract of nanoscale traditional Chinese 48 medicine Huangjing (RP, Rhizoma polygonati) from mount-Tai exhibits fructose-bisphophate 49 aldolase of lyase and nanoscale Goji (Lycium chinense) extract exhibits peroxidase activities. 50 Thus the bioinformatics analysis would provide the additional tool for virtual discovery of natural Nanoscale 51 product nanozyme. 52 2 Page 3 of 25 Nanoscale Advances 53 View Article Online DOI: 10.1039/D1NA00475A 54 55 56 57 Introduction 58 Nanozymes are functional nanomaterials with enzyme-like activity and biocatalytic function that 59 mimic natural enzymes “learning from nature”, while retaining nanomaterial properties [1-2]. Since Manuscript 60 the discovery of iron nanoparticles with intrinsic enzyme activity, more than 540 novel nanozymes 61 have been reported with modifications since the first generation of nanozyme type of peroxidase 62 Creative Commons Attribution-NonCommercial 3.0 Unported Licence. by classical Fe3O4 [3]. Recently Meng et al., for instance, reported an improved peroxidase of pyrite 63 peroxidase nanozyme with super strong binding to substrate hydrogen peroxide (H2O2) compared Accepted 64 to traditional nanozyme Fe3O4 and protein based horseradish peroxidase, with which additional 65 glutathione oxidase-like activity, following with the catalysis of H2O2 generation [4]. The dual This article is licensed under a 66 nanozyme finally targeted KRAS mutation with tumor catalytic therapy [4]. Open Access Article. Published on 12 August 2021. Downloaded 9/24/2021 6:26:24 PM. Advances 67 Currently there are many types of nanozyme reported but mostly concentrated at oxidoreductase, 68 hydrolases and hydrolase [1]. In particular, peroxidase POD is widely studied and reported as a type 69 of oxidoreductase [2]. There are basically 6 or 7 large types of protein-based enzymes, i.e. named 70 as natural enzymes which are classified according to the Enzyme Commission (EC) classification Nanoscale 71 as EC1, 2,3,4,5,6, 7 as oxidoreductases, transferases, hydrolases, lyases, isomerases, ligases, 72 translocase respectively [5]. Frances H Arnold proposed the concept of directed evolution of 73 enzymes by introducing random mutation of genes encoding target protein enzymes and specific 3 Nanoscale Advances Page 4 of 25 74 chemical reaction-based screening of target mutants, with repeated cycles to obtain finally expected View Article Online 75 enzyme [6]. The directed evolution of enzymes are artificially created to simulateDOI: natural 10.1039/D1NA00475A evolution 76 mechanisms with natural selection [7]. For example, cytochrome P450 can be evolved by directed 77 evolution with synthetic carbene and nitrene to improve the catalysis activity via genetic coding 78 changes or mutation [7]. Thus engineered enzyme evolution can be realized by modifications. 79 Nanozyme can combine the advantages of natural enzymes and traditional artificial mimic enzymes 80 and have high-efficiency due to adjustable catalytic activity and nano structure [2]. In addition, 81 nanozymes can function under extreme pH and high temperature [1].The catalytic function of Manuscript 82 nanozyme is related to its own nano effects such as size, surface area, and structure [2]. These 83 effects also provide an operational way for the optimization of simulated enzymes, by manually 84 controlling the nano effects to regulate the catalytic activity and substrate selectivity, which are Creative Commons Attribution-NonCommercial 3.0 Unported Licence. 85 simulating the natural enzyme evolution and selection. For example, Gao et al. synthesized Fe3O4 Accepted 86 nanoparticles by different sizes and the relative catalytic activity can be enhanced 5 times using 30 87 nm rather than 300 nm [8]. This article is licensed under a 88 A type of natural product-based nanozyme and nanoassembly has been arising as a new star in Open Access Article. Published on 12 August 2021. Downloaded 9/24/2021 6:26:24 PM. 89 nano-natural product research. These include the natural compound-based self-assembly and Advances 90 nanozyme, namely herbzyme [9-10]. Recently we reported the natural product-based nanozyme of 91 protease-like, phosphatase-like, and α-Amylases (EC 3.2. 1.1) which are mainly hydrolases [9]. 92 However, whether natural products exert other types of enzyme activity and the evolutions of 93 nanozyme remain elusive. Nanoscale 94 Although numerous nanozymes were discovered and applied in biomedicine, it is still unclear 95 about nanozyme evolution. Here, this paper aims to analyze nano-evolution of nanozyme and 4 Page 5 of 25 Nanoscale Advances 96 natural enzymatic
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