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Herein, we developed an efficient (\n                    <jats:italic>R<\/jats:italic>\n                    )\u2010selective transaminase for the synthesis of (\n                    <jats:italic>R<\/jats:italic>\n                    )\u20101\u2010Boc\u20103\u2010aminopiperidine through an integrated strategy combining phylogeny\u2010guided enzyme mining, structure\u2010guided engineering, and mechanistic analysis. Screening of transaminases identified\n                    <jats:italic>Mb<\/jats:italic>\n                    ATA as the \u201cbest\u201d parent enzyme, affording the target amine from\n                    <jats:italic>N<\/jats:italic>\n                    \u2010Boc\u20103\u2010piperidone (1B3OP) in specific activities of 0.269\u2009U\u00b7g\n                    <jats:sup>\u22121<\/jats:sup>\n                    and &gt;\u200999%\n                    <jats:italic>ee<\/jats:italic>\n                    . Subsequent engineering yielded variant M3 (H61L\/Q57R\/P158Q), which exhibited a 45\u2010fold increase in activity over the wild type (WT) while retaining strict enantioselectivity (&gt;\u200999%\n                    <jats:italic>ee<\/jats:italic>\n                    ). M3 achieved completely aminated 500\u2009mM substrate loading within 4\u2009h, 1000\u2009mM in an extended duration of 24\u2009h. Preparative biotransformation afforded 8.5\u2009g and 15.6\u2009g of product in 85% and 78% isolated yields, respectively. Structural and mechanistic analyses showed that the beneficial mutations enlarged and remodeled the substrate\u2010binding pocket, strengthened favorable substrate interactions, shortened the reactive cofactor\u2013substrate distance, and promoted productive binding conformations. These results establish M3 as a practical biocatalyst for high\u2010substrate\u2010loading synthesis of chiral piperidine amines.\n                  <\/jats:p>","DOI":"10.1002\/bit.70376","type":"journal-article","created":{"date-parts":[[2026,9,21]],"date-time":"2026-09-21T12:04:45Z","timestamp":1789992285000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Engineering an (\n                    <i>R<\/i>\n                    )\u2010Selective Transaminase for High\u2010Substrate\u2010Loading Asymmetric Amination of a Sterically Demanding\n                    <i>N<\/i>\n                    \u2010Heterocyclic Ketone"],"prefix":"10.1002","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-6131-7314","authenticated-orcid":false,"given":"Zhe","family":"Dou","sequence":"first","affiliation":[{"name":"College of Pharmaceutical Sciences, Zhejiang University of Technology Hangzhou Zhejiang China"},{"name":"Key Laboratory of Pharmaceutical Engineering of Zhejiang Province Zhejiang University of Technology Hangzhou China"},{"name":"Key Laboratory for Green Pharmaceutical Technologies and Related Equipment of Ministry of Education Zhejiang University of Technology Hangzhou China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hui\u2010Xuan","family":"Yan","sequence":"additional","affiliation":[{"name":"Key Laboratory of Pharmaceutical Engineering of Zhejiang Province Zhejiang University of Technology Hangzhou China"},{"name":"Key Laboratory for Green Pharmaceutical Technologies and Related Equipment of Ministry of Education Zhejiang University of Technology Hangzhou China"},{"name":"College of Biotechnology and Bioengineering Zhejiang University of Technology Hangzhou Zhejiang China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0007-6122-4461","authenticated-orcid":false,"given":"Cheng\u2010Long","family":"Xu","sequence":"additional","affiliation":[{"name":"Key Laboratory of Pharmaceutical Engineering of Zhejiang Province Zhejiang University of Technology Hangzhou China"},{"name":"Key Laboratory for Green Pharmaceutical Technologies and Related Equipment of Ministry of Education Zhejiang University of Technology Hangzhou China"},{"name":"College of Biotechnology and Bioengineering Zhejiang University of Technology Hangzhou Zhejiang China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jia\u2010Neng","family":"Xu","sequence":"additional","affiliation":[{"name":"College of Pharmaceutical Sciences, Zhejiang University of Technology Hangzhou Zhejiang China"},{"name":"Key Laboratory of Pharmaceutical Engineering of Zhejiang Province Zhejiang University of Technology Hangzhou China"},{"name":"Key Laboratory for Green Pharmaceutical Technologies and Related Equipment of Ministry of Education Zhejiang University of Technology Hangzhou China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Min\u2010Min","family":"Zhao","sequence":"additional","affiliation":[{"name":"Key Laboratory of Pharmaceutical Engineering of Zhejiang Province Zhejiang University of Technology Hangzhou China"},{"name":"Key Laboratory for Green Pharmaceutical Technologies and Related Equipment of Ministry of Education Zhejiang University of Technology Hangzhou China"},{"name":"College of Biotechnology and Bioengineering Zhejiang University of Technology Hangzhou Zhejiang China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2284-0882","authenticated-orcid":false,"given":"Ya\u2010Jun","family":"Wang","sequence":"additional","affiliation":[{"name":"College of Pharmaceutical Sciences, Zhejiang University of Technology Hangzhou Zhejiang China"},{"name":"Key Laboratory of Pharmaceutical Engineering of Zhejiang Province Zhejiang University of Technology Hangzhou China"},{"name":"Key Laboratory for Green Pharmaceutical Technologies and Related Equipment of Ministry of Education Zhejiang University of Technology Hangzhou China"},{"name":"State Key Laboratory of Green Chemical Synthesis and Conversion Deqing Zhejiang 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