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The catalytic performance of ADHs in the presence of organic solvents is often important since most prochiral ketones are highly hydrophobic. Here, the organic solvent tolerance of <jats:italic>Kp<\/jats:italic>ADH from <jats:italic>Kluyveromyces polyspora<\/jats:italic> was semi\u2010rationally evolved. Using tolerant variants obtained, meticulous experiments and computational studies were conducted to explore properties including stability, activity and kinetics in the presence of various organic solvents. Compared with WT, variant V231D exhibited 1.9\u2010fold improvement in ethanol tolerance, while S237G showed a 6\u2010fold increase in catalytic efficiency, a higher , as well as 15% higher tolerance in 7.5% (v\/v) ethanol. Based on 3\u2009\u00d7\u2009100\u2009ns MD simulations, the increased tolerance of V231D and S237G against ethanol may be ascribed to their enhanced ability in retaining water molecules and repelling ethanol molecules. Moreover, 6.3\u2010fold decreased <jats:italic>K<\/jats:italic><jats:sub>M<\/jats:sub> value of V231D toward hydrophilic ketone substrate confirmed its capability of retaining hydration shell. Our results suggest that retaining hydration shell surrounding <jats:italic>Kp<\/jats:italic>ADH is critical for its tolerance to organic solvents, as well as catalytic performance. This study provides useful guidance for engineering organic solvent tolerance of <jats:italic>Kp<\/jats:italic>ADH and other ADHs.<\/jats:p>","DOI":"10.1002\/pro.4933","type":"journal-article","created":{"date-parts":[[2024,3,19]],"date-time":"2024-03-19T07:43:55Z","timestamp":1710834235000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":5,"title":["Engineering diaryl alcohol dehydrogenase <scp><i>Kp<\/i>ADH<\/scp> reveals importance of retaining hydration shell in organic solvent tolerance"],"prefix":"10.1002","volume":"33","author":[{"given":"Lu","family":"Zhang","sequence":"first","affiliation":[{"name":"Key Laboratory of Industrial Biotechnology, Ministry of Education School of Biotechnology, Jiangnan University  Wuxi China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Wei","family":"Dai","sequence":"additional","affiliation":[{"name":"Key Laboratory of Industrial Biotechnology, Ministry of Education 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