title: Co-expression of the recombined alcohol dehydrogenase and glucose dehydrogenase and cross-linked enzyme aggregates stabilization.
abstract: As the key chiral precursor of Crizotinib (S)-1-(2,6-dichloro-3-fluorophenyl) phenethyl alcohol can be prepared from 1-(2,6-dichloro-3-fluorophenyl) acetophenone by the reductive coupling reactions of alcohol dehydrogenase (ADH) and glucose dehydrogenases (GDH). In this work the heterologous expression plasmids harbouring the encoding genes of ADH and GDH were constructed respectively and co-expressed in the same E. coli strain. After optimization, a co-cross-linked enzyme aggregates (co-CLEAs) of both ADH and GDH were prepared from crude enzyme extracts by cross-linking with the mass ratio of Tween 80, glutaraldehyde and total protein (0.6:1:2) which rendered immobilized biocatalysts that retained 81.90% (ADH) and 40.29% (GDH) activity retention. The ADH/GDH co-CLEAs show increased thermal stability and pH stability compared to both enzymes. The ADH/GDH co-CLEAs also show 80% (ADH) and 87% (GDH) residual activity after seven cycles of repeated use. These results make the ADH/GDH co-CLEAs a potential biocatalyst for the industrial preparation of (S)-1-(2,6-dichloro-3-fluorophenyl) phenethyl alcohol.
authors.affiliation: State Key Laboratory of New Drug and Pharmaceutical Process, Shanghai Institute of Pharmaceutical Industry, 285 Gebaini Rd., Shanghai 200040, China; School of Engineering, China Pharmaceutical University, Nanjing 210009, China.
authors.forename: Xiaozhi
authors.initials: X
authors.lastname: Hu
authors.affiliation: State Key Laboratory of New Drug and Pharmaceutical Process, Shanghai Institute of Pharmaceutical Industry, 285 Gebaini Rd., Shanghai 200040, China.
authors.forename: Liqin
authors.initials: L
authors.lastname: Liu
authors.affiliation: State Key Laboratory of New Drug and Pharmaceutical Process, Shanghai Institute of Pharmaceutical Industry, 285 Gebaini Rd., Shanghai 200040, China.
authors.forename: Daijie
authors.initials: D
authors.lastname: Chen
authors.affiliation: School of Life Sciences, Collaborative Innovation Center of Modern Bio-manufacture, Anhui University, Hefei 230039, China.
authors.forename: Yongzhong
authors.initials: Y
authors.lastname: Wang
authors.affiliation: State Key Laboratory of New Drug and Pharmaceutical Process, Shanghai Institute of Pharmaceutical Industry, 285 Gebaini Rd., Shanghai 200040, China.
authors.forename: Junliang
authors.initials: J
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of New Drug and Pharmaceutical Process, Shanghai Institute of Pharmaceutical Industry, 285 Gebaini Rd., Shanghai 200040, China. Electronic address: shaolei00@gmail.com.
authors.forename: Lei
authors.initials: L
authors.lastname: Shao
mesh_terms: D000426:Alcohol Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000077547:Crizotinib; D003432:Cross-Linking Reagents / Q000737:chemistry; D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D042843:Glucose 1-Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005976:Glutaral / Q000737:chemistry; D010626:Phenylethyl Alcohol / Q000031:analogs & derivatives / Q000378:metabolism; D011136:Polysorbates / Q000737:chemistry; D015202:Protein Engineering / Q000379:methods*; D011720:Pyrazoles / Q000737:chemistry; D011725:Pyridines / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D003432:Cross-Linking Reagents; D004800:Enzymes, Immobilized; D011136:Polysorbates; D011720:Pyrazoles; D011725:Pyridines; D011994:Recombinant Proteins; D000077547:Crizotinib; D000426:Alcohol Dehydrogenase; D042843:Glucose 1-Dehydrogenase; D010626:Phenylethyl Alcohol; D005976:Glutaral
keywords: (S)-1-(2,6-dichloro-3-fluorophenyl) phenethyl alcohol; Crizotinib; Recombinant alcohol dehydrogenase; Recombinant glucose dehydrogenase; co-CLEAs