title: Comparative analysis of tertiary alcohol esterase activity in bacterial strains isolated from enrichment cultures and from screening strain libraries.
abstract: The preparation of enantiopure tertiary alcohols is of great contemporary interest due to the application of these versatile building blocks in organic synthesis and as precursors towards high value pharmaceutical compounds. Herein, we describe two approaches taken towards the discovery of novel biocatalysts for the synthesis of these valuable compounds. The first approach was initiated with screening of 47 bacterial strains for hydrolytic activity towards the simple tertiary alcohol ester tert-butyl acetate. In conjunction, a second method focussed on the isolation of strains competent for growth on tert-butyl acetate as the sole source of carbon and energy. From functional screening, 10 Gram-positive Actinomycetes showed hydrolytic activity, whilst enrichment selection resulted in the identification of 14 active strains, of which five belong to the Gram-negative cell-wall type. Bacterial strains obtained from both approaches were viable for enantioselective hydrolysis of pyridine substituted tertiary alcohol esters in addition to bulky aliphatic and keto-derived substrates from the same class. Activity towards each of the test substrates was uncovered, with promising enantioselectivities of up to E = 71 in the hydrolysis of a para-substituted pyridine tertiary alcohol ester using a strain of Rhodococcus ruber. Interestingly strains of Microbacterium and Alcaligenes sp. gave opposite enantiopreference in the hydrolysis of a meta-substituted pyridine tertiary alcohol ester with E values of 17 and 54. These approaches show that via both possibilities, screening established strain collections and performing enrichment selection, it is possible to identify novel species which show activity towards sterically challenging substrates.
authors.affiliation: Institute of Microbiology, Department of Applied Microbiology, University of Greifswald, Friedrich-Ludwig-Jahn-Str. 15a, 17489 Greifswald, Germany.
authors.forename: Susanne
authors.initials: S
authors.lastname: Herter
authors.forename: Giang-Son
authors.initials: GS
authors.lastname: Nguyen
authors.forename: Mark L
authors.initials: ML
authors.lastname: Thompson
authors.forename: Fabian
authors.initials: F
authors.lastname: Steffen-Munsberg
authors.forename: Frieder
authors.initials: F
authors.lastname: Schauer
authors.forename: Uwe T
authors.initials: UT
authors.lastname: Bornscheuer
authors.forename: Robert
authors.initials: R
authors.lastname: Kourist
mesh_terms: D000438:Alcohols / Q000378:metabolism*; D001419:Bacteria / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D004950:Esterases / Q000032:analysis* / Q000235:genetics / Q000378:metabolism*; D015723:Gene Library; D008969:Molecular Sequence Data
chemical_list: D000438:Alcohols; D004950:Esterases