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Nature versus nurture in two highly enantioselective esterases from Bacillus cereus and Thermoanaerobacter tengcongensis.

Grosse S, Bergeron H, Imura A, Boyd J, Wang S, Kubota K, Miyadera A, Sulea T, Lau PC - Microb Biotechnol (2009)

Bottom Line: The principal findings of this study are: (i) the complete sequenced genomes of Bacillus cereus ATCC 14579 and Thermoanaerobacter tengcongensis MB4 contain a hitherto undescribed enantioselective and alkaliphilic esterase (BcEST and TtEST respectively) that is specific for the production of (R)-2-benzyloxy-propionic acid ethyl ester, a key intermediate in the synthesis of levofloxacin, a potent antibiotic; and (ii) directed evolution targeted for increased thermostability of BcEST produced two improved variants, but in either case the 3-5 °C increase in the apparent melting temperature (T(m)) of the mutants over the native BcEST that has a T(m) of 50 °C was outperformed by TtEST, a naturally occurring homologue with a T(m) of 65 °C.Protein modelling of BcEST mapped the S148C and K272R mutations at protein surface and the I88T and Q110L mutations at more buried locations.This work expands the repertoire of characterized members of the α/β-fold hydrolase superfamily.

View Article: PubMed Central - PubMed

Affiliation: Biotechnology Research Institute, National Research Council Canada, Montreal, Quebec H4P 2R2, Canada.

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Chemo‐enzymatic scheme for the production of levofloxacin involving the key intermediate (R)‐BnLAE (O‐benzyl lactic acid ethyl ester or 2‐benzyloxy‐propionic acid ethyl ester). The synthesis of (S)‐benzoxazine derivative is also through enzymatic resolution (Miyadera and Imura, 1999).
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f1: Chemo‐enzymatic scheme for the production of levofloxacin involving the key intermediate (R)‐BnLAE (O‐benzyl lactic acid ethyl ester or 2‐benzyloxy‐propionic acid ethyl ester). The synthesis of (S)‐benzoxazine derivative is also through enzymatic resolution (Miyadera and Imura, 1999).

Mentions: There are at least five synthetic routes to LFC that involve numerous chemical steps and solvents (Kang et al., 1996; 1997; Miyadera and Imura, 1999; Kleemann and Engel, 2001). One of the key chemical intermediates is (R)‐2‐benzyloxy‐propionic acid ethyl ester or O‐benzyl lactic acid ethyl ester (BnLAE, Fig. 1). Chemical synthesis of BnLAE would produce a racemic mixture and therefore additional steps for the separation of the isomers are required. The availability of a biocatalyst, an enantioselective esterase capable of resolving the racemic esters, would provide a green and chemo‐enzymatic route towards the production of LFC.


Nature versus nurture in two highly enantioselective esterases from Bacillus cereus and Thermoanaerobacter tengcongensis.

Grosse S, Bergeron H, Imura A, Boyd J, Wang S, Kubota K, Miyadera A, Sulea T, Lau PC - Microb Biotechnol (2009)

Chemo‐enzymatic scheme for the production of levofloxacin involving the key intermediate (R)‐BnLAE (O‐benzyl lactic acid ethyl ester or 2‐benzyloxy‐propionic acid ethyl ester). The synthesis of (S)‐benzoxazine derivative is also through enzymatic resolution (Miyadera and Imura, 1999).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3815948&req=5

f1: Chemo‐enzymatic scheme for the production of levofloxacin involving the key intermediate (R)‐BnLAE (O‐benzyl lactic acid ethyl ester or 2‐benzyloxy‐propionic acid ethyl ester). The synthesis of (S)‐benzoxazine derivative is also through enzymatic resolution (Miyadera and Imura, 1999).
Mentions: There are at least five synthetic routes to LFC that involve numerous chemical steps and solvents (Kang et al., 1996; 1997; Miyadera and Imura, 1999; Kleemann and Engel, 2001). One of the key chemical intermediates is (R)‐2‐benzyloxy‐propionic acid ethyl ester or O‐benzyl lactic acid ethyl ester (BnLAE, Fig. 1). Chemical synthesis of BnLAE would produce a racemic mixture and therefore additional steps for the separation of the isomers are required. The availability of a biocatalyst, an enantioselective esterase capable of resolving the racemic esters, would provide a green and chemo‐enzymatic route towards the production of LFC.

Bottom Line: The principal findings of this study are: (i) the complete sequenced genomes of Bacillus cereus ATCC 14579 and Thermoanaerobacter tengcongensis MB4 contain a hitherto undescribed enantioselective and alkaliphilic esterase (BcEST and TtEST respectively) that is specific for the production of (R)-2-benzyloxy-propionic acid ethyl ester, a key intermediate in the synthesis of levofloxacin, a potent antibiotic; and (ii) directed evolution targeted for increased thermostability of BcEST produced two improved variants, but in either case the 3-5 °C increase in the apparent melting temperature (T(m)) of the mutants over the native BcEST that has a T(m) of 50 °C was outperformed by TtEST, a naturally occurring homologue with a T(m) of 65 °C.Protein modelling of BcEST mapped the S148C and K272R mutations at protein surface and the I88T and Q110L mutations at more buried locations.This work expands the repertoire of characterized members of the α/β-fold hydrolase superfamily.

View Article: PubMed Central - PubMed

Affiliation: Biotechnology Research Institute, National Research Council Canada, Montreal, Quebec H4P 2R2, Canada.

Show MeSH
Related in: MedlinePlus