A Baeyer-Villiger monooxygenase from Cupriavidus basilensis catalyzes asymmetric synthesis of (R)-lansoprazole and other pharmaco-sulfoxides

Liu, Feng; Shou, Chao; Geng, Qiang; Zhao, Chen; Xu, Jian‐He; Yu, Hui‐Lei

doi:10.1007/s00253-021-11230-0

Cited by 27 publications

(40 citation statements)

References 46 publications

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“…With a feasible HTS method in hand, we subsequently turned to the directed evolution of CbBVMO, a unique biocatalyst for the efficient biosynthesis of (R)-lansoprazole with excellent stereoselectivity. [6] The epPCR libraries (with 50, 75, and 100 mM MnCl 2 ; corresponding to average mutational frequencies of 0.64, 1.26, and 1.91 kb À 1 , respectively) of CbBVMO were collectively cloned into Escherichia coli BL21 (DE3). The HTS procedure consisted of the following steps (Figure 2): (i) Building mutant libraries; (ii) seeding of single colonies into 96-well plates for protein expression; (iii) shaking at 25 °C for 2 h to lyse cells (with 5 h additional incubation at 30 °C for thermostability assays); (iv) addition of reactants (substrate 1 a and NADPH) into the 96-well plates to start the reactions; and (v) extraction of the reactants and products with ethyl acetate, and mixing with an equal volume of FA for spectral analysis.…”

Section: Entrymentioning

confidence: 99%

“…The PCR products were purified, digested, and inserted into the commercially obtained pET-28a expression vector between Nde I and Hind III restriction sites. [6] The recombinant plasmid containing the variant genes were then transformed into E. coli BL21 (DE3) for variants expression.…”

Section: Eppcr-based Library Constructionmentioning

confidence: 99%

“…[5] BVMO-mediated asymmetric sulfoxidation of prazole sulfides under environmentally friendly conditions has been reported as an attractive green alternative to conventional chemical synthesis (Scheme 1). [6][7][8] However, protein engineering against these native BVMOs is necessary for their practical applications due to unsatisfactory activity, thermostability, and/or substrate specificity. [7][8][9][10] Protein engineering approaches have been extensively studied using rational, semi-rational, or random methods.…”

Section: Introductionmentioning

confidence: 99%

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Colorimetric High‐Throughput Screening Method for Directed Evolution of Prazole Sulfide Monooxygenase

et al. 2022

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Baeyer‐Villiger monooxygenases (BVMOs) are important biocatalysts for the enzymatic synthesis of chiral sulfoxides, including chiral sulfoxide‐type proton pump inhibitors for the treatment of gastrointestinal diseases. However, native BVMOs are not yet suitable for practical application due to their unsatisfactory activity and thermostability. Although protein engineering approaches can help address these issues, few feasible high‐throughput methods are available for the engineering of such enzymes. Herein, a colorimetric detection method to distinguish sulfoxides from sulfides and sulfones was developed for prazole sulfide monooxygenases. Directed evolution enabled by this method has identified a prazole sulfide monooxygenase CbBVMO variant with improved activity and thermostability that catalyzes the asymmetric oxidation of lansoprazole sulfide. A 71.3 % increase in conversion and 6 °C enhancement in the melting point were achieved compared with the wild‐type enzyme. This new method is feasible for high‐throughput screening of prazole sulfide monooxygenase variants with improved activity, thermostability, and/or substrate specificity.

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Section: Entrymentioning

confidence: 99%

Section: Eppcr-based Library Constructionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Colorimetric High‐Throughput Screening Method for Directed Evolution of Prazole Sulfide Monooxygenase

et al. 2022

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“…The general biocatalytic approaches for preparing chiral sulfoxides include asymmetric oxidation of sulfides and kinetic resolution of racemic ( rac ) sulfoxides. A great number of oxidases, including Baeyer–Villiger monooxygenases ( de Gonzalo et al, 2010 ; Rioz-Martínez et al, 2010 ; Bisagni et al, 2013 ; Bordewick et al, 2018 ; Zhang et al, 2018 ; Liu et al, 2021 ), cytochrome P450 monooxygenases ( Zhang et al, 2010 ; Wu et al, 2018 ), peroxidases ( Sangar et al, 2012 ; Bassanini et al, 2017 ), and dioxygenases ( Hibi et al, 2011 ; Shainsky et al, 2013 ), have been used for the synthesis of chiral sulfoxides. Meanwhile, biocatalytic kinetic resolution of rac -sulfoxides has also become an attractive approach for preparing chiral sulfoxides ( Anselmi et al, 2021 ), although it was relatively less reported.…”

Section: Introductionmentioning

confidence: 99%

Sulfoxide Reductases and Applications in Biocatalytic Preparation of Chiral Sulfoxides: A Mini-Review

et al. 2021

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Chiral sulfoxides are valuable organosulfur compounds that have been widely used in medicinal and organic synthesis. Biocatalytic approaches for preparing chiral sulfoxides were developed in the past few years, mainly through asymmetric oxidation of prochiral sulfides. Recently, the application of sulfoxide reductase to prepare chiral sulfoxides through kinetic resolution has emerged as a new method, exhibiting extraordinary catalytic properties. This article reviews the chemical and biological functions of these sulfoxide reductases and highlights their applications in chiral sulfoxide preparation.

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“…This mutant enzyme is used to produce esomeprazole ((S)-omeprazole), a popular drug for the clinical treatment of gastroesophageal re ux [Carreno et al 1995;Matsui et al 2014]. Later studies used genomic mining [Liu et al 2021;Zhang et al 2018] or directed evolution to generate several other BVMOs that are capable of transforming sul des with a "prazole" scaffold [Zhang et al 2019;Ren et al 2020;Zhao et al 2021].…”

Section: Introductionmentioning

confidence: 99%

Secretory Expression of Cyclohexanone Monooxygenase by Methylotrophic Yeast for Efficient Omeprazole Sulfide Bio-oxidation

Zheng

Geng

Liu

et al. 2021

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Prochiral pyrmetazole can be asymmetrically oxidized into (S)-omeprazole, a proton pump inhibitor that is used to treat gastroesophageal reflux, by an engineered cyclohexanone monooxygenase (CHMOAcineto-Mut) that has high stereoselectivity. CHMOAcineto-Mut is produced by heterologous expression in Escherichia coli, where it is expressed intracellularly. Thus, isolating this useful biocatalyst requires tedious cell disruption and subsequent purification, which hinders its use for industrial purposes. Here, we report the extracellular production of CHMOAcineto-Mut by a methylotrophic yeast, Pichia pastoris, for the first time. The recombinant CHMOAcineto-Mut expressed by P. pastoris showed a higher flavin occupation rate than that produced by E. coli, and this was accompanied by a 3.2-fold increase in catalytic efficiency. At a cell density of 150 g/L cell dry weight, we achieved a recombinant CHMOAcineto-Mut production rate of 1,700 U/L, representing approximately 85% of the total protein secreted into the fermentation broth. By directly employing the pH adjusted supernatant as a biocatalyst, we were able to almost completely transform 10 g/L of pyrmetazole into the corresponding (S)-sulfoxide, with >99% enantiomeric excess.

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A Baeyer-Villiger monooxygenase from Cupriavidus basilensis catalyzes asymmetric synthesis of (R)-lansoprazole and other pharmaco-sulfoxides

Cited by 27 publications

References 46 publications

Colorimetric High‐Throughput Screening Method for Directed Evolution of Prazole Sulfide Monooxygenase

Colorimetric High‐Throughput Screening Method for Directed Evolution of Prazole Sulfide Monooxygenase

Sulfoxide Reductases and Applications in Biocatalytic Preparation of Chiral Sulfoxides: A Mini-Review

Secretory Expression of Cyclohexanone Monooxygenase by Methylotrophic Yeast for Efficient Omeprazole Sulfide Bio-oxidation

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