Lyophilization conditions for the storage of monooxygenases

Beek, Hugo L. van; Beyer, Nina; Janssen, Dick B.; Fraaije, Marco W.

doi:10.1016/j.jbiotec.2015.03.010

Cited by 12 publications

(12 citation statements)

References 33 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Therefore, we put effort into optimizing the lyophilization conditions of the fusion enzyme, especially for FMO‐E part in the fused protein by using additives, namely, lyoprotectants. Sugars such as sucrose are widely used as lyoprotectants for the lyophilization process . In addition to sugars, there are also other reported lyoprotectants, such as salts, reducing compounds, and amino acids .…”

Section: Methodsmentioning

confidence: 99%

“…Sugars such as sucrose are widely used as lyoprotectants for the lyophilization process . In addition to sugars, there are also other reported lyoprotectants, such as salts, reducing compounds, and amino acids . Based on a literature search, we focused on sucrose and MgSO 4 because they have been shown to stabilize the lyophilization of many Baeyer–Villiger monooxygenases (BVMOs) …”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Convergent Cascade Catalyzed by Monooxygenase–Alcohol Dehydrogenase Fusion Applied in Organic Media

et al. 2019

Self Cite

View full text Add to dashboard Cite

With the aim of applying redox‐neutral cascade reactions in organic media, fusions of a type II flavin‐containing monooxygenase (FMO‐E) and horse liver alcohol dehydrogenase (HLADH) were designed. The enzyme orientation and expression vector were found to influence the overall fusion enzyme activity. The resulting bifunctional enzyme retained the catalytic properties of both individual enzymes. The lyophilized cell‐free extract containing the bifunctional enzyme was applied for the convergent cascade reaction consisting of cyclobutanone and butane‐1,4‐diol in different microaqueous media with only 5 % (v/v) aqueous buffer without any addition of external cofactor. Methyl tert‐butyl ether and cyclopentyl methyl ether were found to be the best organic media for the synthesis of γ‐butyrolactone, resulting in about 27 % analytical yield.

show abstract

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Convergent Cascade Catalyzed by Monooxygenase–Alcohol Dehydrogenase Fusion Applied in Organic Media

et al. 2019

Self Cite

View full text Add to dashboard Cite

show abstract

“…Remaining AS was removed by ion‐exchange chromatography on gravity Econo‐Pac 10DG columns (Bio‐RAD). The columns were equilibrated in Tris ⋅ HCl with 20 mg mL −1 sucrose for storage stabilization of the enzyme . For storage, the enzyme was shock frozen in liquid nitrogen and stored at −80 °C.…”

Section: Methodsmentioning

confidence: 99%

“…The columns were equilibrated in Tris·HCl with 20 mg mL À1 sucrose for storage stabilization of the enzyme. [82] For storage, the enzyme was shock frozen in liquid nitrogen and stored at À80 8C. The concentration of PTDH-P450BM3 variants was determined by measuring CO difference spectra in triplicate.…”

Section: Methodsmentioning

confidence: 99%

Exploring PTDH–P450BM3 Variants for the Synthesis of Drug Metabolites

et al. 2018

Self Cite

View full text Add to dashboard Cite

The conversion of a series of pharmaceutical compounds was examined with three variants of cytochrome P450BM3 fused to phosphite dehydrogenase (PTDH) to enable cofactor recycling. Conditions for enzyme production were optimized, and the purified PTDH-P450BM3 variants were tested against 32 commercial drugs by using rapid UPLC-MS analysis. The sets of mutations (R47L/F87V/L188Q and R47L/F87V/L188Q/E267V/G415S) improved conversion for all compounds, and a variety of products were detected. Product analysis showed that reaction types included C-hydroxylation, N-oxidation, demethylation, and aromatization. Interestingly, enzymatic aromatization could occur independent of the addition of reducing coenzyme. These results identified new conversions catalyzed by P450BM3 variants and showed that a small set of mutations in the oxygenase domain could broaden both substrate range and reaction type.

show abstract

“…Subsequently, the protein was eluted with buffer containing 500 mmol L ‐1 imidazole. 15 mL batches of the purified protein solution were desalted on a HiPrep 26/10 desalting column (GE Healthcare Life Sciences), and then shock frozen in liquid N 2 after addition of 2% (w/v) sucrose for improved retention of enzymatic activity after lyophilization . The lyophilized protein was stored at –20 °C until further use.…”

Section: Methodsmentioning

confidence: 99%

An immobilized and highly stabilized self‐sufficient monooxygenase as biocatalyst for oxidative biotransformations

Valencia

Guillén

Fürst

et al. 2017

J of Chemical Tech & Biotech

View full text Add to dashboard Cite

BACKGROUND The requirement for expensive cofactors that must be efficiently recycled is one of the major factors hindering the wide implementation of industrial biocatalytic oxidation processes. In this research, a sustainable approach based on immobilized self‐sufficient Baeyer–Villiger monooxygenases is discussed. RESULTS A bifunctional biocatalyst composed of an NADPH‐dependent cyclohexanone monooxygenase (CHMO) fused to an NADP+‐accepting phosphite dehydrogenase (PTDH) catalyzes ϵ‐caprolactone synthesis from cyclohexanone, using phosphite as a cheap sacrificial substrate for cofactor regeneration. Several immobilized derivatives of the fused enzyme have been prepared with high immobilization yield (97%); the one obtained by affinity adsorption on Co‐IDA (Co: cobalt chelated; IDA: iminodiacetic acid) support has shown to be highly stable affording average yields of 80% after 18 reaction cycles. CONCLUSIONS The immobilized self‐sufficient monooxygenase has demonstrated to be able to perform Baeyer–Villiger oxidation with efficient cofactor recovery and biocatalyst recycling. The proposed biocatalytic process offers access to valuable molecules with high atom economy and limited waste generation. © 2017 Society of Chemical Industry

show abstract

Lyophilization conditions for the storage of monooxygenases

Cited by 12 publications

References 33 publications

Convergent Cascade Catalyzed by Monooxygenase–Alcohol Dehydrogenase Fusion Applied in Organic Media

Convergent Cascade Catalyzed by Monooxygenase–Alcohol Dehydrogenase Fusion Applied in Organic Media

Exploring PTDH–P450BM3 Variants for the Synthesis of Drug Metabolites

An immobilized and highly stabilized self‐sufficient monooxygenase as biocatalyst for oxidative biotransformations

Contact Info

Product

Resources

About