P450 Enzymes / edited by Hans Renata.

Format:

Book

Contributor:

Renata, Hans, editor.

Series:

Methods in Enzymology Series

Methods in Enzymology Series ; v.Volume 693

Language:

English

Subjects (All):

Enzymes.

Physical Description:

1 online resource (426 pages)

Edition:

First edition.

Place of Publication:

Cambridge, MA : Academic Press, [2023]

Summary:

P450 Enzymes, Volume 692 in the Methods in Enzymology series, highlights new advances in the field with this new volume presenting interesting chapters written by an international board of authors.

Contents:

Front Cover

Series Page

Methods in Enzymology

Copyright

Contents

Contributors

Preface

Chapter One: Directed evolution of P411 enzymes for amination of inert C-H bonds

1 Introduction

2 Generation of mutant libraries

2.1 Cloning and mutagenesis

2.1.1 Equipment

2.1.2 Materials

2.1.3 Protocol for SSM

2.1.4 Protocol for random mutagenesis

2.1.5 Protocol for StEP recombination

2.1.6 Determining the retention of function curve for epPCR

2.2 Plasmid construction with Gibson assembly

2.2.1 Equipment

2.2.2 Materials

2.2.3 Protocol for Gibson Mix™ preparation

2.2.4 Protocol for Gibson assembly

2.3 Transformation of chemically competent E. coli with DNA library

2.3.1 Equipment

2.3.2 Materials

2.3.3 Protocol

3 Protein expression

3.1 Equipment

3.2 Materials

3.3 Protocol for protein expression in 96-well plate

3.4 Protocol for protein expression for analytical-scale validation

3.5 Protocol for protein expression for preparative scale

3.6 Protocol for determining hemeprotein concentration

4 Enzymatic reaction setup

4.1 Equipment

4.2 Materials and chemicals

4.3 Protocol for 96-well plate reaction setup

4.4 Protocol for analytical-scale reaction setup

4.5 Protocol for preparative-scale reaction setup

5 Screening and validation of the enzymatic reactions

5.1 Equipment

5.2 Materials and chemicals

5.3 Protocol for igh-throughput HPLC-MS screening

5.4 Protocol for analytical-scale validation using HPLC-MS screening

5.5 Protocol for chiral GC-FID screening

6 Summary

Acknowledgments

Funding source

Declarations of interest

References

Chapter Two: P450-catalyzed atom transfer radical cyclization

2 Materials

2.1 Cloning

2.2 Enzyme expression in E. coli

2.3 Whole-cell reaction.

2.4 Normal phase HPLC analysis

2.5 Pyridine hemochromagen assay

3 Protocols

3.1 Cloning for a site-saturated mutagenesis screening library

3.2 High-throughput experimentation in 96-well plates

3.3 Analytical scale reactions to validate the screening hits

3.4 Preparative-scale reactions

3.5 HPLC calibration curves

4 Summary

Chapter Three: Self-sufficient P450-reductase chimeras for biocatalysis

1.1 P450s as biocatalysts

2 Protocols

2.1 Developing PtmO5 as a self-sufficient P450 biocatalyst

2.2 Generation of pET28a(+)-PtmO5-RhFRed vector

2.2.1 Materials

2.2.2 Procedure

2.3 Generation of pET15b(+)-PtmO5-RhFRed vector

2.3.1 Materials

2.3.2 Procedure

2.4 Generation of pRSF-Opt13 vector

2.4.1 Materials

2.4.2 Procedure

2.5 Compatible co-expression of PtmO5-RhFRed, GroES/EL, Opt13 in E. coli C41(DE3) cells

2.5.1 Materials

2.5.2 Procedure

3 Biocatalytic reactions with PtmO5-RhFRed

3.1 Materials and equipment

3.2 Procedure

3.3 Product characterization

4 Developing TleB as a self-sufficient P450 biocatalyst

4.1 Generation of pET28a(+)-TleB-RhFRed vector

4.1.1 Materials

4.1.2 Procedure

5 Biocatalytic reaction with TleB-RhFRed

5.1 Materials and equipment

5.2 Procedure

5.3 Product characterization

Chapter Four: The colors of peroxygenase activity: Colorimetric high-throughput screening assays for directed evolution

2 Materials, equipment and reagents

2.1 Strains and chemicals

2.2 Equipment

3 Enzyme production

4 Validation of the assay

4.1 Linearity of the assay

4.2 Coefficient of variation (CV%)

5 High-throughput screening (HTS) assays

5.1 HTS assays for peroxidase activity.

5.1.1 ABTS (2,2′-azino-bis(3ethylbenzothiazoline-6-sulfonic acid)) screening assay

5.1.1.1 Protocol

5.1.1.2 Notes

5.1.2 DMP (2,6-dimethoxyphenol) screening assay

5.1.2.1 Protocol

5.1.2.2 Notes

5.1.3 O-Dianisidine screening assay

5.1.3.1 Protocol

5.1.3.2 Notes

5.1.4 Amplex® Red screening assay

5.1.4.1 Protocol

5.1.4.2 Notes

5.1.5 TMB (3,3′,5,5′-tetramethylbenzidine) screening assay

5.1.5.1 Protocol

5.1.5.2 Notes

5.2 Assays for peroxygenase activity

5.2.1 Purpald® screening assay

5.2.1.1 Protocol

5.2.1.2 Notes

5.2.2 4-Aminoantipyrine screening (4-AAP) assay

5.2.2.1 Protocol

5.2.2.2 Notes

5.2.3 NBD (5-nitro-1,3-benzodioxole) screening assay

5.2.3.1 Protocol

5.2.3.2 Notes

5.2.4 NBP (γ-p-nitrobenzyl-pyridine) screening assay

5.2.4.1 Protocol

5.2.4.2 Notes

5.2.5 Russig's Blue screening assay

5.2.5.1 Protocol

5.2.5.2 Notes

5.2.6 Indoles screening assays

5.2.6.1 Protocol

5.2.6.2 Notes

5.2.7 Fast Red screening assay

5.2.7.1 Protocol

5.2.7.2 Notes

6 Discussion and outlook

Conflicts of Interest

Funding

CRediT authorship contribution statement

Data availability statement

Appendix A Supporting information

Chapter Five: Engineering cytochrome P450s for selective alkene to carbonyl oxidation

2 General methods

3 Library construction

3.2 Reagents

3.3 Procedure

3.4 Notes

4 Recombinant protein expression in 96-well plate format

4.2 Reagents

4.3 Procedure

4.4 Notes

5 Library screening

5.2 Reagents

5.3 Procedure

5.4 Notes

6 Rescreening of variants containing beneficial mutations

6.1 Equipment

6.2 Reagents

6.3 Procedure

6.4 Notes

7 Summary and conclusions

Acknowledgment

References.

Chapter Six: Bacterial cytochrome P450 enzymes: Semi-rational design and screening of mutant libraries in recombinant Escherichia coli cells

2 Cloning of surrogate redox partners for CYP107Z

2.1 Equipment

2.2 Reagents

2.3 Procedure

3 Construction of focused P450 mutant library

4 Expression of site-saturation libraries and whole-cell biotransformation of (−)-deoxypodophyllotoxin in 96-deep-well plates

4.4 Biotransformation

4.5 Notes

5 MISER LC/MS analysis

6 Verification of selected P450 variants for in vitro conversion of (−)-deoxypodophyllotoxin

7 Summary and conclusion

Chapter Seven: Functional analysis of a fungal P450 enzyme

2 In vivo analysis of Trt6 and Trt14

2.1 Overview

2.2 Cloning and overexpression of Trt6 and Trt14

2.2.2 Procedures for gDNA extraction and cloning of trt6 and trt14 into pTAex3

2.2.3 Procedure for cloning trt6 and trt14 in pBARI

2.2.4 Procedure for transformation of plasmids into A. oryzae NSAR1

2.3 In vivo analyses of the Trt6 and Trt14 metabolites

2.3.2 Procedures for the analyses of the metabolites of A. oryzae NSAR1 harboring trt4/trt2/trt5/trt8/trt1/trt9/trt3/trt6 and A. oryzae NSAR1 harboring trt4/trt2/trt5/trt8/trt1/trt9/trt3/trt6/trt14

2.3.3 Isolation and purification of each metabolite

3 In vitro analyses of Trt6 and Trt14

3.1 Overview

3.2 Cloning and overexpression of Trt6 and AtCPR

3.2.1 Materials

3.2.2 Procedure for cloning of Trt6 and AtCPR

3.2.3 Procedure for preparation of Trt6 and AtCPR for enzyme reaction assay.

3.3 Cloning, overexpression, and purification of Trt14

3.3.1 Materials

3.3.2 Procedure for cloning of Trt14

3.3.3 Procedure for Trt14 expression and purification

3.4 In vitro assays of Trt6 and Trt14

3.4.1 Materials

3.4.2 Procedure for assays of Trt6

3.4.3 Procedure for co-incubation assays of Trt6 and Trt14

3.5 Summary

Chapter Eight: Protein engineering using mutability landscapes: Controlling site-selectivity of P450-catalyzed steroid hydroxylation

2 Mutability landscapes

3 Identification of hotspots by mutability landscape and empirical mutations

4 Enzyme engineering by iterative saturation mutation (ISM) and rational design

5 Exploration of the mutants to hydroxylate other steroids

6 Materials, equipment and reagents

6.2 Chemical reagents

6.3 Biological reagents

7 Protocol

7.1 Prepare the following media and buffers

7.2 Perform PCR with appropriately designed DNA primers

7.3 Electrotransformation

7.3.1 Preparation of electrocompetent cells of E. coli BL21 (DE3)

7.3.2 Electrotransformation of E. coli BL21 (DE3)

7.4 The acquisition of the target mutants

7.4.1 Site-directed mutagenesis to construct mutability landscape and rationally-designed mutants

7.4.2 Library construction and quality control

7.5 Library preparation and screening

7.6 Steroid hydroxylation screening by HPLC

7.7 Molecular dynamics simulations

8 Summary

Chapter Nine: P450 in C-C coupling of cyclodipeptides with nucleobases

2 General instruction and cultivation of bacterial strains

2.1 General instruction

2.2 Media used for cultivation of bacterial strains

3 Genomic DNA isolation

3.4 Notes.

4 PCR amplification for P450 gene cloning.

Notes:

Includes bibliographical references.

Description based on print version record.

Description based on publisher supplied metadata and other sources.

ISBN:

0-443-13696-3

0-443-13695-5

OCLC:

1409751421