Enzyme Reprogramming.

Format:

Book

Author/Creator:

Yang, Yang.

Contributor:

Yang Yang

Series:

Methods in Enzymology Series

Methods in Enzymology Series ; v.Volume 720

Language:

English

Subjects (All):

Biocatalysis.

Protein engineering.

Physical Description:

1 online resource (261 pages)

Edition:

1st ed.

Place of Publication:

Chantilly : Elsevier Science & Technology, 2025.

Summary:

Enzyme Reprogramming, Volume 719 in the Methods in Enzymology series, highlights new advances in the field, with this new volume presenting interesting chapters on topics such as Practical Considerations for Reaction Development with Vanadium-Dependent Haloperoxidases, Characterization and Engineering of thermostable PLP-dependent alpha-oxoamine.

Contents:

Front Cover

Series Page

Methods in Enzymology

Copyright

Contents

Contributors

Chapter One: Biaryl coupling reactions catalyzed by cytochrome P450s

1 Introduction

2 Reaction optimization

2.1 Substrate screen

2.2 Cytochrome P450 selection and enzyme engineering

2.3 Scope

3 Protocol

3.1 Equipment

3.2 Bacterial media and buffer

3.3 Procedures

3.3.1 Resting cell assay in 96-deep well plates

3.3.2 Bioconversion assay in flasks

3.3.3 Scalable production in fermenters

3.3.4 Purification

4 Summary and conclusions

Acknowledgments

References

Chapter Two: A metallophotoredox strategy for biocatalytic radical C-N3 and C-SCN bond formatio

2 Constructing site-saturation mutagenesis screening library

2.1 Equipment

2.2 Materials and reagents

2.3 Sequences of template and primers

2.4 Cloning and mutagenesis

3 Library screening

3.2 Materials and reagents

3.3 Protein expression in 96-well plate

3.4 Set up whole-cell screening reactions in 96-well plate

3.5 Reaction workup

4 Small-scale biocatalytic transformation

4.1 Equipment

4.2 Materials and reagents

4.3 Protein purification

4.4 Analytic-scale reactions

4.5 Preparative-scale reactions

5 Summary

Chapter Three: Efficient transferase engineering for SAM analog synthesis using combinatorial library design and high-throughput LC/MS screening

2 General methods

3 Combinatorial library design and construction

3.2 Reagents

3.3 Procedure

3.4 Notes

4 Enzyme overproduction in 96-deep well plates

4.2 Reagents

4.3 Procedure

4.4 Notes

5 Library screening using high-throughput LC/MS

5.1 Equipment

5.2 Reagents

5.3 Procedure

5.4 Notes.

6 Rescreening and characterization of the most active enzyme variants

6.1 Equipment

6.2 Reagents

6.3 Procedure

6.3.1 Purification of selected hits

6.3.2 Rescreening of top 3 hits per substrate

6.3.3 Initial rate experiment

6.3.4 Determination of kcat/KM

6.3.5 Determination of diastereoselectivity

6.4 Notes

7 Conclusion

Chapter Four: Old yellow enzyme-catalyzed desymmetrizing desaturation

2 Cloning for a site-saturation mutagenesis screening library

2.2 Materials

2.3 Protocols

2.4 Notes

3 High-throughput experimentation in 96-well plates

3.2 Materials

3.3 Protocols

4 Analytical scale reactions

4.2 Materials

4.3 Protocols

5 Preparative scale reactions

5.2 Materials

5.3 Protocols

5.4 Notes

Summary

Acknowledgement

Chapter Five: Characterisation and engineering of thermostable PLP-dependent alpha-oxoamine synthases (AOSs)

versatile C-C bond forming biocatalysts

2 Recombinant enzyme expression and purification

2.2 Reagents and consumables

2.3 Protocol for expression

2.4 Ni IMAC purification of WT ThAOS

2.5 Alternate heat precipitation purification of WT ThAOS

3 Activity assays

3.2 Reagents and consumables

3.3 UV-Vis spectroscopy

3.4 Colorimetric DTNB activity assay

3.4.1 Amino acid screen

3.4.2 Enzyme kinetics

3.5 Note

4 Synthesis of pyrroles by the coupled ThAOS/KPR cascade

4.2 Reagents and consumables

4.3 Formation of pyrroles (1-20) using MAA as the KPR reagent (including HPLC analysis)

4.4 Formation of pyrrole product in large scale

4.5 Formation of pyrrole (1) by a biocatalytic cofactor recycling system.

5 Rational engineering of ThAOS for an expanded substrate scope

6 Saturation mutagenesis at ThAOS V79

6.1 Expanded amino acid substrate scope of ThAOS variants

6.1.1 Expanded acyl-CoA substrate scope of ThAOS variants

6.2 2ThAOS variants accepts acetyl-SNAc thioester as a substitute for acyl-CoA

6.3 Increased thermostability of ThAOS variants

6.4 Large scale chemoenzymatic synthesis of pyrrole-1 using acetyl SNAc

7 Summary and conclusions

Chapter Six: Engineering a PLP-dependent Mannichase

2 Equipment and materials

3 Protocols

3.1 Constructing mutant library of LolT by site-saturation mutagenesis

3.1.1 Designing primers for mutant library preparation

3.1.2 PCR amplification and purification

3.1.3 Homemade electrocompetent E. coli strain E. cloni BL21(DE3) cells

3.1.4 Gibson assembly and transformation

3.2 Primary screening to identify LolT variants with enhanced Mannichase activity

3.3 Secondary screening in a 24-well plate to confirm the positive hits

3.4 Biochemical characterization of purified LolT variants

4 Summary

Acknowledgements

Chapter Seven: Repurposing branched chain amino acid aminotransferase in the chemoenzymatic synthesis of azacyclic noncanonical amino acids

2 Co-expression of MtIlvE and GroES/EL in E. coli BL21(DE3) cells

2.1 Materials

2.2 Procedure

3 Chemoenzymatic azacyclic ncAA synthesis with MtIlvE

3.1 Materials and equipment

3.2 Procedure

3.2.1 Prepare MtIlvE stock solution

3.2.2 One-pot chemoenzymatic synthesis of (2S,5S)−5-methyl L-proline with MtIlvE and metal-catalyzed hydrogenation from 2,5-dioxohexanoic acid

3.2.3 Product characterization.

3.2.4 One-pot chemoenzymatic synthesis of (2S,5S)−5-phenyl L-pipecolic acid with MtIlvE and metal catalyzed hydrogenation from ethyl 2,6-dioxo-6-phenylhexanoate

3.2.5 Product characterization

Chapter Eight: Threonine aldolase-catalyzed stereoselective radical α-alkylation of amino acids

2 Materials

2.1 Cloning

2.2 Enzyme expression in E. coli

2.3 Cell-free lysate reaction

2.4 α-Benzylation with threonine aldolase variants

2.5 Reverse-phase HPLC analysis

2.6 Enantiomeric ratio determination using a modified Marfey's method

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 Reverse-phase HPLC calibration curves

3.5 Marfey's derivatization for enantiomeric ratio determination

Chapter Nine: Repurposing thiamine-dependent enzymes for radical biocatalysis

2.2 Expression, purification and concentration determination of enzymes

2.3 Photobiocatalytic reactions

2.4 GC-MS and normal phase HPLC analysis

3.1 Cloning

3.2 Expression, purification and concentration determination of enzymes

3.3 Photobiocatalytic reactions

Chapter Ten: Photobiocatalytic enantioselective C(sp³)-H acylation using thiamine-dependent enzymes

2 Benzaldehyde lyase (PfBAL): Structure, mechanism, and catalytic versatility

3 Expression and purification of PfBAL

3.1 Materials

3.1.1 Strains and plasmids

3.2 Buffers and reagents

3.3 Equipment

4 Step-by-step method details

4.1 Protein expression

4.2 Cell harvesting and lysis

5 Protein purification.

5.1 Immobilized metal affinity chromatography (IMAC)

5.2 Buffer exchange and quantification

6 General considerations

7 Enzymatic reaction setup and product analysis

7.1 Materials and equipment

7.1.1 Buffers and reagents

7.2 Equipment

8 Step-by-step method details

8.1 Analytical-scale enzymatic reactions

8.2 Preparative-scale reactions

8.3 Product extraction and GC-FID analysis of analytical-scale reactions

8.4 Stereoselectivity and quantification of analytical-scale reactions

9 General considerations

10 Summary and conclusions

Chapter Eleven: Genetically encoded artificial photoenzymes for enantioselective [2 + 2] photocycloadditions

2 Construction of mutant libraries

2.3 Protocol for SDM and SSM

2.4 Protocol for epPCR

2.5 Protocol for Gibson assembly

3 Expression and preparation of photoenzymes

3.3 Protocol for protein expression in 48-well plate

3.4 Protocol for protein expression as preparative scale

3.5 Protocol for preparation of crude photoenzyme

3.6 Protocol for purification of photoenzymes

4 Photoenzymatic reaction setup

4.3 Protocol for high-throughput enzymatic reactions in 96-well plates

4.4 Protocol for enzymatic reactions in analytical-scale format

4.5 Protocol for enzymatic reactions in scale-up format

5 Screening photoenzyme variants containing beneficial mutations

5.2 Materials and reagents

5.3 Protocol for construction of HPLC calibration curves

5.4 Protocol for screening variants using HPLC analysis

6 Summary

Back Cover.

Notes:

Description based on publisher supplied metadata and other sources.

Part of the metadata in this record was created by AI, based on the text of the resource.

ISBN:

0-443-41760-1

0-443-41759-8

9780443417603

OCLC:

1547927863