Using Esterase and Laccase Enzymes to Derivatize Bioactive Plant Phenolics for Altered Chemistry

189 Pages·2015·3.53 MB·English

Using Esterase and Laccase Enzymes to Derivatize Bioactive
Plant Phenolics for Altered Chemistry

by

Mohammed Sherif

A thesis submitted in conformity with the requirements
for the degree of Doctor of Philosophy

Department of Cell and Systems Biology
University of Toronto

If someone said
© Copyright by Mohammed Sherif 2015
Using Esterase and Laccase Enzymes to Derivatize Bioactive Plant
Phenolics for Altered Chemistry

Mohammed Sherif
Doctor of Philosophy
Department of Cell and Systems Biology
University of Toronto

2015
ABSTRACT
Plant phenolics have notable antioxidant activity and there is potential to improve their
action by chemical modification. Two enzyme classes carry out reactions that can act on the
hydroxyl moiety of phenolics. Esterase enzymes can be used in non-aqueous solvents to esterify
a long chain acyl group onto the phenolic compound. Laccase enzymes can be used to form
phenoxy radicals that can then couple to form larger molecular weight oligomers. Both
enzymatic modifications may produce a new antioxidant with altered chemistry.
One archaeal esterase (AF1753) from Archaeoglobus fulgidus and one bacterial esterase
(PP3645) from Pseudomonas putida were assayed for activity in organic solvents. Both
enzymes catalyzed hydrolysis of phenyl acetate and vinyl acetate in 98:2 (v/v) (t-amyl
alcohol):buffer; with continued activity up to 96 h of reaction. However, the enzymes were not
able to catalyze transesterification of 4’-hydroxyacetophenone with vinyl acetate in 9:1 (v/v)
cyclohexane:(t-amyl alcohol), which was not explained by enzyme inactivation during
lyophilization. Still, alanine scanning mutagenesis revealed that R37A substitution improved
activity of AF1753 on long-chain p-nitrophenyl (pNP) esters.
A multicopper oxidase (SCO6712) from Streptomyces coelicolor displayed activity on a
variety of phenolics including caffeic acid, ferulic acid, resveratrol, quercetin, morin,
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kaempferol and myricetin. Among the products formed by action on flavonols were dimers of
quercetin, morin, and myricetin. Quercetin and myricetin dimers showed longer retention time
on reversed phase chromatography. All three dimers could be detected by 5 min of reaction but
depleted by 3 h and 24 h. The TRAP and FRAP antioxidant activity of the whole reaction
mixture of modified quercetin, morin, and myricetin decreased, as starting phenolic was
depleted over 24 h. Accordingly, mass spectrometry was used to shed light on the molecular
structure of the dimers produced from quercetin and myricetin. In both cases, mass
spectrometric analyses ruled out dimer formation through the A ring of each monomer. For
myricetin, the most likely linkage structure was determined to be between either two B rings or
a B ring with a C ring. These predicted linkage positions are in agreement to those observed for
quercetin dimers previously extracted from natural plant sources.
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Acknowledgments

I would like to thank my supervisor Professor Emma Master for giving me the
opportunity to work on this project and providing guidance. I will also thank the members of my
Supervisory Committee, Professor Brad Saville and Professor Dinesh Christendat, for their
advice throughout the project. I thank collaborators who gave assistance on this project
including members of the group Structural Proteomics in Toronto (SPiT) in Toronto and
members from Natural Resources Canada (NRCan) in Sault Ste. Marie. BioZone administration
was helpful in making sure things ran smoothly. Colleagues in the Master lab helped with a lot
of theoretical and technical aspects of the project. I thank my family for their support over the
course of my PhD.
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Table of Contents
LIST OF TABLES ................................................................................................................... VIII
LIST OF FIGURES .................................................................................................................... IX
LIST OF ABBREVIATIONS .................................................................................................... XI
CHAPTER 1. OVERVIEW .......................................................................................................... 1
CHAPTER 2. LIERATURE SURVEY........................................................................................ 3
2.1. Plant phenolic compounds ................................................................................................... 3
2.1.1. Types and distribution .............................................................................................. 3
2.1.2. Biosynthesis and role in plants ................................................................................. 7
2.1.3. Health benefits of phenolics ................................................................................... 12
2.1.4. Examples of antioxidant activity ............................................................................ 12
2.1.5. Phenolic antioxidant activity for food preservation .............................................. 14
2.1.6. Structure-functional correlations among phenolics with antioxidant properties 15
2.1.7. In vitro measurement of antioxidant activity ......................................................... 17
2.1.8. Solubility considerations for antioxidant activity .................................................. 18
2.2. Derivatization of plant phenolics ....................................................................................... 20
2.2.1. Enzymatic strategies used in plant phenolic derivatization .................................. 20
2.2.2. Increasing hydrophilicity of phenolic compounds ................................................ 21
2.2.3. Increasing lipophilicity of phenolic compounds ................................................... 23
2.3. Esterases/lipases .................................................................................................................. 26
2.3.2. Structural features .................................................................................................. 27
2.3.3. Catalytic mechanism ............................................................................................... 29
2.3.4. Transesterification reactions .................................................................................. 30
2.3.5. Applied Use ............................................................................................................. 33
2.4. Laccases ............................................................................................................................... 34
2.4.2. Structural features .................................................................................................. 35
2.4.3. Catalytic mechanism ............................................................................................... 37
2.4.4. Effect of Redox potential ........................................................................................ 41
2.4.5. Applied use .............................................................................................................. 42
2.5. Research Hypotheses and Objectives................................................................................ 45
CHAPTER 3. CHARACTERIZATION OF SOLVENT-TOLERANT
CARBOXYLESTERASES WITH ARYLESTERASE ACTIVITY ....................................... 46
3.1. Introduction ........................................................................................................................ 47
3.2. Materials and methods ....................................................................................................... 49
3.2.1. Gene cloning and protein purification ................................................................... 49
3.2.2. Hydrolytic activity of esterases AF1753 and PP3645 in t-amyl alcohol/water
(98:2, v/v) ........................................................................................................................... 49
3.2.3. Transesterification activity of esterases AF1753 and PP3645 in t-amyl
alcohol/cyclohexane (1:9, v/v) .......................................................................................... 50
3.2.4. Protein structure modeling and site-directed mutagenesis of esterase AF1753 .. 51
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3.2.5. Activity of wild type and mutant AF1753 esterases on varying chain-length pNP
esters .................................................................................................................................. 51
3.3. Results and discussion ........................................................................................................ 52
3.3.1. Hydrolytic activity of esterases AF1753 and PP3645 in t-amyl alcohol/water
(98:2, v/v) ........................................................................................................................... 52
3.3.2. Transesterification activity of esterases AF1753 and PP3645 in t-amyl
alcohol/cyclohexane (1:9, v/v) .......................................................................................... 55
3.3.3. Activity of wild type and mutant AF1753 esterases on varying chain-length pNP
esters .................................................................................................................................. 61
3.4. Conclusions .......................................................................................................................... 66
CHAPTER 4. BIOCHEMICAL STUDIES OF THE MULTICOPPER OXIDASE
(SMALL LACCASE) FROM STREPTOMYCES COELICOLOR USING BIOACTIVE
PHYTOCHEMICALS AND SITE-DIRECTED MUTAGENESIS ........................................ 67
4.1. Introduction ........................................................................................................................ 68
4.2. Materials and methods ....................................................................................................... 69
4.2.1. Gene cloning and protein purification ................................................................... 69
4.2.2. Site-directed mutagenesis ....................................................................................... 70
4.2.3. Copper content analysis of wild-type and mutant SCO6712 laccases .................. 70
4.2.4. Substrate profile of wild-type SCO6712 and the Ser292Ala mutant laccases ...... 70
4.2.5. Kinetics of wild-type and Ser292Ala laccases on select substrates ....................... 72
4.2.6. Docking of 2,6-dimethoxyphenol substrate to wild type and Ser292Ala SCO6712
laccase ............................................................................................................................... 72
4.3. Results and discussion ........................................................................................................ 72
4.3.1. Effect of microaerobic cultivation on copper content and activity of SCO6712
laccase ............................................................................................................................... 72
4.3.2. Substrate profile of wild-type SCO6712 laccase .................................................... 73
4.3.3. Kinetics of wild-type SCO6712 laccase on select substrates ................................. 77
4.3.4. Site-directed mutagenesis ....................................................................................... 79
4.4. Conclusions .......................................................................................................................... 82
CHAPTER 5. CHARACTERIZATION OF PRODUCT FORMATION FROM
ENZYMATICALLY OXIDIZED PLANT PHENOLICS AND ASSAY OF
ANTIOXIDANT ACTIVITY ..................................................................................................... 83
5.1. Introduction ........................................................................................................................ 84
5.2. Materials and methods ....................................................................................................... 85
5.2.1. HPLC-MS analysis of flavonol products after SCO6712 laccase treatment ........ 85
5.2.2. HPLC-MS analysis of flavonol dimer presence over laccase reaction time......... 85
5.2.3. Total radical-trapping antioxidant parameter (TRAP) assay of whole laccase
reaction mixture ................................................................................................................ 85
5.2.4. Ferric reducing antioxidant power (FRAP) assay of whole laccase reaction
mixture .............................................................................................................................. 86
5.2.5. HPLC-MS/MS analysis of quercetin dimer and myricetin dimer......................... 86
5.3. Results and discussion ........................................................................................................ 87
5.3.1. HPLC-MS analysis of flavonol products after SCO6712 laccase treatment ........ 87
5.3.2. Antioxidant assay of whole laccase reaction mixture ......................................... 109
5.3.3. HPLC-MS/MS analysis of quercetin dimer and myricetin dimer....................... 120
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5.4. Conclusions ........................................................................................................................ 124
CHAPTER 6. DISCUSSION .................................................................................................... 125
CHAPTER 7. FUTURE RESEARCH ..................................................................................... 137
7.1. Further characterization of esterases for transesterification potential, continuing work
of Chapter 3 .............................................................................................................................. 137
7.2. Further assessment of biochemical potential of laccase SCO6712, continuing work of
Chapter 4 .................................................................................................................................. 138
7.3. Further examination of bioactivity of flavonol dimers, continuing work of Chapter 5
................................................................................................................................................... 139
REFERENCES .......................................................................................................................... 141
APPENDIX 1. SUPPLEMENTAL INFORMATION FOR CHAPTER 3 ......................... 159
APPENDIX 2. SUPPLEMENTAL INFORMATION FOR CHAPTER 4 ......................... 162
APPENDIX 3. SUPPLEMENTAL INFORMATION FOR CHAPTER 5 ......................... 163
APPENDIX 4. TRAP ANTIOXIDANT ASSAY USING LINOLEIC ACID IN PLACE OF
DCFH ........................................................................................................................................ 171
APPENDIX 5. QUANTIFYING SOLUBILITY OF QUERCETIN MONOMER FOR
COMPARISON TO QUERCETIN DIMER ......................................................................... 174
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LIST OF TABLES
Table 2.1. Different classes of plant phenolic compounds (Balasundram et al., 2006). .............. 3
Table 2.2. Major food sources of plant phenolic compounds (Manach et al., 2004). .................. 6
Table 2.3. Tree/shrub sources of plant phenolic compounds. ....................................................... 6
Table 2.4. Popular antioxidant activity assays. ........................................................................... 17
Table 4.1. Kinetic parameters of wild type SCO6712 and the Ser292Ala variant enzyme. ....... 78
Table 5.1. HPLC-MS data for quercetin, morin, myricetin, and their dimers produced after
enzymatic reaction. ...................................................................................................................... 88
Table 5.2. HPLC-MS data for representative intermediate molecular weight products present in
late time point enzymatic reactions of quercetin, morin, and myricetin. .................................... 99
Table 5.3. Top 10 products from late time point enzymatic reactions of quercetin, morin, and
myricetin. ................................................................................................................................... 100
Table 5.4. Top 10 products from 24 h reaction of quercetin without and with laccase enzyme.
................................................................................................................................................... 105

Table A1.1. Sequences of primers used for construction of esterase AF1753 point mutants. . 159
Table A2.1. Sequences of primers used for construction of laccase SCO6712 point mutants. 162
Table A3.1. HPLC-MS data for kaempferol and product produced after enzymatic reaction. 163
Table A3.2. Fragment ions observed for quercetin dimer #1 after HPLC-MS/MS. ................. 163
Table A3.3. Fragment ions observed for myricetin dimer #2 after HPLC-MS/MS. ................ 164
Table A3.4. Fragment ions observed for non-enzymatically produced quercetin dimer after
HPLC-MS/MS. .......................................................................................................................... 166
Table A3.5. Fragment ions observed for non-enzymatically produced myricetin dimer after
HPLC-MS/MS. .......................................................................................................................... 167

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LIST OF FIGURES
Figure 2.1. Structures of some members of A) hydroxybenzoic acids, B) hydroxycinnamic
acids, and C) stilbenes; and D) basic skeleton of all flavonoids, and specifically flavones and
flavonols. ....................................................................................................................................... 5
Figure 2.2. Key steps in biosynthesis pathways for production of hydroxybenzoic acids and
hydroxycinnamic acids. ............................................................................................................... 10
Figure 2.3. Key steps in biosynthesis pathway for production of stilbenes. .............................. 11
Figure 2.4. Key steps in the biosynthesis pathway for production of flavones and flavonols. .. 11
Figure 2.5. Reactions involved in scavenging of peroxyl radicals by a phenolic antioxidant. ... 13
Figure 2.6. Structures of some classes of flavonoids. ................................................................. 16
Figure 2.7. Possible routes to adding long-chain alkyl groups to phenolic compounds to
increase lipophilicity of the phenolic. .......................................................................................... 24
Figure 2.8. Naturally occurring dimers (n=1), trimers (n=2), and tetramers (n=3) made of
successive epicatechin molecules linked to catechin................................................................... 25
Figure 2.9. Reaction catalyzed by esterases and lipases. ............................................................ 27
Figure 2.10. Prototypical α/β hydrolase fold structure. .............................................................. 28
Figure 2.11. Catalytic mechanism of esterases and lipases. ....................................................... 31
Figure 2.12. Reaction catalyzed by laccases............................................................................... 35
Figure 2.13. Structure of laccase TvL from Trametes versicolor. A) One of the cupredoxin
domains of TvL (the protein has three such domains). ............................................................... 37
Figure 2.14. Proposed catalytic mechanisms of oxidation by laccase enzymes. ........................ 40
Figure 2.15. Structures of closely related phenols. ..................................................................... 41
Figure 3.1. Known sites of action of feruloyl esterases and arylesterases.................................. 48
Figure 3.2. Hydrolysis reactions of A) vinyl acetate and B) phenyl acetate carried out in t-amyl
alcohol/water (98:2, v/v) using esterases AF1753 and PP3645. ................................................. 54
Figure 3.3. Transesterification reaction of vinyl acetate and 4’-hydroxyacetophenone carried out
in different solvent mixtures of t-amyl alcohol/co-solvent (1:9, v/v) using commercial lipase PS
(from Amano). ............................................................................................................................. 57
Figure 3.4. Transesterification reaction of vinyl acetate and 4’-hydroxyacetophenone carried out
in t-amyl alcohol/cyclohexane (1:9, v/v) using recombinant esterase AF1753 and PP3645. ..... 58
Figure 3.5. Hydrolysis reaction of ester substrate in transesterification reaction-mix. .............. 59
Figure 3.6. Activity of wild type and mutants of AF1753 on pNP substrates. ........................... 63
Figure 3.7. Images of predicted protein structure of AF1753. ................................................... 65
Figure 4.1. Substrate selectivity of SCO6712. ........................................................................... 76
Figure 4.2. Chemical structures of natural bioactive phenolic substrates. ................................. 77
Figure 4.3. Ribbon image of 2,6-dimethoxyphenol (2,6-DMP) docked in silico to binding
pocket of SCO6712 A) wild type enzyme and B) Ser292Ala mutant. ........................................ 81
Figure 5.1. HPLC-MS chromatograms and m/z spectra for 20 min reaction samples with laccase
enzyme for A) quercetin, B) morin, and C) myricetin. ............................................................... 91
Figure 5.2. HPLC-MS chromatogram and m/z spectrum for 20 min reaction sample with laccase
enzyme for kaempferol. ............................................................................................................... 91
Figure 5.3. HPLC-MS chromatograms for 5 min reaction sample with laccase enzyme for (A)
quercetin, (B) morin, and (C) myricetin. ..................................................................................... 94
Figure 5.4. Mass spectrum for myricetin dimer of m/z 635 (from peak D3 in chromatogram of
Fig. 5.3. C); refer to Table 5.1) for 5 min reaction sample with laccase enzyme. ..................... 94
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Figure 5.5. HPLC-MS analysis of flavonol dimers from A) quercetin (dimer #1), B) morin, C)
myricetin (dimer #2), and D) myricetin (dimer #3). .................................................................... 98
Figure 5.6. Proposed reaction scheme for production of some of the quercetin oxidation
degradation products. ................................................................................................................. 104
Figure 5.7. Structures of the isomers quercetin and morin. ...................................................... 108
Figure 5.8. TRAP antioxidant activity of laccase reaction-mixes from A) quercetin, B) morin,
and C) myricetin reactions. ........................................................................................................ 113
Figure 5.9. TRAP antioxidant activity of laccase reaction-mixes from A) quercetin, B) morin,
and C) myricetin reactions. ........................................................................................................ 117
Figure 5.10. FRAP antioxidant activity of laccase reaction-mixes from A) quercetin, B) morin,
and C) myricetin reactions. ........................................................................................................ 119
Figure 5.11. Fragmentation patterns of flavonols in positive ion mode tandem mass
spectrometry. ............................................................................................................................. 121
Figure 5.12. Proposed fragment structures of quercetin dimer #1 (refer Table 5.1) after tandem
mass spectrometry. .................................................................................................................... 122
Figure 5.13. Proposed fragment structures of myricetin dimer #2 (refer Table 5.1) after tandem
mass spectrometry. .................................................................................................................... 123
Figure 6.1. Two possible mechanisms of quercetin dimer formation. ..................................... 130
Figure 6.2. Mechanism of antioxidant antagonism proposed by Peyrat-Maillard et al. (2003).
................................................................................................................................................... 133
Figure 6.3. Mechanism of antioxidant antagonism that involves reaction of quercetin radicals
with laccase-generated quercetin products. ............................................................................... 135

Figure A1.1. Protein purification of AF1753 mutants. ............................................................. 161
Figure A3.2. Remaining phenolic monomer in laccase reactions, as measured by UV-Vis
spectrophotometry and by intensity of HPLC-MS peak............................................................ 170
Figure A5.1. The maximum amount of quercetin that can be dissolved in A) 50 mM sodium
phosphate buffer (pH 7.4) with 0.1 M NaCl and B) 1-octanol. ................................................. 177

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