Use of highly active and/ or reusable biocatalyst designs for biotransformation

USE OF HIGHLY ACTIVE AND /OR REUSABLE BIOCATALYST DESIGNS FOR

BIOTRANSFORMATIONS

BENU ARORA (nee Monga)

DEPARTMENT OF CHEMISTRY

INDIAN INSTITUTE OF TECHNOLOGY DELHI

FEBRUARY 2016

©Indian Institute of Technology Delhi (IITD), New Delhi, 2016

USE OF HIGHLY ACTIVE AND /OR REUSABLE BIOCATALYST DESIGNS FOR

BIOTRANSFORMATIONS

by

BENU ARORA (nee Monga) DEPARTMENT OF CHEMISTRY

submitted

in fulfilment of the requirements of the degree of DOCTOR OF PHILOSOPHY

to the

INDIAN INSTITUTE OF TECHNOLOGY DELHI

FEBRUARY 2016

CERTIFICATE

This is to certify that the thesis entitled “Use of highly active and/or reusable biocatalyst designs for biotransformations” being submitted by Mrs. Benu Arora (nee Monga) to the Indian Institute of Technology Delhi for the award of the degree of Doctor of Philosophy in Chemistry is a record of bonafide researched work carried out by her. She has worked under our supervision, and has fulfilled the requirements for the submission of the thesis which, to our knowledge, has reached requisite standard.

The results contained in this dissertation have not been submitted in part or in full to any other University or Institute for the award of any degree or diploma.

Dr. S.K. Khare Dr. M. N. Gupta Professor Emeritus Professor

Department of Chemistry Department of Biochemical Engineering Indian Institute of Technology Delhi and Biotechnology

Indian Institute of Technology Delhi

Date:

New Delhi

ACKNOWLEDGEMENTS

First and foremost, I would like to express my deepest sense of gratitude to “THE ONE”

who could foresee this Ph. D. and inspired me to undertake this journey at a point when I was myself unsure of what to do. Although it never went as per my expectations, the unexpected twists and turns have probably been a greater learning experience.

I am extremely grateful to Prof. M.N. Gupta, my supervisor during the first five years of Ph.D., who is now formally my co-supervisor. His words were always full of wisdom, although sometimes I took long to understand the underlying meaning. His never ending enthusiasm for science and ability to persistently carry on with the work, without caring about others’ views are virtues that have always been an inspiration. Often the discussions we had turned out to be an interesting blend of both science and philosophy, and provided an entirely new perspective to problem solving. I am indebted for the continued support he extended during the period when I was going through a tough time both personally as well as in the lab.

I am also grateful to my supervisor, Prof. S.K. Khare for his guidance and support. His encouraging words always instilled a sense of confidence during the low moments.

A special thanks to Prof. P.S. Pandey for all the helpful discussions and suggestions for my research work. His in depth knowledge of organic chemistry has been extremely useful in shaping up this work.

I would also like to thank Head of the Chemistry Department, Prof. A.R. Ramanan for providing all the facilities. I also thank Prof. V. Haridas and Prof. P. Mishra for reviewing this work.

I am also thankful to all the staff members of the Chemistry Department especially the ones who take care of the Instrumentation lab and the NMR lab.

I would also thank all the fellow lab members of the biochemistry lab: Dr. Kusum Solanki who not only introduced me to the art of using enzymes for chemical reactions but also taught me not to be afraid of using them! I must admit that without any previous experience and as a result of a series of initial failed experiments, I had become sceptical about using them. Dr.

Manali Kapoor, who taught me how to use the GC and HPLC. The friendly discussions we

had over lunch and dinner in the hostel were so useful in taking the mind off the day’s failed experiments. Dr. Abir Majumder, for sharing his wealth of practical knowledge about carrying out organic reactions. I learnt most of the work up, purification procedures, and characterization of organic compounds from him. His help in changing the GC cylinders was always invaluable. Veena Ma’am, who was a smiling, friendly neighbour on the bench and shared her superb home cooked food on all those hot summer days when we skipped going to the hostel for lunch. Dr. Gulam Mohamad Rather, for being available for any and every kind of help. On many occasions, starting the reaction would not have been possible without his help in opening up the cap of the solvent bottle, which I alone failed to do many a time!

Joyeeta, with whom it was possible to discuss both work related as well as general issues.

Her feedback was always useful in reaching a better conclusion. Her efforts in taking care of the lab are invaluable. Sarah, Mandeep, Sonali, Aditi, Swati and Deepika for asking me curious questions that made me think deeper and apply better. I also thank Lokenderji, Anjaniji and Piar Chand for taking care of the lab so that we could focus on research work.

I thank my friends Richa, Premlatha, Prasanna and Bhavika for lending an ear to all that I had to say and making me comfortable during the times I felt homesick.

Last but not the least a word of thanks for my family. I thank my husband Raman, who supported and encouraged me whenever I felt dejected and wanted to quit. His criticisms on my shortcomings have enabled me to shape a better self. My grandmother Smt. Kartar Devi and parents-in –law Mr. S.L. Arora and Mrs. Sushma Arora for showing patience all these years when I was not able to completely fulfil my duties as a daughter-in-law. I also thank my brother-in-law Punit, sister-in-law Neha who are more of friends; my niece Tamanna whose queries and stories are a great stress buster. I am indebted to my parents Mr. Lalit Monga and Mrs. Madhu Monga for providing me the opportunity and facilities to focus on

academics right from childhood. I thank my brother Mohit, who inspite of being the younger one often guides and helps me in making choices. I feel sorry for the times when I was needed them but was absent due to this work. I thank God for blessing me with these people and giving me the chance of going through this wonderful experience.

Benu Arora (nee Monga)

Abstract

Use of enzymes as catalysts for synthetic reactions has expanded greatly in the last few decades. The phenomenon of enzyme promiscuity has further increased their application for white biotechnology. Of the various classes of enzymes, hydrolases (particularly lipases and proteases) are more often used for this purpose. This thesis explores the catalytic promiscuity of some high activity biocatalyst designs prepared from these enzymes for catalyzing organic reactions. This thesis is divided into six chapters.

Chapter 1, the introductory chapter reviews the relevant literature related to enzyme promiscuity and the organic reactions covered in this work.

Chapter 2 deals with enhancement of rates for hydrolase-catalyzed aldol reaction. Aldol reaction of p-nitrobenzaldehyde and acetone catalyzed by lipases has been reported to be a slow reaction; often extending over a period of 5-6 days to achieve > 90% conversion. In this chapter, a few simple approaches were used to speed up the same reaction. An initial

optimization of ratio of water: acetone present in the reaction medium revealed that a 1: 1 mixture gave maximum conversion. At very low acetone concentrations, two side products:

p-nitrobenzyl alcohol and p-nitrobenzoic acid were also formed (this reaction investigated in detail is described in the subsequent chapters). Effect addition of a few polar organic co- solvents to the reaction medium at varying concentrations was studied. DMSO at a concentration of 30% (v/v) w.r.t. the total reaction volume was found to be the best co- solvent. Screening of a number of commercially available lipases was then carried out. As a result of these optimizations, >90% conversion was attained after just 24 hours. The same reaction under the optimized conditions was then extended to the proteases. Finally, scope of this reaction was expanded by using various substituted benzaldehydes as substrates.

Chapter 3 describes the lipase catalyzed Cannizzaro-type reaction with substituted benzaldehydes in water. Novozyme 435 (immobilized Candida antarctica lipase B) was found to catalyze the disproportion of p-nitrobenzaldehyde to p-nitobenzyl alcohol and p- nitrobenzoic acid without addition of any external redox reagent. This reaction was found to occur in sodium phosphate buffer, pH 7.0 at 30 ⁰ C. However, the reaction followed an unusual pattern of formation of products. In the beginning alcohol was formed as the major product (around 60% conversion was obtained after 6 hours) while the acid appeared only in traces. Thereafter there was a decline in the amount of alcohol and a simultaneous increase in acid formation, with the two products becoming almost equal after 24 hours. Thereafter, the lipase catalyzed oxidation of the alcohol was found to convert all the alcohol to the acid.

Evaluation of the scope of this reaction was carried out by trying various substituted

benzaldehydes as substrates. The addition of organic solvents such as DMSO, acetonitrile and CPME to the reaction of p-nitrobenzaldehyde catalyzed by Novozyme 435 resulted in almost complete inhibition of the acid formation.

Chapter 4 deals with the reduction reaction of substituted benzaldehydes catalyzed by Novozyme 435. As discussed above, adding organic solvents to the lipase catalyzed

Cannizzaro-type reaction resulted in the exclusive formation of p-nitrobenzyl alcohol from p- nitrobenzaldehyde. To begin with, a wide range of organic solvents was screened to select the one that resulted in maximum conversion to the alcohol.

Cyclopentyl methyl ether was chosen for further work as this solvent gave maximum conversion to p-nitrobenzyl alcohol. CPME is well recognised as a green reaction solvent.

Thus the lipase catalyzed reduction reaction was carried out under environmentally friendly conditions. A stepwise optimization of the substrate concentration, enzyme concentration and reaction temperature was then carried out. Under the optimized conditions, it was possible to convert about 80% of p-nitrobenzaldehyde to p-nitobenzyl alcohol. Finally using the same

optimised reaction conditions, a range of substituted benzaldehydes were reduced to their corresponding benzyl alcohols in high yields.

Chapter 5 describes the lipase catalyzed reactions of phenylglyoxal in aqueous media.

Phenylglyoxal, an α-keto aldehyde is known to undergo intramolecular Cannizzaro reaction in the presence of chemical catalysts forming mandelic acid. In the present chapter the Novozyme 435 catalyzed reaction of phenylglyoxal was carried out in sodium phosphate buffer, pH 7.0. This reaction resulted in formation of mandelic acid along with

phenylglyoxylic acid. The latter was formed as the major product due to the enzyme catalyzed oxidation of phenylglyoxal under the reaction conditions.The influence of polar organic co-solvents on this reaction was also evaluated. Addition of co-solvents such as DMF, DMSO, acetonitrile and dioxane to the reaction medium was carried out. Interestingly, adding the co-solvents increased the amount of mandelic acid formed during the reaction as compared to that formed in totally aqueous condition. However, the oxidation product i.e.

phenylglyoxylic acid continued to be formed as the major product in all these cases as well.

Chapter 6 deals with condition promiscuity of lipases and its application to the synthesis of glucose esters. High activity biocatalyst designs of Candida antarctica lipase B were used to catalyze the transesterification reaction of D-glucose and vinyl acetate in low water media.

Acetylation of D-glucose by vinyl acetate using CAL-B produces both 6-O- acetyl-D-glucose as well as 3, 6-O-diacetyl-D-glucose, with varying degrees of regioselectivity depending on the reaction conditions. Cross-linked enzyme aggregates (CLEAs), protein coated

microcrystals (PCMCs) and cross-linked protein-coated microcrystals (CLPCMs) prepared from Candida antarctica lipase B were used in different solvents. In acetone as the medium, CLEA and CLPCMC gave better overall conversions than PCMC, but the reactions catalyzed by these were less regioselective. With CLPCMC and CLEA, initial rates of 366.1 and 353.5 µmol min^-1g^-1 were obtained respectively.

Next, mixed solvents were used as the reaction medium. Binary mixtures of t-amyl alcohol and DMSO (30% v/v) were initially chosen. The two biocatalyst designs: CLEA and CLPCMC which had performed better earlier were used here. Initial rates of glucose conversion increased to 3636.0 and 3333.0 µmol min^-1g^-1 using CLEA and CLPCMC respectively. A solvent premixing protocol was found to give still higher initial rates of glucose ester formation. Under these conditions initial rates of 7886.9 µmol min^-1g^-1 and 7447.5 µmol min^-1g^-1 were achieved using CLEA and CLPCMC. Overall, >90% conversion of glucose was attained within 1.5 hours of the reaction. Moreover, the monoacetate was formed regioselectively in these cases.

.

TABLE OF CONTENTS

CERTIFICATE i

ACKNOWLEDGEMENTS ii

ABSTRACT v

LIST OF CONTENTS ix

LIST OF FIGURES x

LIST OF TABLES xiii

ABBREVIATIONS AND SYMBOLS xvi

CHAPTER 1 Introduction 1-30 CHAPTER 2 Enhancing the catalytic activity of hydrolases for aldol 31-54

List of Figures

Figure No.

Figure Caption Page

No.

1.1 Creation of a pair of enantiomers during asymmetric aldol reaction 12 1.2 Possible mechanisms for the Cannizzaro reaction via H^-transfer and H ^.

transfer

17

1.3 Schematic representation of preparation of high activity biocatalysts 23

2.1 Hydrolase catalyzed aldol reaction of p-nitrobenzaldehyde and acetone 50 2.2 Influence of organic co-solvents on the lipase catalyzed aldol reaction of

p-nitrobenzaldehyde and acetone

51

2.3 Optimisation of the amount of water present in the reaction medium for alcalse catalyzed aldol reaction

52

2.4(a) HPLC analyses of the aldol reaction catalyzed by Lipase M in reaction media containing different acetone concentrations.

Reaction in medium composed of sodium phosphate buffer, pH 7.0 containing 0.5% (v/v) acetone at time 24 h.

53

2.4 (b) HPLC analyses of the aldol reaction catalyzed by Lipase M in reaction media containing different acetone concentrations.

Reaction in medium composed of sodium phosphate buffer, pH 7.0 containing 50% (v/v) acetone at time 24 h

54

3.1 Novozyme 435 catalyzed Cannizzaro-type reaction of p- nitrobenzaldehyde in water

75

3.2 Time course for the Novozyme 435 catalyzed Cannizzaro-type reaction of p-nitrobenzaldehyde

76

3.3 Novozyme 435 catalyzed oxidation of p-nitrobenzyl alcohol to p-nitrobenzoic acid

77

3.4 Time course for CALB catalyzed Cannizzaro-type reaction of p- nitrobenzaldehyde

78

3.5 Time course for CALA catalyzed Cannizzaro-type reaction of p- nitrobenzaldehyde

79

3.6 Time course for RMIM catalyzed Cannizzaro-type reaction of p- nitrobenzaldehyde

80

3.7 Time course for Palatase catalyzed Cannizzaro-type reaction of p- nitrobenzaldehyde

81

3.8 Effect of addition of co-organic solvents on the Cannizzaro-type reaction of p-nitrobenzaldehyde catalyzed by Novozyme 435.

82

3.9 Novozyme 435 catalyzed Cannizzaro-type reaction of p- nitrobenzaldehyde in presence of 100 ppm FeSO4

83

3.10 Novozyme 435 catalyzed Cannizzaro-type reaction of p-

nitrobenzaldehyde in presence of 10% (v/v) dioxane and 100 ppm FeSO4

84

4.1 Novozyme 435 catalyzed reduction of substituted benzaldehydes to the corresponding benzyl alcohols

95

4.2 Screening of various hydrolases for the reduction reaction of p-nitrobenzaldehyde

96

4.3 Optimization of the enzyme concentration for Novozyme 435 catalyzed reduction of p-nitrobenzaldehyde

97

4.4 HPLC analyses of Novozyme 435 catalyzed reduction reaction of p- nitrobenzaldehyde in CPME/buffer biphasic system. (a) at 1 hour (b) at 24h

98

5.1 Lipase catalyzed reaction of phenylglyoxal in aqueous medium giving phenylglyoxylic acid and mandelic acid

107

5.2 Time course for the Novozyme 435 catalyzed reaction of phenylglyoxal in water.

108

5.3 Effect of DMF (10% v/v) on the Novozyme 435 catalyzed reactions of phenylglyoxal

109

5.4 Effect of DMSO (10% v/v) on the Novozyme 435 catalyzed reactions of phenylglyoxal

110

5.5 Effect of acetonitrile (10% v/v) on the Novozyme 435 catalyzed reactions of phenylglyoxal

111

5.6 Effect of dioxane (10% v/v) on the Novozyme 435 catalyzed reactions of phenylglyoxal

112

6.1 CALB catalyzed acetylation reaction of D-glucose with vinyl acetate producing a mixture of monoacetate and diacetate

134

6.2 Effect of solvent hydrophobicity on the CALB catalyzed acetylation of D-glucose with vinyl acetate

135

6.3 Time course for the CALB CLEA catalyzed regioselective acetylation of D- glucose carried out using ‘solvent-premixing’ protocol

136

6.4 Time course for the CALB CLPCMC catalyzed regioselective acetylation of D-glucose carried out using ‘solvent-premixing’

137

protocol

6.5 Effect of microwave irradiation on CALB CLEA catalyzed acetylation of D-glucose

138

6.6 GC analysis of acetylation reaction of D-glucose catalyzed by CALB CLEA in t-amyl alcohol containing 30% DMSO at 30 min

139

LIST OF TABLES

Table No.

Table Caption Page

No.

1.1 Examples of synthetically useful applications of enzyme condition promiscuity

5

1.2 Some examples of enzymes showing substrate promiscuity 7 1.3 Applications of catalytic promiscuity in organic synthesis 9 1.4 Enzyme catalyzed regioselective sugar fatty acid ester synthesis 20 1.5 CLEAs as efficient biocatalysts for biotechnologically useful

transformations

26

2.1 Effect of variation of acetone concentration on the formation of 4-(4´- nitrophenyl)-4-hydroxy-2-butanone

39

2.2 Lipase catalyzed aldol reaction in presence of varying amounts of DMSO 40 2.3 Screening of lipases for the aldol reaction of p-nitrobenzaldehyde and

acetone

41

2.4 Effect of imidazole on the aldol reaction between p-nitrobenzaldehyde and acetone

42 2.5 Screening different amino-acids as co-catalysts for the aldol reaction of p-

nitrobenzaldehyde and acetone

43

2.6 Effect of variation of enzymes and reaction conditions on the aldol reaction of p- chlorobenzaldehyde and acetone

44

2.7 Aldol reaction of p-nitrobenzaldehyde and acetone catalyzed by alcalase under various conditions

45

2.8 Effect of DMSO concentration on the aldol reaction between p-nitrobenzaldehyde and acetone catalyzed by alcalase

46

2.9 Effect of variation of enzyme concentration on the alcalase catalyzed aldol reaction of p-nitrobenzaldehyde

47

2.10 Catalytic activity of some commercially available proteases for the aldol reaction of p-nitrobenzaldehyde and acetone

48

2.11 Protease catalyzed aldol reaction of various substituted benzaldehydes 49 3.1 Cannizzaro-type reaction of substituted benzaldehydes catalyzed by

Novozyme 435 in aqueous medium

66

3.2 Effect of concentration of Novozyme 435 added on the Cannizzaro-type reaction of p-nitrobenzaldehyde

67

3.3 Effect of concentration of Lipozyme CALB added on the Cannizzaro-type reaction of p-nitrobenzaldehyde

68

3.4 Effect of pH of the reaction medium on the course of Cannizzaro-type reaction of p-nitrobenzaldehyde

69

3.5 Reaction of p-nirobenzaldehyde catalyzed by Novozyme 435 in hydrophobic solvents

70

3.6 Effect of variation in the amount of added Dioxane on the Cannizzaro- type reaction of p-nitrobenzaldehyde

71

3.7 Addition of different concentrations of FeSO4 to the Novozyme 435 catalyzed Cannizzaro-type reaction of p-nitrobenzaldehye in presence of 10% (v/v) dioxane

72

3.8 Addition of different concentrations of Diphenylamineto the Novozyme 435 catalyzed Cannizzaro-type reaction of p-nitrobenzaldehye in presence of 10% (v/v) dioxane.

73

3.9 Time course for the lipase catalyzed, microwave assisted Cannizzaro-type reaction of p-nitrobenzaldehyde

74

4.1 Effect of organic solvents on the reduction reaction of p- nitrobenzaldehyde

91 4.2 Effect of increasing substrate concentration on Novozyme 435 catalyzed

reduction of p-nitrobenzaldehyde

92 4.3 Effect of temperature on the Novozyme 435 catalyzed reduction reaction

of p-nitrobenzaldehyde

93 4.4 Novozyme 435 catalyzed reduction reaction of various benzaldehydes 94 5.1 Effect of variation of DMF on the Novozyme 435 catalyzed reaction of

phenylglyoxal

105 5.2 Screening of a few lipases for the reaction of phenylglyoxal hydrate in

water

106 6.1 Organic solvents with different hydrophobicity that are commonly used as

reaction media for sugar ester synthesis

128 6.2 Acetylation reaction of D-glucose catalyzed by different biocatalyst

designs of CAL-B in acetone

129 6.3 Initial rates for the transesterification of D-glucose and vinyl acetate in

solvents of different polarity

130 6.4 Evaluation of CLEA and CLPCMC as biocatalyst designs for the

regioselective acetylation of D-glucose in t-amyl alcohol/ DMSO mixtures

131

6.5 Initial rates for glucose ester synthesis carried out using solvent- premixing

132 6.6 Effect of varying the acyl donor on the CALB CLEA catalyzed acylation

of D-glucose

133

List of Abbreviations & Symbols

Ala Alanine Arg Arginine CALA Candida antarctica lipase A CALB Candida antarctica lipase B

° C Degree celcius

CLEAs Cross- linked enzyme aggregates

CLPCMCs Cross-linked protein coated microcystals

CPME Cyclopentylmethyl ether

DCM Dichloromethane DME Dimethoxyethane

DMF N, N-Dimethylformamide

DMSO Dimethylsulphoxide

ee Enantiomeric excess

EPRP Enzyme precipitated and rinsed with propanol GA Glutaraldehyde

GC Gas Chromatography

h Hour His Histidine

HPLC High performance liquid chromatography Leu Leucine

Lipase M Mucor javanicus lipase

MCR Multi-component reaction

mM Millimolar

N435 Novozyme 435

NMR Nuclear magnetic resonance

PCMC Protein coated microcrystal

PPL Porcine pancreatic lipase

pNPP p-Nitrophenylpalmitate Pro Proline Ser Serine

THF Tetrahydrofuran TLL Thermomyces lanuginosus lipase Tyr Tyrosine

Val Valine