UNIVERSIDADE FEDERAL DO CEARÁ CENTRO DE TECNOLOGIA
DEPARTAMENTO DE ENGENHARIA QUÍMICA
PROGRAMA DE PÓS-GRADUAÇÃO EM ENGENHARIA QUÍMICA
RAYANNE MENDES BEZERRA
BIOCATALYSTS COMPOSED BY LIPASE FROM THERMOMYCES
LANUGINOSUS IMMOBILIZED ON SUPERPARAMAGNETIC NANOPARTICLES: DESIGN AND APPLICATION OF LIPASE-NANOPARTICLE BIOCATALYSTS IN THE SYNTHESIS OF COMPOUNDS USED IN DIFFERENT INDUSTRIAL FIELDS
RAYANNE MENDES BEZERRA
BIOCATALYSTS COMPOSED BY LIPASE FROM THERMOMYCES LANUGINOSUS IMMOBILIZED ON SUPERPARAMAGNETIC NANOPARTICLES: DESIGN AND APPLICATION OF LIPASE-NANOPARTICLE BIOCATALYSTS IN THE SYNTHESIS OF
COMPOUNDS USED IN DIFFERENT INDUSTRIAL FIELDS
Dissertação apresentada ao Programa de Pós-Graduação em Engenharia Química da Universidade Federal do Ceará, como requisito parcial à obtenção do título de Mestre em Engenharia Química. Área de concentração: Processos Químicos e Bioquímicos.
Orientador: Prof. Dr. José Cleiton Sousa dos Santos.
Coorientadora: Profa. Dra. Luciana Rocha
Barros Gonçalves.
Dados Internacionais de Catalogação na Publicação Universidade Federal do Ceará
Biblioteca Universitária
Gerada automaticamente pelo módulo Catalog, mediante os dados fornecidos pelo(a) autor(a)
B469b Bezerra, Rayanne Mendes.
Biocatalysts composed by lipase from Thermomyces lanuginosus immobilized on superparamagnetic nanoparticles : Design and application of lipase-nanoparticle biocatalysts in the synthesis of compounds used in different industrial fields / Rayanne Mendes Bezerra. – 2018.
123 f. : il. color.
Dissertação (mestrado) – Universidade Federal do Ceará, Centro de Tecnologia, Programa de Pós-Graduação em Engenharia Química, Fortaleza, 2018.
Orientação: Prof. Dr. José Cleiton Sousa dos Santos. Coorientação: Profa. Dra. Luciana Rocha Barros Gonçalves.
1. Biocatalisador. 2. Nanopartículas superparamagnéticas de magnetita. 3. Lipase de Thermomyces lanuginosus. 4. Catálise heterogênea. 5. Síntese enzimática. I. Título.
RAYANNE MENDES BEZERRA
BIOCATALYSTS COMPOSED BY LIPASE FROM THERMOMYCES LANUGINOSUS IMMOBILIZED ON SUPERPARAMAGNETIC NANOPARTICLES: DESIGN AND APPLICATION OF LIPASE-NANOPARTICLE BIOCATALYSTS IN THE SYNTHESIS OF
COMPOUNDS USED IN DIFFERENT INDUSTRIAL FIELDS
Master’s Thesis presented to the postgraduate program in Chemical Engineering of the Federal University of Ceará, as a partial requirement to obtain a Master's degree in Chemical Engineering. Concentration area: Chemical and biochemical processes.
Passed on February 19th, 2018.
EXAMINATION BOARD
PROF. DR. JOSÉ CLEITON SOUSA DOS SANTOS (Advisor)
Universidade da Integração Internacional da Lusofonia Afro-Brasileira (UNILAB)
PROF. DR. PIERRE BASÍLIO ALMEIDA FECHINE Universidade Federal do Ceará (UFC)
PROFA. DRA. MARIA CRISTIANE MARTINS DE SOUZA
To God.
ACKNOWLEDGMENTS
To God, for your love and mercy so present in my life, for the wisdom granted so that I could get so far, for the motivation and perseverance in carrying out this work.
To my parents, sister and brother, for always being the support system I needed, for the trust that they have placed in me, for the attention, for the encouragement, for the understanding, for always being present, for all love, for helping me become who I am today.
To my family; especially to my grandparents: Damiana, Raimunda, Francisco and Luís; for always believing in my abilities and for unconditional love.
To Anthony, for supporting me no matter what, for making me happy, for loving me during my good and my bad times.
To Profa. Dra. Luciana Rocha Barros Gonçalves and to Prof. Dr. José Cleiton Sousa dos Santos, for their confidence in my work, for all the teachings and excellent advices, for the achievements we share in and for the great contribution in my personal and professional growth.
To GPBio (Grupo de Pesquisa e Desenvolvimento de Processos Biotecnológicos), teachers and students, for your support in carrying out this work, for harmony, for making work relaxed and enjoyable, for the advices and for the friendship; specially to Nathalia, Magno, Maísa, Eva, Bruna, Kímberle, Guilherme, Ítalo, Tiago, Juliana, Layanne and Fernando.
To GQMat (Grupo de Química de Materiais Avançados) and to Labs (Laboratório de Biotecnologia e Síntese Orgânica), for the shared knowledge, for your cooperation with us and for the excellent work done by the following collaborators: Davino Andrade, Wesley Galvão, Pierre Fechine, Ana Caroline Carvalho and Marcos Mattos.
To the professors of the Chemical Engineering Department (DEQ) of the Universidade Federal do Ceará (UFC), for the knowledge obtained from the undergraduate to the master's degree; and to the collaborators, Luíz, Jorjão and Danilo.
To Fundação Cearense de Apoio ao Desenvolvimento Científico e Tecnológico (FUNCAP), a Brazilian agency for scientific and technological development, for the financial support.
To the examination board; composed by Professors Maria Cristiane Martins de Souza, Pierre Basílio Almeida Fechine and Marcos Carlos de Mattos; for the time invested in the analysis of my work, for the valuable collaborations and suggestions.
“Our lives begin to end the day we become silent about things that matter”
RESUMO
Lipase de Thermomyces lanuginosus (TLL) foi imobilizada em nanopartículas superparamagnéticas (SPMN) de magnetita (Fe3O4) por diferentes métodos. Inicialmente, as nanopartículas foram sintetizadas pelo método de coprecipitação. Em seguida, a superfície dessas nanopartículas foi funcionalizada com polímeros: 3-aminopropiltrietoxisilano (APTES) ou polietilenoimina ramificada (PEI). Assim sendo, a imobilização da enzima ocorreu de duas formas: por adsorção ou por ligação covalente. Na adsorção, a enzima foi imobilizada por pseudo-afinidade aos polímeros ligados à superfície das SPMN. Antes da imobilização por ligação covalente, o suporte passou por uma etapa de ativação com glutaraldeído (GA) ou com divinilsulfona (DVS). Naturalmente, as lipases catalisam as reações de hidrólise. Logo, os biocatalisadores desenvolvidos foram aplicados em algumas reações de hidrólise de interesse econômico, como na síntese do intermediário de um fármaco a partir da resolução cinética de um racemato, obtendo a máxima conversão (50 %) mesmo após o segundo ciclo reacional. Entretanto, em meios orgânicos, as lipases são capazes de catalisar as reações de esterificação; assim sendo, os biocatalisadores obtidos após a ativação com DVS foram aplicados na reação de esterificação do álcool benzílico, obtendo até 61 % de conversão após o sétimo ciclo reacional. Além disso, os vários derivados foram caracterizados por diferentes análises: difração de raios X, magnetometria de amostra vibrante, espectroscopia vibracional no infravermelha, potencial zeta, entre outras. As SPMN apresentaram comportamento superparamagnético mesmo após os diferentes processos de imobilização enzimática. As condições de trabalho dos biocatalizadores também foram definidas através dos estudos de estabilidades térmica, em solvente orgânico, de estocagem e operacional. Os derivados ativados com DVS tiveram ótima estabilidade operacional, apresentando mais de 50% da atividade catalítica inicial mesmo após dez ciclos reacionais. Por outro lado, os derivados ativados com GA apresentaram maior estabilidade térmica, tendo alcançado mais de 7 horas de tempo de meia-vida em meio de pH 7.0 à 70 °C. De modo geral, esses biocatalisadores são termoestáveis, apresentam alta eficiência catalítica e boa durabilidade operacional quando aplicados dentro das condições ideais de reação. Ademais, as reações de interesse econômico realizadas neste trabalho mostraram o potencial de aplicação dos biocatalisadores desenvolvidos nas indústrias farmacêutica, cosmética, de biotecnologia e de química fina.
ABSTRACT
Lipase from Thermomyces lanuginosus (TLL) was immobilized on superparamagnetic iron oxide (Fe3O4) nanoparticles (SPMN) by different methods. Initially, the nanoparticles were prepared by the coprecipitation method. Then, the surface of magnetite was functionalized with polymers: 3-aminopropyltriethoxysilane (APTES) or branched polyethylenimine (PEI). Therefore, the enzyme immobilization was performed by two ways: by adsorption or by covalent attachment. In the adsorption way, the enzyme was immobilized by ionic affinity to the polymers attached to the SPMN surface. Previously the immobilization by covalent attachment, the support was activated with glutaraldehyde (GA) or divinylsulfone (DVS). Naturally, lipases catalyze the hydrolysis reactions. Thus, the developed biocatalysts were applied in some hydrolysis reactions of economic interest, for example, in the synthesis of a medicament precursor from the kinetic resolution of a racemate, obtaining the maximum conversion (50%) even after the second reaction cycle. However, lipases are able to catalyze esterification reactions in organic media; thus, the biocatalysts obtained after activation with DVS were applied in the esterification reaction of benzyl alcohol, obtaining up to 61% conversion after the seventh reaction cycle. Moreover, the preparations were characterized by different analysis: X-ray diffraction, vibrating sample magnetometry, infrared vibrational spectroscopy, zeta potential, among others. The SPMN presented superparamagnetic behavior even after the different enzymatic immobilization processes. The application conditions of the biocatalysts were also defined through studies of thermal, organic solvent, storage and operational stabilities. The DVS preparations had excellent operational stability, exhibiting more than 50% of the initial catalytic activity even after ten reaction cycles. On the other hand, the GA preparations presented greater thermal stability, reaching more than 7 hours of half-life at pH 7.0 at 70 °C. In general, these biocatalysts are thermostable, have high catalytic efficiency and good operational durability when applied under ideal reaction conditions. In addition, the economic interest reactions carried out in this work showed the potential applications of the developed biocatalysts in the pharmaceutical, cosmetic, biotechnology and fine chemical industries.
LIST OF FIGURES
Figure 2.1 3D structure of the TLL. The catalytic triad consists of amino acid residues: Serine 146 (red), Histidine 201 (yellow) and Aspartate 258 (blue). Lysine residues in green. Protein structure was obtained from the Protein Data Bank (PDB code 1GT6) and drawn using Pymol version 0.99 (DeLano Scientific LLC, Philadelphia, PA, EUA)... 29 Figure 2.2 Enzymatic immobilization in magnetite superparamagnetic nanoparticles... 33 Figure 2.3 Magnetic manipulation with superparamagnetic nanoparticles. (A) SPMN in
solution (B) SPMN in the presence of an external magnetic field………. 34 Figure 2.4 SPMN coated with PEI (this illustration shows only one of the possible
configurations of the coating)……..………. 37 Figure 2.5 SPMN coated with APTES (this illustration shows only one of the possible
configurations of the coating)..………. 38 Figure 2.6 Chemical structure of glutaraldehyde………... 39 Figure 2.7 Chemical structure of divinyl sulfone……….. 39
Figure 3.1 The immobilization procedures of TLL by ionic exchange or by covalent attachment in SPMN recovered with PEI or APTES……… 57 Figure 3.2 XRPD results for the investigated samples. Here, the blue line represents the
relative difference between experimental (YObs, black dots) and calculated (YCalc, red line) intensities obtained through the refinement………...… 63 Figure 3.3 Magnetization curve at room temperature for the studied samples…...………. 64 Figure 3.4 FT-IR spectrum of the samples………...………... 65 Figure 3.5 SDS-PAGE analysis of different TLL preparations. Lane 1: molecular weight
markers (values in kDa), Lane 2: supernatant of the SPMN@APTES-GA-TLL, Lane 3: supernatant of the SPMN@APTES-GA-TLL after reaction cycles, Lane 4: supernatant of the SPMN@APTES-TLL, Lane 5: supernatant of the SPMN@PEI-GA-TLL, Lane 6: supernatant of the SPMN@PEI-GA-TLL after reaction cycles, Lane 7: supernatant of the SPMN@PEI-TLL. (Freeware 1D gel electrophoresis image analysis software GelAnalyzer)……….…………....……….. 67 Figure 3.6 (A) Synthesis reaction of rac-1-methyl-2-(2,6-dimethylphenoxy)ethyl acetate.
(B) Hydrolysis reaction of rac-1-methyl-2-(2,6-dimethylphenoxy)ethyl acetate. 70 Figure 3.7 (A) GC chromatograms of rac- 1-(2,6-dimethylphenoxy)propan-2-ol tR 62,3
Figure 3.8 Operational stability (recycle) of the biocatalysts by hydrolysis of p-NPB (50
mM) at 25 °C. Other specifications are described in Methods section……..…… 74
Figure 4.1 Activation of SPMN@PEI with DVS……….. 87
Figure 4.2 TLL immobilization on SPMN@PEI-DVS... 87
Figure 4.3 Incubation of the biocatalysts at pH 10.0 and blocking of the remaining vinyl sulfone reactive groups……… 88
Figure 4.4 Hydrolysis reaction of ethyl hexanoate……… 91
Figure 4.5 Hydrolysis reaction of (R)- and (S)-methyl mandelate………. 92
Figure 4.6 Transesterification reaction of benzyl alcohol……….. 93
Figure 4.7 Effect of the immobilization pH on the immobilization courses of TLL on SPMN@PEI-DVS. Experiments have been performed at 25 °C, using 25 mM of the different buffers. Other specifications are described in Methods section.... 94
Figure 4.8 SDS-PAGE analysis of different TLL preparations. Lane 1: molecular weight markers (values in kDa), Lane 2: free TLL, Lane 3: SPMN@PEI-DVS-TLL (pH 5), Lane 4: SPMN@PEI-DVS-TLL (pH 5) after incubation at pH 10.0, Lane 5: SPMN@PEI-DVS-TLL (pH 5) after incubation at pH 10.0 and EDA. (Freeware 1D analysis software of gel electrophoresis image, GelAnalyzer). Other specifications are described in Methods section………. 94
Figure 4.9 (A) Size distribution by intensity of the SPMN@PEI measured in distilled water. (B) Zeta potential distribution of the SPMN@PEI measured in distilled water. All experiments were performed by triplicate. Other specifications are described in Methods section... 96
Figure 4.10 Zeta potentials and Hydrodynamic diameters of SPMN@PEI as a function of pH (100 mM). Other specifications are described in Methods section...……… 97
Figure 4.11 Zoom in the magnetization curve at room temperature for the studied samples. Inset: Original graphic. Other specifications are described in Methods section. Black (SPMN@PEI), Red (SPMN@PEI-DVS), Green (SPMN@PEI-DVS-TLL), Blue (SPMN@PEI-DVS-TLL after incubation at alkaline pH) and Yellow (SPMN@PEI-DVS-TLL after incubation at alkaline pH and at EDA)… 99 Figure 4.12 FT-IR spectrum of the samples. Inset: zoom in the FT-IR spectrum from 1800 to 800 cm-1. Other specifications are described in Methods section. Black (SPMN@PEI), Red (SPMN@PEI-DVS), Blue (SPMN@PEI-DVS-TLL)..…... 100
Figure 4.14 Effect of the incubation time at alkaline pH on TLL preparations storage stability. Experiments have been performed at 25 °C. Other specifications are described in Methods section………...……… 102 Figure 4.15 Effect of the incubation time at EDA on TLL preparations operational stability.
Experiments have been performed at 25 °C. Other specifications are described in Methods section………...…… 104 Figure 4.16 Effect of the incubation time at EDA on TLL preparations storage stability.
Experiments have been performed at 25 °C. Other specifications are described in Methods section ………...….... 104 Figure 4.17 Effect of different blocking reagents on TLL preparations storage stability.
Experiments have been performed at 25 °C. Other specifications are described in Methods section……….……….. 105 Figure 4.18 - Effect of different blocking reagents on TLL preparations operational stability.
Experiments have been performed at 25 °C. Other specifications are described in Methods section………... 106 Figure 4.19 Reuse of the immobilized enzyme in the synthesis of benzyl acetate with 24
hours of reaction per cycle. Other specifications are described in Methods section... 110
LIST OF TABLES
Table 2.1 Lipases immobilized on SPMN... 35
Table 3.1 Structural parameters obtained from Rietvield refinement………... 62 Table 3.2 Assignments of the vibrational modes of the FT-IR spectra for immobilized
SPMN….………. 65 Table 3.3 Immobilization parameters. Other specifications are described in Methods
section. IY (immobilization yield), AtT (theoretical activity), AtD (biocatalyst activity), AtR (recovery activity).………...……….. 66 Table 3.4 The intensity of the detected bands (raw volume) were calculated using
GelAnalyzer, a freeware software……… 67 Table 3.5 Effect of different incubation conditions on the enzyme stability of immobilized
TLL lipase biocatalysts. (pH 5–65 °C, pH 7–70 °C, pH 9–60 °C, pH 7–30 °C in the organic solvent) and hydrolytic activity of the immobilized TLL preparations (substrate: tributyrin; reaction conditions: 37 ºC, pH 7). Other specifications are described in Methods section………... 68 Table 3.6 Enzymatic kinetic resolution carried out with
rac-1-methyl-2-(2,6-dimethylphenoxy)ethyl acetate. The detailed experimental procedures are described in the Methods section..……… 72 Table 3.7 Reuse of the immobilized enzyme in the presence of ethyl ether and
biocatalyst/substrate mass ratio of 2:1 at 30 °C and 24 hours of reaction per cycle. Other specifications are described in Methods section.………. 75
Table 4.1 Thermal stability at 60 °C of the enzyme immobilized at different pH values. Other specifications are described in Methods section... 95 Table 4.2 Zeta potential of the different production stages of the biocatalyst measured in
distilled water. Other specifications are described in Methods section…………. 98 Table 4.3 Assignments of the vibrational modes of the FT-IR spectra for SPMN samples.
Other specifications are described in Methods section... 100 Table 4.4 Elemental content (C, H, N and S) of the samples by elemental analysis. Other
specifications are described in Methods section... 101 Table 4.5 Effect of the incubation time at alkaline pH (100 mM) on TLL preparations
thermal stability at pH 7 (25 mM) at 60 °C. Other specifications are described in Methods section………... 103 Table 4.6 Effect of the incubation time at EDA on TLL preparations thermal stability at
Table 4.7 Effect of different blocking reagents on TLL preparations thermal stability at 60 °C and pH 7 (25 mM). Other specifications are described in Methods section 105 Table 4.8 Hydrolytic activity of different TLL preparations versus different substrates
under different conditions. Experiments have been performed at 25 °C. Other specifications are described in Methods section. EH, Ethyl hexanoate; RMM, (R)-methyl mandelate; SMM, (S)-methyl mandelate; ρ-NPB, ρ-nitrophenyl butyrate. The activity is given in µmoles of substrate hydrolyzed per minute and g of immobilized enzyme. ¤Activity (÷103). *Activity (x103)... 108 Table 4.9 Enzymatic esterification catalyzed by TLL preparations of the compound
LIST OF ABBREVIATIONS AND ACRONYMS
APTES 3-Aminopropyltriethoxysilane CTAB Cetyltrimethylammonium Bromide DLS Dynamic Light Scattering
DVS Divinyl Sulfone EDA Ethylenediamine ETA Ethanolamine
FTIR Fourier-Transform Infrared spectroscopy GLY Glycine
GA Glutaraldehyde
PEI Branched Polyethyleneimine ρ-NPB ρ-Nitrophenyl Butyrate
CONTENTS
1 INTRODUCTION...17
1.1 Introduction...18
1.2 Objectives... 21
1.2.1 Specific objectives..... 21
REFERENCES...22
2 LITERATURE REVIEW………...26
2.1 Literature review...27
2.1.1 General overview about enzymes... 27
2.1.1.1 Lipases... 27
2.1.1.2 Thermomyces lanuginosus lipase….…... 29
2.1.2 Enzyme immobilization…...30
2.1.2.1 Immobilization on iron oxide nanoparticles... 33
2.1.2.2 Branched polyethyleneimine coating... 36
2.1.2.3 3-Aminopropyltriethoxysilane coating.…………... 38
2.1.2.4 Activation with Glutaraldehyde...38
2.1.2.5 Activation with Divinyl sulfone…...39
2.1.3 Applications... 40
REFERENCES...41
3 DESIGN OF A LIPASE-NANO PARTICLE BIOCATALYSTS AND ITS USE IN THE KINETIC RESOLUTION OF MEDICAMENT PRECURSORS….………..49
3.1 Abstract...50
3.2 Introduction... 51
3.3 Materials and methods...53
3.3.1 Materials...,...53
3.3.2 Synthesis of Fe3O4 and Functionalization with APTES….………...53
3.3.3 Synthesis of Fe3O4 and Functionalization with PEI………. 54
3.3.4 Activation of SPMN@APTES and SPMN@PEI with glutaraldehyde….…..… 55
3.3.5 Characterization of the supports and biocatalysts….……….55
3.3.5.1 X-ray powder diffraction...55
3.3.5.2 Magnetic characterization...55
3.3.5.3 FTIR analysis...55
3.3.6 Preparation of glyoxyl-agarose beads…....………..………... 56
3.3.7 Immobilization procedure….………..……… 56
3.3.7.1 Covalent immobilization of lipase on SPMN@APTES-GA or SPMN@PEI-GA56 3.3.7.2 Ionic immobilization of lipase on SPMN@APTES or SPMN@PEI….………..56
3.3.7.3 Lipase immobilization on glyoxyl-agarose support...57
3.3.8 Determination of enzyme activity and protein concentration….…...………… 58
3.3.9 Immobilization Parameters…...………..58
3.3.11 Thermal and pH inactivation…... 59
3.3.12 Solvent stability…... 59
3.3.13 Operational stability…...59
3.3.14 Tributyrin hydrolysis…...59
3.3.15 Preparation of the racemic substrates….………60
3.3.15.1 Synthesis of rac-1-(2,6-dimethylphenoxy)propan-2-ol... 60
3.3.15.2 Synthesis of rac-1-methyl-2-(2,6-dimethylphenoxy)ethyl acetate…...60
3.3.16 Kinetic enzymatic resolution of rac-1-methyl-2-(2,6-dimethylphenoxy)ethyl acetate via hydrolysis reaction using TLL immobilized………..…60
3.3.17 Procedure for calculating enantiomeric excess, conversion and enantiomeric ratio………..61
3.3.18 Analysis…..……….. 61
3.4 Results and discussion...62
3.4.1 Characterization of biocatalyst…...62
3.4.1.1 XRPD analysis………...62
3.4.1.2 Magnetic measurement... 63
3.4.1.3 FTIR analysis... 64
3.4.2 Immobilization Parameters…...………..66
3.4.3 SDS-PAGE analysis of the samples…....……….66
3.4.4 Thermal stability at different pH values….……….68
3.4.5 Tributyrin hydrolysis…...69
3.4.6 Synthesis of rac-1-methyl-2-(2,6-dimethylphenoxy)ethyl acetate….…………. 70
3.4.7 Hydrolysis of rac-1-methyl-2-(2,6-dimethylphenoxy)ethyl acetate using lipase….………...…71
3.4.8 Operational stability of the immobilized enzyme….….………..73
3.5 Conclusions...76
REFERENCES….………..……… 77
4 A NEW SUPERPARAMAGNETIC NANOPARTICLES VINYL SULFONE SUPPORT TO IMMOBILIZE ENZYMES: APPLICATION TO LIPASE FROM THERMOMYCES LANUGINOSUS……….…………..81
4.1 Abstract... 82
4.2 Introduction...83
4.3 Materials and methods...85
4.3.1 Materials...85
4.3.2 Synthesis of Fe3O4 and Functionalization with PEI….……….86
4.3.3 Activation of SPMN@PEI with divinylsulfone…....………..…….86
4.3.4 Immobilization procedure….………..…… 87
4.3.5 Determination of enzyme activity and protein concentration….………...88
4.3.6 Immobilization Parameters….………89
4.3.7 Characterization of the supports and biocatalysts….……….89
4.3.7.1 Particle size and zeta potential measurements... 89
4.3.7.2 Magnetic characterization………... 89
4.3.7.4 Elemental analysis….………... 90
4.3.8 SDS-PAGE electrophoresis... 90
4.3.9 Thermal and pH inactivation...90
4.3.10 Operational and storage stability...90
4.3.11 Hydrolysis of ethyl hexanoate...91
4.3.12 Hydrolysis of (R)- and (S)-methyl mandelate….…..……….. 91
4.3.13 Acetylation of benzyl alcohol….………...…..……….92
4.4 Results and discussion...93
4.4.1 Immobilization of TLL on DVS activated support….……….93
4.4.2 Thermal stability of TLL on DVS activated support….………...95
4.4.3 Characterization of biocatalyst…...……..………...96
4.4.3.1 Particle size and zeta potential measurements….………...…... 96
4.4.3.2 Magnetic measurement….………...………...98
4.4.3.3 FT-IR analysis…………...………...………....99
4.4.3.4 Elemental analysis….…...………...………...100
4.4.4 Effect of incubation time at alkaline pH on immobilized enzyme performance…..………... 101
4.4.5 Effect of the blocking reagents...103
4.4.5.1 Effect of incubation time at EDA on immobilized enzyme performance….…....103
4.4.5.2 Effect of the different blocking reagents on immobilized enzyme performance...104
4.4.6 Biocatalysis preparations versus different substrates - Hydrolytic activity…....106
4.4.7 Enzymatic synthesis of benzyl acetate and Reuse of the biocatalysts…..…….. 109
4.5 Conclusions... 111
REFERENCES….………..………112
5 FINAL CONSIDERATIONS AND WORK PROSPECTS...117
5.1 Final considerations... 118
5.2 Work prospects... 118
5.2.1 Reactors for SPMN biocatalysts... 119
