Applications of enzymes to the preparation of optically active compounds

The introduction to this thesis is in the form of a review entitled 'Aspects of Selectivity in Lipase Catalysed Biotransformations'. Each of the most widely used lipases have been discussed. The reactions of each lipase have been extensively reviewed with the aim of establishing whether any trends have appeared in the characteristics of compounds accepted as substrates. The remaining chapters cover five unrelated studies in areas of resolutions of chiral compounds using biotransformations. In Chapter 2 the resolution of a P-blocker precursor was attempted via lipase-catalysed hydrolysis. l-Chloro-2- hydroxy-3[4(2-acetoxyethyl) phenoxy] propane was obtained in high enantiomeric excess from hydrolysis of the corresponding butyrate ester with lipases from Mucor or Rhizopus sp. Yields were low however, owing to enzyme inhibition by the butyric acid byproduct. In Chapter 3 the resolution of methyl 3-hydroxy-4-(p- chlorophenylthio)-butanoate was carried out. Hydrolysis of the corresponding butanoate ester with lipase P gave the R enantiomer of the desired compound in high enantiomeric excess. Transesterification of the racemic alcohol with vinyl acetate, again catalysed by lipase P, furnished the opposite enantiomer in 62%ee. Chapter 4 is concerned with the resolution of a chiral acid, namely 3-methyl-4-oxo-4(4-aminobenzyl) butanoic acid. This was attempted by hydrolysis of an ester using pig liver esterase and various lipases and via microbial reduction of the corresponding unsaturated compound. The reactions were all found to be non-stereoselective. Chapters 5 and 6 discuss novel methods for the enzymatic resolution of ketones. The enantioselective enzymatic hydrolysis of oxime esters is discussed in Chapter 5. The resulting optically enriched oximes may readily be cleaved to the ketones. This method was unsuccessful in the resolution of the 2-methyl- and 2,6- dimethylcyclohexanones. A low enantiomeric excess was achieved in the resolution of norcamphor, and attempts to improve this using a purified enzyme and by variation of the ester chain were unsuccessful. However, this represents the first example of the indirect enzymatic resolution of ketones. In Chapter 6 the enantioselective hydrolysis of enol acetates of three ketones was attempted. This method of resolution was unsuccessful in the resolution of 2-methyl and 2,6- dimethylcyclohexanones. In the case of norcamphor the ketone was obtained in low enantiomeric excess

Novozym 435 : the “perfect” lipase immobilized biocatalyst?

Novozym 435 (N435) is a commercially available immobilized lipase produced by Novozymes. It is based on immobilization via interfacial activation of lipase B from Candida antarctica on a resin, Lewatit VP OC 1600. This resin is a macroporous support formed by polyIJmethyl methacrylate) crosslinked with divinylbenzene. N435 is perhaps the most widely used commercial biocatalyst in both academy and industry. Here, we review some of the success stories of N435 (in chemistry, energy and lipid manipulation), but we focus on some of the problems that the use of this biocatalyst may generate. Some of these problems are just based on the mechanism of immobilization (interfacial activation) that may facilitate enzyme desorption under certain conditions. Other problems are specific to the support: mechanical fragility, moderate hydrophilicity that permits the accumulation of hydrophilic compounds (e.g., water or glycerin) and the most critical one, support dissolution in some organic media. Finally, some solutions (N435 coating with silicone, enzyme physical or chemical crosslinking, and use of alternative supports) are proposed. However, the N435 history, even with these problems, may continue in the coming future due to its very good properties if some simpler alternative biocatalysts are not developed

Applications of enzymes to the preparation of optically active compounds

The introduction to this thesis is in the form of a review entitled 'Aspects of Selectivity in Lipase Catalysed Biotransformations'. Each of the most widely used lipases have been discussed. The reactions of each lipase have been extensively reviewed with the aim of establishing whether any trends have appeared in the characteristics of compounds accepted as substrates. The remaining chapters cover five unrelated studies in areas of resolutions of chiral compounds using biotransformations. In Chapter 2 the resolution of a P-blocker precursor was attempted via lipase-catalysed hydrolysis. l-Chloro-2- hydroxy-3[4(2-acetoxyethyl) phenoxy] propane was obtained in high enantiomeric excess from hydrolysis of the corresponding butyrate ester with lipases from Mucor or Rhizopus sp. Yields were low however, owing to enzyme inhibition by the butyric acid byproduct. In Chapter 3 the resolution of methyl 3-hydroxy-4-(p- chlorophenylthio)-butanoate was carried out. Hydrolysis of the corresponding butanoate ester with lipase P gave the R enantiomer of the desired compound in high enantiomeric excess. Transesterification of the racemic alcohol with vinyl acetate, again catalysed by lipase P, furnished the opposite enantiomer in 62%ee. Chapter 4 is concerned with the resolution of a chiral acid, namely 3-methyl-4-oxo-4(4-aminobenzyl) butanoic acid. This was attempted by hydrolysis of an ester using pig liver esterase and various lipases and via microbial reduction of the corresponding unsaturated compound. The reactions were all found to be non-stereoselective. Chapters 5 and 6 discuss novel methods for the enzymatic resolution of ketones. The enantioselective enzymatic hydrolysis of oxime esters is discussed in Chapter 5. The resulting optically enriched oximes may readily be cleaved to the ketones. This method was unsuccessful in the resolution of the 2-methyl- and 2,6- dimethylcyclohexanones. A low enantiomeric excess was achieved in the resolution of norcamphor, and attempts to improve this using a purified enzyme and by variation of the ester chain were unsuccessful. However, this represents the first example of the indirect enzymatic resolution of ketones. In Chapter 6 the enantioselective hydrolysis of enol acetates of three ketones was attempted. This method of resolution was unsuccessful in the resolution of 2-methyl and 2,6- dimethylcyclohexanones. In the case of norcamphor the ketone was obtained in low enantiomeric excess

Strategies for the enhancement of the catalytic performance of cutinase in nonaqueous media

Dissertação para obtenção do Grau de Doutor em Engenharia Química e Bioquímic

Solvent stable microbial lipases: Current understanding and biotechnological applications

Objective: This review examines on our current understanding of microbial lipase solvent tolerance, with a specific focus on the molecular strategies employed to improve lipase stability in a non-aqueous environment. Results: It provides an overview of known solvent tolerant lipases and of approaches to improving solvent stability such as; enhancing stabilising interactions, modification of residue flexibility and surface charge alteration. It shows that judicious selection of lipase source supplemented by appropriate enzyme stabilisation, can lead to a wide application spectrum for lipases. Conclusion: Organic solvent stable lipases are, and will continue to be, versatile and adaptable biocatalytic workhorses commonly employed for industrial applications in the food, pharmaceutical and green manufacturing industries

Lipase-catalyzed kinetic resolution of branched chain fatty acids and their esters : a study towards the production of enantiopure 4-methyloctanoic acid = Lipase-gekatalyseerde kinetische resolutie van vertakte vetzuren en hun esters : een studie naar de productie van enantiomeer zuiver 4-methyloctaanzuur

Flavors and fragrances make an important contribution to the taste and smell of all kinds of food products both as natural occurring components and as additional flavors or fragrances. One of these flavor components is 4-methyloctanoic acid (4-MOA). This branched chain fatty acid contributes to the characteristic smell and taste of mutton and sheep's cheese. The enantiomers of 4-MOA have a different smell and taste; it is therefore a challenge to separate both stereoisomers. One possible way of separation, which is designated as food-grade, is enzyme catalyzed kinetic resolution. Since the chiral center is positioned at theγ-carbon atom, a low enantioselectivity was expected. Therefore, sequential kinetic resolution (esterification followed by hydrolysis) was investigated. To find a suitable enzyme to catalyze these reactions, a group of 25 hydrolases was screened in the transesterification of 4-methyloctanoic acid methyl ester, resulting in the selection of Novozym 435 ® .This enzyme was used to esterify 4-MOA with polyethylene glycol (PEG, food grade). Unfortunately PEG appeared not to be suitable since no stable two-phase system could be formed, which was necessary for the continuous production of enantiomerically pure 4-MOA in a diffusion membrane reactor. Therefore, PEG was replaced by ethanol (food grade) in the esterification of 4-MOA. In this reaction, part of the substrate appeared to sorb into the polymer matrix of the immobilization material of Novozym 435 ® affecting the enantiomeric ratio estimation. Therefore correction for sorption was needed. The enantioselectivity of the enzyme was dependent on the alcohol concentration used, both for esterification (optimum E = 57 ± 11, obtained for the ratio 1:8 (mol fatty acid: mol EtOH)) and for hydrolysis (optimum E =12 ± 1, in the presence of 20% (v/v) EtOH). When combining these results, sequential kinetic resolution of 4-methyloctanoic acid will result in an enantiomeric ratio of approximately 350.Since integration of esterification and product separation resulted in a low enantiomeric ratio ( E = 15) in comparison with the batch reaction ( E = 57), and simulation and measurements showed no improvement of the eep , the batch reaction is recommended to obtain enantiopure 4-methyloctanoic acid.</p

Novozym 435: the “perfect” lipase immobilized biocatalyst?

Novozym 435 (N435) is a commercially available immobilized lipase produced by Novozymes. It is based on immobilization via interfacial activation of lipase B from Candida antarctica on a resin, Lewatit VP OC 1600. This resin is a macroporous support formed by poly(methyl methacrylate) crosslinked with divinylbenzene. N435 is perhaps the most widely used commercial biocatalyst in both academy and industry. Here, we review some of the success stories of N435 (in chemistry, energy and lipid manipulation), but we focus on some of the problems that the use of this biocatalyst may generate. Some of these problems are just based on the mechanism of immobilization (interfacial activation) that may facilitate enzyme desorption under certain conditions. Other problems are specific to the support: mechanical fragility, moderate hydrophilicity that permits the accumulation of hydrophilic compounds (e.g., water or glycerin) and the most critical one, support dissolution in some organic media. Finally, some solutions (N435 coating with silicone, enzyme physical or chemical crosslinking, and use of alternative supports) are proposed. However, the N435 history, even with these problems, may continue in the coming future due to its very good properties if some simpler alternative biocatalysts are not developed.We gratefully recognize the financial support from MINECO from the Spanish Government (project number CTQ2017-86170-R, Colciencias, Ministerio de Educación Nacional, Ministerio de Industria, Comercio y Turismo e ICETEX, Convocatoria Ecosistema Científico – Colombia Científica. Fondo Francisco José de Caldas, Contrato RC-FP44842-212-2018 and Colciencias (Colombia) (project number FP44842-076-2016), Generalitat Valenciana (PROMETEO/2018/076), FAPERGS (project number 17/2551-0000939-8), CONICET (R. Argentina), FUNCAP (project number BP3-0139-00005.01.00/18) and ANPCyT (PICT 2015-0932 and PICT CABBIO 4687)

Evaluation of Designed Immobilized Catalytic Systems: Activity Enhancement of Lipase B from Candida antarctica

Publisher's version (útgefin grein)Immobilized enzymatic catalysts are widely used in the chemical and pharmaceutical industries. As Candida antarctica lipase B (CALB) is one of the more commonly used biocatalysts, we attempted to design an optimal lipase-catalytic system. In order to do that, we investigated the enantioselectivity and lipolytic activity of CALB immobilized on 12 different supports. Immobilization of lipase on IB-D152 allowed us to achieve hyperactivation (178%) in lipolytic activity tests. Moreover, the conversion in enantioselective esterification increased 43-fold, when proceeding with lipase-immobilized on IB-S861. The immobilized form exhibited a constant high catalytic activity in the temperature range of 25 to 55°C. Additionally, the lipase immobilized on IBD152 exhibited a higher lipolytic activity in the pH range of 6 to 9 compared with the native form. Interestingly, our investigations showed that IB-S500 and IB-S60S offered a possibility of application in catalysis in both organic and aqueous solvents. A significant link between the reaction media, the substrates, the supports and the lipase was confirmed. In our enzymatic investigations, highperformance liquid chromatography (HPLC) and the titrimetric method, as well as the Bradford method were employed.This work was supported by the National Science Centre Poland grant DEC-2013/09/N/NZ7/03557.Peer Reviewe

Synthesis of Zr-beta zeolite in fluoride medium and its applications in catalytic liquid-phase reactions

Ph.DDOCTOR OF PHILOSOPH