DEVELOPMENT OF RECOMBINANT ENZYMES TOWARDS THE PRODUCTION OF PHARMACEUTICAL INTERMEDIATES USING BIOTRANSFORMATIONS

Bragança, Caio Roberto Soares (2022) DEVELOPMENT OF RECOMBINANT ENZYMES TOWARDS THE PRODUCTION OF PHARMACEUTICAL INTERMEDIATES USING BIOTRANSFORMATIONS. Doctoral thesis, SETU Waterford.

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Abstract

Nitrile compounds are versatile and can be converted into amides, amines, imines, oximes, carboxylic acids, esters and alcohols, encompassing a large group of economically important synthetic intermediates. The pharmaceutical industry particularly requires amides and acids for use as intermediates in the manufacture of many drugs and chemicals. The biotransformation of nitriles mediated by microorganisms has therefore attracted considerable attention in academia and industry as a sustainable alternative to the conventional chemical reactions that require drastic conditions of pH, temperature and pressure, use of metal catalysts, high-energy consumption and low selectivity in the process. Certain bacterial cells contain a nitrile-metabolizing gene; when the corresponding enzyme is incubated in a reaction-mixture containing a nitrile, the nitrile-metabolizing enzyme catalyses the conversion of the nitrile to the corresponding amide or acid. The amide or acid may then be extracted from the reaction mixture. This biological conversion is referred to as a biotransformation and is considered “Green Chemistry”. As a result, the search for microorganisms which contain the enzymes responsible for these biotransformations (nitrilases, NHase and amidases) is crucial. The main goals of this research were to; isolate bacteria with activity towards three pharmaceutically relevant β-hydroxynitriles from environmental samples collected worldwide; develop a high throughput screening strategy for filamentous fungi with potential for nitrile biotransformation; apply functional metagenomics to search for novel nitrile hydrolyzing enzymes using environmental samples collected in Ireland; and to formulate an appropriate production medium using statistical optimization that can substantially increase nitrilase production. In this study, we have found three promising bacterial isolates which are source of genes for nitrile-degrading enzymes including, Nocardia coeliaca strain DSM, Klebsiella oxytoca strain JCM1665 and R. erythropolis PR4. The three strains presented with enantiomeric excess of >90 % towards 3-hydroxybutyronitrile (3HBN). Of the three promising isolates, one showed exceptional >99.99 ee % towards 3HBN, indicating that the bacterial isolate is highly enantioselective and possibly enantiospecific with 100 % ee of (S)-acid. In contrast, while most industrial nitrilase enzymes are derived from bacterial sources, the potential of filamentous fungi was explored with a view to industrial use. The fungus Fusarium solani strain F3 was isolated and exhibited exceptional enantioselectivity towards 3-phenylpropionitrile with >99.99 % ee and enantioselectivity towards 3-hydroxybutyronitrile with 98.03 %, indicating the presence of a highly enantioselective enzyme using the whole mycelial cells. Once an enzyme is characterized and chosen for its desired properties, large scale production in heterologous hosts often becomes essential, with growth conditions requiring optimisation even before scale-up can begin. Acknowledging this frequent need, providing an approach that addresses these technical challenges was demonstrated in this work by integrating two science fields: statistics with microbiology laboratory experiments to optimize the fermentation process parameters. Our results showed that an average of 5.54 mmol/L of nitrilase activity was attained using whole cells in the validation experiment under optimized conditions, which was 66 % higher than the prior yield of 3.33 mmol/L.

Item Type:	Thesis (Doctoral)
Uncontrolled Keywords:	Enzymes
Departments or Groups:	*NONE OF THESE*
Divisions:	School of Science > Department of Chemical and Life Sciences
Depositing User:	Derek Langford
Date Deposited:	06 Oct 2022 10:53
Last Modified:	06 Oct 2022 10:53
URI:	https://repository.wit.ie/id/eprint/3545

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