Novel Lipases: Expression and Improvement for Applied Biocatalysis = Nuevas lipasas: expresión y mejoras para biocatálisis aplicada
This thesis is focused in the identification and improvement of lipases for biotechnological application. The importance of lipases is increasing in several industries. However, the commercial use of lipases is still a drawback in the economics of the lipase-based industrial applications. There are...
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| Format: | doctoral thesis |
| Status: | Published version |
| Publication Date: | 2017 |
| Country: | España |
| Institution: | CBUC, CESCA |
| Repository: | TDR. Tesis Doctorales en Red |
| OAI Identifier: | oai:www.tdx.cat:10803/456674 |
| Online Access: | http://hdl.handle.net/10803/456674 |
| Access Level: | Open access |
| Keyword: | Lipases Lipasas Lipase Biocatàlisi Biocatálisis Biocatalysis Ciències Experimentals i Matemàtiques 579 |
| Summary: | This thesis is focused in the identification and improvement of lipases for biotechnological application. The importance of lipases is increasing in several industries. However, the commercial use of lipases is still a drawback in the economics of the lipase-based industrial applications. There are many tools for improving and adapting the enzyme properties to the desired requirements of a process that could lead lipase catalysis through a cost-effective process. In this context, the main objective of this work was: “To characterize, express and to improve novel bacterial lipases for sustainable industrial processes”. The first activity done was to explore and to characterize a new esterase from P. barcinonensis. It was isolate from P. barcinonensis the gene corresponding to Est23, and its cloning in a proper vector to perform expression and purification for biochemical characterization. Est23 showed preference for mid-chain substrates and having maximum activity at 37 °C and pH 7. It also includes in silico analysis of the 3D model structure and phylogeny. Moreover, a phylogenetic tree was constructed to assign Est23 to one of the bacterial hydrolase families described by Arpingy and Jaeger. Est23 could not be assigned to any bacterial hydrolases described till that moment, suggesting that Est23 could be part of a new group of bacterial lipases. Because Est23 displays a GGG(A)X-type putative oxyanion hole, widely described as a motif involved in tertiary alcohol resolution, the ability of Est23 for conversion and resolution of tertiary alcohols was evaluated. However, no conversion was detected using the esters linalyl and terpinyl acetate alcohols as substrates. To improve LipR activity by protein engineering LipR was then desired. LipR was isolated from Rhodococcus sp. strain CR-53 in a previous and showed an unusual fungal-like oxyanion-hole never found among bacterial lipases, close to the Y-type oxyanion hole described for Candida antartica lipase A (CalA), a lipase widely used in industry. In order to improve LipR activity on long-chain substrates, several enzyme-engineering approaches were done to change the amino acids constituting the rare oxyanion hole of LipR for further industrial application. These mutations also allowed studying the role of the amino acids forming the oxyanion hole of LipR. Hydrolytic activity over short, mid and long- chain substrates was assayed with the variants obtained. The LipR variant Asp111Gly produced a change on the chain- length- substrate preference of LipR, displaying a 5.6 fold increase of activity on muf-oleate. This improvement of activity on longer chain length substrates makes of this LipR variant a very attractive candidate for testing activity on biodiesel synthesis, a process requiring activity on long-chain substrates. Nevertheless, LipR and LipR_YGS variant need a clear expression enhancement in order to apply them to transesterification reactions using oily feedstocks. The stabilization Pseudomonas lipases LipA, LipC and LipCmut was improved by immobilization in order to applied these enzymes in transesterification reactions. Therefore, a fast and economic immobilization procedure by adsorption was set up. Finally, the three immobilized lipase preparations and a commercial lipase were used to test alternative feedstocks for triglyceride transesterification. A total of four oils were tested: commercial triolein, degummed soybean oil, waste cooking oil, and Mucor circinelloides oil. Moreover, the characterization of the tested raw materials in terms of FFAs, tri, di and monoglyceride contents measure was also of interest. In a global analysis, a good increase of FAMEs percent was obtained with LipA, LipC and LipCmut immobilized on Accurel MP1000. But better results were achieved when the reactions were catalyzed by Novozym® 435 commercial enzyme. |
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