Optimizing the Production of Recombinant Hydroperoxide Lyase in Escherichia coli Using Statistical Design
| Autor: | Jacques Maury, Jessica Croce, Sophie Vincenti, Virginie Brunini-Bronzini de Caraffa, Eva Faillace, Magali Mariani, Liliane Berti |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2021 |
| Předmět: |
0106 biological sciences
food.ingredient medicine.disease_cause lcsh:Chemical technology 01 natural sciences Catalysis law.invention response surface methodology lcsh:Chemistry 03 medical and health sciences food Erlenmeyer flask law 010608 biotechnology medicine lcsh:TP1-1185 Response surface methodology Food science hydroperoxide lyase Physical and Theoretical Chemistry Escherichia coli Aroma 030304 developmental biology chemistry.chemical_classification 0303 health sciences E. coli heterologous protein production biology Food additive Green leaf volatiles protein expression optimization biology.organism_classification Enzyme Box–mBehnken design chemistry lcsh:QD1-999 Recombinant DNA |
| Zdroj: | Catalysts, Vol 11, Iss 176, p 176 (2021) Catalysts Volume 11 Issue 2 |
| ISSN: | 2073-4344 |
| Popis: | Hydroperoxide lyase (HPL) catalyzes the synthesis of volatiles C6 or C9 aldehydes from fatty acid hydroperoxides. These short carbon chain aldehydes, known as green leaf volatiles (GLV), are widely used in cosmetic industries and as food additives because of their &ldquo fresh green&rdquo aroma. To meet the growing demand for natural GLVs, the use of recombinant HPL as a biocatalyst in enzyme-catalyzed processes appears to be an interesting application. Previously, we cloned and expressed a 13-HPL from olive fruit in Escherichia coli and showed high conversion rates (up to 94%) during the synthesis of C6 aldehydes. To consider a scale-up of this process, optimization of the recombinant enzyme production is necessary. In this study, four host-vector combinations were tested. Experimental design and response surface methodology (RSM) were used to optimize the expression conditions. Three factors were considered, i.e., temperature, inducer concentration and induction duration. The Box&ndash Behnken design consisted of 45 assays for each expression system performed in deep-well microplates. The regression models were built and fitted well to the experimental data (R2 coefficient > 97%). The best response (production level of the soluble enzyme) was obtained with E. coli BL21 DE3 cells. Using the optimal conditions, 2277 U L&minus 1of culture of the soluble enzyme was produced in microliter plates and 21,920 U L&minus 1of culture in an Erlenmeyer flask, which represents a 79-fold increase compared to the production levels previously reported. |
| Databáze: | OpenAIRE |
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