PLANEJAMENTO EXPERIMENTAL E AVALIAÇÃO METROLÓGICA DA PRODUÇÃO DE LIPASES POR YARROWIA LIPOLYTICA / EXPERIMENTAL DESIGN AND METROLOGICAL EVALUATION OF LIPASE PRODUCTION BY YARROWIA LIPOLYTICA

AUTOR(ES)
DATA DE PUBLICAÇÃO

2007

RESUMO

Objective: Optimization of lipase production by Yarrowia lipolytica cells through experimental design and evaluation of metrological aspects related to process. Motivation: Although several published papers are found describing lipase production by microorganisms, few of them refer to process optimization through experimental design and none of them refer to metrological evaluation and the estimate of the uncertainty in measurement. Context: Microbial lipases are an important group of biotechnologically valuable enzymes that present widely diversified catalytic properties. The interest in production of these versatile enzymes increased significantly, due to the vast amplitude of their industrial applications. There are many microorganisms able to produce lipases, but yeasts present some operational advantages compared to other microorganisms, and for this reason a strain of Yarrowia lipolytica was used in this work. A serie of factors, individually or in association, can affect production of such substances leading to different levels of product concentration and productivity. Empirical studies have been traditionally used to determine the effect of these factors on process parameters. A possible approach is to vary one factor while keeping the other at a constant level, but this approach is time consuming, does not include the interaction effects among variables, and does not necessarily lead to optimized results. Nowadays, different strategies are employed to optimize production parameters. These strategies not only allow process optimization but can also establish the dependent and independent variables. Methodology: Ten experiments were planned with the D-optimal design in which concentrations of inoculum, carbon source and nitrogen source were changed. The estimate of the uncertainty in measurement of the lipase activity was developed based on the Guide to the Expression of Uncertainty in Measurement (GUM) and on the Guide CG 4 published by EURACHEM. Results: The highest lipolytic activity obtained was 4815 U/L (80,3 (mi)kat/L) with an expanded uncertainty of 137 U/L (2,3 (mi)kat/L), under the following conditions: 0.5% of olive oil (carbon source),0.7% of peptone (nitrogen source) and 10 mg/mL of inoculum. In terms of volumetric productivity, these conditions provided 69.28 U/L h. Conclusions: It is possible to conclude that the D-optimal design usage enhanced lipase production in shaken-flasks. Moreover the activity obtained in this work was higher than others previously reported with the same microorganism, without experimental design, in shaken-flasks (2700 U/L) or even in bench-fermenter (4240 U/L). The production of lipases by Yarrowia lipolytica cells aiming the classification as certified reference material can be recommended after further purification and stability studies.

ASSUNTO(S)

uncertainty of analytical results lipase lipase biometrology biometrologia experimental design planejamento experimental incerteza de medicao

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