Estratégias para obtenção da enzima recombinante L-arabinose isomerase (L-AI) por expressão heteróloga em Escherichia coli BL21 (DE3) através do método de autoindução.

Abstract

The constant changes in eating habits led to the generation of products that add health benefits to those who consume them, as improvements of a physiological nature when related to the products available on the market. Among these products, D-tagatose appears as a promising substitute for sucrose, presenting 90% sweetness and low caloric value (1.5 kcal. g-1), when compared to common sugar (4.0 kcal. g-1). This sweetener is poorly absorbed by the body, does not raise blood sugar levels, and has prebiotic, antioxidant and anti-diabetic properties. One of the ways to obtain D-tagatose is through the isomerization of D-galactose catalyzed by the enzyme L-arabinose isomerase (L-AI). This study evaluates different strategies for obtaining the recombinant enzyme L-AI from Enterococcus faecium DBFIQ E36 in E. coli aiming to replace the induction method, which uses 0.5 mM of Isopropyl β-1-D-thiogalactopyranoside (IPTG) by the auto-induction method, aspiring to enable its availability at commercial level. For the process of obtaining the recombinant protein, glucose and glycerol were used as carbon sources, lactose (commercial) and lactose from whey as induction agents at concentrations of 2 and 4 g.L-1. Whey is considered a by-product of dairy industry and has stood out as a promising raw material applied in biotechnological processes. Cultivations by the auto-induction method are considered low cost, do not present cytotoxicity and since the bacteria select the sources of carbon and the inducer available in the medium, there is no need to monitor the process for adding lactose. The L-AI extracts achieved catalytic activities of 1.67 ± 0.14 U. mL-1 and 0.7 ± 0.04 U. mL-1, when produced using commercial lactose and IPTG, respectively. The extracts induced with lactose derived from whey showed catalytic activity of 3.8 U. mL-1. Evaluating the scale increase, the expression of the enzyme using commercial lactose in a bioreactor at 300 rpm with a useful volume of 3L and an oxygen transfer flow of 0.6 L.min-1, showed its maximum enzymatic activity with 8h of culture (2.7 ± 0.01 U.mL-1). L-AI exhibited the best catalytic activities at 50 °C, pH 5.6 and greater bioconversion of D-galactose isomerization reaction to D-tagatose when reaction occurred using 500 mM D-galactose reaching a concentration of 97.7 mM and 20% yield, after 28h. Thus, this study presented promising and innovative protocols for the effective expression of recombinant protein through auto-induction methods using commercial lactose and lactose from whey.