Estudo da produção de poligalacturonases em processo submerso em biorreatores de agitação mecânica e airlift

Abstract

In this work, the production of endo and exo-polygalacturonase (PG) by Aspergillus oryzae IPT 301 was studied in a stirred tank bioreactor (STR) and in airlift bioreactors with internal and external circulation loop. Using a factorial experimental design, a soluble culture medium (WBE) was defined which allowed the production of exo and endo-PG comparable to that obtained in a medium containing suspended wheat bran. With the WBE medium, the production of PG was compared in STR and internal-circulation-airlift bioreactor. In these tests, superior enzymatic activities for both endo-PG (91.3 units per mL of medium, U/mL) and exo-PG (65.2 U/mL) were obtained in the STR, whereas in the airlift reactor the maximum activities were 86.2 U/mL and 60.6 U/mL, respectively. These results indicate the feasibility of airlift reactors for producing polygalacturonases by A. oryzae. In STR cultivations, the influence of different concentrations of pectin (0, 10, and 20 g/L) and glucose (0, 5, 10, 15, 20, 25 e 30 g/L), in WBE medium, on the process was assessed. Furthermore, in media containing 20 g/L pectin and 20, 30, and 50 g/L glucose, tests where pH was controlled to a minimum value of 2.7 were carried out. In tests where pH was controlled to this minimum value, strong improvement of both endo and exo-PG activities was attained, especially in the run with 30 g/L glucose in which enzyme activities of 175.0 U/mL for endo-PG and 103.0 U/mL for exo-PG. In STR, cultivation media formulated with regular pectin (WBE) and with partially enzyme-hydrolysed pectin to reduce the viscosity of medium and as such to improve the oxygen transfer to the culture were evaluated. In one condition (WBE5), the medium was formulated with half of the pectin partially hydrolysed and half of the pectin untreated. In this condition, activities of endo-PG of 149.0 U/mL and exo-PG of 82.2 U/mL were achieved, whereas with the non-hydrolysed-pectin medium statistically inferior activities 130.0 and 78.0 U/mL, respectively were measured. PG production was compared in STR and in both airlift bioreactors (internal and external circulation loop), using the condition WBE5 and a further condition (WBE6), with non-hydrolysed pectin medium, in which the nutrient salts were fed to the medium in lots during the cultivation. After 96 h of process, a higher activity of endo-PG (145.0 U/mL) was attained in STR followed by the condition WBE6 in the same reactor (140.0 U/mL). In airlift bioreactors, superior titres of endo-PG (116.0 U/mL) and exo-PG (80.4 U/mL) were obtained in the condition WBE6, with the external loop configuration, the activity of exo-PG being similar to that measured in STR. The process was evaluated in media with different concentrations of KH2PO4, (NH4)2SO4, FeSO4, MnSO4, MgSO4 e ZnSO4. In individual analysis of each salt, performed in shake-flasks experiments, the influence of the concentrations of NH4+, Fe2+, Mn2+, Mg2+, Zn2+ sulphates on the production of polygalacturonases was evidenced. In STR, under controlled conditions, the enzyme production in WBE medium was compared with different conditions of salt feeding to the culture. Among the conditions tested in STR, the highest activities for endo-PG (175.0 U/mL) and exo-PG (86.5 U/mL) were achieved when KH2PO4, (NH4)2SO4, FeSO4, MnSO4, MgSO4 e ZnSO4 were fed to the medium in lots, with a significant improvement in comparison to the use of WBE medium that resulted in activities of 130.0 and 78.6 U/mL, respectively. The results of this work reveal the high potential of airlift bioreactors as a relatively simple building operating system to be used in large-scale polygalacturonase production.