Immobilization of laccase enzyme on supports derived from peanut shell

SERBENT, PILAR; RODRIGUEZ, M. D. ; FERMANELLI, CARLA; MAGARIO, IVANA; SAUX, CLARA

Ciudad Autónoma de Buenos Aires

Simposio; Biotecnología para un mundo en cambio? XIV SIMPOSIO REDBIO ARGENTINA; 2023

REDBIO Argentina

Fungal ligninolytic enzymes have the ability to transform a large spectrum of contaminants; this makes their application in biotechnological processes very attractive. Supported enzymes are more stable than free ones and can be reused, making them even more interesting. Synthetic supports for enzyme immobilization are expensive, for this reason there is an interest in exploring the use of low-cost materials such as residual biomass from agricultural activities. Peanut shells (PS) represent an interesting resource in the central region of Argentina, since an annual volume of more than 250 thousand tons is generated. The objective of this work was to evaluate the use of PS as an adsorbent support for the fungal enzyme laccase (E). The enzyme was obtained from the cultivation of Phlebia brevispora BAFC 633 in liquid media composed by malt extract (1.27% P/V), corn soluble extract (5% V/V) and CuSO4 (0.5 mM). Laccase activity was determined using 5 mM 2,6-dimetoxifenol (DMP) as substrate in 0.1 M buffer sodium acetate (pH 3.6). The change of absorbance was monitored by spectrophotometry at 469 nm (ε469 = 27.5 mM−1cm−1) at 3 and 5 minutes of reaction. The enzymatic activity was expressed in enzymatic units per gram of support (U g-1), where 1 U is equivalent to 1 µM min-1 product at 30° C. The PS were washed with distilled water, oven-dried at 105 °C to constant weight and milled to a particle size < 3.35 mm. An exploratory screening design was carried out (factorial 2^4, with five center points) using four factors; namely, contact time (1, 3.5 and 6 h) between PS and E; enzyme concentration (1000, 2500 and 4000 U L-1); consistency –given by the ratio between mass of PS (1 g) and different volumes of enzymatic extract (5, 7.5 and 10 ml) – and agitation (0, 100, 200 rpm). The response variable was the enzymatic activity of the obtained solid. Each factor was studied at three levels: low (‐1), intermediate (0) and high (+1). Thereafter, the identified statistically significant parameters were optimized by a central composite design (2^2 start points) and the procedure was validated. Data were analyzed using the software STATGRAPHICS Centurion XVI. In the exploratory screening design, the supported laccase values ranged from ~55 to 98 U g-1. Analysis of variance showed that the factors with statistically significant influence (p-valor