Comparison of extraction methods for recovery of extracellular β-glucosidase in two different forest soils

Abstract

A study was made of the efficiency of three different extractants, 0.1 M sodium pyrophosphate (pH 7), 67 mM phosphate buffer (pH 6) and 0.5 M potassium sulphate (pH 6.6), in recovering the protein quantity and the β-glucosidase enzyme activity from two natural forest soils: (1) an Inceptisoil located in Tuscany (Italy) in a mild Mediterranean climate, and (2) a Lithic Calcixeroll soil located in Murcia (south-east of Spain) in a dry-semiarid climate. The pyrophosphate was used to determine the activity of extracellular-humic-bound proteins, while the phosphate buffer and potassium sulphate were used to extract dissolved extracellular proteins. The latter extractant, after chloroform fumigation, was also used to measure total proteins in soil. A preliminary screening, using SDS-PAGE in one dimension, was also carried out in order to optimize the separation condition of soil proteins extracted with different buffers. To remove the interfering co-extracted substances (humic acid) a purification step using a column packed with insoluble polyvinylpyrrolidone was performed. The highest β-glucosidase activity was recovered in the pyrophosphate extract, thus confirming its capability of extracting humic-bound β-glucosidase enzyme in a stable and active form. The extractants performed differently with the two soil types and band patterns obtained with SDS-PAGE were extractant-specific, demonstrating that each was selective for a particular class of proteins. Surprisingly, protein bands were also obtained using pyrophosphate, in spite of the very dark extract colour due to the presence of humic substances.