Isolation of extracellular protein from greenhouse soil

Although extracellular proteins may play an important role in the soil environment, these proteins are difficult to isolate because they are immediately degraded by soil microbes, or become associated with clay mineral and humic substances. We developed a method of isolating extracellular proteins from greenhouse soils. Phosphate buffer (pH 6.0) was used to extract protein from soil. A phosphate buffer with higher pH was not recommended because it extracted a large amount of non-proteinaceous organic matter as well as protein and, as a result, the extracted protein was difficult to separate by sodium dodecylsulfate–polyacrylamide gel electrophoresis (SDS–PAGE). After removing cells by filtration, proteins dissolved in the soil extract were recovered by precipitation with 5% trichloroacetic acid (TCA) and isolated by SDS–PAGE. Proteins were detected in 10 of 32 soil samples derived from different greenhouses and the protein bands ranged in apparent molecular mass from 35 to 68 kDa, suggesting that some of soils derived from greenhouse culture contained significant amounts of a specific protein soluble in 67 mM phosphate buffer (pH 6.0). N-terminal amino acid sequence of one of the isolated proteins was found to be a homologue of thermostable cellulase produced by the genus Humicola, a thermophilic fungus.