Abstract
Proteins are a substantial nitrogen source in soils provided that they can be hydrolyzed into bioavailable small peptides or amino acids. However, the strong associations between proteins and soil minerals restrict such proteolytic reactions. This study focused on how an extracellular fungal protease (Rhizopus sp.) hydrolyzed iron oxide-associated bovine serum albumin (BSA) and the factors that affected the proteolysis. We combined batch experiments with size-exclusion and reversed phase liquid chromatography and in situ infrared spectroscopic measurements to monitor the generation of proteolytic products in solution as well as the real-time changes of the adsorbed BSA during 24 h. Results showed that protease hydrolyzed the iron oxide-associated BSA directly at the surface without an initial desorption of BSA. Concurrently, the protease was adsorbed to vacant surface sites at the iron oxides, which significantly slowed down the rate of proteolysis. This inhibiting effect was counteracted by the presence of preadsorbed phosphate or by increasing the BSA coverage, which prevented protease adsorption. Fast initial rates of iron oxide-associated BSA proteolysis, comparable to proteolysis of BSA in solution, and very slow rates at prolonged proteolysis suggest a large variability in mineral-associated proteins as a nitrogen source in soils and that only a fraction of the protein is bioavailable.
Highlights
The primary production in boreal forest ecosystems is limited by the availability of nitrogen (N).[1]
To address these research questions, we examined the proteolysis of iron oxideassociated bovine serum albumin (BSA) by an aspartic protease isolated from the filamentous fungus Rhizopus sp. that is omnipresent in soils.[21]
BSA and a protease from Rhizopus sp. were purchased from Sigma-Aldrich ( Merck KGaA, Darmstadt, Germany). This protease is an aspartic endopeptidase with optimum activity in acidic environments, and its isoelectric point is suggested to be in the pH range 4−6,26 similar to that of BSA at ca. pH 4.7.27 The autoproteolysis of the protease in solution was minor within the timeframe of our experiments (Supporting Information Figure S1)
Summary
The primary production in boreal forest ecosystems is limited by the availability of nitrogen (N).[1]. In the absence of pepstatin A, the peak intensity decreased even further (Supporting Information Figure S8), which likely was a result of increased protease adsorption caused by proteolysis of iron oxide-associated BSA that created vacant surface sites.
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