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Proteases from actinomycetes interfere in solid media plate assays of hyaluronidase activity
L A de Azeredo1, S G Leite, D M Freire
1Departamento de Microbiologia Geral, Instituto de Microbiologia Prof. Paulo de Góes, Bloco I, Centro de Ciências da Saúde, Cidade Universitária, Ilha do Fundão, Universidade Federal do Rio de Janeiro, 21941-590, RJ, Rio de Janeiro, Brazil.
Abstract:
Four hundred and fifteen actinomycete strains were screened for hyaluronidase activity in two plate assays media. In the first one, using hyaluronic acid as substrate and bovine serum albumin (BSA) to help precipitation of the nondegraded substrate, only strain 594 and hyaluronidase control were positive. In the second assay, plates with hyaluronic acid, but not BSA, gave the same results. For plates containing only BSA, proteinase activity was detected in strain 594. When hyaluronic acid was treated with pronase, the only clear zones, in the second assay without BSA, were those around hyaluronidase controls. Protease activity, commonly found in actinomycetes, was detected only in strain 594, among the 415 studied, when tested in hyaluronidase assay using hyaluronate plus BSA. This may be due to the composition of the growth medium, since media with different composition gave different results for protease activity in each of the 15 strains analyzed. These data suggest that proteases can affect an accurate detection of hyaluronidase in media containing proteins, not only from hyaluronate preparations, but also from other medium ingredients. Thus, for a correct interpretation of the method, they must be excluded. Commercial Hyaluronidase used as controls must be also tested for the presence of protease contamination.
Insights
Accurate hyaluronidase detection requires excluding proteases, which can interfere with assays. Screening 415 actinomycete strains revealed that proteases, common in these microbes, can yield false positives in hyaluronidase tests.
Area of Science:
- Microbiology
- Enzymology
Background:
- Hyaluronidase is an enzyme that degrades hyaluronic acid.
- Actinomycetes are known to produce various enzymes, including proteases.
- Accurate detection of microbial enzymes is crucial for various applications.
Purpose of the Study:
- To screen a large collection of actinomycete strains for hyaluronidase activity.
- To investigate the potential interference of proteases in hyaluronidase detection assays.
- To optimize methods for reliable hyaluronidase screening in actinomycetes.
Main Methods:
- Screening of 415 actinomycete strains using two plate assays with hyaluronic acid as substrate.
- Assays were conducted with and without bovine serum albumin (BSA) to differentiate hyaluronidase and protease activity.
- Protease activity was specifically tested in strain 594 and 15 other selected strains using different media compositions.
Main Results:
- Only one strain (594) showed positive results for hyaluronidase in initial assays, which was later attributed to protease activity.
- Protease activity was detected in strain 594 and was influenced by the growth medium composition.
- When hyaluronic acid was pre-treated with pronase, hyaluronidase controls showed clear zones, indicating protease interference in the absence of BSA.
Conclusions:
- Protease activity commonly found in actinomycetes can interfere with the accurate detection of hyaluronidase.
- Media formulations and the presence of proteins (like BSA) can influence protease activity and assay results.
- It is essential to exclude proteases from assays and test commercial enzyme preparations for protease contamination to ensure reliable hyaluronidase detection.