Purification and Preliminary Characterization of a Cold-Adapted Extracellular Proteinase from Trichoderma atroviride
Eleven cold-tolerant Trichoderma isolates were screened for the production of proteolytic activities at 10 °C. Based on the activity profiles determined with paranitroanilide substrates at 5 °C, strain T221 identified as Trichoderma atroviride was selected for further investigations. The culture broth of the strain grown at 10 °C in casein-containing culture medium was concentrated by lyophilization and subjected to gel filtration, which was followed by chromatofocusing of the fraction showing the highest activity on JV-benzoyl-Phe-Val-Arg-paranitroanilide. The purified enzyme had a molecular weight of 24 kDa, an isoelectric point of 7.3 and a pH optimum of 6.2. The temperature optimum of 25 °C and the low thermal stability suggested that it is a true cold-adapted enzyme. Substrate specificity data indicate that the enzyme is a proteinase with a preference for Arg or Lys at the P1 position. The effect of proteinase inhibitors suggests that the enzyme has a binding pocket similar to the one present in trypsin.
KeywordsCold tolerance psychrophilic enzymes Trichoderma trypsin-like proteinase
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We thank Miss Mária Lele for her technical help. L. Kredics is a grantee of the János Bolyai Research Scholarship.
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