Kredics L, Terecskei Kata, Antal Zsuzsanna, Szekeres A, Hatvani L, Manczinger L, Vágvölgyi Cs
Department of Microbiology, Faculty of Sciences and Informatics, University of Szeged, Szeged, Hungary.
Acta Biol Hung. 2008 Jun;59(2):259-68. doi: 10.1556/ABiol.59.2008.2.11.
Eleven cold-tolerant Trichoderma isolates were screened for the production of proteolytic activities at 10 degrees C. Based on the activity profiles determined with paranitroanilide substrates at 5 degrees C, strain T221 identified as Trichoderma atroviride was selected for further investigations. The culture broth of the strain grown at 10 degrees 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 N-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 degrees 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.
筛选了11株耐冷木霉菌株，以检测其在10℃下产生蛋白水解活性的能力。根据在5℃下用对硝基苯胺底物测定的活性谱，选择鉴定为深绿木霉的T221菌株进行进一步研究。将该菌株在含酪蛋白的培养基中于10℃培养的培养液冻干浓缩，然后进行凝胶过滤，接着对在N-苯甲酰-Phe-Val-Arg-对硝基苯胺上显示最高活性的级分进行层析聚焦。纯化后的酶分子量为24 kDa，等电点为7.3，最适pH为6.2。最适温度为25℃且热稳定性低，表明它是一种真正的冷适应酶。底物特异性数据表明该酶是一种蛋白酶，对P1位的精氨酸或赖氨酸有偏好。蛋白酶抑制剂的作用表明该酶具有与胰蛋白酶相似的结合口袋。
