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These variants also exhibited reduced K and V values. An increase in their Km values, leading to a decreased catalytic efficiency (the K /K ratio) of autolytic and mixed variants. Moreover, in the variant with A59T mutation, K and V values have reduced compared to wild type. The mix variants showed the most increase in Km value (almost 2-fold) as well as reduced enzymatic affinity to the substrate. Thus, the results indicated that combined mutations at the ocriplasmin sequence were more effective compared with single mutations. The resul
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