Proteolytic activity of Vibrio harveyi YeaZ is related with resuscitation on the viable but non-culturable state.

The YeaZ protein of Vibrio harveyi was expressed in Escherichia coli and purified. The purified recombinant protein YeaZ exhibited the protease activity. The proteolytic activities with azocasein as substrate were 39 130 U mg-1 . The mutation of the amino acid in active sites such as Asp88 , Ser185 and Trp169 was performed. The enzyme activities of the purified mutant proteins with Asp88 -Ala, Ser185 -Leu and Trp169 -Glu were decreased to 24·28, 35·27 and 41·66%, respectively. The mutant protein with two amino acid residues (Asp88 -Ala/Ser185 -Leu) lost the protease activity completely. Addition of the purified recombinant YeaZ increased resuscitation of the viable but non-culturable state (VBNC) cells to culturable state, and the culturable cell count increased from 1·35 × 102 to 3·10 × 106  CFU per ml. While addition of the mutant YeaZ without protease activities did not show obvious promoting effect on resuscitation of VBNC cells. Moreover, the purified YeaZ also showed lower muralytic activity, and the activities of proteins with single amino acids mutation (Thr71 and Asp112 ) were reduced from 7·05 to 4·75 and 2·50 U mg-1 , the resuscitation-promoting effect on VBNC cells was not affected by these mutant proteins. These results implied that resuscitation-promoting effect of YeaZ on VBNC cell was partly related to its protease activities, but not with the muralytic activity.