Cloning, expression and characterization of the recombinant Yersinia pseudotuberculosis l-asparaginase

► We created Escherichia coli strain with Yersinia pseudotuberculosis l-asparaginase expression. ► The enzyme has similar biochemical properties with E. coli type II l-asparaginase. ► Y. pseudotuberculosis l-asparaginase has low activity towards l-glutamine. ► Received l-asparaginase inhibits growth of tumor cells both in vitro and in vivo. We have cloned ansB (YPTB1411) gene from Yersinia pseudotuberculosis Q66CJ2 and constructed stable inducible expression system that overproduce l-asparaginase from Y. pseudotuberculosis (YpA) in Escherichia coli BL21 (DE3) cells. For purification of YpA we used Q-Sepharose and DEAE-Toyopearl column chromatography. We examined kinetics of the enzyme reaction, catalytic activity as a function of pH, temperature and ionic strength, thermostability and other enzyme properties. Biochemical properties of YpA are similar with those of E. coli type II l-asparaginase. Km for l-asparagine is 17±0.9μM and pI 5.4±0.3. Enzyme demonstrates maximum activity at pH 8.0 and 60°C. YpA l-glutaminase activity is relatively low and more than 15 times less than specific activity towards l-asn. We evaluated also the antiproliferative effect of YpA in vitro and in vivo with E. coli l-asparaginase (EcA) as the reference substance at similar conditions.