We have cloned ansB (YPTB1411) gene from Yersinia pseudotuberculosis Q66C12 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. K-m for L-asparagine is 17 +/- 0.9 mu M and pI 5.4 +/- 0.3. Enzyme demonstrates maximum activity at pH 8.0 and 60 degrees C. YpA L-glutaminase activity is relatively low and more than 15 times less than specific activity towards L-asn. We evaluated also the anti-proliferative effect of YpA in vitro and in vivo with E. coli L-asparaginase (EcA) as the reference substance at similar conditions. (C) 2012 Published by Elsevier Inc.