We describe a simple and effective procedure to screen for active prot
eases among a large number of mutants. First, the mutants are genetica
lly tested by the protease activity produced in the periplasm of trans
formed bacteria which supplies the cells with a nitrogen source by hyd
rolyzing a protein applied to plates, Then a less sensitive activity s
taining and an X-ray film digestion assay are used to verify and estim
ate the activity of the mutants that proved to be positive in the firs
t step, Depending essentially on the level of periplasmic protease act
ivity, the method can detect both the activity and the stability of th
e expressed enzymes. We calibrated the method with transformants that
produce wild-type trypsin, chymotrypsin and trypsin mutants of known a
ctivity, Using this method we found two active revertants of the inact
ive Asn102 trypsin mutant, by screening similar to 4.4X10(4) random mu
tants that were generated by the polymerase chain reaction on a cDNA f
ragment. This procedure should be useful in searching for proteases of
novel specificity and/or reaction chemistry engineered by random muta
genesis, and also for in vitro evolution studies.