The active site of Trichoderma reesei cellobiohydrolase II: the role of tyrosine 169

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Abstract
We describe a simple and effective procedure to screen for active proteases among a large number of mutants. First, the mutants are genetically tested by the protease activity produced in the periplasm of transformed bacteria which supplies the cells with a nitrogen source by hydrolyzing a protein applied to plates. Then a less sensitive activity staining and an X-ray film digestion assay are used to verify and estimate the activity of the mutants that proved to be positive in the first step. Depending essentially on the level of periplasmic protease activity, the method can detect both the activity and the stability of the expressed enzymes. We calibrated the method with transformants that produce wild-type trypsin, chymotrypsin and trypsin mutants of known activity. Using this method we found two active revertants of the inactive Asn102 trypsin mutant, by screening ˜4.4×104 random mutants that were generated by the polymerase chain reaction on a cDNA fragment. This procedure should be useful in searching for proteases of novel specificity and/or reaction chemistry engineered by random mutagenesis, and also for in vitro evolution studies.

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