SUBSTITUTIONS OF SER FOR ASN-163 AND PRO FOR LEU-264 ARE IMPORTANT FOR STABILIZATION OF LIPASE FROM PSEUDOMONAS-AERUGINOSA

The lipase gene from Pseudomonas aeruginosa was randomly mutated by er ror-prone PCR to obtain thermostable mutants, followed by screening fo r thermostable mutant lipases, Out of about 2,600 transformants, four thermostable clones were obtained, Their nucleotide sequences showed t hat they had two or three amino acid substitutions, Analysis of the th ermal stabilization of these mutant lipases indicated that Asn-163 to Ser and Leu-264 to Pro mutations were essential for the increased stab ility of the lipase, We expressed a mutant lipase (StLipA-5) having on ly the Asn-163 to Ser mutation and another (StLipA-6) having only the Leu-264 to Pro mutation in P. aeruginosa PAO 1161, purified them, and then confirmed that the temperature which causes a 50% decrease in the activity of the non-treated enzyme on treatment for 30 min was increa sed by 1.5 and 3 degrees C, respectively, compared to the wild-type en zyme, However, the thermal stability of the mutant lipase (StLipA-7) h aving both mutations was increased only by 2.5 degrees C, These mutant lipases were stabilized through a decrease in activation entropy, Kin etic studies showed that the k(cat)/K-m values of StLipA-5, StLipA-6, and StLipA-7 were decreased by 14.4, 52.9, and 26.0%, respectively, In terestingly, the pH-stabilities of StLipA-6 and StLipA-7 were also inc reased, especially at alkaline pH, Based on these results, the tertiar y structure and mechanism of stabilization of the lipase were discusse d.