Substitutions of Ser for Asn-163 and Pro for Leu-264 are important for stabilization of lipase from Pseudomonas aeruginosa.

Abstract

The lipase gene from Pseudomonas aeruginosa was randomly mutated by error-prone PCR to obtain thermostable mutants, followed by screening for thermostable mutant lipases. Out of about 2,600 transformants, four thermostable clones were obtained. Their nucleotide sequences showed that they had two or three amino acid substitutions. Analysis of the thermal stabilization of these mutant lipases indicated that Asn-163 to Ser and Leu-264 to Pro mutations were essential for the increased stability of the lipase. We expressed a mutant lipase (StLipA-5) having only the Asn-163 to Ser mutation and another (StLipA-6) having only the Leu-264 to Pro mutation in P. aeruginosa PAO1161, 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 increased by 1.5 and 3 degrees C, respectively, compared to the wild-type enzyme. However, the thermal stability of the mutant lipase (StLipA-7) having both mutations was increased only by 2.5 degrees C. These mutant lipases were stabilized through a decrease in activation entropy. Kinetic studies showed that the Kcat/K(m) values of StLipA-5, StLipA-6, and StLipA-7 were decreased by 14.4, 52.9, and 26.0%, respectively. Interestingly, the pH-stabilities of StLipA-6 and StLipA-7 were also increased, especially at alkaline pH. Based on these results, the tertiary structure and mechanism of stabilization of the lipase were discussed.