caa a22 4500 397558619 CHVBK 20180308164810.0 cr unu---uuuuu 161202e199611 xx s 000 0 eng 10.1093/oxfordjournals.jbchem.a021506 doi (NATIONALLICENCE)oxford-10.1093/oxfordjournals.jbchem.a021506 Substitutions of Ser for Asn-163 and Pro for Leu-264 Are Important for Stabilization of Lipase from Pseudomonas aeruginosa [Elektronische Daten] [Akeo Shinkai, Akiko Hirano, Kazuo Aisaka] 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 inP. 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 mm was increased by 1.5 and 3°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°C. These mutant lipases were stabilized through a decrease in activation entropy. Kinetic studies showed that the kcet/Km 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. © 1996 BY THE JOURNAL OF BIOCHEMISTRY Regular Papers nationallicence lipase nationallicence pH-stabilization nationallicence Pseudomonas aeruginosa nationallicence random mutagenesis nationallicence thermal stabilization nationallicence Shinkai Akeo Tokyo Research Laboratories, Kyowa Hakko Kogyo Co., Ltd., 3-6-6, Asahi-machi, Machida, Tokyo 194 aut Hirano Akiko Tokyo Research Laboratories, Kyowa Hakko Kogyo Co., Ltd., 3-6-6, Asahi-machi, Machida, Tokyo 194 aut Aisaka Kazuo Tokyo Research Laboratories, Kyowa Hakko Kogyo Co., Ltd., 3-6-6, Asahi-machi, Machida, Tokyo 194 aut The Journal of Biochemistry Oxford University Press 120/5(1996-11), 915-921 0021-924X 120:5<915 1996 120 jbchem https://doi.org/10.1093/oxfordjournals.jbchem.a021506 text/html Onlinezugriff via DOI 1 other jats NATIONALLICENCE 856 40 https://doi.org/10.1093/oxfordjournals.jbchem.a021506 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Shinkai Akeo Tokyo Research Laboratories, Kyowa Hakko Kogyo Co., Ltd., 3-6-6, Asahi-machi, Machida, Tokyo 194 aut NATIONALLICENCE 700 1- Hirano Akiko Tokyo Research Laboratories, Kyowa Hakko Kogyo Co., Ltd., 3-6-6, Asahi-machi, Machida, Tokyo 194 aut NATIONALLICENCE 700 1- Aisaka Kazuo Tokyo Research Laboratories, Kyowa Hakko Kogyo Co., Ltd., 3-6-6, Asahi-machi, Machida, Tokyo 194 aut NATIONALLICENCE 773 0- The Journal of Biochemistry Oxford University Press 120/5(1996-11), 915-921 0021-924X 120:5<915 1996 120 jbchem Metadata rights reserved CC BY-NC-4.0 http://creativecommons.org/licenses/by-nc/4.0 nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-oxford