Improving the activity and stability of thermolysin by site-directed mutagenesis.
In previous site-directed mutagenesis study on thermolysin, mutations which increase the catalytic activity or the thermal stability have been identified. In this study, we attempted to generate highly active and stable thermolysin by combining the mutations so far revealed to be effective. Three mutant enzymes, L144S (Leu144 in the central alpha-helix located at the bottom of the active site cleft is replaced with Ser), G8C/N60C/S65P (Gly8, Asn60, and Ser65 in the N-terminal region are replaced with Cys, Cys, and Pro, respectively, to introduce a disulfide bridge between the positions 8 and 60), and G8C/N60C/S65P/L144S, were constructed by site-directed mutagenesis. In the hydrolysis of N-[3-(2-furyl)acryloyl]-glycyl-L-leucine amide (FAGLA) and N-carbobenzoxy-L-aspartyl-L-phenylalanine methyl ester (ZDFM), the k(cat)/K(m) values of L144S and G8C/N60C/S65P/L144S were 5- to 10-fold higher than that of the wild-type enzyme. The rate constants for thermal inactivation at 70 degrees C and 80 degrees C of G8C/N60C/S65P and G8C/N60C/S65P/L144S decreased to 50% of that of the wild-type enzyme. These results indicate that G8C/N60C/S65P/L144S is more active and stable than the wild-type thermolysin. Thermodynamic analysis suggests that the single mutation of Leu144-->Ser and the triple mutation of Gly8-->Cys, Asn60-->Cys, and Ser65-->Pro are independent.
Yasukawa
Kiyoshi
K
Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan.
Inouye
Kuniyo
K
D019943:Amino Acid Substitution / Q000235:genetics*; D001407:Bacillus / Q000201:enzymology; D002364:Caseins / Q000378:metabolism; D002384:Catalysis; D004789:Enzyme Activation / Q000235:genetics; D004795:Enzyme Stability / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006868:Hydrolysis; D016297:Mutagenesis, Site-Directed; D013379:Substrate Specificity / Q000235:genetics; D013820:Thermolysin / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
D002364:Caseins; D013820:Thermolysin