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Effects of the mutational combinations on the activity and stability of thermolysin

Paper ID Volume ID Publish Year Pages File Format Full-Text
24395 43512 2010 10 PDF Available
Title
Effects of the mutational combinations on the activity and stability of thermolysin
Abstract

We have previously indicated that three single mutations (Leu144  →  Ser, Asp150 → Glu, and Ile168 → Ala) in the site-directed mutagenesis of thermolysin increase the activity and two single (Ser53 → Asp and Leu155 → Ala) and one triple (Gly8 → Cys/Asn60 → Cys/Ser65 → Pro) mutations increase the stability. In the present study, aiming to generate highly active and stable thermolysin variants, we combined these mutations and analyzed the effect of combinations on the activity and stability of thermolysin. The combination of the mutations of Leu144 → Ser and Asp150 → Glu yielded the most significant increase in the hydrolytic activities for N-[3-(2-furyl)acryloyl]-Gly-l-Leu amide (FAGLA) and N-carbobenzoxy-l-Asp-l-Phe methyl ester (ZDFM), while that of Leu144 → Ser and Ile168 → Ala abolished the activity. The combination of Ser53 → Asp and Leu155 → Ala yielded the greatest increase in the thermal stability, while that of Ser53 → Asp and Gly8 → Cys/Asn60 → Cys/Ser65 → Pro increased the stability as high as the individual mutations do. The combination of three mutations of Leu144 → Ser, Asp150 → Glu, and Ser53 → Asp yielded a variant L144S/D150E/S53D with improved activity and stability. Its kcat/Km values in the hydrolysis of FAGLA and ZDFM were 8.6 and 10.2 times higher than those of wild-type thermolysin (WT), respectively, and its rate constant for thermal inactivation at 80 °C was 60% of that of WT.

Keywords
FAGLA, N-[3-(2-furyl)acryloyl]-glycyl-l-leucine amide; ZDFM, N-carbobenzoxy-l-aspartyl-l-phenylalanine methyl esterActivity–stability relationship; Mutational combination; Site-directed mutagenesis; Thermal stability; Thermolysin
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Effects of the mutational combinations on the activity and stability of thermolysin
Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 147, Issue 1, 3 May 2010, Pages 7–16
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering