Substitution of Asp189 residue alters the activity and thermostability of <Emphasis Type="Italic">Geobacillus</Emphasis> sp. NTU 03 lipase |
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Authors: | Tsung-Wei Shih Tzu-Ming Pan |
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Institution: | (1) Department of Biochemical Science and Technology, College of Life Science, National Taiwan University, 1 Roosevelt Rd. Sec. 4, Taipei, 106, Taiwan, ROC |
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Abstract: | Error-prone PCR was used to create more thermoactive and/or thermostable variants of thermoalkalophilic lipases. A variant
of the α6 helix (lid domain), with an 189E to V substitution at residue 189, lost its thermostability but exhibited higher
activity than that of its wild-type predecessor (r03Lip). Site-saturation mutagenesis was used to explore the sequence-function
relationship. Five other mutants also lost thermostability (20–40%) but exhibited enhanced thermoactivity (6.3–79-fold). The
mutant E189I showed the highest activity retaining 50% activity after maintaining it at 65°C for 24 h. In comparison to r03Lip,
the mutant E189I had a higher affinity for p-nitrophenyl palmitate and p-nitrophenyl stearate (61 and 56% decreased Km) and catalytic efficiency (42-fold and 18-fold increased kcat/Km). The mutant
lipase retained its tolerance to n-hexane, but had an improved transesterification activity. The results suggest that residue Glu189 plays a significant role
in the thermostability and activity of this thermoalkalophilic lipase. |
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Keywords: | |
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