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Biotechnol Lett. 2011 Sep;33(9):1841-6. doi: 10.1007/s10529-011-0635-3. Epub 2011 May 5.

Substitution of Asp189 residue alters the activity and thermostability of Geobacillus sp. NTU 03 lipase.

Author information

1
Department of Biochemical Science and Technology, College of Life Science, National Taiwan University, 1 Roosevelt Rd. Sec. 4, Taipei 106, Taiwan, ROC. d93b47106@ntu.edu.tw

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.

PMID:
21544610
DOI:
10.1007/s10529-011-0635-3
[Indexed for MEDLINE]

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