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

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

    Tsung-Wei Shih & Tzu-Ming Pan

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  1. Tsung-Wei Shih
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  2. Tzu-Ming Pan
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Correspondence to Tzu-Ming Pan.

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Shih, TW., Pan, TM. Substitution of Asp189 residue alters the activity and thermostability of Geobacillus sp. NTU 03 lipase. Biotechnol Lett 33, 1841–1846 (2011). https://doi.org/10.1007/s10529-011-0635-3

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  • DOI: https://doi.org/10.1007/s10529-011-0635-3

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