Engineering of Bacillus lipase by directed evolution for enhanced thermal stability: effect of isoleucine to threonine mutation at protein surface.

Molecular Biology Reports
Jyoti KhuranaJagdeep Kaur

Abstract

A lip gene from a Bacillus isolate was cloned and expressed in E. coli. By thermal denaturation analysis, T(1/2) of lipase was observed to be 7 min at 50°C with less than 10% activity after 1 h incubation at 50°C. To expand the functionality of cloned lipase, attempts have been made to create thermostable variants of lip gene. A lipase variant with an isoleucine to threonine amino acid substitution at the protein surface was isolated that demonstrated higher thermostability than its wild type predecessor. To explore the structure-function relationship, the lip gene product of wild type (WT) and mutant was characterized in detail. The mutation enhanced the specific activity of enzyme by 2-folds when compared with WT. The mutant enzyme showed enhanced T(1/2) of 21 min at 50°C. The kinetic parameters of the mutant enzyme were significantly altered. The mutant enzyme displayed higher affinity for substrate (decreased K ( m )) in comparison to the wild type. The k (cat) and catalytic efficiency (k (cat)/K ( m )) of mutant were also enhanced by two and five times, respectively, as compared with the WT. The mutation resides on the part of helix which is exposed to the solvent and away from the catalytic triad. The replacement of a sol...Continue Reading

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Citations

Nov 5, 2014·Journal of Molecular Modeling·Parameswaran SaravananSanjukta Patra
Jul 20, 2017·Biotechnology and Applied Biochemistry·Parisa FarrokhAli Asghar Karkhane
Aug 14, 2019·Protein Engineering, Design & Selection : PEDS·Ana L Alfaro-ChávezDavid L Ollis
Nov 29, 2013·Biotechnology and Bioengineering·Hyun Tae HwangArvind Varma
Jan 14, 2021·Biotechnology and Applied Biochemistry·Surabhi Soni
Dec 29, 2020·International Journal of Biological Macromolecules·Karen Rodríguez-NúñezRonny Martínez
Jul 24, 2021·International Journal of Biological Macromolecules·Abdallah R IsmailKwang-Hyun Baek
Jul 27, 2017·Chemical Reviews·Toshiyuki Itoh
Sep 7, 2021·Applied Microbiology and Biotechnology·Siti Hajar HamdanThean Chor Leow

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