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Improvement of thermal stability via outer-loop ion pair interaction of mutated T1 lipase from Geobacillus zalihae strain T1.

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Ruslan R, Abd Rahman RN, Leow TC, Ali MS, Basri M, Salleh AB
OpenAccess logo Int J Mol Sci 13 943-60 (2012)
Cited: 17 times
EuropePMC logo PMID: 22312296

Abstract

Mutant D311E and K344R were constructed using site-directed mutagenesis to introduce an additional ion pair at the inter-loop and the intra-loop, respectively, to determine the effect of ion pairs on the stability of T1 lipase isolated from Geobacillus zalihae. A series of purification steps was applied, and the pure lipases of T1, D311E and K344R were obtained. The wild-type and mutant lipases were analyzed using circular dichroism. The T(m) for T1 lipase, D311E lipase and K344R lipase were approximately 68.52 °C, 70.59 °C and 68.54 °C, respectively. Mutation at D311 increases the stability of T1 lipase and exhibited higher T(m) as compared to the wild-type and K344R. Based on the above, D311E lipase was chosen for further study. D311E lipase was successfully crystallized using the sitting drop vapor diffusion method. The crystal was diffracted at 2.1 Å using an in-house X-ray beam and belonged to the monoclinic space group C2 with the unit cell parameters a = 117.32 Å, b = 81.16 Å and c = 100.14 Å. Structural analysis showed the existence of an additional ion pair around E311 in the structure of D311E. The additional ion pair in D311E may regulate the stability of this mutant lipase at high temperatures as predicted in silico and spectroscopically.

Articles - 3umj mentioned but not cited (3)

  1. Improvement of thermal stability via outer-loop ion pair interaction of mutated T1 lipase from Geobacillus zalihae strain T1. Ruslan R, Rahman RNZRA, Leow TC, Ali MSM, Basri M, Salleh AB. Int J Mol Sci 13 943-960 (2012)
  2. The conserved lid tryptophan, W211, potentiates thermostability and thermoactivity in bacterial thermoalkalophilic lipases. Timucin E, Sezerman OU. PLoS One 8 e85186 (2013)
  3. Crystal structure of pathogenic Staphylococcus aureus lipase complex with the anti-obesity drug orlistat. Kitadokoro K, Tanaka M, Hikima T, Okuno Y, Yamamoto M, Kamitani S. Sci Rep 10 5469 (2020)


Reviews citing this publication (5)

  1. From structure to catalysis: recent developments in the biotechnological applications of lipases. Anobom CD, Pinheiro AS, De-Andrade RA, Aguieiras EC, Andrade GC, Andrade GC, Moura MV, Almeida RV, Freire DM. Biomed Res Int 2014 684506 (2014)
  2. Cold Active Lipases: Biocatalytic Tools for Greener Technology. Mhetras N, Mapare V, Gokhale D. Appl Biochem Biotechnol 193 2245-2266 (2021)
  3. Lipase improvement: goals and strategies. Bassegoda A, Cesarini S, Diaz P. Comput Struct Biotechnol J 2 e201209005 (2012)
  4. An Appraisal on Prominent Industrial and Biotechnological Applications of Bacterial Lipases. Akram F, Mir AS, Haq IU, Roohi A. Mol Biotechnol 65 521-543 (2023)
  5. Realm of Thermoalkaline Lipases in Bioprocess Commodities. Lajis AFB. J Lipids 2018 5659683 (2018)

Articles citing this publication (9)

  1. Enhancing the Thermostability of Rhizomucor miehei Lipase with a Limited Screening Library by Rational-Design Point Mutations and Disulfide Bonds. Li G, Fang X, Su F, Chen Y, Xu L, Yan Y. Appl Environ Microbiol 84 e02129-17 (2018)
  2. Combinatorial reshaping of a lipase structure for thermostability: additive role of surface stabilizing single point mutations. Kumar R, Singh R, Kaur J. Biochem Biophys Res Commun 447 626-632 (2014)
  3. Ability of T1 Lipase to Degrade Amorphous P(3HB): Structural and Functional Study. Mohamed RA, Salleh AB, Leow ATC, Yahaya NM, Abdul Rahman MB. Mol Biotechnol 59 284-293 (2017)
  4. Increasing thermal stability and catalytic activity of glutamate decarboxylase in E. coli: An in silico study. Tavakoli Y, Esmaeili A, Saber H. Comput Biol Chem 64 74-81 (2016)
  5. Decoding the intricate network of molecular interactions of a hyperstable engineered biocatalyst. Markova K, Chmelova K, Marques SM, Carpentier P, Bednar D, Damborsky J, Marek M. Chem Sci 11 11162-11178 (2020)
  6. New engineered Geobacillus lipase GD-95RM for industry focusing on the cleaner production of fatty esters and household washing product formulations. Druteika G, Sadauskas M, Malunavicius V, Lastauskiene E, Statkeviciute R, Savickaite A, Gudiukaite R. World J Microbiol Biotechnol 36 41 (2020)
  7. Probing the roles of two tryptophans surrounding the unique zinc coordination site in lipase family I.5. Timucin E, Cousido-Siah A, Mitschler A, Podjarny A, Sezerman OU. Proteins 84 129-142 (2016)
  8. Single Residue Substitution at N-Terminal Affects Temperature Stability and Activity of L2 Lipase. Bukhari N, Leow ATC, Abd Rahman RNZR, Mohd Shariff F. Molecules 25 E3433 (2020)
  9. Identification, Characterization, and Computer-Aided Rational Design of a Novel Thermophilic Esterase from Geobacillus subterraneus, and Application in the Synthesis of Cinnamyl Acetate. Zhang J, Lin L, Wei W, Wei D. Appl Biochem Biotechnol (2023)


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