КОМПЬЮТЕРНЫЙ АНАЛИЗ СТРУКТУРЫ ЛИПАЗ БАКТЕРИЙ pода Geobacillus И ВЫЯВЛЕНИЕ МОТИВОВ, ВЛИЯЮЩИХ НА ИХ ТЕРМОСТАБИЛЬНОСТЬ

В работе проведен анализ свойств термостабильных липаз бактерий рода Geobacillus. Проведена классификация ферментов по группам термостабильности. Показано наличие согласованных аминокислотных замен у группы липаз с высокой термостабильностью (со временем полуинактивации свыше 1000 мин): V198A, Q203E, V204I, Q217E и V294I, P306A, T307A, D312S, R313H, E316G, V324I, S334N, A343T. Наибольшее достоверное влияние на термостабильность ферментов оказывал гидрофильный момент α-спиралей, который коррелировал с зарядом и полярностью аминокислотных остатков данных регионов. Показано, что у липаз с наибольшей термостабильностью происходит стабилизация «lid»-домена на структурном уровне в районе 198А-217Е.

1. Abdel-Fattah Y.R., Gaballa A.A. Identification and overexpression of a thermostable lipase from Geobacillus thermoleovorans Toshki in Escherichia coli // Microbiol. Res. 2008. V. 163. No. 1. P. 13–20.

2. Chakravorty D., Parameswaran S., Dubey V.K., Patra S. In silico characterization of thermostable lipases // Extremophiles. 2011. V. 15. P. 89–103.

3. Cho A.R., Yoo S.K., Kim E.J. Cloning, sequencing and expression in Escherichia coli of a thermophilic lipase from Bacillus thermoleovorans ID-1 // FEMS Microbiol. Lett. 2000. V. 186. No. 2. P. 235–238.

4. Choi W.C., Kim M.H., Ro H.S. et al. Zinc in lipase L1 from Geobacillus stearothermophilus L1 and structural implications on thermal stability // FEBS Lett. 2005. V. 579. No. 16. P. 3461–3466.

5. Ebrahimpour A., Rahman R.N.Z.R.A., Basri M., Salleh A.B. High level expression and characterization of a novel thermostable, organic solvent tolerant, 1,3-regioselective lipase from Geobacillus sp. strain ARM // Bioresource Technol. 2011. V. 102. No. 13. P. 6972–6981.

6. Guex N., Peitsch M.C. SWISS-MODEL and the Swiss-Pdb-Viewer: An environment for comparative protein modeling // Electrophoresis. 1997. V. 18. P. 2714–2723.

7. Hebin L., Xiaobo Z. Characterization of thermostable lipase from thermophilic Geobacillus sp. TW1 // Protein Expres. Purif. 2005. V. 42. No. 1. P. 153–159.

8. Ivanisenko V.A., Eroshkin A.M., Kolchanov N.A. WebPro-Analyst: an interactive tool for analysis of quantitative structure-activity relationships in protein families // Nucl. Acids Res. 2005. V. 33. P. 99–104.

9. Jaeger K.E., Eggert T. Lipases for biotechnology // Curr. Opin. Biotechnol. 2002. V. 13. P. 390–397.

10. Karkhane А.А., Yakhchali B., Jazii F.R., Bambai B. The effect of substitution of Phe181 and Phe182 with Ala on activity, substrate specificity and stabilization of substrate at the active site of Bacillus thermocatenulatus lipase // J. Mol. Catalysis B: Enzymatic. 2009. V. 61. No. 3/4. P. 162–167.

11. Kim H.K., Park S.Y., Lee J.K., Oh.T.K. Gene cloning and characterization of thermostable lipase from Bacillus stearothermophilus L1 // Biosci., Biotechnol. Biochem. 1998. V. 62. No. 1. P. 66–71.

12. Quintana-Castro R., Dнaz P., Valerio-Alfaro G. et al. Gene cloning, expression, and characterization of the Geobacillus thermoleovorans CCR11 thermoalkaliphilic lipase // Mol. Biotechnol. 2009. V. 42. No. 1. P. 75–83.

13. Quyen D.T., Schmidt-Dannert C., Schmid R.D. High-level expression of a lipase from Bacillus thermocatenulatus BTL2 in Pichia pastoris and some properties of the recombinant lipase // Protein Expres. Purif. 2003. V. 28. No. 1. P. 102–110.

14. Ruslan R., Rahman R.N.Z.R.A., Leow T.C. et al. Improvement of thermal stability via outer-loop ion pair interaction of mutated T1 lipase from Geobacillus zalihae strain T1 // Intern. J. Mol. Sci. 2012. V. 13. No. 1. P. 943–960.

15. Sabri S., Rahman R.N.Z.R.A., Leow T.C. et al. Secretory expression and characterization of a highly Ca2+-activated thermostable L2 lipase // Protein Expres. Purif. 2009. V. 68. No. 2. P. 161–166.

16. Sharma P.K., Kumar R., Kumar R. et al. Engineering of a metagenome derived lipase toward thermal tolerance: Effect of asparagine to lysine mutation on the protein surface // Gene. 2012. V. 491. No. 2. P. 264–271.

17. Shih T.W., Pan T.M. Substitution of Asp189 residue alters the activity and thermostability of Geobacillus sp. NTU 03 lipase // Biotechnol. Lett. 2011. V. 33. Nо. 9. P. 1841–1846.

18. Baldessari A. Lipases as catalysts in synthesis of fine chemicals // Methods Mol. Biol. 2012. V. 861. P. 445–456.

19. Soliman N.A., Knoll M., Abdel-Fattah Y.R. et al. Molecular cloning and characterization of thermostable esterase and lipase from Geobacillus thermoleovorans YN isolated from desert soil in Egypt // Process Biochem. 2007. V. 42. No. 7. P. 1090–1100.

20. Tayyab M., Rashid N., Akhtar M. Isolation and identification of lipase producing thermophilic Geobacillus sp. SBS-4S: Cloning and characterization of the lipase // J. Biosci. Bioengineer. 2011. V. 111. No. 3. P. 272–278.

21. Tyndall J.D.A., Sinchaikul S., Fothergill-Gilmore L.A., Taylor P. Crystal structure of a thermostable lipase from Bacillus stearothermophilus P1 // J. Mol. Biol. 2002. V. 323. No. 5. P. 859–869.

22. Wu L., Liu B., Hong Y. et al. Residue Tyr224 is critical for the thermostability of Geobacillus sp. RD-2 lipase // Biotechnol. Lett. 2010. V. 32. No. 1. P. 107–112.