Microbiology and Biotechnology Letters (한국미생물·생명공학회지)

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Enhancement of Pseudomonas savastanoi pv. glycinea Levansucrase Thermostability by Site-Directed Mutagenesis

  • Received : 2024.03.05
  • Accepted : 2024.04.17
  • Published : 2024.06.28
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Abstract

Levan is a functional fructooligosaccharide that belongs to the class of fructans and displays a range of physiological and dietary benefits. Levansucrase, classified as a fructosyltransferase, catalyzes the synthesis of levan fructans through the transfer of d-fructosyl residues from sucrose. Recombinant levansucrase exhibits a significantly high level of expression and activity in E. coli, however, its industrial applicability is limited due to its relatively poor thermal stability. In this study, we generated six single mutants by targeting either the high flexibility regions (242-250) or the 382Glu residue within the -TEAP- motif, and further developed two double mutants featuring concurrent mutations encompassing both regions. We expressed multiple levansucrase mutant proteins in E. coli DH5α using site-directed mutagenesis and measured the enzymatic activity and thermal stability of each mutant by levan formation reactions. The E246Y mutant displayed half-time 5.02-fold and 1.89-fold higher at 55℃ and 56℃, respectively, when compared to the WT, whereas the E382L mutant exhibited a 1.06-fold enhancement in half-time at 55℃. Furthermore, E246Y/E382L exhibited 1.95-fold and 1.86-fold higher thermal stability at 55℃ and 56℃, respectively. These findings suggest the potential for the developed thermally stable levansucrases to be applied in industrial settings, highlighting the effectiveness of our targeted mutagenesis approach.

Keywords

Acknowledgement

This paper was supported by the Konyang University Research Fund in 2022.

References

  1. Hernandez L, Banguela A. 2006. Fructans from natural sources to transgenic plants. Aplicada Biotechnol. 23: 202-210. 
  2. Van Geel-Schutten GH, Faber EJ, Smit E, Bonting K, Smith MR, Ten Brink B, et al. 1999. Biochemical and structural characterization of the glucan and fructan exopolysaccharides synthesized by the Lactobacillus reuteri wild-type strain and by mutant strains. Appl. Environ. Microbiol. 65: 3008-3014. 
  3. Korakli M, Pavlovic M, Ganzle MG, Vogel RF. 2003. Exopolysaccharide and kestose production by Lactobacillus sanfranciscensis LTH2590. Appl. Environ. Microbiol. 69: 2073-2079. 
  4. Kang HK, Seo MY, Seo ES, Kim D, Chung SY, Kimura A, et al. 2005. Cloning and expression of levansucrase from Leuconostoc mesenteroides B-512 FMC in Escherichia coli. Biochim. Biophys. Acta 1727: 5-15. 
  5. Yanase H, Maeda M, Hagiwara E, Yagi H, Taniguchi K, Okamato K. 2002. Identification of functionally important amino acid residues in Zymomonas mobilis levansucrase. J. Biochem. 132: 565-572. 
  6. Ragab TIM, Shalaby ASG, Awdan SAE, El-Bassyouni GT, Salama BM, Helmy WA, et al. 2020. Role of levan extracted from bacterial honey isolates in curing peptic ulcer: In vivo. Int. J. Biol. Macromol. 142: 564-573. 
  7. Porras-Dominguez JR, Avila-Fernandez A, Rodriguez-Alegria ME, Miranda-Molina A, Escalante A, Gonzalez-Cervantes R, et al. 2014. Levan-type FOS production using a Bacillus licheniformis endolevanase. Process. Biochem. 49: 783-790. 
  8. Adamberg K, Adamberg S, Ernits K, Larionova A, Voor T, Jaagura M, et al. 2018. Composition and metabolism of fecal microbiota from normal and overweight children are differentially affected by melibiose, raffinose and raffinose-derived fructans. Anaerobe 52: 100-110. 
  9. Dahech I, Harrabi B, Hamden K, Feki A, Mejdoub H, Belghith H, et al. 2013. Antioxidant effect of nondigestible levan and its impact on cardiovascular disease and atherosclerosis. Int. J. Biol. Macromol. 58: 281-286. 
  10. Oh J, Lee SR, Hwang KT, Ji GE. 2014. The anti-obesity effects of the dietary combination of fermented red ginseng with levan in high fat diet mouse model. Phytother. Res. 28: 617-622. 
  11. Song B, Zhu W, Song R, Yan F, Wang Y. 2019. Exopolysaccharide from Bacillus vallismortis WF4 as an emulsifier for antifungal and antipruritic peppermint oil emulsion. Int. J. Biol. Macromol. 125: 436-444. 
  12. Cantarel BL, Coutinho PM, Rancurel C, Bernard T, Lombard V, Henrissat B. 2008. The carbohydrate-active enzymes database (CAZy): an expert resource for glycogenomics. Nucleic Acids Res. 37 (Suppl): D233-D238. 
  13. Ni D, Xu W, Bai Y, Zhang W, Zhang T, Mu W. 2018. Biosynthesis of levan from sucrose using a thermostable levansucrase from Lactobacillus reuteri LTH5448. Int. J. Biol. Macromol. 113: 29-37. 
  14. Rairakhwada D, Seo J, Seo M, Kwon O, Rhee S, Kim CH. 2010. Gene cloning, characterization, and heterologous expression of levansucrase from Bacillus amyloliquefaciens. J. Ind. Microbiol. Biotechnol. 37: 195-204. 
  15. Nakapong S, Pichyangkura R, Ito K, Iizuka M, Pongsawasdi P. 2013. High expression level of levansucrase from Bacillus licheniformis RN-01 and synthesis of levan nanoparticles. Int. J. Biol. Macromol. 54: 30-36. 
  16. Li R, Zhang T, Jiang B, Mu W, Miao M. 2015. Purification and characterization of an intracellular levansucrase derived from Bacillus methylotrophicus SK 21.002. Biotechnol. Appl. Biochem. 62: 815-822. 
  17. Li WJ, Yu SH, Zhang T, Jiang B, Mu WM. 2015. Efficient biosynthesis of lactosucrose from sucrose and lactose by the purified recombinant levansucrase from Leuconostoc mesenteroides B512 FMC. J. Agric. Food Chem. 63: 9755-9763. 
  18. Li WJ, Yu SH, Zhang T, Jiang B, Mu WM. 2015. Recent novel applications of levansucrases. Appl. Microbiol. Biotechnol. 99: 6959-6969. 
  19. El-Refai HA, Abdel-Fattah AF, Mostafa FA. 2009. Enzymic synthesis of levan and fructo-oligosaccharides by Bacillus circulans and improvement of levansucrase stability by carbohydrate coupling. World J. Microbiol. Biotechnol. 25: 821-827. 
  20. Reetz MT, Carballeira JD, Vogel A. 2006. Iterative saturation mutagenesis on the basis of B factors as a strategy for increasing protein thermostability. Angew. Chem. Int. Ed. 45: 7745-7751. 
  21. Sadeghi M, Naderi-Manesh H, Zarrabi M, Ranjbar B. 2006. Effective factors in thermostability of thermophilic proteins. Biophys. Chem. 119: 256-270. 
  22. Sun Z, Liu Q, Qu G, Feng Y, Reetz MT. 2019. Utility of B-factors in protein science: interpreting rigidity, flexibility, and internal motion and engineering thermostability. Chem. Rev. 119: 1626-1665. 
  23. Yu H, Huang H. 2014. Engineering proteins for thermostability through rigidifying flexible sites. Biotechnol. Adv. 32: 308-315. 
  24. Camps J, Carrillo O, Emperador A, Orellana L, Hospital A, Rueda M, et al. 2009. FlexServ: an integrated tool for the analysis of protein flexibility. Bioinformatics 25: 1709-1710. 
  25. Fei B, Xu H, Cao Y, Ma S, Guo H, Song T, et al. 2013. A multi-factors rational design strategy for enhancing the thermostability of Escherichia coli AppA phytase. J. Ind. Microbiol. Biotechnol. 40: 457-464. 
  26. Chen J, Yu H, Liu C, Liu J, Shen Z. 2013. Improving stability of nitrile hydratase by bridging the salt-bridges in specific thermal-sensitive regions. J. Biotechnol. 164: 354-362. 
  27. Fei B, Xu H, Cao Y, Ma S, Guo H, Song T, et al. 2013. A multi-factors rational design strategy for enhancing the thermostability of Escherichia coli AppA phytase. J. Ind. Microbiol. Biotechnol. 40: 457-464. 
  28. Joo JC, Pack SP, Kim YH, Yoo YJ. 2011. Thermostabilization of Bacillus circulans xylanase: computational optimization of unstable residues based on thermal fluctuation analysis. J. Biotechnol. 151: 56-65. 
  29. Chen P, Wang B, Wong HS, Huang DS. 2007. Prediction of protein B-factors using multi-class bounded SVM. Protein Pept. Lett. 14: 185-190. 
  30. Yuan Z, Bailey TL, Teasdale RD. 2005. Prediction of protein B-factor profiles. Proteins Struct. Funct. Bioinform 58: 905-912. 
  31. Pan XY, Shen HB. 2009. Robust prediction of B-factor profile from sequence using two-stage SVR based on random forest feature selection. Protein Pept. Lett. 16: 1447-1454. 
  32. Vander Meersche Y, Cretin G, de Brevern AG, Gelly JC, Galochkina T. 2021. MEDUSA: Prediction of protein flexibility from sequence. J. Mol. Biol. 433: 166882. 
  33. Anonymous. 2009. A new prediction strategy for long local protein structures using an original description. Proteins 76: 570-587. 
  34. Xu W, Peng J, Zhang W, Zhang T, Guang C, Mu W. 2019. Enhancement of the Brenneria sp. levansucrase thermostability by site-directed mutagenesis at Glu404 located at the "-TEAP-" residue motif. J. Biotechnol. 290: 1-9. 
  35. Biedendieck R, Beine R, Gamer M, Jordan E, Buchholz K, Seibel J, et al. 2007. Export, purification, and activities of affinity tagged Lactobacillus reuteri levansucrase produced by Bacillus megaterium. Appl. Microbiol. Biotechnol. 74: 1062-1073. 
  36. Park H, Park NH, Kim M, Lee TH, Lee HG, Yang J, et al. 2003. Enzymatic synthesis of fructosyl oligosaccharides by levansucrase from Microbacterium laevaniformans ATCC 15953. Enzyme Microb. Technol. 32: 820.