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Applied Microbiology and Biotechnology

, Volume 100, Issue 18, pp 8063–8074 | Cite as

Enhanced exo-inulinase activity and stability by fusion of an inulin-binding module

  • Shun-Hua Zhou
  • Yuan Liu
  • Yu-Juan Zhao
  • Zhe Chi
  • Zhen-Ming Chi
  • Guang-Lei Liu
Biotechnologically relevant enzymes and proteins

Abstract

In this study, an inulin-binding module from Bacillus macerans was successfully fused to an exo-inulinase from Kluyveromyces marxianus, creating a hybrid functional enzyme. The recombinant exo-inulinase (rINU), the hybrid enzyme (rINUIBM), and the recombinant inulin-binding module (rIBM) were, respectively, heterologously expressed and biochemically characterized. It was found that both the inulinase activity and the catalytic efficiency (k cat/K m(app)) of the rINUIBM were considerably higher than those of rINU. Though the rINU and the rINUIBM shared the same optimum pH of 4.5, the optimum temperature of the rINUIBM (60 °C) was 5 °C higher than that of the rINU. Notably, the fused IBM significantly enhanced both the pH stability and the thermostability of the rINUIBM, suggesting that the rINUIBM obtained would have more extensive potential applications. Furthermore, the fusion of the IBM could substantially improve the inulin-binding capability of the rINUIBM, which was consistent with the determination of the K m(app). This meant that the fused IBM could play a critical role in the recognition of polysaccharides and enhanced the hydrolase activity of the associated inulinase by increasing enzyme-substrate proximity. Besides, the extra supplement of the independent non-catalytic rIBM could also improve the inulinase activity of the rINU. However, this improvement was much better in case of the fusion. Consequently, the IBM could be designated as a multifunctional domain that was responsible for the activity enhancement, the stabilization, and the substrate binding of the rINUIBM. All these features obtained in this study make the rINUIBM become an attractive candidate for an efficient inulin hydrolysis.

Keywords

Inulin-binding module Exo-inulinase pH stability Thermostability Inulin-binding capability Protein engineering 

Notes

Acknowledgments

This research was supported by the National Natural Science Foundation of China (Grant no. 31400047) and Key Research and Development Plan of Shandong Province (Grant no. 2015GSF121024).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no competing interests.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Supplementary material

253_2016_7587_MOESM1_ESM.pdf (750 kb)
ESM 1 (PDF 749 kb)

References

  1. Boivin S, Kozak S, Meijers R (2013) Optimization of protein purification and characterization using Thermofluor screens. Protein Expr Purif 91(2):192–206. doi: 10.1016/j.pep.2013.08.002 CrossRefPubMedGoogle Scholar
  2. Boraston AB, Bolam DN, Gilbert HJ, Davies GJ (2004) Carbohydrate-binding modules: fine-tuning polysaccharide recognition. Biochem J 382(Pt 3):769–781. doi: 10.1042/BJ20040892 CrossRefPubMedPubMedCentralGoogle Scholar
  3. Chi ZM, Zhang T, Cao TS, Liu XY, Cui W, Zhao CH (2011) Biotechnological potential of inulin for bioprocesses. Bioresour Technol 102(6):4295–4303. doi: 10.1016/j.biortech.2010.12.086 CrossRefPubMedGoogle Scholar
  4. Codera V, Gilbert HJ, Faijes M, Planas A (2015) Carbohydrate-binding module assisting glycosynthase-catalysed polymerizations. Biochem J 470:15–22. doi: 10.1042/Bj20150420 CrossRefPubMedGoogle Scholar
  5. Foumani M, Vuong TV, MacCormick B, Master ER (2015) Enhanced polysaccharide binding and activity on linear beta-glucans through addition of carbohydrate-binding modules to either terminus of a glucooligosaccharide oxidase. PLoS One 10(5):e0125398. doi: 10.1371/journal.pone.0125398 CrossRefPubMedPubMedCentralGoogle Scholar
  6. Gao J, Yuan W, Li Y, Xiang R, Hou S, Zhong S, Bai F (2015) Transcriptional analysis of Kluyveromyces marxianus for ethanol production from inulin using consolidated bioprocessing technology. Biotechnol Biofuels 8:115. doi: 10.1186/s13068-015-0295-y CrossRefPubMedPubMedCentralGoogle Scholar
  7. Gietz RD, Schiestl RH (2007) High-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method. Nat Protoc 2(1):31–34. doi: 10.1038/nprot.2007.13 CrossRefPubMedGoogle Scholar
  8. Gilbert HJ, Knox JP, Boraston AB (2013) Advances in understanding the molecular basis of plant cell wall polysaccharide recognition by carbohydrate-binding modules. Curr Opin Struct Biol 23(5):669–677. doi: 10.1016/j.sbi.2013.05.005 CrossRefPubMedGoogle Scholar
  9. Gusakov AV, Kondratyeva EG, Sinitsyn AP (2011) Comparison of two methods for assaying reducing sugars in the determination of carbohydrase activities. Int J Anal Chem 2011:283658. doi: 10.1155/2011/283658 CrossRefPubMedPubMedCentralGoogle Scholar
  10. He M, Wu D, Wu J, Chen J (2014) Enhanced expression of endoinulinase from Aspergillus niger by codon optimization in Pichia pastoris and its application in inulooligosaccharide production. J Ind Microbiol Biot 41(1):105–114. doi: 10.1007/s10295-013-1341-z CrossRefGoogle Scholar
  11. Hong SJ, Kim HJ, Kim JW, Lee DH, Seo JH (2015) Optimizing promoters and secretory signal sequences for producing ethanol from inulin by recombinant Saccharomyces cerevisiae carrying Kluyveromyces marxianus inulinase. Bioprocess Biosyst Eng 38(2):263–272. doi: 10.1007/s00449-014-1265-7 CrossRefPubMedGoogle Scholar
  12. Isaksen T, Westereng B, Aachmann FL, Agger JW, Kracher D, Kittl R, Ludwig R, Haltrich D, Eijsink VGH, Horn SJ (2014) A C4-oxidizing lytic polysaccharide monooxygenase cleaving both cellulose and cello-oligosaccharides. J Biol Chem 289(5):2632–2642. doi: 10.1074/jbc.M113.530196 CrossRefPubMedGoogle Scholar
  13. Jun H, Bing Y, Keying Z, Xuemei D, Daiwen C (2009) Thermostable carbohydrate binding module increases the thermostability and substrate-binding capacity of Trichoderma reesei xylanase 2. New Biotechnol 26(1–2):53–59. doi: 10.1016/j.nbt.2009.04.002 CrossRefGoogle Scholar
  14. Kango N, Jain SC (2011) Production and properties of microbial inulinases: recent advances. Food Biotechnol 25(3):165–212. doi: 10.1080/08905436.2011.590763 CrossRefGoogle Scholar
  15. Karimi M, Habibi-Rezaei M, Safari M, Moosavi-Movahedi AA, Sayyah M, Sadeghi R, Kokini J (2014) Immobilization of endo-inulinase on poly-D-lysine coated CaCO3 micro-particles. Food Res Int 66:485–492. doi: 10.1016/j.foodres.2014.08.041 CrossRefGoogle Scholar
  16. Lee JH, Kim KN, Choi YJ (2004) Identification and characterization of a novel inulin binding module (IBM) from the CFTase of Bacillus macerans CFC1. FEMS Microbiol Lett 234(1):105–110. doi: 10.1016/j.femsle.2004.03.013 CrossRefPubMedGoogle Scholar
  17. Leufken CM, Moerschbacher BM, Dirks-Hofmeister ME (2015) Dandelion PPO-1/PPO-2 domain-swaps: the C-terminal domain modulates the pH optimum and the linker affects SDS-mediated activation and stability. Biochim Biophys Acta 1854(2):178–186. doi: 10.1016/j.bbapap.2014.11.007 CrossRefPubMedGoogle Scholar
  18. Liu GL, Chi Z, Chi ZM (2013) Molecular characterization and expression of microbial inulinase genes. Crit Rev Microbiol 39(2):152–165. doi: 10.3109/1040841X.2012.694411 CrossRefPubMedGoogle Scholar
  19. Liu GL, Fu GY, Chi Z, Chi ZM (2014) Enhanced expression of the codon-optimized exo-inulinase gene from the yeast Meyerozyma guilliermondii in Saccharomyces sp. W0 and bioethanol production from inulin. Appl Microbiol Biotechnol 98(21):9129–9138. doi: 10.1007/s00253-014-6079-7 CrossRefPubMedGoogle Scholar
  20. Liu LW, Cheng J, Chen HG, Li XQ, Wang SY, Song AD, Wang MD, Wang B, Shen JW (2011) Directed evolution of a mesophilic fungal xylanase by fusion of a thermophilic bacterial carbohydrate-binding module. Process Biochem 46(1):395–398. doi: 10.1016/j.procbio.2010.07.026 CrossRefGoogle Scholar
  21. Liu XY, Chi Z, Liu GL, Wang F, Madzak C, Chi ZM (2010) Inulin hydrolysis and citric acid production from inulin using the surface-engineered Yarrowia lipolytica displaying inulinase. Metab Eng 12(5):469–476. doi: 10.1016/j.ymben.2010.04.004 CrossRefPubMedGoogle Scholar
  22. Lu WD, Li AX, Guo QL (2014) Production of novel alkalitolerant and thermostable inulinase from marine actinomycete Nocardiopsis sp. DN-K15 and inulin hydrolysis by the enzyme. Ann Microbiol 64(2):441–449. doi: 10.1007/s13213-013-0674-1 CrossRefGoogle Scholar
  23. Lyu QQ, Wang S, Xu WH, Han BQ, Liu WS, Junes DNM, Liu WZ (2014) Structural insights into the substrate-binding mechanism for a novel chitosanase. Biochem J 461:335–345. doi: 10.1042/Bj20140159 CrossRefPubMedGoogle Scholar
  24. Madzak C (2015) Yarrowia lipolytica: recent achievements in heterologous protein expression and pathway engineering. Appl Microbiol Biotechnol 99(11):4559–4577. doi: 10.1007/s00253-015-6624-z CrossRefPubMedGoogle Scholar
  25. Mai-Gisondi G, Turunen O, Pastinen O, Pahimanolis N, Master ER (2015) Enhancement of acetyl xylan esterase activity on cellulose acetate through fusion to a family 3 cellulose binding module. Enzym Microb Technol 79-80:27–33. doi: 10.1016/j.enzmictec.2015.07.001 CrossRefGoogle Scholar
  26. Niesen FH, Berglund H, Vedadi M (2007) The use of differential scanning fluorimetry to detect ligand interactions that promote protein stability. Nat Protoc 2(9):2212–2221. doi: 10.1038/nprot.2007.321 CrossRefPubMedGoogle Scholar
  27. Oliveira C, Carvalho V, Domingues L, Gama FM (2015) Recombinant CBM-fusion technology—applications overview. Biotechnol Adv 33(3–4):358–369. doi: 10.1016/j.biotechadv.2015.02.006 CrossRefPubMedGoogle Scholar
  28. Qiao WB, Tang SG, Mi SF, Jia XJ, Peng XW, Han YJ (2014) Biochemical characterization of a novel thermostable GH11 xylanase with CBM6 domain from Caldicellulosiruptor kronotskyensis. J Mol Catal B Enzym 107:8–16. doi: 10.1016/j.molcatb.2014.05.009 CrossRefGoogle Scholar
  29. Quinlan RJ, Sweeney MD, Lo Leggio L, Otten H, Poulsen JCN, Johansen KS, Krogh KBRM, Jorgensen CI, Tovborg M, Anthonsen A, Tryfona T, Walter CP, Dupree P, Xu F, Davies GJ, Walton PH (2011) Insights into the oxidative degradation of cellulose by a copper metalloenzyme that exploits biomass components. Proc Natl Acad Sci U S A 108(37):15079–15084. doi: 10.1073/pnas.1105776108 CrossRefPubMedPubMedCentralGoogle Scholar
  30. Sharma AD, Gill PK (2007) Purification and characterization of heat-stable exo-inulinase from Streptomyces sp. J Food Eng 79(4):1172–1178. doi: 10.1016/j.jfoodeng.2006.04.008 CrossRefGoogle Scholar
  31. Tang ZZ, Chen H, Chen LJ, Liu S, Han XY, Wu Q (2014) Improving endoglucanase activity by adding the carbohydrate-binding module from Corticium rolfsii. J Microbiol Biotechnol 24(4):440–446. doi: 10.4014/jmb.1311.11007 CrossRefPubMedGoogle Scholar
  32. Telke AA, Ghatge SS, Kang SH, Thangapandian S, Lee KW, Shin HD, Um Y, Kim SW (2012) Construction and characterization of chimeric cellulases with enhanced catalytic activity towards insoluble cellulosic substrates. Bioresour Technol 112:10–17. doi: 10.1016/j.biortech.2012.02.066 CrossRefPubMedGoogle Scholar
  33. Torabizadeh H, Habibi-Rezaei M, Safari M, Moosavi-Movahedi AA, Sharifizadeh A, Azizian H, Amanlou M (2011) Endo-inulinase stabilization by pyridoxal phosphate modification: a kinetics, thermodynamics, and simulation approach. Appl Biochem Biotechnol 165(7–8):1661–1673. doi: 10.1007/s12010-011-9385-x CrossRefPubMedGoogle Scholar
  34. Vaaje-Kolstad G, Bohle LA, Gaseidnes S, Dalhus B, Bjoras M, Mathiesen G, Eijsink VGH (2012) Characterization of the chitinolytic machinery of Enterococcus faecalis V583 and high-resolution structure of its oxidative CBM33 enzyme. J Mol Biol 416(2):239–254. doi: 10.1016/j.jmb.2011.12.033 CrossRefPubMedGoogle Scholar
  35. Wang YG, Tang RT, Tao J, Gao G, Wang XN, Mu Y, Feng Y (2011) Quantitative investigation of non-hydrolytic disruptive activity on crystalline cellulose and application to recombinant swollenin. Appl Microbiol Biotechnol 91(5):1353–1363. doi: 10.1007/s00253-011-3421-1 CrossRefPubMedGoogle Scholar
  36. Wilkins MR, Gasteiger E, Bairoch A, Sanchez JC, Williams KL, Appel RD, Hochstrasser DF (1999) Protein identification and analysis tools in the ExPASy server. Methods Mol Biol 112:531–552PubMedGoogle Scholar
  37. Zemolin GP, Gazoni M, Zabot GL, Golunski SM, Astolfi V, Dal Pra V, Foletto EL, Meili L, Da Rosa MB, Rosa CD, Mossi AJ, Treichel H, Mazutti MA (2012) Immobilization of inulinase obtained by solid-state fermentation using spray-drying technology. Biocatal Biotransformation 30(4):409–416. doi: 10.3109/10242422.2012.715635 CrossRefGoogle Scholar
  38. Zhao CH, Cui W, Liu XY, Chi ZM, Madzak C (2010) Expression of inulinase gene in the oleaginous yeast Yarrowia lipolytica and single cell oil production from inulin-containing materials. Metab Eng 12(6):510–517. doi: 10.1016/j.ymben.2010.09.001 CrossRefPubMedGoogle Scholar
  39. Zhou HX, Xin FH, Chi Z, Liu GL, Chi ZM (2014) Inulinase production by the yeast Kluyveromyces marxianus with the disrupted MIG1 gene and the over-expressed inulinase gene. Process Biochem 49(11):1867–1874. doi: 10.1016/j.procbio.2014.08.001 CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  • Shun-Hua Zhou
    • 1
  • Yuan Liu
    • 1
  • Yu-Juan Zhao
    • 1
  • Zhe Chi
    • 1
  • Zhen-Ming Chi
    • 1
  • Guang-Lei Liu
    • 1
  1. 1.College of Marine Life ScienceOcean University of ChinaQingdaoChina

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