Enhanced exo-inulinase activity and stability by fusion of an inulin-binding module
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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.
KeywordsInulin-binding module Exo-inulinase pH stability Thermostability Inulin-binding capability Protein engineering
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.
This article does not contain any studies with human participants or animals performed by any of the authors.
- 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
- 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
- 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
- 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
- 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
- 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
- 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
- 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
- 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
- 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