Amino Acids

, Volume 51, Issue 4, pp 599–610 | Cite as

Low-resolution structure, oligomerization and its role on the enzymatic activity of a sucrose-6-phosphate hydrolase from Bacillus licheniformis

  • Alain Mera
  • Mariana Zuliani Theodoro de Lima
  • Amanda Bernardes
  • Wanius Garcia
  • João Renato Carvalho MunizEmail author
Original Article


Knowing the key features of the structure and the biochemistry of proteins is crucial to improving enzymes of industrial interest like β-fructofuranosidase. Gene sacA from Bacillus licheniformis ATCC 14580 codifies a sucrose-6-phosphate hydrolase, a β-fructofuranosidase (E.C., protein BlsacA), which has no crystallographic structure available. In this study, we report the results from numerous biochemical and biophysical techniques applied to the investigation of BlsacA in solution. BlsacA was successfully expressed in E. coli in soluble form and purified using affinity and size-exclusion chromatographies. Results showed that the optimum activity of BlsacA occurred at 30 °C around neutrality (pH 6.0–7.5) with a tendency to alkalinity. Circular dichroism spectrum confirmed that BlsacA contains elements of a β-sheet secondary structure at the optimum pH range and the maintenance of these elements is related to BlsacA enzymatic stability. Dynamic light scattering and small-angle X-ray scattering measurements showed that BlsacA forms stable and elongated homodimers which displays negligible flexibility in solution at optimum pH range. The BlsacA homodimeric nature is strictly related to its optimum activity and is responsible for the generation of biphasic curves during differential scanning fluorimetry analyses. The homodimer is formed through the contact of the N-terminal β-propeller domain of each BlsacA unit. The results presented here resemble the key importance of the homodimeric form of BlsacA for the enzyme stability and the optimum enzymatic activity.


Bacillus licheniformis β-Fructofuranosidase Sucrose-6-phosphate hydrolase Biochemical properties 



We would like to thank the National Synchrotron Light Laboratory (LNLS, Brazil) and Central Experimental Multiusuário da Universidade Federal do ABC (CEM/UFABC); Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) for the financial support via Grants # 2017/16291-5 (JRCM); # 2017/17275-3 (WG) São Carlos Institute of Physics/University of Sao Paulo; Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for the financial support via Grants # 309767/2015-6 (JRCM) and 486546/2013-6 (JRCM); and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for fellowship to AM, MZTL.

Compliance with ethical standards

Conflict of interests

All authors of this work declare that they have no potential conflict of interest and that there is no financial, consultant, institutional or other relationships that might lead to bias or conflicts of interest in this research. Financial grants, infrastructure and fellowships supporting this work are described in the acknowledgements section.

Human and animal rights statement

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

Informed consent

Consent to submit this work has been received explicitly from all co-authors, as well as from the institute and the university where the work has been carried out. All authors contributed to the scientific work and, therefore, share collective responsibility and accountability for the results.

Supplementary material

726_2018_2690_MOESM1_ESM.docx (557 kb)
Supplementary material 1 (DOCX 557 kb)


  1. Alberto F, Jordi E, Henrissat B, Czjzek M (2006) Crystal structure of inactivated Thermotoga maritima invertase in complex with the trisaccharide substrate raffinose. Biochem J 395:457–462. CrossRefGoogle Scholar
  2. Alvaro-Benito M, Polo A, Gonzalez B et al (2010) Structural and kinetic analysis of Schwanniomyces occidentalis invertase reveals a new oligomerization pattern and the role of its supplementary domain in substrate binding. J Biol Chem 285:13930–13941. CrossRefGoogle Scholar
  3. Álvaro-Benito M, Sainz-polo MA, González-pérez D et al (2012) Structural and kinetic insights reveal that the Amino Acid Pair Gln-228/Asn-254 Modulates the Transfructosylating specificity of Schwanniomyces occidentalis beta-fructofuranosidase, an enzyme that produces prebiotics. J Biol Chem. Google Scholar
  4. Bujacz A, Jedrzejczak-Krzepkowska M, Bielecki S et al (2011) Crystal structures of the apo form of β-fructofuranosidase from Bifidobacterium longum and its complex with fructose. FEBS J 278:1728–1744. CrossRefGoogle Scholar
  5. Camilo CM, Polikarpov I (2014) High-throughput cloning, expression and purification of glycoside hydrolases using ligation-independent cloning (LIC). Protein Expr Purif 99:35–42. CrossRefGoogle Scholar
  6. Chen C, Huang H, Wu CH (2017) Protein bioinformatics databases and resources. In: Methods in molecular biology (Clifton, N.J.). pp 3–39Google Scholar
  7. Cuskin F, Flint JE, Gloster TM et al (2012) How nature can exploit nonspecific catalytic and carbohydrate binding modules to create enzymatic specificity. Proc Natl Acad Sci USA 109:20889–20894. CrossRefGoogle Scholar
  8. Davies G, Henrissat B (1995) Structures and mechanisms of glycosyl hydrolases. Structure 3:853–859. CrossRefGoogle Scholar
  9. de Oliveira LC, da Silva VM, Colussi F et al (2015) Conformational changes in a hyperthermostable glycoside hydrolase: enzymatic activity is a consequence of the loop dynamics and protonation balance. PLoS ONE 10:e0118225. CrossRefGoogle Scholar
  10. Di Bartolomeo F, Van den Ende W (2015) Fructose and fructans: opposite effects on health? Plant Foods Hum Nutr. Google Scholar
  11. Durand D, Vivès C, Cannella D et al (2010) NADPH oxidase activator p67phox behaves in solution as a multidomain protein with semi-flexible linkers. J Struct Biol 169:45–53. CrossRefGoogle Scholar
  12. Engels V, Georgi T, Wendisch VF (2008) ScrB (Cg2927) is a sucrose-6-phosphate hydrolase essential for sucrose utilization by Corynebacterium glutamicum. FEMS Microbiol Lett. 289:80–89. CrossRefGoogle Scholar
  13. Ericsson UB, Hallberg BM, DeTitta GT et al (2006) Thermofluor-based high-throughput stability optimization of proteins for structural studies. Anal Biochem 357:289–298CrossRefGoogle Scholar
  14. Gimeno-Pérez M, Linde D, Fernández-Arrojo L et al (2015) Heterologous overproduction of β-fructofuranosidase from yeast Xanthophyllomyces dendrorhous, an enzyme producing prebiotic sugars. Appl Microbiol Biotechnol 99:3459–3467. CrossRefGoogle Scholar
  15. Hall M, Rubin J, Behrens SH, Bommarius AS (2011) The cellulose-binding domain of cellobiohydrolase Cel7A from Trichoderma reesei is also a thermostabilizing domain. J Biotechnol 155:370–376. CrossRefGoogle Scholar
  16. Hammersley AP (2016) FIT2D: a multi-purpose data reduction, analysis and visualization program. J Appl Crystallogr 49:646–652. CrossRefGoogle Scholar
  17. Henrissat B (1991) A classification of glycosyl hydrolases based on amino acid sequence similarities. Biochem J 280(Pt 2):309–316CrossRefGoogle Scholar
  18. Henrissat B, Bairoch A (1993) New families in the classification of glycosyl hydrolases based on amino acid sequence similarities. Biochem J 293(Pt 3):781–788CrossRefGoogle Scholar
  19. Henrissat B, Davies G (1997) Structural and sequence-based classification of glycoside hydrolases. Curr Opin Struct Biol 7:637–644CrossRefGoogle Scholar
  20. Jachimska B, Wasilewska M, Adamczyk Z (2008) Characterization of globular protein solutions by dynamic light scattering, electrophoretic mobility, and viscosity measurements. Langmuir 24:6866–6872. CrossRefGoogle Scholar
  21. Jedrzejczak-Krzepkowska M, Tkaczuk KL, Bielecki S (2011) Biosynthesis, purification and characterization of β-fructofuranosidase from Bifidobacterium longum KN29.1. Process Biochem 46:1963–1972. CrossRefGoogle Scholar
  22. Kanehisa M, Goto S (2000) KEGG: kyoto encyclopedia of genes and genomes. Nucleic Acids Res 28:27–30CrossRefGoogle Scholar
  23. Kanehisa M, Sato Y, Kawashima M et al (2016) KEGG as a reference resource for gene and protein annotation. Nucleic Acids Res 44:D457–D462. CrossRefGoogle Scholar
  24. Kanehisa M, Furumichi M, Tanabe M et al (2017) KEGG: new perspectives on genomes, pathways, diseases and drugs. Nucleic Acids Res 45:D353–D361. CrossRefGoogle Scholar
  25. Kim Y, Dementieva I, Zhou M et al (2004) Automation of protein purification for structural genomics. J Struct Funct Genomics 5:111–118. CrossRefGoogle Scholar
  26. Kozin MB, Svergun DI (2001) research papers automated matching of high- and low-resolution structural models research papers. J Appl Crystallogr. 34:33–41. CrossRefGoogle Scholar
  27. Kunst F, Pascal M, Lepesant J-A et al (1974) Purification and some properties of an endocellular sucrase from a constitutive mutant of Bacillus subtilis Marburg 168. Eur J Biochem 42:611–620. CrossRefGoogle Scholar
  28. Lammens W, Le Roy K, Van Laere A et al (2008) Crystal structures of Arabidopsis thaliana cell-wall invertase mutants in complex with sucrose. J Mol Biol 377:378–385. CrossRefGoogle Scholar
  29. Li Y, Ferenci T (1996) The Bacillus stearothermophilus NUB36 surA gene encodes a thermophilic sucrase related to Bacillus subtilis SacA. Microbiology 142:1651–1657CrossRefGoogle Scholar
  30. Lombard V, Golaconda Ramulu H, Drula E et al (2013) The carbohydrate-active enzymes database (CAZy) in 2013. Nucleic Acids Res 42:1–6. Google Scholar
  31. Micsonai A, Wien F, Kernya L et al (2015) Accurate secondary structure prediction and fold recognition for circular dichroism spectroscopy. Proc Natl Acad Sci 112:E3095–E3103. CrossRefGoogle Scholar
  32. Miller GL (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 31(3):426–428CrossRefGoogle Scholar
  33. Mohandesi N, Haghbeen K, Ranaei O et al (2017) Catalytic efficiency and thermostability improvement of Suc2 invertase through rational site-directed mutagenesis. Enzyme Microb Technol 96:14–22. CrossRefGoogle Scholar
  34. Nagaya M, Kimura M, Gozu Y, Sato S, Hirano K, Tochio T et al (2017) Crystal structure of a β-fructofuranosidase with high transfructosylation activity from Aspergillus kawachii. Biosci Biotechnol Biochem 81(9):1786–1795. CrossRefGoogle Scholar
  35. Ohta Y, Hatada Y, Hidaka Y et al (2014) Enhancing thermostability and the structural characterization of Microbacterium saccharophilum K-1 β-fructofuranosidase. Appl Microbiol Biotechnol 98:6667–6677. CrossRefGoogle Scholar
  36. Pascal M, Kunst F, Lepesant J (1971) Characterization of two sucrase activities in Bacillus subtilis Marburg. Biochimie 53:1059–1066CrossRefGoogle Scholar
  37. Phillips K, De la Peña AH (2011) The combined use of the Thermofluor assay and ThermoQ analytical software for the determination of protein stability and buffer optimization as an aid in protein crystallization. Curr Protoc Mol Biol Chapter 10(Unit10):28. Google Scholar
  38. Rambo RP, Tainer JA (2011) Characterizing flexible and intrinsically unstructured biological macromolecules by SAS using the Porod-Debye law. Biopolymers 95(8):559–571. CrossRefGoogle Scholar
  39. Ramírez-Escudero M, Gimeno-pe M, Gonza B et al (2016) Structural Analysis of Fructofuranosidase from Xanthophyllomyces dendrorhous reveals unique features and the crucial role of N-glycosylation in oligomerization and activity. J Biol Chem 291:6843–6857. CrossRefGoogle Scholar
  40. Reid SJ, Abratt VR (2005) Sucrose utilisation in bacteria: genetic organisation and regulation. Appl Microbiol Biotechnol 67:312–321. CrossRefGoogle Scholar
  41. Sainz-polo MA, Ramírez-escudero M, Lafraya A et al (2013) Three-dimensional structure of Saccharomyces invertase role of a non-catalytic domain in oligomerization and substrate. J Biol Chem 288:9755–9766. CrossRefGoogle Scholar
  42. Svergun DI (1992) Determination of the regularization parameter in indirect-transform methods using perceptual criteria. J Appl Crystallogr 25:495–503. CrossRefGoogle Scholar
  43. Svergun DI (1999) Restoring low resolution structure of biological macromolecules from solution scattering using simulated annealing. Biophys J 76:2879–2886. CrossRefGoogle Scholar
  44. Svergun D, Barberato C, Koch MHJ (1995) CRYSOL—a program to evaluate X-ray solution scattering of biological macromolecules from atomic coordinates. J Appl Crystallogr 28:768–773. CrossRefGoogle Scholar
  45. Veith B, Herzberg C, Steckel S et al (2004) The complete genome sequence of Bacillus licheniformis DSM13, an organism with great industrial potential. J Mol Microbiol Biotechnol 7:204–211. CrossRefGoogle Scholar
  46. Voigt B, Schweder T, Dörte B et al (2004) A proteomic view of cell physiology of Bacillus licheniformis. Proteomics 4:1465–1490. CrossRefGoogle Scholar
  47. Volkov VV, Svergun DI (2003) Uniqueness of ab initio shape determination in small-angle scattering. J Appl Cryst 36:860–864CrossRefGoogle Scholar
  48. Wang HL, Shi M, Xu X et al (2016) Effects of Flavomycin, Bacillus licheniformis and enramycin on performance, nutrient digestibility, gut morphology and the intestinal microflora of broilers. J Poult Sci 53:128–135. CrossRefGoogle Scholar
  49. Wanker E, Huber A, Schwab H (1995) Purification and characterization of the Bacillus subtilis levanase produced in Escherichia coli. Appl Environ Microbiol 61:1953–1958Google Scholar
  50. Williams RS, Trumbly RJ, MacColl R et al (1985) Comparative properties of amplified external and internal invertase from the yeast SUC2 gene. J Biol Chem 260(24):13334–13341Google Scholar
  51. Wlodawer A, Minor W, Dauter Z, Jaskolski M (2008) Protein crystallography for non-crystallographers, or how to get the best (but not more) from published macromolecular structures. FEBS J 275:1–21. CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Austria, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Sao Carlos Institute of Physics (IFSC)University of Sao Paulo (USP)São CarlosBrazil
  2. 2.Mackenzie Presbyterian UniversityCampinasBrazil
  3. 3.Centro de Ciências Naturais e HumanasUniversidade Federal do ABC (UFABC)Santo AndréBrazil

Personalised recommendations