The production, properties, and applications of thermostable steryl glucosidases

  • Andres Aguirre
  • Florencia Eberhardt
  • Guillermo Hails
  • Sebastian Cerminati
  • María Eugenia Castelli
  • Rodolfo M. Rasia
  • Luciana Paoletti
  • Hugo G. Menzella
  • Salvador Peiru


Extremophilic microorganisms are a rich source of enzymes, the enzymes which can serve as industrial catalysts that can withstand harsh processing conditions. An example is thermostable β-glucosidases that are addressing a challenging problem in the biodiesel industry: removing steryl glucosides (SGs) from biodiesel. Steryl glucosidases (SGases) must be tolerant to heat and solvents in order to function efficiently in biodiesel. The amphipathic nature of SGs also requires enzymes with an affinity for water/solvent interfaces in order to achieve efficient hydrolysis. Additionally, the development of an enzymatic process involving a commodity such as soybean biodiesel must be cost-effective, necessitating an efficient manufacturing process for SGases. This review summarizes the identification of microbial SGases and their applications, discusses biodiesel refining processes and the development of analytical methods for identifying and quantifying SGs in foods and biodiesel, and considers technologies for strain engineering and process optimization for the heterologous production of a SGase from Thermococcus litoralis. All of these technologies might be used for the production of other thermostable enzymes. Structural features of SGases and the feasibility of protein engineering for novel applications are explored.


Extremozymes Biofuels Steryl glucosidases 



The funding was provided by ANCYT PICT (Grant Nos. 2010-1157, 2013-2134, 2013-2726).


  1. Adams MW, Perler FB, Kelly RM (1995) Extremozymes: expanding the limits of biocatalysis. Nat Biotechnol 13:662–668CrossRefGoogle Scholar
  2. Aguirre A, Peiru S, Eberhardt F, Vetcher L, Cabrera R, Menzella HG (2014) Enzymatic hydrolysis of steryl glucosides, major contaminants of vegetable oil-derived biodiesel. Appl Microbiol Biotechnol 98:4033–4040. CrossRefGoogle Scholar
  3. Aguirre A, Cabruja M, Cabrera R, Eberhardt F, Peirú S, Menzella HG, Rasia RM (2015) A fluorometric enzymatic assay for quantification of steryl glucosides in biodiesel. J Am Oil Chem Soc 92:47–53CrossRefGoogle Scholar
  4. Akiba T, Nishio M, Matsui I, Harata K (2004) X-ray structure of a membrane-bound beta-glycosidase from the hyperthermophilic archaeon Pyrococcus horikoshii. Proteins 57:422–431. CrossRefGoogle Scholar
  5. Aloulou A, Rodriguez JA, Fernandez S, van Oosterhout D, Puccinelli D, Carrière F (2006) Exploring the specific features of interfacial enzymology based on lipase studies. Biochim Biophys Acta (BBA)-Mol Cell Biol Lipids 1761:995–1013CrossRefGoogle Scholar
  6. Al-Zuhair S, Ramachandran K, Hasan M (2008) Effect of enzyme molecules covering of oil–water interfacial area on the kinetic of oil hydrolysis. Chem Eng J 139:540–548CrossRefGoogle Scholar
  7. An DS et al (2010) Identification and characterization of a novel Terrabacter ginsenosidimutans sp. nov. beta-glucosidase that transforms ginsenoside Rb1 into the rare gypenosides XVII and LXXV. Appl Environ Microbiol 76:5827–5836. CrossRefGoogle Scholar
  8. Anbarasan S, Timoharju T, Barthomeuf J, Pastinen O, Rouvinen J, Leisola M, Turunen O (2015) Effect of active site mutation on pH activity and transglycosylation of Sulfolobus acidocaldarius β-glycosidase. J Mol Catal B: Enzym 118:62–69CrossRefGoogle Scholar
  9. Badenes SM, Lemos F, Cabral JM (2011) Kinetics and mechanism of the cutinase-catalyzed transesterification of oils in AOT reversed micellar system. Bioprocess Biosyst Eng 34:1133–1142CrossRefGoogle Scholar
  10. Bernaudat F et al. (2011) Heterologous expression of membrane proteins: choosing the appropriate host. PLoS ONE 6:e29191CrossRefGoogle Scholar
  11. Bouic PJ, Etsebeth S, Liebenberg RW, Albrecht CF, Pegel K, Van Jaarsveld PP (1996) beta-Sitosterol and beta-sitosterol glucoside stimulate human peripheral blood lymphocyte proliferation: implications for their use as an immunomodulatory vitamin combination. Int J Immunopharmacol 18:693–700CrossRefGoogle Scholar
  12. Brask J, Nielsen R (2010) Enzymatic removal of steryl glycosides in fatty acid alkyl esters WO2010102952 A1Google Scholar
  13. Camerlynck S, Chandler J, Hornby B, van Zuylen I (2012) FAME filterability: understanding and solutions SAE. Int J Fuels Lubr 5:968–976CrossRefGoogle Scholar
  14. Davies G, Henrissat B (1995) Structures and mechanisms of glycosyl hydrolases. Structure 3:853–859 CrossRefGoogle Scholar
  15. Deive FJ, López E, Rodriguez A, Longo MA, Sanromán M (2012) Targeting the production of biomolecules by extremophiles at bioreactor scale. Chem Eng Technol 35:1565–1575CrossRefGoogle Scholar
  16. Doukyu N, Ogino H (2010) Organic solvent-tolerant enzymes. Biochem Eng J 48:270–282CrossRefGoogle Scholar
  17. Eberhardt F, Aguirre A, Menzella HG, Peiru S (2017) Strain engineering and process optimization for enhancing the production of a thermostable steryl glucosidase in Escherichia coli. J Ind Microbiol Biotechnol 44:141–147. CrossRefGoogle Scholar
  18. Eberhardt F et al. (2018) Pilot-scale process development for low-cost production of a thermostable biodiesel refining enzyme in Escherichia coli. Bioprocess Biosyst Eng 1–10Google Scholar
  19. Elleuche S, Schroder C, Sahm K, Antranikian G (2014) Extremozymes–biocatalysts with unique properties from extremophilic microorganisms. Curr Opin Biotechnol 29:116–123. CrossRefGoogle Scholar
  20. Gómez-Coca RB, Pérez-Camino MdC, Moreda W (2012) Specific procedure for analysing steryl glucosides in olive oil European. J Lipid Sci Technol 114:1417–1426CrossRefGoogle Scholar
  21. Gupta A, Khare S (2009) Enzymes from solvent-tolerant microbes: useful biocatalysts for non-aqueous enzymology. Crit Rev Biotechnol 29:44–54CrossRefGoogle Scholar
  22. Hermansyah H, Kubo M, Shibasaki-Kitakawa N, Yonemoto T (2006) Mathematical model for stepwise hydrolysis of triolein using Candida rugosa lipase in biphasic oil–water system. Biochem Eng J 31:125–132CrossRefGoogle Scholar
  23. Hermansyah H, Wijanarko A, Kubo M, Shibasaki-Kitakawa N, Yonemoto T (2010) Rigorous kinetic model considering positional specificity of lipase for enzymatic stepwise hydrolysis of triolein in biphasic oil–water system. Bioprocess Biosyst Eng 33:787–796CrossRefGoogle Scholar
  24. Isorna P, Polaina J, Latorre-Garcia L, Canada FJ, Gonzalez B, Sanz-Aparicio J (2007) Crystal structures of Paenibacillus polymyxa beta-glucosidase B complexes reveal the molecular basis of substrate specificity and give new insights into the catalytic machinery of family I glycosidases. J Mol Biol 371:1204–1218. CrossRefGoogle Scholar
  25. Junge F, Schneider B, Reckel S, Schwarz D, Dötsch V, Bernhard F (2008) Large-scale production of functional membrane proteins. Cell Mol Life Sci 65:1729–1755CrossRefGoogle Scholar
  26. Kalinowska M, Wojciechowski ZA (1978) Purification and some properties of steryl beta-D-glucoside hydrolase from Sinapis alba seedlings. Phytochemistry 17:1533–1537CrossRefGoogle Scholar
  27. Lacoste F, Dejean F, Griffon H, Rouquette C (2009) Quantification of free and esterified steryl glucosides in vegetable oils and biodiesel European. J Lipid Sci Technol 111:822–828. CrossRefGoogle Scholar
  28. Lee I, Pfalzgraf L, Poppe G, Powers E, Haines T (2007) The role of sterol glucosides on filter plugging. Biodiesel Mag 4:105–112Google Scholar
  29. Leung DY, Wu X, Leung M (2010) A review on biodiesel production using catalyzed transesterification. Appl Energy 87:1083–1095CrossRefGoogle Scholar
  30. Lin X, Ma L, Racette SB, Anderson Spearie CL, Ostlund RE Jr (2009) Phytosterol glycosides reduce cholesterol absorption in humans. Am J Physiol Gastrointest Liver Physiol 296:G931-935. Google Scholar
  31. Lombard V, Golaconda Ramulu H, Drula E, Coutinho PM, Henrissat B (2013) The carbohydrate-active enzymes database (CAZy) in 2013. Nucleic Acids Res 42:D490–D495CrossRefGoogle Scholar
  32. Matsui I, Sakai Y, Matsui E, Kikuchi H, Kawarabayasi Y, Honda K (2000) Novel substrate specificity of a membrane-bound beta-glycosidase from the hyperthermophilic archaeon Pyrococcus horikoshii. FEBS Lett 467:195–200CrossRefGoogle Scholar
  33. Menzella H, Peiru S, Vetcher L (2012) Enzymatic Removal of Steryl Glycosides PCT/US2013/031769Google Scholar
  34. Middelberg AP, O’Neill BK, ID LB, Snoswell MA (1991) A novel technique for the measurement of disruption in high-pressure homogenization: studies on E. coli containing recombinant inclusion bodies. Biotechnol Bioeng 38:363–370. CrossRefGoogle Scholar
  35. Miroux B, Walker JE (1996) Over-production of proteins in Escherichia coli: mutant hosts that allow synthesis of some membrane proteins and globular proteins at high levels. J Mol Biol 260(3):289–298CrossRefGoogle Scholar
  36. Munger LH, Nystrom L (2014) Enzymatic hydrolysis of steryl glycosides for their analysis in foods. Food Chem 163:202–211. CrossRefGoogle Scholar
  37. Na-Ranong D, Kitchaiya P (2014) Precipitation above cloud point in palm oil based biodiesel during production and storage. Fuel 122:287–293. CrossRefGoogle Scholar
  38. Noh KH, Oh DK (2009) Production of the rare ginsenosides compound K, compound Y, and compound Mc by a thermostable beta-glycosidase from Sulfolobus acidocaldarius. Biol Pharm Bull 32:1830–1835CrossRefGoogle Scholar
  39. Noh KH, Son JW, Kim HJ, Oh DK (2009) Ginsenoside compound K production from ginseng root extract by a thermostable beta-glycosidase from Sulfolobus solfataricus. Biosci Biotechnol Biochem 73:316–321CrossRefGoogle Scholar
  40. Nystrom L (2008) Enzymatic hydrolisis of steryl ferulates and steryl glycosides. Eur Food Res Technol 227:727–733CrossRefGoogle Scholar
  41. Oppliger SR, Munger LH, Nystrom L (2014) Rapid and highly accurate detection of steryl glycosides by ultraperformance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS). J Agric Food Chem 62:9410–9419. CrossRefGoogle Scholar
  42. Peiru S, Aguirre A, Eberhardt F, Braia M, Cabrera R, Menzella HG (2015) An industrial scale process for the enzymatic removal of steryl glucosides from biodiesel. Biotechnol Biofuels 8:223. CrossRefGoogle Scholar
  43. Pfalzgraf L, Lee I, Foster J, Poppe G (2007) The effect of minor components on cloud point and filterability inform. Suppl Biorenewable Resour 4:17–21Google Scholar
  44. Plata V, Gauthier-Maradei P, Kafarov V (2014) Influence of minor components on precipitate formation and filterability of palm oil biodiesel. Fuel 144:130–136CrossRefGoogle Scholar
  45. Reis P, Holmberg K, Watzke H, Leser M, Miller R (2009) Lipases at interfaces: a review. Adv Colloid Interface Sci 147:237–250CrossRefGoogle Scholar
  46. Ringwald SC (2007) Biodiesel characterization in the QC environment. The 98th AOCS Annual Meeting Abstracts AOCS Press, Urbana:15Google Scholar
  47. Rozzell JD (1999) Commercial scale biocatalysis: myths and realities. Bioorg Med Chem 7:2253–2261CrossRefGoogle Scholar
  48. Sarmiento F, Peralta R, Blamey JM (2015) Cold and hot extremozymes: industrial relevance and current trends. Front Bioeng Biotechnol 3:148 CrossRefGoogle Scholar
  49. Schröter S, Stahmann K-P, Schnitzlein K (2015) Impact of mass transport on the enzymatic hydrolysis of rapeseed oil. Appl Microbiol Biotechnol 99:293–300CrossRefGoogle Scholar
  50. Soe JB (2010) Method WO 2010004423 p A2Google Scholar
  51. Sugawara T, Miyazawa T (1999) Separation and determination of glycolipids from edible plant sources by high-performance liquid chromatography and evaporative light- scattering detection Lipids 34:1231–1237CrossRefGoogle Scholar
  52. Tang H, De Guzman R, Salley S, Simon Ng KY (2010) Comparing process efficiency in reducing steryl glucosides in biodiesel. J Am Oil Chem Soc 87:337–345CrossRefGoogle Scholar
  53. Umetsu M, Tsumoto K, Ashish K, Nitta S, Tanaka Y, Adschiri T, Kumagai I (2004) Structural characteristics and refolding of in vivo aggregated hyperthermophilic archaeon proteins. FEBS Lett 557:49–56CrossRefGoogle Scholar
  54. Van Hoed V, Zyaykina N, de Greyt W, Maes J, Verhé R, Demeestere K (2008) Identification and occurrence of steryl glucosides in palm and soy biodiesel. J Am Oil Chem Soc 85:701–709CrossRefGoogle Scholar
  55. Verger R, De Haas GH (1976) Interfacial enzyme kinetics of lipolysis. Ann Rev Biophys Bioeng 5:77–117CrossRefGoogle Scholar
  56. Webb B, Sali A (2014) Protein structure modeling with MODELLER. In: Protein structure prediction. Springer, New York, pp 1–15Google Scholar

Copyright information

© Springer Science+Business Media B.V., part of Springer Nature 2018

Authors and Affiliations

  • Andres Aguirre
    • 1
    • 2
  • Florencia Eberhardt
    • 1
  • Guillermo Hails
    • 1
  • Sebastian Cerminati
    • 1
  • María Eugenia Castelli
    • 1
  • Rodolfo M. Rasia
    • 3
  • Luciana Paoletti
    • 1
  • Hugo G. Menzella
    • 1
    • 2
  • Salvador Peiru
    • 1
    • 2
  1. 1.Instituto de Procesos Biotecnológicos y Químicos (IPROBYQ), Facultad de Ciencias Bioquímicas y FarmacéuticasUniversidad Nacional de Rosario (UNR), CONICETRosarioArgentina
  2. 2.Keclon S.A.RosarioArgentina
  3. 3.Instituto de Biología Molecular y Celular de Rosario (IBR-CONICET-UNR), Area Biofísica, Facultad de Ciencias Bioquímicas y FarmacéuticasUniversidad Nacional de Rosario, Ocampo y Esmeralda, predio CONICETRosarioArgentina

Personalised recommendations