Applied Microbiology and Biotechnology

, Volume 99, Issue 1, pp 301–308 | Cite as

High-level expression and characterization of recombinant acid urease for enzymatic degradation of urea in rice wine

Biotechnologically relevant enzymes and proteins


Ethylcarbamate, a carcinogenic compound, is formed from urea and ethanol in rice wine, and enzymatic elimination of urea is always attractive. In the present work, we amplified the acid urease gene cluster ureABCEFGD from Lactobacillus reuteri CICC6124 and constructed robust Lactococcus lactis cell factories for the production of acid urease. The titer of the recombinant acid urease was increased from 1,550 to 11,560 U/L by optimization of the cultivation process. Meanwhile, the enzyme showed satisfied properties toward urea elimination in the rice wine model system. By incubating the enzyme (50 U/L) at 20 °C for 60 h, about 95.8 % of urea in rice wine was removed. Interestingly, this acid urease also exhibited activity toward ethylcarbamate. The results demonstrated that this recombinant acid urease has great potential in the elimination of urea in rice wine.


Acid urease Lactococcus lactis Lactobacillus reuteri Nisin-induced system 


  1. Andrich L, Esti M, Moresi M (2010) Urea degradation in some white wines by immobilized acid urease in a stirred bioreactor. J Agric Food Chem 58:6747–6753PubMedCrossRefGoogle Scholar
  2. Battaglia R, Conacher HB, Page BD (1990) Ethyl carbamate (urethane) in alcoholic beverages and foods: a review. Food Addit Contam 7:477–496PubMedCrossRefGoogle Scholar
  3. Chen YY, Clancy KA, Burne RA (1996) Streptococcus salivarius urease: genetic and biochemical characterization and expression in a dental plaque Streptococcus. Infect Immun 64:585–592PubMedCentralPubMedGoogle Scholar
  4. Dahabieh MS, Husnik JI, Van Vuuren HJ (2010) Functional enhancement of sake yeast strains to minimize the production of ethylcarbamate in sake wine. J Appl Microbiol 109:963–973PubMedCrossRefGoogle Scholar
  5. de Ruyter PG, Kuipers OP, de Vos WM (1996) Controlled gene expression systems for Lactococcus lactis with the food-grade inducer nisin. Appl Environ Microbiol 62:3662–3667PubMedCentralPubMedGoogle Scholar
  6. de Vos WM (1999) Gene expression systems for lactic acid bacteria. Curr Opin Microbiol 2:289–295PubMedCrossRefGoogle Scholar
  7. Esti M, Fidaleo M, Moresi M, Tamborra P (2007) Modeling of urea degradation in white and rose wines by acid urease. J Agric Food Chem 55:2590–2596PubMedCrossRefGoogle Scholar
  8. Fidaleo M, Esti M, Moresi M (2006) Assessment of urea degradation rate in model wine solutions by acid urease from Lactobacillus fermentum. J Agric Food Chem 54:6226–6235PubMedCrossRefGoogle Scholar
  9. Hu LT, Mobley HLT (1993) Expression of catalytically active recombinant helicobacter-pylori urease at wild-type levels in Escherichia coli. Infect Immun 61:2563–2569PubMedCentralPubMedGoogle Scholar
  10. Jabri E, Carr MB, Hausinger RP, Karplus PA (1995) The crystal structure of urease from Klebsiella aerogenes. Science 268:998–1004PubMedCrossRefGoogle Scholar
  11. Kakimoto S, Sumino Y, Kawahara K, Yamazaki E, Nakatsui I (1989) Purification and characterization of acid urease from Lactobacillus reuteri. Agric Biol Chem 53:1119–1125CrossRefGoogle Scholar
  12. Kakimoto S, Sumino Y, Kawahara K, Yamazaki E, Nakatsui I (1990a) Properties of acid ureases from Lactobacillus and Streptococcus strains. Agric Biol Chem 54:381–386CrossRefGoogle Scholar
  13. Kakimoto S, Sumino Y, Kawahara K, Yamazaki E, Nakatsui I (1990b) Purification and characterization of acid urease from Lactobacillus fermentum. Appl Microbiol Biotechnol 32:538–543PubMedCrossRefGoogle Scholar
  14. Kleerebezem M, Beerthuyzen MM, Vaughan EE, de Vos WM, Kuipers OP (1997) Controlled gene expression systems for lactic acid bacteria: transferable nisin-inducible expression cassettes for Lactococcus, Leuconostoc, and Lactobacillus spp. Appl Environ Microbiol 63:4581–4584PubMedCentralPubMedGoogle Scholar
  15. Kuipers OP, de Ruyter PGGA, Kleerebezem M, de Vos WM (1998) Quorum sensing-controlled gene expression in lactic acid bacteria. J Biotechnol 64:15–21CrossRefGoogle Scholar
  16. Liang X, Zhang L, Zhong J, Huan L (2007) Secretory expression of a heterologous nattokinase in Lactococcus lactis. Appl Microbiol Biotechnol 75:95–101PubMedCrossRefGoogle Scholar
  17. Liu J, Xu Y, Nie Y, Zhao GA (2012) Optimization production of acid urease by Enterobacter sp in an approach to reduce urea in Chinese rice wine. Bioproc Biosyst Eng 35:651–657CrossRefGoogle Scholar
  18. Maischberger T, Mierau I, Peterbauer CK, Hugenholtz J, Haltrich D (2010) High-level expression of Lactobacillus beta-galactosidases in Lactococcus lactis using the food-Grade, nisin-controlled expression system NICE. J Agric Food Chem 58:2279–2287PubMedCrossRefGoogle Scholar
  19. Mierau I, Kleerebezem M (2005) 10 years of the nisin-controlled gene expression system (NICE) in Lactococcus lactis. Appl Microbiol Biotechnol 68:705–717PubMedCrossRefGoogle Scholar
  20. Mierau I, Leij P, van Swam I, Blommestein B, Floris E, Mond J, Smid EJ (2005) Industrial-scale production and purification of a heterologous protein in Lactococcus lactis using the nisin-controlled gene expression system NICE: The case of lysostaphin. Microb Cell Factories 4:15CrossRefGoogle Scholar
  21. Miyagawa K, Sumida M, Nakao M, Harada M, Yamamoto H, Kusumi T, Yoshizawa K, Amachi T, Nakayama T (1999) Purification, characterization, and application of an acid urease from Arthrobacter mobilis. J Biotechnol 68:227–236PubMedCrossRefGoogle Scholar
  22. Mobley HL, Hausinger RP (1989) Microbial ureases: significance, regulation, and molecular characterization. Microbiol Rev 53:85–108PubMedCentralPubMedGoogle Scholar
  23. Mobley HL, Island MD, Hausinger RP (1995) Molecular biology of microbial ureases. Microbiol Rev 59:451–480PubMedCentralPubMedGoogle Scholar
  24. Mohapatra BR, Bapuji M (1997) Characterization of urethanase from Micrococcus species associated with the marine sponge (Spirastrella species). Lett Appl Microbiol 25:393–396CrossRefGoogle Scholar
  25. Mora D, Fortina MG, Parini C, Ricci G, Gatti M, Giraffa G, Manachini PL (2002) Genetic diversity and technological properties of Streptococcus thermophilus strains isolated from dairy products. J Appl Microbiol 93:278–287PubMedCrossRefGoogle Scholar
  26. Mora D, Maguin E, Masiero M, Parini C, Ricci G, Manachini PL, Daffonchio D (2004) Characterization of urease genes cluster of Streptococcus thermophilus. J Appl Microbiol 96:209–219PubMedCrossRefGoogle Scholar
  27. Mora D, Monnet C, Parini C, Guglielmetti S, Mariani A, Pintus P, Molinari F, Daffonchio D, Manachini PL (2005) Urease biogenesis in Streptococcus thermophilus. Res Microbiol 156:897–903PubMedCrossRefGoogle Scholar
  28. Ough CS (1976) Ethylcarbamate in fermented beverages and foods. J Agric Food Chem 24:328–331PubMedCrossRefGoogle Scholar
  29. Ough CS, Trioli G (1988) Urea removal from wine by an acid urease. Am J Enol Vitic 39:303–307Google Scholar
  30. Peterbauer C, Maischberger T, Haltrich D (2011) Food-grade gene expression in lactic acid bacteria. Biotechnol J 6:1147–1161PubMedCrossRefGoogle Scholar
  31. Sandine BE, Terzaghi WE (1975) Improved medium for lactic Streptococci and their bacteriophages. Appl Microbiol 29:807–813PubMedCentralPubMedGoogle Scholar
  32. Schehl B, Senn T, Lachenmeier DW, Rodicio R, Heinisch JJ (2007) Contribution of the fermenting yeast strain to ethyl carbamate generation in stone fruit spirits. Appl Microbiol Biotechnol 74:843–850PubMedCrossRefGoogle Scholar
  33. Shaw AJ, Covalla SF, Miller BB, Firliet BT, Hogsett DA, Herring CD (2012) Urease expression in a Thermoanaero bacterium Saccharolyticum ethanologen allows high titer ethanol production. Metab Eng 14:528–532PubMedCrossRefGoogle Scholar
  34. Siren N, Salonen K, Leisola M, Nyyssola A (2008) A new and efficient phosphate starvation inducible expression system for Lactococcus lactis. Appl Microbiol Biotechnol 79:803–810PubMedCrossRefGoogle Scholar
  35. Suzuki K, Benno Y, Mitsuoka T, Takebe S, Kobashi K, Hase J (1979) Urease-producing species of intestinal anaerobes and their activities. Appl Environ Microbiol 37:379–382PubMedCentralPubMedGoogle Scholar
  36. Wozny MA, Bryant MP, Holdeman LV, Moore WE (1977) Urease assay and urease-producing species of anaerobes in the bovine rumen and human feces. Appl Environ Microbiol 33:1097–1104PubMedCentralPubMedGoogle Scholar
  37. Yamazaki E, Kakimoto S, Sumino Y, Nakatsui I (1990) Characteristics of acid urease from Streptococcus mitior. Agric Biol Chem 54:2433–2435CrossRefGoogle Scholar
  38. Yang LQ, Wang SH, Tian YP (2010) Purification, properties, and application of a novel acid urease from Enterobacter sp. Appl Biochem Biotechnol 160:303–313PubMedCrossRefGoogle Scholar
  39. Zhao X, Zou H, Fu J, Zhou J, Du G, Chen J (2014) Metabolic engineering of the regulators in nitrogen catabolite repression to reduce the production of ethylcarbamate in a model rice wine system. Appl Environ Microbiol 80:392–398PubMedCentralPubMedCrossRefGoogle Scholar
  40. Zotta T, Ricciardi A, Rossano R, Parente E (2008) Urease production by Streptococcus thermophilus. Food Microbiol 25:113–119PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  1. 1.Synergetic Innovation Center of Food Safety and NutritionJiangnan UniversityWuxiChina
  2. 2.The Key Laboratory of Industrial Biotechnology, Ministry of EducationJiangnan UniversityWuxiChina
  3. 3.School of BiotechnologyJiangnan UniversityWuxiChina
  4. 4.National Engineering Laboratory for Cereal Fermentation TechnologyJiangnan UniversityWuxiChina
  5. 5.The Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of EducationJiangnan UniversityWuxiChina

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