The impact of heterologous catalase expression and superoxide dismutase overexpression on enhancing the oxidative resistance in Lactobacillus casei

  • Jinzhong Lin
  • Yexia Zou
  • Kunlin Cao
  • Chengjie MaEmail author
  • Zhengjun ChenEmail author
Genetics and Molecular Biology of Industrial Organisms


Two heme-dependent catalase genes were amplified from genomic DNA of Lactobacillus plantarum WCFS1 (KatE1) and Lactobacillus brevis ATCC 367 (KatE2), respectively, and a manganese-containing superoxide dismutase from Lactobacillus casei MCJΔ1 (MnSOD) were cloned into plasmid pELX1, yielding pELX1-KatE1, pELX1-KatE2 and pELX1-MnSOD, then the recombinant plasmids were transferred into L. casei MCJΔ1. The strains of L. casei MCJΔ1/pELX1-KatE1 and L. casei MCJΔ1/pELX1-KatE2 were tolerant at 2 mM H2O2. The survival rates of L. casei MCJΔ1/pELX1-KatE1 and L. casei MCJΔ1/pELX1-KatE2 were 270-fold and 300-fold higher than that of the control strain on a short-term H2O2 exposure, and in aerated condition, the survival cells counts were 146- and 190-fold higher than that of the control strain after 96 h of incubation. Furthermore, L. casei MCJΔ1/pELX1-MnSOD was the best in three recombinants which was superior in the living cell viability during storage when co-storage with Lactobacillus delbrueckii subsp. lactis LBCH-1.


Lactobacillus casei Superoxide dismutase (SOD) Catalase Heterologous expression Oxidative resistance 


  1. 1.
    Abriouel H, Herrmann A, Starke J, Yousif NM, Wijaya A, Tauscher B, Holzapfel W, Franz CM (2004) Cloning and heterologous expression of hematin-dependent catalase produced by Lactobacillus plantarum CNRZ 1228. Appl Environ Microbiol 70(1):603–606. doi: 10.1128/AEM.70.1.603-606.2004 CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Ai L, Chen C, Zhou F, Wang L, Zhang H, Chen W, Guo B (2011) Complete genome sequence of the probiotic strain Lactobacillus casei BD-II. J Bacteriol 193(12):3160–3161. doi: 10.1128/JB.00421-11 CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    An H, Zhai Z, Yin S, Luo Y, Han B, Hao Y (2011) Coexpression of the superoxide dismutase and the catalase provides remarkable oxidative stress resistance in Lactobacillus rhamnosus. J Agric Food Chem 59(8):3851–3856. doi: 10.1021/jf200251k CrossRefPubMedGoogle Scholar
  4. 4.
    Ashraf R, Shah NP (2011) Selective and differential enumerations of Lactobacillus delbrueckii subsp. bulgaricus, Streptococcus thermophilus, Lactobacillus acidophilus, Lactobacillus casei and Bifidobacterium spp. in yoghurt—a review. Int J Food Microbiol 149(3):194–208. doi: 10.1016/j.ijfoodmicro.2011.07.008 CrossRefPubMedGoogle Scholar
  5. 5.
    Bove P, Capozzi V, Garofalo C, Rieu A, Spano G, Fiocco D (2012) Inactivation of the ftsH gene of Lactobacillus plantarum WCFS1: effects on growth, stress tolerance, cell surface properties and biofilm formation. Microbiol Res 167(4):187–193. doi: 10.1016/j.micres.2011.07.001 CrossRefPubMedGoogle Scholar
  6. 6.
    Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254. doi: 10.1016/0003-2697(76)90527-3 CrossRefPubMedGoogle Scholar
  7. 7.
    Bruno-Barcena JM, Andrus JM, Libby SL, Klaenhammer TR, Hassan HM (2004) Expression of a heterologous manganese superoxide dismutase gene in intestinal Lactobacillus provides protection against hydrogen peroxide toxicity. Appl Environ Microbiol 70(8):4702–4710. doi: 10.1128/AEM.70.8.4702-4710.2004 CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Chen Z, Lin J, Ma C, Zhao S, She Q, Liang Y (2014) Characterization of pMC11, a plasmid with dual origins of replication isolated from Lactobacillus casei MCJ and construction of shuttle vectors with each replicon. Appl Microbiol Biotechnol 98(13):5977–5989. doi: 10.1007/s00253-014-5649-z CrossRefPubMedGoogle Scholar
  9. 9.
    Condon S (1987) Responses of lactic acid bacteria to oxygen. FEMS Microbiol Lett 46(3):269–280. doi: 10.1111/j.1574-6968.1987.tb02465.x CrossRefGoogle Scholar
  10. 10.
    Culotta VC, Daly MJ (2013) Manganese complexes: diverse metabolic routes to oxidative stress resistance in prokaryotes and yeast. Antioxid Redox Signal 19(9):933–944. doi: 10.1089/ars.2012.5093 CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Davidson LE, Fiorino AM, Snydman DR, Hibberd PL (2011) Lactobacillus GG as an immune adjuvant for live-attenuated influenza vaccine in healthy adults: a randomized double-blind placebo-controlled trial. Eur J Clin Nutr 65(4):501–507. doi: 10.1038/ejcn.2010.289 CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Fiocco D, Collins M, Muscariello L, Hols P, Kleerebezem M, Msadek T, Spano G (2009) The Lactobacillus plantarum ftsH gene is a novel member of the CtsR stress response regulon. J Bacteriol 191(5):1688–1694. doi: 10.1128/JB.01551-08 CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Fu L, Kong J, Sun Z, Zhang L, Zhang X, Guo T (2013) Enhancing the oxidative resistance of yoghurt starter bacteria with heterologous catalase expression in Streptococcus thermophilus. Int Dairy J 30(2):68–72. doi: 10.1016/j.idairyj.2012.11.012 CrossRefGoogle Scholar
  14. 14.
    Guidone A, Ianniello RG, Ricciardi A, Zotta T, Parente E (2013) Aerobic metabolism and oxidative stress tolerance in the Lactobacillus plantarum group. World J Microbiol Biotechnol 29(9):1713–1722. doi: 10.1007/s11274-013-1334-0 CrossRefPubMedGoogle Scholar
  15. 15.
    Imlay JA (2003) Pathways of oxidative damage. Annu Rev Microbiol 57:395–418. doi: 10.1146/annurev.micro.57.030502.090938 CrossRefPubMedGoogle Scholar
  16. 16.
    Knauf HJ, Vogel RF, Hammes WP (1992) Cloning, sequence, and phenotypic expression of katA, which encodes the catalase of Lactobacillus sake LTH677. Appl Environ Microbiol 58(3):832–839PubMedPubMedCentralGoogle Scholar
  17. 17.
    Kono Y, Fridovich I (1983) Isolation and characterization of the pseudocatalase of Lactobacillus plantarum. J Biol Chem 258(10):6015–6019PubMedGoogle Scholar
  18. 18.
    Kullisaar T, Songisepp E, Aunapuu M, Kilk K, Arend A, Mikelsaar M, Rehema A, Zilmer M (2010) Complete glutathione system in probiotic Lactobacillus fermentum ME-3. Prikl Biokhim Mikrobiol 46(5):527–531. doi: 10.1134/S0003683810050030 PubMedGoogle Scholar
  19. 19.
    Lin J, Zou Y, Ma C, She Q, Liang Y, Chen Z, Ge X (2015) Heterologous expression of mannanase and developing a new reporter gene system in Lactobacillus casei and Escherichia coli. PLoS One 10(11):e0142886. doi: 10.1371/journal.pone.0142886 CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Liong MT (2007) Probiotics: a critical review of their potential role as antihypertensives, immune modulators, hypocholesterolemics, and perimenopausal treatments. Nut Rev 65(7):316–328. doi: 10.1111/j.1753-4887.2007.tb00309.x CrossRefGoogle Scholar
  21. 21.
    Liu Q, Hang X, Liu X, Tan J, Li D, Yang H (2012) Cloning and heterologous expression of the manganese superoxide dismutase gene from Lactobacillus casei Lc18. Ann Microbiol 62(1):129–137. doi: 10.1007/s13213-011-0237-2 CrossRefGoogle Scholar
  22. 22.
    Makarova K, Slesarev A, Wolf Y, Sorokin A, Mirkin B et al (2006) Comparative genomics of the lactic acid bacteria. Proc Natl Acad Sci USA 103(42):15611–15616. doi: 10.1073/pnas.0607117103 CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Marty-Teysset C, de la Torre F, Garel J (2000) Increased production of hydrogen peroxide by Lactobacillus delbrueckii subsp. bulgaricus upon aeration: involvement of an NADH oxidase in oxidative stress. Appl Environ Microbiol 66(1):262–267. doi: 10.1128/AEM.66.1.262-267.2000 CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Mills S, Stanton C, Fitzgerald GF, Ross RP (2011) Enhancing the stress responses of probiotics for a lifestyle from gut to product and back again. Microb Cell Fact 10(Suppl 1):S19. doi: 10.1186/1475-2859-10-S1-S19 CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Peskin AV, Winterbourn CC (2000) A microtiter plate assay for superoxide dismutase using a water-soluble tetrazolium salt (WST-1). Clin Chim Acta 293(1–2):157–166. doi: 10.1016/S0009-8981(99)00246-6 CrossRefPubMedGoogle Scholar
  26. 26.
    Rochat T, Gratadoux JJ, Gruss A, Corthier G, Maguin E, Langella P, van de Guchte M (2006) Production of a heterologous nonheme catalase by Lactobacillus casei: an efficient tool for removal of H2O2 and protection of Lactobacillus bulgaricus from oxidative stress in milk. Appl Environ Microbiol 72(8):5143–5149. doi: 10.1128/AEM.00482-06 CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor, NewYorkGoogle Scholar
  28. 28.
    Sanders ME, Klaenhammer TR (2001) Invited review: the scientific basis of Lactobacillus acidophilus NCFM functionality as a probiotic. J Dairy Sci 84(2):319–331. doi: 10.3168/jds.S0022-0302(01)74481-5 CrossRefPubMedGoogle Scholar
  29. 29.
    Singh VP, Sharma J, Babu S, Rizwanulla Singla A (2013) Role of probiotics in health and disease: a review. J Pak Med Assoc 63(2):253–257PubMedGoogle Scholar
  30. 30.
    Sinha AK (1972) Colorimetric assay of catalase. Anal Biochem 47(2):389–394CrossRefPubMedGoogle Scholar
  31. 31.
    Tharmaraj N, Shah NP (2003) Selective enumeration of Lactobacillus delbrueckii ssp. bulgaricus, Streptococcus thermophilus, Lactobacillus acidophilus, Bifidobacteria, Lactobacillus casei, Lactobacillus rhamnosus, and Propionibacteria. J Dairy Sci 86(7):2288–2296. doi: 10.3168/jds.S0022-0302(03)73821-1 CrossRefPubMedGoogle Scholar
  32. 32.
    van de Guchte M, Ehrlich SD, Maguin E (2001) Production of growth-inhibiting factors by Lactobacillus delbrueckii. J Appl Microbiol 91(1):147–153. doi: 10.1046/j.1365-2672.2001.01369.x CrossRefPubMedGoogle Scholar
  33. 33.
    Zhang W, Yu D, Sun Z, Wu R, Chen X, Chen W, Meng H, Hu S, Zhang H (2010) Complete genome sequence of Lactobacillus casei Zhang, a new probiotic strain isolated from traditional homemade koumiss in Inner Mongolia. China. J Bacteriol 192(19):5268–5269. doi: 10.1128/JB.00802-10 CrossRefPubMedGoogle Scholar

Copyright information

© Society for Industrial Microbiology and Biotechnology 2016

Authors and Affiliations

  1. 1.State Key Laboratory of Dairy BiotechnologyTechnology Center of Bright Dairy and Food Co., Ltd.ShanghaiChina
  2. 2.State Key Laboratory of Agricultural MicrobiologyHuazhong Agricultural UniversityWuhanChina
  3. 3.Synergetic Innovation Center of Food Safety and NutritionJiangnan UniversityWuxiChina
  4. 4.Technology Center of Wuhan Xinxingdi Biotechnology Development Co., Ltd.WuhanChina

Personalised recommendations