Skip to main content
SpringerLink
Log in

Functional Identification of a Putative β-Galactosidase Gene in the Special lac Gene Cluster of Lactobacillus acidophilus

  • Published:
Current Microbiology Aims and scope Submit manuscript

Abstract

The putative β-galactosidase gene (lacZ) of Lactobacillus acidophilus has a very low degree of homology to the Escherichia coli β-galactosidase gene (lacZ) and locates in a special lac gene cluster which contains two β-galactosidase genes. No functional characteristic of the putative β-galactosidase has been described so far. In this study, the lacZ gene of L. acidophilus was hetero-expressed in E. coli and the recombinant protein was purified by a three-step procedure. The product of the lacZ gene was also extracted from L. acidophilus ATCC 4356 and active staining was carried out. The enzymatic properties of the purified recombinant LacZ were assayed. The results of hetero-expression showed the recombinant LacZ without tag had β-galactosidase activity. The purified recombinant LacZ had a specific activity of 43.2 U/mg protein. The result of active staining showed that the functional product of the lacZ gene did exist in L. acidophilus. The L. acidophilus β-galactosidase (LacZ) had an optimal pH of 6, an optimal temperature of 37°C and could hydrolyze 73% of lactose in milk in 30 h at 10°C. The L. acidophilus β-galactosidase (LacZ) was identified as cold-adapted β-galactosidase in this study for the first time, and may be useful for lactose removal from dairy products at low temperatures.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

References

  1. Altermann E, Russell WM, Azcarate-Peril MA, Barrangou R, Buck BL, McAuliffe O, Souther N, Dobson A, Duong T, Callanan M, Lick S, Hamrick A, Cano R, Klaenhammer TR (2005) Complete genome sequence of the probiotic lactic acid bacterium Lactobacillus acidophilus NCFM. Proc Natl Acad Sci USA 102:3906–3912

    Article  CAS  PubMed  Google Scholar 

  2. Barrangou R, Azcarate-Peril MA, Duong T, Conners SB, Kelly RM, Klaenhammer TR (2006) Global analysis of carbohydrate utilization by Lactobacillus acidophilus using cDNA microarrays. Proc Natl Acad Sci USA 103:3816–3821

    Article  CAS  PubMed  Google Scholar 

  3. Beckwith JR (1967) Regulation of the lac operon. Recent studies on the regulation of lactose metabolism in Escherichia coli support the operon model. Science 156:597–604

    Article  CAS  PubMed  Google Scholar 

  4. 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

    Article  CAS  PubMed  Google Scholar 

  5. Callanan MJ, Beresford TP, Ross RP (2005) Genetic diversity in the lactose operons of Lactobacillus helveticus strains and its relationship to the role of these strains as commercial starter cultures. Appl Environ Microbiol 71:1655–1658

    Article  CAS  PubMed  Google Scholar 

  6. Callanan M, Kaleta P, O’Callaghan J, O’Sullivan O, Jordan K, McAuliffe O, Sangrador-Vegas A, Slattery L, Fitzgerald GF, Beresford T, Ross RP (2008) Genome sequence of Lactobacillus helveticus, an organism distinguished by selective gene loss and insertion sequence element expansion. J Bacteriol 190:727–735

    Article  CAS  PubMed  Google Scholar 

  7. Coombs JM, Brenchley JE (1999) Biochemical and phylogenetic analyses of a cold-active beta-galactosidase from the lactic acid bacterium Carnobacterium piscicola BA. Appl Environ Microbiol 65:5443–5450

    CAS  PubMed  Google Scholar 

  8. Di LB, Strazzulli A, Perugino G, La CF, Bedini E, Corsaro MM, Rossi M, Moracci M (2008) Isolation and characterization of a new family 42 beta-galactosidase from the thermoacidophilic bacterium Alicyclobacillus acidocaldarius: identification of the active site residues. Biochim Biophys Acta 1784:292–301

    Google Scholar 

  9. Goldin BR, Gorbach SL (1984) The effect of milk and lactobacillus feeding on human intestinal bacterial enzyme activity. Am J Clin Nutr 39:756–761

    CAS  PubMed  Google Scholar 

  10. Hidaka M, Fushinobu S, Ohtsu N, Motoshima H, Matsuzawa H, Shoun H, Wakagi T (2002) Trimeric crystal structure of the glycoside hydrolase family 42 beta-galactosidase from Thermus thermophilus A4 and the structure of its complex with galactose. J Mol Biol 322:79–91

    Article  CAS  PubMed  Google Scholar 

  11. Holmes ML, Dyall-Smith ML (2000) Sequence and expression of a halobacterial beta-galactosidase gene. Mol Microbiol 36:114–122

    Article  CAS  PubMed  Google Scholar 

  12. Holmes ML, Scopes RK, Moritz RL, Simpson RJ, Englert C, Pfeifer F, Dyall-Smith ML (1997) Purification and analysis of an extremely halophilic beta-galactosidase from Haloferax alicantei. Biochim Biophys Acta 1337:276–286

    CAS  PubMed  Google Scholar 

  13. Hu JM, Li H, Cao LX, Wu PC, Zhang CT, Sang SL, Zhang XY, Chen MJ, Lu JQ, Liu YH (2007) Molecular cloning and characterization of the gene encoding cold-active beta-galactosidase from a psychotropic and halotolerant Planococcus sp. L4. J Agric Food Chem 55:2217–2224

    Article  CAS  PubMed  Google Scholar 

  14. Jain S, Yadav H, Sinha PR, Naito Y, Marotta F (2008) Dahi containing probiotic Lactobacillus acidophilus and Lactobacillus casei has a protective effect against Salmonella enteritidis infection in mice. Int J Immunopathol Pharmacol 21:1021–1029

    CAS  PubMed  Google Scholar 

  15. Jiang T, Savaiano DA (1997) In vitro lactose fermentation by human colonic bacteria is modified by Lactobacillus acidophilus supplementation. J Nutr 127:1489–1495

    CAS  PubMed  Google Scholar 

  16. Kang SK, Cho KK, Ahn JK, Bok JD, Kang SH, Woo JH, Lee HG, You SK, Choi YJ (2005) Three forms of thermostable lactose-hydrolase from Thermus sp. IB-21: cloning, expression, and enzyme characterization. J Biotechnol 116:337–346

    Article  PubMed  CAS  Google Scholar 

  17. Kim CS, Ji ES, Oh DK (2003) Expression and characterization of Kluyveromyces lactis beta-galactosidase in Escherichia coli. Biotechnol Lett 25:1769–1774

    Article  CAS  PubMed  Google Scholar 

  18. Lee NK, Yun CW, Kim SW, Chang HI, Kang CW, Paik HD (2008) Screening of Lactobacilli derived from chicken feces and partial characterization of Lactobacillus acidophilus A12 as an animal probiotics. J Microbiol Biotechnol 18:338–342

    CAS  PubMed  Google Scholar 

  19. Mountzouris KC, Kotzampassi K, Tsirtsikos P, Kapoutzis K, Fegeros K (2009) Effects of Lactobacillus acidophilus on gut microflora metabolic biomarkers in fed and fasted rats. Clin Nutr 28:318–324

    Article  CAS  PubMed  Google Scholar 

  20. Mustapha A, Jiang T, Savaiano DA (1997) Improvement of lactose digestion by humans following ingestion of unfermented acidophilus milk: influence of bile sensitivity, lactose transport, and acid tolerance of Lactobacillus acidophilus. J Dairy Sci 80:1537–1545

    Article  CAS  PubMed  Google Scholar 

  21. Nakagawa T, Ikehata R, Myoda T, Miyaji T, Tomizuka N (2007) Overexpression and functional analysis of cold-active beta-galactosidase from Arthrobacter psychrolactophilus strain F2. Protein Expr Purif 54:295–299

    Article  CAS  PubMed  Google Scholar 

  22. Nguyen TH, Splechtna B, Steinbock M, Kneifel W, Lettner HP, Kulbe KD, Haltrich D (2006) Purification and characterization of two novel beta-galactosidases from Lactobacillus reuteri. J Agric Food Chem 54:4989–4998

    Article  CAS  PubMed  Google Scholar 

  23. Nguyen TH, Splechtna B, Krasteva S, Kneifel W, Kulbe KD, Divne C, Haltrich D (2007) Characterization and molecular cloning of a heterodimeric beta-galactosidase from the probiotic strain Lactobacillus acidophilus R22. FEMS Microbiol Lett 269:136–144

    Article  CAS  PubMed  Google Scholar 

  24. Nguyen TH, Splechtna B, Yamabhai M, Haltrich D, Peterbauer C (2007) Cloning and expression of the beta-galactosidase genes from Lactobacillus reuteri in Escherichia coli. J Biotechnol 129:581–591

    Article  CAS  PubMed  Google Scholar 

  25. Pridmore RD, Berger B, Desiere F, Vilanova D, Barretto C, Pittet AC, Zwahlen MC, Rouvet M, Altermann E, Barrangou R, Mollet B, Mercenier A, Klaenhammer T, Arigoni F, Schell MA (2004) The genome sequence of the probiotic intestinal bacterium Lactobacillus johnsonii NCC 533. Proc Natl Acad Sci USA 101:2512–2517

    Article  CAS  PubMed  Google Scholar 

  26. Reyhan GG, Kemal G, Annarita P, Barbara N (2007) Purification and some properties of a β-galactosidase from the thermoacidophilic Alicyclobacillus acidocaldarius subsp. rittmannii isolated from Antarctica. Enzyme Microb Technol 40:1570–1577

    Article  CAS  Google Scholar 

  27. Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory Press, New York

    Google Scholar 

  28. Sheridan PP, Brenchley JE (2000) Characterization of a salt-tolerant family 42 beta-galactosidase from a psychrophilic antarctic Planococcus isolate. Appl Environ Microbiol 66:2438–2444

    Article  CAS  PubMed  Google Scholar 

  29. Siddiqui KS, Cavicchioli R (2006) Cold-adapted enzymes. Annu Rev Biochem 75:403–433

    Article  CAS  PubMed  Google Scholar 

  30. Smith DK, Radivojac P, Obradovic Z, Dunker AK, Zhu G (2003) Improved amino acid flexibility parameters. Protein Sci 12:1060–1072

    Article  CAS  PubMed  Google Scholar 

  31. Yuan T, Yang P, Wang Y, Meng K, Luo H, Zhang W, Wu N, Fan Y, Yao B (2008) Heterologous expression of a gene encoding a thermostable beta-galactosidase from Alicyclobacillus acidocaldarius. Biotechnol Lett 30:343–348

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

This research was supported by grant (05SG022-019) from Science and Technology Committee of Sichuan Province, China. We would like to thank Dr. Ming Li and Brian Keyser at Tulane University for critical reading the manuscript.

Author information

Authors and Affiliations

  1. Department of Microbiology, Third Military Medical University, Chongqing, 400038, China

    Qu Pan, Junmin Zhu, Lina Liu, Yanguang Cong & Fuquan Hu

  2. Department of Microbiology, Chengdu Medical College, Chengdu, 610083, China

    Qu Pan, Jinchuan Li & Xiaoping Yu

Authors
  1. Qu Pan
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Junmin Zhu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Lina Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Yanguang Cong
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Fuquan Hu
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Jinchuan Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Xiaoping Yu
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Fuquan Hu.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Pan, Q., Zhu, J., Liu, L. et al. Functional Identification of a Putative β-Galactosidase Gene in the Special lac Gene Cluster of Lactobacillus acidophilus . Curr Microbiol 60, 172–178 (2010). https://doi.org/10.1007/s00284-009-9521-9

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00284-009-9521-9

Keywords

Search

Navigation