Functional Identification of a Putative β-Galactosidase Gene in the Special lac Gene Cluster of Lactobacillus acidophilus
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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.
Keywords
Lactose lacZ Gene Lactobacillus Acidophilus Lactose Intolerance Active StainingNotes
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.
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 102:3906–3912CrossRefPubMedGoogle 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 103:3816–3821CrossRefPubMedGoogle 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 156:597–604CrossRefPubMedGoogle 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 72:248–254CrossRefPubMedGoogle 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 71:1655–1658CrossRefPubMedGoogle 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 190:727–735CrossRefPubMedGoogle 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 65:5443–5450PubMedGoogle 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 1784:292–301Google Scholar
- 9.Goldin BR, Gorbach SL (1984) The effect of milk and lactobacillus feeding on human intestinal bacterial enzyme activity. Am J Clin Nutr 39:756–761PubMedGoogle 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 322:79–91CrossRefPubMedGoogle Scholar
- 11.Holmes ML, Dyall-Smith ML (2000) Sequence and expression of a halobacterial beta-galactosidase gene. Mol Microbiol 36:114–122CrossRefPubMedGoogle 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 1337:276–286PubMedGoogle 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 55:2217–2224CrossRefPubMedGoogle 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 21:1021–1029PubMedGoogle Scholar
- 15.Jiang T, Savaiano DA (1997) In vitro lactose fermentation by human colonic bacteria is modified by Lactobacillus acidophilus supplementation. J Nutr 127:1489–1495PubMedGoogle 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 116:337–346CrossRefPubMedGoogle Scholar
- 17.Kim CS, Ji ES, Oh DK (2003) Expression and characterization of Kluyveromyces lactis beta-galactosidase in Escherichia coli. Biotechnol Lett 25:1769–1774CrossRefPubMedGoogle 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 18:338–342PubMedGoogle 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 28:318–324CrossRefPubMedGoogle 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 80:1537–1545PubMedCrossRefGoogle 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 54:295–299CrossRefPubMedGoogle 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 54:4989–4998CrossRefPubMedGoogle 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 269:136–144CrossRefPubMedGoogle 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 129:581–591CrossRefPubMedGoogle 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 101:2512–2517CrossRefPubMedGoogle 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 40:1570–1577CrossRefGoogle Scholar
- 27.Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory Press, New YorkGoogle 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 66:2438–2444CrossRefPubMedGoogle Scholar
- 29.Siddiqui KS, Cavicchioli R (2006) Cold-adapted enzymes. Annu Rev Biochem 75:403–433CrossRefPubMedGoogle Scholar
- 30.Smith DK, Radivojac P, Obradovic Z, Dunker AK, Zhu G (2003) Improved amino acid flexibility parameters. Protein Sci 12:1060–1072CrossRefPubMedGoogle 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 30:343–348CrossRefPubMedGoogle Scholar