Skip to main content
SpringerLink
Log in

A novel metagenome-derived β-galactosidase: gene cloning, overexpression, purification and characterization

  • Biotechnologically Relevant Enzymes and Proteins
  • Published:
Applied Microbiology and Biotechnology Aims and scope Submit manuscript

Abstract

A novel β-galactosidase gene, zd410, was isolated by screening a soil metagenomic library. Sequence analysis revealed that zd410 encodes a protein of 672 amino acids with a predicted molecular weight of 78.6 kDa. The recombinant ZD410 was expressed and purified in Pichia pastoris, with a yield of ca. 300 mg from 1 L culture. The purified enzyme displayed optimal activity at 38°C and pH 7.0. Given that the enzyme had 54% of the maximal activity at 20°C and 11% of the maximal activity at close to 0°C, ZD410 was regarded as a cold-adapted β-galactosidase. ZD410 displays high enzymatic activity for its synthetic substrate-ONPG (o-nitrophenyl-β-d-galactopyranoside, 243 U/mg) and its natural substrate-lactose (25.4 U/mg), while its activity was slightly stimulated by addition of Na+, K+, or Ca2+ at low concentrations. ZD410 is a good candidate of β-galactosidases for food industry after further study.

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

  • Amann RI, Ludwig W, Schleifer KH (1995) Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiol Rev 59:143–169

    CAS  Google Scholar 

  • Białkowska AM, Cieśliński H, Nowakowska KM, Kur J, Turkiewicz M (2009) A new β-galactosidase with a low temperature optimum isolated from the Antarctic Arthrobacter sp. 20B: gene cloning, purification and characterization. Arch Microbiol 191:825–835

    Article  Google Scholar 

  • Bradford MM (1976) A rapid and sensitive for the quantitation of microgram quantitites of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

    Article  CAS  Google Scholar 

  • Cereghino JL, Cregg JM (2000) Heterologous protein expression in the methylotrophic yeast Pichia pastoris. FEMS microbiol Rev 24:45–66

    Article  CAS  Google Scholar 

  • Cieśliński H, Kur J, Białkowska A, Baran I, Makowski K, Turkiewicz M (2005) Cloning, expression, and purification of a recombinant cold-adapted β-Galactosidase from Antarctic bacterium Pseudoalteromonas sp. 22b. Protein Expr Purif 39:27–34

    Article  Google Scholar 

  • Coker JA, Sheridan PP, Loveland-Curtze J, Gutshall KR, Auman AJ, Brenchley JE (2003) Biochemical characterization of a β-galactosidase with a low temperature optimum obtained from an Antarctic Arthrobacter isolate. J Bacteriol 185:5473–5482

    Article  CAS  Google Scholar 

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

    CAS  Google Scholar 

  • Curtis TP, Sloan WT (2005) Exploring microbial diversity—a vast below. Science 309:1331–1333

    Article  CAS  Google Scholar 

  • Daniel R (2005) The metagenomics of soil. Nat Rev Microbiol 3:470–478

    Article  CAS  Google Scholar 

  • Fernandes S, Geueke B, Delgado O, Coleman J, Hatti-Kaul R (2002) β-Galactosidase from a cold-adapted bacterium: purification, characterization and application for lactose hydrolysis. Appl Microbiol Biotechnol 58:313–321

    Article  CAS  Google Scholar 

  • Gans J, Wolinsky M, Dunbar J (2005) Computational improvements reveal great bacterial diversity and high metal toxicity in soil. Science 309:1387–1390

    Article  CAS  Google Scholar 

  • Gutshall KR, Trimbur DE, Kasmir JJ, Brenchley JE (1995) Analysis of a novel gene and β-galactosidase isozyme from a psychrotrophic Arthrobacter isolate. J Bacteriol 177:1981–1988

    CAS  Google Scholar 

  • Gutshall K, Wang K, Brenchley JE (1997) A novel Arthrobacter β-galactosidase with homology to eucaryotic β-galactosidases. J Bacteriol 179:3064–3067

    CAS  Google Scholar 

  • Handelsman J, Rondon MR, Brady SF, Clardy J, Goodman RM (1998) Molecular biological access to the chemistry of unknown soil microbes: a new frontier for natural products. Chem Biol 5:R245–R249

    Article  CAS  Google Scholar 

  • Hårdeman F, Sjöling S (2007) Metagenomic approach for the isolation of a novel low-temperature-active lipase from uncultured bacteria of marine sediment. FEMS Microbiol Ecol 59:524–534

    Article  Google Scholar 

  • Hildebrandt P, Wanarska M, Kur J (2009) A new cold-adapted β-D-galactosidase from the Antarctic Arthrobacter sp. 32c-gene cloning, overexpression, purification and properties. BMC microbiol 9:151

    Article  Google Scholar 

  • Hoyoux A, Jennes I, Dubois P, Genicot S, Dubail F, Francois JM, Baise E, Feller G, Gerday C (2001) Cold-adapted β-Galactosidase from the Antarctic psychrophile Pseudoalteromonas haloplanktis. Appl Environ Microbiol 67:1529–1535

    Article  CAS  Google Scholar 

  • 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 psychrotrophic and halotolerant Planococcus sp. L4. J Agric Food Chem 55:2217–2224

    Article  CAS  Google Scholar 

  • Karasová-Lipovová P, Strnad H, Spiwok V, Malá S, Králová B, Russell NJ (2003) The cloning, purification and characterization of a cold-active β-Galactosidase from the psychrotolerant Antarctic bacterium Arthrobacter sp. C2-2. Enzyme Microb Technol 33:836–844

    Article  Google Scholar 

  • Margesin R, Schinner F (1994) Properties of cold-adapted microorganisms and their potential role in biotechnology. J Biotechnol 33:1–14

    Article  CAS  Google Scholar 

  • Nakagawa T, Fujimoto Y, Ikehata R, Miyaji T, Tomizuka N (2006) Purification and molecular characterization of cold-active beta-galactosidase from Arthrobacter psychrolactophilus strain F2. Appl Microbiol Biotechnol 72:720–725

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  • Nakayama T, Amachi T (1999) β-Galactosidase, enzymology. In: Flickinger MC, Drew SW (eds) Encyclopedia of bioprocess technology: fermentation, biocatalysis, and bioseparation, vol 3. Wiley, New York, pp 1291–1305

    Google Scholar 

  • Rahim KAA, Lee BH (1991) Specificity, inhibitory studies, and oligosaccharide formation by β-Galactosidase from psychrotrophic Bacillus subtilis KL88. J Dairy Sci 74:1773–1778

    Article  CAS  Google Scholar 

  • Rappé MS, Giovannoni SJ (2003) The uncultured microbial majority. Annu Rev Microbiol 57:369–394

    Article  Google Scholar 

  • Rhee JK, Ahn DG, Kim YG, Oh JW (2005) New thermophilic and thermostable esterase with sequence similarity to the hormone-sensitive lipase family, cloned from a metagenomic library. Appl Environ Microbiol 71:817–825

    Article  CAS  Google Scholar 

  • Russell NJ (1998) Molecular adaptations in psychrophilic bacteria: potential for biotechnological applications. Adv Biochem Eng Biotechnol 61:1–21

    CAS  Google Scholar 

  • Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406–425

    CAS  Google Scholar 

  • Sambrook J, Frisch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laborotory Press, New York

    Google Scholar 

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

    Article  CAS  Google Scholar 

  • Steele HL, Jaeger KE, Daniel R, Streit WR (2009) Advances in recovery of novel biocatalysts from metagenomes. J Mol Microbiol Biotechnol 16:25–37

    Article  CAS  Google Scholar 

  • Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol 24:1596–1599

    Article  CAS  Google Scholar 

  • Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The CLUSTAL X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25:4876–4882

    Article  CAS  Google Scholar 

  • Timbur DE, Gutshall KR, Prema P, Brenchley JE (1994) Characterization of a psychrotrophic Arthrobacter gene and its cold-active β-galactosidase. Appl Environ Microbiol 60:4544–4552

    Google Scholar 

Download references

Acknowledgements

We are grateful to the National High Technology Research and Development Program of China (863 Program) (2007AA10Z308), National Natural Science Foundation of China (30970107), the Science and Technology Plan Project of Guangdong province (2007A010900001), and the Reserve Key Project of Sun Yat-sen University (2007-33000-1132628) for their financial support.

Author information

Authors and Affiliations

  1. State Key Laboratory of Biocontrol, Sun Yat-sen University, Guangzhou, 510275, People’s Republic of China

    Kui Wang, Gang Li, Shi Qin Yu, Chen Ting Zhang & Yu Huan Liu

  2. Key Laboratory of Gene Engineering of the Ministry of Education, Sun Yat-sen University, Guangzhou, 510275, People’s Republic of China

    Gang Li

Authors
  1. Kui Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Gang Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Shi Qin Yu
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Chen Ting Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Yu Huan Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Yu Huan Liu.

Additional information

Kui Wang and Gang Li contributed equally to this work.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Wang, K., Li, G., Yu, S.Q. et al. A novel metagenome-derived β-galactosidase: gene cloning, overexpression, purification and characterization. Appl Microbiol Biotechnol 88, 155–165 (2010). https://doi.org/10.1007/s00253-010-2744-7

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00253-010-2744-7

Keywords

Search

Navigation