A new β-galactosidase extracted from the infant feces with high hydrolytic and transgalactosylation activity

  • Yongping Xin
  • Tingting Guo
  • Yi Zhang
  • Jiapeng Wu
  • Jian KongEmail author
Biotechnologically relevant enzymes and proteins


A β-galactosidase (β-GalINF) was directly isolated from feces of an 8-month-old infant. Mass spectrum analysis showed β-GalINF with coverage over 50% to the β-galactosidase from Bifidobacterium longum EK3. Accordingly, the β-galINF was amplified from the feces metagenomic DNA by degenerate primers. After overexpressed in Escherichia coli, the β-GalINF was purified and biochemical characterized. β-GalINF existed as homotetramer and homodimer, whose activity (optimal at 50 °C, pH 6.5) was exhilaratingly increased to 484% by artificial intestinal juice. The Km and Vmax values for oNPG and lactose were 20.95 ± 2.76 mM, 5004.50 ± 318.8 μmol min−1 mg−1 and 140.2 ± 17.7 mM, 293.1 ± 14.7 μmol min−1 mg−1, respectively. The production rate of galacto-oligosaccharides by β-GalINF from 20% lactose at 50 °C was 33.4 ± 0.67%. These results suggested the β-GalINF with high hydrolytic and transgalactosylation activity from the infant intestinal has great potential as infant lactase preparation. Moreover, this study provided a new way for exploring undetected enzymes by uncultured-dependent methods.


Infant β-Galactosidase Hydrolytic activity Transgalactosylation 


Funding information

This work was supported by grants from the National Key Research and Development Program of China (2017YFD0400300), the National Natural Science Foundation of China (31871767), and the Public Service Sectors (Agriculture) Special and Scientific Research Projects (201503134).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interests.

Ethical approval

All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

Informed consent

Informed consent was obtained from all parents of the infants included in the study.

Supplementary material

253_2019_10092_MOESM1_ESM.pdf (156 kb)
ESM 1 (PDF 155 kb)


  1. Aehle W (2007) Enzymes in industry: production and applications. John Wiley & SonsGoogle Scholar
  2. Arreola SL, Intanon M, Suljic J, Kittl R, Pham NH, Kosma P, Haltrich D, Nguyen TH (2014) Two beta-galactosidases from the human isolate Bifidobacterium breve DSM 20213: molecular cloning and expression, biochemical characterization and synthesis of galacto-oligosaccharides. PLoS One 9(8):e104056. CrossRefGoogle Scholar
  3. Bibbo S, Ianiro G, Giorgio V, Scaldaferri F, Masucci L, Gasbarrini A, Cammarota G (2016) The role of diet on gut microbiota composition. Eur Rev Med Pharmacol Sci 20(22):4742–4749Google Scholar
  4. Boon MA, Janssen AE, van’t Riet K (2000) Effect of temperature and enzyme origin on the enzymatic synthesis of oligosaccharides. Enzym Microb Technol 26(2-4):271–281CrossRefGoogle Scholar
  5. Carevic M, Bezbradica D, Banjanac K, Milivojevic A, Fanuel M, Rogniaux H, Ropartz D, Velickovic D (2016) Structural elucidation of enzymatically synthesized galacto-oligosaccharides using ion-mobility spectrometry-tandem mass spectrometry. J Agric Food Chem 64(18):3609–3615. CrossRefGoogle Scholar
  6. Carević M, Vukašinović-Sekulić M, Ćorović M, Rogniaux H, Ropartz D, Veličković D, Bezbradica D (2018) Evaluation of β-galactosidase from Lactobacillus acidophilus as biocatalyst for galacto-oligosaccharides synthesis: Product structural characterization and enzyme immobilization. J Biosci Bioeng 126(6):697–704. CrossRefGoogle Scholar
  7. Cheng J, Romantsov T, Engel K, Doxey AC, Rose DR, Neufeld JD, Charles TC (2017) Functional metagenomics reveals novel beta-galactosidases not predictable from gene sequences. PLoS One 12(3):e0172545. CrossRefGoogle Scholar
  8. Clarke G, Stilling RM, Kennedy PJ, Stanton C, Cryan JF, Dinan TG (2014) Minireview: Gut microbiota: the neglected endocrine organ. Mol Endocrinol 28(8):1221–1238. CrossRefGoogle Scholar
  9. Davis LM, Martinez I, Walter J, Goin C, Hutkins RW (2011) Barcoded pyrosequencing reveals that consumption of galactooligosaccharides results in a highly specific bifidogenic response in humans. PLoS One 6(9):e25200. CrossRefGoogle Scholar
  10. Di Rienzo T, D’Angelo G, D’Aversa F, Campanale MC, Cesario V, Montalto M, Gasbarrini A, Ojetti V (2013) Lactose intolerance: from diagnosis to correct management. Eur Rev Med Pharmacol Sci 17(Suppl 2):18–25Google Scholar
  11. Felicilda-Reynaldo RF, Kenneally M (2016) Digestive enzyme replacement therapy: pancreatic enzymes and lactase. Medsurg Nurs 25(3):182–185Google Scholar
  12. Gibson MK, Crofts TS, Dantas G (2015) Antibiotics and the developing infant gut microbiota and resistome. Curr Opin Microbiol 27:51–56. CrossRefGoogle Scholar
  13. Goulas T, Goulas A, Tzortzis G, Gibson GR (2009) Comparative analysis of four β-galactosidases from Bifidobacterium bifidum NCIMB41171: purification and biochemical characterisation. Appl Microbiol Biotechnol 82(6):1079–1088. CrossRefGoogle Scholar
  14. Hsu CA, Lee SL, Chou CC (2007) Enzymatic production of galactooligosaccharides by beta-galactosidase from Bifidobacterium longum BCRC 15708. J Agric Food Chem 55(6):2225–2230. CrossRefGoogle Scholar
  15. Hung MN, Lee BH (2002) Purification and characterization of a recombinant beta-galactosidase with transgalactosylation activity from Bifidobacterium infantis HL96. Appl Microbiol Biotechnol 58(4):439–445. CrossRefGoogle Scholar
  16. Hung MN, Xia Z, Hu NT, Lee BH (2001) Molecular and biochemical analysis of two -galactosidases from Bifidobacterium infantis HL96. Appl Microbiol Biotechnol 67(9):4256–4263. Google Scholar
  17. Husain Q (2010) Beta galactosidases and their potential applications: a review. Crit Rev Biotechnol 30(1):41–62. CrossRefGoogle Scholar
  18. Iqbal S, Nguyen TH, Nguyen HA, Nguyen TT, Maischberger T, Kittl R, Haltrich D (2011) Characterization of a heterodimeric GH2 beta-galactosidase from Lactobacillus sakei Lb790 and formation of prebiotic galacto-oligosaccharides. J Agric Food Chem 59(8):3803–3811. CrossRefGoogle Scholar
  19. Iqbal S, Nguyen TH, Nguyen TT, Maischberger T, Haltrich D (2010) beta-Galactosidase from Lactobacillus plantarum WCFS1: biochemical characterization and formation of prebiotic galacto-oligosaccharides. Carbohydr Res 345(10):1408–1416. CrossRefGoogle Scholar
  20. Kumar S, Stecher G, Tamura K (2016) MEGA7: Molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol 33(7):1870–1874. CrossRefGoogle Scholar
  21. Liu L, Gong W, Sun X, Chen G, Wang L (2018) Extracellular enzyme composition and functional characteristics of Aspergillus niger An-76 induced by food processing byproducts and based on integrated functional omics. J Agric Food Chem 66(5):1285–1295. CrossRefGoogle Scholar
  22. Liu YG, Whittier RF (1995) Thermal asymmetric interlaced PCR: automatable amplification and sequencing of insert end fragments from P1 and YAC clones for chromosome walking. Genomics 25(3):674–681CrossRefGoogle Scholar
  23. Lukito W, Malik SG, Surono IS, Wahlqvist ML (2015) From ‘lactose intolerance’ to ‘lactose nutrition’. Asia Pac J Clin Nutr 24(Suppl 1):S1–S8. Google Scholar
  24. Macfarlane GT, Steed H, Macfarlane S (2008) Bacterial metabolism and health-related effects of galacto-oligosaccharides and other prebiotics. J Appl Microbiol 104(2):305–344. Google Scholar
  25. Maischberger T, Leitner E, Nitisinprasert S, Juajun O, Yamabhai M, Nguyen TH, Haltrich D (2010) Beta-galactosidase from Lactobacillus pentosus: purification, characterization and formation of galacto-oligosaccharides. Biotechnol J 5(8):838–847. CrossRefGoogle Scholar
  26. Marchler-Bauer A, Derbyshire MK, Gonzales NR, Lu S, Chitsaz F, Geer LY, Geer RC, He J, Gwadz M, Hurwitz DI, Lanczycki CJ, Lu F, Marchler GH, Song JS, Thanki N, Wang Z, Yamashita RA, Zhang D, Zheng C, Bryant SH (2015) CDD: NCBI’s conserved domain database. Nucleic Acids Res 43(Database issue):D222–D226. CrossRefGoogle Scholar
  27. Neri DF, Balcão VM, Costa RS, Rocha IC, Ferreira EM, Torres DP, Rodrigues LR, Carvalho LB Jr, Teixeira JA (2009) Galacto-oligosaccharides production during lactose hydrolysis by free Aspergillus oryzae β-galactosidase and immobilized on magnetic polysiloxane-polyvinyl alcohol. Food Chem 115(1):92–99CrossRefGoogle Scholar
  28. 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(1):136–144. CrossRefGoogle Scholar
  29. Nguyen TT, Nguyen HA, Arreola SL, Mlynek G, Djinovic-Carugo K, Mathiesen G, Nguyen TH, Haltrich D (2012) Homodimeric beta-galactosidase from Lactobacillus delbrueckii subsp. bulgaricus DSM 20081: expression in Lactobacillus plantarum and biochemical characterization. J Agric Food Chem 60(7):1713–1721. CrossRefGoogle Scholar
  30. Nijpels H (1981) Lactases and their applications Enzymes and food processing. Springer, pp 89-104Google Scholar
  31. Nyonyo T, Shinkai T, Tajima A, Mitsumori M (2013) Effect of media composition, including gelling agents, on isolation of previously uncultured rumen bacteria. Lett Appl Microbiol 56(1):63–70. CrossRefGoogle Scholar
  32. Oliveira C, Guimarães PMR, Domingues L (2011) Recombinant microbial systems for improved β-galactosidase production and biotechnological applications. Biotechnol Adv 29(6):600–609. CrossRefGoogle Scholar
  33. Park AR, Oh DK (2010) Galacto-oligosaccharide production using microbial beta-galactosidase: current state and perspectives. Appl Microbiol Biotechnol 85(5):1279–1286. CrossRefGoogle Scholar
  34. Quigley EM (2013) Gut bacteria in health and disease. Gastroenterol Hepatol 9(9):560–569Google Scholar
  35. Rada V, Petr J (2000) A new selective medium for the isolation of glucose non-fermenting bifidobacteria from hen caeca. J Microbiol Methods 43(2):127–132CrossRefGoogle Scholar
  36. Rettedal EA, Gumpert H, Sommer MO (2014) Cultivation-based multiplex phenotyping of human gut microbiota allows targeted recovery of previously uncultured bacteria. Nat Commun 5:4714. CrossRefGoogle Scholar
  37. Saqib S, Akram A, Halim SA, Tassaduq R (2017) Sources of beta-galactosidase and its applications in food industry. 3 Biotech 7(1):79. CrossRefGoogle Scholar
  38. Shaukat A, Levitt MD, Taylor BC, MacDonald R, Shamliyan TA, Kane RL, Wilt TJ (2010) Systematic review: effective management strategies for lactose intolerance. Ann Intern Med 152(12):797–803. CrossRefGoogle Scholar
  39. Urrutia P, Rodriguez-Colinas B, Fernandez-Arrojo L, Ballesteros AO, Wilson L, Illanes A, Plou FJ (2013) Detailed analysis of galactooligosaccharides synthesis with β-galactosidase from Aspergillus oryzae. J Agric Food Chem 61(5):1081–1087. CrossRefGoogle Scholar
  40. Vandenplas Y (2015) Lactose intolerance. Asia Pac J Clin Nutr 24 Suppl 1:S9-13 doi:
  41. Zhang X, Li H, Li C-J, Ma T, Li G, Liu Y-H (2013) Metagenomic approach for the isolation of a thermostable β-galactosidase with high tolerance of galactose and glucose from soil samples of Turpan Basin. BMC Microbiol 13:237. CrossRefGoogle Scholar
  42. Zhou JY, Schepmoes AA, Zhang X, Moore RJ, Monroe ME, Lee JH, Camp DG, Smith RD, Qian WJ (2010) Improved LC-MS/MS spectral counting statistics by recovering low-scoring spectra matched to confidently identified peptide sequences. J Proteome Res 9(11):5698–5704. CrossRefGoogle Scholar
  43. Zhou QZ, Chen XD (2001) Effects of temperature and pH on the catalytic activity of the immobilized β-galactosidase from Kluyveromyces lactis. Biochem Eng J 9(1):33–40CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.State Key Laboratory of Microbial TechnologyShandong UniversityQingdaoPeople’s Republic of China
  2. 2.QingdaoPeople’s Republic of China

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