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. 2021 Mar 18;20(1):71.
doi: 10.1186/s12934-021-01553-y.

Prebiotic properties of Bacillus coagulans MA-13: production of galactoside hydrolyzing enzymes and characterization of the transglycosylation properties of a GH42 β-galactosidase

Martina Aulitto  1   2 , Andrea Strazzulli  1   3 , Ferdinando Sansone  1 , Flora Cozzolino  4   5 , Maria Monti  4   5 , Marco Moracci  1   3   6 , Gabriella Fiorentino  1   7 , Danila Limauro  1   7 , Simonetta Bartolucci  1 , Patrizia Contursi  8   9   10
Affiliations

Prebiotic properties of Bacillus coagulans MA-13: production of galactoside hydrolyzing enzymes and characterization of the transglycosylation properties of a GH42 β-galactosidase

Martina Aulitto et al. Microb Cell Fact. .

Abstract

Background: The spore-forming lactic acid bacterium Bacillus coagulans MA-13 has been isolated from canned beans manufacturing and successfully employed for the sustainable production of lactic acid from lignocellulosic biomass. Among lactic acid bacteria, B. coagulans strains are generally recognized as safe (GRAS) for human consumption. Low-cost microbial production of industrially valuable products such as lactic acid and various enzymes devoted to the hydrolysis of oligosaccharides and lactose, is of great importance to the food industry. Specifically, α- and β-galactosidases are attractive for their ability to hydrolyze not-digestible galactosides present in the food matrix as well as in the human gastrointestinal tract.

Results: In this work we have explored the potential of B. coagulans MA-13 as a source of metabolites and enzymes to improve the digestibility and the nutritional value of food. A combination of mass spectrometry analysis with conventional biochemical approaches has been employed to unveil the intra- and extra- cellular glycosyl hydrolase (GH) repertoire of B. coagulans MA-13 under diverse growth conditions. The highest enzymatic activity was detected on β-1,4 and α-1,6-glycosidic linkages and the enzymes responsible for these activities were unambiguously identified as β-galactosidase (GH42) and α-galactosidase (GH36), respectively. Whilst the former has been found only in the cytosol, the latter is localized also extracellularly. The export of this enzyme may occur through a not yet identified secretion mechanism, since a typical signal peptide is missing in the α-galactosidase sequence. A full biochemical characterization of the recombinant β-galactosidase has been carried out and the ability of this enzyme to perform homo- and hetero-condensation reactions to produce galacto-oligosaccharides, has been demonstrated.

Conclusions: Probiotics which are safe for human use and are capable of producing high levels of both α-galactosidase and β-galactosidase are of great importance to the food industry. In this work we have proven the ability of B. coagulans MA-13 to over-produce these two enzymes thus paving the way for its potential use in treatment of gastrointestinal diseases.

Keywords: Bacillus coagulans; Galacto-oligosaccharides; Prebiotics; Thermophilic; Transgalactosylation; α-galactosidase; β-galactosidase.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Detection of enzymatic activities on different artificial substrates
Fig. 2
Fig. 2
Detection of α-galactosidase activity. a Enzymatic assays of intracellular and extracellular extract of B. coagulans MA-13 on PNP-α-gal, after growth on selective medium containing locust bean gum. b Zymogram of supernatants from B. coagulans MA-13 cells grown on locust bean gum medium, using PNP-α-gal as substrate
Fig. 3
Fig. 3
β-galactosidase genomic context in Bacillus coagulans MA-13
Fig. 4
Fig. 4
Effect of pH and temperature on the enzymatic activity. a The pH dependence was evaluated in different buffers ranging from pH 4.0 to pH 10.0. b The pH stability was studied by incubating BcGalB in different buffers ranging from pH 4.0 to 8.0 up to 24 h. c Temperature optimum was determined by testing the enzyme in the range 30–80 °C. d For thermostability studies, the recombinant enzyme was incubated at different temperatures ranging from 4 to 60 °C up to 24 h
Fig. 5
Fig. 5
Inhibitory effect of sugars on BcGalB hydrolytic activity
Fig. 6
Fig. 6
Time course of transgalactosylation reaction detected by TLC analysis. Homo-condensation reactions using ONP-β-gal as donor and acceptor (a). Hetero-condensation reactions performed with PNP-β-glu (b) and PNP-β-xyl (c) as acceptors and ONP-β-gal as donor. Transgalactosylation reactions using D-lactose as substrate (d). Red, blue and green circles show the UV signals obtained from the arylic group of ONP-β-gal, PNP-β-glu, and PNP-β-xyl respectively. STD1: Standard with ONP-β-gal and D-galactose, STD2: Standard with PNP-β-glu and D-glucose, STD3: Standard with PNP-β-xyl and D-xylose, STD4: Standard with D-glucose, STD5: Standard with D-galactose and D-lactose, S: Samples collected at different times (min), B: Blank at different times, SD: Sample diluted, BD: Blank diluted, TP: Transgalactosylation products
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