In Vitro Production of Galactooligosaccharides by a Novel β-Galactosidase of <i>Lactobacillus bulgaricus</i>
β-galactosidase is an enzyme with dual activity and important industrial application. As a hydrolase, the enzyme eliminates lactose in milk, while as a trans-galactosidase it produces prebiotic galactooligosaccharides (GOS) with various degrees of polymerization (DP). The aim of the present study is...
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MDPI AG
2022-11-01
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| Series: | International Journal of Molecular Sciences |
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| Online Access: | https://www.mdpi.com/1422-0067/23/22/14308 |
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| author | Alexander Arsov Ivan Ivanov Lidia Tsigoriyna Kaloyan Petrov Penka Petrova |
| author_facet | Alexander Arsov Ivan Ivanov Lidia Tsigoriyna Kaloyan Petrov Penka Petrova |
| author_sort | Alexander Arsov |
| collection | DOAJ |
| description | β-galactosidase is an enzyme with dual activity and important industrial application. As a hydrolase, the enzyme eliminates lactose in milk, while as a trans-galactosidase it produces prebiotic galactooligosaccharides (GOS) with various degrees of polymerization (DP). The aim of the present study is the molecular characterization of β-galactosidase from a Bulgarian isolate, <i>Lactobacillus delbrueckii</i> subsp. <i>bulgaricus</i> 43. The sequencing of the <i>β-gal</i> gene showed that it encodes a new enzyme with 21 amino acid replacements compared to all other β-galactosidases of this species. The molecular model revealed that the new β-galactosidase acts as a tetramer. The amino acids D207, H386, N464, E465, Y510, E532, H535, W562, N593, and W980 form the catalytic center and interact with Mg<sup>2+</sup> ions and substrate. The <i>β-gal</i> gene was cloned into a vector allowing heterologous expression of <i>E. coli</i> BL21(DE3) with high efficiency, as the crude enzyme reached 3015 U/mL of the culture or 2011 U/mg of protein. The enzyme’s temperature optimum at 55 °C, a pH optimum of 6.5, and a positive influence of Mg<sup>2+</sup>, Mn<sup>2+</sup>, and Ca<sup>2+</sup> on its activity were observed. From lactose, β-Gal produced a large amount of GOS with DP3 containing β-(1→3) and β-(1→4) linkages, as the latter bond is particularly atypical for the <i>L. bulgaricus</i> enzymes. DP3-GOS formation was positively affected by high lactose concentrations. The process of lactose conversion was rapid, with a 34% yield of DP3-GOS in 6 h, and complete degradation of 200 g/L of lactose for 12 h. On the other hand, the enzyme was quite stable at 55 °C and retained about 20% of its activity after 24 h of incubation at this temperature. These properties expand our horizons as regards the use of β-galactosidases in industrial processes for the production of lactose-free milk and GOS-enriched foods. |
| first_indexed | 2024-03-09T18:15:43Z |
| format | Article |
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| institution | Directory Open Access Journal |
| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-09T18:15:43Z |
| publishDate | 2022-11-01 |
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| spelling | doaj.art-6ff488ea92094eeebafbf0b0c8873eb02023-11-24T08:42:04ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672022-11-0123221430810.3390/ijms232214308In Vitro Production of Galactooligosaccharides by a Novel β-Galactosidase of <i>Lactobacillus bulgaricus</i>Alexander Arsov0Ivan Ivanov1Lidia Tsigoriyna2Kaloyan Petrov3Penka Petrova4Institute of Microbiology, Bulgarian Academy of Sciences, 1113 Sofia, BulgariaInstitute of Microbiology, Bulgarian Academy of Sciences, 1113 Sofia, BulgariaInstitute of Chemical Engineering, Bulgarian Academy of Sciences, 1113 Sofia, BulgariaInstitute of Chemical Engineering, Bulgarian Academy of Sciences, 1113 Sofia, BulgariaInstitute of Microbiology, Bulgarian Academy of Sciences, 1113 Sofia, Bulgariaβ-galactosidase is an enzyme with dual activity and important industrial application. As a hydrolase, the enzyme eliminates lactose in milk, while as a trans-galactosidase it produces prebiotic galactooligosaccharides (GOS) with various degrees of polymerization (DP). The aim of the present study is the molecular characterization of β-galactosidase from a Bulgarian isolate, <i>Lactobacillus delbrueckii</i> subsp. <i>bulgaricus</i> 43. The sequencing of the <i>β-gal</i> gene showed that it encodes a new enzyme with 21 amino acid replacements compared to all other β-galactosidases of this species. The molecular model revealed that the new β-galactosidase acts as a tetramer. The amino acids D207, H386, N464, E465, Y510, E532, H535, W562, N593, and W980 form the catalytic center and interact with Mg<sup>2+</sup> ions and substrate. The <i>β-gal</i> gene was cloned into a vector allowing heterologous expression of <i>E. coli</i> BL21(DE3) with high efficiency, as the crude enzyme reached 3015 U/mL of the culture or 2011 U/mg of protein. The enzyme’s temperature optimum at 55 °C, a pH optimum of 6.5, and a positive influence of Mg<sup>2+</sup>, Mn<sup>2+</sup>, and Ca<sup>2+</sup> on its activity were observed. From lactose, β-Gal produced a large amount of GOS with DP3 containing β-(1→3) and β-(1→4) linkages, as the latter bond is particularly atypical for the <i>L. bulgaricus</i> enzymes. DP3-GOS formation was positively affected by high lactose concentrations. The process of lactose conversion was rapid, with a 34% yield of DP3-GOS in 6 h, and complete degradation of 200 g/L of lactose for 12 h. On the other hand, the enzyme was quite stable at 55 °C and retained about 20% of its activity after 24 h of incubation at this temperature. These properties expand our horizons as regards the use of β-galactosidases in industrial processes for the production of lactose-free milk and GOS-enriched foods.https://www.mdpi.com/1422-0067/23/22/14308β-galactosidase<i>Lactobacillus bulgaricus</i>prebiotic |
| spellingShingle | Alexander Arsov Ivan Ivanov Lidia Tsigoriyna Kaloyan Petrov Penka Petrova In Vitro Production of Galactooligosaccharides by a Novel β-Galactosidase of <i>Lactobacillus bulgaricus</i> International Journal of Molecular Sciences β-galactosidase <i>Lactobacillus bulgaricus</i> prebiotic |
| title | In Vitro Production of Galactooligosaccharides by a Novel β-Galactosidase of <i>Lactobacillus bulgaricus</i> |
| title_full | In Vitro Production of Galactooligosaccharides by a Novel β-Galactosidase of <i>Lactobacillus bulgaricus</i> |
| title_fullStr | In Vitro Production of Galactooligosaccharides by a Novel β-Galactosidase of <i>Lactobacillus bulgaricus</i> |
| title_full_unstemmed | In Vitro Production of Galactooligosaccharides by a Novel β-Galactosidase of <i>Lactobacillus bulgaricus</i> |
| title_short | In Vitro Production of Galactooligosaccharides by a Novel β-Galactosidase of <i>Lactobacillus bulgaricus</i> |
| title_sort | in vitro production of galactooligosaccharides by a novel β galactosidase of i lactobacillus bulgaricus i |
| topic | β-galactosidase <i>Lactobacillus bulgaricus</i> prebiotic |
| url | https://www.mdpi.com/1422-0067/23/22/14308 |
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