Heterologous Expression of a Thermostable β-1,3-Galactosidase and Its Potential in Synthesis of Galactooligosaccharides
A thermostable β-1,3-galactosidase from <i>Marinomonas</i> sp. BSi20414 was successfully heterologously expressed in <i>Escherichia coli</i> BL21 (DE3), with optimum over-expression conditions as follows: the recombinant cells were induced by adding 0.1 mM of IPTG to...
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MDPI AG
2018-10-01
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| Series: | Marine Drugs |
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| Online Access: | https://www.mdpi.com/1660-3397/16/11/415 |
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doaj-c860a8a3c1a44a6fb79d2a55ce81f9fd2020-11-25T01:01:28ZengMDPI AGMarine Drugs1660-33972018-10-01161141510.3390/md16110415md16110415Heterologous Expression of a Thermostable β-1,3-Galactosidase and Its Potential in Synthesis of GalactooligosaccharidesHaitao Ding0Lili Zhou1Qian Zeng2Yong Yu3Bo Chen4SOA Key Laboratory for Polar Science, Polar Research Institute of China, Shanghai 200136, ChinaSOA Key Laboratory for Polar Science, Polar Research Institute of China, Shanghai 200136, ChinaSOA Key Laboratory for Polar Science, Polar Research Institute of China, Shanghai 200136, ChinaSOA Key Laboratory for Polar Science, Polar Research Institute of China, Shanghai 200136, ChinaSOA Key Laboratory for Polar Science, Polar Research Institute of China, Shanghai 200136, ChinaA thermostable β-1,3-galactosidase from <i>Marinomonas</i> sp. BSi20414 was successfully heterologously expressed in <i>Escherichia coli</i> BL21 (DE3), with optimum over-expression conditions as follows: the recombinant cells were induced by adding 0.1 mM of IPTG to the medium when the OD<sub>600</sub> of the culture reached between 0.6 and 0.9, followed by 22 h incubation at 20 °C. The recombinant enzyme β-1,3-galactosidase (rMaBGA) was further purified to electrophoretic purity by immobilized metal affinity chromatography and size exclusion chromatography. The specific activity of the purified enzyme was 126.4 U mg<sup>−1</sup> at 37 °C using ONPG (<i>o</i>-nitrophenyl-β-galactoside) as a substrate. The optimum temperature and pH of rMaBGA were determined as 60 °C and 6.0, respectively, resembling with its wild-type counterpart, wild type (wt)MaBGA. However, rMaBGA and wtMaBGA displayed different thermal stability and steady-state kinetics, although they share identical primary structures. It is postulated that the stability of the enzyme was altered by heterologous expression with the absence of post-translational modifications such as glycosylation, as well as the steady-state kinetics. To evaluate the potential of the enzyme in synthesis of galactooligosaccharides (GOS), the purified recombinant enzyme was employed to catalyze the transgalactosylation reaction at the lab scale. One of the transgalactosylation products was resolved as 3′-galactosyl-lactose, which had been proven to be a better bifidogenic effector than GOS with β-1,4 linkage and β-1,6 linkages. The results indicated that the recombinant enzyme would be a promising alternative for biosynthesis of GOS mainly with β-1,3 linkage.https://www.mdpi.com/1660-3397/16/11/415β-galactosidaserecombinantthermostabletransglycosylationgalactooligosaccharides<i>Marinomonas</i> |
| collection |
DOAJ |
| language |
English |
| format |
Article |
| sources |
DOAJ |
| author |
Haitao Ding Lili Zhou Qian Zeng Yong Yu Bo Chen |
| spellingShingle |
Haitao Ding Lili Zhou Qian Zeng Yong Yu Bo Chen Heterologous Expression of a Thermostable β-1,3-Galactosidase and Its Potential in Synthesis of Galactooligosaccharides Marine Drugs β-galactosidase recombinant thermostable transglycosylation galactooligosaccharides <i>Marinomonas</i> |
| author_facet |
Haitao Ding Lili Zhou Qian Zeng Yong Yu Bo Chen |
| author_sort |
Haitao Ding |
| title |
Heterologous Expression of a Thermostable β-1,3-Galactosidase and Its Potential in Synthesis of Galactooligosaccharides |
| title_short |
Heterologous Expression of a Thermostable β-1,3-Galactosidase and Its Potential in Synthesis of Galactooligosaccharides |
| title_full |
Heterologous Expression of a Thermostable β-1,3-Galactosidase and Its Potential in Synthesis of Galactooligosaccharides |
| title_fullStr |
Heterologous Expression of a Thermostable β-1,3-Galactosidase and Its Potential in Synthesis of Galactooligosaccharides |
| title_full_unstemmed |
Heterologous Expression of a Thermostable β-1,3-Galactosidase and Its Potential in Synthesis of Galactooligosaccharides |
| title_sort |
heterologous expression of a thermostable β-1,3-galactosidase and its potential in synthesis of galactooligosaccharides |
| publisher |
MDPI AG |
| series |
Marine Drugs |
| issn |
1660-3397 |
| publishDate |
2018-10-01 |
| description |
A thermostable β-1,3-galactosidase from <i>Marinomonas</i> sp. BSi20414 was successfully heterologously expressed in <i>Escherichia coli</i> BL21 (DE3), with optimum over-expression conditions as follows: the recombinant cells were induced by adding 0.1 mM of IPTG to the medium when the OD<sub>600</sub> of the culture reached between 0.6 and 0.9, followed by 22 h incubation at 20 °C. The recombinant enzyme β-1,3-galactosidase (rMaBGA) was further purified to electrophoretic purity by immobilized metal affinity chromatography and size exclusion chromatography. The specific activity of the purified enzyme was 126.4 U mg<sup>−1</sup> at 37 °C using ONPG (<i>o</i>-nitrophenyl-β-galactoside) as a substrate. The optimum temperature and pH of rMaBGA were determined as 60 °C and 6.0, respectively, resembling with its wild-type counterpart, wild type (wt)MaBGA. However, rMaBGA and wtMaBGA displayed different thermal stability and steady-state kinetics, although they share identical primary structures. It is postulated that the stability of the enzyme was altered by heterologous expression with the absence of post-translational modifications such as glycosylation, as well as the steady-state kinetics. To evaluate the potential of the enzyme in synthesis of galactooligosaccharides (GOS), the purified recombinant enzyme was employed to catalyze the transgalactosylation reaction at the lab scale. One of the transgalactosylation products was resolved as 3′-galactosyl-lactose, which had been proven to be a better bifidogenic effector than GOS with β-1,4 linkage and β-1,6 linkages. The results indicated that the recombinant enzyme would be a promising alternative for biosynthesis of GOS mainly with β-1,3 linkage. |
| topic |
β-galactosidase recombinant thermostable transglycosylation galactooligosaccharides <i>Marinomonas</i> |
| url |
https://www.mdpi.com/1660-3397/16/11/415 |
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