Characterization of a β-galactosidase from Bacillus subtilis with transgalactosylation activity

Carneiro, Lara Aparecida Buffoni Campos; Yu, Li; Dupree, Paul; Ward, Richard J.

doi:10.1016/j.ijbiomac.2018.07.116

Cited by 26 publications

(27 citation statements)

References 42 publications

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“…The preference for β-linked galactosidic substrates such as ONPG or p-Nitrophenyl-β-d-fucopyranoside over lactose has been frequently described in the characterization of β-galactosidases 99,100 . Similar to our results with pTsbg, other studies have reported β-galactosidases with activity towards ONPG but unable to hydrolyze their natural substrate lactose in vitro such as YesZ β-galactosidase from Bacillus subtilis 101 or the β-Gal II from Bifidobacterium adolescentis DSM 20083 102 . The lack of β-galactosidase activity towards lactose reduces considerably the biotechnological potential of pTsbg, as it could not be applied to produce GOS from lactose and to generate lactose-free dairy products.…”

Section: Determination Of Substrate Specificity Of Ptsbgsupporting

confidence: 91%

Exploring the taxonomical and functional profile of As Burgas hot spring focusing on thermostable β-galactosidases

DeCastro

Doane

Dinsdale

et al. 2021

Sci Rep

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In the present study we investigate the microbial community inhabiting As Burgas geothermal spring, located in Ourense (Galicia, Spain). The approximately 23 Gbp of Illumina sequences generated for each replicate revealed a complex microbial community dominated by Bacteria in which Proteobacteria and Aquificae were the two prevalent phyla. An association between the two most prevalent genera, Thermus and Hydrogenobacter, was suggested by the relationship of their metabolism. The high relative abundance of sequences involved in the Calvin–Benson cycle and the reductive TCA cycle unveils the dominance of an autotrophic population. Important pathways from the nitrogen and sulfur cycle are potentially taking place in As Burgas hot spring. In the assembled reads, two complete ORFs matching GH2 beta-galactosidases were found. To assess their functional characterization, the two ORFs were cloned and overexpressed in E. coli. The pTsbg enzyme had activity towards o-Nitrophenyl-β-d-galactopyranoside (ONPG) and p-Nitrophenyl-β-d-fucopyranoside, with high thermal stability and showing maximal activity at 85 °C and pH 6, nevertheless the enzyme failed to hydrolyze lactose. The other enzyme, Tsbg, was unable to hydrolyze even ONPG or lactose. This finding highlights the challenge of finding novel active enzymes based only on their sequence.

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Section: Determination Of Substrate Specificity Of Ptsbgsupporting

confidence: 91%

Exploring the taxonomical and functional profile of As Burgas hot spring focusing on thermostable β-galactosidases

DeCastro

Doane

Dinsdale

et al. 2021

Sci Rep

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“…In agreement with our results [38], the recombinant β-galactosidase from Bacillus licheniformis was purified by a single-step purification protocol using a Ni-Sepharose 6 fast-flow column, and the purified enzyme shows a molecular mass of 75 kDa when analyzed by SDS-PAGE. Consistently, [16] also purified the recombinant β-galactosidase from Bacillus subtilis with a Superdex G-200 column step and the purified recombinant enzyme was exhibiting a single-protein band with an apparent molecular mass of 75 kDa, in agreement with the theoretical molecular weight of 75,164.0 kDa calculated for the YesZ amino acid sequence involving the C-terminal extension [39]. cloned the β-galactosidase of Thermotoganaph thophila and expressed it in E. coli, and the SDS-PAEG of the purified recombinant enzyme exhibited a molecular weight of 70 kDa.…”

Section: Purification Of β-Galactosidasementioning

confidence: 58%

“…Also, [38] shows that the presence of Na + or Mn +2 could enhance the enzyme activity, but when their synergistic effect together was tested, there were no any stimulation. It is worth mentioning that Ca +2 and Zn +2 were known as an inhibitor of some β-galactosidase [16,35,40]. However, Ca +2 did not display any change in the enzymatic activity even at 10 mM while a slightly decrease in the activity was noticed at 10 mM of Zn +2 .…”

Section: Effect Of Ph Temperature and Salinity On β-Galactosidasementioning

confidence: 99%

“…Indeed, until now, the kinetic properties of βgalactosidase used in the dairy industry having some limitations. One of them is the inhibition of βgalactosidase caused by the hydrolysis-formed product D-galactose which was regarded as a big barrier to its utilization in the industrial sector [2,16]. Therefore, it is of great economic interest to explore a new source to generate β-galactosidase with improved processing characteristics for their utilization in dairy industries.…”

Section: Introductionmentioning

confidence: 99%

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Activation of LacZ gene in Escherichia coli DH5α via α-complementation mechanism for β-galactosidase production and its biochemical characterizations

Hamed

Khedr

Abdelraof

2020

Journal of Genetic Engineering and Biotechnology

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Background Plasmid propagation in recombination strains such as Escherichia coli DH5α is regarded as a beneficial instrument for stable amplification of the DNA materials. Here, we show trans-conjugation of pGEM-T cloning vector (modified Promega PCR product cloning vector with tra genes, transposable element (Tn5)) and M13 sequence via α-complementation mechanism in order to activate β-d-galactosidase gene in DH5α strain (non-lactose-fermenting host). Results Trans-conjugation with pGEM-T allows correction of LacZ gene deletion through Tn5, and successful trans-conjugants in DH5α host cells can be able to produce active enzyme, thus described as lactose fermenting strain. The intracellular β-galactosidase was subjected to precipitation by ammonium sulfate and subsequently gel filtration, and the purified enzyme showed a molecular weight of approximately 72-kDa sodium dodecyl sulfate-polyacrylamid gel electrophoresis. The purified enzyme activity showed an optimal pH and temperature of 7.5 and 40 °C, respectively; it had a high stability within pH 6–8.5 and moderate thermal stability up to 50 °C. Conclusion Trans-conjugant of E. coli DH5α- lacZ∆M15 was successfully implemented. UV mutagenesis of the potent trans-conjugant isolate provides an improvement of the enzyme productivity. The enzymatic competitive inhibition by d-galactose and hydrolysis of lactose at ambient temperatures could make this enzyme a promising candidate for use in the dairy industry.

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“…As a rule of thumb, thermal stability is imperative for the application of enzymes in practice. To obtain quantified BGAs for GOS manufacturing, BGAs from various sources had been extensively studied [ 11 , 12 , 13 ]. In our previous study, a novel β-1,3-galactosidase (MaBGA) from Marinomonas sp.…”

Section: Introductionmentioning

confidence: 99%

Heterologous Expression of a Thermostable β-1,3-Galactosidase and Its Potential in Synthesis of Galactooligosaccharides

et al. 2018

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A thermostable β-1,3-galactosidase from Marinomonas sp. BSi20414 was successfully heterologously expressed in Escherichia coli 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 OD600 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−1 at 37 °C using ONPG (o-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.

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Characterization of a β-galactosidase from Bacillus subtilis with transgalactosylation activity

Cited by 26 publications

References 42 publications

Exploring the taxonomical and functional profile of As Burgas hot spring focusing on thermostable β-galactosidases

Exploring the taxonomical and functional profile of As Burgas hot spring focusing on thermostable β-galactosidases

Activation of LacZ gene in Escherichia coli DH5α via α-complementation mechanism for β-galactosidase production and its biochemical characterizations

Heterologous Expression of a Thermostable β-1,3-Galactosidase and Its Potential in Synthesis of Galactooligosaccharides

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