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Identification and characterization of a novel ?-D-galactosidase that releases pyruvylated galactose.

ABSTRACT: Pyruvyl modification of oligosaccharides is widely seen in both prokaryotes and eukaryotes. Although the biosynthetic mechanisms of pyruvylation have been investigated, enzymes that metabolize and degrade pyruvylated oligosaccharides are not well known. Here, we searched for a pyruvylated galactose (PvGal)-releasing enzyme by screening soil samples. We identified a Bacillus strain, as confirmed by the 16S ribosomal RNA gene analysis, that exhibited PvGal-ase activity toward p-nitrophenyl-?-D-pyruvylated galactopyranose (pNP-?-D-PvGal). Draft genome sequencing of this strain, named HMA207, identified three candidate genes encoding potential PvGal-ases, among which only the recombinant protein encoded by ORF1119 exhibited PvGal-ase activity. Although ORF1119 protein displayed broad substrate specificity for pNP sugars, pNP-?-D-PvGal was the most favorable substrate. The optimum pH for the ORF1119 PvGal-ase was determined as 7.5. A BLAST search suggested that ORF1119 homologs exist widely in bacteria. Among two homologs tested, BglC from Clostridium but not BglH from Bacillus showed PvGal-ase activity. Crystal structural analysis together with point mutation analysis revealed crucial amino acids for PvGal-ase activity. Moreover, ORF1119 protein catalyzed the hydrolysis of PvGal from galactomannan of Schizosaccharomyces pombe, suggesting that natural polysaccharides might be substrates of the PvGal-ase. This novel PvGal-catalyzing enzyme might be useful for glycoengineering projects to produce new oligosaccharide structures.

SUBMITTER: Higuchi Y

PROVIDER: S-EPMC6090015 | biostudies-literature | 2018 Aug

REPOSITORIES: biostudies-literature

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Identification and characterization of a novel β-D-galactosidase that releases pyruvylated galactose.

Higuchi Yujiro Y Matsufuji Hitomi H Tanuma Masanari M Arakawa Takatoshi T Mori Kazuki K Yamada Chihaya C Shofia Risa R Matsunaga Emiko E Tashiro Kosuke K Fushinobu Shinya S Takegawa Kaoru K

Scientific reports 20180813 1

Pyruvyl modification of oligosaccharides is widely seen in both prokaryotes and eukaryotes. Although the biosynthetic mechanisms of pyruvylation have been investigated, enzymes that metabolize and degrade pyruvylated oligosaccharides are not well known. Here, we searched for a pyruvylated galactose (PvGal)-releasing enzyme by screening soil samples. We identified a Bacillus strain, as confirmed by the 16S ribosomal RNA gene analysis, that exhibited PvGal-ase activity toward p-nitrophenyl-β-D-pyr ...[more]

PMID: 30104607

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Project description:Backgroundβ-D-Galactosidases (EC 3.2.1.23) catalyze the hydrolysis of terminal non-reducing β-D-galactose residues in β-D-galactosides. Cold-active β-D-galactosidases have recently become a focus of attention of researchers and dairy product manufactures owing to theirs ability to: (i) eliminate of lactose from refrigerated milk for people afflicted with lactose intolerance, (ii) convert lactose to glucose and galactose which increase the sweetness of milk and decreases its hydroscopicity, and (iii) eliminate lactose from dairy industry pollutants associated with environmental problems. Moreover, in contrast to commercially available mesophilic β-D-galactosidase from Kluyveromyces lactis the cold-active counterparts could make it possible both to reduce the risk of mesophiles contamination and save energy during the industrial process connected with lactose hydrolysis.ResultsA genomic DNA library was constructed from soil bacterium Paracoccus sp. 32d. Through screening of the genomic DNA library on LB agar plates supplemented with X-Gal, a novel gene encoding a cold-active β-D-galactosidase was isolated. The in silico analysis of the enzyme amino acid sequence revealed that the β-D-galactosidase Paracoccus sp. 32d is a novel member of Glycoside Hydrolase Family 2. However, owing to the lack of a BGal_small_N domain, the domain characteristic for the LacZ enzymes of the GH2 family, it was decided to call the enzyme under study 'BgaL'. The bgaL gene was cloned and expressed in Escherichia coli using the pBAD Expression System. The purified recombinant BgaL consists of two identical subunits with a combined molecular weight of about 160 kDa. The BgaL was optimally active at 40°C and pH 7.5. Moreover, BgaL was able to hydrolyze both lactose and o-nitrophenyl-β-D-galactopyranoside at 10°C with Km values of 2.94 and 1.17 mM and kcat values 43.23 and 71.81 s-1, respectively. One U of the recombinant BgaL would thus be capable hydrolyzing about 97% of the lactose in 1 ml of milk in 24 h at 10°C.ConclusionsA novel bgaL gene was isolated from Paracoccus sp. 32d encoded a novel cold-active β-D-galactosidase. An E. coli expression system has enabled efficient production of soluble form of BgaL Paracoccus sp. 32d. The amino acid sequence analysis of the BgaL enzyme revealed notable differences in comparison to the result of the amino acid sequences analysis of well-characterized cold-active β-D-galactosidases belonging to Glycoside Hydrolase Family 2. Finally, the enzymatic properties of Paracoccus sp. 32d β-D-galactosidase shows its potential for being applied to development of a new industrial biocatalyst for efficient lactose hydrolysis in milk.

Project description:Backgroundβ-Galactosidases can be used to produce low-lactose milk and dairy products for lactose intolerant people. Although commercial β-galactosidases have outstanding lactose hydrolysis ability, their thermostability is low, and reaction products have strong inhibition to these enzymes. In addition, the β-galactosidases possessing simultaneously high thermostability and tolerance of galactose and glucose are still seldom reported until now. Therefore, identification of novel β-galactosidases with high thermostability and tolerance to reaction products from unculturable microorganisms accounting for over 99% of microorganisms in the environment via metagenomic strategy is still urgently in demand.ResultsIn the present study, a novel β-galactosidase (Gal308) consisting of 658 amino acids was identified from a metagenomic library from soil samples of Turpan Basin in China by functional screening. After being overexpressed in Escherichia coli and purified to homogeneity, the enzymatic properties of Gal308 with N-terminal fusion tag were investigated. The recombinant enzyme displayed a pH optimum of 6.8 and a temperature optimum of 78 °C, and was considerably stable in the temperature range of 40 °C - 70 °C with almost unchangeable activity after incubation for 60 min. Furthermore, Gal308 displayed a very high tolerance of galactose and glucose, with the highest inhibition constant K(i,gal) (238 mM) and K(i,glu) (1725 mM) among β-galactosidases. In addition, Gal308 also exhibited high enzymatic activity for its synthetic substrate o-nitrophenyl-β-D-galactopyranoside (ONPG, 185 U/mg) and natural substrate lactose (47.6 U/mg).ConclusionThis study will enrich the source of β-galactosidases, and attract some attentions to β-galactosidases from extreme habitats and metagenomic library. Furthermore, the recombinant Gal308 fused with 156 amino acids exhibits many novel properties including high activity and thermostability at high temperatures, the pH optimum of 6.8, high enzyme activity for lactose, as well as high tolerance of galactose and glucose. These properties make it a good candidate in the production of low-lactose milk and dairy products after further study.

Dataset Information

Identification and characterization of a novel ?-D-galactosidase that releases pyruvylated galactose.

Publications

Identification and characterization of a novel β-D-galactosidase that releases pyruvylated galactose.

Similar Datasets

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