Dataset Information

Exploring a Highly D-Galactose Specific L-Arabinose Isomerase From Bifidobacterium adolescentis for D-Tagatose Production.

ABSTRACT: D-Galactose-specific L-arabinose isomerase (L-AI) would have much potential for the enzymatic conversion of D-Galactose into D-tagatose, while most of the reported L-AIs are L-arabinose specific. This study explored a highly D-Galactose-specific L-AI from Bifidobacterium adolescentis (BAAI) for the production of D-tagatose. In the comparative protein-substrate docking for D-Galactose and L-arabinose, BAAI showed higher numbers of hydrogen bonds in D-Galactose-BAAI bonding site than those found in L-arabinose-BAAI bonding site. The activity of BAAI was 24.47 U/mg, and it showed good stability at temperatures up to 65°C and a pH range 6.0-7.5. The K m, V max, and K cat/K m of BAAI were found to be 22.4 mM, 489 U/mg and 9.3 mM-1 min-1, respectively for D-Galactose, while the respective values for L-arabinose were 40.2 mM, 275.1 U/mg, and 8.6 mM-1 min-1. Enzymatic conversion of D-Galactose into D-tagatose by BAAI showed 56.7% conversion efficiency at 55°C and pH 6.5 after 10 h.

SUBMITTER: Zhang G

PROVIDER: S-EPMC7201074 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Exploring a Highly D-Galactose Specific L-Arabinose Isomerase From <i>Bifidobacterium adolescentis</i> for D-Tagatose Production.

Zhang Guoyan G An Yingfeng Y Parvez Amreesh A Zabed Hossain M HM Yun Junhua J Qi Xianghui X

Frontiers in bioengineering and biotechnology 20200429

D-Galactose-specific L-arabinose isomerase (L-AI) would have much potential for the enzymatic conversion of D-Galactose into D-tagatose, while most of the reported L-AIs are L-arabinose specific. This study explored a highly D-Galactose-specific L-AI from <i>Bifidobacterium adolescentis</i> (BAAI) for the production of D-tagatose. In the comparative protein-substrate docking for D-Galactose and L-arabinose, BAAI showed higher numbers of hydrogen bonds in D-Galactose-BAAI bonding site than those ...[more]

PMID: 32411693

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Project description:BackgroundL-arabinose isomerase (AI) is a crucial catalyst for the biotransformation of D-galactose to D-tagatose. In previous reports, AIs from thermophilic bacterial strains had been wildly researched, but the browning reaction and by-products formed at high temperatures restricted their applications. By contrast, AIs from mesophilic Bacillus strains have some different features including lower optimal temperatures and lower requirements of metallic cofactors. These characters will be beneficial to the development of a more energy-efficient and safer production process. However, the relevant data about the kinetics and reaction properties of Bacillus AIs in D-tagatose production are still insufficient. Thus, in order to support further applications of these AIs, a comprehensive characterization of a Bacillus AI is needed.ResultsThe coding gene (1422 bp) of Bacillus coagulans NL01 AI (BCAI) was cloned and overexpressed in the Escherichia coli BL21 (DE3) strain. The enzymatic property test showed that the optimal temperature and pH of BCAI were 60 °C and 7.5 respectively. The raw purified BCAI originally showed high activity in absence of outsourcing metallic ions and its thermostability did not change in a low concentration (0.5 mM) of Mn(2+) at temperatures from 70 °C to 90 °C. Besides these, the catalytic efficiencies (k cat/K m) for L-arabinose and D-galactose were 8.7 mM(-1) min(-1) and 1.0 mM(-1) min(-1) respectively. Under optimal conditions, the recombinant E. coli cell containing BCAI could convert 150 g L(-1) and 250 g L(-1) D-galactose to D-tagatose with attractive conversion rates of 32 % (32 h) and 27 % (48 h).ConclusionsIn this study, a novel AI from B. coagulans NL01was cloned, purified and characterized. Compared with other reported AIs, this AI could retain high proportions of activity at a broader range of temperatures and was less dependent on metallic cofactors such as Mn(2+). Its substrate specificity was understood deeply by carrying out molecular modelling and docking studies. When the recombinant E. coli expressing the AI was used as a biocatalyst, D-tagatose could be produced efficiently in a simple one-pot biotransformation system.

Project description:BackgroundBifidobacteria are gram-positive, probiotic, and generally regarded as safe bacteria. Techniques such as transformation, gene knockout, and heterologous gene expression have been established for Bifidobacterium, indicating that this bacterium can be used as a cell factory platform. However, there are limited previous reports in this field, likely because of factors such as the highly anaerobic nature of this bacterium. Bifidobacterium adolescentis is among the most oxygen-sensitive Bifidobacterium species. It shows strain-specific gamma-aminobutyric acid (GABA) production. GABA is a potent bioactive compound with numerous physiological and psychological functions. In this study, we investigated whether B. adolesentis could be used for mass production of GABA.ResultsThe B. adolescentis 4-2 strain isolated from a healthy adult human produced approximately 14 mM GABA. It carried gadB and gadC, which encode glutamate decarboxylase and glutamate GABA antiporter, respectively. We constructed pKKT427::Pori-gadBC and pKKT427::Pgap-gadBC plasmids carrying gadBC driven by the original gadB (ori) and gap promoters, respectively. Recombinants of Bifidobacterium were then constructed. Two recombinants with high production abilities, monitored by two different promoters, were investigated. GABA production was improved by adjusting the fermentation parameters, including the substrate concentration, initial culture pH, and co-factor supplementation, using response surface methodology. The optimum initial cultivation pH varied when the promoter region was changed. The ori promoter was induced under acidic conditions (pH 5.2:4.4), whereas the constitutive gap promoter showed enhanced GABA production at pH 6.0. Fed-batch fermentation was used to validate the optimum fermentation parameters, in which approximately 415 mM GABA was produced. The conversion ratio of glutamate to GABA was 92-100%.ConclusionWe report high GABA production in recombinant B. adolescentis. This study provides a foundation for using Bifidobacterium as a cell factory platform for industrial production of GABA.

Project description:D-Tagatose is a natural monosaccharide which can be used as a low-calorie sugar substitute in food, beverages and pharmaceutical products. It is also currently being tested as an anti-diabetic and obesity control drug. D-Tagatose is a rare sugar, but it can be manufactured by the chemical or enzymatic isomerization of D-galactose obtained by a ?-D-galactosidase-catalyzed hydrolysis of milk sugar lactose and the separation of D-glucose and D-galactose. L-Arabinose isomerases catalyze in vitro the conversion of D-galactose to D-tagatose and are the most promising enzymes for the large-scale production of D-tagatose.In this study, the araA gene from psychrotolerant Antarctic bacterium Arthrobacter sp. 22c was isolated, cloned and expressed in Escherichia coli. The active form of recombinant Arthrobacter sp. 22c L-arabinose isomerase consists of six subunits with a combined molecular weight of approximately 335 kDa. The maximum activity of this enzyme towards D-galactose was determined as occurring at 52°C; however, it exhibited over 60% of maximum activity at 30°C. The recombinant Arthrobacter sp. 22c L-arabinose isomerase was optimally active at a broad pH range of 5 to 9. This enzyme is not dependent on divalent metal ions, since it was only marginally activated by Mg2+, Mn2+ or Ca2+ and slightly inhibited by Co2+ or Ni2+. The bioconversion yield of D-galactose to D-tagatose by the purified L-arabinose isomerase reached 30% after 36 h at 50°C. In this study, a recombinant Pichia pastoris yeast strain secreting ?-D-galactosidase Arthrobacter chlorophenolicus was also constructed. During cultivation of this strain in a whey permeate, lactose was hydrolyzed and D-glucose was metabolized, whereas D-galactose was accumulated in the medium. Moreover, cultivation of the P. pastoris strain secreting ?-D-galactosidase in a whey permeate supplemented with Arthrobacter sp. 22c L-arabinose isomerase resulted in a 90% yield of lactose hydrolysis, the complete utilization of D-glucose and a 30% conversion of D-galactose to D-tagatose.The method developed for the simultaneous hydrolysis of lactose, utilization of D-glucose and isomerization of D-galactose using a P. pastoris strain secreting ?-D-galactosidase and recombinant L-arabinose isomerase seems to offer an interesting alternative for the production of D-tagatose from lactose-containing feedstock.

Dataset Information

Exploring a Highly D-Galactose Specific L-Arabinose Isomerase From Bifidobacterium adolescentis for D-Tagatose Production.

Publications

Exploring a Highly D-Galactose Specific L-Arabinose Isomerase From <i>Bifidobacterium adolescentis</i> for D-Tagatose Production.

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