Dataset Information

Enhanced Antioxidant Activity in Streptococcus thermophilus by High-Level Expression of Superoxide Dismutase.

ABSTRACT: Superoxide dismutase (SOD) plays an essential role in eliminating oxidative damage of lactic acid bacteria. Streptococcus thermophilus, an important probiotic lactic acid bacterium, often inevitably suffers from various oxidative stress during dairy fermentation. In this study, to confer high-level oxidative resistance, the sod gene from Lactobacillus casei was heterologous expressed in S. thermophilus S-3 using our previous constructed native constitutive promoter library. The enzyme activity of SOD was significantly enhanced in engineered S. thermophilus by promoter #14 (2070 U/mg). Furthermore, the strategy of multi-copy sod-expressing cassettes was employed to improve SOD activity. The maximum activity (2750 U/mg) was obtained by the two-copy sod recombinant, which was 1.5-fold higher than that of one-copy recombinant. In addition, the survival rate of multi-copy sod recombinants was increased about 97-fold with 3.5 mmol/L H₂O₂ treatment. To our knowledge, this is the first report of multi-copy sod gene expression in S. thermophilus, which exerts a positive effect on coping with oxidative stress to enhance the potential of industrial application.

SUBMITTER: Kong L

PROVIDER: S-EPMC7688587 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Enhanced Antioxidant Activity in Streptococcus thermophilus by High-Level Expression of Superoxide Dismutase.

Kong Linghui L Xiong Zhiqiang Z Song Xin X Xia Yongjun Y Zhang Hui H Yang Ying Y Ai Lianzhong L

Frontiers in microbiology 20201112

Superoxide dismutase (SOD) plays an essential role in eliminating oxidative damage of lactic acid bacteria. Streptococcus thermophilus, an important probiotic lactic acid bacterium, often inevitably suffers from various oxidative stress during dairy fermentation. In this study, to confer high-level oxidative resistance, the sod gene from Lactobacillus casei was heterologous expressed in S. thermophilus S-3 using our previous constructed native constitutive promoter li ...[more]

PMID: 33281773

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Project description:ImportanceX-linked adrenoleukodystrophy (ALD) may switch phenotype to the fatal cerebral form (ie, cerebral ALD [cALD]), the cause of which is unknown. Determining differences in antioxidant capacity and superoxide dismutase (SOD) levels between phenotypes may allow for the generation of a clinical biomarker for predicting the onset of cALD, as well as initiating a more timely lifesaving therapy.ObjectiveTo identify variations in the levels of antioxidant capacity and SOD activity between ALD phenotypes in patients with cALD or adrenomyeloneuropathy (AMN), heterozygote female carriers, and healthy controls and, in addition, correlate antioxidant levels with clinical outcome scores to determine a possible predictive value.Design, setting, and participantsSamples of monocytes and blood plasma were prospectively collected from healthy controls, heterozygote female carriers, and patients with AMN or cALD. We are counting each patient as 1 sample in our study. Because adrenoleukodystrophy is an X-linked disease, the affected group populations of cALD and AMN are all male. The heterozygote carriers are all female. The samples were assayed for total antioxidant capacity and SOD activity. The data were collected in an academic hospital setting. Eligibility criteria included patients who received a diagnosis of ALD and heterozygote female carriers, both of which groups were compared with age-matched controls. The prospective samples (n?=?30) were collected between January 2015 to January 2016, and existing samples were collected from tissue storage banks at the Kennedy Krieger Institute (n?=?30). The analyses were performed during the first 3 months of 2016.Main outcome and measuresCommercially available total antioxidant capacity and SOD assays were performed on samples of monocytes and blood plasma and correlated with magnetic resonance imaging severity score.ResultsA reduction in antioxidant capacity was shown between the healthy controls (0.225 mmol trolox equivalent) and heterozygote carriers (0.181 mmol trolox equivalent), and significant reductions were seen between healthy controls and patients with AMN (0.102 mmol trolox equivalent; P?<?.01), as well as healthy controls and patients with cALD (0.042 mmol trolox equivalent; P?<?.01). Superoxide dismutase activity in human blood plasma mirrored these reductions between prospectively collected samples from healthy controls (2.66 units/mg protein) and samples from heterozygote female carriers (1.91 units/mg protein), patients with AMN (1.39 units/mg protein; P?=?.01), and patients with cALD (0.8 units/mg protein; P?<?.01). Further analysis of SOD activity in biobank samples showed significant reductions between patients with AMN (0.89 units/mg protein) and patients with cALD (0.18 units/mg protein) (P?=?.03). Plasma SOD levels from patients with cALD demonstrated an inverse correlation to brain magnetic resonance imaging severity score (R2?=?0.75, P?<?.002). Longitudinal plasma SOD samples from the same patients (n?=?4) showed decreased activity prior to and at the time of cerebral diagnosis over a period of 13 to 42 months (mean period, 24 months).Conclusions and relevancePlasma SOD may serve as a potential biomarker for cerebral disease in ALD following future prospective studies.

Project description:Superoxide dismutase 1 (SOD1) is known to be involved in the pathogenesis of Amyotrophic Lateral Sclerosis (ALS) and is therefore considered to be an important ALS drug target. Identifying potential drug leads that bind to SOD1 and characterizing their interactions by nuclear magnetic resonance (NMR) spectroscopy is complicated by the fact that SOD1 is a homodimer. Creating a monomeric version of SOD1 could alleviate these issues. A specially designed monomeric form of human superoxide dismutase (T2M4SOD1) was cloned into E. coli and its expression significantly enhanced using a number of novel DNA sequence, leader peptide and growth condition optimizations. Uniformly 15N-labeled T2M4SOD1 was prepared from minimal media using 15NH4Cl as the 15N source. The T2M4SOD1 monomer (both 15N labeled and unlabeled) was correctly folded as confirmed by 1H-NMR spectroscopy and active as confirmed by an in-gel enzymatic assay. To demonstrate the utility of this new SOD1 expression system for NMR-based drug screening, eight pyrimidine compounds were tested for binding to T2M4SOD1 by monitoring changes in their 1H NMR and/or 19F-NMR spectra. Weak binding to 5-fluorouridine (FUrd) was observed via line broadening, but very minimal spectral changes were seen with uridine, 5-bromouridine or trifluridine. On the other hand, 1H-NMR spectra of T2M4SOD1 with uracil or three halogenated derivatives of uracil changed dramatically suggesting that the pyrimidine moiety is the crucial binding component of FUrd. Interestingly, no change in tryptophan 32 (Trp32), the putative receptor for FUrd, was detected in the 15N-NMR spectra of 15N-T2M4SOD1 when mixed with these uracil analogs. Molecular docking and molecular dynamic (MD) studies indicate that interaction with Trp32 of SOD1 is predicted to be weak and that there was hydrogen bonding with the nearby aspartate (Asp96), potentiating the Trp32-uracil interaction. These studies demonstrate that monomeric T2M4SOD1 can be readily used to explore small molecule interactions via NMR.

Dataset Information

Enhanced Antioxidant Activity in Streptococcus thermophilus by High-Level Expression of Superoxide Dismutase.

Publications

Enhanced Antioxidant Activity in <i>Streptococcus thermophilus</i> by High-Level Expression of Superoxide Dismutase.

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