Purification, characterization and probiotic proliferation effect of exopolysaccharides produced by Lactiplantibacillus plantarum HDC-01 isolated from sauerkraut

In this study, an exopolysaccharide (EPS)-producing strain of Lactiplantibacillus plantarum HDC-01 was isolated from sauerkraut, and the structure, properties and biological activity of the studied EPS were assessed. The molecular weight of the isolated EPS is 2.505 × 106 Da. Fourier transform infrared spectrometry (FT-IR) and nuclear magnetic resonance (NMR) results showed that the EPS was composed of glucose/glucopyranose subunits linked by an α-(1 → 6) glycosidic bond and contained an α-(1 → 3) branching structure. X-ray diffraction (XRD) analysis revealed the amorphous nature of the EPS. Scanning electron microscopy (SEM) and atomic force microscopy (AFM) showed that the isolated EPS had a smooth and compact surface with several protrusions of varying lengths and irregularly shaped material. Moreover, the studied EPS showed good thermal stability, water holding capacity, and milk coagulation ability and promoted the growth of probiotics. L. plantarum EPS may be used as prebiotics in the fields of food and medicine

Research Progress of Glucansucrase from Lactic Acid Bacteria

Glucansucrase (EC.2.4.5.1) is a class of α-glucosyltransferases, mainly produced by lactic acid bacteria such as Leuconostoc, Streptococcus and Lactobacillus. The structure and catalytic mechanism of glucansucrase are diverse, and it is an important tool enzyme for the biosynthesis of exopolysaccharide. This article mainly reviews the source, classification, structure and reaction mechanism of glucansucrase and the effects of medium composition, culture conditions on the production of glucansucrase, with a focus on the optimization methods, separation and purification processes, and enzymatic properties of glucansucrase, and prospects for its development trend. This article aims to provide reference for the research of glucansucrase in related fields

Glucansucrase Produced by Lactic Acid Bacteria: Structure, Properties, and Applications

Glucansucrase (GS) belongs to the GH70 family, which not only can synthesize exopolysaccharides (EPSs) with different physicochemical properties through glucosyl transglycosylation (by hydrolyzing sucrose) but can also produce oligosaccharides. Different strains produce different GSs, which catalyze the synthesis of EPS with different glycosidic bond structures; these EPSs have different biological functions. As an important enzymatic tool, GS has great potential in health care medicine, biological materials, ecological protection, the food chemical industry, etc. GS is mainly produced by lactic acid bacteria (LAB), including Leuconostoc, Streptococcus, Lactobacillus, and Weissella species. With the elucidation of the crystal structure of GS and the advancement of genome sequencing technology, its synthesis reaction mechanism and specific structural characteristics are gradually becoming clear. This review summarizes the isolation, purification, physical and chemical properties, detection methods, sources, and applications of GS in order to provide a reference for the research and development of GS

The Purification and Biochemical Characterization of a <i>Weissella cibaria</i> F1 Derived <i>β</i>-Mannanase for Its Use in the Preparation of Konjac Oligo-Glucomannan with Immunomodulatory Properties

Mannanase with a molecular weight of 33.1 kDa was purified from Weissella cibaria F1. The F1 mannanase contained 289 amino acid residues and shared 70.0% similarity with mannanase from Bacillus subtilis (P55278 (MANB_BACIU)). The optimum reaction conditions of F1 mannanase were 50 °C and pH 6.5. After incubation at pH 4.5–8.0 and 30–60 °C for 2 h, the enzyme activity remained above 60%. The effects of metal ions on mannanase enzyme activity were measured, and Mn2+, Mg2+, and Cu2+ increased enzyme activity. The Km (16.96 ± 0.01 μmol·mL−1) and Vmax (1119.05 ± 0.14 μmol·min−1) values showed that the enzyme exhibited high affinity for locust bean gum. Mannanase was used to hydrolyze konjac glucomannan to produce konjac oligo-glucomannan (KOGM). KOGM increased the proliferation and phagocytosis of RAW264.7 macrophages and enhanced nitric oxide, and cytokine production in macrophages, which showed potent immunostimulatory activity. In this study, the advantages of mannanase derived from lactic acid bacteria were utilized to expand the application of KOGM in the medical field, which is helpful to explore the broad prospects of KOGM in functional food or medicine

The Purification and Biochemical Characterization of a Weissella cibaria F1 Derived &beta;-Mannanase for Its Use in the Preparation of Konjac Oligo-Glucomannan with Immunomodulatory Properties

Mannanase with a molecular weight of 33.1 kDa was purified from Weissella cibaria F1. The F1 mannanase contained 289 amino acid residues and shared 70.0% similarity with mannanase from Bacillus subtilis (P55278 (MANB_BACIU)). The optimum reaction conditions of F1 mannanase were 50 &deg;C and pH 6.5. After incubation at pH 4.5&ndash;8.0 and 30&ndash;60 &deg;C for 2 h, the enzyme activity remained above 60%. The effects of metal ions on mannanase enzyme activity were measured, and Mn2+, Mg2+, and Cu2+ increased enzyme activity. The Km (16.96 &plusmn; 0.01 &mu;mol&middot;mL&minus;1) and Vmax (1119.05 &plusmn; 0.14 &mu;mol&middot;min&minus;1) values showed that the enzyme exhibited high affinity for locust bean gum. Mannanase was used to hydrolyze konjac glucomannan to produce konjac oligo-glucomannan (KOGM). KOGM increased the proliferation and phagocytosis of RAW264.7 macrophages and enhanced nitric oxide, and cytokine production in macrophages, which showed potent immunostimulatory activity. In this study, the advantages of mannanase derived from lactic acid bacteria were utilized to expand the application of KOGM in the medical field, which is helpful to explore the broad prospects of KOGM in functional food or medicine

ROS play an important role in ATPR inducing differentiation and inhibiting proliferation of leukemia cells by regulating the PTEN/PI3K/AKT signaling pathway

Abstract Background Acute myeloid leukemia (AML) is an aggressive and mostly incurable hematological malignancy with frequent relapses after an initial response to standard chemotherapy. Therefore, novel therapies are urgently required to improve AML clinical outcomes. 4-Amino-2-trifluoromethyl-phenyl retinate (ATPR), a novel all-trans retinoic acid (ATRA) derivative designed and synthesized by our team, has been proven to show biological anti-tumor characteristics in our previous studies. However, its potential effect on leukemia remains unknown. The present research aims to investigate the underlying mechanism of treating leukemia with ATPR in vitro. Methods In this study, the AML cell lines NB4 and THP-1 were treated with ATPR. Cell proliferation was analyzed by the CCK-8 assay. Flow cytometry was used to measure the cell cycle distribution and cell differentiation. The expression levels of cell cycle and differentiation-related proteins were detected by western blotting and immunofluorescence staining. The NBT reduction assay was used to detect cell differentiation. Results ATPR inhibited cell proliferation, induced cell differentiation and arrested the cell cycle at the G0/G1 phase. Moreover, ATPR treatment induced a time-dependent release of reactive oxygen species (ROS). Additionally, the PTEN/PI3K/Akt pathway was downregulated 24 h after ATPR treatment, which might account for the anti-AML effects of ATPR that result from the ROS-mediated regulation of the PTEN/PI3K/AKT signaling pathway. Conclusions Our observations could help to develop new drugs targeting the ROS/PTEN/PI3K/Akt pathway for the treatment of AML

Characterization of Dextran Biosynthesized by Glucansucrase from <i>Leuconostoc pseudomesenteroides</i> and Their Potential Biotechnological Applications

Glucansucrase was purified from Leuconostoc pseudomesenteroides. The glucansucrase exhibited maximum activity at pH 5.5 and 30 °C. Ca2+ significantly promoted enzyme activity. An exopolysaccharide (EPS) was synthesized by this glucansucrase in vitro and purified. The molecular weight of the EPS was 3.083 × 106 Da. Fourier transform infrared (FT-IR) and nuclear magnetic resonance (NMR) spectroscopy showed that the main structure of glucan was 97.3% α-(1→6)-linked D-glucopyranose units, and α-(1→3) branched chain accounted for 2.7%. Scanning electron microscopy (SEM) observation of dextran showed that its surface was smooth and flaky. Atomic force microscopy (AFM) of dextran revealed a chain-like microstructure with many irregular protuberances in aqueous solution. The results showed that dextran had good thermal stability, water holding capacity, water solubility and emulsifying ability (EA), as well as good antioxidant activity; thus it has broad prospects for development in the fields of food, biomedicine, and medicine

Obtaining a Panel of Cascade Promoter-5′-UTR Complexes in <i>Escherichia coli</i>

A promoter is one of the most important and basic tools used to achieve diverse synthetic biology goals. Escherichia coli is one of the most commonly used model organisms in synthetic biology to produce useful target products and establish complicated regulation networks. During the fine-tuning of metabolic or regulation networks, the limited number of well-characterized inducible promoters has made implementing complicated strategies difficult. In this study, 104 native promoter-5′-UTR complexes (PUTR) from <i>E. coli</i> were screened and characterized based on a series of RNA-seq data. The strength of the 104 PUTRs varied from 0.007% to 4630% of that of the P_BAD promoter in the transcriptional level and from 0.1% to 137% in the translational level. To further upregulate gene expression, a series of combinatorial PUTRs and cascade PUTRs were constructed by integrating strong transcriptional promoters with strong translational 5′-UTRs. Finally, two combinatorial PUTRs (P_ssrA-UTR_rpsT and P_dnaKJ-UTR_rpsT) and two cascade PUTRs (PUTR_ssrA-PUTR_infC‑rplT and PUTR_alsRBACE-PUTR_infC‑rplT) were identified as having the highest activity, with expression outputs of 170%, 137%, 409%, and 203% of that of the P_BAD promoter, respectively. These engineered PUTRs are stable for the expression of different genes, such as the red fluorescence protein gene and the β-galactosidase gene. These results show that the PUTRs characterized and constructed in this study may be useful as a plug-and-play synthetic biology toolbox to achieve complicated metabolic engineering goals in fine-tuning metabolic networks to produce target products

Optimization and purification of glucansucrase produced by <i>Leuconostoc mesenteroides</i> DRP2-19 isolated from Chinese Sauerkraut

<p>Strain DRP2-19 was detected to produce high yield of glucansucrase in MRS broth, which was identified to be <i>Leuconostoc mesenteroides</i>. In order for industrial glucansucrase production of <i>L. mesenteroides</i> DRP2-19, a one-factor test was conducted, then response surface method was applied to optimize its yield and discover the best production condition. Based on Plackett–Burman (PB) experiment, sucrose, Ca²⁺, and initial pH were found to be the most significant factors for glucansucrase production. Afterwards, effects of the three main factors on glucansucrase activity were further investigated by central composite design and the optimum composition was sucrose 35.87 g/L, Ca²⁺ 0.21 mmol/L, and initial pH 5.56. Optimum results showed that glucansucrase activity was increased to 3.94 ± 0.43 U/mL in 24 hr fermentation, 2.66-fold higher than before. In addition, the crude enzyme was purified using ammonium sulfate precipitation, ion-exchange chromatography, and gel filtration. The molecular weight of glucansucrase was determined as approximately 170 kDa by Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme was purified 15.77-fold and showed a final specific activity of 338.56 U/mg protein.</p