Recombinant expression and tryptophan-assisted analysis of human sweet taste receptor T1R3's extracellular domain in sweetener interaction studies.

The human palate can discern multiple tastes; however, it predominantly perceives five fundamental flavors: sweetness, saltiness, sourness, bitterness, and umami. Sweetness is primarily mediated through the sweet taste receptor, a membrane-bound heterodimeric structure comprising T1R2-T1R3. However, unraveling the structural and mechanistic intricacies of the sweet taste receptor has proven challenging.

Functional significance of serine 13 in the active site of glutathione S-transferase F3 from Oryza sativa.

Plant glutathione S-transferase (GST, EC 2.5.1.

A quantitative assay for agarase activity determination using agarose-iodine complex.

Rapid and simple spectrophotometric methods are required to detect various oligosaccharides produced by agar-hydrolysing enzymes. Herein, we present a quantitative agarose-iodine assay for agarase activity determination via the detection of the extent of agarose degradation. The agarose-iodine complex becomes reddish orange upon the addition of Lugol solution, and the enzymatic activity can be detected with ultraviolet-visible spectroscopy at 600 nm.

Efficient brazzein production in yeast (Kluyveromyces lactis) using a chemically defined medium.

The sweet-tasting protein brazzein offers considerable potential as a functional sweetener with antioxidant, anti-inflammatory, and anti-allergic properties. Here, we optimized a chemically defined medium to produce secretory recombinant brazzein in Kluyveromyces lactis, with applications in mass production. Compositions of defined media were investigated for two phases of fermentation: the first phase for cell growth, and the second for maximum brazzein secretory production.

Histidine residues at the copper-binding site in human tyrosinase are essential for its catalytic activities.

Tyrosinase is a copper-binding enzyme involved in melanin biosynthesis. However, the detailed structure of human tyrosinase has not yet been solved, along with the identification of the key sites responsible for its catalytic activity. We used site-directed mutagenesis to identify the residues critical for the copper binding of human tyrosinase.

3M-Brazzein as a Natural Sugar Substitute Attenuates Obesity, Metabolic Disorder, and Inflammation.

Obesity is a global chronic disease linked to various diseases. Increased consumption of added sugars, especially in beverages, is a key contributor to the obesity epidemic. It is essential to reduce or replace sugar intake with low-calorie sweeteners.

Optimized production and quantification of the tryptophan-deficient sweet-tasting protein brazzein in .

The sweet-tasting protein brazzein is a candidate sugar substitute owing to its sweet, sugar-like taste and good stability. To commercialize brazzein as a sweetener, optimization of fermentation and purification procedure is necessary. Here, we report the expression conditions of brazzein in the yeast and purification method for maximum yield.

Antioxidant, anti-inflammatory, and anti-allergic activities of the sweet-tasting protein brazzein.

Sweet-tasting proteins may be useful as low-calorie sugar substitutes in foods, beverages, and medicines. Brazzein is an attractive sweetener because of its high sweetness, sugar-like taste, and good stability at high temperature and wide pH ranges. To investigate the bioactivities of brazzein, the antibacterial, antifungal, antioxidant, anti-inflammatory, and anti-allergic activities were determined in vitro.

Secretory expression and enzymatic characterization of recombinant Agarivorans albus β-agarase in Escherichia coli.

Agarase catalyzes the hydrolysis of agar, which is primarily used as a medium for microbiology, various food additives, and new biomass materials. In this study, we described the expression of the synthetic gene encoding β-agarase from Agarivorans albus (Aaβ-agarase) in Escherichia coli. The synthetic β-agarase gene was designed based on the biased codons of E.

Improved Secretory Production of the Sweet-Tasting Protein, Brazzein, in Kluyveromyces lactis.

Brazzein is an intensely sweet protein with high stability over a wide range of pH values and temperatures, due to its four disulfide bridges. Recombinant brazzein production through secretory expression in Kluyveromyces lactis is reported, but is inefficient due to incorrect disulfide formation, which is crucial for achieving the final protein structure and stability. Protein disulfide bond formation requires protein disulfide isomerase (PDI) and Ero1p.

Importance of Glu53 in the C-terminal region of brazzein, a sweet-tasting protein.

Background: The sweetness of brazzein, one of the known sweet proteins, is dependent on charges and/or structures of its specific amino acid side chains. As the residues in the C-terminus of brazzein are known to play a critical role in sweetness, the currently unknown function of Glu53 requires further study.

Results: To identify important residues responsible for the sweetness of the protein brazzein, four mutants of the Glu53 residue in the C-terminal region of des-pE1M-brazzein, which lacks the N-terminal pyroglutamate, were constructed using site-directed mutagenesis.

Efficient secretory expression of the sweet-tasting protein brazzein in the yeast Kluyveromyces lactis.

Brazzein is an intensely sweet-tasting protein with high water solubility, heat stability, and taste properties resembling those of carbohydrate sweeteners. In the present study, we describe the expression of the synthetic gene encoding brazzein, a sweet protein in the yeast Kluyveromyces lactis. The synthetic brazzein gene was designed based on the biased codons of the yeast, so as to optimize its expression, as well as on the extracellular secretion for expression in an active, soluble form.

Multiple mutations of the critical amino acid residues for the sweetness of the sweet-tasting protein, brazzein.

We have previously identified critical residues important for sweetness of the sweet protein brazzein by site-directed mutagenesis (Yoon, Kong, Jo, & Kong, 2011). In order to elucidate the interaction mechanisms of brazzein with the sweet taste receptor, we made multiple mutations of three residues (His31 in loop 30-33, Glu36 in β-strand III, and Glu41 in loop 40-43). We found that all double mutations (H31R/E36D, H31R/E41A and E36D/E41A) made the molecules sweeter than des-pE1M-brazzein and three single mutants.

An efficient method for the expression and reconstitution of thermostable Mn/Fe superoxide dismutase from Aeropyrum pernix K1.

The gene APE0743 encoding the superoxide dismutase (ApSOD) of a hyperthermophilic archaeon Aeropyrum pernix K1 was cloned and over-expressed as a GST fusion protein at a high level in Escherichia coli. The expressed protein was simply purified by the process of glutathione affinity chromatography and thrombin treatment. The ApSOD was a homodimer of 25 kDa subunits and a cambialistic SOD which was active with either Fe(II) or Mn(II) as a cofactor.

Characterization of human tyrosinase ectodomain expressed in Escherichia coli.

The human tyrosinase ectodomain has been expressed in Escherichia coli as a soluble form and purified by immobilized metal affinity column chromatography. The ectodomain exhibited tyrosinase activities toward the hydroxylation and oxidation reactions. Biochemical properties of the ectodomain appeared to be distinct from those of the human tyrosinase, although common features were retained.

Study on the biochemical characterization of herbicide detoxification enzyme, glutathione S-transferase.

To gain further insight into herbicide detoxification, we studied the herbicide activity and specificity toward glutathione S-transferases from human and rice. In this study, the genes of the plant specific phi and tau class GST enzymes from Oryza sativa (OsGST) and human pi class GST enzyme (hGSTP1-1) were cloned and expressed in Escherichia coli with the pET and pKK vector systems, respectively. The gene products were purified to homogeneity by GSH Sepharose affinity column chromatography.

Site-specific methylation of CpG nucleotides in the hTERT promoter region can control the expression of hTERT during malignant progression of colorectal carcinoma.

Expression of hTERT has been recognized an important factor in cellular aging and immortalization. Therefore, to analyze regulatory mechanism of hTERT expression, we investigated the CpG methylation pattern of the hTERT promoter as an epigenetic mechanism and its implication in transcriptional regulation of hTERT using tissues of colorectal carcinoma. As a result, we were able to observe an increased pattern of hTERT expression according to the malignant progression of colorectal carcinoma.

A phi class glutathione S-transferase from Oryza sativa (OsGSTF5): molecular cloning, expression and biochemical characteristics.

A glutathione S-transferase (GST) related to the phi (F) class of enzymes only found in plants has been cloned from the Oryza sativa. The GST cDNA was cloned by PCR using oligonucleotide primers based on the OsGSTF5 (GenBank Accession No. AF309382) sequences.

Purification and biochemical properties of glutathione S-transferase from Lactuca sativa.

A glutathione S-transferase (GST) from Lactuca sativa was purified to electrophoretic homogeneity approximately 403-fold with a 9.6% activity yield by DEAE-Sephacel and glutathione (GSH)-Sepharose column chromatography. The molecular weight of the enzyme was determined to be approximately 23,000 by SDS-polyacrylamide gel electrophoresis and 48,000 by gel chromatography, indicating a homodimeric structure.

The highly stable alcohol dehydrogenase of Thermomicrobium roseum: purification and molecular characterization.

An alcohol dehydrogenase (ADH) was purified to electrophoretic homogeneity from an extremely thermophilic bacterium, Thermomicrobium roseum. The native enzyme was found to be a homo-dimer of 43-kDa subunits. The pI of the enzyme was determined to be 6.