Competitive adsorption of α-lactalbumin in the molten globule state

The molten globule state of α-lactalbumin is a partially denatured form with native-like secondary structure and disordered tertiary structure. Using circular dichroism measurements, it was demonstrated that the molten globule state was produced by decreasing the pH to 2.0 at 25°C or by removing bound Ca²⁺ by treatment with ethylenediamine—tetraacetic acid (EDTA) at pH 7.5 and 40°C. Tension measurements showed that α-lactalbumin in the molten globule state is more easily unfolded at liquid interfaces than is the native protein. Results of competitive adsorption experiments involving α-lactalbumin and β-lactoglobulin at the oil droplet surface in emulsions are consistent with preferential adsorption of α-lactalbumin during emulsification when it is in the molten globule state. In contrast to the difficulty of exchange between α-lactalbumin and β-lactoglobulin at the oil-water interface in emulsions at 25°C, it has been found that the two whey proteins are able partially to displace one another from the oil—water interface at 40°C. While native α-lactalbumin was found to be readily displaced from the oil—water interface by β-lactoglobulin at 40°C, it was found that α-lactalbumin in the molten globule state in the presence of EDTA at 40°C had itself the capacity for displacing β-lactoglobulin from the interface.     

Association of remnant lipoprotein levels with impairment of endothelium-dependent vasomotor function in human coronary arteries

BACKGROUND: It remains undetermined whether triglyceride-rich lipoproteins are an independent risk factor for atherosclerosis. METHODS AND RESULTS: The correlation of responses of coronary arterial diameter (quantitative coronary angiography) and coronary blood flow (intracoronary flow wire technique) to intracoronary infusion of acetylcholine (10 and 50 microg/min) with coronary risk factors including remnant lipoprotein levels was statistically analyzed in 106 consecutive subjects with normal coronary angiograms. Remnant lipoproteins were isolated from fasting blood with an immunoaffinity mixed gel containing anti-apolipoprotein (apo) A-1 and anti-apoB-100 monoclonal antibodies. In multivariate stepwise regression analysis, remnant lipoprotein levels had the most significant correlation with abnormal epicardial coronary vasomotor responses to acetylcholine infusion, reflected by impaired dilation or constriction of the epicardial coronary arteries, and the levels also had an inverse and independent correlation with the coronary blood flow increase in response to acetylcholine. In a subgroup of 53 consecutive subjects, constrictor responses of epicardial coronary diameters to intracoronary infusion of NG-monomethyl-L-arginine (50 micromol/min for 4 minutes) at baseline, reflecting the presence of coronary nitric oxide bioactivity, had an inverse and independent correlation with remnant lipoprotein levels by use of multivariate analysis. CONCLUSIONS: Remnant lipoprotein levels were independently associated with abnormal endothelium-dependent vasomotor function in large and resistance coronary arteries in humans, indicating that remnant lipoproteins may impair endothelial vasomotor function in human coronary arteries. The decrease in coronary nitric oxide bioactivity may be responsible in part for the inhibitory effects of remnant lipoproteins.     

Application of femtosecond laser ablation for detaching grown protein crystals from glass capillary tube

We developed a novel technique for detaching protein crystals from glass capillary tube using the counter diffusion crystallization technique by femtosecond laser irradiation. X-ray diffraction analysis demonstrated that femtosecond laser irradiation has little effect on crystallinity. This technique will contribute to progress in structural genomics as a powerful tool.     

Isolation and characterization of a novel gene encoding alpha-L-arabinofuranosidase from Aspergillus oryzae

We cloned and characterized a novel gene (abfA) encoding alpha-L-arabinofuranosidase (alpha-L-AFase) from Aspergillus oryzae. One clone homologous to the alpha-L-AFase gene of Thermotoga maritima was found in an expressed sequence tag (EST) library of A. oryzae and a corresponding gene was isolated. Molecular analysis showed that the abfA gene carried six exons interrupted by five introns and had an open reading frame encoding 481 amino acid residues. The amino acid sequence similarity at active sites to the alpha-L-AFases from other organisms indicated that the alpha-L-AFase encoded by abfA was classified as a family 51 glycoside hydrolase. When the abfA was overexpressed in the homologous hyperexpression system of A. oryzae, a large amount of alpha-L-AFase was produced as intracellular protein. The apparent molecular mass of the purified enzyme was estimated to be 228,000 by gel filtration and that of its subunit as 55,000 by SDS-PAGE, suggesting that the enzyme is a tetramer. The enzyme hydrolyzed p-nitrophenyl-alpha-L-arabinofuranoside but not other p-nitrophenyl glycosides. These results demonstrated that the abfA gene encodes a functional alpha-L-AFase.     

Breeding of high malate‐producing diploid sake yeast with a homozygous mutation in the VID24 gene

Malate is an important taste component of sake (a Japanese alcoholic beverage) that is produced by the yeast Saccharomyces cerevisiae during alcoholic fermentation. A variety of methods for generating high malate‐producing yeast strains have been developed to date. We recently reported that a high malate‐producing strain was isolated as a mutant sensitive to dimethyl succinate (DMS), and that a mutation in the vacuolar import and degradation protein (VID) 24 gene was responsible for high malate productivity and DMS sensitivity. In this work, the relationships between heterozygous and homozygous mutants of VID24 and malate productivity in diploid sake yeast were examined and a method was developed for breeding a higher malate‐producing strain. First a diploid yeast was generated with a homozygous VID24 mutation by genetic engineering. The homozygous integrants produced more malate during sake brewing and grew more slowly in DMS medium than wild‐type and heterozygous integrants. Thus, the genotype of the VID24 mutation influenced the level of malate production and sensitivity to DMS in diploid yeast. Then a homozygous mutant from a heterozygous mutant was obtained without genetic engineering by ultraviolet irradiation and culturing in DMS with nystatin enrichment. The non‐genetically modified sake yeast with a homozygous VID24 mutation exhibited a higher level of malate productivity than the parent heterozygous mutant strain. These findings provide a basis for controlling malate production in yeast, and thereby regulating malate levels in sake. Copyright © 2016 The Institute of Brewing & Distilling     

Polymerization of β-lactoglobulin and bovine serum albumin at oil-water interfaces in emulsions by transglutaminase

Polymerization of β-lactoglobulin and bovine serum albumin at the oil—water interfaces in n-tetradecane-in-water emulsions induced by the transglutaminase reaction was studied. The emulsions were incubated with transglutaminase for various times, and adsorbed and unadsorbed protein fractions at the oil—water interfaces were analyzed by sodium dodecyl sulfate—polyacrylamide gel electrophoresis. While only monomers were detected in the unadsorbed fractions, polymers were observed in the adsorbed fractions of the both proteins. The sizes and amounts of the polymers increased with incubation time. The incubation with transglutaminase caused much flocculation of the emulsion stabilized by β-lactoglobulin. An increase in viscosity was also observed with the flocculation. The flocculation was probably initiated by the formation of ε-(γ-glutamyl)-lysyl isopeptide bonds between β-lactoglobulin molecules adsorbed on different oil droplets. In the case of the emulsion stabilized by bovine serum albumin, however, the flocculation and the increase in viscosity occurred to only limited extents by the transglutaminase reaction. This suggests that ε-(γ-glutamyl)-lysyl isopeptide bonds induced by the transglutaminase reaction were formed only between neighboring molecules of bovine serum albumin on the same droplet.     

A PCR assay for detection of acetic acid-tolerant lactic acid bacteria in acidic food products

A PCR assay for the detection of acetic acid-tolerant lactic acid bacteria in the genera of Lactobacillus and Pediococcus was developed in this study. Primers targeting the bacterial 16S rRNA gene were newly designed and used in this PCR assay. To determine the specificity of the assay, 56 different bacterial strains (of 33 genera), 2 fungi, 3 animals, and 4 plants were tested. Results were positive for most tested bacterial members of 16S rRNA gene-based phylogenetic groups (classified in the Lactobacillus casei and Pediococcus group), including Lactobacillus fructivorans, Lactobacillus brevis, Lactobacillus buchneri, Lactobacillus plantarum, and Lactobacillus paracasei. For all other bacterial strains and eukaryote tested, results were negative. Bacterial DNA for PCR was prepared with a simple procedure with the use of Chelex 100 resin from culture after growth in deMan Rogosa Sharpe broth (pH 6.0). To test this PCR assay for the monitoring of the acetic acid-tolerant lactic acid bacteria, L. fructivorans was inoculated into several acidic food as an indicator. Before the PCR, the inoculation of 10 to 50 CFU of bacteria per g of food was followed by a 28-h enrichment culture step, and the PCR assay allowed the detection of bacterial cells. Including the enrichment culture step, the entire PCR detection process can be completed within 30 h.