Soy peptides enhance heterologous membrane protein productivity during the exponential growth phase of Saccharomyces cerevisiae

In this study, the production of eight G protein-coupled receptors by Saccharomyces cerevisiae was compared using two types of media, one of which contained soy peptides and the other free amino acids. Yeast cell growth improved in the medium with soy peptides, and the expression levels of six of the receptors increased during the exponential phase by an average of 2.3-fold as against the free amino acid-based medium. The enhancement of protein expression by soy peptides can be explained by alleviation of metabolite stress due to amino acid source depletion caused by heterologous protein expression.     

The high expression of Aspergillus pseudoglaucus protease in Escherichia coli for hydrolysis of soy protein and milk protein

Abstract

The hydrolysates of soy protein and milk protein are nutritional and functional food ingredients. Aspergillus pseudoglaucus aspergillopepsin I (App) is an acidic protease, including signal peptide, propeptide, and catalytic domain. Here, we cloned the catalytic domain App with or without propeptide in Escherichia coli. The results showed that the App without propeptide was not expressed or did not exhibit activity and App with propeptide (proApp) was highly expressed with a specific activity of 903?U/mg. Moreover, the denaturation temperature of proApp was 4.1?°C higher than App’s. The proApp showed 104?U/mg and 252?U/mg hydrolysis activities towards soy protein and milk protein under acidic conditions. By RP-HPLC analysis, the peptides obtained from the hydrolysates of soy protein and milk protein were hydrophilic peptides. This work first demonstrates efficient proteolysis of soy protein and milk protein through the functional expression of full-length proApp, which will likely have valuable industrial applications.     

Modification of yeast characteristics by soy peptides: cultivation with soy peptides represses the formation of lipid bodies

We have previously reported that the cultivation of yeast cells with soy peptides can improve the tolerance of yeast to freeze–thaw stress (Izawa et al. Appl Microbiol Biotechnol 75:533–538, 2007), indicating that soy peptides can modify the characteristics of yeast cells. To gain a greater understanding of the potencies of soy peptides, we further investigated the effects of cultivation with soy peptides on yeast physiology and found that soy peptides repress the formation of lipid bodies (also called lipid droplets or lipid particles), in which neutral lipids are accumulated. Compared with casein peptone, bacto peptone, yeast nitrogen base, and free amino acid mixtures having the same amino acid composition as soy peptides, cultivation with soy peptides caused decreased levels of mRNAs of neutral lipid synthesis-related genes, such as DGA1, and repressed the formation of lipid bodies and accumulation of triacylglycerol. These results indicate that soy peptides affect the lipid metabolism in yeast cells, and also demonstrate a potentiality of edible natural ingredients as modifiers of the characteristics of food microorganisms.     

Soy peptide ingestion augments the synthesis and metabolism of noradrenaline in the mouse brain

To examine whether edible peptide intake affects neurotransmitter metabolism in the brain, we evaluated the effect of peptides derived from soy proteins or fish collagen on free amino acids and monoamines in the mouse brain. Ingestion of soy peptides led to markedly higher levels of tyrosine, a catecholamine precursor, in the serum, and cerebral cortex compared to those following ingestion of vehicle alone or collagen peptides. Soy peptide ingestion also effectively increased 3-methoxy-4-hydroxyphenylethyleneglycol and normetanephrine, the principal metabolites of noradrenaline, in the cerebral cortex, hippocampus, and brainstem, whereas collagen peptides did not exert such effects. Further, soy peptide ingestion led to a significant increase in noradrenaline itself in the brainstem, where noradrenergic neurons are present. Noradrenergic turnover was also markedly stimulated in these regions after soy peptide ingestion. These in vivo observations suggest that soy peptide ingestion can maintain and promote the synthesis and metabolism of noradrenaline in the brain.     

地衣芽孢杆菌E7氨肽酶的异源表达及其在高效蛋白水解中的应用

【目的】将地衣芽孢杆菌(Bacilluslicheniformis)E7氨肽酶基因pepN克隆到大肠杆菌(Escherichia coli) BL21中,实现氨肽酶Ec PepN的异源表达,研究重组酶的酶学性质及其与碱性蛋白酶协同作用,高效水解大豆蛋白和酪蛋白,产生小分子活性肽和游离氨基酸。【方法】以地衣芽孢杆菌E7基因组DNA为模板,将氨肽酶基因pepN克隆到载体pET28a中,构建重组表达载体pET28-pepN,转化到大肠杆菌BL21感受态细胞中,经DNA测序验证,获得重组菌E. coli BL21/pET28-pepN。利用镍离子亲和层析柱对重组酶进行分离纯化,研究纯酶的pH和温度稳定性、半衰期和NaCl的耐受性等酶学性质。以商品化氨肽酶与碱性蛋白酶协同作用为对照,重组酶Ec PepN与碱性蛋白酶协同水解大豆蛋白和酪蛋白,测定水解产物中小分子活性肽和游离氨基酸的组成。【结果】Ec PepN在大肠杆菌BL21中可溶性表达,SDS-PAGE分析表明纯化的重组酶在52kDa左右显示单一条带。在7种测定底物中,Ec PepN的最适底物为Ala-pNA。在最适条件(pH 9.0和50°C...     

Binding of phosphorylated peptides and inhibition of their interaction with disease‐relevant human proteins by synthetic metal‐chelate receptors

The modulation of biological signal transduction pathways by masking phosphorylated amino acid residues represents a viable route toward pharmacologic protein regulation. Binding of phosphorylated amino acid residues has been achieved with synthetic metal‐chelate receptors. The affinity and selectivity of such receptors can be enhanced if combined with a second binding site. We demonstrate this principle with a series of synthetic ditopic metal‐chelate receptors, which were synthesized and investigated for their binding affinity to phosphorylated short peptides under conditions of physiological pH. The compounds showing highest affinity were subsequently used to inhibit the interaction of the human STAT1 protein to a peptide derived from the interferon‐γ receptor, and between the checkpoint kinase Chk2 and its preferred binding motif. Two of the investigated ditopic synthetic receptors show a significant increase in inhibition activity. The results show that regulation of protein function by binding to phosphorylated amino acids is possible. The introduction of additional binding sites into the synthetic receptors increases their affinity, but the flexibility of the structures investigated so far prohibited stringent amino acid sequence selectivity in peptide binding. Copyright © 2009 John Wiley & Sons, Ltd.     

Covalent immobilization of alkaline proteinase on amino-functionalized magnetic nanoparticles and application in soy protein hydrolysis

Magnetic nanoparticles (MNPs) were synthesized and surface modified with (3-Aminopropyl)triethoxysilane (APTES). The alkaline proteinase (AP) was covalently immobilized on the APTES-modified MNPs through glutaraldehyde linkage. The resulting AP-loaded MNPs have an average size of 84 nm in aqueous solution, and a magnetization of 40 emu/g, endowing the immobilized enzyme with excellent magnetic responsively and dispersity. The maximum amount of AP and catalytic activity immobilized 1.0 mg MNPs was 120 μg and 25.3 units, respectively. Immobilized AP showed maximum activity at pH 10.0 and 50°C. Compared with free enzyme, the immobilized AP exhibited better storage stability. Moreover, immobilized AP can be reused 10 times and still maintained about 50% of its initial activity. The degree of hydrolysis of soy protein hydrolysates for immobilized AP could reach 19.0%, which was closer to the value of free enzyme. The molecular weight (M.W.) analysis showed that the soy protein was hydrolyzed successfully into small peptides of two main fractions with an average M.W. of 742 and 2126 Da. This study indicated that the immobilized AP could be used to hydrolyze continuously soy protein for potential industry application. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2756, 2019.     

Relationship between relative protein value and somein vitro indices of protein quality

Besides amino acid composition of a protein, their bioavailability is an important determinant of the protein quality. In view of the observations over the last decade or two, implicating the small peptide uptake by the mammalian intestine as a major route of protein absorption, a few animal and plant proteins were subjected to sequential enzymatic digestionin vitro with pepsin, pancreatin + trypsin and erepsin and the release of amino acids as small (including dipeptides) and large peptides and free amino acids, was determined. The relative protein values of α-lactalbumin, egg whites, casein, gluten, zein and protein isolates of soyabeans and groundnuts was determined using rat growth method. It was observed that relative protein value were positively correlated with the essential amino acid index of protein, quantity of essential amino acids released as small peptides and the dipeptide content of enzymatic digests, while there was a negative correlation between relative protein value and essential amino acid content of large peptide fraction.     

Oligopeptide assimilation and transport by Oenococcus oeni

Aims: Oenococcus oeni is a slow‐growing wine bacterium with a low growth yield. It thrives better on complex nitrogen sources than on free amino‐acid medium. We aimed to characterize the oligopeptide use of this micro‐organism. Methods and Results: Several peptides of two to eight amino‐acid residues were able to provide essential amino acids. The disappearance of various peptides from extracellular medium was assessed with whole cells. Initial rates of utilization varied with the peptide, and free amino acids were released into the medium. Conclusions: Oenococcus oeni was able to transport the oligopeptides with two to five amino‐acid residues tested and to hydrolyse them further. Significance and Impact of the Study: This study has clear implications for the relationship between wine nitrogen composition and the ability of O. oeni to cope with its environment.     

Development of a glutathione production process from proteinaceous biomass resources using protease-displaying <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Glutathione is a valuable tri-peptide that is widely used in the pharmaceutical, food, and cosmetic industries. Glutathione is produced industrially by fermentation using Saccharomyces cerevisiae, and supplementation of fermentation with several amino acids can increase intracellular GSH content. More recently, however, focus has been given to protein as a resource for biofuel and fine chemical production. We demonstrate that expression of a protease on the cell surface of S. cerevisiae enables the direct use of keratin and soy protein as a source of amino acids and that these substrates enhanced intracellular GSH content. Furthermore, fermentation using soy protein also enhanced cell concentration. GSH fermentation from keratin and to a greater extent from soy protein using protease-displaying yeast yielded greater GSH productivity compared to GSH fermentation with amino acid supplementation. This protease-displaying yeast is potentially applicable to a variety of processes for the bio-production of value-added chemicals from proteinaceous biomass resources.     

Whey protein hydrolysates enhance water absorption in the perfused small intestine of anesthetized rats

We evaluated the effect of whey protein hydrolysates (WPH) on the water absorption rate in the small intestine using a rat small intestine perfusion model. The rate was significantly higher with 5 g/L WPH than with 5 g/L soy protein hydrolysates or physiological saline (p?p?p?r?=?0.82, p?相似文献   

Identification and Quantitation of New Glutamic Acid Derivatives in Soy Sauce by UPLC/MS/MS

Glutamic acid is an abundant amino acid that lends a characteristic umami taste to foods. In fermented foods, glutamic acid can be found as a free amino acid formed by proteolysis or as a non‐proteolytic derivative formed by microorganisms. The aim of the present study was to identify different structures of glutamic acid derivatives in a typical fermented protein‐based food product, soy sauce. An acidic fraction was prepared with anion‐exchange solid‐phase extraction (SPE) and analyzed by UPLC/MS/MS and UPLC/TOF‐MS. α‐Glutamyl, γ‐glutamyl, and pyroglutamyl dipeptides, as well as lactoyl amino acids, were identified in the acidic fraction of soy sauce. They were chemically synthesized for confirmation of their occurrence and quantified in the selected reaction monitoring (SRM) mode. Pyroglutamyl dipeptides accounted for 770 mg/kg of soy sauce, followed by lactoyl amino acids (135 mg/kg) and γ‐glutamyl dipeptides (70 mg/kg). In addition, N‐succinoylglutamic acid was identified for the first time in food as a minor compound in soy sauce (5 mg/kg).     

Methodology for identification of pore forming antimicrobial peptides from soy protein subunits β-conglycinin and glycinin

Antimicrobial peptides (AMPs) inactivate microbial cells through pore formation in cell membrane. Because of their different mode of action compared to antibiotics, AMPs can be effectively used to combat drug resistant bacteria in human health. AMPs can also be used to replace antibiotics in animal feed and immobilized on food packaging films. In this research, we developed a methodology based on mechanistic evaluation of peptide-lipid bilayer interaction to identify AMPs from soy protein. Production of AMPs from soy protein is an attractive, cost-saving alternative for commercial consideration, because soy protein is an abundant and common protein resource. This methodology is also applicable for identification of AMPs from any protein. Initial screening of peptide segments from soy glycinin (11S) and soy β-conglycinin (7S) subunits was based on their hydrophobicity, hydrophobic moment and net charge. Delicate balance between hydrophilic and hydrophobic interactions is necessary for pore formation. High hydrophobicity decreases the peptide solubility in aqueous phase whereas high hydrophilicity limits binding of the peptide to the bilayer. Out of several candidates chosen from the initial screening, two peptides satisfied the criteria for antimicrobial activity, viz. (i) lipid-peptide binding in surface state and (ii) pore formation in transmembrane state of the aggregate. This method of identification of antimicrobial activity via molecular dynamics simulation was shown to be robust in that it is insensitive to the number of peptides employed in the simulation, initial peptide structure and force field. Their antimicrobial activity against Listeria monocytogenes and Escherichia coli was further confirmed by spot-on-lawn test.     

Evidence for the Existence of a Soybean Resistant Protein That Captures Bile Acid and Stimulates Its Fecal Excretion

Feeding HMF, an insoluble “high-molecular-weight fraction” from an industrial enzymatic digest of a soy protein isolate, increased the fecal excretion of bile acid concomitant with increased fecal nitrogen. An amino acid analysis revealed that this increased fecal nitrogen could be explained by an increase in the insoluble protein fraction. This suggests the existence of an indigestable protein or peptide that can be called a “resistant protein” in the feces. The presumed resistant protein was rich in hydrophobic amino acids and bound bile acid by hydrophobic interaction. The residual fraction of HMF obtained after in vitro pepsin and pancreatin digestion, showed higher in vitro bile acid-binding capacity and excreted more bile acid in vivo than HMF. Its amino acid composition was similar to that of the feces of rat fed with HMF. These results suggest that the fecal resistant protein with bile acid-binding ability could be derived from the indigestable fraction of HMF.     

Amino acid fermentation byBacteroides melaninogenicus

Each of four strains ofBacteroides melaninogenicus grew well in a trypticaseyeast extract medium, without carbohydrate. Addition of glucose did not increase growth, and the sugar was fermented to only a limited extent. However, growth decreased when the trypticase concentration of the medium was reduced. These observations suggest that amino acid fermentation is of major importance in the energy metabolism ofB. melaninogenicus. Acetic, butyric and isovaleric acids were produced by all four strains. Two of the strains also formed propionic and isobutyric acids. Experiments using media containing either labeled glucose or labeled protein indicated that these acids are primarily derived from the proteinaceous substrates in the medium, rather than from glucose, indicating that amino acid fermentation byB. melaninogenicus is not subject to glucose repression. Resting-cell suspensions ofB. melaninogenicus possessed a limited ability to ferment free acids, as judged by the liberation of ammonia. However the organisms readily fermented amino acids when present as peptides, suggesting that peptides are more readily transported into the cell. Three of the 4 strains studied grew well when incubated under an atmosphere containing 2 to 4 % oxygen in cultures possessing a high surface to volume ratio. Hence not all strains ofB. melaninogenicus can be considered strict anaerobes.This investigation was supported in part by grant DE-02847 from the National Institute of Dental Research, and in part by a grant from the Colgate-Palmolive Co.     

Identification of Rad's effector-binding domain,intracellular localization,and analysis of expression in Pima Indians

In order to characterize the endogenous gene product for rad (ras-related protein associated with diabetes), we prepared antibodies to synthetic peptides that correspond to amino acids (109–121, 178–195, 254–271) within the protein. These antibodies were used to analyze the expression, structure, and function of rad. Western analysis with these antibodies revealed that rad was a 46 kDa protein which was expressed during myotube formation. Further, immunolocalization studies showed that rad localized to thin filamentous regions in skeletal muscle. Interestingly, when muscle biopsies from diabetic and control Pima Indians were compared, no differences in rad protein or mRNA expression were observed. Similarly, no differences were observed in protein expression in diabetic and control Zucker diabetic fatty (ZDF) rats. Functional analysis of muscle rad revealed that its GTP-binding activity was inhibited by the addition of N-ethylmaliemide, GTP, GTPγS, and GDPβS but not ATP or dithiothreitol. Moreover, cytosol-dependent rad-GTPase activity was stimulated by the peptide corresponding to amino acids 109–121. Antibodies corresponding to this epitope inhibited cytosol-dependent rad-GTPase activity. Taken together, the results indicate that 1) rad is a 46 kDa GTP-binding protein localized to thin filaments in muscle and its expression increases during myoblast fusion, 2) expression of rad in Pima Indians and ZDF rats does not correlate with diabetes, and 3) the amino acids (109–121) may be involved in regulating rad-GTPase activity, perhaps by interacting with a cytosolic factor(s) regulating nucleotide exchange and/or hydrolysis. J. Cell. Biochem. 65:527–541. © 1997 Wiley-Liss Inc.     

The role of dietary protein on lipotoxicity

Lipotoxicity is a metabolic abnormality frequently observed during the development of obesity and is the main cause of several changes in the metabolic observed during metabolic syndrome. Consistent consumption of diets high in saturated fat or simple carbohydrates combined with low physical activity are the main causes of obesity and its comorbidities. However, the contribution of dietary protein and, in particular, the contribution due to the type of dietary protein, to the process of obesity and its metabolic consequences are less well-understood. In this review, we showed that the type of dietary protein has a significant contribution to the process of lipotoxicity through the modulation of insulin secretion and the regulation of adipocyte metabolic function. Consumption of soy protein stimulates insulin secretion to a lower extent than casein despite the fact that both are high-quality proteins. The amino acid profiles of soy protein and its isoflavones are responsible for the reduced insulin secretion. Also, soy protein increases insulin sensitivity, whereas casein has the opposite effect. Consequently, soy protein reduces SREBP-1 expression in the liver leading to low accumulation of hepatic triglycerides, despite the consumption of a high-fat diet. Furthermore, soy protein reduces adipocyte hypertrophy, hyperleptinemia, and free fatty acid concentration. Thus, the influx of FA into the liver decreases, and hepatic oxidation of FA increases. These metabolic changes result in a decrease in lipid depots and ceramide which reduce hepatic lipotoxicity, whereas casein produces the opposite effect. This study emphasizes that the type of dietary protein has an important effect on lipotoxicity.     

In vitro substrate specificity of protein tyrosine kinases

Synthetic peptides such as P60^stc autophosphorylation site peptides and angiotensin are indiscriminately phosphorylated by protein tyrosine kinases. The observation has led to the general belief that protein tyrosine kinases are highly promiscuous, displaying littlein vitro site specificity. In recent years, evidence has been accumulating to indicate that such a belief requires close examination. Synthetic peptides showing high substrate activity for specific groups of protein tyrosine kinases have been obtained. Systematic modification of certain substrate peptides suggests that kinase substrate determinants reside with specific amino acid residues proximal to the target tyrosine. A number of protein kinases have been shown to be regulated by tyrosine phosphorylation at specific sites by highly specific protein tyrosine kinases. These and other selected biochemical studies that contribute to the evolving view ofin vitro substrate specificity of protein tyrosine kinases are reviewed.     

The complete amino-acid sequence of the rieske FeS-precursor protein from spinach chloroplasts deduced from cDNA analysis

Summary SummarySeveral cDNA clones encoding the entire Rieske FeS-precursor protein of the chloroplast cytochrome b ₆ f-complex have been isolated by high density plaque immunoscreening of a phage lambda gt11 cDNA expression library, made from poly A⁺-RNA of spinach seedlings. The identity of the cDNAs has been confirmed by N-terminal amino acid sequencing of the purified protein. The nucleotide sequence indicates a protein of 247 amino acid residues including a putative transit sequence of 68 amino acids corresponding to molecular masses of 26.3 kDa (precursor) and 18.8 kDa (mature protein; 179 amino acid residues). Alignteins of the sequence with sequences from Rieske FeS-proteins of respiratory electron transport chains, two of bacterial and three of mitochondrial origin, shows little sequence homology, but remarkable similarity in secondary structure including a putative N-terminal transmembrane segment of about 25 residues and the peptides CTHLGCV and CPCHGS in the C-terminal region of the protein that are involved in the binding of the Fe₂S₂-cluster.     

Efficient expression of isotopically labeled peptides for high resolution NMR studies: application to the Cdc42/Rac binding domains of virulent kinases in Candida albicans

The production of bioactive peptides and small protein fragments is commonly achieved via solid-phase chemical synthesis. However, such techniques become unviable and prohibitively expensive when the peptides are large (e.g., >30 amino acids) or when isotope labeling is required for NMR studies. Expression and purification of large quantities of unfolded peptides in E. coli have also proved to be difficult even when the desired peptides are carried by fusion proteins such as GST. We have developed a peptide expression system that utilizes a novel fusion protein (SFC120) which is highly expressed and directs the peptides to inclusion bodies, thereby minimizing in-cell proteolysis whilst maintaining high yields of peptide expression. The expressed peptides can be liberated from the carrier protein by CNBr cleavage at engineered methionine sites or through proteolysis by specific proteases for peptides containing methionine residues. In the present systems, we use CNBr, due to the absence of methionine residues in the target peptides, although other cleavage sites can be easily inserted. We report the production of six unfolded protein fragments of different composition and lengths (19 to 48 residues) derived from the virulent effector kinases, Cla4 and Ste20 of Candida albicans. All six peptides were produced with high yields of purified material (30–40 mg/l in LB, 15–20 mg/l in M9 medium), pointing to the general applicability of this expression system for peptide production. The enrichment of these peptides with ¹⁵N, ¹⁵N/¹³C and even ¹⁵N/¹³C/²H isotopes is presented allowing speedy assignment of poorly-resolved resonances of flexible peptides.