Fermentation products of the fungus Monascus spp. impairs the physiological activities of toxin-producing Vibrio cholerae.

Monascus spp. are filamentous fungi used in fermented foods. They are also natural colorants and food preservatives. Certain metabolites of Monascus spp. lower cholesterol and have other health-promoting effects in humans. In the present study, we demonstrated that the fermentation products of Monascus spp. inhibited ATP synthesis and motility in toxigenic Vibrio cholerae. Single-cell tracking and rotation assays on single flagella showed that Monascus fermentation extract (MFE) significantly impaired V. cholerae swimming by disrupting flagellar rotation. A membrane potential-sensitive carbocyanine dye revealed that MFE depolarized the V. cholerae cell membrane which, in turn, lowered the membrane potential and, by extension, restricted ATP synthesis and flagellar rotation. MFE also severely hindered the motility of other pathogenic bacteria such as V. parahaemolyticus, Pseudomonas aeruginosa, Salmonella enterica Typhimurium, and Leptospira interrogans. The foregoing findings indicate that Monascus fermentation extract could potentially preventing infection caused by multiple pathogenic bacteria as the conventional prophylaxes and slow their progression and lower mortality and morbidity.

Proteomic study of bioactive peptides from tempe.

Tempe is a traditional Indonesian fermented soybean mostly produced in small industries and sold locally throughout the country. Studies on the bioactive peptides in tempe are rare. Here, we studied bioactive peptides in samples from three tempe producers with different degrees of sanitation. The peptide sub-fractions of tempe from each producer were collected following water extraction, ultrafiltration (<3 kDa), gel filtration chromatography, and reversed phase-high performance liquid chromatography (RP-HPLC) separation followed by liquid chromatography-mass spectrometry (LC-MS). The MS spectra were then predicted using FindPept tools, and their biofunctionalities were confirmed with BIOPEP databases. There were few similar peptides found in tempe from the three producers. Peptides Val-His and Ala-Leu-Glu-Pro were found in tempe from all producers. Producers having a good sanitation level had more bioactive peptides than those with moderate or poor sanitation levels (58%, 43% and 35%, from good to poor sanitation). This work showed that the tempe from the three producers had antihypertensive, antidiabetic, antioxidative and antitumor peptides.

Taste and chemical characteristics of low molecular weight fractions from tofuyo - Japanese fermented soybean curd.

Tofuyo, a Japanese traditional food, is a fermented soybean curd manufactured in Okinawa region. Due to its original cheese-like flavor, the current study was designed to evaluate the sensory and chemical characteristics of three stepwise ultrafiltration fractions, using 10,000, 3000 and 500 Da membranes and further chromatographic fractions from tofuyo. The results showed that umami, sweet and salty were the characteristic tastes of all fractions, with umami intensity evaluated for the fraction with MW less than 500 Da (F-500) as the most prominent among the three fractions. Subsequent Sephadex G-25 SF fractions and RP-HPLC fractions were subjected to sensory and chemical analyses. The tastiest fraction contained sodium chloride, sugars, organic acids, umami and sweet free amino acids, at concentrations above their thresholds. The abundant presence of umami and sweet free amino acids with certain concentrations of sodium chloride and glucose might provide the typical savory taste of tofuyo.

Characterization and in vitro biological activities of Thai traditional fermented shrimp pastes.

In this work, chemical and biological characteristics of two types of Thai fermented shrimp paste, Kapi Ta Dam and Kapi Ta Deang, at different fermentation periods and their raw materials were investigated. Kapi had low water activity and high proteins with high glutamic acid and lysine. Both Kapis, which had different sources, showed similar characteristics. The number of lactic acid bacteria in the products increased during the early stages of fermentation. Free α-amino acid contents in the products increased with the fermentation time. The water extracts from Kapi products showed strong antioxidative activities against ABTS(+) radical, and ACE inhibitory activity but they did not exhibit antimicrobial activity against Staphylococcus aureus, Bacillus cereus, Escherichia coli and Salmonella Typhimurium. Biological activities in Kapi could be developed by fermentation process, enzymatic hydrolysis of proteins and non-enzymatic browning reactions. Kapi could, thus, serve as a potential source of natural bioactive substances.

Biochemical aspects of red koji and tofuyo prepared using Monascus fungi.

Red koji or red mold rice is prepared by growing a genus Monascus on steamed rice. For centuries, it has been used in Asia for the production of fermented foods including red rice wine and fermented tofu. Although red koji is an important source of various hydrolytic enzymes critical for food fermentation, information on the enzymatic properties in red koji has been limited. Hydrolytic enzymes produced by Monascus fungi may play important roles in ripening of tofuyo (Japanese fermented tofu) regarding the chemical and physical properties of the product. This review provides an introduction of red koji, its properties, and the application of hydrolytic enzymes, especially aspartic proteinases and carboxypeptidases from Monascus fungi. We also describe tofuyo and a novel fermented soybean protein food using a microbial action originating from red koji.

Determination of L-methionine using methionine-specific dehydrogenase for diagnosis of homocystinuria due to cystathionine ß-synthase deficiency.

To determine the L-methionine (L-Met) concentration in an extract from dried blood spots (DBSs) for newborn mass screening for homocystinuria (HCU) due to cystathionine ß-synthase (CBS) deficiency, a new fluorometric microplate assay using a methionine-specific dehydrogenase (MetDH) and the diaphorase/reazusrin system was established. We created by directed mutagenesis an NAD⁺-dependent MetDH from phenylalanine dehydrogenase (PheDH) showing higher substrate specificity toward L-Met than L-phenylalanine (L-Phe). However, it also exhibited notable activity for branched-chain amino acids (BCAAs). BCAAs in blood clearly interfered with the determination of L-Met in the DBS specimens using a single application of MetDH. To measure L-Met selectively, we used a branched-chain amino acid transaminase (BCAT) to eliminate the BCAAs in the specimens and screened for a BCAT with low activity toward L-Met. In microplate assays using MetDH, pretreatment of specimens with the BCAT from Lactobacillus delbrueckii subsp. bulgaricus coupled with L-glutamate oxidase minimized the effects of BCAAs, and L-Met concentrations were determined with high accuracy even at elevated BCAA concentrations. This enzymatic end-point assay is suitable for determining L-Met concentrations in DBSs for neonatal screening for HCU due to CBS deficiency.

Toxicity monitoring with primary cultured hepatocytes underestimates the acetaminophen-induced inflammatory responses of the mouse liver.

In vitro gene expression profiling with isolated hepatocytes has been used to assess the hepatotoxicity of certain chemicals because of animal welfare issues. However, whether an in vitro system can completely replace the in vivo system has yet to be elucidated in detail. Using a focused microarray established in our laboratory, we examined gene expression profiles in the mouse liver and primary cultured hepatocytes after treatment with different doses of acetaminophen, a widely used analgesic that frequently causes liver injury. The acute hepatotoxicity of acetaminophen was confirmed by showing the induction of an oxidative stress marker, heme oxygenase-1, elevated levels of serum transaminase, and histopathological findings. In vivo microarray and network analysis showed that acetaminophen treatment provoked alterations in relation to the inflammatory response, and that tumor necrosis factor-α plays a central role in related pathway alterations. By contrast, pathway analyses in in vitro isolated hepatocytes did not find such prominent changes in the inflammation-related networks compared with the in vivo situation. Thus, although in vitro gene expression profiles are useful for evaluating the direct toxicity of chemicals, indirect toxicities including inflammatory responses mediated by cell-cell interactions or secondary toxicity due to pathophysiological changes in the whole body may be overlooked. Our results indicate that the in vitro hepatotoxicity prediction system using isolated hepatocytes does not fully reflect the in vivo cellular response. An in vitro system may be appropriate, therefore, for high throughput screening to detect the direct hepatotoxicity of a test compound.

Application of an acid proteinase from Monascus purpureus to reduce antigenicity of bovine milk whey protein.

An acid proteinase from Monascus purpureus No. 3403, MpuAP, was previously purified and some characterized in our laboratory (Agric Biol Chem 48:1637-1639, 1984). However, further information about this enzyme is lacking. In this study, we investigated MpuAP's comprehensive substrate specificity, storage stability, and prospects for reducing antigenicity of whey proteins for application in the food industry. MpuAP hydrolyzed primarily five peptide bonds, Gln(4)-His(5), His(10)-Leu(11), Ala(14)-Leu(15), Gly(23)-Phe(24) and Phe(24)-Phe(25) in the oxidized insulin B-chain. The lyophilized form of the enzyme was well preserved at 30-40°C for 7 days without stabilizers. To investigate the possibility of reducing the antigenicity of the milk whey protein, enzymatic hydrolysates of the whey protein were evaluated by inhibition ELISA. Out of the three main components of whey protein, casein and α-lactalbumin were efficiently degraded by MpuAP. The sequential reaction of MpuAP and trypsin against the whey protein successfully degraded casein, α-lactalbumin and ß-lactoglobulin with the highest degree of hydrolysis. As a result, the hydrolysates obtained by using the MpuAP-trypsin combination showed the lowest antigenicity compared with the single application of pepsin, trypsin or pepsin-trypsin combination. Therefore, the overall result suggested that the storage-stable MpuAP and trypsin combination will be a productive approach for making hypoallergic bovine milk whey protein hydrolysates.

Hyperglycemia perturbs biochemical networks in human trophoblast BeWo cells.

Determining the effects of hyperglycemia on gene expression in placental trophoblast is important to gain a better understanding of how diabetes adversely affects pregnancy. In this study, we examined whether exposure to high glucose during forskolin-induced differentiation affects gene expression in differentiated trophoblasts. Human trophoblast BeWo cells were differentiated under low glucose (LG: 11 mM) or high glucose (HG: 25 mM) conditions. Gene expression was analyzed using a GeneChip system and the obtained data were analyzed using Ingenuity Pathways Analysis. In HG conditions, there were marked alterations in gene expression in differentiated BeWo cells compared with LG conditions. In particular, BeWo cells responded to HG with major changes in the expression levels of cell cycle- and metabolism-related genes. We selected the aromatase gene for further investigation of the molecular mechanisms. Mannitol or 3-O-methylglucose did not mimic the expression changes caused by HG, indicating that the effect of glucose was not due to a difference in osmotic pressure, and that glucose metabolism plays an essential role in inducing the HG effects. Cotreatment with N-acetylcysteine reduced the effect of HG on aromatase gene expression, suggesting that hyperglycemia may perturb biochemical networks because of the elevation of oxidative stress. Overall, our results will aid further understanding of the effect of diabetes on the regulation of trophoblast differentiation and function.

Purification and characterization of a halotolerant serine proteinase from thermotolerant Bacillus licheniformis RKK-04 isolated from Thai fish sauce.

A gram-positive thermotolerant bacterium, designated strain RKK-04, was isolated from a fermented Thai fish sauce broth as it demonstrated high proteolytic activity. A phylogenetic analysis based on comparisons of 16S rRNA gene sequences showed that strain RKK-04 is Bacillus licheniformis. The proteolytic enzyme, which was purified 80-fold with 18% yield, has a molecular mass of 31 kDa and an isoelectric point higher than 9.3. The optimum pH and temperature of the enzyme activity were found to be 10.0 and 50 degrees C, respectively. The addition of diisopropyl fluorophosphate and phenylmethanesulfonyl fluoride completely inhibited enzymatic activity. These results showed that the enzyme is a subtilisin-like alkaline serine proteinase. On the other hand, the enzyme exhibited unique cleavage sites in oxidized insulin B-chain that differed from those of other subtilisin-like proteases. High enzymatic activity was also retained under high salt conditions (30% NaCl). The myosin heavy chain of fish protein was completely digested by reaction with this enzyme. Thus the halotolerant proteinase from B. licheniformis RKK-04 is a key enzyme for fish sauce fermentation.

Carbon tetrachloride affects inflammation-related biochemical networks in the mouse liver as identified by a customized cDNA microarray system.

OBJECTIVES: We have attempted to upgrade and validate an in-house cDNA microarray system developed by our group for the evaluation of chemical toxicity. METHODS: To establish an in-house microarray, we selected genes that play pivotal roles in detoxifying exogenous substances and maintaining homeostasis in the liver. To validate the system, we examined gene expression profiles in mouse liver following treatment with different doses of carbon tetrachloride (CCl(4)). The data were also analyzed by pathway analysis tools. RESULTS: We upgraded our array system by collecting genes that are responsive to xenobiotic receptors, apoptosis-related genes, and stress-responsive genes. The acute toxicity of CCl(4) was confirmed by elevated levels of serum transaminase and histopathological findings. The microarray data showed the CCl(4) treatment induced significant changes in gene expression in the mouse liver, and the ingenuity pathways analysis revealed alterations in gene expression in inflammation-related networks. CONCLUSIONS: We have established a focused microarray system that may be useful for use in toxicogenomics studies. Using this array system, we gained insight into the mechanisms by which CCl(4) exerts its toxic effects. The results of our study also indicate that the combination of focused arrays and bioinformatics tools is helpful in the mechanistic analysis of chemical toxicity.

Discrimination of aliphatic substrates by a single amino acid substitution in Bacillus badius and Bacillus sphaericus phenylalanine dehydrogenases.

Replacement of glycine by serine at positions 123 and 124 of phenylalanine dehydrogenases from Bacillus badius and Bacillus sphaericus respectively strikingly decreased enzyme activity toward aromatic amino acids and resulted in an elevation of relative activity toward aliphatic amino acids. The mutant from B. badius preferentially dehydrogenated branched-chain amino acids, while that from B. sphaericus acted on amino acids with straight-chain amino acids.

Effect of Wnt-1 inducible signaling pathway protein-2 (WISP-2/CCN5), a downstream protein of Wnt signaling, on adipocyte differentiation.

Wnt signaling negatively regulates adipocyte differentiation, and ectopic expression of Wnt-1 in 3T3-L1 cells induces several downstream molecules of Wnt signaling, including Wnt-1 inducible signaling pathway protein (WISP)-2. In this study, we examined the role of WISP-2 in the process of adipocyte differentiation using an in vitro cell culture system. In the differentiation of 3T3-L1 cells, WISP-2 expression was observed in growing cells and declined thereafter. In the mitotic clonal expansion phase of adipocyte differentiation, WISP-2 expression was transiently down-regulated concurrently with up-regulation of CCAAT/enhancer-binding protein delta expression. Treatment of 3T3-L1 cells in the differentiation medium with lithium, an activator of Wnt signaling, inhibited the differentiation process with concomitant induction of WISP-2. Treatment of differentiated cells with lithium induced de-differentiation as evidenced by profound reduction of peroxisome proliferator-activator receptor gamma expression and concomitant induction of WISP-2. However, de-differentiation of differentiated cells induced by tumor necrosis factor-alpha did not induce WISP-2 expression. To directly examine the effect of WISP-2 on adipocyte differentiation, 3T3-L1 cells were infected with a retrovirus carrying WISP-2. Although forced expression of WISP-2 inhibited preadipocyte proliferation, it had no effect on adipocyte differentiation. Thus, although WISP-2 is a downstream protein of Wnt signaling, the role of WISP-2 on adipocyte differentiation may be marginal, at least in this in vitro culture model.

Purification and characterization of cytoplasmic NAD-dependent polypropylene glycol dehydrogenase from Stenotrophomonas maltophilia.

The oxidizing enzyme NAD(+)-dependent polypropylene glycol dehydrogenase (PPG-DH) was purified to homogeneity from the cytoplasmic fraction of Stenotrophomonas maltophilia grown on polypropylene glycol (diol type) 2000. The purified enzyme consisted of a homotetrameric protein (37 kDa subunit) with a molecular mass of around 154 kDa. The N-terminal amino acid sequence (25 residues) showed similarity to the sequences of NAD(+)-dependent secondary alcohol dehydrogenases and NADH-dependent reductases. The enzyme preferentially oxidized medium-chain secondary alcohols, di- and tri-propylene glycols and polypropylene glycols including those with secondary alcohol groups in their molecular structure. Consequently, the enzyme was classified into a group of NAD(+)-dependent secondary alcohol dehydrogenases.

Expression profile of liver genes in response to hepatotoxicants identified using a SAGE-based customized DNA microarray system.

Gene expression analysis using customized or focused DNA microarrays is favorable because of a reduction in the cost and time needed for the analysis. To examine the effect of chemicals on the liver, we developed an in-house cDNA microarray system, mouse Liver Stress Array ver. 1.0, containing 355 unique genes involved in drug metabolism, inflammation and liver-related proteins. These genes were selected for sensing the homeostasis of the liver and based on the information of liver transcriptome revealed by serial analysis of gene expression. By using this customized microarray, we analyzed gene expression changes in the mouse liver treated by 11 known hepatotoxicants. Gene expression measurements corresponding to the in vivo response to known hepatotoxicants revealed that profiles of chemicals with similar mechanisms clustered together. For each of the chemicals tested, several genes that were induced or repressed were common in each chemical exposure, whereas other genes were unique for the specific class compound. Ingenuity pathways analysis revealed that significant alterations in gene expression occurred in a number of biological networks by these treatments. Although the genes spotted on our array was limited to a highly focused set for toxicity classification, this work provides proof of concept that patterns of gene regulation assessed by a focused array system are useful to classify unknown chemicals.

Purification and characterization of heterogeneous glucoamylases from Monascus purpureus.

Two forms of an extracellular glucoamylase, MpuGA-I and MpuGA-II, were purified to homogeneity from Monascus purpureus RY3410. The molecular weights of these enzymes were estimated to be 60,000 (MpuGA-I) and 89,000 (MpuGA-II). These enzymes were glycoproteins with a carbohydrate content of 15.0% (MpuGA-I) and 16.2% (MpuGA-II) respectively. The pH optima were 5.0 for both enzymes, and the optimal temperatures were 50 degrees C (MpuGA-I) and 65 degrees C (MpuGA-II). The Km values for soluble starch were calculated to be 4.0+/-0.8 mg/ml (MpuGA-I) and 1.1+/-0.2 mg/ml (MpuGA-II) respectively.

Supramolecular-mediated immobilization of L-phenylalanine dehydrogenase on cyclodextrin-coated Au electrodes for biosensor applications.

A supramolecular approach was used for adsorbing a monolayer of adamantane-modified phenylalanine dehydrogenase on beta-cyclodextrin-coated Au electrodes. The enzyme electrode (poised at +200 mV vs. Ag/AgCl) showed a linear amperometric response up to 3 mM L-phenylalanine (L-Phe) with a lower detection limit of 15 microM. The reversible nature of this immobilization approach was confirmed.

Application of an enzyme chip to the microquantification of l-phenylalanine.

We describe here a new microquantification method of l-phenylalanine concentration in an extract from a dried blood spot by using the diaphorase-resazurin system. To miniaturize the fluorometric enzymatic microplate assay for the diagnosis of phenylketonuria, an enzyme chip immobilized with His-tag fused phenylalanine dehydrogenase (PheDH) was developed. His-tag fused PheDH was immobilized on the surface of nickel-coated slide glass. A microarray sheet (8 x 30 well) was fabricated with poly(dimethylsiloxane) (PDMS) using the photolithographic technique. An enzyme reaction chamber in a double-layered structure was constructed with different types of microarray PDMS sheets on the surface of Ni-coated slide glass immobilized with His-tagged PheDH. To evaluate the affinity toward the Ni-chelating ligand, eight kinds of His-tagged PheDH variants were constructed and expressed. (His)(6)- and (His)(9)-PheDH variants at the N terminus showed high adsorption ratio to Ni-chelating ligand. The V(max) and k(cat) values of the (His)(6)-PheDH variant at the N terminus for l-phenylalanine were higher than those of the (His)(9)-PheDH variant, and the (His)(6)-PheDH variant was found to be most suitable for immobilization onto nickel-coated slide glass. Fluorescence formed by resazurin-coupled enzymatic reaction (in a 0.2-microl reaction mixture) on the enzyme chip exhibited good linearity and a correlation coefficient up to 12.8 mg/dl of the l-phenylalanine-containing sample extracted from a dried blood spot on filter paper.

Cloning and expression of the gene for periplasmic poly(vinyl alcohol) dehydrogenase from Sphingomonas sp. strain 113P3, a novel-type quinohaemoprotein alcohol dehydrogenase.

A gene for periplasmic poly(vinyl alcohol) (PVA) dehydrogenase (PVADH) was cloned, based on the N-terminal amino acid sequence of the purified PVADH from Sphingomonas sp. 113P3 and the sequence of the gene for PVADH (pvaA, GenBank accession no. AB190288). The recombinant PVADH tagged with hexahistidine was expressed in Escherichia coli and purified to homogeneity. The recombinant enzyme had the same characteristics as the purified enzyme from Sphingomonas sp. strain 113P. In addition to PVA, the recombinant PVADH could oxidize glycols such as polypropylene glycols and 1,3-butane/cyclohexanediol and 2,4-pentanediol, but neither primary nor secondary alcohols. The amino acid sequence of the recombinant PVADH showed similarity with those of PVADH from Pseudomonas sp. strain VM15C, putative PVADHs from Azoarcus sp. EbN1, and Xanthomonas species (54-25 % identity), and the quinohaemoprotein alcohol dehydrogenases (QH-ADHs) from Comamonas testosteroni, Ralstonia eutropha and Pseudomonas putida (25-29 % identity). PVADHs from strains 113P3 and VM15C have a conserved superbarrel domain (SD), probable PQQ-binding amino acids in the SD and a haem-binding domain (HBD) (they should be designated QH-PVADHs), but the positions of the amino acid sequences for the HBD and SD are the reverse of those of QH-ADHs. A protein structure of QH-PVADHs is proposed. Results of dot-blot hybridization and RT-PCR indicated that the three genes encoding oxidized PVA hydrolase, PVADH and cytochrome c are expressed constitutively and form an operon.

Increased conformational and thermal stability properties for phenylalanine dehydrogenase by chemical glycosidation with end-group activated dextran.

A mono-aminated dextran derivative was attached to Bacillus badius phenylalanine dehydrogenase via a carbodiimide-catalyzed reaction. The optimum temperature for the conjugate was 10 degrees C higher than for native enzyme, and its thermostability was improved by 8 degrees C. The activation free energy of thermal inactivation at 45 degrees C was increased by 16.8 kJ/mol. The improved conformational stability of the modified enzyme was confirmed by fluorescence spectroscopy.