Inhibitory activity (IA) against porcine pancreatic alpha-amylase (PPA) has been found in the water extract of Eisenia fetida. IA is recovered mostly in a fraction (U3EE) containing compounds of low molecular mass under 3 kDa. U3EE is treated with 85 % ethanol (EtOH) and the obtained EtOH extract is applied to the solid-phase extraction (SPE) system. An IA fraction named AI is separated from SPE by elution with water, and then a fraction AI* has been obtained by elution with 5 % EtOH. In the previous study, PPA inhibitors of guanine (Gua), guanosine (Guo), and inosine (Ino) have been isolated from AI. In the present study, IA of AI* was examined and a new inhibitor 2'-deoxyguanosine (2'-dGuo) was isolated by RP-HPLC. It was a mixed-type inhibitor showing IC50 of 0.7 +/- 0.1 mM and inhibitor constants Ki and Ki' of 2.0 +/- 0.5 and 0.9 +/- 0.2 mM, respectively, at pH 6 and at 37 degrees C. PPA inhibitors of U3EE were arranged in the order according to the inhibitory efficiency as Gua> 2'dGuo> Guo> Ino. This study reports that compounds related to nucleic acids could be PPA inhibitors. These inhibitors as well as U3EE might be suitable for functional foods to prevent obesity and type 2 diabetes.

We have reported previously that the water extract of the earthworm Eisenia fetida has inhibitory effect on human dipeptidyl-peptidase IV (DPP IV) in vitro. Here we studied to identify DPP IV inhibitors in a low-molecular mass extract (designated U3EE) under 3 kDa prepared from the water extract. U3EE showed 50 % inhibition (IC50) at the concentration of 5.3 +/- 0.3 mg/mL. An inhibitory active fraction obtained by solid-phase extraction of U3EE was separated into three parts by reversed-phase HPLC. These parts were shown by GC/MS to be composed of ten (Ala, Gly, Thr, Ser, Asn, Asp, Lys, His, Orn, and cystine), two (Leu and Ile), and one (Met) amino acids, respectively. Among them, Met, Leu, and His showed strong inhibition with IC50 values of 3.4 +/- 0.3, 6.1 +/- 0.3 and 14.7 +/- 1.2 mM, respectively; Ala, Lys, Orn, and Ile showed rather weaker inhibition than those, while the others showed no inhibition. Met, Leu, and Ile were competitive inhibitors and His was a mixed-type one. DPP IV inhibition by U3EE might be due to additive and/or synergistic effects of the inhibitory amino acids, suggesting that it could be useful as pharmaceutical and supplement for diabetes prevention.

Earthworms are known as a source of a traditional medicine, and bioactive components have been reported. We have reported that a fraction (U3EE) with molecular mass under 3 kDa from the water extract of Eisenia fetida inhibits porcine pancreatic alpha-amylase (PPA) activity with a half-maximal inhibitory concentration (IC50) of 73.7 +/- 4.0 mg/mL. Here we purified PPA-inhibitory components from U3EE by sequential procedures of 85 %-ethanol (EtOH) extraction, solid-phase extraction (SPE), and RP-HPLC. The water eluate from SPE of the 85 %-EtOH extract was a major inhibitory fraction, from which three components were separated by 2nd RP-HPLC and identified with MS, TLC, and UV spectroscopy as guanine (Gua), inosine (Ino), and guanosine (Guo). Kinetic analysis showed that Gua and Guo were non-competitive inhibitors and Ino a mixed-type one, suggesting a key role of the purine ring in inhibition. The inhibitor constants (Ki) of Gua and Guo were 0.28 +/- 0.07 and 1.64 +/- 0.14 mM, respectively, and Ki and Ki' of Ino in the EI and ESI complexes were 5.8 +/- 1.1 and 59 +/- 12 mM, respectively. U3EE might be useful for food supplements to prevent obesity and diabetes.

The vitamin D-3 25-hydroxylase (CYP27A1), 25-hydroxyvitamin D-3 1alpha-hydroxylase (CYP27B1) and 1alpha, 25-dihydroxyvitamin D-3 24-hydroxylase (CYP24A1) are members of the cytochrome P450 superfamily, and key enzymes of vitamin D-3 metabolism. Using the heterologous expression in E. coli, enzymatic properties of the P450s were recently investigated in detail. Upon analyses of the metabolites of vitamin D-3 by the reconstituted system, CYP27A1 surprisingly produced at least seven forms of minor metabolites including 1alpha, 25( OH)(2)D-3 in addition to the major metabolite 25( OH)D-3. These results indicated that human CYP27A1 catalyzes multiple reactions involved in the vitamin D-3 metabolism. In contrast, CYP27B1 only catalyzes the hydroxylation at C-1alpha position of 25(OH) D-3 and 24R, 25(OH)(2)D-3. Enzymatic studies on substrate specificity of CYP27B1 suggest that the 1alpha-hydroxylase activity of CYP27B1 requires the presence of 25-hydroxyl group of vitamin D-3 and is enhanced by 24-hydroxyl group while the presence of 23-hydroxyl group greatly reduced the activity. Eight types of missense mutations in the CYP27B1 gene found in vitamin D-dependent rickets type I (VDDR-I) patients completely abolished the 1alpha-hydroxylase activity. A three-dimensional model of CYP27B1 structure simulated on the basis of the crystal structure of rabbit CYP2C5 support s the experimental data from mutagenesis study of CYP27B1 that the mutated amino acid residues may be involved in protein folding, heme-propionate binding or activation of molecular oxygen. CYP24A1 expressed in E. coli showed a remarkable metabolic processes of 25(OH) D-3 and 1alpha, 25( OH)(2)D-3. Rat CYP24A1 catalyzed six sequential monooxygenation reactions that convert 1alpha, 25(OH)(2)D-3 into calcitroic acid, a known final metabolite of C-24 oxidation pathway. In addition to the C-24 oxidation pathway, human CYP24A1 catalyzed also C-23 oxidation pathway to produce 1alpha, 25(OH)(2)D-3- 26,23-lactone. Surprisingly, more than 70 % of the vitamin D metabolites observed in a living body were found to be the products formed by the activities of CYP27A1, CYP27B1 and CYP24A1. The species-based difference was also observed in the metabolism of vitamin D analogs by CYP24A1, suggesting that the recombinant system for human CYP24A1 may be of great use for the prediction of the metabolism of vitamin D analogs in humans.

A fraction (designated as U3EE) with molecular mass under 3 kDa from the water extract of the earthworm Eisenia fetida was prepared and its effects on mammalian digestive enzymes were examined. U3EE itself showed no relevant enzyme activities. However, it increased the activities of trypsin, α-chymotrypsin, and lipase, while decreased those of α-amylase and α-glucosidase. The trypsin activation and α-amylase inhibition were analyzed precisely by enzyme kinetics. The former was solely dependent on increase in the molecular activity kcat without any change in the Michaelis constant Km, and the latter was mainly dependent on decrease in Km with a slight change in kcat. Effects of the treatments of U3EE with freezing-and-thawing, heat, acid, and hexane were examined. The treated U3EE showed the effects on the enzymes with the same potencies as those provided by the non-treated one, indicating that U3EE was enough stable toward the harsh treatments to hold the modulating effects on the enzyme activities. U3EE was also shown to be highly hydrophilic. The results obtained in this paper suggested that U3EE could be applicable as a novel constituent for pharmaceuticals and functional foods.

In this chapter, we describe the contents of chlorogenic acids (CGAs) of various species of green and roasted coffee beans. CGAs are a family of esters that are structural analogs of quinic acid with various cinnamate derivatives. A total of 82 CGAs have been detected in green coffee beans. The contents of CGAs of Coffea arabica and Coffea canephora, two of the major cultivated species, are in the range of 3.40–7.24% and 5.17–14.4% w/w dry matter, respectively, although those of some Coffea species, such as Coffea pseudozanguebariae and Coffea rhamnifolia, are <1%. The contents may decrease with roasting of green coffee beans, and the extent of this decrease depends on the degree of roasting. Reportedly, the average contents of total CGAs, caffeoylquinic acids, feruloylquinic acids, and dicaffeoylquinic acids in 12 commercial roasted coffee beans are 2.66%, 2.26%, 0.21%, and 0.19% w/w dry matter, respectively. The contents of CGAs in commercial instant coffee are in the range of 3.61–10.73% w/w dry matter (instant coffee powder).

α-Amylase inhibitors appear helpful in the prevention and medical treatment of metabolic syndromes such as type 2 diabetes and obesity. Here, we focus on the inhibitory effects on porcine pancreas α-amylase by nine types of chlorogenic acids (CGAs) from green coffee beans: three subgroups of caffeoylquinic acids (CQAs), feruloylquinic acids (FQAs), and dicaffeoylquinic acids. The inhibition modes of six types of CQAs and FQAs were mixed-type, suggesting that the inhibitor-binding site (I-site) for CGAs exists separately from the substrate-binding site on the enzyme surface. CQAs (esters of caffeic acid (CA) and quinic acid (QA)) and FQAs (esters of ferulic acid (FA)) and QA bound to the same I-site, which was separated into two subsites (i.e., CA/FA subsite and QA subsite), thereby accommodating the CA or FA substructures and QA substructure of CQA or FQA, respectively, based on a comparison of the inhibitor constants for each CGA.

Parasporin-4 (PS4) is an aerolysin-type β-pore-forming toxin produced by Bacillus thuringiensis strain A1470. It exhibits specific cytotoxicity against human cancer cell lines; therefore, it is expected to be useful for the diagnosis and treatment of particular types of cancer cells. We examined the acute toxicity of PS4 on ICR mice. The LD50 value was 160 μg/kg by a subcutaneous route. Potassium, ammonium, magnesium ion, creatinine, and urea nitrogen decreased in urine by the injection of PS4. Simultaneously, creatinine and urea nitrogen in mice serum increased. These results imply that PS4 impairs kidney function in mice. PS4 is obtained from Pro-parasporin-4 (ProPS4) by processing, and ProPS4 is produced by recombinant Escherichia coli as the inclusion body. The inclusion body of ProPS4 can be solubilized in a weak acid solution and activated by pepsin, implying that it would be solubilized and activated in the stomach of mammals after oral administration. Thus, the influence of the oral administration of it by C57BL/6J mice was examined. Although ProPS4 was activated to PS4 in the mouse digestive tract, any serious health hazard was not observed and there was no significant difference in body weight change.

Sulfated glycosaminoglycans and sulfated lipids are involved in the biological functions of human matrix metalloproteinase 7 (MMP-7). In this study, the effects of heparin and cholesterol sulfate (CS) on the activity and stability of MMP-7 in the hydrolysis of a synthetic substrate, (7-methoxycoumarin-4-yl)acetyl-l-Pro-l-Leu-Gly-l-Leu-[N-3-(2,4-dinitrophenyl)-l-2,3-diaminopropionyl]-l-Ala-l-Arg-NH2, were examined. Heparin increased activity by decreasing K-m, and the K-m values for 0 and 50 mu M heparin were 57 +/- 8 and 19 +/- 5 mu M, respectively. CS decreased activity in a non-competitive inhibitory manner with a K-i value of 11 +/- 3 mu M. In thermal incubation at 50-70 degrees C, heparin increased relative activity (the ratio of k(cat)/K-m of MMP-7 with incubation to that without it), while CS decreased relative activity. These results indicate that heparin increases the activity and stability of MMP-7, while CS decreases them.

Coffee is one of the most frequently consumed drinks in the world. Coffee silverskin (CS) is the only by-product produced during the coffee beans roasting process, and large amounts of CS are produced by roasters in coffee-consuming countries. However, methods for the effective utilization of CS have not been developed. Reuse of CS, which is the primary residue from the coffee industry, is important for the environment and economy. Recently, there have been some attempts to reuse CS for biological materials and as a nutrient source for solid-state fermentation. The purpose of this review is to provide an overview about CS, its chemical composition, biological activity, and attempts at its reuse.

Biofuel production from plant-derived lignocellulosic material using fungal cellulases is facing cost-effective challenges related to high temperature requirements. The present study identified a cold-adapted cellulase named endo-1,4-beta-glucanase (EF-EG2) from the earthworm Eisenia fetida. The gene was cloned in the cold-shock expression vector (pCold I) and functionally expressed in Escherichia coli ArcticExpress RT (DE3). The gene consists of 1368 bp encoding 456 amino acid residues. The amino acid sequence shares sequence homology with the endo-1,4-beta-glucanases of Eisenia andrei (98%), Pheretima hilgendorfi (79%), Perineresis brevicirris (63%), and Strongylocentrotus nudus (58%), which all belong to glycoside hydrolase family 9. Purified recombinant EF-EG2 hydrolyzed soluble cellulose (carboxymethyl cellulose), but not insoluble (powdered cellulose) or crystalline (Avicel) cellulose substrates. Thin-layer chromatography analysis of the reaction products from 1,4-beta-linked oligosaccharides of various lengths revealed a cleavage mechanism consistent with endoglucanases (not exoglucanases). The enzyme exhibited significant activity at 10 degrees C (38% of the activity at optimal 40 degrees C) and was stable at pH 5.0-9.0, with an optimum pH of 5.5. This new cold-adapted cellulase could potentially improve the cost effectiveness of biofuel production. (C) 2013 Elsevier Ltd. All rights reserved.

Neutral salts activate and stabilize thermolysin. In this study, to explore the mechanism, we analyzed the interaction of 8-anilinonaphthalene 1-sulphonate (ANS) and thermolysin by ANS fluorescence. At pH 7.5, the fluorescence of ANS increased and blue-shifted with increasing concentrations (0-2.0 mu M) of thermolysin, indicating that the anilinonaphthalene group of ANS binds with thermolysin through hydrophobic interaction. ANS did not alter thermolysin activity. The dissociation constants (K-d) of the complex between ANS and thermolysin was 33 +/- 2 mu M at 0M NaCl at pH 7.5, decreased with increasing NaCl concentrations, and reached 9 +/- 3 mu M at 4M NaCl. The K-d values were not varied (31-34 mu M) in a pH range of 5.5-8.5. This suggests that at high NaCl concentrations, Na+ and/or Cl- ions bind with thermolysin and affect the binding of ANS with thermolysin. Our results also suggest that the activation and stabilization of thermolysin by NaCl are partially brought about by the binding of Na+ and/or Cl- ions with thermolysin.

Acrylamide (AA) is classified as a Group 2A carcinogen according to the International Agency for Research on Cancer. Although coffee contains a small amount of AA, it is a popular beverage worldwide. Approximately 10 billion canned coffees are consumed each year in Japan. In this study, we investigated how to decrease AA contained in canned coffee by modifying the heat treatment used for sterilization during the manufacturing process. The AA content of both types of canned coffee (black and milk) was decreased by approximately 95% by heat treatment with adding cysteine at 121 °C for 6 min. The content was also decreased by heat treatment with dithiothreitol, although that with cystine had no effect. Therefore, it is shown that thiol groups in cysteine and dithiothreitol might play an important role in decreasing the AA content.

We constructed a simple method for discrimination of single nucleotide polymorphism (SNP) with a surface plasmon resonance (SPR)-based sensor by determining the binding volume (BV) between a target SNP allele and a probe complementary to the target. In the method, a novel additive termed "blocker," which is a short single-stranded DNA complementary to the target, was used. The blocker enhanced the BV of the target only to the full-match probe 10-fold or more, so the SNP alleles could be discriminated readily. The effect of the blocker concentration was also examined. The BV to only a full-match probe increased with increasing the blocker concentration and reached a plateau at the concentration of 300-500 nM. To assess the effectiveness of this method, the SNP associated with progressive rod-cone degeneration in dog was determined. The results of genotyping with the method were in good agreement with those obtained by direct sequencing.

Fluorescence of wheat β-amylase (WBA) was quenched by the interaction with maltose or glucose, which are competitive inhibitors of WBA, suggesting that the states of tryptophan and tyrosine residues could be changed by the interaction. The fluorescence emitted by excitation at 280 and 295 nm was titrated by changing the concentrations of maltose and glucose. The dissociation constant (Kd) values of the WBA-maltose and WBA-glucose complexes were determined to be 0.20 ± 0.12 M for maltose and 0.36 ± 0.11 M for glucose at 25°C, pH 5.4. Maltose exhibited additional binding mode at higher concentration with a distinct Kd value (1.5 ± 0.4 M). The Kd values at various temperatures and pHs are in agreement with the inhibitor constant (Ki) values previously reported. The negative standard enthalpy changes (ΔH°) of the WBA association with glucose and maltose indicate that the associations are exothermic. The association constant (Ka) and ΔG° values of the maltose and glucose binding to WBA decreased slightly with increasing temperature from 25°C to 45°C but not dependent on pH change (pH 3.0, 5.4 and 9.0). Fluorescence of WBA could be used as a structural probe to examine the inhibitory interaction with the products of starch hydrolysis.

The amino groups of wheat β-amylase (WBA) were modified by 2,4,6-trinitrobenzenesulfonic acid (TNBS), 2,4-bis (O-methoxypolyethylene glycol)-6-chloro-s-triazine (mPEG), and glutaraldehyde (GA) to improve its thermal stability and activity. Modification of WBA by 5mM TNBS, 4.8μM mPEG and 11 mM GA improved its T50 (the temperature at which 50% of its activity is lost after 30 min of incubation) from 47 ± 1°C to 48 ± 2, 55 ± 2, and 54 ± 2°C, respectively. The catalytic activity of WBA was reduced by 15% and 59% with modification by 5mM TNBS and 11mM GA, respectively. In all cases, the enhancement of thermostability of modified WBA was entropically driven. The activity of WBA modified by 4.8μM mPEG was enhanced by 39% at 25°C. Therefore, the thermal stability of WBA was significantly improved by modification with mPEG, GA and slightly by TNBS and its catalytic activity was enhanced by mPEG.

A new protease was purified from the culture filtrate of a plant worm, Nomuraea atypicola. The activity of the protease was suppressed by metalloprotease inhibitors such as EDTA and 1,10-phenanthroline, suggesting that it might be a metalloprotease. Its molecular mass was estimated to be 48 kDa by SDS-PAGE, and its optimal pH and temperature were pH 8.5-9.0 and 40 C, respectively. The N-terminal amino acid sequence of the metalloprotease was similar to those of fungalysin metallopeptidases of the M36 family from fungi such as Coccidioides posadasii, Pyrenophora tritici-repentis, and Arthroderma gypseum, supporting the idea that it is a fungalysin-like metallopeptidase. Crown Copyright (C) 2012 Published by Elsevier Ltd. All rights reserved.

Inhibition of wheat β-amylase (WBA) by glucose and maltose was studied by kinetics and thermodynamics. The inhibitory effects of fructose, difructose, sucrose, trehalose, cellobiose, acarbose, and 1-deoxynojirimycin on WBA were also evaluated. The half maximal inhibitory concentrations (IC50) of acarbose, maltose and glucose were 0.06±0.01M, 0.22±0.09M, and 1.41±0.17M, respectively. The inhibitor constant (Ki) and the thermodynamic parameters such as changes in Gibbs energy (ΔG), enthalpy (ΔH), and entropy (ΔS) of the dissociation reactions of the WBA-glucose and WBA-maltose complexes were temperature and pH-dependent. The dissociation reactions were endothermic and enthalpy-driven. Both glucose and maltose behaved as competitive inhibitors at pH 3.0 and 5.4 at a temperature of 25°C with respective Ki values of 0.33±0.02M and 0.12±0.03M. In contrast, both sugars exhibited uncompetitive inhibition at pH 9 at a temperature of 25°C with Ki values of 0.21±0.03M for glucose and 0.11±0.04M for maltose. The pH-dependence of the inhibition type and Ki values indicate that the ionizing groups of WBA influence drastically the interaction with these carbohydrates. This evidence enables us to consider temperature and pH in the WBA-catalyzed hydrolysis to manipulate the inhibition by end-product, maltose, and even by glucose.