High antioxidant activity of coffee silverskin extracts obtained by the treatment of coffee silverskin with subcritical water.

Coffee silverskin (CS) is a thin tegument of the outer layer of green coffee beans and a major by-product of the roasting process to produce roasted coffee beans. CS extracts obtained by the treatment of CS with subcritical water at 25-270°C were investigated for their antioxidant activity using hydrophilic oxygen radical absorption capacity (H-ORAC) and DPPH radical scavenging capacity assays. The antioxidant activity increased with increasing the extraction temperature and the highest activity was observed with the extracts obtained at 270°C. The H-ORAC and DPPH values of the extracts were 2629±193 and 379±36μmol TE/g of CS extract, respectively. High correlation (R=0.999) was observed between H-ORAC and DPPH values for the CS extracts. High correlation of the antioxidant activity was also observed with protein and phenolic contents in the extracts. The CS extracts could be useful as a good source of antioxidative materials

Biochemical Properties of Cytochrome P-450 in Relation to Steroid Oxygenation a

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/73858/1/j.1749-6632.1985.tb14606.x.pd

Properties of the tryptophan residue in rabbit liver microsomal cytochrome P-450 isozyme 2 as determined by fluorescence

Cytochrome P-450 isozyme 2 from rabbit liver microsomes fluoresces upon excitation at 295 nm due to the single tryptophyl residue (Trp121) in the protein. The fluorescence spectrum, which is not altered by the presence of phospholipid or substrates, has a maximum at 335 nm, which suggests that the environment of the residue is hydrophobic. The fluorescence intensity decreases linearly with increase of specific content of the cytochrome preparations, and the holoenzyme was estimated to exhibit, at most, 6% as much fluorescence as the apoenzyme. This indicates that the fluorescence of the tryptophan is quenched by energy transfer to the heme. The distance between the tryptophyl residue and the heme was estimated to be less than 40 A. From enhancement of the fluorescence by methanol and ethanol, 30 to 50% of the Trp residue was found to be accessible to these solvents. On the other hand, the accessibility to iodide and cesium ions, as estimated by quenching effects, is less than 14%. From such evidence, the tryptophyl residue is believed to be partly buried. Since Trp121 is conserved at or near the same position in all mammalian P-450's so far sequenced, the results obtained may be applicable to these related cytochromes as well.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/25697/1/0000251.pd

Review on utilization and composition of coffee silverskin

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

Inhibitory effects of chlorogenic acids from green coffee beans and cinnamate derivatives on the activity of porcine pancreas α-amylase isozyme I

Nine kinds of chlorogenic acids (CGAs) account for 80% of the total CGA content in green coffee beans. They consist of three subgroups of caffeoylquinic acids (CQAs), feruloylquinic acids (FQAs), and dicaffeoylquinic acids (diCQAs). We previously reported the inhibitory effects of 5-CQA on porcine pancreas α-amylase (PPA) isozymes, PPA-I and PPA-II. In this paper, we investigated the PPA-I inhibition by eight kinds of CGAs. The IC50 values of CQAs, FQAs, and diCQAs against the PPA-I-catalysed hydrolysis of p-nitrophenyl-α-D-maltoside were 0.08–0.23 mM, 1.09–2.55 mM, and 0.02–0.03 mM, respectively. All CQAs and FQAs and 3, 5-diCQA showed mixed-type inhibition with binding to the enzyme–substrate complex (ES) being stronger than to the enzyme (E). 3, 4-DiCQA and 4, 5-diCQA showed mixed-type inhibition, but, conversely are suggested to bind to E stronger than ES

Effects of the mutational combinations on the activity and stability of thermolysin.

We have previously indicated that three single mutations (Leu144-->Ser, Asp150-->Glu, and Ile168-->Ala) in the site-directed mutagenesis of thermolysin increase the activity and two single (Ser53-->Asp and Leu155-->Ala) and one triple (Gly8-->Cys/Asn60-->Cys/Ser65-->Pro) mutations increase the stability. In the present study, aiming to generate highly active and stable thermolysin variants, we combined these mutations and analyzed the effect of combinations on the activity and stability of thermolysin. The combination of the mutations of Leu144-->Ser and Asp150-->Glu yielded the most significant increase in the hydrolytic activities for N-[3-(2-furyl)acryloyl]-Gly-L-Leu amide (FAGLA) and N-carbobenzoxy-L-Asp-L-Phe methyl ester (ZDFM), while that of Leu144-->Ser and Ile168-->Ala abolished the activity. The combination of Ser53-->Asp and Leu155-->Ala yielded the greatest increase in the thermal stability, while that of Ser53-->Asp and Gly8-->Cys/Asn60-->Cys/Ser65-->Pro increased the stability as high as the individual mutations do. The combination of three mutations of Leu144-->Ser, Asp150-->Glu, and Ser53-->Asp yielded a variant L144S/D150E/S53D with improved activity and stability. Its k(cat)/K(m) values in the hydrolysis of FAGLA and ZDFM were 8.6 and 10.2 times higher than those of wild-type thermolysin (WT), respectively, and its rate constant for thermal inactivation at 80 degrees C was 60% of that of WT

Thermodynamic analysis of ionizable groups involved in the catalytic mechanism of human matrix metalloproteinase 7 (MMP-7).

Human matrix metalloproteinase 7 (MMP-7) exhibits a broad bell-shaped pH-dependence with the acidic and alkaline pK(e) (pK(e1) and pK(e2)) values of about 4 and 10. In this study, we estimated the ionizable groups involved in its catalytic mechanism by thermodynamic analysis. pK(a) of side chains of L-Asp, L-Glu, L-His, L-Cys, L-Tyr, L-Lys, and L-Arg at 25-45°C were determined by the pH titration of amino-acid solutions, from which their enthalpy changes, ∆H°, of deprotonation were calculated. pK(e1) and pK(e2) of MMP-7 at 15-45°C were determined in the hydrolysis of (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-NH(2), from which ∆H(o) for pK(e1) and pK(e2) was calculated. The ∆H(o) for pK(e1) (-20.6±6.1kJmol(-1)) was similar to that for L-Glu (-23.6±5.8kJmol(-1)), and the ∆H(o) for pK(e2) (89.9±4.0kJmol(-1)) was similar to those for L-Arg (87.6±5.5kJmol(-1)) and L-Lys (70.4±4.4kJmol(-1)). The mutation of the active-site residue Glu198 into Ala completely abolished the activity, suggesting that Glu198 is the ionizable group for pK(e1). On the other hand, no arginine or lysine residues are found in the active site of MMP-7. We proposed a possibility that a protein-bound water is the ionizable group for pK(e2)

Interaction of wheat β-amylase with maltose and glucose as examined by fluorescence.

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

Characterization and solvent engineering of wheat β-amylase for enhancing its activity and stability.

The kinetic and thermodynamic parameters of wheat β-amylase (WBA) were characterized and various additives were evaluated for enhancing its activity and thermostability. WBA activity was examined by neocuproine method using soluble starch as substrate. The Michaelis constant (K(m)) and molecular activity (k(cat)) were determined to be 1.0±0.1% (w/v) and 94±3s(-1), respectively, at pH 5.4 and at 25°C. The optimum reaction temperature (T(opt)) for WBA activity was 55°C and the temperature (T(50)) at which it loses half of the activity after 30-min incubation was 50±1°C. Modifications of the solvent with 182mM glycine and 0.18% (w/v) gelatin have increased the T(50) by 5°C. Glycerol, ethylene glycol, dimethylformamide (DMF) and dimethyl sulfoxide have also slightly enhanced the thermostability plausibly through weakening the water structure and decreasing the water shell around the WBA protein. Ethanol and DMF activated WBA by up to 24% at 25°C probably by inducing favorable conformation for the active site or changing the substrate structure by weakening the hydrogen bonding. Its half-life in the inactivation at 55°C was improved from 23 to 48min by 182mM glycine. The thermodynamic parameters indicate that WBA is thermo-labile and sufficient stabilization was achieved through solvent modification with additives and that the heat inactivation of WBA is entropic-driven. It is suggested that WBA could be applied more widely in starch-saccharification industries with employing suitable additives

Involvement of Val 315 located in the C-terminal region of thermolysin in its expression in Escherichia coli and its thermal stability.

Thermolysin is a thermophilic and halophilic zinc metalloproteinase that consists of β-rich N-terminal (residues 1-157) and α-rich C-terminal (residues 158-316) domains. Expression of thermolysin variants truncated from the C-terminus was examined in E. coli culture. The C-terminal Lys316 residue was not significant in the expression, but Val315 was critical. Variants in which Val315 was substituted with fourteen amino acids were prepared. The variants substituted with hydrophobic amino acids such as Leu and Ile were almost the same as wild-type thermolysin (WT) in the expression amount, α-helix content, and stability. Variants with charged (Asp, Glu, Lys, and Arg), bulky (Trp), or small (Gly) amino acids were lower in these characteristics than WT. All variants exhibited considerably high activities (50-100% of WT) in hydrolyzing protein and peptide substrates. The expression amount, helix content, and stability of variants showed good correlation with hydropathy indexes of the amino acids substituted for Val315. Crystallographic study of thermolysin has indicated that V315 is a member of the C-terminal hydrophobic cluster. The results obtained in the present study indicate that stabilization of the cluster increases thermolysin stability and that the variants with higher stability are expressed more in the culture. Although thermolysin activity was not severely affected by the variation at position 315, the stability and specificity were modified significantly, suggesting the long-range interaction between the C-terminal region and active site