Secondary Hydrolysis Research Articles

A new correction method for determination on carbohydrates in lignocellulosic biomass

The accurate determination on the key components in lignocellulosic biomass is the premise of pretreatment and bioconversion. Currently, the widely used 72% H2SO4 two-step hydrolysis quantitative saccharification (QS) procedure uses loss coefficient of monosaccharide standards to correct monosaccharide loss in the secondary hydrolysis (SH) of QS and may result in excessive correction. By studying the quantitative relationships of glucose and xylose losses during special hydrolysis conditions and the HMF and furfural productions, a simple correction on the monosaccharide loss from both PH and SH was established by using HMF and furfural as the calibrators. This method was used to the component determination on corn stover, Miscanthus and cotton stalk (raw materials and pretreated) and compared to the NREL method. It has been proved that this method can avoid excessive correction on the samples with high-carbohydrate contents.

Production of reducing sugar from corn stover by dilute acid hydrolysis co-catalyzed with metal salts under microwave radiation

Acid hydrolysis of corn stover under microwave radiation by adding four metal salt co-catalysts (ferrous sulfate, copper sulfate, zinc sulfate, and manganese sulfate) to produce reducing sugar was investigated. The influences of microwave power, reaction time, sulfuric acid concentration, and co-catalyst concentration on the yield of reducing sugar were studied. The results show that the catalytic effects of ferrous sulfite and copper sulfate are more obvious for the yield of reducing sugar compared with only dilute acid hydrolysis with microwave radiation. Long-time microwave irradiation resulted in secondary hydrolysis, generating small-molecule compounds and decreasing the yield of reducing sugar. The reducing sugar yield was increased by decreasing the hydrolysis reaction time and microwave radiation power. In terms of reducing sugar yield, the four kinds of metal salt co-catalyst act in the order: ferrous sulfate > copper sulfate > sulfuric acid zinc sulfate > manganese sulfate. At concentration of 3.0 %, the best was ferrous sulfate, producing yield of 35.10 %.

Sodium Caseinate 가수분해물의 Angiotensin-I Converting Enzyme 저해효과에 관한 연구

This study was carried out to identify the ACE (Angiotensin converting enzyme) inhibitory activity of casein hydrolysates for development of anti-hypertensive hydrolysates. Sodium caseinate was treated with six kinds of commercial proteases such as Flavourzyme, Protamex, Neutrase 1.5, Alcalase, Protease M, and Protease S for 8 h individually, and was then treated with the enzyme combination for 4 h at 45 o C. The hydrolysate which had the highest ACE inhibitory effect was then hydrolysed successively with three digestive enzymes: pepsin, trypsin, and α-chymotrypsin, at 37 o C for 4 h under conditions mimicking those of the gastrointestinal tract. UF (ultra filtration) treatment was applied to one of the secondary hydrolysates to determine ACE inhibitory activity. When sodium caseinate was hydrolysed by commercial proteases, the degree of hydrolysis (DH) showed 2.54 to 4.25% and after secondary hydrolysis, DH showed 4.30 to 5.22%. ACE inhibitory activity and IC50 values decreased, and inhibition rates increased during hydrolysis. Protamex treatment showed the lowest IC50 value (516 µg/mL) and Flavourzyme hydrolysate showed the highest IC50 value (866 µg/mL). As the first hydrolysate was treated with Flavourzyme, the ACE inhibitory activity increased. Neutrase hydrolysate had the highest activity with an IC50 value (282 µg/mL). When Neutrase plus Flavourzyme treatment was hydrolyzed by digestive enzymes, the IC50 value (597 µg/mL) was decreased statistically (p<0.05). As Neutrase plus Flavourzyme hydrolysate is treated by UF with MW cut-off 10,000, permeate showed 273 µg/mL of IC50 value, showed no difference, but retentate which has over MW 10,000 showed statistically different IC50 value, 635 µg/mL (p<0.05).

Evaluation of Secondary Acid and Enzymatic Hydrolysis of Hemicellulose in Hot Water Pre-Pulping Extract of Mixed Hardwoods

Pre-pulping extracts were found to contain a dilute amount of xylo-oligosaccharides and acetic acid as the major components, and many minor components including other organic acids, lignin-derived phenolics, and sugar degradation products. Once separated from the pulp, a secondary hydrolysis step was required to hydrolyze oligomeric hemicellulose sugars into monomeric sugars before fermentation. The following study detailed the extent of hemicellulose recovery by pre-pulping using hot water extraction and characterized the hydrolysis of the extract with respect to comparing acid and enzymatic hydrolysis. The secondaryhydrolysis of hot water extracts made at an H-Factor of 800 was tested for a variety of acid and enzyme loading levels using the sulfuric acid and xylanases. The maximum fermentable sugar yield from acid and enzyme hydrolysis of the extract was 18.7 g/<TEX>${\ell}$</TEX> and 17.7 g/<TEX>${\ell}$</TEX> representing 84.6% and 80.1% of the maximum possible yield, respectively.

Improving the diastereoselectivity of penicillin G acylase for ampicillin synthesis from racemic substrates

Semi-synthetic β-lactam antibiotics are synthesized enzymatically with the use of penicillin G acylase (PGA). Currently, PGA only exhibits weak diastereoselectivity with respect to the alpha amino group of rac-phenylglycine methyl ester (rac-PGME) when it is coupled with 6-aminopenicillanic acid to synthesize ampicillin. Therefore, we sought to improve the diastereoselectivity of PGA by targeting residues for site-saturation based on the proximity to the substrate's chiral center. Four variants with improved selectivity for (R)-ampicillin synthesis were identified, all resulting from a mutation at the β24 position. βPhe24Ala, while not identified from our library screening, was obtained with site-directed mutagenesis because it has been previously shown to be selective for (R)-enantiomers with substituents other than an amino group. The diastereomeric excess (d.e.(R)) value of 37% for the wild-type enzyme was improved to a d.e.(R) value of 98% for our most selective mutant, βPhe24Ala. Also, four mutations at the α146 position that resulted in (S)-selective PGA variants were identified. βPhe24 and αPhe146 are on opposite sides of the alpha carbon of the substrate, and we have shown that altering these residues results in enhanced selectivity in opposite directions. All variants that showed selectivity for (S)-ampicillin synthesis showed decreased synthetic activity for pure substrates and a decreased synthesis-to-hydrolysis ratio. In contrast, the mutants that were selective for (R)-ampicillin showed significantly decreased primary and secondary hydrolysis when synthesizing ampicillin from pure (R)-PGME, resulting in up to 4-fold decrease in the synthesis to hydrolysis ratio and up to 2-fold increase in the yield achieved. Finally, it was discovered that the selective PGA variants have racemase or epimerase activity, a fascinating phenomenon that has never been reported.

Study of the kinetic parameters for synthesis and hydrolysis of pharmacologically active salicin isomer catalyzed by baker’s yeast maltase

One of the key elements for understanding enzyme reactions is determination of its kinetic parameters. Since transglucosylation is kinetically controlled reaction, besides the reaction of synthesis, very important is the reaction of enzymatic hydrolysis of created product. Therefore, in this study, kinetic parameters for synthesis and secondary hydrolysis of pharmacologically active α isosalicin by baker’s yeast maltase were calculated, and it was shown that specifity of maltase for hydrolysis is approximately 150 times higher then for synthesis.

Hydrolysis of Substance P in the Presence of the Osteosarcoma Cell Line SaOS-2: Release of Free Amino Acids

The possible hydrolysis of substance P (Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met) in presence of the osteoblastic cell line SaOS-2 was measured by capillary electrophoresis coupled to mass detection. The results obtained indicate that a very rapid disappearance of the intact undecapeptide was associated to a slower appearance of seven of its eight component amino acids. These results can be interpreted as indicating that an extremely fast hydrolysis of substance P by endopeptidases, which released peptidic by-products, was followed by a noticeably slower secondary degradation which released free amino acids. In decreasing quantitative importance, these phenomena appear to originate by the hydrolysis of the Pro(4)-Gln(5) bond, followed by C-terminal sequential degradation of the Arg(1)-Pro(4) tetrapeptide; by the hydrolysis of or Phe(7)-Phe(8) bond (or, possibly, of Gln(6)-Phe(7)) leading to release of free Phe and Gln; by hydrolysis of the Gly(9)-Leu(10) bond with subsequent release of Met and Leu. Results obtained appear to be compatible with the expression by SaOS-2 cells of enzymes already known to catalyze substance P hydrolysis, together with an apparent low efficiency of aminopeptidases. Because of the activity of C-terminal fragments on NK1 receptors, the delay between primary hydrolysis of substance P and secondary hydrolysis of its peptidic fragments indicated by the data shown implies a possible persistence of substance P physiological effects even after degradation of the intact peptide.

Kinetics and Mechanism of S-Nitrosothiol Acid-Catalyzed Hydrolysis: Sulfur Activation Promotes Facile NO+ Release

The denitrosation of three primary S-nitrosothiols (RSNO; S-nitrosocysteine, S-nitroso-N-acetylcysteine, and S-nitrosoglutathione) and two tertiary RSNOs (S-nitrosopenicillamine and S-nitroso-N-acetylpenicillamine) was investigated in 3.75 M H(2)SO(4) to probe the mechanism of acid-catalyzed RSNO hydrolysis and its dependence on RSNO structure. This reversible reaction was forced to proceed in the denitrosation direction by trapping the nitrosating agent with HN(3). The primary RSNOs exhibited hydrolysis k(obs) values of ∼2 × 10(-4) s(-1), and the tertiary RSNO k(obs) values were an order of magnitude higher. Product analysis by HPLC revealed that the parent thiols (RSHs) were formed in 90-100% yield on 79-99% RSNO denitrosation. Possible hydrolysis mechanisms were studied computationally at the CBS-QB3 level using S-nitrosomethanethiol (MeSNO) as a model RSNO. Consideration of RSNOs as a combination of conventional R-S-N═O, zwitterionic R-S(+)═N-O(-), and RS(-)/NO(+) ion-pair resonance structures was key in understanding the mechanistic details of acid-catalyzed hydrolysis. Protonation of the S-nitroso oxygen or nitrogen activates the sulfur and nucleophilic attack by H(2)O at this atom leads to the formation of the sulfoxide-protonated N-hydroxysulfinamide, MeS(+)(OH)NHOH, with barriers of 19 and 29 kcal/mol, respectively. Proton loss and reprotonation at the nitrogen lead to secondary hydrolysis that produces the sulfinic acid MeS(═O)OH and NH(2)OH. Notably, no low-energy RSNO hydrolysis pathway for HNO release was found in the computational analysis. Protonation of the S-nitroso sulfur gives rise to NO(+) release with a low activation barrier (ΔH(double dagger)(calc) ≈ 6 kcal/mol) and the formation of MeSH in agreement with experiment. The experimental k(obs) can be expressed as K(a)k(1), where K(a) is the acid dissociation constant for protonation of the S-nitroso sulfur and k(1) the pseudo-first-order hydrolysis rate constant. Given the low ΔH(double dagger)(calc) for denitrosation of the S-protonated isomer, the observed slow rates of acid-catalyzed RSNO hydrolysis must be controlled by the magnitude of K(a). The 10-fold higher K(a) calculated for Me(3)CS(H(+))NO (∼10(-15)) compared to MeS(H(+))NO (10(-16)) is consistent with the order of magnitude larger k(obs) reported here for the tertiary vs primary RSNOs.

Peptide synthesis catalysed by a haloalkaliphilic serine protease from the archaeon Natrialba magadii (Nep)

Haloarchaeal proteases function optimally in high salt (low water activity); thus, they offer an advantage over the nonhalophilic counterparts as biocatalysts for protease-catalysed peptide synthesis. The haloalkaliphilic archaeon Natrialba magadii secretes a solvent-tolerant protease, Nep (Natrialba magadii extracellular protease). In this work, the ability of Nep to catalyse peptide synthesis was examined. The tripeptide Ac-Phe-Gly-Phe-NH(2) was synthesized using Ac-Phe-OEt and Gly-Phe-NH(2) substrates as building blocks in the presence of Nep, 30% (v/v) dimethyl sulfoxide (DMSO) and 1.5 or 0.5 mol l(-1) NaCl. Purification and identification of the peptide product was achieved by RP-HPLC and ESI-MS, respectively. The native as well as the recombinant enzyme produced in Haloferax volcanii (HvNep) was similarly effective as catalysts for the synthesis of this model tripeptide with yields of up to 60% and without secondary hydrolysis of the product. HvNep catalysed the synthesis of various tripeptides with preference for those having aromatic amino acids in the P1 site. Nep is able to catalyse peptide synthesis under different salt concentrations in the presence of DMSO. The catalytic property of Nep in peptide synthesis combined with overproduction of this protease in Hfx. volcanii anticipates the potential applicability of this haloarchaeal protease in biotechnology.

Optimized enzymatic synthesis of C-terminal peptide amides using subtilisin A from Bacillus licheniformis

Abstract A mild and efficient method for the conversion of C-terminal esters of side-chain protected peptides into an amide function via enzyme-catalysed ammonolysis in organic media with low water content is described. Subtilisin A, the alkaline serine protease from Bacillus licheniformis , was used as biocatalyst and ammonium carbamate as source of ammonia. Response surface methodology (RSM) and central composite design were employed to estimate the effects of reaction parameters such as molar ratio of ammonia source to peptide methyl ester (2:1–10:1), composition of the solvent system (Bu t OH/DMF, % v/v, 70:30–95:5) and water concentration (0.2–0.8%) at different temperatures (30–50 °C) for the preparation of Z-Ala-Phe-NH 2 starting from Z-Ala-Phe-OMe. Optimum reaction conditions for maximum amide yield and minimum secondary hydrolysis were determined from cross-section analysis: temperature 30 °C, solvent composition Bu t OH/DMF 82.5:17.5 (v/v) containing 0.2% water (v/v) and molar ratio of ammonia source to peptide methyl ester of 10:1. The maximum yield of Z-Ala-Phe-NH 2 was 87% after 21 h for a quantitative substrate conversion. The method proved to be generally applicable for the synthesis of C-terminal amides of dipeptides with different terminal amino acids and sequence.

Ethanol Production from Orange Peels: Two-Stage Hydrolysis and Fermentation Studies Using Optimized Parameters through Experimental Design

Orange peels were evaluated as a fermentation feedstock, and process conditions for enhanced ethanol production were determined. Primary hydrolysis of orange peel powder (OPP) was carried out at acid concentrations from 0 to 1.0% (w/v) at 121 degrees C and 15 psi for 15 min. High-performance liquid chromatography analysis of sugars and inhibitory compounds showed a higher production of hydroxymethyfurfural and acetic acid and a decrease in sugar concentration when the acid level was beyond 0.5% (w/v). Secondary hydrolysis of pretreated biomass obtained from primary hydrolysis was carried out at 0.5% (w/v) acid. Response surface methodology using three factors and a two-level central composite design was employed to optimize the effect of pH, temperature, and fermentation time on ethanol production from OPP hydrolysate at the shake flask level. On the basis of results obtained from the optimization experiment and numerical optimization software, a validation study was carried out in a 2 L batch fermenter at pH 5.4 and a temperature of 34 degrees C for 15 h. The hydrolysate obtained from primary and secondary hydrolysis processes was fermented separately employing parameters optimized through RSM. Ethanol yields of 0.25 g/g on a biomass basis (YP/X) and 0.46 g/g on a substrate-consumed basis (YP/S) and a promising volumetric ethanol productivity of 3.37 g/L/h were attained using this process at the fermenter level, which shows promise for further scale-up studies.

Valorisation of hardwood hemicelluloses in the kraft pulping process by using an integrated biorefinery concept

A primary hydrolysis treatment (auto or acid-catalysed) of Eucalyptus globulus wood was performed before the cooking stage to extract part of the hemicelluloses that otherwise would be dissolved in the kraft liquor and burned. As xylose was the main monosaccharide Pichia stipitis was selected to produce bioethanol. Two methods were tested, with different alkalis, to reduce hydrolysates toxicity and adjust pH. A two-step method using Ca(OH) 2 leads to better fermentation results. Acid hydrolysates promoted higher ethanol concentrations (12 g L −1) with high productivity and yield values (0.22 g eth L −1h −1 and 0.48 g eth/g xyl eqs), whilst auto-hydrolysates, even after a secondary hydrolysis, gave low ethanol concentrations (2–4 g L −1). The impacts on kraft cooking and pulp quality were also studied in order to fully understand the feasibility of this biorefinery concept (combining ethanol production and hardwood pulping). A decrease of the overall pulp yield (10% for auto- and 15% for acid-hydrolysis) was observed. However, a decrease on bleaching requirements (up to 15%) and on brightness reversion was registered. Moreover, auto-hydrolysis improves pulp viscosity, whilst acid-hydrolysis is more efficient in metals leaching. Overall, from the pulp production point of view, auto-hydrolysis conditions are more attractive than the acid-catalysed ones.

Combined Supercritical and Subcritical Process for Cellulose Hydrolysis to Fermentable Hexoses

A combined supercritical and subcritical process for pretreatment and hydrolysis of lignocellulosic waste has been proposed. Batch experiments and kinetic analysis were performed to investigate the secondary hydrolysis of cellulose in subcritical water as well as the feasibility of combining the whole process. A 60 mg amount of microcrystalline cellulose and 2.5 mL of deionized water were mixed in each reactor and reacted at 380 degrees C and 16 s as the primary hydrolysis, followed by different subcritical conditions for secondary hydrolysis. The products were analyzed by HPLC, which showed that the primary hydrolysis produced 28.1% oligosaccharides and 26.3% hexoses. For the secondary hydrolysis in subcritical reactions, the highest yield of hexoses, obtained at 280 degrees C and 44 s, was 39.5%. At lower temperatures, higher maximum yield of hexoses was obtained, but the reaction times were longer. Kinetic analysis showed smaller reaction rate constants for hexoses decomposition compared to those for oligosaccharides hydrolysis, and the difference between these increased with the decreasing reaction temperature. This explains the observed hexose accumulations in the subcritical reaction and indicates the feasibility and advantage of combined supercritical and subcritical processes for biomass conversion.

Alcalase®‐catalysed synthesis of the precursor tetrapeptide N‐benzoylarginylglycylaspartylserinamide (Bz‐RGDS‐NH2) of the cell‐adhesion peptide arginylglycylaspartylserine (RGDS)

In the present study, a precursor tetrapeptide Bz-RGDS-NH2 (N-benzoylarginylglycylaspartylserinamide) of cell-adhesion peptide RGDS (arginylglycylaspartylserine) was synthesized by a novel route. First of all, the precursor tripeptide GDS-NH2 (glycylaspartylserinamide) was synthesized by a chemical method only using aspartic acid and serine at gram scale in four steps. The linkage of the fourth amino acid Bz-Arg-OEt (N-benzoyl-L-arginine ethyl ester) to GDS-NH2 was completed by an enzymatic method under kinetic control in water-miscible organic media. An industrial alkaline protease, Alcalase, with a wide substrate specificity, was used as the catalyst. The effects of organic solvents, pH value, reaction temperature, water content and molar ratio of substrates on the yield of Bz-RGDS-NH2 synthesis were examined. The optimum reaction conditions were found to be pH 10.0, 35 degrees C, 8 h, in an acetonitrile/(Na2CO3/NaHCO3) buffer system (93:7, v/v) with a maximal yield of 65.2%. We found that secondary hydrolysis of the peptide product did not take place in these water-miscible organic solvents.

More Accurate Determination of Acid-Labile Carbohydrates in Lignocellulose by Modified Quantitative Saccharification

Production of biofuels and biobased products from lignocellulosic materials is an emerging industry. Quantitative saccharification (QS) is a widely used method for determining carbohydrate composition in lignocellulosic materials. The NREL methods (versions 1996 and 2006) involve a primary hydrolysis in 72% w/w sulfuric acid at 30 °C, converting polysaccharides into oligosaccharides, and a secondary hydrolysis in 4% w/w sulfuric acid at 121 °C, converting all oligosaccharides to monomeric sugars. Because some polysaccharides are degraded during the harsh hydrolysis processes, a sugar control set of monomeric sugars was run in parallel, and the correction coefficients of monomeric sugars were used to compensate the polysaccharide degradation. This assumption may be invalid because polysaccharide and monosaccharide sugars have different degradation rates, especially for acid-labile carbohydrates. Here we propose a modified quantitative saccharification involving a primary hydrolysis (72% sulfuric acid, 30 °C, 1 h), followed by a secondary hydrolysis (4% sulfuric acid, 121 °C, 1 h, for glucose, galactose, and mannose) and a parallel secondary hydrolysis (1% sulfuric acid, 121 °C, 1 h, for xylose and arabinose). The weaker secondary hydrolysis can decrease degradation of acid-labile xylose by ∼4.4-fold. The data of acid-labile hemicellulose carbohydrates for all of the five tested lignocellulosic materials from herbs to hardwood to softwood suggest that the current QS protocol results in a 4.2%–9.1% overestimation of xylan contents. Such statistically significant overestimation is attributed to theoretical errors from the methods’ assumption.

Protease-catalyzed Synthesis of Bz-Arg-Gly-Asp-OMe in Full Aqueous Medium

Synthesis of N-benzoyl-argininylglycylasparagine methyl ester (Bz-Arg-Gly-Asp-OMe), a precursor tripeptide of Arg-Gly-Asp) was catalyzed by papain under kinetic control, at alkaline pH, in a full aqueous medium. The substrates were N-benzoyl-argininylglycine ethyl ester and asparagine dimethyl ester. An aqueous solution of 0.1 mol/L KCl/NaOH containing 8 mmol/L EDTA and 2 mmol/L DTT was selected as the reaction medium. The synthesized hydrophilic tripeptide was soluble in the reaction medium during the reaction process, however, the secondary hydrolysis of the tripeptide product was not considerable. The effects of different factors, including water content, temperature, reaction time, and molar ratio of the substrates, on the yield of Bz-Arg-Gly-Asp-OMe were examined. The optimal reaction conditions were 0.05 mol/L Bz-Arg-Gly-OEt and 0.15 mol/L Asp(-OMe)2 . HCl in 0.1 mol/L KCl/NaOH solution (pH 8.5), at 40°C, and a reaction time of 60 min, with a maximum conversion yield of 62.4%.

Mitigation of cellulose recalcitrance to enzymatic hydrolysis by ionic liquid pretreatment

Efficient hydrolysis of cellulose-to-glucose is critically important in producing fuels and chemicals from renewable feedstocks. Cellulose hydrolysis in aqueous media suffers from slow reaction rates because cellulose is a water-insoluble crystalline biopolymer. The high-crystallinity of cellulose fibrils renders the internal surface of cellulose inaccessible to the hydrolyzing enzymes (cellulases) as well as water. Pretreatment methods, which increase the surface area accessible to water and cellulases are vital to improving the hydrolysis kinetics and conversion of cellulose to glucose. In a novel technique, the microcrystalline cellulose was first subjected to an ionic liquid (IL) treatment and then recovered as essentially amorphous or as a mixture of amorphous and partially crystalline cellulose by rapidly quenching the solution with an antisolvent. Because of their extremely low-volatility, ILs are expected to have minimal environmental impact. Two different ILs, 1-n-butyl-3-methylimidazolium chloride (BMIMC1) and 1-allyl-3-methylimidazolium chloride (AMIMC1) were investigated. Hydrolysis kinetics of the IL-treated cellulose is significantly enhanced. With appropriate selection of IL treatment conditions and enzymes, the initial hydrolysis rates for IL-treated cellulose were up to 90 times greater than those of untreated cellulose. We infer that this drastic improvement in the "overall hydrolysis rates" with IL-treated cellulose is mainly because of a significant enhancement in the kinetics of the "primary hydrolysis step" (conversion of solid cellulose to soluble oligomers), which is the rate-limiting step for untreated cellulose. Thus, with IL-treated cellulose, primary hydrolysis rates increase and become comparable with the rates of inherently faster "secondary hydrolysis" (conversion of soluble oligomers to glucose).

The inhibitory effects of freshwater clam ( Corbicula fluminea, Muller) muscle protein hydrolysates on angiotensin I converting enzyme

The muscle of golden freshwater clam was extracted using hot water. The residual meat was freeze-dried then hydrolyzed at 50 °C first by Protamex (PX) as primary hydrolysis followed by a secondary hydrolysis (Flavourzyme, F). The inhibitory effects of hot water extracts and hydrolysates against angiotensin I converting enzyme (ACE) were investigated. The soluble protein content and peptide content of the hot water extracts was 85 and 84 mg/g, respectively. The IC 50 on ACE was 1.95 mg/ml. The highest values of soluble protein (571 mg/g) and peptide content (272 mg/g) of hydrolysate prepared by PX hydrolysis for 5 h (PX5) or PX hydrolysis for 5 h followed by F hydrolysis for 0.5 h (PX5F0.5). PX5 showed the lowest IC 50 on ACE (0.043 mg/ml), and a mixed-type inhibition kinetics while Captopril showed competitive inhibition. Their K i values were 0.032 mg/ml and 0.0067 μg/ml, respectively. The PX5 hydrolysate was fractionated by size exclusion chromatography on a Sephadex G-25. A fraction of the PX5 had molecular weight ranged 420–380 Da showed the highest inhibitory efficiency ratio (IER) being 1314.7%/mg/ml. The amino acid sequences of the two peaks of this fraction PX-D1 and PX-D2 were Val-Lys-Pro and Val-Lys-Lys, of which IC 50 value were 3.7 and 1045 μM, respectively. This freshwater clam hydrolysate (PX5) (peptide concentration 5 mg/ml) was used as drinks administered to spontaneously hypertensive rats (SHR) for 8 weeks. The systolic blood pressure and diastolic blood pressure of the SHR were significantly reduced by 22.0 and 13.2 mmHg, respectively, indicating a hypotensive effect by oral administration.

Synthesis of tetrapeptide Bz-RGDS-NH2 by a combination of chemical and enzymatic methods

The tetrapeptide Bz-Arg-Gly-Asp-Ser-NH(2) (Bz-RGDS-NH(2)) was successfully synthesized by a combination of chemical and enzymatic methods in this study. Firstly, the precursor tripeptide Gly-Asp-Ser-NH(2) (GDS-NH(2)) was synthesized by a novel chemical method in four steps including chloroacetylation of l-aspartic acid, synthesis of chloroacetyl l-aspartic acid anhydride, the synthesis of ClCH(2)COAsp-SerOMe and ammonolysis of ClCH(2)COAsp-SerOMe. Secondly, lipase (PPL) was used to catalyze the formation of Bz-RGDS-NH(2) in aqueous water-miscible organic cosolvent systems using Bz-Arg-OEt as the acyl donor and GDS-NH(2) as the nucleophile. The optimum conditions were Bz-Arg-OEt 50 mM; GDS-NH(2) 400 mM; 10 degrees C, 0.1M phosphate buffer, pH 7.5; 60% DMF or 58% DMSO, PPL: 10 mg ml(-1) with the maximum yields of the tetrapeptide of 73.6% for DMF and 70.4% for DMSO, respectively. The secondary hydrolysis of the tetrapeptide product did not take place due to the absence of amidase activity of lipase.

Chemo-enzymatic synthesis of precursor tetrapeptide Bz–RGDS–NH 2 of cellular adhesion motif in low-water organic media

The tetrapeptide Bz–Arg–Gly–Asp–Ser–NH 2 (Bz–RGDS–NH 2) as a precursor of cellular adhesion motif of RGDS was synthesized by a combination of chemical and enzymatic methods in this study for the first time. First of all, the precursor tripeptide Gly–Asp–Ser–NH 2 (GDS–NH 2) was synthesized by a novel chemical method in four steps including chloroacetylation of l-aspartic acid, synthesis of chloroacetyl l-aspartic acid anhydride, synthesis of ClCH 2COAsp–SerOMe and ammonolysis of ClCH 2COAsp–SerOMe. Secondly, trypsin-catalyzed synthesis of Bz–RGDS–NH 2 using Bz–Arg–OEt (Bz–R–OEt) as the acyl donor and GDS–NH 2 as the nucleophile under kinetic control in low-water organic media was carried out. The reaction conditions optimized are pH 8.0, 30 °C, 14 h, in ethanol/Tris–HCl buffer system (97:3, v/v). The yield of Bz–RGDS–NH 2 is 68.3 ± 1.74% for pre-treated trypsin prepared by dissolving trypsin powder in 0.1 M Tris–HCl buffer (pH 8.0) and lyophilizing for 24 h in a freeze-drier with −55 °C, 4 × 10 −4–1 × 10 −2 mbar. Compared to trypsin powder, pre-treated trypsin (pH memory) displays higher catalytic activity and stability in low-water organic media, the yield at pH 8.0 increased by 7%. The secondary hydrolysis of the tetrapeptide product did not take place in this low-water organic medium.