Automated phenylthiocarbamyl amino acid analysis of carboxypeptidase/aminopeptidase digests and acid hydrolysates

A fully automated exopeptidase digestion procedure for the partial determination of N- and C-terminal peptide/protein sequence is described. The digestion of various substrates with aminopeptidase M, carboxypeptidase A, P or Y was accomplished with the Varian 9090 autosampler's robotic automix routines. The released free amino acids, in addition to free amino acids from acid hydrolysates, were derivatized with phenylisothiocyanate in an automated fashion and subsequently chromatographed on a C18 column for separation and quantitation. The advantages of automating this precolumn phenylisothiocyanate derivatization are the virtual elimination of sample manipulation errors and very reproducible data due to the precise control of the reaction conditions both of which, facilitate the interpretation of the exopeptidase reaction kinetic data.     

Exopeptidase degradation for the analysis of phosphorylation site in a mono-phosphorylated peptide with matrix-assisted laser desorption/ionization mass spectrometry.

The utility of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) coupled with a peptide ladder sequencing method employing exopeptidase degradation for the analysis of phosphorylation site in a mono-phosphorylated peptide is investigated. MALDI-TOFMS analysis of time-dependent exopeptidase digestion using carboxypeptidase W and aminopeptidase M of the mono-phosphorylated 33-48 fragment isolated from a beta-casein tryptic digestion mixture allowed for the sequencing analysis from both the C-terminus and N-terminus. Negative ion detection MALDI-TOFMS made it possible to clearly measure the peptide ladder of mono-phosphorylated peptide by the strong negative charge localized at the phosphoric acid group. Since exopeptidase activity was suppressed by the existence of a phosphorylated amino acid residue, the termination exopeptidase degradation therefore suggested the existence of a phosphorylated amino acid residue at that site. This peptide ladder sequencing method using exopeptidases was effective for the identification of the site of a phosphorylated amino acid residue by a simple MALDI-TOFMS analysis in the negative ion detection mode.     

Rapid Protein Hydrolysis by Microwave Irradiation Using Heat-Resistant Teflon-Pyrex Tubes

A rapid peptide-bond hydrolysis by means of microwave irradiation is introduced for the facile preparation of protein hydrolysates used for amino acid analysis. The optimal hydrolysis condition has been determined using several enzymes with known amino acid compositions. The effects of hydrolysis time on the recovery of various labile and hydrophobic amino acids are also exemplified in the microwave heating of standard amino acids. The method has been applied to the complete amino acid analysis with a single nonvolatile solvent of methanesulfonic acid with good recovery of tryptophan and half-cystine. It provides a radical expedition of protein and peptide hydrolysis via commercial microwave ovens and specially-designed Teflon-Pyrex tubes, circumventing the tedious procedures using vacuum-sealed pyrex lubes heating at 110°C for more than 24 h. This novel type of microwave chemistry associated with rapid peptide-bond cleavage is of great potential in the automation of the complete process of amino acid analysis starting from the preparation of protein hydrolysates.     

Enzymatic Preparation of Bioactive Peptides Exhibiting ACE Inhibitory Activity from Soybean and Velvet Bean: A Systematic Review

The Angiotensin-I-converting enzyme (ACE) is a peptidase with a significant role in the regulation of blood pressure. Within this work, a systematic review on the enzymatic preparation of Angiotensin-I-Converting Enzyme inhibitory (ACEi) peptides is presented. The systematic review is conducted by following PRISMA guidelines. Soybeans and velvet beans are known to have high protein contents that make them suitable as sources of parent proteins for the production of ACEi peptides. Endopeptidase is commonly used in the preparation of soybean-based ACEi peptides, whereas for velvet bean, a combination of both endo- and exopeptidase is frequently used. Soybean glycinin is the preferred substrate for the preparation of ACEi peptides. It contains proline as one of its major amino acids, which exhibits a potent significance in inhibiting ACE. The best enzymatic treatments for producing ACEi peptides from soybean are as follows: proteolytic activity by Protease P (Amano-P from Aspergillus sp.), a temperature of 37 °C, a reaction time of 18 h, pH 8.2, and an E/S ratio of 2%. On the other hand, the best enzymatic conditions for producing peptide hydrolysates with high ACEi activity are through sequential hydrolytic activity by the combination of pepsin-pancreatic, an E/S ratio for each enzyme is 10%, the temperature and reaction time for each proteolysis are 37 °C and 0.74 h, respectively, pH for pepsin is 2.0, whereas for pancreatin it is 7.0. As an underutilized pulse, the studies on the enzymatic hydrolysis of velvet bean proteins in producing ACEi peptides are limited. Conclusively, the activity of soybean-based ACEi peptides is found to depend on their molecular sizes, the amino acid residues, and positions. Hydrophobic amino acids with nonpolar side chains, positively charged, branched, and cyclic or aromatic residues are generally preferred for ACEi peptides.     

Nonionic micellar liquid chromatography coupled to immobilized enzyme reactors

Immobilized enzyme reactors are used as post-column reactors to modify the detectability of analytes. An immobilized amino acid oxidase reactor was prepared and coupled to an immobilized peroxidase reactor to detect low level of amino acids by fluorescence of the homovanilic dimer produced. A cholesterol oxidase reactor was prepared to detect cholesterol and metabolites by 241 nm UV absorbance of the enone produced. The preparation of the porous glass beads with the immobilized enzymes is described. Micellar liquid chromatography is used with non-ionic micellar phases to separate the amino acids or cholesterol derivatives. It is demonstrated that the non ionic Brij 35 micellar phases are very gentle for the enzyme activity allowing the reactor activity to remain at a higher level and for a much longer time than with hydro-organic classical chromatographic mobile phases or aqueous buffers. The coupling of nonionic micellar phases with enzymatic detection gave limits of detection of 32 pmol (4.8 ng injected) of methionine and 50 pmol (19 ng injected) of 20alpha-hydroxy cholesterol. The immobilized enzyme reactors could be used continuously for a week without losing their activity. It is shown that the low efficiency obtained with micellar liquid chromatography is compensated by the possibility offered by the technique to easily adjust selectivity.     

One-step separation-free amperometric biosensor for the detection of protein

A novel enzyme biosensor for the detection of protein is presented. The biosensor was made from a screen-printed three-electrode configuration. Amino acid oxidase was immobilized with glutaraldehyde and polyethylenimine on a working electrode made of rodinised carbon. A protease was immobilized on an immunodyne membrane and was placed on the electrode. A protein sample was deposited on the membrane, and was subsequently hydrolyzed to amino acids in the presence of the protease. This in turn produced hydrogen peroxide by the immobilized amino acid oxidase. The oxidation of hydrogen peroxide was then detected at +400 mV vs. an Ag/AgCl reference electrode. The method was very effective at detecting a very low level of protein. The sensor does not require any washing step. The sensor works with only 40 μl of sample per detection, and may be used on-site as a disposable sensor using a hand-held meter. The electrodes are also stable for more than 6 weeks.     

Specificity of Carboxypeptidases from Actinomucor elegans and Their Debittering Effect on Soybean Protein Hydrolysates

The specificities of carboxypeptidases from Actinomucor elegans were investigated by determining enzymatic activities at pH 7.0 and pH 4.0 with 16 Z-dipeptides and three Z-tripeptides as substrates. The debittering effect was evaluated and the free amino acid compositions of the soybean protein hydrolysates were analyzed before and after treatment with A. elegans extract at pH 7.0 and pH 4.0, with carboxypeptidases from Aspergillus oryzae as control. The results of the enzyme activity determinations indicated that carboxypeptidases from A. elegans prefer hydrophobic substrates, such as Z-Phe-Leu, Z-Phe-Tyr-Leu, and Z-Phe-Tyr. The sensory evaluation and free amino acid composition analysis showed that these carboxypeptidases are efficient tools for decreasing the bitterness of peptides because they liberated the fewest free amino acids, which consisted of 73% hydrophobic amino acids, under acidic conditions. Carboxypeptidases from A. elegans display promising prospects for future applications in the protein hydrolysate industry.     

Comparison of digestibility and quality of intact proteins with their respective hydrolysates

Quality of proteins depends on their composition in essential amino acids and on the availability of amino acids. Great interest has been shown in the role played by hydrolysates of proteins in clinical diets for pathologies with reduced absorptive capacity and food allergies caused by intact protein epitopes. Milk proteins are the most important protein source used in the development of protein hydrolysates designed for nutritional support of patients. Several studies have shown that casein and whey hydrolysates have a composition in amino acids equivalent to that in native milk proteins and that digestibility is similar or better. Among plant proteins, soy is the major source of hydrolysates. Soy hydrolysates are also used in infant formulas. Plant hydrolysates have good functional properties and a nutritional quality similar to that of starting material. Some technical improvements in production of hydrolysates, particularly for plants, are nevertheless necessary to improve product palatability.     

A Two-Step,One-Pot Enzymatic Method for Preparation of Duck Egg White Protein Hydrolysates with High Antioxidant Activity

Biocatalytic hydrolysis reactions were designed for preparation of bioactive hydrolysate of duck egg white protein (DEWP) employing two enzymes in one pot. Firstly, the fresh DEWP was thermal treated at 95 °C, for 40 min at pH 10, to effectively deactivate enzyme inhibitors thus facilitating the following two-step enzymatic hydrolysis. Compared with single-enzyme processes, the two-step enzymatic procedures showed much higher reaction efficiency. The first enzymatic step (in the presence of Alcalase or hydrolase SEEP) allowed to hydrolyze DEWP with degree of hydrolysis (DH) of 8.8–10 % and soluble peptide yield (SEP) of 60.5–70.2 % in a short period (4 h). The second enzymatic step (in the presence of Trypsin or Alcalase) gave a further degradation of DEWP with DH and SEP being more than 26.2 % and 90.4 %, respectively. The final hydrolysates exhibited high antioxidant activity in an evident DH dependent manner. The hydrolysates achieved by sequential addition of the proteinase SEEP and Alcalase at DH value 21 % gave the highest antioxidant activity, which was mainly ascribed to the changes in the amino acid compositions that the contents of some key amino acids and total hydrophobic amino acids were significantly improved by the enzymatic hydrolysis.     

Capillary gas chromatographic analysis of amino acids in blood and protein hydrolysates as tert.-butyldimethylsilyl derivatives

A method is given for a one-step derivatization and gas chromatography of amino acids in blood and protein hydrolysates. Blood samples are partially purified by solvent extraction. Protein hydrolysates are neutralized with a triethylamine solution. Then tert.-butyldimethylsilyl derivatives of the amino acids are prepared in a one-step procedure and separated on a 30-m fused-silica SE-30 capillary column. Except for tryptophan and cystine, amino acids are eluted within 30 min. Amino acids are derivatized more rapidly than their corresponding trimethylsilyl derivatives and do not degrade on the long fused-silica columns.     

STUDY ON IMMOBILIZED PORCINE PANCREATIC LIPASE CATALYZING TRANSESTERIFICATION BETWEEN METHYL－BUTYRATE AND 1－BUTANOL IN NONAQUEOUS SYSTEMS

ＳＴＵＤＹＯＮＩＭＭＯＢＩＬＩＺＥＤＰＯＲＣＩＮＥＰＡＮＣＲＥＡＴＩＣＬＩＰＡＳＥＣＡＴＡＬＹＺＩＮＧＴＲＡＮＳＥＳＴＥＲＩＦＩＣＡＴＩＯＮＢＥＴＷＥＥＮＭＥＴＨＹＬ－ＢＵＴＹＲＡＴＥＡＮＤ１－ＢＵＴＡＮＯＬＩＮＮＯＮＡＱＵＥＯＵＳＳＹＳＴＥＭＳＸｉ...     

Rapid classification of enzymes in cleaning products by hydrolysis, mass spectrometry and linear discriminant analysis

A method for the rapid classification of proteases, lipases, amylases and cellulases used as enhancers in cleaning products, based on precipitation with acetone, hydrolysis with HCl, dilution of the hydrolysates with ethanol, and direct infusion into the electrospray ion source of an ion-trap mass spectrometer, has been developed. The abundances of the ([M+H]+ ions of the amino acids, from the hydrolysates of both the enzyme industrial concentrates and the detergent bases spiked with them, were used to construct linear discriminant analysis models, capable of distinguishing between the enzyme classes. For this purpose, the variables were normalized as follows: (A) the ion abundance of each amino acid was divided by the sum of the ion abundances of all the amino acids in the corresponding mass spectrum; (B) the ratios of pairs of ion abundances were obtained by dividing the ion abundance of each amino acid by each one of the ion abundances of the other 17 amino acids in the corresponding mass spectrum. Using normalization procedure B, excellent class-resolution between proteases, lipases, amylases and cellulases was achieved. In all cases, enzymes in industrial concentrates and manufactured cleaning products were correctly classified with >98% assignment probability.     

STUDY ON IMMOBILIZED PORCINE PANCREATIC LIPASE CATALYZING TRANSESTERIFICATION BETWEEN METHYL—BUTYRATE AND 1—BUTANOL IN NONAQUEOUS SYSTEMS

Transesterification between methyl-butyrate and 1-butanol in nonaqueous systems was catalyzed by porcine pancreatic lipase which was immobilized on cross-linked polystyrene.Organic solvents,substrate concentration,contents of water and other parameters which affect the immobilized enzyme activity were studied.Lipase immobilized on hydrophobic crosslinked polystyrene can reduce its diffusion limit in the reaction.It was found that the activity of immobilized lipase in organic systems was two times as high as that of free lipase.     

Affinity purification and enzymatic cleavage of inter-alpha inhibitor proteins using antibody and elastase immobilized on CIM monolithic disks

Epoxy-activated monolithic CIM disks seem to be excellent supports for immobilization of protein ligands. The potential use of enzymes, immobilized on monolithic disks for rapid preparative cleavage proteins in solution was investigated. Digestion of complex plasma proteins was demonstrated by using inter-alpha inhibitors with elastase, immobilized on epoxy-activated CIM disks. Recently, a monoclonal antibody against human inter-alpha inhibitor proteins (MAb 69.31) was developed. MAb 69.31 blocks the inhibitory activity of inter-alpha inhibitor proteins to serine proteases. These results suggest that the epitope defined by this antibody is located within or proximal to the active site of the inhibitor molecule. This antibody, immobilized on monolithic disk, was used for very rapid isolation of inter-alpha proteins. The isolated complex protein was used for enzymatic digestion and isolation of cleavage products, especially from inter-alpha inhibitor light chain to elucidate precisely the target sequence for MAb 69.31 by N-terminal amino acid sequencing. Bovine pancreatic elastase immobilized on monolithic disk cleaves inter-alpha inhibitor protein complex into small fragments which are still reactive with MAb 69.31. One of these proteolytic fragments was isolated and partially sequenced. It could be shown that this sequence is located at the beginning of two proteinase inhibitor domains of the inter-alpha inhibitor light chain (bikunin). Elastase immobilized on monolithic disk offers a simple and rapid method for preparative isolation of protease cleavage fragments. The immobilized enzyme is stable and still active after repeated runs. A partial or complete digestion can be achieved by varying the flow rate.     

Adsorptive detagging of poly-histidine tagged protein using hexa-histidine tagged exopeptidase

The ubiquitous use of poly-histidine fusion tags has made the purification of the recombinant target proteins much simpler, although the presence of residual fusion tags can generate immunogenic products or products with changed biological activities. This work presents a generic method of removing poly-histidine fusion tags from recombinant proteins through the use of a hexa-histidine tagged exopeptidase (DAPase) when both tagged species are adsorbed to the immobilized metal affinity chromatography (IMAC) adsorbent. Adsorptive detagging was performed in the presence of 50mM imidazole in order to allow the cleavage reaction by the hexa-histidine tagged DAPase to occur. The progress of batch and adsorptive detagging by DAPase of maltose binding protein (MBP) tagged with two variants of hexa-histidine fusion tag was successfully monitored using cationic exchange chromatography. A single-step, column-based detagging strategy was then optimized to maximize the recovery of native MBP. The kinetics of batch and on-column digestion for both HT6 and HT15 fusion tags were investigated. The process involved the sequential removal of dipeptides during the digestion of full-length fusion protein down to its fully detagged native form. During the course of tag digestion, 4 and 7 different intermediates were detected for HT6 and HT15 tagged MBP respectively. The characteristics of on-column cleavage of poly-histidine fusion tags by DAPase as a function of incubation temperature and amount of protease activity used were examined. It was found that the influence of fusion tag design on the batch and column-based detagging yield and efficiency was substantial. In addition, the structural difference of fusion tags affects the binding strength of the fusion protein, which can influence the resulting product purity. Despite being a longer tag, HT15 fusion tag was the preferred sequence for shortening the time needed for on-column detagging. These results can be applied to the wider use of the proposed platform protocol for the on-column cleavage of poly-histidine tagged proteins using exopeptidases.     

Magnetic Bead Cellulose as a Suitable Support for Immobilization of α-Chymotrypsin

Magnetic bead cellulose was prepared by a suspension method from the mixture of viscose and magnetite using thermal sol?Cgel transition and regeneration of cellulose. The prepared magnetic particles after their activation with divinyl sulfone were shown to be suitable magnetic carrier for immobilization of ??-chymotrypsin and for its application in proteomic studies. The specific activity of the immobilized proteinase was high; its activity did not change in the course of storage. The following properties of the immobilized proteinase were compared with those of the soluble enzyme: pH and temperature dependence of the activity, self-cleavage activity, and possibility of repeated use. ??-Chymotrypsin immobilized to magnetic bead cellulose was used for the proteolytic digestion of porcine pepsin A and human gastric juice and a possibility of direct use of enzyme reaction products for matrix-assisted laser desorption/ionization time of flight mass spectrometry analysis was shown.     

氨基酸分子改性的SiO2杂化材料用于胰蛋白酶固定化

为改善二氧化硅载体材料本身的生物相容性和疏水性,维持包埋生物分子的活性,本文对水解前驱体3-氨基丙基三甲氧基硅烷进行氨基酸分子改性。具体过程包括N-Fmoc-L-缬氨酸和氯化亚砜反应生成N-Fmoc-L-缬氨酰氯,再和3-氨基丙基三甲氧基硅烷反应生成N-（3-三甲氧基硅基）丙基-N′-Fmoc-L-缬氨酰胺后。然后去除Fmoc,得到N-（3-三甲氧基硅基）丙基-L-缬氨酰胺作为氨基酸修饰的硅源前驱体。通过IR、MS、1H-NMR等分析测试手段对合成得到的各个化合物的结构进行了表征。利用正硅酸甲酯（TMOS）和N-（3-三甲氧基硅基）丙基-L-缬氨酰胺为复合硅源,经过溶胶-凝胶过程来包埋了胰蛋白酶,研究得到最适的固定化条件为,N-（3-三甲氧基硅基）丙基-L-缬氨酰胺的含量为15mol%。在该条件下,固定化胰蛋白酶活力的绝对值是199U,游离酶的酶活力的绝对值是103U, 四甲氧基硅烷直接包埋的固定化酶活力的活性是38 U。在该条件下,杂化硅源得到的固定化酶的活性是以四甲氧基硅烷水解前驱体的固定化酶活性的5倍,杂化硅源固定化胰蛋白酶的最相比游离酶,酶的最高活力提高的几乎2倍。这些结果表明氨基酸分子对水解前驱体修饰以后,水解产生的固定化载体具有良好的生物相容性。通过改性载体制备的固定化酶,对甲醇变性剂的稳定性,对酸碱的抵抗性及热稳定性也有明显地提高。     

Fractionation of casein hydrolysates using polysulfone ultrafiltration hollow fiber membranes

The objective of this study was to characterize the fractionation profile of casein hydrolysates obtained with polysulfone hollow fiber ultrafiltration membranes. The two-step ultrafiltration process developed by Turgeon and Gauthier [J. Food Sci., 55 (1990) 106] was used: a caseinate solution was submitted to proteolysis with chymotrypsin or trypsin, and the reaction mixture (RM) was subsequently ultrafiltered using a 30 kDa (MWCO) hollow-fiber polysulfone membrane. The total hydrolysate permeating from this first step was further fractionated using a 1 kDa (MWCO) membrane, producing the mixture of polypeptides (retentate) and the amino acid fraction (permeate). The effect of enzyme specificity and of membrane retentivitiy on the total composition (total nitrogen, fat, lactose, minerals) and amino acid profile of the fractions was studied. The overall composition of the fractions was not significantly affected by the nature of the enzyme but the degree of hydrolysis and the molecular weight distribution profile analyses showed a marked effect of the enzyme specificity, with trypsin giving a larger proportion of small peptides (< 200 Da) in the mixture of polypeptides. Amino acid profile analyses provided useful information on the phenomena governing the fractionation of amino acids with a polysulfone membrane: (1) the target amino acids of the enzyme are concentrated in the permeate as a result of their presence in all peptides produced by hydrolysis, (2) polar amino acids are retained by the membrane, (3) non-polar amino acids are not selectively rejected by the membrane. Our results suggest that the charge/hydrophobicity balance of the peptides produced is the predominant factor determining the fractionation of casein hydrolysates.     

Determining protease substrate selectivity and inhibition by label-free supramolecular tandem enzyme assays

An analytical method has been developed for the continuous monitoring of protease activity on unlabeled peptides in real time by fluorescence spectroscopy. The assay is enabled by a reporter pair comprising the macrocycle cucurbit[7]uril (CB7) and the fluorescent dye acridine orange (AO). CB7 functions by selectively recognizing N-terminal phenylalanine residues as they are produced during the enzymatic cleavage of enkephalin-type peptides by the metalloendopeptidase thermolysin. The substrate peptides (e.g., Thr-Gly-Ala-Phe-Met-NH(2)) bind to CB7 with moderately high affinity (K ≈ 10(4) M(-1)), while their cleavage products (e.g., Phe-Met-NH(2)) bind very tightly (K > 10(6) M(-1)). AO signals the reaction upon its selective displacement from the macrocycle by the high affinity product of proteolysis. The resulting supramolecular tandem enzyme assay effectively measures the kinetics of thermolysin, including the accurate determination of sequence specificity (Ser and Gly instead of Ala), stereospecificity (d-Ala instead of l-Ala), endo- versus exopeptidase activity (indicated by differences in absolute fluorescence response), and sensitivity to terminal charges (-CONH(2) vs -COOH). The capability of the tandem assay to measure protease inhibition constants was demonstrated on phosphoramidon as a known inhibitor to afford an inhibition constant of (17.8 ± 0.4) nM. This robust and label-free approach to the study of protease activity and inhibition should be transferable to other endo- and exopeptidases that afford products with N-terminal aromatic amino acids.     

Determination of amino acids by precolumn derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate and high performance liquid chromatography with ultraviolet detection

A precolumn derivatization method for the determination of amino acids using 6-aminoquinolyl-N-hyroxy-succinimidyl carbamate (AQC) followed by high-performance liquid chromatography is described. Ultraviolet detection was used for the assay of AQC derivatives of amino acids with the detection wavelength set at 248 nm. The reagent peak interference was minimized by optimizing the pH of the eluent and the gradient elution profile to improve the resolution between the reagent peak and amino acid derivatives. All nineteen amino acids were separated in 35 min with resolutions 1.6. The correlation coefficients of the calibration graphs for seventeen amino acids were fairly good (r 0.9999) at concentrations of 25–500 μM. The detection limits for all common amino acids including cystine and trytophan were at the range of 0.07–0.3 pmol. Good reproducibility and accuracy of the method were demonstrated by the determination of amino acids in three typical kinds of samples (protein, peptide and feed.) The average relative standard deviations for bovine serum albumin (BSA) and neuromedin were 0.86% and 1.36, respectively, and the average relative errors were 3.2% and 2.3%, respectively. The results of the analysis of feed hydrolysates agreed with those obtained by an ion-exchange method and the average recovery of the method for feed hydrolysates was 98%.