Trypsin as a catalyst for peptide synthesis

Trypsin-catalyzed syntheses of peptides were performed using various N-acylated amino acid or peptide esters as donors and amino acid derivatives, peptides, or their derivatives as acceptors. The synthesis was almost quantitative under optimal conditions. Considerably more enzyme and a more alkaline pH were necessary for synthesis than hydrolysis. Another very important condition was the concentration of the starting materials; higher concentrations resulted in much better product yields. The nucleophile specificity for synthesis was also important; hydrophobic or bulky amino acid residues were most efficient at the P1' position, and L-proline as well as D-amino acid residues were the worst choices. The synthesis was also dependent on the solubility of the products synthesized; the yield was higher with products of lower solubility. As donor esters, good substrates were all useful. Accordingly, fragment condensation was possible using N-acylated peptide esters and various peptides. The present study suggests that trypsin may become a useful tool for peptide synthesis.     

A PDZ domain-based assay for measuring HIV protease activity: assay design considerations

We have recently described a biochemical detection method for peptide products of enzymatic reactions based on the formation of PDZ domain*peptide ligand complexes. The product sensor is based on using masked or cryptic PDZ domain peptide ligands as enzyme substrates. Upon enzymatic processing, a PDZ-binding motif is exposed, and the product sequence bound specifically by a Eu(3+)chelate-labeled GST-PDZ ([Eu(3+)]GST-PDZ). The practical applicability of this PDZ-based detection method is determined by the affinity of the PDZ domain*peptide ligand interaction, and the efficiency of the enzyme to process the masked peptide ligand. To expand the use of this PDZ-based detection strategy to a broader range of enzymatic assays, we have taken advantage of the plasticity in ligand recognition by the variety of PDZ domains found in nature. In the original work, the PDZ3 of PSD-95 was used, which preferentially recognizes the consensus sequence Ser-X-Val-COOH. Here, we show that NHERF PDZ1, which binds to the consensus sequence Thr/Ser-X-Leu-COOH, can be used to extend the flexibility in the recognition of the carboxy-terminal amino acid of the ligand, and monitor the enzymatic activity of HIV protease. The choices of detection format, for example, TRET or ALPHA, were also investigated and influenced assay design.     

Protease-catalyzed fragment condensation via substrate mimetic strategy: a useful combination of solid-phase peptide synthesis with enzymatic methods.

The concept of substrate mimetic strategy represents a new powerful method in the field of enzymatic peptide synthesis. This strategy takes advantage of the shift in the site-specific amino acid moiety from the acyl residue to the ester-leaving group of the carboxyl component enabling acylation of the enzyme by nonspecific acyl residues. As a result, peptide bond formation occurs independently of the primary specificity of proteases. Moreover, because of the coupling of nonspecific acyl residues, the newly formed peptide bond is not subject to secondary hydrolysis achieving irreversible peptide synthesis. Here, we report the combination of solid-phase peptide synthesis with substrate mimetic-mediated enzymatic peptide fragment condensations. First, the utility of the oxime resin strategy for the synthesis of peptide fragments in the form of substrate mimetics esterified as 4-guanidinophenyl-, phenyl- and mercaptopropionic acid esters was investigated. The study was completed by using the resulting N(alpha)-protected peptide esters as acyl donors in trypsin-, alpha-chymotrypsin- and V8 protease-catalyzed fragment condensations.     

Enzymatic peptide synthesis by the recombinant proline-specific endopeptidase from Flavobacterium meningosepticum and its mutationally altered Cys-556 variant

Proline-specific endopeptidase (PSE) (EC 3.4.21.26) was investigated for its potential as a catalyst in peptide synthesis. Using an activated peptide ester or a peptide amide as the acyl component, the enzyme catalyzed kinetically controlled aminolysis and transpeptidation respectively, with various amino acid amides as acyl acceptors. To a certain extent the nucleophile preference reflected the amino acid preference in the S₁-position of the enzyme in peptide hydrolysis: the highest fractions of aminolysis were obtained using amino acid amides with hydrophobic side-chains (e.g. Leu-NH₂, Phe-NH₂). PSE also catalyzed the thermodynamically controlled condensation of short peptides with a free carboxyterminus and various amino acid amides. This enabled us to examine the acceptance of different acyl components in the substrate-binding site of the enzyme with regard to their amino acid composition: In the S₁ position proline was clearly favored, but alanine was also accepted, whereas the S₂ subsite accepted various amino acids rather unspecifically. Since PSE was shown to be extremely sensitive against water-miscible organic solvents, an alternative approach was used to increase yields in enzymatic peptide synthesis: a derivative of PSE in which the catalytic Ser-556 is converted to a Cys was constructed by protein engineering. This mutant (PSE_cys) exhibited a dramatically increased peptide ligase activity in aqueous solution.     

Pepsin-catalyzed peptide synthesis

The use of pepsin as a catalyst for the synthesis of peptide bonds was investigated. It is shown that the enzyme enables the preparation of several protected dipeptides and tripeptides containing two adjacent aromatic residues of the type P-Al-Phe-Y, P-PHe-Ar-Y, or P-AR-Phe-Y where P and Y are amino and carboxyl protecting groups, AL is an aliphatic amino acid residue, and Ar is an aromatic, amino acid residue. They yields are in the rang 25–97%. The high yields, combined with the enzyme's stereospecificity, permit the isolation of optically pure enantiomers from racemic mixtures. For example, when Z-DL -Ph-OH is allowed to react with an excess of H-L -Phe-NH₂, the stereoisomer Z-L -Phe-L -Phe-NH₂ is obtained in practically quantitative yield. At the same time, the unreacted, optically pure Z-D -Phe-OH can be recovered (Z = carbobenzyloxy, Phe = phenylalanine). The advantages and disadvantages of the enzymatic coupling procedure as a possible routine method for peptide synthesis are discussed.     

Continuous production of kyotorphin

The continuous alpha-chymotrypsin-catalyzed peptide synthesis of kyotorphin, tyrosyl-arginine, via the N(alpha) formyltyrosyl-arginine propyl ester is described. For continuous process development, two reaction systems were studied: immobilized alpha-chymotrypsin covalently bound to Eupergit C packed in a column, and soluble alpha-chymotrypsin utilizing an enzyme membrane reactor. Selectivities and kinetic parameters are discussed. The use of soluble enzyme in an enzyme membrane reactor proved superior to the covalently immobilized enzyme. A significant loss of enzyme activity and a certain decrease of selectivity was observed during immobilization. It was shown that the addition of organic solvent, in this case n-propanol, causes a severe diminuation of the enzyme activity.     

The applicability of subtilisin Carlsberg in peptide synthesis.

The synthesis of peptide bonds catalysed by subtilisin Carlsberg was studied in different hydrophilic organic solvents with variable H2O concentration. Z-Val-Trp-OMe and Z-Ala-Phe-OMe were used as acyl donors, and a series of amino acid derivatives, di- and tripeptides of the general structure Xaa-Gly, Gly-Xaa, Gly-Gly-Xaa (Xaa represents all natural L-amino acids except cysteine) and other peptides were used as nucleophiles. A comparative study of the enzymatic synthesis in aqueous DMF (50%, v/v) and acetonitrile containing 10% (v/v) of H2O demonstrated that the yields of peptide products were higher in most cases when acetonitrile with low H2O concentration was used. The acylation of weak nucleophiles was improved in organic solvents with very low H2O concentration (2%). The reactions in anhydrous Bu(t)-OH proceeded with substantially lower velocity. Generally, the restricted nucleophile specificity of the enzyme for glycine and hydrophilic amino acid residues in P1' position, as well as numerous side reactions, limit the utilization of subtilisin in peptide synthesis, especially in the case of the segment condensations. Contrary to the published data, we have proved that proline derivatives were not acylated in any media with the help of subtilisin Carlsberg. Effective ester hydrolysis of a protected nonapeptide corresponding to the N-terminal sequence of dicarba-eel-calcitonin catalysed by subtilisin was achieved.     

alpha-Chymotrypsin as the catalyst for peptide synthesis.

alpha-Chymotrypsin (EC 3.4.21.1)-catalysed syntheses of peptides were performed with various N-acylated amino acid or peptide esters as donors, and amino acid derivatives, peptides or their derivatives as acceptors. Under optimal conditions the synthesis was almost quantitative. As acceptor nucleophiles, free amino acids or the ester derivatives were inadequate, but amino acid amides or hydrazides, di- or tri-peptides, or the amides, hydrazides and esters of the peptides were useful. The nucleophile specificity for synthesis was markedly similar to the leaving-group specificity in hydrolysis; hydrophobic or bulky amino acid residues were most effecient at both P1' and P2' positions [notation of Schechter & Berger (1967) Biochem. Biophys. Res. Commun. 27, 157-162], but L-proline as well as D-amino acid residues were the worst choices. The synthesis was further dependent on the solubility of the products synthesized; a higher yield of products was expected with lower solubility. As donor esters, good substrates were all useful. Accordingly, fragment condensation was possible by using N-acylated peptide esters and various peptides. The present study suggested that alpha-chymotrypsin may become a useful tool for peptide synthesis.     

Isolation and expression in Escherichia coli of a cDNA clone encoding porcine pancreatic elastase

We have cloned a DNA that is complementary to the messenger RNA that encodes porcine pancreatic elastase 1 from pancreas using rat pancreatic elastase 1 cDNA as a probe. This complementary DNA contains the entire protein coding region of 798 nucleotides which encodes an elastase of 266 amino acids, and 22 and 136 nucleotides of the 5' and 3'-untranslated sequences. When this deduced amino acid sequence was compared with known amino acid sequences, a carboxy-terminal 240 amino acids long peptide was found to be identical with a mature form of porcine pancreatic elastase 1, except for two amino acids. The porcine enzyme contains the same number of amino acid residues as the rat enzyme, and their amino acid sequences are 85% homologous. Taking the above findings together, we conclude that the cloned cDNA encodes a mature enzyme of 240 amino acids including a leader and activation peptide of 26 amino acids. We expressed the cloned porcine pancreatic elastase 1 cDNA in E. coli as a lac-fused protein. The resulting fused protein showed enzymatic activity and immunoreactivity toward anti-elastase serum.     

Glutamine-181 is crucial in the enzymatic activity and substrate specificity of human endoplasmic-reticulum aminopeptidase-1

ERAP-1 (endoplasmic-reticulum aminopeptidase-1) is a multifunctional enzyme with roles in the regulation of blood pressure, angiogenesis and the presentation of antigens to MHC class I molecules. Whereas the enzyme shows restricted specificity toward synthetic substrates, its substrate specificity toward natural peptides is rather broad. Because of the pathophysiological significance of ERAP-1, it is important to elucidate the molecular basis of its enzymatic action. In the present study we used site-directed mutagenesis to identify residues affecting the substrate specificity of human ERAP-1 and identified Gln(181) as important for enzymatic activity and substrate specificity. Replacement of Gln(181) by aspartic acid resulted in a significant change in substrate specificity, with Q181D ERAP-1 showing a preference for basic amino acids. In addition, Q181D ERAP-1 cleaved natural peptides possessing a basic amino acid at the N-terminal end more efficiently than did the wild-type enzyme, whereas its cleavage of peptides with a non-basic amino acid was significantly reduced. Another mutant enzyme, Q181E, also revealed some preference for peptides with a basic N-terminal amino acid, although it had little hydrolytic activity toward the synthetic peptides tested. Other mutant enzymes, including Q181N and Q181A ERAP-1s, revealed little enzymatic activity toward synthetic or peptide substrates. These results indicate that Gln(181) is critical for the enzymatic activity and substrate specificity of ERAP-1.     

New convenient synthesis of immunostimulating peptides containing meso-diaminopimelic acid. Syntheses of FK-565 and FK-156.

A new improved synthesis of two immunostimulating peptides: FK-156 (D-lactyl-alanyl-gamma-D-glutamyl-(L)-meso-2,6-diaminopimelyl-(L)- glycine) and FK-565 (heptanoyl-gamma-D-glutamyl-(L)-meso-2,6-diaminopimelyl-(L)- D-alanine) is described. A proper differentiation between the two chiral amino acid moieties of diaminopimelic acid was accomplished by selective enzymatic hydrolysis of one methyl ester group of the L-centre of Z2-meso-A2pm(OMe)2 (2). Utilization of a commercially available protease and diester 2 as an enzyme substrate made possible the relatively simple synthesis of a key intermediate 4 and considerably simplified the final deprotection steps. Aminolysis of the N-carboxyanhydride (4) with D-AlaONBzl or GlyONBzl was chosen to obtain the appropriate dipeptides with one free amino group as convenient intermediates for further peptide synthesis. The BOP reagent, used for peptide bond formation, secured good yields and high chemical and chiral purity of the peptides. A modification of alanine deamination procedure leading to a significant increase of D-Lac(OAc) yield is presented.     

A practical approach to obtain high yield lipase-mediated synthesis of octyl caprylate with Novozym 435

Abstract

This study evaluated coupled effects of molar ratio of substrates and enzyme loading in a solvent-free system using a simple mathematical approach to obtain high conversions on octyl caprylate synthesis with Novozym 435. When molar ratios of caprylic acid to n-octanol (1:1 and 1:3) were evaluated with enzyme loadings of 1% to 4% (wt/wt acid), an interdependence between the masses of reagents and the enzymes was observed, that was expressed as a mathematical relation. The study of this relation, named as SER, indicated a specific range of reaction conditions that resulted in conversions above 90%. The most suitable condition corresponded to an acid:alcohol molar ratio of 1:1.3 and an enzyme loading of 1.5%, resulting in 94.5% of conversion at 65?°C in 3?hours of reaction. A different reaction system (bottle reactor) was used to evaluate the influence of reagents mixture and heat distribution. The use of a bottle reactor allowed yield improvement that reached 99.3%. At this condition, Novozym 435 was reused, without washing steps, in three subsequent batches keeping high conversion. A possible balance between the shift of chemical equilibrium by stoichiometric excess of reagents and enzymatic inhibition effects by substrates can be expressed mathematically in a convenient way, helping to predict the behaviour of synthesis in different conditions. The mathematical relation proposed, SER, allowed the achievement of 99% of conversion on enzymatic synthesis of octyl caprylate.     

Protease-catalyzed synthesis of melanocyte-stimulating hormone (MSH) fragments

In the present study on enzymatic peptide bond formation the proteosynthetic potential of several proteases was explored. Trypsin, α-chymotrypsin, papain, carboxypeptidase Y (CPD-Y), and thermolysin served as catalysts for the protease-controlled synthesis of some fragments of melanocyte-stimulating hormones. To obviate possible proteolytic cleavage of preexisting peptide bonds—a drawback often encountered during enzymatic peptide syntheses—several expedients leading to the target peptides were developed. The enzymatic procedure enabled under mild conditions the preparation of the desired peptides whose amino acid composition may give rise to severe complications during conventional syntheses.     

Peptide enzymatic microsynthesis, using carboxypeptidase Y as the catalyst: application to stepwise synthesis of Leuenkephalin

In order to develop the use of carboxypeptidase Y (CPD-Y, EC 3.4.12.1) as a catalyst for radioactive hormonal synthesis, the stepwise synthesis of a pentapeptide, Leu-enkephalin, was studied on a microscale. Each peptide bond was formed by enzymatic catalysis, using microquantities of the precursors (amino acid or peptide esters as acyl-components and amino acid ester or amide as nucleophiles). The high condensation yields obtained suggests that CPD-Y can be a useful tool for preparation of radioactive hormonal peptides.     

Proline-specific endopeptidases from microbial sources: isolation of an enzyme from a Xanthomonas sp.

An extensive screening among microorganisms for the presence of post-proline-specific endopeptidase activity was performed. This activity was found among ordinary bacteria from soil samples but not among fungi and actinomycetes. This result is in contrast to the previous notion that this activity is confined to the genus Flavobacterium. A proline endopeptidase was isolated from a Xanthomonas sp. and characterized with respect to physicochemical and enzymatic properties. The enzyme is composed of a single peptide chain with a molecular weight of 75,000. The isoelectric point is 6.2. It is inhibited by diisopropylfluorophosphate and may therefore be classified as a serine endopeptidase. The activity profile is bell shaped with an optimum at pH 7.5. By using synthetic peptide substrates and intramolecular fluorescence quenching it was possible to study the influence of substrate structure on the rate of hydrolysis. The enzyme specifically hydrolyzed Pro-X peptide bonds. With Glu at position X, low rates of hydrolysis were observed; otherwise the enzyme exhibited little preference for particular amino acid residues at position X. A similar substrate preference was observed with respect to the amino acid residue preceding the prolyl residue in the substrate. The enzyme required a minimum of two amino acid residues toward the N terminus from the scissile bond, but further elongation of the peptide chain by up to six amino acid residues caused only a threefold increase in the rate of hydrolysis. Attempts to cleave at the prolyl residues in oxidized RNase failed, indicating that the enzyme does not hydrolyze long peptides, a peculiar property it shares with other proline-specific endopeptidases.     

Properties of ACE inhibitory peptide prepared from protein in green tea residue and evaluation of its anti-hypertensive activity

Green tea contains active ingredients which are beneficial for health. While numerous studies have been conducted on the components extracted from green tea, few studies have investigated the active ingredients in tea residue. In this study, proteins were extracted from green tea residue via an optimised alkaline extraction combined with enzymatic hydrolysis, of which, an acidic protease was selected to prepare an enzymatic hydrolysate because of its high angiotensin converting enzyme (ACE) inhibitory activity. The composition characteristics of extracted green tea proteolysis products were elucidated, including amino acid composition, molecular weight distribution and possible amino acid sequences. In addition, the protein hydrolysate had anti-digestive properties, maintained its activity of inhibiting ACE enzyme at different temperatures, pH and metal ions, and exhibited antihypertensive activity in animals. In conclusion, the optimised alkaline extraction and enzymatic hydrolysis conditions of a ACE inhibitory peptide from green tea residue is an optimal extraction method to maintain its antihypertensive activity, providing the basis for the clinical application of green tea for blood pressure reduction.     

The expansion of mechanistic and organismic diversity associated with non-ribosomal peptides

Non-ribosomal peptides are a group of secondary metabolites with a wide range of bioactivities, produced by prokaryotes and lower eukaryotes. Recently, non-ribosomal synthesis has been detected in diverse microorganisms, including the myxobacteria and cyanobacteria. Peptides biosynthesized non-ribosomally may often play a primary or secondary role in the producing organism. Non-ribosomal peptides are often small in size and contain unusual or modified amino acids. Biosynthesis occurs via large modular enzyme complexes, with each module responsible for the activation and thiolation of each amino acid, followed by peptide bond formation between activated amino acids. Modules may also be responsible for the enzymatic modification of the substrate amino acid. Recent analysis of biosynthetic gene clusters has identified novel integrated, mixed and hybrid enzyme systems. These diverse mechanisms of biosynthesis result in the wide variety of non-ribosomal peptide structures and bioactivities seen today. Knowledge of these biosynthetic systems is rapidly increasing and methods of genetically engineering these systems are being developed. In the future, this may lead to rational drug design through combinatorial biosynthesis of these enzyme systems.     

Inactivation of brain myo-inositol monophosphate phosphatase by pyridoxal-5'-phosphate

Myo-inositol monophosphate phosphatase (IMPP) is a key enzyme in the phosphoinositide cell-signaling system. This study found that incubating the IMPP from a porcine brain with pyridoxal-5'-phosphate (PLP) resulted in a time-dependent enzymatic inactivation. Spectral evidence showed that the inactivation proceeds via the formation of a Schiff's base with the amino groups of the enzyme. After the sodium borohydride reduction of the inactivated enzyme, it was observed that 1.8 mol phosphopyridoxyl residues per mole of the enzyme dimer were incorporated. The substrate, myo-inositol-1-phosphate, protected the enzyme against inactivation by PLP. After tryptic digestion of the enzyme modified with PLP, a radioactive peptide absorbing at 210 nm was isolated by reverse-phase HPLC. Amino acid sequencing of the peptide identified a portion of the PLP-binding site as being the region containing the sequence L-Q-V-S-Q-Q-E-D-I-T-X, where X indicates that phenylthiohydantoin amino acid could not be assigned. However, the result of amino acid composition of the peptide indicated that the missing residue could be designated as a phosphopyridoxyl lysine. This suggests that the catalytic function of IMPP is modulated by the binding of PLP to a specific lysyl residue at or near its substrate-binding site of the protein.     

Integrated process for the enzymatic synthesis of the octapeptide PhAcCCK-8

A process for the enzymatic synthesis of PhAcCCK-8 is presented. The CCK-8 (CCK(26-33)) peptide fragment is the minimum sequence with biological activity of the cholecystokinin hormone. A synthetic convergent strategy has been developed starting from amino acid derivatives as raw materials, employing proteases as biocatalysts for each peptide coupling. The enzymes have been immobilized by deposition onto solid supports in order to be employed in organic media at low water activity. N-terminal protecting groups such as PhAc, which can be introduced and removed enzymatically, have been employed. The synthesis process has been set up at preparative level with focus in the integration of reaction and separation steps with an overall yield of 15%.     

Nucleotide sequence and expression in Escherichia coli of the gene coding for sphingomyelinase of Bacillus cereus

Bacillus cereus secretes phospholipases C, which hydrolyze phosphatidylcholine, sphingomyelin and phosphatidylinositol. A 7.5-kb HindIII fragment of B. cereus DNA cloned into Escherichia coli, with pUC18 as a vector, directed the synthesis of the sphingomyelin-hydrolyzing phospholipase C, sphingomyelinase. Nucleotide sequence analysis of the subfragment revealed that it contained two open reading frames in tandem. The upstream truncated open reading frame corresponds to the carboxy-terminal portion of the phosphatidylcholine-hydrolyzing phospholipase C, and the downstream open reading frame to the entire translational portion of the sphingomyelinase. The two phospholipase C genes form a gene cluster. As inferred from the DNA sequence, the B. cereus sphingomyelinase has a signal peptide of 27 amino acid residues and the mature enzyme comprises 306 amino acid residues, with a molecular mass of 34233 Da. The signal peptide of the enzyme was found to be functional in protein transport across the membrane of E. coli. The enzymatic properties of the sphingomyelinase synthesized in E. coli resemble those of the donor strain sphingomyelinase. The enzymatic activity toward sphingomyelin was enhanced 20-30-fold in the presence of MgCl2, and the adsorption of the enzyme onto erythrocyte membranes was accelerated in the presence of CaCl2.