Propionic acid fermentation of glycerol and glucose by Propionibacterium acidipropionici and Propionibacterium freudenreichii ssp.shermanii

A comparative study was carried out in anaerobic batch cultures on 20 g/l of either glycerol or glucose using two propionibacteria strains, Propionibacterium acidipropionici and Propionibacterium freudenreichii ssp. shermanii. In all cases, fermentation end-products were the same and consisted of propionic acid as the major product, acetic acid as the main by-product and two minor metabolites, n-propanol and succinic acid. Evidence was provided that greater production of propionic acid by propionibacteria was obtained with glycerol as carbon and energy sources. P. acidipropionici showed higher efficiency in glycerol conversion to propionic acid with a faster substrate consumption (0.64 g l⁻¹ h⁻¹) and a higher propionic acid production (0.42 g l⁻¹ h⁻¹ and 0.79 mol/mol). The almost exclusive production of propionic acid from glycerol by this bacterium suggested an homopropionic tendency of this fermentation. Acetic acid final concentration was two times lower on glycerol (2 g/l) than on glucose (4 g/l) for both micro-organisms. P. freudenreichii ssp. shermanii exhibited a glycerol fermentation pattern typical of non-associated glycerol-consumption-product formation. This could indicate a particular metabolism for P. freudenreichii ssp. shermanii oriented towards the production of other specific components. These results tend to show that glycerol could be an excellent alternative to conventional carbon sources such as carbohydrates for propionic acid production. Received: 21 May 1999 / Accepted: 1 November 1999     

Gene Cloning and Heterologous Expression of Glycoside Hydrolase Family 55 β-1,3-Glucanase from the Basidiomycete Phanerochaete Chrysosporium

The basidiomycete Phanerochaete chrysosporium produces several β-1,3-glucanases when grown on laminarin, a β-1,3/1,6-glucan, as the sole carbon source. To characterize one of the major unknown β-1, 3-glucanases with a molecular mass of 83 kDa, identification, cloning, and heterologous over-expression were carried out using the total genomic information of P. chrysosporium. The cDNA encoding this enzyme included an ORF of 2337 bp and the deduced amino acid sequence contains a predicted signal peptide of 26 amino acids and the mature protein of 752 amino acids. The amino acid sequence showed a significant similarity with glycoside hydrolase family 55 enzymes from filamentous fungi and was named Lam55A. Since the recombinant Lam55A expressed in the methylotrophic yeast Pichia pastoris degraded branched β-1,3/1,6-glucan as well as linear β-1,3-glucan, the kinetic features of the enzyme were compared with those of other β-1,3-glucanases.     

Molecular and Genetic Characterization of Propionicin F, a Bacteriocin from Propionibacterium freudenreichii

This work describes the purification and characterization of propionicin F, the first bacteriocin isolated from Propionibacterium freudenreichii. The bacteriocin has a bactericidal activity and is only active against strains of P. freudenreichii. Propionicin F appears to be formed through a processing pathway new to bacteriocins. The mass of the purified bacteriocin was determined by mass spectrometry, and the N-terminal amino acid sequence was determined by Edman degradation. Sequencing of pcfA, the bacteriocin structural gene, revealed that propionicin F corresponds to a 43-amino-acid peptide in the central part of a 255-amino-acid open reading frame, suggesting that mature propionicin F is excised from the probacteriocin by N- and C-terminal proteolytic modifications. DNA sequencing and Northern blot hybridizations revealed that pcfA is cotranscribed with genes encoding a putative proline peptidase and a protein from the radical S-adenosylmethionine family. A gene encoding an ABC transporter was also identified in close proximity to the bacteriocin structural gene. The potential role of these genes in propionicin F maturation and secretion is discussed.     

Spectroscopic Detection of Pseudo-Turns in Homodetic Cyclic Penta- and Hexapeptides Comprising β-Homoproline

β-Amino acids with side chains at C² and/or at C³ are of growing interest in drug design, as they may induce astonishing and unusual peptide conformations. Therefore it is of eminent importance to gather information on the consequences of β-amino acid incorporation on the three-dimensional structure of a peptide. This paper describes the synthesis and conformational analysis of cyclic penta- and hexapeptides comprising either (S)-Pro or (S)-β-Hpro. The conformational influence of the β-homoproline building block was analyzed by the combined application of CD, FT-IR and NMR. While the CD spectra of the proline containing peptides indicate the presence of inverse γ-turns and βII-turns, the CD spectra of the β-homoamino acid analogs are dominated by an unprecedented negative band near 205 nm associated with a pseudo-β-turn (Ψβ) or pseudo-γ-turn (Ψγ). These results were confirmed by FT-IR spectroscopy, which also indicates the formation of two internal hydrogen bonds in the cyclic peptides containing the β-homoproline. The conformations of the β-homoproline containing pentapeptides were additionally determined by NMR in combination with MD simulations in two different solvents. The conformation in trifluoroethanol (TFE) is characterized by a bifurcated hydrogen bond stabilizing a pseudo-γ-turn with β-homoproline in the central position, nested with a pseudo-β-turn with β-homoproline in the i+1 position. The combined CD/FT-IR studies clearly show that the replacement of proline by β-homoproline gives rise to a more flexible peptide backbone, and CD spectroscopy hints towards the presence of pseudo-β- or pseudo-γ-turns.     

Mass spectrometry proteomic analysis of stress adaptation reveals both common and distinct response pathways in<Emphasis Type="Italic"> Propionibacterium freudenreichii</Emphasis>

Microorganisms used in food technology and probiotics are exposed to technological and digestive stresses, respectively. Traditionally used as Swiss-type cheese starters, propionibacteria also constitute promising human probiotics. Stress tolerance and cross-protection in Propionibacterium freudenreichii were thus examined after exposure to heat, acid, or bile salts stresses. Adapted cells demonstrated acquired homologous tolerance. Cross-protection between bile salts and heat adaptation was demonstrated. By contrast, bile salts pretreatment sensitized cells to acid challenge and vice versa. Surprisingly, heat and acid responses did not present significant cross-protection in P. freudenreichii. During adaptations, important changes in cellular protein synthesis were observed using two-dimensional electrophoresis. While global protein synthesis decreased, several proteins were overexpressed during stress adaptations. Thirty-four proteins were induced by acid pretreatment, 34 by bile salts pretreatment, and 26 by heat pretreatment. Six proteins are common to all stresses and represent general stress-response components. Among these polypeptides, general stress chaperones, and proteins involved in energetic metabolism, oxidative stress response, or SOS response were identified. These results bring new insight into the tolerance of P. freudenreichii to heat, acid, and bile salts, and should be taken into consideration in the development of probiotic preparations.     

Studies on amino acid replacement and inhibitory activity of a β-lactamase inhibitory peptide

An SHV β-lactamase gene was amplified from a β-lactam resistant Klebsiella pneumoniae K-71 genomic DNA. After expression and purification, we demonstrated that peptide P1 could inhibit the hydrolysis activity of both TEM-1 and SHV β-lactamase in vitro. Three mutations were introduced into P1 in which the first residue S was replaced by F, the 18th residue V was mutated to Y, and the 15th residue Y was substituted with A, C, G, and R to obtain the mutants of P1-A, P1- C, P1-G, and P1-R, respectively. The mutant peptides were purified and their inhibitory constants against TEM-1 and SHV β-lactamase were determined. All these β-lactamase inhibitory peptides could inhibit the activity of both β-lactamases, while the mutant peptides showed stronger inhibitory activities against TEM-1 β-lactamase than against SHV β-lactamase. Inhibition data suggested that P1-A improved the β-lactamase inhibitory activity by over 3-fold compare to P1. When P1-A was incubated with K. pneumoniae K-71 in Luria-Bertani medium containing ampicillin, it showed a much stronger growth of inhibition ratio over P1. This study gives us a good candidate for development of novel β-lactamase inhibitors.     

High-level expression of a truncated 1,3-1,4-β-<Emphasis Type="SmallCaps">d</Emphasis>-glucanase from <Emphasis Type="Italic">Fibrobacter succinogenes</Emphasis> in <Emphasis Type="Italic">Pichia pastoris</Emphasis> by optimization of codons and fermentation

1,3-1,4-β-d-glucanase is an important endoglycosidase in the brewing and animal feed industries. To achieve high-level expression of recombinant glucanase in Pichia pastoris, we designed sequences encoding the α-factor signal peptide from Saccharomyces cerevisiae and the truncated 1,3-1,4-β-d-glucanase from Fibrobacter succinogenes as a whole. The codons encoding the 52 amino acids of the signal peptide and 106 residues of the glucanase protein were optimized for expression in P. pastoris; 189 nucleotides were changed. The G + C content was adjusted to 48–49%, and AT-rich stretches were eliminated to avoid premature termination. The messenger ribonucleic acid secondary structure near the AUG start codon was also adjusted to ensure efficient translation; the resulting glucanase production was twofold higher compared with that achieved with gene structure optimization alone. We also propose a new fermentation strategy for the induction phase, in which 5/95% glycerol/methanol mixed feed was used in days 1–3 and 100% methanol was used on days 4–6. By comparison with methanol feed and glycerol/methanol-mixed feed alone, the yield of recombinant glucanase increased by 38.5 and 16.5%, respectively. The expressed optimized recombinant 1,3-1,4-β-d-glucanase constituted ~90% of the total secreted protein, reaching up to 3 g l⁻¹ in the medium.     

New aspects of genes and enzymes for β-lactam antibiotic biosynthesis

Penicillins, cephalosporins and cephamycins are peptide antibiotics synthesized by condensation of l-α-aminoadipic acid, l-cysteine and l-valine to form the tripeptide δ(l-α-aminoadipyl)-l-cysteinyl-d-valine (Aad-Cys-Val) by a non-ribosomal peptide synthetase. The genes pcbAB and pcbC, common to all penicillin and cephalosporin producers, that encode the Aad-Cys-Val synthetase¹ and isopenicillin N (IPN) synthase¹ respectively, have been cloned and the encoded enzymes studied in detail. The IPN synthase has been crystallized and its active center identified, providing evidence for the molecular mechanism of cyclization of the tripeptide Aad-Cys-Val to isopenicillin N. The late genes of the penicillin and cephalosporin pathways have also been characterized although some of the molecular mechanisms catalyzed by the encoded enzymes (e.g. IPN acyltransferase) are still obscure. In cephamycin-producing organisms, biosynthesis of the α-aminoadipic acid precursor proceeds in two steps catalyzed by lysine 6-aminotransferase and piperideine-6-carboxylic acid dehydrogenase. The gene lat for the first of these enzymes is located in the cephamycin gene cluster, providing an interesting example of association of genes encoding enzymes for the formation of a precursor with genes involved in assembly of the antibiotics. Novel enzymes involved in methoxylation at C-7 and carbamoylation at C-3′ of the cephem nucleus were isolated from Nocardia lactamdurans and Streptomyces clavuligerus. The methoxylation system is encoded by two linked genes cmcI-cmcJ and their products (proteins P₇ and P₈) form a complex that is required for hydroxylation at C-7 and for the subsequent methylation of the 7-hydroxycephem derivative to form the methoxyl group. Carbamoylation at the C-3′-hydroxyl group of the cephem nucleus is catalyzed by a specific carbamoyltransferase encoded by the gene cmcH. Finally, genes for a β-lactamase (bla), a penicillin-binding protein (pbp) and a transmembrane protein (cmcT) that appears to be involved in cephamycin exportation, are clustered together with the biosynthetic genes in the cephamycin clusters of S. clavuligerus and N. lactamdurans. Availability of the cloned genes allows metabolic engineering of the β-lactam biosynthetic pathways such as a channelling precursors and directed removal of bottlenecks in the β-lactam biosynthetic pathways. Several new β-lactam antibiotics have been discovered in gram-positive and gram-negative bacteria that will provide new genes for combinatorial synthesis of new molecules. Received: 2 December 1997 / Received revision: 20 February 1998 / Accepted: 24 February 1998     

Effect of Phenolic Compounds Against A<Emphasis Type="Italic">β</Emphasis> Aggregation and A<Emphasis Type="Italic">β</Emphasis>-Induced Toxicity in Transgenic <Emphasis Type="Italic">C. elegans</Emphasis>

Substantial evidence suggests that the aggregation of amyloid-β (Aβ) peptide into fibrillar structures that is rich in β-sheets is implicated as the cause of Alzheimer’s disease. Therefore, an attractive therapeutic strategy is to prevent or alter Aβ aggregation. Phenolic compounds are natural substances that are composed of one or more aromatic phenolic rings and present in wine, tea, fruits, vegetables and a wide variety of plants. In this work, we investigated the effects of ferulic acid, morin, quercetin and gossypol against Aβ aggregation. From the ThT and turbidity assays, it is observed that in addition to the fibril aggregate, another type of aggregate is formed in the presence of morin, quercetin, and gossypol. On the other hand, ferulic acid did not prevent fibril formation, but it did appear to reduce the average length of fibrils compared to Aβ alone. To study the protective effects of phenolic compounds on Aβ-induced toxicity, we utilized the nematode Caenorhabditis elegans (C. elegans) as an in vivo model organism, human Aβ is expressed intracellularly in the body wall muscle. We found that exposure of Caenorhabditis elegans to ferulic acid give more protection against Aβ toxicity than morin, quercetin and gossypol.     

Structural and thermodynamical properties of CuII amyloid-β16/28 complexes associated with Alzheimer’s disease

The aggregation of the peptide amyloid-β (Aβ) to form amyloid plaques is a key event in Alzheimer’s disease. It has been shown that Cu^II can bind to soluble Aβ and influence its aggregation properties. Three histidines and the N-terminal amine have been proposed to be involved in its coordination. Here, for the first time, we show isothermal titration calorimetry (ITC) measurements of the Cu^II binding to Aβ16 and Aβ28, models of the soluble Aβ. Moreover, different spectroscopic methods were applied. The studies revealed new insights into these Cu^II–Aβ complexes: (1) ITC showed two Cu^II binding sites, with an apparent K _d of 10⁻⁷ and 10⁻⁵ M, respectively; (2) the high-affinity site has a smaller enthalpic contribution but a larger entropic contribution than the low-affinity binding site; (3) azide did not bind to Cu^II in the higher-affinity binding site, suggesting the absence of a weak, labile ligand; (4) azide could bind to the Cu^II in the low-affinity binding site in Aβ28 but not in Aβ16; (5) ¹H-NMR suggests that the carboxylate of aspartic acid in position 1 is involved in the ligation to Cu^II in the high-affinity binding site; (6) the pK _a of 11.3 of tyrosine in position 10 was not influenced by the binding of 2 equivalents of Cu^II.Electronic Supplementary Material Supplementary material is available to authorized users in the online version of this article at .     

Heterologous Production of Antimicrobial Peptides in Propionibacterium freudenreichii

Heterologous bacteriocin production in Propionibacterium freudenreichii is described. We developed an efficient system for DNA shuttling between Escherichia coli and P. freudenreichii using vector pAMT1. It is based on the P. freudenreichii rolling-circle replicating plasmid pLME108 and carries the cml(A)/cmx(A) chloramphenicol resistance marker. Introduction of the propionicin T1 structural gene (pctA) into pAMT1 under the control of the constitutive promoter (P₄) yielded bacteriocin in amounts equal to those of the wild-type producer Propionibacterium thoenii 419. The P. freudenreichii clone showed propionicin T1 activity in coculture, killing 90% of sensitive bacteria within 48 h. The pamA gene from P. thoenii 419 encoding the protease-activated antimicrobial peptide (PAMP) was cloned and expressed in P. freudenreichii, resulting in secretion of the pro-PAMP protein. Like in the wild type, PAMP activation was dependent on externally added protease. Secretion of the antimicrobial peptide was obtained from a clone in which the pamA signal peptide and PAMP were fused in frame. The promoter region of pamA was identified by fusion of putative promoter fragments to the coding sequence of the pctA gene. The P₄ and P_pamp promoters directed constitutive gene expression, and activity of both promoters was enhanced by elements upstream of the promoter core region.     

Phylogenetic analysis of condensation domains in NRPS sheds light on their functional evolution

Background  

Non-ribosomal peptide synthetases (NRPSs) are large multimodular enzymes that synthesize a wide range of biologically active natural peptide compounds, of which many are pharmacologically important. Peptide bond formation is catalyzed by the Condensation (C) domain. Various functional subtypes of the C domain exist: An^LC_L domain catalyzes a peptide bond between two L-amino acids, a^DC_L domain links an L-amino acid to a growing peptide ending with a D-amino acid, a Starter C domain (first denominated and classified as a separate subtype here) acylates the first amino acid with a β -hydroxy-carboxylic acid (typically a β -hydroxyl fatty acid), and Heterocyclization (Cyc) domains catalyze both peptide bond formation and subsequent cyclization of cysteine, serine or threonine residues. The homologous Epimerization (E) domain flips the chirality of the last amino acid in the growing peptide; Dual E/C domains catalyze both epimerization and condensation.     

Identification,cloning, and characterization of β-glucosidase from <Emphasis Type="Italic">Ustilago esculenta</Emphasis>

Hydrolytic enzymes responsible for laminarin degradation were found to be secreted during growth of Ustilago esculenta on laminarin. An enzyme involved in laminarin degradation was purified by assaying release of glucose from laminaribiose. Ion-exchange chromatography of the culture filtrate followed by size-exclusion chromatography yielded a 110-kDa protein associated with laminaribiose hydrolysis. LC/MS/MS analysis of the 110-kDa protein identified three peptide sequences that shared significant similarity with a putative glucoside hydrolase family (GH) 3 β-glucosidase in Ustilago maydis. Based on the DNA sequence of the U. maydis GH3 β-glucosidase, a gene encoding a putative GH3 β-glucosidase in U. esculenta (Uebgl3A) was cloned by PCR. Based on the deduced amino acid sequence, the protein encoded by Uebgl3A has a molecular mass of 91 kDa and shares 90% identity with U. maydis GH3 β-glucosidase. Recombinant UeBgl3A expressed in Aspergillus oryzae released glucose from β-1,3-, β-1,4-, and β-1,6-linked oligosaccharides, and from 1,3-1,4-β-glucan and laminarin polysaccharides, indicating that UeBgl3A is a β-glucosidase. Kinetic analysis showed that UeBgl3A preferentially hydrolyzed laminaritriose and laminaritetraose. These results suggest that UeBgl3A is a key enzyme that produces glucose from laminarioligosaccharides during growth of U. esculenta on laminarin.     

The Complete Genome of Propionibacterium freudenreichii CIRM-BIA1T,a Hardy Actinobacterium with Food and Probiotic Applications

Background

Propionibacterium freudenreichii is essential as a ripening culture in Swiss-type cheeses and is also considered for its probiotic use [1]. This species exhibits slow growth, low nutritional requirements, and hardiness in many habitats. It belongs to the taxonomic group of dairy propionibacteria, in contrast to the cutaneous species P. acnes. The genome of the type strain, P. freudenreichii subsp. shermanii CIRM-BIA1 (CIP 103027^T), was sequenced with an 11-fold coverage.

Methodology/Principal Findings

The circular chromosome of 2.7 Mb of the CIRM-BIA1 strain has a GC-content of 67% and contains 22 different insertion sequences (3.5% of the genome in base pairs). Using a proteomic approach, 490 of the 2439 predicted proteins were confirmed. The annotation revealed the genetic basis for the hardiness of P. freudenreichii, as the bacterium possesses a complete enzymatic arsenal for de novo biosynthesis of aminoacids and vitamins (except panthotenate and biotin) as well as sequences involved in metabolism of various carbon sources, immunity against phages, duplicated chaperone genes and, interestingly, genes involved in the management of polyphosphate, glycogen and trehalose storage. The complete biosynthesis pathway for a bifidogenic compound is described, as well as a high number of surface proteins involved in interactions with the host and present in other probiotic bacteria. By comparative genomics, no pathogenicity factors found in P. acnes or in other pathogenic microbial species were identified in P. freudenreichii, which is consistent with the Generally Recognized As Safe and Qualified Presumption of Safety status of P. freudenreichii. Various pathways for formation of cheese flavor compounds were identified: the Wood-Werkman cycle for propionic acid formation, amino acid degradation pathways resulting in the formation of volatile branched chain fatty acids, and esterases involved in the formation of free fatty acids and esters.

Conclusions/Significance

With the exception of its ability to degrade lactose, P. freudenreichii seems poorly adapted to dairy niches. This genome annotation opens up new prospects for the understanding of the P. freudenreichii probiotic activity.     

Modulation of aggregate size- and shape-distributions of the amyloid-β peptide by a designed β-sheet breaker

A peptide with 42 amino acid residues (Aβ42) plays a key role in the pathogenesis of the Alzheimer’s disease. It is highly prone to self aggregation leading to the formation of fibrils which are deposited in amyloid plaques in the brain of diseased individuals. In our study we established a method to analyze the aggregation behavior of the Aβ peptide with a combination of sedimentation velocity centrifugation and enhanced data evaluation software as implemented in the software package UltraScan. Important information which becomes accessible by this methodology is the s-value distribution and concomitantly also the shape-distribution of the Aβ peptide aggregates generated by self-association. With this method we characterized the aggregation modifying effect of a designed β-sheet breaker molecule. This compound is built from three head-to-tail connected aminopyrazole moieties and represents a derivative of the already described Tripyrazole. By addition of this compound to a solution of the Aβ42 peptide the maximum of the s-value distribution was clearly shifted to smaller s-values as compared to solutions where only the vehicle DMSO was added. This shift to smaller s-values was stable for at least 7 days. The information about size- and shape-distributions present in aggregated Aβ42 solutions was confirmed by transmission electron microscopy and by measurement of amyloid formation by thioflavin T fluorescence.     

Analysis of beta-tubulin cDNAs from taxol-resistant Pestalotiopsis microspora and taxol-sensitive Pythium ultimum and comparison of the taxol-binding properties of their products

The anti-cancer drug taxol binds to β-tubulin in assembled microtubules and causes cell cycle arrest in animal cells; in contrast, in fungi, the effect of taxol varies. For instance, the taxol-producer Pestalotiopsis microspora Ne32, an ascomycete, is resistant to taxol (IC₅₀ greater than 11.7 μM), whereas Pythium ultimum, an oomycete, is sensitive to taxol (IC₅₀ 0.1 μM). In order to understand the differential fungal response to taxol, we isolated cDNAs encoding β-tubulin from both P. microspora and P. ultimum. The deduced amino acid sequence of β-tubulin from P. microspora is very similar to those from other Ascomycetes, many of which are resistant to taxol. The sequence of β-tubulin from P. ultimum is very similar to those from Oomycetes and non-fungal organisms, many of which are sensitive to taxol. To examine the interaction between taxol and fungal microtubules, binding studies were performed with fungal cells, using [³H]taxol. The labeled taxol was found to bind specifically to P. ultimum, but not to P. microspora. In addition, the amount of [³H]taxol specifically bound to P. ultimum was reduced by the microtubule-depolymerizing drug thiabendazole, in a dose-dependent manner. These results suggest efficient binding of taxol to microtubules in P. ultimum, but not in P. microspora, and are consistent with the differential taxol sensitivity of these two organisms. Finally a comparison of previously characterized taxol binding sites in various β-tubulin sequences showed that β-tubulins of taxol-sensitive organisms, including P. ultimum, contain Thr219, but β-tubulins of resistant organisms, including P. microspora, contain Asn or Gln at this position, suggesting an important role for residue 219 in the interaction between taxol and β-tubulin. Received: 16 March 1999 / Accepted: 21 August 1999     

Identification of binding peptides of the ADAM15 disintegrin domain using phage display

ADAM15 plays an important role in tumour development by interacting with integrins. In this study, we investigated the target peptides of the ADAM15 disintegrin domain. First, we successfully produced the recombinant human ADAM15 disintegrin domain (RADD) that could inhibit melanoma cell adhesion by using Escherichia coli. Second, four specific binding peptides (peptides A, B, C, and D) were selected using a phage display 12-mer peptide library. The screening protocol involved 4 rounds of positive panning on RADD and 2 rounds of subtractive selection with streptavidin. By using the BLAST software and a relevant protein database, integrin α _ν β ₃ was found to be homologous to peptide A. Synthetic peptide A had a highly inhibitory effect on RADD-integrin α _v β ₃ binding. The results demonstrate the potential application of short peptides for disrupting high-affinity ADAM-integrin interactions.     

Cloning and characterization of an endo-β-1,3(4)glucanase and an aspartic protease from Phaffia rhodozyma CBS 6938

We describe the identification and expression cloning of two novel enzymes, a β-glucanase and an aspartic protease, secreted from the basidiomycetous yeast Phaffia rhodozyma. A cDNA library from P. rhodozyma CBS 6938 was constructed, and full-length cDNA encoding an endo-1,3(4)-β-glucanase (bg1) and an aspartic protease (pr1) were cloned by expression cloning in Saccharomyces cerevisiae W3124. The bg1 cDNA encodes a 424-residue precursor protein with a putative signal peptide. The pr1 cDNA encodes a 405-residue prepropolypeptide with an 81-residue leader peptide. The aspartic protease was purified and characterized. It has a molecular mass of 36 kDa, an isoelectric point of pH 7.5, a pH activity optimum at 4.0–6.0, and a temperature activity optimum around 40 °C. Both enzymes show only low sequence identity to other known enzymes. Received: 6 August 1998 / Received revision: 29 October 1998 / Accepted: 30 October 1998     

Synthesis of bis-amino acid derivatives by Suzuki cross-coupling,Michael addition and substitution reactions

Several bis-amino acids were prepared using a bis-Suzuki coupling (compounds 4–8, 10), a sequential Michael addition and bis-Suzuki coupling (compounds 12, 13) and a Michael addition followed by a substitution reaction (compounds 18, 19). Thus, the pure stereoisomer of the methyl esters of N-(tert-butoxycarbonyl)-β-bromodehydroaminobutyric acid and dehydrophenylalanine and of N-benzyloxycarbonyl-β-bromodehydroaminobutyric acid were reacted with 1,4-phenylene-bis-boronic acid or 9,9-dioctyl-9H-fluorene-2,7-bis-boronic acid using modified Suzuki coupling conditions. The corresponding bis-dehydroamino acid derivatives were obtained in good to high yields maintaining the stereochemistry of the starting materials. This reaction was also applied successfully to a brominated dehydrodipeptide and 1,4-phenylene-bis-boronic acid showing that it could be used to create cross-links in peptide chains. An N,N-diacyldehydroalanine derivative was used in a sequential Michael addition and bis-Suzuki coupling giving a p-terphenyl bis-amino acid and a fluorenyl bis-amino acid in good yields. Two bis-α,β-diamino acids were obtained by a Michael addition of 1,2,4-triazole to the methyl esters of N-(4-toluenesulfonyl), N-(tert-butoxycarbonyl) dehydroamino acids followed by treatment with ethylenediamine.