Screening of environmental samples for bacteria producing 1,3-propanediol from glycerol.

Twenty nine environmental samples were screened for the presence of anaerobic microorganisms fermenting glycerol with 1,3-propanediol as a final product. Seven samples were then selected for the next step of our research and eight bacteria strains were cultured anaerobically. Seven of them produced 1,3-propanediol with a yield of 0.47-0.58. Six of the the isolated microorganisms were then classified as Clostridium butyricum (four strains), C. lituseburense (one strain), and C. sartagoforme (one strain). We suggest that of all these strains C. butyricum 2CR371.5 is the best 1,3-propanediol producer as producing no lactate as a by-product and growing well on a glycerol-containing medium.

1,3-propanediol production by Escherichia coli expressing genes of dha operon from Clostridium butyricum 2CR371.5.

1,3-propanediol is used as a monomer in the production of some polymers e.g. polytrimethylene terephthalate used in the production of carpets and textile fibers and in the thermoplastics engineering. However, the traditional chemical synthesis is expensive, generates some toxic intermediates and requires a reduction step under high hydrogen pressure. Biological production of 1,3-propanediol could be an attractive alternative to the traditional chemical methods. Moreover, crude glycerol which is a by-product of biodiesel production, can be used. We constructed a recombinant Escherichia coli strain producing 1,3-propanediol from glycerol by introducing genes of the dha operon from Clostridium butyricum 2CR371.5, a strain from our collection of environmental samples and strains. The E. coli strain produced 3.7 g of 1,3-propanediol per one litre of culture with the yield of 0.3 g per 1 g of glycerol consumed.

Design of serine proteinase inhibitors by combinatorial chemistry using trypsin inhibitor SFTI-1 as a starting structure.

A small peptide library of monocyclic SFTI-1 trypsin inhibitor from sunflower seeds modified in positions P(1) and P(4)' was synthesized using a portioning-mixing method. The peptide library was deconvoluted by the iterative approach in solution. Two trypsin ([Met(9)]-SFTI-1 and [Arg(5), Abu(9)]-SFTI-1), one chymotrypsin ([Phe(5)]-SFTI-1) and one human elastase ([Leu(5), Trp(9)]-SFTI-1) inhibitors were selected and resynthesized. The values of their association equilibrium constants (K(a)) with target enzymes indicate that they are potent inhibitors. In addition, the last two analoges belong to the most active inhibitors of this size. The results obtained show that the conserved Pro(9) residue in the Bowman-Birk inhibitor (BBI)s is not essential for inhibitory activity.

Trypsin inhibitors from the garden four o'clock (Mirabilis jalapa) and spinach (Spinacia oleracea) seeds: isolation, characterization and chemical synthesis.

Five serine proteinase inhibitors (Mirabilis jalapa trypsin inhibitors, MJTI I and II and Spinacia oleracea trypsin inhibitors, SOTI I, II, and III) from the garden four-o'clock (M. jalapa) and spinach (S. oleracea) seeds were isolated. The purification procedures included affinity chromatography on immobilized methylchymotrypsin in the presence of 5M NaCl, ion exchange chromatography and/or preparative electrophoresis, and finally RP-HPLC on a C-18 column. The inhibitors, crosslinked by three disulfide bridges, are built of 35 to 37 amino-acid residues. Their primary structures differ from those of known trypsin inhibitors, but showed significant similarity to the antimicrobial peptides isolated from the seeds of M. jalapa (MJ-AMP1, MJ-AMP2), Mesembryanthemum crystallinum (AMP1), and Phytolacca americana (AMP-2 and PAFP-S) and from the hemolymph of Acrocinus longimanus (Alo-1, 2 and 3). The association equilibrium constants (K(a)) with bovine beta-trypsin for the inhibitors from M. jalapa (MJTI I and II) and S. oleracea (SOTI I-III) were found to be about 10(7)M(-1). Fully active MJTI I and SOTI I were obtained by solid-phase peptide synthesis. The disulfide bridge pattern in both inhibitors (Cys1-Cys4, Cys2-Cys5 and Cys3-Cys6) was established after their digestion with thermolysin and proteinase K followed by the MALDI-TOF analysis.

Introduction of alpha-hydroxymethyamino acid residues in substrate specificity P1 position of trypsin inhibitor SFTI-1 from sunflower seeds retains its activity.

In many complexes formed by serine proteinases and their inhibitors, the hydroxyl group provided by water molecule or by the inhibitor Ser residue is located close to the inhibitor P1-P1' reactive site. In order to investigate the role of this group, we synthesized analogues of trypsin inhibitor SFTI-1 isolated from the seeds of sunflower modified in P1 by alpha-hydroxymethylserine (HmSer) and both enantiomers of alpha-hydroxymethylvaline (HmVal). All the synthesized analogues inhibited bovine beta-trypsin and human leukocyte elastase. SFTI-1 analogues with HmVal and HmSer appear to be potent inhibitors of bovine beta-trypsin, whereas [Val5]SFTI-1 is practically inactive. Also trypsin inhibitory activity of [Ser5]SFTI-1 is significantly lower. Since the electrostatic interaction between protonated epsilon-NH2 group of the inhibitor P1 position and beta-carboxylate of trypsin Asp189 is the main driving force for interaction of both molecules, the results obtained are very interesting. We believe that these SFTI-1 analogues belong to a novel class of serine proteinase inhibitors.

Examples of peptide-peptoid hybrid serine protease inhibitors based on the trypsin inhibitor SFTI-1 with complete protease resistance at the P1-P1' reactive site.

Research in the field of protease inhibitors is focused on obtaining potent, specific and protease-resistant inhibitors. To our knowledge, there are no reports in the literature that consider the application of N-substituted glycine residues (peptoid monomers) for the design of peptidomimetic protease inhibitors. We hereby present the chemical synthesis and kinetic properties of two new analogues of the trypsin inhibitor SFTI-1 modified at the P1 position. Substitution of Lys5 in SFTI-1 by N-(4-aminobutyl)-glycine and N-benzylglycine, which mimic Lys and Phe, respectively, made these analogues completely protease-resistant at their P1-P1' reactive sites. The analogues synthesised appeared to be potent inhibitors of bovine beta-trypsin and alpha-chymotrypsin. These noncovalent, competitive and selective peptide-peptoid hybrid (peptomeric) inhibitors might open the way to targeting unwanted proteolysis.

Monoubiquitinated histone H1B is required for antiviral protection in CD4(+)T cells resistant to HIV-1.

Linker histone H1B (H1B) coeluted with an antiviral activity during the purification of HIV-1 resistance factor (HRF) from supernatants of HRF(+) cells. Western blot analysis of the supernatant using alpha-H1 and alpha-ubiquitin antibodies detected the same band of roughly 46 kDa; this band was absent from the control supernatant. Depletion of histone from biologically active material did not affect its potential, suggesting that ubiquitinated H1B is not required for the HRF-mediated antiviral protection in HIV-1 susceptible target cells; however, specific silencing of histone H1B via RNAi in HRF(+) cells reduced the biological activity of cell culture supernatants by 96% and reversed the HIV-1 resistance phenotype of HRF(+) cells. Exposure to HRF induced ubiquitination and secretion of H1B from target HIV-1 susceptible cells, suggesting that ubiquitinated H1B is a cofactor of HRF, possibly regulating its expression and secretion from CD4(+)T cells induced to resist HIV-1 infection.

A simple method for selection of trypsin chromogenic substrates using combinatorial chemistry approach.

A tetrapeptide combinatorial library, considered as chromogenic substrates of bovine beta-trypsin, was synthesized by the solid phase method. The peptides contain an analog of p-nitroanilide, obtained by attaching 5-amino-2-nitrobenzoic acid (Anb(5,2)) to the C-termini. Deconvolution of the peptide library, performed in solution using an iterative method, yielded four efficient trypsin substrates. The most active one, Phe-Val-Pro-Arg-Anb(5,2)-NH(2), appeared to be 125-fold more active than Bz-D,L-Arg-pNA (BAPNA) used as a reference compound. The reported method of designing trypsin chromogenic substrate libraries is straightforward. Such p-nitroanilides may be useful for the investigation of any protease substrate specificity.

Selection of low-molecular-mass trypsin and chymotrypsin inhibitors based on the binding loop of CMTI-III using combinatorial chemistry methods.

Using a combinatorial chemistry approach, a decapaptide library containing the N-terminal fragment of trypsin inhibitor CMTI-III was synthesized by the solid-phase method. The peptide library was screened for trypsin and chymotrypsin inhibitory activity applying the iterative method in solution. Two decapeptides were selected and resynthesized for each enzyme. The association equilibrium constants ((1.1+/-0.2)x10(8) and (7.3+/-1.6)x10(7)) determined for peptides with trypsin inhibitory activity indicate that they are 3-4-fold less active than the CMTI inhibitors. On the other hand, they are significantly more effective as compared with the starting sequence. Two peptides selected as chymotrypsin inhibitors displayed about 10 times higher activity (1.7+/-0.4)x10(7) and (1.1+/-0.2)x10(7), respectively) than those monosubstituted in position P(1) of the CMTI-III analogue. Considering low molecular weight of peptides selected and the lack of conformational constraints in their structures, the results are promising. They are good templates as starting sequences for further selection of small, peptidomimetic proteinase inhibitors.

Chemical synthesis and kinetic study of the smallest naturally occurring trypsin inhibitor SFTI-1 isolated from sunflower seeds and its analogues.

The smallest known naturally occurring trypsin inhibitor SFTI-1 (14 amino acid residues head-to-tail cyclic peptide containing one disulfide bridge) and its two analogues with one cycle each were synthesized by the solid phase method. Their trypsin inhibitory activity was determined as association equilibrium constants (K(a)). Additionally, hydrolysis rates with bovine beta-trypsin were measured. Among all three peptides, the wild SFTI-1 and the analogue with the disulfide bridge only had, within the experimental error, the same activity (the K(a) values 1.1 x 10(10) and 9.9 x 10(9) M(-1), respectively). Both peptides displayed unchanged inhibitory activity up to 6 h. The trypsin inhibitory activity of the analogue with the head-to-tail cycle only was 2.4-fold lower. It was also remarkably faster hydrolyzed (k = 1.1 x 10(-4) mol(peptide) x mol(enzyme)(-1) x s(-1)) upon the incubation with the enzyme than the other two peptides. This indicates that the head-to-tail cyclization is significantly less important than the disulfide bridge for maintaining trypsin inhibitory activity.