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.

Identification and molecular modeling of a novel lipase from an Antarctic soil metagenomic library.

In this work, we present the construction of a metagenomic library in Escherichia coli using pUC19 vector and environmental DNA directly isolated from Antarctic topsoil and screened for lipolytic enzymes. Screening on agar supplemented with olive oil and rhodamine B revealed one clone with lipolytic activity (Lip1) out of 1000 E. coli clones. This clone harbored a plasmid, pLip1, which has an insert of 4722 bp that was completely sequenced from both directions. Further analysis of the insert showed three open reading frames (ORFs). ORF2 encoded a protein (Lip1 ) of 469 amino acids with 93% identity to the uncultured Pseudomonas sp. lipase LipJ03. Amino acid sequence comparison and phylogenetic analysis indicated that Lip1 lipase was closely related to family I subfamily 3. Furthermore, we present a three-dimensional model of lipase Lip1 which was generated based on the two known structures of mesophilic lipases from Pseudomonas sp. MIS 38 (PML lipase, PDB; 2Z8X) and Serratia marcescens (SML lipase, PDB: 2QUB). Finally, we report the results of comparisons between lipase Lip1 and mesophilic lipases and point out similarities and differences in the catalytic site and in other parts of the analyzed structures.

Purification and biochemical characteristic of a cold-active recombinant esterase from Pseudoalteromonas sp. 643A under denaturing conditions.

In this paper production of a cold-active esterase EstA from the Antarctic bacterium Pseudoalteromonas sp. 643A in E. coli expression system was described. The purification and biochemical characteristic of EstA were performed in the presence of urea and then compared with results obtained for the esterase with no addition of urea and isolated from the native source. In both cases the cold-active enzyme displayed similar properties. However, the differences concerning thermal activity were observed. The optimal temperature for recombinant esterase in the presence of urea (1 M) was about 15 degrees C lower in comparison with enzyme isolated from the native source. Furthermore, the EstA was found to be more thermolabile in denaturant conditions. The differences were presumably caused by slightly changed protein structure in the presence of urea. The preservation of activity of EstA dissolved in buffer containing 8 M urea suggests that the protein structure is retained and it does not undergo dramatic changes due to high urea concentration. This thesis was confirmed with FT-IR data.

An MTA phosphorylase gene discovered in the metagenomic library derived from Antarctic top soil during screening for lipolytic active clones confers strong pink fluorescence in the presence of rhodamine B.

In this work, we present the construction of a metagenomic library in Escherichia coli using the pUC19 vector and environmental DNA directly isolated from Antarctic topsoil and screened for lipolytic enzymes. Unexpectedly, the screening on agar supplemented with olive oil and rhodamine B revealed one unusual pink fluorescent clone (PINKuv) out of 85 000 clones. This clone harbored a plasmid, pPINKuv, which has an insert of 8317 bp that has been completely sequenced. Further analysis of the insert showed eight ORFs. Three ORFs among these exhibited similarities to Psychrobacter arcticus genes. A nucleotide sequence designated as ORF4 encoded a protein with 93% identity to the methylthioadenosine phosphorylase of P. arcticus. This protein was responsible for the observed pink fluorescence of the PINKuv clone in the presence of rhodamine B. We found that colonies of recombinant E. coli TOP10F'/pUC19-ORF4 strain showed pink fluorescence under UV illumination on the Luria-Bertani agar supplemented with rhodamine B after culturing at 25, 30 and 37 degrees C. The same effect was achieved using other E. coli strains such as DH5alpha, LMG194, JM101 and BL21(DE3) pLysS. The results presented here will provide the basis for further studies on the use of the discovered gene as a new reporter gene for molecular biology applications.

Structure of EstA esterase from psychrotrophic Pseudoalteromonas sp. 643A covalently inhibited by monoethylphosphonate.

The crystal structure of the esterase EstA from the cold-adapted bacterium Pseudoalteromonas sp. 643A was determined in a covalently inhibited form at a resolution of 1.35 A. The enzyme has a typical SGNH hydrolase structure consisting of a single domain containing a five-stranded beta-sheet, with three helices at the convex side and two helices at the concave side of the sheet, and is ornamented with a couple of very short helices at the domain edges. The active site is located in a groove and contains the classic catalytic triad of Ser, His and Asp. In the structure of the crystal soaked in diethyl p-nitrophenyl phosphate (DNP), the catalytic serine is covalently connected to a phosphonate moiety that clearly has only one ethyl group. This is the only example in the Protein Data Bank of a DNP-inhibited enzyme with covalently bound monoethylphosphate.

Extracellular secretion of Pseudoalteromonas sp. cold-adapted esterase in Escherichia coli in the presence of Pseudoalteromonas sp. components of ABC transport system.

Recently we described identification and characterization of GDSL esterase EstA from psychrotrophic bacterium Pseudoalteromonas sp. 643A. Attempts to obtain heterologous overexpression of this enzyme in Escherichia coli system were not satisfactory. The EstA protein was expressed as inclusion bodies, most of that were inactive after purification step, and the recovery of esterolytic activity was very low after refolding. Based on the sequence analysis we found that the esterase EstA gene is clustered with three genes encoding components of ABC transport system. These genes, designated abc1, abc2, and abc3 encode an ATP-binding protein (ABC1) and two permease proteins (ABC2 and ABC3). In present study, to obtain larger amounts of the active cold-adapted EstA esterase from Pseudoalteromonas sp. 643A, we designed a two-plasmid E. coli expression system where the gene encoding EstA enzyme was cloned into pET30b(+) expression vector and three genes encoding components of ABC transport system were cloned into pACYC-pBAD vector. It was shown that the created expression system was useful for extracellular production of active EstA enzyme which was purified from the culture medium. In the presence of all the three transporter proteins the secretion of EstA was at the highest level. When one or two of these components were missing, EstA secretion was also possible, but not so effective. It indicates that ABC2 and ABC3 proteins of Pseudoalteromonas sp. 643A could be replaced with their homologous proteins of E. coli.

A cold-adapted esterase from psychrotrophic Pseudoalteromas sp. strain 643A.

A psychrotrophic bacterium producing a cold-adapted esterase upon growth at low temperatures was isolated from the alimentary tract of Antarctic krill Euphasia superba Dana, and classified as Pseudoalteromonas sp. strain 643A. A genomic DNA library of strain 643A was introduced into Escherichia coli TOP10F', and screening on tributyrin-containing agar plates led to the isolation of esterase gene. The esterase gene (estA, 621 bp) encoded a protein (EstA) of 207 amino acid residues with molecular mass of 23,036 Da. Analysis of the amino acid sequence of EstA suggests that it is a member of the GDSL-lipolytic enzymes family. The purification and characterization of native EstA esterase were performed. The enzyme displayed 20-50% of maximum activity at 0-20 degrees C. The optimal temperature for EstA was 35 degrees C. EstA was stable between pH 9 and 11.5. The enzyme showed activity for esters of short- to medium-chain (C(4) and C(10)) fatty acids, and exhibited no activity for long-chain fatty acid esters like that of palmitate and stearate. EstA was strongly inhibited by phenylmethylsulfonyl fluoride, 2-mercaptoethanol, dithiothreitol and glutathione. Addition of selected divalent ions e.g. Mg(2+), Co(2+) and Cu(2+) led to the reduction of enzymatic activity and the enzyme was slightly activated ( approximately 30%) by Ca(2+) ions.

Structure of the single-stranded DNA-binding protein SSB from Thermus aquaticus.

The crystal structure of the single-stranded DNA-binding protein from Thermus aquaticus has been solved and refined at 1.85 A resolution. Two monomers, each encompassing two oligonucleotide/oligosaccharide-binding (OB) domains and a number of flexible beta-hairpin loops, form an oligomer of approximate D(2) symmetry typical of bacterial SSBs. Comparison with other SSB structures confirms considerable variability in the mode of oligomerization and aggregation of SSB oligomers.

Single-stranded DNA-binding proteins (SSBs) -- sources and applications in molecular biology.

Single-stranded DNA-binding proteins (SSBs) play essential roles in DNA replication, recombination, and repair in bacteria, archaea and eukarya. The SSBs share a common core ssDNA-binding domain with a conserved OB (oligonucleotide/oligosaccharide binding) fold. This ssDNA-binding domain was presumably present in the common ancestor to all three major branches of life. In recent years, there has been an increasing interest in SSBs because they are useful for molecular biology methods and for analytical purposes. In this review, we concentrate on recent advances in the discovery of new sources of SSBs as well as certain aspects of their applications in analytical sciences.