A New Expression System Based on Psychrotolerant Debaryomyces macquariensis Yeast and Its Application to the Production of Cold-Active ß-d-Galactosidase from Paracoccus sp. 32d.

Yeasts provide attractive host/vector systems for heterologous gene expression. The currently used yeast-based expression platforms include mesophilic and thermotolerant species. A eukaryotic expression system working at low temperatures could be particularly useful for the production of thermolabile proteins and proteins that tend to form insoluble aggregates. For this purpose, an expression system based on an Antarctic psychrotolerant yeast Debaryomyces macquariensis strain D50 that is capable of growing at temperatures ranging from 0 to 30 °C has been developed. The optimal physical culture conditions for D. macquariensis D50 in a fermenter are as follows: temperature 20 °C, pH 5.5, aeration rate of 1.5 vvm, and a stirring speed of 300 rpm. Four integrative plasmid vectors equipped with an expression cassette containing the constitutive GAP promoter and CYC1 transcriptional terminator from D. macquariensis D50 were constructed and used to clone and express a gene-encoding cold-active ß-d-galactosidase of Paracoccus sp. 32d. The yield was 1150 U/L of recombinant yeast culture. Recombinant D. macquariensis D50 strains were mitotically stable under both selective and non-selective conditions. The D. macquariensis D50 host/vector system has been successfully utilized for the synthesis of heterologous thermolabile protein, and it can be an alternative to other microbial expression systems.

The psychrotrophic yeast Sporobolomyces roseus LOCK 1119 as a source of a highly active aspartic protease for the in vitro production of antioxidant peptides.

A psychrotrophic yeast strain producing a cold-adapted protease at low temperature was classified as Sporobolomyces roseus. In standard YPG medium, S. roseus LOCK 1119 synthesized an extracellular protease with an activity of approximately 560 U/L. Optimization of medium composition and process temperature considerably enhanced enzyme biosynthesis; an approximate 70% increase in activity (2060 U/L). The native enzyme was purified to homogeneity by cation exchange chromatography followed by a size exclusion step, resulting in a 103-fold increase in specific activity (660 U/mg) with 25% recovery. The enzyme displayed 10%-30% of its maximum activity at 0-25 °C, with the optimum temperature being 50°C. Protease G8 was strongly inactivated by pepstatin A, an aspartic protease inhibitor. The enzyme was used to hydrolyze four natural substrates, and their antioxidant activities were evaluated against 1,1-diphenyl-2-picrylhydrazyl. The highest antioxidant activity (69%) was recorded for beef casein.

Application of byproducts from food processing for production of 2,3-butanediol using Bacillus amyloliquefaciens TUL 308.

A nonpathogenic bacterial strain Bacillus amyloliquefaciens TUL 308 synthesized minor 2,3-butanediol (2,3-BD) amounts from glucose, fructose, sucrose, and glycerol, and efficiently produced the diol from molasses and hydrolysates of food processing residues. Batch fermentations yielded 16.53, 10.72, and 5 g/L 2,3-BD from enzymatic hydrolysates of apple pomace, dried sugar beet pulp, and potato pulp (at initial concentrations equivalent to 45, 20, and 30 g/L glucose, respectively), and 25.3 g/L 2,3-BD from molasses (at its initial concentration equivalent to 60 g/L saccharose). Fed-batch fermentations in the molasses-based medium with four feedings with either glucose or sucrose (in doses increasing their concentration by 25 g/L) resulted in around twice higher maximum 2,3-BD concentration (of about 60 and 50 g/L, respectively). The GRAS Bacillus strain is an efficient 2,3-BD producer from food industry byproducts.

Effects of genetic modifications and fermentation conditions on 2,3-butanediol production by alkaliphilic Bacillus subtilis.

Two recombinants of alkaliphilic Bacillus subtilis LOCK 1086, constructed via different strategies such as cloning the gene encoding bacterial hemoglobin from Vitreoscilla stercoraria (vhb) and overexpression of the gene encoding acetoin reductase/2,3-butanediol dehydrogenase (bdhA) from B. subtilis LOCK 1086, did not produce more 2,3-butanediol (2,3-BD) than the parental strain. In batch fermentations, this strain synthesized 9.46 g/L in 24 h and 12.80 g/L 2,3-BD in 46 h from sugar beet molasses and an apple pomace hydrolysate, respectively. 2,3-BD production by B. subtilis LOCK 1086 was significantly enhanced in fed-batch fermentations. The highest 2,3-BD concentration (75.73 g/L in 114 h, productivity of 0.66 g/L × h) was obtained in the sugar beet molasses-based medium with four feedings with glucose. In a medium based on the apple pomace hydrolysate with three feedings with sucrose, B. subtilis LOCK 1086 produced up to 51.53 g/L 2,3-BD (in 120 h, productivity of 0.43 g/L × h).

Application of enzymatic apple pomace hydrolysate to production of 2,3-butanediol by alkaliphilic Bacillus licheniformis NCIMB 8059.

2,3-Butanediol (2,3-BD) synthesis by a nonpathogenic bacterium Bacillus licheniformis NCIMB 8059 from enzymatic hydrolysate of depectinized apple pomace and its blend with glucose was studied. In shake flasks, the maximum diol concentration in fed-batch fermentations was 113 g/L (in 163 h, from the hydrolysate, feedings with glucose) while in batch processes it was around 27 g/L (in 32 h, from the hydrolysate and glucose blend). Fed-batch fermentations in the 0.75 and 30 L fermenters yielded 87.71 g/L 2,3-BD in 160 h, and 72.39 g/L 2,3-BD in 94 h, respectively (from the hydrolysate and glucose blend, feedings with glucose). The hydrolysate of apple pomace, which was for the first time used for microbial 2,3-BD production is not only a source of sugars but also essential minerals.

Crystal structure and enzymatic properties of a broad substrate-specificity psychrophilic aminotransferase from the Antarctic soil bacterium Psychrobacter sp. B6.

Aminotransferases (ATs) are enzymes that are commonly used in the chemical and pharmaceutical industries for the synthesis of natural and non-natural amino acids by transamination reactions. Currently, the easily accessible enzymes from mesophilic organisms are most commonly used; however, for economical and ecological reasons the utilization of aminotransferases from psychrophiles would be more advantageous, as their optimum reaction temperature is usually significantly lower than for the mesophilic ATs. Here, gene isolation, protein expression, purification, enzymatic properties and structural studies are reported for the cold-active aromatic amino-acid aminotransferase (PsyArAT) from Psychrobacter sp. B6, a psychrotrophic, Gram-negative strain from Antarctic soil. Preliminary computational analysis indicated dual functionality of the enzyme through the ability to utilize both aromatic amino acids and aspartate as substrates. This postulation was confirmed by enzymatic activity tests, which showed that it belonged to the class EC 2.6.1.57. The first crystal structures of a psychrophilic aromatic amino-acid aminotransferase have been determined at resolutions of 2.19 Šfor the native enzyme (PsyArAT) and 2.76 Šfor its complex with aspartic acid (PsyArAT/D). Both types of crystals grew in the monoclinic space group P21 under slightly different crystallization conditions. The PsyArAT crystals contained a dimer (90 kDa) in the asymmetric unit, which corresponds to the active form of this enzyme, whereas the crystals of the PsyArAT/D complex included four dimers showing different stages of the transamination reaction.

Purification, characterisation and expression in Saccharomyces cerevisiae of LipG7 an enantioselective, cold-adapted lipase from the Antarctic filamentous fungus Geomyces sp. P7 with unusual thermostability characteristics.

A lipase, LipG7, has been purified from the Antarctic filamentous fungus Geomyces sp. P7 which was found to be cold-adapted and able to retain/regain its activity after heat denaturation. The LipG7 exhibits 100% residual activity following 1h incubation at 100°C whilst simultaneously showing kinetic adaptations to cold temperatures. LipG7 was also found to have industrial potential as an enantioselective biocatalyst as it is able to effectively catalyse the enantioselective transesterification of a secondary alcohol. The LipG7 coding sequence has been identified and cloned using 454 pyrosequencing of the transcriptome and inverse PCR. The LipG7 protein has been heterologously expressed in Saccharomyces cerevisiae BJ5465 and shown to exhibit the same characteristics as the native protein.

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.

A new beta-galactosidase with a low temperature optimum isolated from the Antarctic Arthrobacter sp. 20B: gene cloning, purification and characterization.

A psychrotrophic bacterium producing a cold-adapted beta-galactosidase upon growth at low temperatures was classified as Arthrobacter sp. 20B. A genomic DNA library of strain 20B introduced into Escherichia coli TOP10F' and screening on X-Gal (5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside)-containing agar plates led to the isolation of beta-galactosidase gene. The beta-galactosidase gene (bgaS) encoding a protein of 1,053 amino acids, with a calculated molecular mass of 113,695 kDa. Analysis of the amino acid sequence of BgaS protein, deduced from the bgaS ORF, suggested that it is a member of the glycosyl hydrolase family 2. A native cold-adapted beta-galactosidase was purified to homogeneity and characterized. It is a homotetrameric enzyme, each subunit being approximately 116 kDa polypeptide as deduced from native and SDS-PAGE, respectively. The beta-galactosidase was optimally active at pH 6.0-8.0 and 25 degrees Celsius. P-nitrophenyl-beta-D-galactopyranoside (PNPG) is its preferred substrate (three times higher activity than for ONPG-o-nitrophenyl-beta-D-galactopyranoside). The Arthrobacter sp. 20B beta-galactosidase is activated by thiol compounds (53% rise in activity in the presence of 10 mM 2-mercaptoethanol), some metal ions (activity increased by 50% for Na(+), K(+) and by 11% for Mn(2+)) and inactivated by pCMB (4-chloro-mercuribenzoic acid) and heavy metal ions (Pb(2+), Zn(2+), Cu(2+)).

Identification of the csp gene and molecular modelling of the CspA-like protein from Antarctic soil-dwelling psychrotrophic bacterium Psychrobacter sp. B6.

We cloned and sequenced the cspA-like gene from a psychrotrophic Antarctic soil-dwelling bacterial strain Psychrobacter sp. B6. The gene is 213 bp long and shows 99% and 98% sequence identity with the Psychrobacter cryohalolentis K5 gene encoding a cold-shock DNA-binding domain protein and the Psychrobacter arcticus transcriptional regulator-CspA gene, respectively. The protein encoded by the Psychrobacter sp. B6 cspA-like gene shows 100% identity with the two proteins mentioned above, and also 61% sequence identity with CspB from Bacillus subtilis and Csp from Bacillus caldolyticus, and 56% - with Escherichia coli CspA protein. A three-dimensional model of the CspA-like protein from Psychrobacter sp. B6 was generated based on three known structures of cold shock proteins: the crystal structure of the major cold shock protein from Escherichia coli (CspA), the NMR structure of the latter protein, and the NMR structure of Csp from Thermotoga maritima. The deduced structure of the CspA-like protein from Psychrobacter sp. B6 was found to be very similar to these known structures of Csp-like proteins.

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.

Molecular characterization of a cryptic plasmid from the psychrotrophic antarctic bacterium Pseudoalteromonas sp. 643A.

We report the identification and nucleotide sequence analysis of pKW1, a plasmid of the psychrotrophic bacterium Pseudoalteromonas sp. 643A isolated from the stomach of Antarctic krill Euphasia superba. pKW1 consists of 4583 bp, has a G+C content of 43% and seven putative open reading frames (ORFs). The deduced amino acid sequence from ORF-1 shared significant similarity with the plasmid replicase protein of Psychrobacter cryohalolentis, strain K5. The DNA region immediately downstream of the ORF-1 showed some homology with the Rep-binding sequence of the theta-replicating ColE2-type plasmids. The ORF-3 amino acid sequence revealed amino acid sequence homology with the mobilization protein of Psychrobacter sp. PRwf-1 and Moraxella catarrhalis, with identities of 28% and 25%, respectively. The ORF-4 showed 46% amino acid sequence homology with the putative relaxase/mobilization nuclease MobA of Hafnia alvei and 44% homology with the putative mobilization protein A of Pasterulla multocida. The copy number of pKW1 in Pseudoalteromonas sp. 643A was estimated of 15 copies per chromosome.

Purification and characterization of two cold-adapted extracellular tannin acyl hydrolases from an Antarctic strain Verticillium sp. P9.

Two extracellular tannin acyl hydrolases (TAH I and TAH II) produced by an Antarctic filamentous fungus Verticillium sp. P9 were purified to homogeneity (7.9- and 10.5-fold with a yield of 1.6 and 0.9%, respectively) and characterized. TAH I and TAH II are multimeric (each consisting of approximately 40 and 46 kDa sub-units) glycoproteins containing 11 and 26% carbohydrates, respectively, and their molecular mass is approximately 155 kDa. TAH I and TAH II are optimally active at pH of 5.5 and 25 and 20 degrees C, respectively. Both the enzymes were activated by Mg(2+)and Br(-) ions and 0.5-2.0 M urea and inhibited by other metal ions (Zn(2+), Cu(2+), K(+), Cd(2+), Ag(+), Fe(3+), Mn(2+), Co(2+), Hg(2+), Pb(2+) and Sn(2+)),[Formula: see text] anions, Tween 20, Tween 60, Tween 80, Triton X-100, sodium dodecyl sulphate, beta-mercaptoethanol, alpha-glutathione and 4-chloromercuribenzoate. Both tannases more efficiently hydrolyzed tannic acid than methyl gallate. E (a) of these reactions and temperature dependence (at 0-30 degrees C) of k (cat), k (cat)/K (m), DeltaG*, DeltaH* and DeltaS* for both the enzymes and substrates were determined. The k (cat) and k (cat)/K (m) values (for both the substrates) were considerably higher for the combined preparation of TAH I and TAH II.

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.

Immobilized preparation of cold-adapted and halotolerant Antarctic beta-galactosidase as a highly stable catalyst in lactose hydrolysis.

A cold-active beta-galactosidase of Antarctic marine bacterium Pseudoalteromonas sp. 22b was synthesized by an Escherichia coli transformant harboring its gene and immobilized on glutaraldehyde-treated chitosan beads. Unlike the soluble enzyme the immobilized preparation was not inhibited by glucose, its apparent optimum temperature for activity was 10 degrees C higher (50 vs. 40 degrees C, respectively), optimum pH range was wider (pH 6-9 and 6-8, respectively) and stability at 50 degrees C was increased whilst its pH-stability remained unchanged. Soluble and immobilized preparations of Antarctic beta-galactosidase were active and stable in a broad range of NaCl concentrations (up to 3 M) and affected neither by calcium ions nor by galactose. The activity of immobilized beta-galactosidase was maintained for at least 40 days of continuous lactose hydrolysis at 15 degrees C and its shelf life at 4 degrees C exceeded 12 months. Lactose content in milk was reduced by more than 90% over a temperature range of 4-30 degrees C in continuous and batch systems employing the immobilized enzyme.

Thermostable Pyrococcus woesei beta-D-galactosidase--high level expression, purification and biochemical properties.

The gene encoding beta-D-galactosidase from Pyrococcus woesei was PCR amplified, cloned, expressed in Escherichia coli under the control of an inducible T7 promoter, purified and characterized. The expression system was developed by the construction of recombinant plasmid, based on the high copy number pUET1 vector, giving four times more efficient expression of P. woesei beta-D-galactosidase (20 mg of enzyme from 1 liter of culture) than that obtained from a previously constructed one. The recombinant enzymes were purified in a two-step procedure: double heat-denaturation of E. coli cell proteins and affinity chromatography on p-aminobenzyl 1-thio-beta-D-galactopyranoside-agarose. To achieve efficient purification of P. woesei beta-D-galactosidase by immobilized metal-ion affinity chromatography (IMAC), a His-tag was placed either at the N- or the C-terminal of the coding sequence. The obtained fusion proteins revealed the same specific activity of approximately 5400 U/mg, which was 10 times lower than the wild-type beta-D-galactosidase (51100 U/mg). The activity of P. woesei beta-D-galactosidase was enhanced by thiol compounds, Mg(2+) ions and D-galactose, and was inhibited by heavy metal ions and D-glucose, while Ca(2+) ions had no effect.

Cloning, expression, and purification of a recombinant cold-adapted beta-galactosidase from antarctic bacterium Pseudoalteromonas sp. 22b.

The gram-negative antarctic bacterium Pseudoalteromonas sp. 22b, isolated from the alimentary tract of krill Thyssanoessa macrura, synthesizes an intracellular cold-adapted beta-galactosidase. The gene encoding this beta-galactosidase has been PCR amplified, cloned, expressed in Escherichia coli, purified, and characterized. The enzyme is active as a homotetrameric protein, and each monomer consists of 1028 amino acid residues. The enzyme was purified to homogeneity (50% recovery of activity) by using the fast, two-step procedure, including affinity chromatography on PABTG-Sepharose. Enzymatic properties of the recombinant protein are identical to those of native Pseudoalteromonas sp. 22b beta-galactosidase. The enzyme is cold-adapted and at 10 degrees C retains 20% of maximum activity. The purified enzyme displayed maximum activity close to 40 degrees C and at pH of 6.0-8.0. PNPG was its preferred substrate (58% higher activity than against ONPG). The enzyme was particularly thermolabile, losing all activities within 10 min at 50 degrees C. The hydrolysis of lactose in a milk assay revealed that 90% of milk lactose was hydrolyzed during 6 h at 30 degrees C and during 28 h at 15 degrees C. Because of its attributes, the recombinant Pseudoalteromonas sp. 22b beta-galactosidase could be applied at refrigeration temperatures for production of lactose-reduced dairy products.