Production of N-acetyl-D-neuraminic Acid by Recombinant Single Whole Cells Co-expressing N-acetyl-D-glucosamine-2-epimerase and N-acetyl-D-neuraminic Acid Aldolase.

N-acetyl-D-neuraminic acid (Neu5Ac) is a costly precursor for many drugs such as anti-influenza antivirals. In a previous study, a whole-cell process for Neu5Ac production was developed using a combination of two Escherichia coli cells expressing Anabaena sp. CH1 N-acetyl-D-glucosamine-2-epimerase (bage) and E. coli N-acetyl-D-neuraminic acid aldolase (nanA), respectively. In this study, we constructed a bAGE and NanA co-expression system to improve Neu5Ac production. Two recombinant E. coli strains, E. coli BL21 (DE3) pET-bage-nanA (HA) and E. coli BL21 (DE3) pET-bage-2nanA (HAA), synchronously expressing bAGE and NanA were used as biocatalysts to generate Neu5Ac from N-acetyl-D-glucosamine (GlcNAc) and pyruvate. The HA biocatalysts produced 187.5 mM Neu5Ac within 8 h. The yield of GlcNAc was 15.6%, and the Neu5Ac production rate was 7.25 g/L/h. The most active HAA biocatalysts generated 412.6 mM Neu5Ac and a GlcNAc yield of 34.4%. HAA achieved a Neu5Ac production rate of 15.9 g/L/h, which surpassed those for all reported Neu5Ac production processes so far. The present study demonstrates that using recombinant E. coli cells synchronously expressing bAGE and NanA as biocatalysts could potentially be used in the industrial mass production of Neu5Ac.

Purification and characterization of an aspartic protease from the Rhizopus oryzae protease extract, Peptidase R

Background Aspartic proteases are a subfamily of endopeptidases that are useful in a variety of applications, especially in the food processing industry. Here we describe a novel aspartic protease that was purified from Peptidase R, a commercial protease preparation derived from Rhizopus oryzae. Results An aspartic protease sourced from Peptidase R was purified to homogeneity by anion exchange chromatography followed by polishing with a hydrophobic interaction chromatography column, resulting in a 3.4-fold increase in specific activity (57.5 × 10³ U/mg) and 58.8% recovery. The estimated molecular weight of the purified enzyme was 39 kDa. The N-terminal sequence of the purified protein exhibited 63-75% identity to rhizopuspepsins from various Rhizopus species. The enzyme exhibited maximal activity at 75°C in glycine-HCl buffer, pH 3.4 with casein as the substrate. The protease was stable at 35°C for 60 min and had an observed half-life of approximately 30 min at 45°C. Enzyme activity was not significantly inhibited by chelation with ethylenediamine tetraacetic acid (EDTA), and the addition of metal ions to EDTA-treated protease did not significantly change enzyme activity, indicating that proteolysis is not metal ion-dependent. The purified enzyme was completely inactivated by the aspartic protease inhibitor Pepstatin A. Conclusion Based on the observed enzyme activity, inhibition profile with Pepstatin A, and sequence similarity to other rhizopuspepsins, we have classified this enzyme as an aspartic protease.

Tailored nanoparticles for tumour therapy.

Gd doped iron-oxide nanoparticles were developed for use in tumour therapy via magnetic fluid hyperthermia (MFH). The effect of the Gd3+ dopant on the particle size and magnetic properties was investigated. The final particle composition varied from Gd0.01Fe2.99O4 to Gd0.04Fe2.96O4 as determined by Inductively coupled plasma atomic emission spectroscopy (ICP-AES). TEM image analysis showed the average magnetic core diameters to be 12 nm and 33 nm for the lowest and highest Gd levels respectively. The specific power adsorption rate (SAR) determined with a field strength of 246 Oe and 52 kHz had a maximum of 38Wg(-1) [Fe] for the Gd0.03Fe2.97O4 sample. This value is about 4 times higher than the reported SAR values for Fe3O4. The potential for in vivo tumour therapy was investigated using a mouse model. The mouse models treated with Gd0.02Fe2.98O4 displayed much slower tumour growth after the first treatment cycle, the tumour had increased its mass by 25% after 7 days post treatment compared to a 79% mass increase over the same period for those models treated with standard iron-oxide or saline solution. After a second treatment cycle the mouse treated with Gd0.02Fe2.98O4 showed complete tumour regression with no tumour found for at least 5 days post treatment.

Expression, purification, crystallization and preliminary X-ray analysis of the D-alanyl carrier protein DltC from Staphylococcus epidermidis.

The D-alanyl lipoteichoic acids (D-alanyl LTAs) present in the cell walls of Gram-positive bacteria play crucial roles in autolysis, cation homeostasis and biofilm formation. The alanylation of LTAs requires the D-alanyl carrier protein DltC to transfer D-Ala onto a membrane-associated LTA. Here, DltC from Staphylococcus epidermidis (SeDltC) was purified and crystallized using the sitting-drop vapour-diffusion method. The crystals diffracted to a resolution of 1.83 Šand belonged to space group P2, with unit-cell parameters a = 66.26, b = 53.28, c = 88.05 Å, ß = 98.22°. The results give a preliminary crystallographic analysis of SeDltC and shed light on the functional role of DltC in the alanylation of LTAs.

Crystallization and preliminary X-ray diffraction analysis of a novel wild-type blue fluorescent protein from Vibrio vulnificus CKM-1.

The use of green fluorescent protein (GFP) for non-invasive in vivo imaging is limited to aerobic systems, as chromophore formation requires oxygen. However, a novel NADPH-dependent blue fluorescent protein from Vibrio vulnificus CKM-1 (BFPvv) that emits blue fluorescence in both aerobic and anaerobic systems has recently been discovered. Wild-type BFPvv was overexpressed in Escherichia coli, purified and crystallized using the sitting-drop vapour-diffusion method. The resulting BFPvv crystals diffracted to a resolution of 1.9 Å and belonged to space group P3, with unit-cell parameters a = b = 96.62, c = 214.511 Å. Assuming the presence of eight molecules in the unit cell, the solvent content was estimated to be ~56.16%.

Purification and characterization of a novel extracellular tripeptidyl peptidase from Rhizopus oligosporus.

A novel extracellular tripeptidyl peptidase (TPP) was homogenously purified from the culture supernatant of Rhizopus oligosporus by sequential fast protein liquid chromatography. The purified enzyme was a 136.5 kDa dimer composed of identical subunits. The effects of inhibitors and metal ions indicated that TPP is a metallo- and serine protease. TPP was activated by divalent cations, such as Co(2+) and Mn(2+), and completely inhibited by Cu(2+). Enzyme activity was optimal at pH 7.0 and 45 °C with a specific activity of 281.9 units/mg for the substrate Ala-Ala-Phe-pNA. The purified enzyme catalyzed cleavage of various synthetic tripeptides but not when proline occupied the P1 position. Purified TPP cleaved the pentapeptide Ala-Ala-Phe-Tyr-Tyr and tripeptide Ala-Ala-Phe, confirming the TPP activity of the enzyme.

A novel hydantoinase process using recombinant Escherichia coli cells with dihydropyrimidinase and L-N-carbamoylase activities as biocatalyst for the production of L-homophenylalanine.

A dihydropyrimidinase gene (pydB) was cloned from the moderate thermophilic Brevibacillus agri NCHU1002 and expressed in Escherichia coli. The purified dihydropyrimidinase exhibited strict d-enantioselectivity for D,L-p-hydroxyphenylhydantoin and D,L-5-[2-(methylthio)ethyl]hydantoin, and non-enantiospecificity for D,L-homophenylalanylhydantoin (D,L-HPAH). The hydrolytic activity of PydB was enhanced notably by Mn2+, with a maximal activity at 60 degrees C and pH 8.0. This enzyme was completely thermostable at 50 degrees C for 20 days. A whole cell biocatalyst for the production of L-homophenylalanine (L-HPA) from D,L-HPAH by coexpression of the pydB gene and a thermostable L-N-carbamoylase gene from Bacillus kaustophilus CCRC11223 in E. coli JM109 was developed. The expression levels of dihydropyrimidinase and L-N-carbamoylase in the recombinant E. coli cells were estimated to be about 20% of the respective total soluble proteins. When 1% (w/v) isopropyl-beta-D-thiogalactopyranoside-induced cells were used as biocatalysts, a conversion yield of 49% for L-HPA with more than 99% ee could be reached in 16 h at pH 7.0 from 10mM D,L-HPAH. The cells can be reused for at least eight cycles at a conversion yield of more than 43%. Our results revealed that coexpression of pydB and lnc in E. coli might be a potential biocatalyst for L-HPA production.

Enantioselective synthesis of L-homophenylalanine by whole cells of recombinant Escherichia coli expressing L-aminoacylase and N-acylamino acid racemase genes from Deinococcus radiodurans BCRC12827.

L-Homophenylalanine (l-HPA) is a chiral unnatural amino acid used in the synthesis of angiotensin converting enzyme inhibitors and many pharmaceuticals. To develop a bioconversion process with dynamic resolution of N-acylamino acids for the l-HPA production, N-acylamino acid racemase (NAAAR) and l-aminoacylase (LAA) genes were cloned from Deinococcus radiodurans BCRC12827 and expressed in Escherichia coli XLIBlue. The recombinant enzymes were purified by nickel-chelate chromatography, and their biochemical properties were determined. The NAAAR had high racemization activity toward chiral N-acetyl-homophenylalanine (NAc-HPA). The LAA exhibited strict l-enantioselection to hydrolyze the NAc-l-HPA. A stirred glass vessel containing transformed E. coli cells expressing D. radiodurans NAAAR and LAA was used for the conversion of NAc-d-HPA to l-HPA. Unbalance activities of LAA and NAAAR were found in E. coli cell coexpressing laa and naaar genes, which resulted in the accumulation of an intermediate, NAc-l-HPA, in the early stage of conversion and a low productivity of 0.83 mmol l-HPA/L h. The results indicated that low activity of LAA present in the biomass is the rate-limiting factor in l-HPA production. In the case of two whole cells with separately expressed enzyme, the enzymatic activities of LAA and NAAAR could be balanced by changing the loading of individual cells. When the activities of two enzymes were fixed at 3600 U/L, 99.9% yield of l-HPA could be reached in 1 h, with a productivity of 10 mmol l-HPA/L h. The cells can be reused at least six cycles at a conversion yield of more than 96%. This is the first NAAAR/LAA process using NAc-HPA as substrate and recombinant whole cells containing Deinococcus enzymes as catalysts for the production of l-HPA to be reported.

Enantioselective synthesis of (S)-2-amino-4-phenylbutanoic acid by the hydantoinase method.

Biosynthesis of (S)-(+)-2-amino-4-phenylbutanoic acid (1) was performed by nonenantioselective hydantoinase and L-N-carbamoylase using racemic 5-[2-phenylethyl]-imidazolidine-2,4-dione (rac-2) as a substrate. The compounds involved in this biocatalysis process could be simultaneously resolved by high-performance liquid chromatography using Chirobiotic T column with a mobile phase of EtOH/H(2)O = 10/90 at pH 4.2-4.5. To our knowledge, this is the first report of the successful production of 1 by the combination of recombinant hydantoinase and L-N-carbamoylase.

A gene cluster involved in pyrimidine reductive catabolism from Brevibacillus agri NCHU1002.

Genes involved in pyrimidine reductive catabolism (pyd) were isolated from a moderate thermophile, Brevibacillus agri NCHU1002, and nine ORFs in an 8.2-kb DNA fragment were identified by DNA sequence analysis. The pyd gene cluster included three closely spaced ORFs, designated pydA, pydB, and pydC, transcribed in the same orientation. Based on their amino acid sequence identity and enzyme activity assay, the gene products were identified as dihydropyrimidine dehydrogenase (PydA), dihydropyrimidinase (PydB), and beta-alanine synthase (PydC). Northern blot and primer extension analyses revealed that the pydBC genes are induced by dihydrouracil and regulated under the control of sigma(54) recognized promoter at transcriptional level as a polycistronic operon. All results indicate that the pydABC genes participate in the pathway of the pyrimidine reductive catabolism. This is the first bacterial pyd gene cluster to be reported.