The phenotypic and genomic diversity of Aspergillus strains producing glucose dehydrogenase.

Twelve Aspergillus sp. strains producing glucose dehydrogenase were identified using ITS region sequencing. Based on the sequences obtained, the genomic relationship of the analyzed strains was investigated. Moreover, partial gdh gene sequences were determined and aligned. The amplified fragment length polymorphism (AFLP) method was applied for genomic fingerprinting of twelve Aspergillus isolates. Using one PstI restriction endonuclease and five selective primers in an AFLP assay, 556 DNA fragments were generated, including 532 polymorphic bands. The AFLP profiles were found to be highly specific for each strain and they unambiguously distinguished twelve Aspergilli fungi. The AFLP-based dendrogram generated by the UPGMA method grouped all the Aspergillus fungi studied into two major clusters. All the Aspergillus strains were also characterized using Biolog FF MicroPlates to obtain data on C-substrate utilization and mitochondrial activity. The ability to decompose various substrates differed among the analyzed strains up to three folds. All of the studied strains mainly decomposed carbohydrates.

Response surface methodology to optimize partition and purification of two recombinant oxidoreductase enzymes, glucose dehydrogenase and d-galactose dehydrogenase in aqueous two-phase systems.

Oxidoreductases are an important family of enzymes that are used in many biotechnological processes. An experimental design was applied to optimize partition and purification of two recombinant oxidoreductases, glucose dehydrogenase (GDH) from Bacillus subtilis and d-galactose dehydrogenase (GalDH) from Pseudomonas fluorescens AK92 in aqueous two-phase systems (ATPS). Response surface methodology (RSM) with a central composite rotatable design (CCRD) was performed to optimize critical factors like polyethylene glycol (PEG) concentration, concentration of salt and pH value. The best partitioning conditions was achieved in an ATPS composed of 12% PEG-6000, 15% K2HPO4 with pH 7.5 at 25°C, which ensured partition coefficient (KE) of 66.6 and 45.7 for GDH and GalDH, respectively. Under these experimental conditions, the activity of GDH and GalDH was 569.5U/ml and 673.7U/ml, respectively. It was found that these enzymes preferentially partitioned into the top PEG-rich phase and appeared as single bands on SDS-PAGE gel. Meanwhile the validity of the response model was confirmed by a good agreement between predicted and experimental results. Collectively, according to the obtained data it can be inferred that the ATPS optimization using RSM approach can be applied for recovery and purification of any enzyme from oxidoreductase family.

Engineering of recombinant E. coli cells co-expressing P450pyrTM monooxygenase and glucose dehydrogenase for highly regio- and stereoselective hydroxylation of alicycles with cofactor recycling.

E. coli (P450pyrTM-GDH) with dual plasmids, pETDuet containing P450pyr triple mutant I83H/M305Q/A77S (P450pyrTM) and ferredoxin reductase (FdR) genes and pRSFDuet containing glucose dehydrogenase (GDH) and ferredoxin (Fdx) genes, was engineered to show a high activity (12.7 U g⁻¹ cdw) for the biohydroxylation of N-benzylpyrrolidine 1 and a GDH activity of 106 U g⁻¹ protein. The E. coli cells were used as efficient biocatalysts for highly regio- and stereoselective hydroxylation of alicyclic substrates at non-activated carbon atom with enhanced productivity via intracellular recycling of NAD(P)H. Hydroxylation of N-benzylpyrrolidine 1 with resting cells in the presence of glucose showed excellent regio- and stereoselectivity, giving (S)-N-benzyl-3-hydroxypyrrolidine 2 in 98% ee as the sole product in 9.8 mM. The productivity is much higher than that of the same biohydroxylation using E. coli (P450pyrTM)b without expressing GDH. E. coli (P450pyrTM-GDH) was found to be highly regio- and stereoselective for the hydroxylation of N-benzylpyrrolidin-2-one 3, improving the regioselectivity from 90% of the wild-type P450pyr to 100% and giving (S)-N-benzyl-4-hydroxylpyrrolidin-2-one 4 in 99% ee as the sole product. A high activity of 15.5 U g⁻¹ cdw was achieved and (S)-4 was obtained in 19.4 mM. E. coli (P450pyrTM-GDH) was also found to be highly regio- and stereoselective for the hydroxylation of N-benzylpiperidin-2-one 5, increasing the ee of the product (S)-N-benzyl-4-hydroxy-piperidin-2-one 6 to 94% from 33% of the wild-type P450pyr. A high activity of 15.8 U g⁻¹ cdw was obtained and (S)-6 was produced in 3.3 mM as the sole product. E. coli (P450pyrTM-GDH) represents the most productive system known thus far for P450-catalyzed hydroxylations with cofactor recycling, and the hydroxylations with E. coli (P450pyrTM-GDH) provide with simple and useful syntheses of (S)-2, (S)-4, and (S)-6 that are valuable pharmaceutical intermediates and difficult to prepare.

Transgenic expression of glucose dehydrogenase in Azotobacter vinelandii enhances mineral phosphate solubilization and growth of sorghum seedlings.

The enzyme quinoprotein glucose dehydrogenase (GDH) catalyses the oxidation of glucose to gluconic acid by direct oxidation in the periplasmic space of several Gram-negative bacteria. Acidification of the external environment with the release of gluconic acid contributes to the solubilization of the inorganic phosphate by biofertilizer strains of the phosphate-solubilizing bacteria. Glucose dehydrogenase (gcd) gene from Escherichia coli, and Azotobacter-specific glutamine synthetase (glnA) and phosphate transport system (pts) promoters were isolated using sequence-specific primers in a PCR-based approach. Escherichia coli gcd, cloned under the control of glnA and pts promoters, was mobilized into Azotobacter vinelandii AvOP and expressed. Sorghum seeds were bacterized with the transgenic azotobacters and raised in earthen pots in green house. The transgenic azotobacters, expressing E. coli gcd, showed improved biofertilizer potential in terms of mineral phosphate solubilization and plant growth-promoting activity with a small reduction in nitrogen fixation ability.

Over-expression of glucose dehydrogenase improves cell growth and riboflavin production in Bacillus subtilis.

Ribulose 5-phosphate is a precursor for riboflavin biosynthesis. Alteration of carbon flow into the pentose phosphate pathway will affect the availability of ribulose 5-phosphate and the riboflavin yield. We have modulated carbon flow in Bacillus subtilis through the gluconate bypass by over-expression of glucose dehydrogenase under the control of the constitutively expressed P43 promoter. Over-expression of glucose dehydrogenase resulted in low acid production (acetate and pyruvate). The substantial reduction in acid production is accompanied by increased riboflavin production and an increased rate of growth while glucose consumption remained unchanged. Metabolic analysis indicated that over-expression of glucose dehydrogenase increased intracellular pool of ribulose 5-phosphate. The high concentrations of ribulose 5-phosphate could explain the increased riboflavin production.

A fast method for quantitative proteomics based on a combination between two-dimensional electrophoresis and 15N-metabolic labelling.

We provide a method for accurate protein quantitation that uses two-dimensional (2-D) gel electrophoresis for protein separation, but does not require extensive statistical analysis of staining intensities on gels. Instead, accurate quantitation occurs on the mass spectrometer (MAS) on multiple peptides to provide statistical evidence. In an example study, Sulfolobus solfataricus cells were grown on the carbon sources glucose, fructose and glutamate. The glucose phenotype (reference) was grown on (15)N-enriched medium. Next, the glutamate and the fructose phenotypes are mixed with the reference and two 2-D gels are created. Staining intensities of gel spots in this case are used for initial, semiquantitative assessment of differential expression. On this basis, spots are selected for accurate quantitation on the MAS. A number of differentially expressed proteins were found, for example: a (25.2 +/- 8.2)-fold upregulation of isocitrate lyase and a (7.14 +/- 0.82)-fold downregulation of glucose dehydrogenase on glutamate compared to glucose. With this protocol, intergel and interlaboratory comparisons are facilitated, since the light and heavy versions of a protein are equally affected by variations in sample preparation and buffer composition. Because the statistical evidence is gathered on the MAS, the need to run vast numbers of gels is removed.

Essential role of the small subunit of thermostable glucose dehydrogenase from Burkholderia cepacia.

The co-expression in Escherichia coli of the gamma-subunit and the catalytic alpha-subunit of the thermostable glucose dehydrogenase (GDH) from Burkholderia cepacia sp. SM4 produced 12.7 U GDH activity mg(-1) protein. A 47-amino acid, twin-arginine translocase signal peptide was identified at the amino terminus of the gamma-subunit. The expression of the alpha-subunit in the absence of the gamma-subunit or the gamma-subunit signal peptide failed to produce any detectable GDH protein or activity. The gamma-subunit may be a chaperone-like component that assists folding of the alpha-subunit polypeptide to the active form and its translocation to the periplasm.