Micromol Min Research Articles

Purification and characterization of an intracellular β-glucosidase from the protoplast fusant of Aspergillus oryzae and Aspergillus niger

Protoplasts of Aspergillus oryzae 3.481 and Aspergillus niger 3.316 were prepared using cellulose and snail enzyme with 0.6 M NaCl as osmotic stabilizer. Protoplast fusion has been performed using 35% polyethylene glycol 4.000 with 0.01 mM CaCl2. The fused protoplasts have been regenerated on regeneration medium and fusants were selected for further studies. An intracellular beta-glucosidase (EC 3.2.1.21) was purified from the protoplast fusant of Aspergillus oryzae 3.481 and Aspergillus niger 3.316 and characterized. The enzyme was purified 138.85-fold by ammonium sulphate precipitation, DE-22 ion exchange and Sephadex G-150 gel filtration chromatography with a specific activity of 297.14 U/mg of protein. The molecular mass of the purified enzyme was determined to be about 125 kDa by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The enzyme had an optimum pH of 5.4 and temperature of 65 degrees C, respectively. This enzyme showed relatively high stability against pH and temperature and was stable in the pH range of 3.0-6.6. Na+, K+, Ca2+, Mg2+ and EDTA completely inhibited the enzyme activity at a concentration of 10 mM. The enzyme activity was accelerated by Fe3+. The enzyme activity was strongly inhibited by glucose, the end product ofglucoside hydrolysis. The K(m) and V(max) values against salicin as substrate were 0.035 mM and 1.7215 micromol min(-1), respectively.

Cloning, biochemical characterisation, tissue localisation and possible post-translational regulatory mechanism of the cytosolic phosphoglucose isomerase from developing sunflower seeds

Lipid biosynthesis in developing sunflower (Helianthus annuus L.) seeds requires reducing power. One of the main sources of cellular NADPH is the oxidative pentose phosphate pathway (OPPP), generated from the oxidation of glucose-6-phosphate. This glycolytic intermediate, which can be imported to the plastid and enter in the OPPP, is the substrate and product of cytosolic phosphoglucose isomerase (cPGI, EC 5.3.1.9). In this report, we describe the cloning of a full-length cDNA encoding cPGI from developing sunflower seeds. The sequence was predicted to code for a protein of 566 residues characterised by the presence of two sugar isomerase domains. This cDNA was heterologously expressed in Escherichia coli as a His-tagged protein. The recombinant protein was purified using immobilised metal ion affinity chromatography and biochemically characterised. The enzyme had a specific activity of 1,436 micromol min(-1) mg(-1) and 1,011 micromol min(-1) mg(-1) protein when the reaction was initiated with glucose-6-phosphate and fructose-6-phosphate, respectively. Activity was not affected by erythrose-4-phosphate, but was inhibited by 6-P gluconate and glyceraldehyde-3-phosphate. A polyclonal immune serum was raised against the purified enzyme, allowing the study of protein levels during the period of active lipid synthesis in seeds. These results were compared with PGI activity profiles and mRNA expression levels obtained from Q-PCR studies. Our results point to the existence of a possible post-translational regulatory mechanism during seed development. Immunolocalisation of the protein in seed tissues further indicated that cPGI is highly expressed in the procambial ring.

Characterization of Xyn10A, a highly active xylanase from the human gut bacterium Bacteroides xylanisolvens XB1A

A xylanase gene xyn10A was isolated from the human gut bacterium Bacteroides xylanisolvens XB1A and the gene product was characterized. Xyn10A is a 40-kDa xylanase composed of a glycoside hydrolase family 10 catalytic domain with a signal peptide. A recombinant His-tagged Xyn10A was produced in Escherichia coli and purified. It was active on oat spelt and birchwood xylans and on wheat arabinoxylans. It cleaved xylotetraose, xylopentaose, and xylohexaose but not xylobiose, clearly indicating that Xyn10A is a xylanase. Surprisingly, it showed a low activity against carboxymethylcellulose but no activity at all against aryl-cellobioside and cellooligosaccharides. The enzyme exhibited K (m) and V (max) of 1.6 mg ml(-1) and 118 micromol min(-1) mg(-1) on oat spelt xylan, and its optimal temperature and pH for activity were 37 degrees C and pH 6.0, respectively. Its catalytic properties (k (cat)/K (m) = 3,300 ml mg(-1) min(-1)) suggested that Xyn10A is one of the most active GH10 xylanase described to date. Phylogenetic analyses showed that Xyn10A was closely related to other GH10 xylanases from human Bacteroides. The xyn10A gene was expressed in B. xylanisolvens XB1A cultured with glucose, xylose or xylans, and the protein was associated with the cells. Xyn10A is the first family 10 xylanase characterized from B. xylanisolvens XB1A.

Engineering of a fungal β-galactosidase to remove product inhibition by galactose

Beta-galactosidase is an enzyme administered as a digestive supplement to treat lactose intolerance, a genetic condition prevalent in most world regions. The gene encoding an acid-stable beta-galactosidase potentially suited for use as a digestive supplement was cloned from Aspergillus niger van Tiegh, sequenced and expressed in Pichia pastoris. The purified recombinant protein exhibited kinetic properties similar to those of the native enzyme and thus was also competitively inhibited by its product, galactose, at application-relevant concentrations. In order to alleviate this product inhibition, a model of the enzyme structure was generated based on a Penicillium sp. beta-galactosidase crystal structure with bound beta-galactose. This led to targeted mutagenesis of an Asp(258)-Ser-Tyr-Pro-Leu-Gly-Phe amino acid motif in the A. niger van Tiegh enzyme and isolation from the resultant library of a mutant beta-galactosidase enzyme with reduced sensitivity to inhibition by galactose (K (i) of 6.46 mM galactose, compared with 0.76 mM for the wildtype recombinant enzyme). The mutated enzyme also exhibited an increased K (m) (3.76 mM compared to 2.21 mM) and reduced V (max) (110.8 micromol min(-1) mg(-1) compared to 172.6 micromol min(-1) mg(-1)) relative to the wild-type enzyme, however, and its stability under simulated fasting gastric conditions was significantly reduced. The study nevertheless demonstrates the potential to rationally engineer the A. niger van Tiegh enzyme to relieve product inhibition and create mutants with improved, application-relevant kinetic properties for treatment of lactose intolerance.

Hydrolysis of cyclic poly(ethylene terephthalate) trimers by a carboxylesterase from Thermobifida fusca KW3

We have identified a carboxylesterase produced in liquid cultures of the thermophilic actinomycete Thermobifida fusca KW3 that were supplemented with poly(ethylene terephthalate) fibers. The enzyme hydrolyzed highly hydrophobic, synthetic cyclic poly(ethylene terephthalate) trimers with an optimal activity at 60 degrees C and a pH of 6. V (max) and K (m) values for the hydrolysis were 9.3 micromol(-1) min(-1) mg(-1) and 0.5 mM, respectively. The esterase showed high specificity towards short and middle chain-length fatty acyl esters of p-nitrophenol. The enzyme retained 37% of its activity after 96 h of incubation at 50 degrees C and a pH of 8. Enzyme inhibition studies and analysis of substitution mutants of the carboxylesterase revealed the typical catalytic mechanism of a serine hydrolase with a catalytic triad composed of serine, glutamic acid, and histidine.

A family history of type 2 diabetes increases risk factors associated with overfeeding

The purpose of the study was to test prospectively whether healthy individuals with a family history of type 2 diabetes are more susceptible to adverse metabolic effects during experimental overfeeding. We studied the effects of 3 and 28 days of overfeeding by 5,200 kJ/day in 41 sedentary individuals with and without a family history of type 2 diabetes (FH+ and FH- respectively). Measures included body weight, fat distribution (computed tomography) and insulin sensitivity (hyperinsulinaemic-euglycaemic clamp). Body weight was increased compared with baseline at 3 and 28 days in both groups (p < 0.001), FH+ individuals having gained significantly more weight than FH- individuals at 28 days (3.4 +/- 1.6 vs 2.2 +/- 1.4 kg, p < 0.05). Fasting serum insulin and C-peptide were increased at 3 and 28 days compared with baseline in both groups, with greater increases in FH+ than in FH- for insulin at +3 and +28 days (p < 0.01) and C-peptide at +28 days (p < 0.05). Fasting glucose also increased at both time points, but without a significant group effect (p = 0.1). Peripheral insulin sensitivity decreased in the whole cohort at +28 days (54.8 +/- 17.7 to 50.3 +/- 15.6 micromol min(-1) [kg fat-free mass](-1), p = 0.03), and insulin sensitivity by HOMA-IR decreased at both time points (p < 0.001) and to a greater extent in FH+ than in FH- (p = 0.008). Liver fat, subcutaneous and visceral fat increased similarly in the two groups (p < 0.001). Overfeeding induced weight and fat gain, insulin resistance and hepatic fat deposition in healthy individuals. However, individuals with a family history of type 2 diabetes gained more weight and greater insulin resistance by HOMA-IR. The results of this study suggest that healthy individuals with a family history of type 2 diabetes are predisposed to adverse effects of overfeeding. ClinicalTrials.gov NCT00562393 The study was funded by the National Health and Medical Research Council (NHMRC), Australia (no. #427639).

TRIB3 R84 variant affects glucose homeostasis by altering the interplay between insulin sensitivity and secretion

The results of studies on the genetics of complex traits need to be replicated and to reach robust statistical significance before they can be considered as established. We here tried to replicate the previously reported association between the TRIB3 Q84R polymorphism (rs2295490) and glucose homeostasis. Three samples of Europeans with fasting glucose <7.0 mmol/l were studied. In sample 1 (n=791), the association between TRIB3 Q84R and impaired glucose regulation (IGR; defined as impaired fasting glucose and/or impaired glucose tolerance and/or type 2 diabetes by OGTT) and insulin sensitivity (ISI), and its interplay with early-phase insulin secretion (i.e. disposition index [DI]) were analysed. Sample 2 (n=374) and sample 3 (n=394) were used to replicate the association with IGR and insulin sensitivity (by glucose clamp), respectively. Genotyping was performed by TaqMan allele discrimination. R84 carriers were at higher risk of IGR: OR for the additive model 1.54, p=0.004, and 1.63, p=0.027, in samples 1 and 2, respectively. In sample 1, both ISI (p=0.005) and DI (p=0.043) were progressively lower from QQ to QR and RR individuals. A 'triangulation approach' indicated that the association with IGR was mostly mediated by DI rather than by ISI changes (i.e. being the expected ORs 1.51 and 1.25, respectively). In sample 3, glucose disposal was 38.8+/-17.7, 33.8+/-14.4, and 31.6+/-13.3 micromol min(-1)kg(-1), p=0.022, in QQ, QR and RR individuals, respectively. Our data confirm that the TRIB3 R84 variant affects glucose homeostasis and suggest this effect is due to an alteration of the interplay between insulin sensitivity and secretion.

Novel biosynthesis of (R)-ethyl-3-hydroxyglutarate with (R)-enantioselective hydrolysis of racemic ethyl 4-cyano-3-hydroxybutyate by Rhodococcus erythropolis

(R)-ethyl-3-hydroxyglutarate with highly optical purity (> or = 99%) can be used as a novel precursor for synthesis of chiral side chain of rosuvastatin. In this study, a novel synthesis route of (R)-ethyl-3-hydroxyglutarate by whole microorganism cells from racemic ethyl 4-cyano-3-hydroxybutyate was created. A strain ZJB-0910 capable of transforming racemic beta-hydroxy aliphatic nitrile was isolated by employing a screening method based on a colorimetric reaction of Co(2+) ion with ammonia, and identified as Rhodococcus erythropolis based on its morphology, physiological tests, Biolog, and the 16S rDNA sequence. After cultivation in a sterilized medium with composition of 20 g glucose, 5 g yeast extract, 0.5 g KH(2)PO(4), 0.5 g K(2)HPO(4), 0.2 g MgSO(4).7H(2)O per liter at 30 degrees C and 150 rpm for 48 h, the whole cells of R. erythropolis ZJB-0910 were prepared as a catalyst in (R)-enantioselective hydrolysis of racemic ethyl 4-cyano-3-hydroxybutyate for synthesis of (R)-ethyl-3-hydroxyglutarate, without bearing hydrolase activity for the ester bond of ethyl 4-cyano-3-hydroxybutyate. Under the optimized biotransformation conditions of pH 7.5, 30 degrees C,and 20 mM substrate concentration, (R)-ethyl-3-hydroxyglutarate with 46.2% yield (ee>99%) was afforded, and its chemical structure was determined by ESI-MS, NMR, and IR. The apparent Michaelis constant K(m) and maximum rate Vmax for this biocatalytic reaction were 0.01 M and 85.6 micromol min(-1)g(-1), respectively.

Arginine Metabolising Enzymes as Therapeutic Tools for Alzheimer’s Disease: Peptidyl Arginine Deiminase Catalyses Fibrillogenesis of β-amyloid Peptides

The accumulation of arginine in the cerebrospinal fluid and brains of patients suffering from acute neurodegenerative diseases like Alzheimer's disease, point to defects in the metabolic pathways involving this amino acids. The deposits of neurofibrillary tangles and senile plaques perhaps as a consequence of fibrillogenesis of beta-amyloid peptides has also been shown to be a hallmark in the aetiology of certain neurodegenerative diseases. Peptidylarginine deiminase (PAD II) is an enzyme that uses arginine as a substrate and we now show that PAD II not only binds with the peptides Abeta(1-40), Abeta(22-35), Abeta(17-28), Abeta(25-35) and Abeta(32-35) but assists in the proteolytic degradation of these peptides with the concomitant formation of insoluble fibrils. PAD was purified in 12.5% yield and 137 fold with a specific activity of 59 micromol min(-1) mg(-1) from bovine brain by chromatography on diethylaminoethyl (DEAE)-Sephacel. Characterisation of the enzyme gave a pH and temperature optima of 7.5 degrees C and 68 degrees C, respectively, and the enzyme lost 50% activity within 38 min at this temperature. Michaelis-Menten kinetics established a V(max) and K(m) of 1.57 micromol min(-1) ml(-1) and 1.35 mM, respectively, with N-benzoyl arginine ethyl ester as substrate. Kinetic analysis was used to measure the affinity (K(i)) of the amyloid peptides to PAD with values between 1.4 and 4.6 microM. The formation of Abeta fibrils was rate limiting involving an initial lag time of about 24 h that was dependent on the concentration of the amyloid peptides. Turbidity measurements at 400 nm, Congo Red assay and Thioflavin-T staining fluorescence were used to establish the aggregation kinetics of PAD-induced fibril formation.

Decreased leg glucose uptake during exercise contributes to the hyperglycaemic effect of octreotide

During prolonged infusion of somatostatin, there is an increase in arterial glucose concentration, and this increase persists even during prolonged exercise. The aim of the study was to measure glucose uptake in the leg muscles during infusion of the somatostatin analogue octreotide before and during leg exercise. Eight healthy male subjects were investigated twice in the fasting state: during 3 h infusion of octreotide [30 ng (kg min)(-1)] or sodium chloride with exercise at 50% of maximal VO(2) in the last hour. Glucose uptake and oxygen uptake in the leg were measured using Fick's principle by blood sampling from an artery and a femoral vein. Blood flow in the leg was measured using the indicator (indocyanine green) dilution technique. After an initial decrease during rest, octreotide infusion resulted in a significant increase in arterial glucose concentrations compared to control conditions during exercise (mean +/- SEM: 7.6 +/- 0.6 versus 5.6 +/- 0.1 mmol l(-1), P<0.01). During rest, octreotide did not change the leg glucose uptake (59 +/- 10 versus 55 +/- 11 micromol min(-1)). In contrast, leg glucose uptake was significantly lower during exercise compared to control conditions (208 +/- 79 versus 423 +/- 87 micromol min(-1), P<0.05). During exercise, leg oxygen uptake was not different in the two experiments (20.4 +/- 1.3 versus 19.5 +/- 1.1 micromol min(-1)). In conclusion, infusion of octreotide reduced leg glucose uptake during exercise, despite the same leg oxygen consumption and blood flow compared to control conditions. The hyperglycaemic effect of octreotide can partly be explained by the reduction in leg glucose uptake. Furthermore, the results suggest that a certain level of circulating insulin is necessary to obtain sufficient stimulation of glucose uptake in the exercising muscles.

Molecular cloning and characterization of the novel acidic xylanase XYLD from Bispora sp. MEY-1 that is homologous to family 30 glycosyl hydrolases

We cloned and sequenced a xylanase gene named xylD from the acidophilic fungus Bispora sp. MEY-1 and expressed the gene in Pichia pastoris. The 1,422-bp full-length complementary DNA fragment encoded a 457-amino acid xylanase with a calculated molecular mass of 49.8 kDa. The mature protein of XYLD showed high sequence similarity to both glycosyl hydrolase (GH) families 5 and 30 but was more homologous to members of GH 30 based on phylogenetic analysis. XYLD shared the highest identity (49.9%) with a putative endo-1,6-beta-D-glucanase from Talaromyces stipitatus and exhibited 21.1% identity and 34.3% similarity to the well-characterized GH family 5 xylanase from Erwinia chrysanthemi. Purified recombinant XYLD showed maximal activity at pH 3.0 and 60 degrees C, maintained more than 60% of maximal activity when assayed at pH 1.5-4.0, and had good thermal stability at 60 degrees C and remained stable at pH 1.0-6.0. The enzyme activity was enhanced in the presence of Ni(2+) and beta-mercaptoethanol and inhibited by some metal irons (Hg(2+), Cu(2+), Pb(2+), Mn(2+), Li(+), and Fe(3+)) and sodium dodecyl sulfate. The specific activity of XYLD for beechwood xylan, birchwood xylan, 4-O-methyl-D-glucuronoxylan, and oat spelt xylan was 2,463, 2,144, 2,020, and 1,429 U mg(-1), respectively. The apparent K (m) and V (max) values for beechwood xylan were 5.6 mg ml(-1) and 3,622 micromol min(-1) mg(-1), respectively. The hydrolysis products of different xylans were mainly xylose and xylobiose.

Biochemical analysis of a recombinant glutathione transferase from the cestode Echinococcus granulosus

Glutathione transferases (GSTs) are believed to be a major detoxification system in helminths. We describe the expression and functional analysis of EgGST, a cytosolic GST from Echinococcus granulosus, related to the Mu-class of mammalian enzymes. EgGST was produced as an enzymatically active dimeric protein (rEgGST), with highest specific activity towards the standard substrate 1-chloro-2,4-dinitrobenzene (CDNB; 2.5 micromol min(-1)mg(-1)), followed by ethacrynic acid. Interestingly, rEgGST displayed glutathione peroxidase activity (towards cumene hydroperoxide), and conjugated reactive carbonyls (trans-2-nonenal and trans,trans-2,4-decadienal), indicating that it may intercept damaging products of lipid peroxidation. In addition, classical GST inhibitors (cybacron blue, triphenylthin chloride and ellagic acid) and a number of anthelmintic drugs (mainly, hexachlorophene and rafoxanide) were found to interfere with glutathione-conjugation to CDNB; suggesting that they may bind to EgGST. Considered globally, the functional properties of rEgGST are similar to those of putative orthologs from Echinococcus multilcularis and Taenia solium, the other medically important cestodes. Interestingly, our results also indicate that differences exist between these closely related cestode GSTs, which probably reflect specific biological functions of the molecules in each parasitic organism.

Gene Cloning, Expression, and Characterization of a Thermostable Xylanase from Nesterenkonia xinjiangensis CCTCC AA001025

An endo-beta-1,4-xylanase-encoding gene, xyn11NX, was cloned from Nesterenkonia xinjiangensis CCTCC AA001025 and expressed in Escherichia coli. The gene encoded a 192-amino acid polypeptide and a putative 50-amino acid signal peptide. The deduced amino acid sequence exhibited a high degree of similarity with the xylanases from Streptomyces thermocyaneoviolaceus (68%) and Thermobifida fusca (66%) belonging to glycoside hydrolase family 11. After purification to homogeneity, the recombinant Xyn11NX exhibited optimal activity at pH 7.0 and 55 degrees C and remained stable at weakly acidic to alkaline pH (pH 5.0-11.0). The enzyme was thermostable, retaining more than 80% of the initial activity after incubation at 60 degrees C for 1 h and more than 40% of the activity at 90 degrees C for 15 min. The K (m) and V (max) values for oat spelt xylan and birchwood xylan were 16.08 mg ml(-1) and 45.66 micromol min(-1) mg(-1) and 9.22 mg ml(-1) and 16.05 micromol min(-1) mg(-1), respectively. The predominant hydrolysis products were xylobiose and xylotriose when using oat spelt xylan or birchwood xylan as substrate.

A novel Ca2+-dependent phospholipase D from Streptomyces tendae, possessing only hydrolytic activity

An extracellular phospholipase D (PLD(St)) was purified from Streptomyces tendae by two successive chromatographic steps on Sepharose CL-6B and DEAE-Sepharose CL-6B. Molecular weight of the PLD(St) was estimated to be approximately 43 kDa by sodium dodecyl sulfatepolyacrylamide gel electrophoresis. Maximal activity was at pH 8 and 60 degrees C, and the enzyme was stable at or below 60 degrees C and between pH 8 and 10, when assayed after 1.5 and 24 h, respectively. The enzyme activity had an absolute requirement of Ca(2+), and the maximum activity was at 2 mM CaCl(2). The Km and Vmax values for phosphatidyl choline were 0.95 mM and 810 micromol min(-1) mg(-1), respectively. More importantly, PLD(St) could not catalyze transphosphatidylation of glycerol, L-serine, myo-inositol and ethanolamine, which have been extensively used to evaluate the activity. The result strongly suggests that PLD( St ) does not have the transphosphatidylation activity, thereby making it the first Streptomyces PLD possessing only hydrolytic activity. PLD(St) may therefore be a novel type of PLD enzyme.

Expression of a Glucose-tolerant β-glucosidase from Humicola grisea var. thermoidea in Saccharomyces cerevisiae

A beta-glucosidase gene (bgl4) from Humicola grisea var thermoidea was successfully expressed in Saccharomyces cerevisiae. The recombinant protein (BGL4(Sc)) was initially detected associated with yeast cells and later in the culture medium. BGL4(Sc) showed optimal pH and temperature of 6.0 and 40 degrees C, respectively, and an apparent molecular mass of 57 kDa. The enzyme showed activity against cellobiose and synthetic substrates, and was inhibited more than 80% by Fe2+, Cu2+, Zn2+, and Al3+. Using p-nitrophenyl-beta-D-glucopyranoside (pNPG) as substrate, BGL4(Sc) presented a V(max) of 6.72 micromol min(-1) mg total protein(-1) and a K (m) of 0.16 mM under optimal conditions. Most important, BGL4(Sc) is resistant to inhibition by glucose and the calculated K (i) value for this sugar is 70 mM. This feature prompts BLG4(Sc) as an ideal enzyme to be used in the saccharification process of lignocellulosic materials for ethanol production.

Microbial fuel cell with an azo-dye-feeding cathode

Microbial fuel cells (MFCs) were constructed using azo dyes as the cathode oxidants to accept the electrons produced from the respiration of Klebsiella pneumoniae strain L17 in the anode. Experimental results showed that a methyl orange (MO)-feeding MFC produced a comparable performance against that of an air-based one at pH 3.0 and that azo dyes including MO, Orange I, and Orange II could be successfully degraded in such cathodes. The reaction rate constant (k) of azo dye reduction was positively correlated with the power output which was highly dependent on the catholyte pH and the dye molecular structure. When pH was varied from 3.0 to 9.0, the k value in relation to MO degradation decreased from 0.298 to 0.016 micromol min(-1), and the maximum power density decreased from 34.77 to 1.51 mW m(-2). The performances of the MFC fed with different azo dyes can be ranked from good to poor as MO>Orange I>Orange II. Furthermore, the cyclic voltammograms of azo dyes disclosed that the pH and the dye structure determined their redox potentials. A higher redox potential corresponded to a higher reaction rate.

Gene Cloning, Overexpression, and Characterization of a Xylanase from Penicillium sp. CGMCC 1669

A xylanase-encoding gene, xyn11F63, was isolated from Penicillium sp. F63 CGMCC1669 using degenerated polymerase chain reaction (PCR) and thermal asymmetric interlaced (TAIL)-PCR techniques. The full-length chromosomal gene consists of 724 bp, including a 73-bp intron, and encodes a 217 amino acid polypeptide. The deduced amino acid sequence of xyn11F63 shows the highest identity of 70% to the xylanase from Penicillium sp. strain 40, which belongs to glycosyl hydrolases family 11. The gene was overexpressed in Pichia pastoris, and its activity in the culture medium reached 516 U ml(-1). After purification to electrophoretic homogeneity, the enzyme showed maximal activity at pH 4.5 and 40 degrees C, was stable at acidic buffers of pH 4.5-9.0, and was resistant to proteases (proteinase K, trypsin, subtilisin A, and alpha-chymotrypsin). The specific activity, K (m), and V (max) for oat spelt xylan substrate was 7,988 U mg(-1), 22.2 mg ml(-1), and 15,105.7 micromol min(-1) mg(-1), respectively. These properties make XYN11F63 a potential economical candidate for use in feed and food industrial applications.

Co-expression of the lipase and foldase of Pseudomonas aeruginosa to a functional lipase in Escherichia coli

The lipA gene, a structural gene encoding for protein of molecular mass 48 kDa, and lipB gene, encoding for a lipase-specific chaperone with molecular mass of 35 kDa, of Pseudomonas aeruginosa B2264 were co-expressed in heterologous host Escherichia coli BL21 (DE3) to obtain in vivo expression of functional lipase. The recombinant lipase was expressed with histidine tag at its N terminus and was purified to homogeneity using nickel affinity chromatography. The amino acid sequence of LipA and LipB of P. aeruginosa B2264 was 99-100% identical with the corresponding sequence of LipA and LipB of P. aeruginosa LST-03 and P. aeruginosa PA01, but it has less identity with Pseudomonas cepacia (Burkholderia cepacia) as it showed only 37.6% and 23.3% identity with the B. cepacia LipA and LipB sequence, respectively. The molecular mass of the recombinant lipase was found to be 48 kDa. The recombinant lipase exhibited optimal activity at pH 8.0 and 37 degrees C, though it was active between pH 5.0 and pH 9.0 and up to 45 degrees C. K (m) and V (max) values for recombinant P. aeruginosa lipase were found to be 151.5 +/- 29 microM and 217 +/- 22.5 micromol min(-1) mg(-1) protein, respectively.

Revisiting the IspH Catalytic System in the Deoxyxylulose Phosphate Pathway: Achieving High Activity

From two C(5) isoprene building blocks, isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP), the more than 30 000 members of the isoprenoid family are constructed in nature using two biosynthetic pathways, the mevalonate (MVA) pathway and the deoxyxylulose phosphate (DXP) pathway. IspH of the DXP pathway is a protein containing an iron-sulfur cluster and catalyzes a reductive dehydration reaction of the DXP pathway. In the literature, a wide range of Escherichia coli IspH activities have been reported (2.0 nmol min(-1) mg(-1) to 3.4 micromol min(-1) mg(-1)). For such a broad range of activities, reaction assays were carried out under many different conditions, preventing direct comparison of the activities and determination of the key factor responsible for such a dramatic difference in IspH activities. In this work, we systematically examined the role of redox mediators in IspH catalysis using E. coli IspH as the enzyme and dithionite as the ultimate electron source. Our studies not only suggest the importance of the iron-sulfur cluster but also improve the E. coli IspH activity by nearly 97-fold relative to that from the E. coli NADPH-flavodoxin reductase-flavodoxin system.

Characterization of a neutral ceramidase orthologue fromAspergillus oryzae

Ceramide is an important molecule not only structurally but also regulationally as a modulator of various cellular events. Ceramidase (CDase) are classified into three different types (acid, alkaline, and neutral CDases). Neutral CDase could play an important role in the regulation of ceramide levels in the extracellular space. In this study, we describe the characterization of a neutral CDase orthologue from the filamentous fungus Aspergillus oryzae. The gene encoding the neutral CDase orthologue was cloned and overexpressed in A. oryzae. The purified recombinant enzyme was optimally active at pH 4.0-4.5 and 40 degrees C. The apparent K(m) and V(max) values of the enzyme for C12-NBD-ceramide were 3.32 microM and 0.085 micromol min(-1) mg(-1), respectively.