Cloning and Sequence Analysis of the estA gene encoding enzyme for producing (R)-β-acetylmercaptoisobutyric acid from Pseudomonas aeruginosa 1001

The estA gene encoding the enzyme that catalyzes the production of (R)-β-acetylmercaptoisobutyric acid from (R,S)-ester from Pseudomonas aeruginosa 1001, was cloned in Escherichia coli and its nucleotide sequence was determined, revealing the presumed open reading frame encoding a polypeptide of 316 amino acid residues (948 nucleotides). The overall A+T and C+G compositions were 32.59% and 67.41%, respectively. The amino acid sequence of the estA gene product showed a significant similarity with that of the triacylglycerol lipase from Psychrobacter immobilis (38% identity), triacylglycerol lipase from Moraxella sp. (36% identity), and two forms of carboxyl esterases from Acinetobacter calcoaceticus (17% and 17% identities). The deduced amino acid sequences have a pentapeptide consensus sequence, G-X-S-X-G, having an active serine residue, and another active site, dipeptides H-G, located at 70–100 amino acids upstream of the G-X-S-X-G consensus sequence.     

Fermentative production of (R)-(-)-3-hydroxybutyrate using 3-hydroxybutyrate dehydrogenase null mutant of Ralstonia eutropha and recombinant Escherichia coli

Two systems, one using an (R)-(-)-3-hydroxybutyrate dehydrogenase (BDH) null mutant of Ralstonia eutropha and the other using a recombinant Escherichia coli strain containing a synthetic poly[(R)-(-)-3-hydroxybutyrate] (PHB) operon and an extracellular PHB depolymerase gene, were used for the fermentative production of (R)-(-)-3-hydroxybutyrate (3HB). The concentration of 3HB in the culture supernatant of the mutant R. eutropha system reached about 30 mM after 5 d under anaerobic conditions, although it was about 4-10 mM under aerobic conditions. On the other hand, the 3HB concentration in the culture supernatant of the recombinant E. coli system reached about 70 mM after 4 d, indicating that about 70% of the glucose added was converted to 3HB.     

Radiation-inducible TNF-α gene expression under stress-inducible promoter gadd 153 for cancer therapy

We demonstrated the effectiveness of radiation-inducible expression of the TNF-α gene for cancer therapy in vitro. The TNF-α gene under the control of the stress-inducible promoter, gadd 153, was introduced into the human glioma cell line, U251-SP. Without cobalt-60 gamma irradiation, no cytotoxicity against the transfected cells was observed. When the transfected cells were irradiated with 10 or 20 gray (Gy), the gadd 153 promoter was highly induced and the expression level of TNF-α increased. Five days after the irradiation, the TNF-α productions of each cell irradiated with 10 and 20 Gy were 30 and 100 times higher than the basal level, respectively. The cytotoxicities against the transfected cells 5 d after irradiation with 10 and 20 Gy were 79% or 91%, respectively, which are much higher than those against the nontransfected cells that were irradiated at the same dose (43% and 78%, respectively). These results demonstrate that the gadd 153-TNF-α system may be an effective tool for radiosurgery of malignant brain tumors.     

Investigation of the interaction between (−)‐epigallocatechin‐3‐gallate with trypsin and α‐chymotrypsin

Tea polyphenol (TP) inhibits digestive enzymes and reduces food digestibility. To explore the interaction between TP with digestive enzymes, bindings of ‐epigallocatechin‐3‐gallate (EGCG) to trypsin and α‐chymotrypsin were studied in detail using fluorescence, resonance light‐scattering, circular dichroism, fourier transform infrared spectroscopy methods and protein‐ligand docking. The binding parameters were calculated according to Stern–Volmer equation, and the thermodynamic parameters were determined by the van't Hoff equation. The results indicated that EGCG was capable of binding trypsin and α‐chymotrypsin with high affinity, resulting in a change of native conformation of these enzymes. EGCG had a greater influence on the structure of α‐chymotrypsin than trypsin. This study can be used to explain the binding interaction mechanism between TP and digestive enzymes.     

ENZYMIC QUANTIFICATION OF (1→3) (1→4)-β-D-GLUCAN IN BARLEY AND MALT

A simple and quantitative method for the determination of (1→3) (1→4)-β-D-glucan in barley flour and malt is described. The method allows direct analysis of β-glucan in flour and malt slurries. Mixed-linkage β-glucan is specifically depolymerized with a highly purified (1→3) (1→4)-β-D-glucanase (lichenase), from Bacillus subtilis, to tri-, tetra- and higher degree of polymerization (d.p.) oligosaccharides. These oligosaccharides are then specifically and quantitatively hydrolysed to glucose using purified β-D-glucosidase. The glucose is then specifically determined using glucose oxidase/peroxidase reagent. Since barley flours contain only low levels of glucose, and maltosaccharides do not interfere with the assay, removal of low d.p. sugars is not necessary. Blank values are determined for each sample allowing the direct measurement of β-glucan in maltsamples.α-Amylasedoes not interfere with the assay. The method issuitable for the routineanalysis of β-glucan in barley samples derived from breeding programs; 50 samples can be analysed by a single operator in a day. Evaluation of the technique on different days has indicated a mean standard error of 0–1 for barley flour samples containing 3–8 and 4–6% (w/w) β-glucan content.     

Preparation and characterization of molecular weight standards of low polydispersity from oat and barley (1→3)(1→4)-β- -glucan

Purified (1→3)(1→4)-β- -glucans (β-glucans) from oat and barley with broad molecular weight (MW) distribution were separated into seven fractions using gradient precipitation with ammonium sulfate (NH₄)₂SO₄. The MW of each fraction decreased consecutively with the concentration of (NH₄)₂SO₄ at which it was precipitated. The MW distribution of each fraction was much narrower compared to the parent sample and is comparable to commercially available pullulan MW standards. To determine whether the fractionation process was separating sub-fractions of different structure, the original β-glucan sample and each fraction were hydrolyzed by a (1→3)(1→4)-D-β-glucan-4-glucanohydrolase (lichenase, E.C.3.2.1.73) and the liberated oligosaccharides were analyzed by high performance anion exchange chromatography. The analysis revealed no differences in oligosaccharide pattern (DP 2–9) derived from each fraction and the parent sample. In particular, the tri/tetra oligosaccharide ratio remained constant for all fractions, indicating no fractionation based on structural features had taken place. The effect of starting β-glucan concentration on the fractionation process was studied. The results showed that it was possible to achieve good separation at overlapping parameter c[η] lower than 3.5. Further increase in starting β-glucan concentration hindered clear separation of the fractions. Temperature also affected the fractionation efficiency. The higher the temperature, the lower the amount of (NH₄)₂SO₄ that was necessary to precipitate the samples of same MW. A Mark Houwink relationship was derived from the measured MW and intrinsic viscosity for fractions from oat and barley, respectively.     

Structural modification and characterization of rice starch treated by Thermus aquaticus 4-α-glucanotransferase

Rice starch was modified using Thermus aquaticus 4-α-glucanotransferase (TAαGTase) in this study. The changes in the molecular structure and the effect on the starch retrogradation by TAαGTase treatment were investigated on isolated rice starch. By treating TAαGTase, molecular weight profile of amylopectins shifted to higher elution time from 1.0 × 10⁸ to 2.4 × 10⁷ or 0.8 × 10⁷, depending on the level of enzyme dosage. Meanwhile, there were huge increases in the proportions of content corresponding to amylose size and even smaller molecules. On treating with TAαGTase, short branch chains (DP 1–8) increased, and longer branch chains (>DP 19) increased significantly as well, with a broader distribution up to DP 46 compared to the control rice starch. Amylose content decreased from 30.0 to 21.8–23.7%. This indicated that the amylose could be transferred to the amylopectin branch chain by the disproportionation of TAαGTase, resulting in lowering the amylose content and the formation of amylopectin with a broader branch-chain length distribution. TAαGTase modified rice starch showed that X-ray diffraction pattern of the B-type crystalline even before cold storage, and that a variety of cyclic glucans (DP 5–19) were produced by enzymatic reaction. In particular, the accelerated rate of starch retrogradation was clearly observed compared to the control due to an overall increase in the number of elongated long-branch chains, decrease in the amount of amylose–lipid complex, and the possible synergistic effects of these factors.     

Effects of different food commodities on larval development and α-amylase activity of Plodia interpunctella (Hübner) (Lepidoptera: Pyralidae)

The present work was undertaken to study the influence of four commodities (wheat flour, dates, sorghum and barley) on Plodia interpunctella post-embryonic development. Larval weight, larval mortality, pupation and adult emergence were recorded. The study also aimed to find out the effect of these commodities on protein and glycogen production as well as on α-amylase activity. Results indicated that the weight of fourth instar larvae placed on dates increased gradually. Percentage mortality was low. Pupation and adult emergence were delayed. In contrast, the weight of larvae placed on wheat flour, sorghum or barley remained low. Pupation and adult emergence occurred sooner than among those placed on dates and the percentage mortality was highest for larvae placed on barley. Results also showed that protein content and α-amylase activity were lower for larvae placed on dates than for those placed on other commodities. The biochemical composition of different commodities showed that dates are a rich source of glucose, while their protein and starch contents were very low as compared to the other commodities. In contrast, wheat flour, sorghum and barley contained large amounts of starch and protein and low amounts of glucose. Thus, the reduction in α-amylase activity was probably due to the high levels of glucose in dates.     

Measurement of (1 → 3),(1 → 4)-β-D-glucan in barley and oats: A streamlined enzymic procedure

A commercially available enzymic method for the quantitative measurement of (1 → 3),(1 → 4)-β-glucan has been simplified to allow analysis of up to 10 grain samples in 70 min or of 100–200 samples by a single operator in a day. These improvements have been achieved with no loss in accuracy or precision and with an increase in reliability. The glucose oxidase/peroxidase reagent has been significantly improved to ensure colour stability for periods of up to 1 h after development. Some problems experienced with the original method have been addressed and resolved, and further experiments to demonstrate the quantitative nature of the assay have been designed and performed.     

Production of triacylglycerol and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) by the toluene-degrading bacterium Rhodococcus aetherivorans IAR1

Poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) produced by various bacteria has been intensively investigated as a promising biodegradable plastic, but required a supply of an expensive precursor as a secondary carbon source for its production. In a previous study, we identified a new bacterial strain, Rhodococcus aetherivorans IAR1, which synthesizes PHBV from toluene without the supply of a precursor. Toluene is the volatile organic compound most abundantly emitted to the environment. In the present paper, we show that R. aetherivorans IAR1 produces triacylglycerols (TAGs) simultaneously with PHBV. Both PHBV and TAGs were synthesized before the nitrogen source is completely exhausted. The cellular content of PHBV reached 10% of cell dry weight (CDW) and its synthesis ceased even during intermittent supply of toluene. However, accumulation of TAGs continued during cultivation and their cellular content reached 24% of CDW at the end of cultivation. Cerulenin inhibited TAG production and increased PHBV cellular content up to 30% of CDW. The mole fraction of 3-hydroxyvalerate (3HV) in PHBV produced from toluene increased from 60% to 80% during its accumulation. Fatty acid compositions of TAGs produced from acetate and toluene were different. At the end of cultivation, the mole fraction of C17:0, one of odd-carbon number fatty acids, was 5% on toluene or 10% on acetate while the mole fraction of 3HV in PHBV from toluene was as high as that in PHBV from acetate, suggesting that a C5 intermediate of toluene degradation might directly become a precursor of 3HV whereas propionyl-CoA is required for the incorporation of C17:0 into TAGs.     

Influence of high pressure on interactions of 11S globulin Vicia faba with ι-carrageenan in bulk solution and at interfaces

The influence of ι-carrageenan (ι-CAR) on the solution, interfacial and emulsifying properties of 11S globulin Vicia faba at low ionic strength and pH 8 has been investigated before and after high-pressure processing at 200 MPa for 20 min. The total calorimetric enthalpy (ΔH) and size exclusion chromatography studies for the pure 11S indicate that there is subunit dissociation and extensive aggregation of the protein during or following treatment. Under the same treatment conditions, 1-anilinonaphthalene-8-sulphonate (ANS) data has shown increased protein surface hydrophobicity. Pressure treatment of 11S gives much lower values of the surface tension, and apparent surface shear rheology experiments show that the molecules in the film adsorbed from the pressurised 11S are much more strongly interacting than those adsorbed from the native 11S. However, emulsions prepared with pressure processed 11S give substantially bigger droplets than those made with the untreated pure protein. Addition of ι-CAR to 11S reduces the denaturation temperature (T_m), the ΔH value, and protein surface hydrophobicity. Size exclusion chromatography at low ionic strength is indicative of complex formation. Tension measurements at the air–water interface are also consistent with the presence of a complex. Emulsions made with the simple 1:0.33 mixture of 11S+ι-CAR give emulsions with smaller droplets and pressure processing of the biopolymer mixture leads to emulsions with even smaller droplets. The presence of ι-CAR at low ionic strength appears to protect the globulin against pressure-induced aggregation.     

Towards a better understanding of the relationship between the β-carotene in vitro bio-accessibility and pectin structural changes: A case study on carrots

The quality of fruit and vegetables based products is affected by processing. Two important parameters to consider are the structural characteristics and the nutritional value. As pectin is a major constituent of plant cell walls, pectin structure engineering can be used as a tool to affect the structural quality of plant based food products. During thermal processing, pectin characteristics are influenced. Recently, it has been highlighted that nutrient bio-accessibility is affected by food structure. The intracellular localization of nutrients implies that their accessibility can be hindered by several structural elements.Therefore, this investigation focused on the relation between the structural quality and the nutritional value of carrots. Texture was measured as an indication for the structural quality, while the β-carotene in vitro bio-accessibility was selected as a parameter reflecting the nutritional value. The effect of thermal (pre)processing on this relationship was investigated. The results clearly indicate that the structural quality of carrots and the β-carotene in vitro bio-accessibility are inversely correlated. Moreover, it was hypothesized that pectin changes during thermal processing play a key role in this inverse relationship.