Characterization of an omega-6 linoleate lipoxygenase from Burkholderia thailandensis and its application in the production of 13-hydroxyoctadecadienoic acid.

A recombinant putative lipoxygenase from Burkholderia thailandensis with a specific activity of 26.4 U mg(-1) was purified using HisTrap affinity chromatography. The native enzyme was a 75-kDa dimer with a molecular mass of 150 kDa. The enzyme activity and catalytic efficiency (k cat/K m) were the highest for linoleic acid (k cat of 93.7 s(-1) and K m of 41.5 µM), followed by arachidonic acid, α-linolenic acid, and γ-linolenic acid. The enzyme was identified as an omega-6 linoleate lipoxygenase (or a linoleate 13S-lipoxygenase) based on genetic and HPLC analyses as well as substrate specificity. The reaction conditions for the enzymatic production of 13-hydroxy-9,11(Z,E)-octadecadienoic acid (13-HODE) were optimal at pH 7.5, 25 °C, 20 g l(-1) linoleic acid, 2.5 g l(-1) enzyme, 0.1 mM Cu(2+), and 6% (v/v) methanol. Under these conditions, linoleate 13-lipoxygenase from B. thailandensis produced 20.8 g l(-1) 13-HODE (70.2 mM) from 20 g l(-1) linoleic acid (71.3 mM) for 120 min, with a molar conversion yield of 98.5% and productivity of 10.4 g l(-1) h(-1). The molar conversion yield and productivity of 13-HODE obtained using B. thailandensis lipoxygenase were 151 and 158% higher, respectively, than those obtained using commercial soybean lipoxygenase under the optimum conditions for each enzyme at the same concentrations of substrate and enzyme.

Characterization of a F280N variant of L-arabinose isomerase from Geobacillus thermodenitrificans identified as a D-galactose isomerase.

The double-site variant (C450S-N475K) L-arabinose isomerase (L-AI) from Geobacillus thermodenitrificans catalyzes the isomerization of D-galactose to D-tagatose, a functional sweetener. Using a substrate-docking homology model, the residues near to D-galactose O6 were identified as Met186, Phe280, and Ile371. Several variants obtained by site-directed mutagenesis of these three residues were analyzed, and a triple-site (F280N) variant enzyme exhibited the highest activity for D-galactose isomerization. The k cat/K m of the triple-site variant enzyme for D-galactose was 2.1-fold higher than for L-arabinose, whereas the k cat/K m of the double-site variant enzyme for L-arabinose was 43.9-fold higher than for D-galactose. These results suggest that the triple-site variant enzyme is a D-galactose isomerase. The conversion rate of D-galactose to D-tagatose by the triple-site variant enzyme was approximately 3-fold higher than that of the double-site variant enzyme for 30 min. However, the conversion yields of L-arabinose to L-ribulose by the triple-site and double-site variant enzymes were 10.6 and 16.0 % after 20 min, respectively. The triple-site variant enzyme exhibited increased specific activity, turnover number, catalytic efficiency, and conversion rate for D-galactose isomerization compared to the double-site variant enzyme. Therefore, the amino acid at position 280 determines the substrate specificity for D-galactose and L-arabinose, and the triple-site variant enzyme has the potential to produce D-tagatose on an industrial scale.

Production of D-Allose From D-Allulose Using Commercial Immobilized Glucose Isomerase.

Rare sugars are regarded as functional biological materials due to their potential applications as low-calorie sweeteners, antioxidants, nucleoside analogs, and immunosuppressants. D-Allose is a rare sugar that has attracted substantial attention in recent years, owing to its pharmaceutical activities, but it is still not widely available. To address this limitation, we continuously produced D-allose from D-allulose using a packed bed reactor with commercial glucose isomerase (Sweetzyme IT). The optimal conditions for D-allose production were determined to be pH 8.0 and 60°C, with 500 g/L D-allulose as a substrate at a dilution rate of 0.24/h. Using these optimum conditions, the commercial glucose isomerase produced an average of 150 g/L D-allose over 20 days, with a productivity of 36 g/L/h and a conversion yield of 30%. This is the first report of the successful continuous production of D-allose from D-allulose by commercial glucose isomerase using a packed bed reactor, which can potentially provide a continuous production system for industrial applications of D-allose.

Mechanical Properties of Epoxy Resin Mortar with Sand Washing Waste as Filler.

The objective of this study was to investigate the potential use of sand washing waste as filler for epoxy resin mortar. The mechanical properties of four series of mortars containing epoxy binder at 10, 15, 20, and 25 wt. % mixed with sand blended with sand washing waste filler in the range of 0-20 wt. % were examined. The compressive and flexural strength increased with the increase in epoxy and filler content; however, above epoxy 20 wt. %, slight change was seen in strength due to increase in epoxy and filler content. Modulus of elasticity also linearly increased with the increase in filler content, but the use of epoxy content beyond 20 wt. % decreased the modulus of elasticity of the mortar. For epoxy content at 10 wt. %, poor bond strength lower than 0.8 MPa was observed, and adding filler at 20 wt. % adversely affected the bond strength, in contrast to the mortars containing epoxy at 15, 20, 25 wt. %. The results indicate that the sand washing waste can be used as potential filler for epoxy resin mortar to obtain better mechanical properties by adding the optimum level of sand washing waste filler.

Methylsulfonylmethane Induces G1 Arrest and Mitochondrial Apoptosis in YD-38 Gingival Cancer Cells.

Gingival squamous cell carcinoma is a rare form of cancer that accounts for less than 10% of all head and neck cancers. Targeted therapies with natural compounds are of interest because they possess high efficacy with fewer side-effects. Methylsulfonylmethane (MSM) is an organic sulfur-containing compound with anticancer activities. The main goal of this study was to induce proliferation inhibition and apoptosis in the metastatic YD-38 cell line. MSM up-regulated expression of P21Waf1/Cip1 and P27Kip1 genes and down-regulated expression of cyclin D1 (CCND1) and CDK4. Moreover, treatment with MSM induced apoptosis and up-regulation of BAX in YD-38 cells. In accordance, the expression of the BCL-2 and BCL-XL, were inhibited, indicating the role of mitochondria in MSM-induced apoptosis. Analysis of mitochondrial integrity showed a loss of mitochondrial potential with an increased level of cytochrome c in the cytosol compared to mitochondria. Active CASPASE-3 (CASP3) was also observed, confirming that MSM-induced apoptosis is caspase-mediated.

Enzymatic production of 15-hydroxyeicosatetraenoic acid from arachidonic acid by using soybean lipoxygenase.

15-Hydroxyeicosatetraenoic acid (HETE), as a mammalian biologically active metabolite, has anticarcinogenic effect. The conditions of producing 15-HETE from arachidonic acid by using soybean lipoxygenase were optimal at pH 8.5 and 20°C with 9 g/l arachidonic acid, 54.4 U/ml soybean lipoxygenase, and 4% methanol. Under these optimized conditions, the enzyme produced 9.5 g/l 15-HETE after 25 min, with a molar conversion yield of 99% and a productivity of 22.8 g l(-1) h(-1). To the best of our knowledge, this is the first biotechnological production of 15-HETE.

Complete conversion of major protopanaxadiol ginsenosides to compound K by the combined use of α-L-arabinofuranosidase and ß-galactosidase from Caldicellulosiruptor saccharolyticus and ß-glucosidase from Sulfolobus acidocaldarius.

The ginsenoside compound K has pharmaceutical activities, including anti-tumor, anti-inflammatory, anti-allergic, and hepatoprotective effects. To increase the production of compound K, the α-L-arabinofuranoside-hydrolyzing α-L-arabinofuranosidase (CS-abf) and/or the α-L-arabinopyranoside-hydrolyzing ß-galactosidase from Caldicellulosiruptor saccharolyticus (CS-bgal) were mixed with the ß-D-glucopyranoside-hydrolyzing ß-glucosidase from Sulfolobus acidocaldarius (SA-bglu). The optimum conditions for the production of ginsenoside compound K from ginsenoside Rc or Rb2, or from major protopanaxadiol ginsenosides in ginseng root extract were determined to be pH 6.0 and 75°C with 8 mg ml⁻¹ ginsenoside Rc, 8 mg ml⁻¹ Rb2, or 10% (w/v) ginseng root extract; and 10.5 U ml⁻¹ CS-abf or CS-bgal supplemented with 4.5 U ml⁻¹ SA-bglu, or 10.5 U ml⁻¹ CS-abf and 10.5 U ml⁻¹ CS-bgal supplemented with 4.5 U ml⁻¹ SA-bglu, respectively. Under optimum conditions, ginsenosides Rc and Rb2, and major protopanaxadiol ginsenosides in ginseng root extract were completely converted to compound K after 12, 14, and 20 h, respectively, with the respective productivities of 388, 328, and 144 mg l⁻¹ h⁻¹. This is the first report of the complete conversion of major protopanaxadiol ginsenosides to compound K.