Kinetic and Transesterification Properties of Lipase from Sprouted Melon (Cucumeropsis manni) Seeds

Crude lipase (acetone powder) was extracted from freshly sprouted melon seeds (Cucumeropsis manni). The activity, kinetic properties (effect of time, pH, and enzyme and substrate concentration, respectively) as well as the ability of the crude lipase to catalyze the production of methyl esters (biodiesel) were examined. The enzyme activity was determined using n-hexane as the solvent (1:2 v/w solvent: substrate ratio) and the transesterification product was analyzed by HPLC. A linear relationship was observed between reaction time and rate of lipolysis with the optimal activity at 2hr of incubation. Furthermore, the lipase was optimally active at acid pH 5 and lipolysis was achieved optimally when the amount of enzyme was 2.0g. Rate of lipolysis was observed to increase linearly at concentrations up to 5.0g of substrate above which a drop in the rate, with no apparent decrease in activity, was observed. The Km (6.25g) and Vmax (13.33%FFA/hr) were also determined. Analysis of the transesterification product yielded 0.61% alkyl ester, 0.81 %FFA, 93.17% TAG, 4.15% 1, 3-DAG and 1.26% 1, 2-DAG while transesterification efficiency was determined to be at 0.588%. Biodiesel (alkyl esters) prepared with the crude lipase was had a density of 0.872 g/mL while its cloud and pour points were 22°C and 12°C, respectively. The results from this research showed that an active lipase was isolated from sprouted melon seeds. However, the fuel properties of the biodiesel produced did not meet international transportation fuel standards. In order to be used industrially, better reaction conditions need to be established for the lipase.

In Vitro Genotoxic Effects of Sarcocystis gigantea Cystizoites Acetone Powder Extract on Sheep Lymphocytes

The toxin of the protozoan intracellular parasite of sheep Sarcocystis gigantea is associated with many clinical and pathological signs. The aim of the study was to investigate In Vitro various chromosomal aberrations due to sarcocystosis infection. Macrocysts of Sarcocystis gigantea were isolated from local karadi sheep, homogenized with glass Dounce homogenizer; acetone powder was prepared from it and used in various concentration to investigate the chromosomal aberration in vitro against sheep lymphocytes. The direct effects of parasite cystizoites acetone powder revealed various genotoxicity effects. These effects included chromosomal aberration (Isogap, Breaks and Dicentrics) and chromatids aberration (Gap and Deletion). It had also an effect on the mitotic index of the lymphocyte cells division. These genotoxicities were studied for the first time with in vitro technique using sheep lymphocytes. These results reflected that Sarcocystis gigantean parasite could cause structural and internal aberration in the chromosomes of their hosts.

Beta-Glucan From Barley Attenuates Post-prandial Glycemic Response by Inhibiting the Activities of Glucose Transporters but Not Intestinal Brush Border Enzymes and Amylolysis of Starch

Beta (β)-glucan (BG) from cereal grains is associated with lowering post-prandial blood glucose but the precise mechanism is not well-elucidated. The main aim of this study was to understand the mechanism through which BG from barley affects post-prandial glycemic response. Waffles containing 0, 1, 2, and 3 g barley BG and the same amount of available carbohydrate (15 g) were fed to the TIM-1 dynamic gastrointestinal digestion system to study the effect of BG on starch hydrolysis. Intestinal acetone powder and Xenopus laevis oocytes were used to study BG's effect on mammalian intestinal α-glucosidase and glucose transporters. The presence of BG did not significantly affect the in vitro starch digestion profiles of waffles suggesting that BG does not affect α-amylase activity. Intestinal α-glucosidase and glucose transport activities were significantly (p < 0.0001) inhibited in the presence of barley BG. Interestingly, BG viscosity did not influence α-amylase, α-glucosidase, GLUT2, and SGLT1 activities. This study provides the first evidence for the mechanism by which BG from barley attenuates post-prandial glycemic response is via alteration of α-glucosidase, GLUT2, and SGLT1 activity, but not amylolysis of starch. The decrease in post-prandial blood glucose in the presence of BG is likely a consequence of the interaction between BG and membrane active proteins (brush border enzymes and glucose transporters) as opposed to the commonly held hypothesis that increased viscosity caused by BG inhibits starch digestion.

Comprehensive analysis of the isozyme composition of laccase derived from Japanese lacquer tree, Toxicodendron vernicifluum

We performed an analysis using isoelectric focusing to comprehensively clarify the isozyme composition of laccase derived from Japanese lacquer tree, Toxicodendron vernicifluum. When water extracts of acetone powder obtained from lacquer were subjected to isoelectric focusing, five bands within pI 7.35–9.30 and nine bands within pI 3.50–5.25 were detected using Coomassie staining. Similarly, laccase activity staining using guaiacol showed five bands within pI 7.35–9.30 and three bands within pI 3.50–4.25. However, laccase activity staining using gallic acid showed remarkable staining within pI 3.50–5.85, whereas staining was very weak within pI 7.35–9.30. When the water extracts of acetone powder were fractionated into the fractions containing bands within pI 7.35–9.30 and pI 3.50–5.85 by SP-Sepharose column chromatography, the former had a blue color and the latter a yellow color. The laccase activity was measured for each of the fractions in buffer solution in the pH range of 2.5–8.0. When syringaldazine, guaiacol, and 2,6-dimethoxyphenol were used as substrates, the yellow fraction showed considerably higher activity than the blue fraction for pH 5.5–7.5. When 3-methylcatechol and 4-methylcatechol were used as substrates, the yellow fraction showed higher activity for pH 4.5–6.5, and the blue fraction showed higher activity for pH 7.0–8.0. When 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) was used as the substrate, both fractions showed maximum activity at optimum pH of 3.0–4.0. Conventionally, in research on blue laccase derived from lacquer, the non-blue fraction corresponding to the yellow fraction lower than pI 6 has been removed during the purification process and thus has not been analyzed. Our results indicated that yellow laccase was present in the non-blue components of lacquer and that it may play a role in urushiol polymerization with previously reported blue laccase.

Preparation of Ganglioside GM1 by Supercritical CO2 Extraction and Immobilized Sialidase

Monosialotetrahexosylganglioside (GM1) has good activity on brain diseases and was developed to be a drug applied in clinics for neurological disorders and nerve injury. It is difficult to isolate GM1 in industry scale from the brains directly. In this work, a simple and highly efficient method with high yield was developed for the isolation, conversion, and purification of GM1 from a pig brain. Gangliosides (GLS) were first extracted by supercritical CO2 (SCE). The optimum extraction time of GLS by SCE was 4 h, and the ratio of entrainer to acetone powder from the pig brain was 3:1 (v/w). GM1 was then prepared from GLS by immobilized sialidase and purified by reverse-phase silica gel. Sodium alginate embedding was used for the immobilization of sialidase. Under the optimized method, the yield of high-purity GM1 was around 0.056%. This method has the potential to be applied in the production of GM1 in the industry.

The capability of the red seaweed Gracilaria vermiculophylla in producing prostaglandins

The red seaweed G. vermiculophylla is rich in polyunsaturated fatty acids with 20 carbon atoms, mainly arachidonic acid (AA) and eicosapentaenoic acid, which are precursors of prostaglandins (PGs). The present study aimed to elucidate the capability of the seaweed in releasing PGs using acetone powder as the crude enzyme. Crude enzyme was prepared using cold acetone. The crude enzyme was incubated with AA at different concentrations (0.1– 4 mg). For determination of PG contents, 5 µL of sample as the test solution corresponding to 0.2 g wet mass of the seaweed was injected into the HPLC. For mass spectrometer analysis, an HPLC system connected with mass spectrometer was used. Results of the study showed that t he released PGs from incubation of acetone powder and AA analyzed by HPLC consisted of PGE 2 , 15-keto-PGE 2 , 15-hydroperoxy-PGE 2 , PGA 2 , and AA while PGs detected by LC-MS were PGF 2α , PGE 2 , 15-keto-PGE 2 , 15-hydroperoxy-PGE 2 , and PGA 2 . The capability of the red algae in producing PGs was affected by available oxygen, aspirin, a cyclooxygenase inhibitor, and AA concentration. The crude enzyme of the red alga (250 mg) was capable to produce 1.63 µg and 1.32 µg of PG 2 and 15-keto-PGE 2 from incubation with 0.25 mg of AA. This method could be the one way to provide PGs in vitro to fulfill demands of PGs in the pharmaceutical industry.

The capability of the red seaweed Gracilaria vermiculophylla in producing prostaglandins

The red seaweed G. vermiculophylla is rich in polyunsaturated fatty acids with 20 carbon atoms, mainly arachidonic acid (AA) and eicosapentaenoic acid, which are precursors of prostaglandins (PGs). The present study aimed to elucidate the capability of the seaweed in releasing PGs using acetone powder as the crude enzyme. Crude enzyme was prepared using cold acetone. The crude enzyme was incubated with AA at different concentrations (0.1– 4 mg). For determination of PG contents, 5 µL of sample as the test solution corresponding to 0.2 g wet mass of the seaweed was injected into the HPLC. For mass spectrometer analysis, an HPLC system connected with mass spectrometer was used. Results of the study showed that t he released PGs from incubation of acetone powder and AA analyzed by HPLC consisted of PGE 2 , 15-keto-PGE 2 , 15-hydroperoxy-PGE 2 , PGA 2 , and AA while PGs detected by LC-MS were PGF 2α , PGE 2 , 15-keto-PGE 2 , 15-hydroperoxy-PGE 2 , and PGA 2 . The capability of the red algae in producing PGs was affected by available oxygen, aspirin, a cyclooxygenase inhibitor, and AA concentration. The crude enzyme of the red alga (250 mg) was capable to produce 1.63 µg and 1.32 µg of PG 2 and 15-keto-PGE 2 from incubation with 0.25 mg of AA. This method could be the one way to provide PGs in vitro to fulfill demands of PGs in the pharmaceutical industry.

Optimization of the asymmetric synthesis of (S)-1-phenylethanol using Ispir bean as whole-cell biocatalyst

In this study, enantiomerically pure (S)-1-phenylethanol was produced via asymmetric bioreduction of acetophenone. Ispir bean (Phaseolus vulgaris) was used as an alcohol dehydrogenase (ADH) source since whole cells are cheaper than isolated enzymes. Acetone powder methodology was applied for biocatalyst. Glucose was used as a cosubstrate in-order to regenerate cofactor (NADPH). The reactions were carried out in an orbital shaker whose temperature and agitation rate can be controlled. (S)-1-phenylethanol concentration was analyzed by HPLC using a Chiralcel OB column. Effects of the reaction time, substrate concentration, cosubstrate concentration and biocatalyst concentration on the (S)-1-phenylethanol production were investigated using Response Surface Methodology (RSM). 36 h bioreduction time, 6 mM acetophenone concentration, 25.15 mM glucose concentration, and 175 mg/mL biocatalyst concentration were determined as optimum values. In these conditions, 2.4 mM (S)-1-phenylethanol was obtained in phosphate buffer (pH=7.0) at 30°C with >99% enantiomeric excess.

Inhibition of Intestinalα-Glucosidase and Glucose Absorption by Feruloylated Arabinoxylan Mono- and Oligosaccharides from Corn Bran and Wheat Aleurone

The effect of feruloylated arabinoxylan mono- and oligosaccharides (FAXmo) on mammalianα-glucosidase and glucose transporters was investigated using human Caco-2 cells, rat intestinal acetone powder, andXenopus laevisoocytes. The isolated FAXmo from wheat aleurone and corn bran were identified to have degree of polymerization (DP) of 4 and 1, respectively, by HPLC-MS. Both FAXmo extracts were effective inhibitors of sucrase and maltase functions of theα-glucosidase. The IC50 for FAXmo extracts on Caco-2 cells and rat intestinalα-glucosidase was 1.03–1.65 mg/mL and 2.6–6.5 mg/mL, respectively. Similarly, glucose uptake in Caco-2 cells was inhibited up to 40%. The inhibitory effect of FAXmo was dependent on their ferulic acid (FA) content (R= 0.95). Sodium independent glucose transporter 2 (GLUT2) activity was completely inhibited by FAXmo in oocytes injected to express GLUT2. Our results suggest that ferulic acid and feruloylated arabinoxylan mono-/oligosaccharides have potential for use in diabetes management.