利用TAIL-PCR技术克隆猴头菇β-glucosidase基因

根据已知的β-glucosidase基因的保守序列设计引物,从猴头菇的菌丝体中克隆到β-glucosidase基因片段B1,再利用TAIL-PCR技术扩增B1两端的侧翼序列B2和B3,B2和B3序列经Blastn、ORF和Primer5·0软件分析后,再设计B2和B3侧翼序列的特异引物对猴头菇的基因组DNA进行扩增,最终扩增到全长的β-glucosidase基因,其大小为2226bp。

好氧堆肥高温期纤维素酶活及β-glucosidase GH1家族阳性微生物群落的相关研究

以固相酶活试验方法检测牛粪-稻草堆肥高温期的CMC酶活性和β-葡萄糖苷水解酶活性,分析结果表明,两种酶活变化存在相关性,并受堆肥过程中的理化因素影响。设计简并引物,应用PCR-DGGE技术对β-葡萄糖苷水解酶GH1家族阳性的微生物群落的组成,及其时空分布进行研究,试验结果显示β-葡萄糖苷水解酶相关的功能性微生物群落在堆肥的进程中存在演替现象。

Isolation and screening of glucosidase inhibitors from Chinese medicines

Objective: To search for glucosidase inhibitors from Chinese medicines. Methods: Six kinds of widely-used Chinese medicines with the activity of decreasing blood glucose were prepared by the process of boiling, condensing, precipitating, exchanging with resins and rinsing. In vitro glucosidase inhibitory activities were examined by photometric bioassay derived from rats, yeast and almond of all the Chinese medicine extracts. Diabetic ICR mice models were established by intraperitoneal injection of STZ (200 mg/kg). To investigate the in vivo effect of lowering blood glucose, the mouse blood glucose level was assayed at 30 min after being given 2.5 g/kg starch and acarbose or varied concentrations of different constituents of some Chinese medicines by stomach tube. Results: The constituents of Sangye, Sangzhi, Sangbaipi, Dihuang and Yuzhu showed potent inhibitory activities against glucosidase. Furthermore, the first kind of constituents was proved to be beneficial in reducing blood glucose by in vivo glucose tolerance experiments. Conclusion: The constituents of Chinese medicines with reducing blood glucose effect have been discovered, thus providing a clue to novel drugs.

Updated clinical and glycomic features of mannosyl-oligosaccharide glucosidase deficiency:Two case reports

BACKGROUND Mannosyl-oligosaccharide glucosidase(MOGS)deficiency is an extremely rare type of congenital disorder of glycosylation(CDG),with only 12 reported cases.Its clinical,genetic,and glycomic features are still expanding.Our aim is to update the novel clinical and glycosylation features of 2 previously reported patients with MOGS-CDG.CASE SUMMARY We collected comprehensive clinical information,and conducted the immunoglobulin G1 glycosylation assay using nano-electrospray ionization source quadruple time-of-flight mass spectrometry.Novel dysmorphic features included an enlarged tongue,forwardly rotated earlobes,a birth mark,overlapped toes,and abnormal fat distribution.Novel imaging findings included pericardial effusion,a deep interarytenoid groove,mild congenital subglottic stenosis,and laryngomalacia.Novel laboratory findings included peripheral leukocytosis with neutrophil predominance,elevated C-reactive protein and creatine kinase,dyslipidemia,coagulopathy,complement 3 and complement 4 deficiencies,decreased proportions of T lymphocytes and natural killer cells,and increased serum interleukin 6.Glycosylation studies showed a significant increase of hypermannosylated glycopeptides(Glc3Man7GlcNAc2/N2H10 and Man5GlcNAc2/N2H5)and hypersialylated glycopeptides.A compensatory glycosylation pathway leading to an increase in Man5GlcNAc2/N2H5 was indicated with the glycosylation profile.CONCLUSION We confirmed abnormal glycomics in 1 patient,expanding the clinical and glycomic spectrum of MOGS-CDG.We also postulated a compensatory glycosylation pathway,leading to a possible serum biomarker for future diagnosis.

Inhibitory activities of microalgal fucoxanthin againstα-amylase,α-glucosidase, and glucose oxidase in 3T3-L1cells linked to type 2 diabetes

Postprandial hyperglycemia is an early indication of type 2 diabetes and the target of many anti-diabetic and anti-obesity studies.α-Glucosidase and α-amylase are the crucial factors in regulating starch digestion and glucose absorption,making them key targets for many studies to treat postprandial hyperglycemia.We studied the inhibitory activities of microalgal fucoxanthin against rat-intestinalα-glucosidase and pancreaticα-amylase along with the antidiabetic eff ect to induce diff erentiation in 3T3-L1 pre-adipocytes using Oil Red-O staining.Fucoxanthin displayed strong hindrance activities towardα-amylase in a concentration-dependent manner,with an IC50 value of 0.68mmol/L,whereas weak inhibitory activity against α-glucosidase,with an IC 50 value of 4.75 mmol/L.Fucoxanthin also considerably elevated glucose oxidase activity in 3T3-L1 cells by 31.3%at 5μmol/L.During adipocyte differentiation,fucoxanthin showed lipid accumulation in 3T3-L1 cells with no cytotoxicity up to 20μmol/L.However,fucoxanthin had no inhibitory activity on glucose-6-phosphate dehydrogenase.These results suggest that fucoxanthin might be useful for the prevention of obesity or diabetes by inhibiting carbohydrate-hydrolyzing enzymes and lipid accumulation and be utilized as an ingredient for a functional food or dietary supplement.

α-Glucosidase Inhibitory Activities of Lutein and Zeaxanthin Purified from Green Alga Chlorella ellipsoidea

α-Glucosidase inhibitors are used therapeutically to treat type-2 diabetes mellitus. Through a bioassay-guided fractionation technique, three carotenoids,(all-E)-lutein,(all-E)-zeaxanthin and(9-Z)-zeaxanthin, were purified from the green alga Chlorella ellipsoidea, in which(all-E)-lutein and(9-Z)-zeaxanthin had potent α-glucosidase inhibitory activity. IC_(50) values of(all-E)-lutein and(9-Z)-zeaxanthin were 70 and 53.5 μmol L^(-1) against Saccharomyces cerevisiae α-glucosidase, respectively, with non-competitive inhibition. In addition, IC_(50) values of(9-Z)-zeaxanthin against Bacillus stearothermophilus and rat-intestinal α-glucosidase were 805.1 and 671.2 μmol L^(-1), respectively. The K_i values of(all-E)-lutein and(9-Z)-zeaxanthin against S. cerevisiae α-glucosidase were 78.1 and 16.5 μmol L^(-1), respectively. Therefore, C. ellipsoidea carotenoids might be utilized as a novel candidate to prevent type-2 diabetes mellitus related disorders in food and medical industry.

Antioxidant and α-glucosidase inhibitor activities of natural compounds isolated from Quercus gilva Blume leaves

Objective: To isolate and investigate antioxidant and α-glucosidase inhibitor compounds in the leaves of Quercus gilva Blume(Q. gilva).Methods: Dry leaves of Q. gilva were extracted with methanol and the methanolic extract was further separated by silica gel column chromatography using several solvents with increasing polarity. The antioxidant activities of the isolated compounds were evaluated using various in vitro assays: 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity, hydrogen peroxide radical scavenging activity, β-carotene bleaching assay, and reducing power assay. The α-glucosidase inhibitory assay was conducted against α-glucosidase from Saccharomyces cerevisiae.Results: Three compounds were isolated and their structures were identii ed as catechin(1), epicatechin(2), and tiliroside(3) using an instrumental analysis. Compound 2 had higher antioxidant activity with inhibitory concentrations(IC50) of(22.55 ± 2.23) μmol/L than that of quercetin, which was used as the standard, with an IC50 of(28.08 ± 2.39) μmol/L, followed by compound 1 with IC50 of(40.86 ± 3.45) μmol/L. On the other hand, compound 3 had the lowest antioxidant activity with an IC50 of(160.24 ± 8.15) μmol/L. However, compound 3 had the highest α-glucosidase inhibitory activity with an IC50 of(28.36 ± 0.11) μmol/L, followed by compounds 1 and 2 with(168.60 ± 5.15) and(920.60 ± 10.10) μmol/L, respectively.Conclusions: The results obtained for the antioxidant activities and α-glucosidase inhibitory activities in a methanolic extract from the leaves of Q. gilva coni rmed the potential of this plant as a source of natural antioxidants and antidiabetic medicine.

1-Deoxynojirimycin Content and Alfa-Glucosidase Inhibitory Activity and Heat Stability of 1-Deoxynojirimycin in Silkworm Powder

Silkworm powder containing 1-deoxynojirimycin (DNJ) has α-glucosidase inhibitory activity and is promising as a complementary and alternative medicine (CAM) agent in Japan. Silkworm powder produced in Korea was extracted with 75% ethanol. The extract was derivatized with 9-fluorenylmethyl chloroformate (FMOC-Cl), and DNJ-FMOC content was measured by HPLC. Then, alfa-glucosidase inhibition by silkworm and mulberry powder in pig liver crude enzyme was assayed using 4-nitrophenyl-alfa-D-glucopyranoside as a substrate. Silkworm powder DNJ content (0.39 to 0.58%) was higher than that in mulberry powder (0.08 to 0.12%). The alfa-glucosidase inhibitory activity of silkworm powder was more potent than that of mulberry leaves and green tea. Silkworm powder DNJ was stable upon heating to 121?C for up to 15 min.

Effects of Hibiscus sabdariffa Linn. fruit extracts on α-glucosidase enzyme,glucose diffusion and wound healing activities

Objective:To provide in vitro evidence for antidiabetic activity through potential inhibition of α-glucosidase enzyme,glucose diffusion and enhancement in the wound healing using methanolic extract and fractions from Hibiscus sabdariffa Linn.fruit.Methods:The inhibitory action of methanolic extract and fractions of such fruit on aglucosidase enzyme and glucose movement through in vitro assay assessment was reported.Their activities on wound healing were tested using the scratch assay.Results:Ethyl acetate fraction at 50 mg/m L concentration exhibited significant aglucosidase inhibition(95.79 mg/m L) with P < 0.05.At the same concentration,the methanolic extract as well as other fractions revealed lower α-glucosidase inhibition and higher glucose diffusion retardation across the dialysis tube than the control.Ethyl acetate and butanol fractions displayed notably higher glucose diffusion inhibitory activity of 5.21 mmol/L and 5.2 mmol/L,respectively as compared to methanolic extract and n-hexane fraction of 6.58 mmol/L and 6.49 mmol/L,respectively.Conversely,compared to other fractions the methanolic extract and ethyl acetate fraction manifested proliferative effect at the incubation time of 6 h during the wound healing study.Conclusions:It is established that methanolic extract and fractions from H.sabdariffa Linn.fruit can inhibit the α-glucosidase enzyme and glucose movement as well as influence the wound healing activity positively.

Identification of α-glucosidase inhibitors from Clinacanthus nutans leaf extract using liquid chromatography-mass spectrometry-based metabolomics and protein-ligand interaction with molecular docking

The present study used in vitro and in silico techniques, as well as the metabolomics approach to characterise α-glucosidase inhibitors from different fractions of Clinacanthus nutans. C. nutans is a medicinal plant belonging to the Acanthaceae family, and is traditionally used to treat diabetes in Malaysia. nHexane, n-hexane: ethyl acetate(1:1, v/v), ethyl acetate, ethyl acetate: methanol(1:1, v/v), and methanol fractions were obtained via partitioning of the 80% methanolic crude extract. The in vitro α-glucosidase inhibitory activity was analyzed using all the fractions collected, followed by profiling of the metabolites using liquid chromatography combined with mass spectrometry. The partial least square(PLS) statistical model was developed using the SIMCA P^+14.0 software and the following four inhibitors were obtained:(1) 4,6,8-Megastigmatrien-3-one;(2) N-Isobutyl-2-nonen-6,8-diynamide;(3) 1′,2′-bis(acetyloxy)-3′,4′-didehydro-2′-hydro-β, ψ-carotene; and(4) 22-acetate-3-hydroxy-21-(6-methyl-2,4-octadienoate)-olean-12-en-28-oic acid. The in silico study performed via molecular docking with the crystal structure of yeast isomaltase(PDB code: 3 A4 A) involved a hydrogen bond and some hydrophobic interactions between the inhibitors and protein. The residues that interacted include ASN259, HID295, LYS156, ARG335,and GLY209 with a hydrogen bond, while TRP15, TYR158, VAL232, HIE280, ALA292, PRO312, LEU313,VAL313, PHE314, ARG315, TYR316, VAL319, and TRP343 with other forms of bonding.

Oleuropein-Specific-β-Glucosidase Activity Marks the Early Response of Olive Fruits (Olea europaea) to Mimed Insect Attack

Olive fruits are seriously deteriorated by pre and postharvest damage due to the attack of insects, such as Bactrocera olaea, which strongly alters the quality of olives. Defence response in olive fruits injured both by pathogens and by mechanical damages has been associated with the enzyme β-glucosidase, which specifically hydrolyses oleuropein, producing highly reactive aldehyde molecules. In situ detection of ^-glucosidase activity in olive fruit tissues following injury, which simulates Bactrocera oleae punctures, is reported. The assay was performed in two cultivars showing different degrees of susceptibilities to fly infestation. In both cultivars, the histochemical assay for β-glucosidase showed that within 20 min after the injury, a strong ^-glucosidase activity could be observed in the damaged tissues. Thereafter a progressive enzyme inactivation occurred starting from tissues around the boundary of the injury with decrease of the enzyme activity and stopped after 3 h. Whereas the mass of active cells reached a distance of （300±50） μm from the edge of the injury. Biochemical analyses showed that in extracts of the injured fruit, β-glucosidase activity rapidly increased within 20 min from injury, thereafter decreasing and reaching values comparable with those in intact fruits. Following puncture, the oleuropein contents did not change significantly in the high susceptibility cultivar, whereas it rapidly decreased in the cultivar showing low susceptibility. The results strongly suggest that olive fruits susceptible towards fly infestation could be related to the ability of the oleuropein-degrading-β-glucosidase to produce the highly reactive molecules in the damaged tissues. As a consequence of puncture, high level of peroxidase activity was detected. This feature also suggested that this enzyme could play a key role in the defence response against insect injuries.

Characterization of purified <i>β</i>-glucosidase produced from <i>Trichoderma viride</i>through bio-processing of orange peel waste

In the present study, solid state fermentation was carried out using orange peel waste to produce β-glucosidase from Trichoderma viride. A locally isolated fungal strain T. viride was cultured in the solid state medium of orange peel (50% w/w moisture) under optimized fermentation conditions and maximum activity of 515 ± 12.4 U/mL was recorded after 4th day of incubation at pH 5.5 and 30℃. Indigenously produced β-glucosidase was subjected to the ammonium sulfate precipitation and Sephadex-G-100 gel filtration chromatography. In comparison to the crude extract β-glucosidase was 5.1-fold purified with specific activity of 758 U/mg. The enzyme was shown to have a relative molecular weight of 62 kDa as evidenced by sodium dodecyl sulphate polyacrylamide gel electrophoresis. The purified β-glucosidase displayed 6 and 60℃ as an optimum pH and temperature respectively.

Efficiency of Oryza punctata extract on glucose regulation: Inhibition of α-amylase and α-glucosidase activities

Red rice(Oryza punctata) is a type of unpolished rice which has higher nutritional value compared to white rice or even polished rice. Owing to higher nutritive content and metabolites, dieticians strongly advise red rice for peoples with metabolic disorders including diabetics. However, the mechanism of action and contents of secondary metabolites in Indian red rice variety not reported scientifically. Therefore, the present study aimed to evaluate its mechanism of action through inhibitory effect of α-amylase and α-glucosidase. Initially, the whole grain of red rice was macerated with methanol at room temperature for 2 weeks. Then, the dried and powdered, samples at different concentrationfi(2.5, 10, 40, and 80 μg/m L) were employed to nd out in vitro inhibitory effects on α-amylase and α-glucosidase. In addition, an enzyme kinetics of effective extract was calculated by Line-weaver Burk(LWB) plot analysis. Moreover,the valuable metabolites in the efficient methanolic extract were quantified using high performance liquid chromatography(HPLC). The results demonstrated that red rice methanolic extract(RRMEt) possess strong inhibitory activity onα-amylase and α-glucosidase compared with acarbose(P < 0.01). The IC50 values of RRMEt was found to be 29.7 ±7.43 μg/m L for α-amylase and 20.4 ± 0.25 μg/m L for α-glucosidase. LWB indicated that RRMEt is an uncompetitive inhibitor. Further, HPLC analysis revealed protocatechuic acid, catechin, and chlorogenic acids were more abundant in RRMEt among the fourteen metabolites. We conclude, the efficiency of enzyme inhibition through the influence of phenolic compounds in RRMEt.

β-GLUCOSIDASE BY THERM OPHILIC AND ANAEROBIC CLOSTRIDIOM THERMOCOPRIAE

In this report,the β-glucosidase from the C.thermocopriae JT3-3 strain was studied.By purifying,the enzyme specific activity was increased about 30 times,and the yield was about 2%.The molecular weight of β-glucosidase is 50000 by gel filtration chromatography,and about 46000 by SDS polyacrylamide eIectrophoresis.Next the effects of pH and temperature on enzyme activity were studied and the Km value for β-glucosidase was calculated from Lineweaver-Burk.In addition,we succeeded in the cloning and expression of β-glucosidase gene from C.thermocopriae to E.coli cells using pBR322 as a vector.

Antioxidant and ɑ-glucosidase activities and phytochemical constituents of Chrysanthoglossum trifurcatum(Desf.)

Objective: To investigate the antioxidant and ɑ-glucosidase properties and phytochemical constituents of roots, stems, leaves and flowers extracts and aerial parts oil of Chrysanthoglossum trifurcatum(Desf.)(C. trifurcatum). Methods: For extraction from roots, stems, leaves and flowers of C. trifurcatum, methanol, ethyl acetate and petroleum ether solvents were used. Phenols, flavonoids, flavonols and tannins contents were evaluated. More, C. trifurcatum aerial parts oil composition was determined using chromatography/mass spectrometry. The antioxidant effect was estimated by DPPH, ABTS and reducing power test systems. The ɑ-glucosidase inhibition was determined by colorimetric assay using the enzyme from Aspergillus niger and the p-nitrophenyl glucopyranoside(pNPG) as substrate. Results: The highest amounts of polyphenols, flavonoids, flavonols and tannins were shown by the methanolic extract of leaves. The main components of the aerial parts oil were limonene(29.21%), γ-terpinene(12.96%), 4-terpenyl acetate(12.18%) and ɑ-pinene(5.76%). The activity evaluated by DPPH, ABTS and reducing power tests was important for stems(IC_(50)=0.68 mg/mL) and flowers(IC_(50)=0.67 mg/mL) methanolic extracts and essential oil(IC_(50)=0.72 mg/mL). Findings of ɑ-glucosidase activity revealed that petroleum ether extracts of leaves and roots together with aerial parts oil showed a highest activity with IC_(50) of 0.044, 0.045 and 0.049 mg/m L, respectively. Conclusions: Observed antioxidant and ɑ-glucosidase activities of oil and extracts are attributed to the presence of the active phytochemicals in C. trifurcatum organs. Thus, the C. trifurcatum can be used as a source of antioxidant compounds and dietary supplement to treat patients with type 2 diabetes.

Inhibition of α-amylase and α-glucosidase activities by ethanolic extract of Telfairia occidentalis (fluted pumpkin) leaf

Objective:To investigate the inhibitory effect of Telfairia occidentalis Hook f.(Curcubitaceae)(T.occidentalis)leaf on key enzyme linked to type-2 diabetes(α-amylase andα-glucosidase)as well as assess the effect of blanching(a commonly practiced food processing technique)of the vegetable on these key enzymes.Methods:Fresh leaves of T.occidentalis were blanched in hot water for 10 minutes,and the extracts of both the fresh and blanched vegetables were prepared and used for subsequent analysis.The inhibitory effect of the extract onα-amylase andα-glucosidase activities as well as some antioxidant parameter was determined in vitro.Results:The result revealed that unprocessed T.occidentalis leaf reduce Fe^(3+)to Fe^(2+)and also inhibitedα-amylase andα-glucosidase activities in a dose dependent manner.However,blanching of the leafy vegetables caused a significant(P<0.05)increase in the antioxidant properties but decrease their ability to inhibitα-amylase andα-glucosidase activities.Conclusions:This antioxidant properties and enzyme inhibition could be part of the mechanism by which they are used in the treatment/prevention of type-2 diabetes.However,the blanched vegetable reduces their ability to inhibit bothα-amylase andα-glucosidase activity in vitro.

Purification and Characterization of β-Glucosidase from a Newly Isolated Strain Tolypocladium cylindrosporum Syzx4

A higher β-glucosidase-producing strain was isolated and identified as Tolypocladium cylindrosporum syzx4 based on its morphology and internal transcribed spacer（ITS） rDNA gene sequence.The present study is to ferment,purify and characterize a β-glucosidase from T.cylindrosporum gams.The enzyme was purified to homogeneity by sulfate precipitataion,diethylaminoethyl cellulose anion exchange chromatography and Sephadex G-100 gel filtration with a 9.47-fold increase in specific activity and a recovery of 12.27%.The molecular weight（Mw） of the β-glucosidase was estimated to be 58600 by SDS-PAGE,which is much lower than that of the enzyme from other fungi.The β-glucosidase shows high activity over a wide range of temperature from 35℃ to 70℃ and the optimum pH is approximately 2.4.Then the kinetic parameters Km（0.85 mmol/L） and vmax[85.23 mmol/（L·s）] of the β-glucosidase were studied,respectively with high affinity p-nitrophenyl-β-D-glucopyranoside（p-NPG） as the substrate,inhibition constant Ki（39.5 mmol/L） with the tolerance of glucose.Although β-glucosidases have been purified and characterized from several other sources,T.cylindrosporum β-glucosidase with the unique optimal pH and higher tolerance of glucose distinguished from others makes the β-glucosidase a potential application in simultaneous saccharification and fermentation（SSF）.

Predicting pH Optimum for Activity of Beta-Glucosidases

The working conditions for enzymatic reaction are elegant, but not many optimal conditions are documented in literatures. For newly mutated and newly found enzymes, the optimal working conditions can only be extrapolated from our previous experience. Therefore a question raised here is whether we can use the knowledge on enzyme structure to predict the optimal working conditions. Although working conditions for enzymes can be easily measured in experiments, the predictions of working conditions for enzymes are still important because they can minimize the experimental cost and time. In this study, we develop a 20-1 feedforward backpropagation neural network with information on amino acid sequence to predict the pH optimum for the activity of beta-glucosidase, because this enzyme has drawn much attention for its role in bio-fuel industries. Among 25 features of amino acids being screened, the results show that 11 features can be used as predictors in this model and the amino-acid distribution probability is the best in predicting the pH optimum for the activity of beta-glucosidases. Our study paves the way for predicting the optimal working conditions of enzymes based on the amino-acid features.

Predictors for Predicting Temperature Optimum in Beta-Glucosidases

This is the continuation of our studies on beta-glucosidase, which plays an important role in biological processes and recently strong interests focus on their potential role in biofeul production. In order to develop simple methods to predict the optimal working condition for beta-glucosidase, we used a 20-1 feedforward backpropagation neural network to screen possible predictors to predict the temperature optimum of beta-glucosidase from 25 amino-acid properties related to the primary structure of beta-glucosidases. The results show that the normalized polarizability index and amino-acid distribution probability can predict the temperature optimum of beta-glucosidase, which highlights a cost-effective way to predict various enzymatic parameters of beta-glucosidase.

A STUDY OF α-1,4-GLUCOSIDASE ACTIVITY IN HUMAN SEMINAL PLASMA

The activity of α-1, 4-glucosidase in seminal plasma from 105 fertile Chinese was 42.7±2.0 mIU/ml and 136.5±8.1 mIU/total. The specific activity was 874.3±84.0 mIU/g protein. There was a significantly positive correlation between the activity (mIU/ml) and the number of spermatozoa. The activity for infertile group was lower than that of the fertile group. For the azoospermia group, the activity was 28.7±3.4 mIU/ml and 47.8±4.9 mIU/total (n=29). There was a significant difference as compared with that of fertile group. The enzyme activity of varicocele group was 35.6±5.1 mIU/ml and 120.3±13.6 mIU/total (n=18). It was higher than that of the azoospermia group (P【0.001). There was no significant difference between the varicocele group and the fertile group. The possible role of the enzyme in seminal plasma and the significance of measurement of the activity in clinical investigation are discussed briefly.