Sephadex G-100 Column Chromatography Research Articles

Characterization of a novel keratinase from Chrysosporium tropicum

A keratinophilic fungus Chrysosporium tropicum was isolated from poultry farm soil and screened for extracellular keratinase activity. The fungus was cultured in basal salts medium and keratinase production was assessed. The novel keratinase was purified by Sephadex G-100 column chromatography and characterized. The molecular weight of the enzyme was estimated to be 14.5 KDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The optimum pH and temperature of the keratinase were found to be 7.5 and 40oC respectively. The Km for keratin powder from human hair was 6.67 mg and the Vmax of novel keratinase was determined to be 0.33 mg ml-1 by the Lineweaver-Burk plot. The enzyme activity was almost completely inhibited by phenylmethylsulphonyl fluoride (PMSF) suggesting that the keratinase belongs to the serine protease family.

Chitinolytic activity of Streptomyces sanglieri TBG AG-28 isolated from Agasthyarkoodam region of Western Ghats, Kerala

Chitinase enzyme is the N-acetyl D-glucosamine residue group of enzyme responsible for chitin hydrolysis, a linear polymer of β(1,4)nitrogen. Chitinases are wide spread in nature and play a key role in chitin. This study focuses on the isolation of efficient chitinase producing Streptomyces sp. from Western Ghats, a global biodiversity hotspot. A Streptomyces strain TBG AG-28 isolated from the soil sample of Agasthyarkoodam region of Western Ghats, Kerala was found to be an efficient chitinase producer. The strain was identified as Streptomyces sanglieri based on conventional morphological, micro-morphological and 16s rDNA sequence analysis. Different physiological parameters like incubation time, pH and temperature were optimized for maximum production of chitinase under submerged conditions. The highest activity of 42.5 U/mL was attained at seventh day of incubation in colloidal chitin broth with 1% sucrose as carbon source, pH 7 at 32°C. The optimized media showed an increase in activity from 27.5U/mL to 42.5U/mL. Chitinase enzyme was purified using Sephadex G100 column chromatography to 1.84 folds with 6.1% activity recovery. The molecular weight of purified enzyme was found to be 38 kDa by SDS-PAGE analysis and was subsequently characterized.

Xylanase from thermotolerant Bacillus haynesii strain, synthesis, characterization, optimization using Box-Behnken Design, and biobleaching activity

AbstractThe economic value of xylanolytic enzymes is derived from their use in a variety of industrial processes, which necessitates a cost-effective manufacturing procedure. In the current study, forty bacterial isolates were isolated from water samples and investigate their efficacy to producing xylanase enzyme. The most potent bacterial isolate was identified by sequencing and amplifications of 16Sr RNA gene as Bacillus haynesii strain K6. The impacts of various culture conditions on the productivity of xylane were examined. Data showed that the highest xylanase production was achieved at pH 7, in presence of 3 g/L xylan, 5 g/L peptone, and incubated at 40 °C for 24 h. The Box-Behnken model was used to find the best parameters for the relevant variables, and the results revealed an increase in xylanase production with values of 35.02 U/mL. The maximum precipitation of xylanase from the optimized culture was attained by ammonium sulfate (60%) followed by purification using dialysis and sephadex G100 column chromatography. The purified xylanase had a 12-fold enrichment, with a specific activity of 84 U/mg and a molecular weight approximately 439 KDa determined by thin-layer chromatography (TLC)/mass spectrometry. The amino acid analysis of the purified xylanase enzyme revealed the presence of 15 amino acids, with the highest concentrations of 1940 and 1520 mg/L for proline and cysteine, respectively. Finally, the physical properties of wastepaper pulp were improved after treatment with xylanase enzyme. The whiteness and softness of xylanase-treated wastepaper were improved with percentages of 34.6% and 16.2%, respectively. Therefore, we recommend the use of xylanase enzyme in the bleaching process as it is a biologically synthetic material, safe, and suitable for industrial use, and it reduces the use of harmful chemicals.

Effects of an Armillaria mellea Polysaccharide on Learning and Memory of D-Galactose-Induced Aging Mice.

Arm illaria mellea has been known and used in traditional medicine in East Asia for hundreds of years. It has already been reported that A. mellea extracts have various pharmacological effects, and the polysaccharides of A. mellea exhibit antioxidant and anti-apoptotic activities. In this study, a water-soluble polysaccharide (AMP-N-a-1), with an average molecular weight of 17 kD, was isolated and purified from the water extract of A. mellea using DEAE-52, Sepharose CL-4B, and Sephadex G-100 column chromatography. AMP-N-a-1 was mainly composed of Man (1.65%), Glca (1.64%), Rha (1.82%), Gala (2.49%), Glc (90.48%), Gal (0.89%), Xyl (0.42%), and Ara (0.61%). AMP-N-a-1 was used to study the effect on the learning and memory of mice and its underlying mechanisms. The results showed that AMP-N-a-1 could significantly increase the activities of catalase (CAT) and superoxide dismutase (SOD) and reduce the content of nitric oxide (NO) in mouse brain tissue. Meanwhile, AMP-N-a-1 could reduce the contents of norepinephrine (NE) and dopamine (DA) but could increase the content of 5-hydroxytryptamine (5-HT) in mouse brain tissue. In addition, the immunofluorescence experiment showed that AMP-N-a-1 could promote the proliferation of hippocampal dentate gyrus neurons. The above results indicate that AMP-N-a-1 can significantly improve the learning and memory of mice, and the mechanism may be that AMP-N-a-1 can participate in the regulation of learning and memory through a variety of ways.

Screening, characterization and anti-cancer application of purified intracellular MGL

L-methionine-γ-lyase (MGL) producing bacterial isolates were screened from soil samples that further characterized as ‘Klebsiella oxytoca BLM-1’ by biochemical and 16S rDNA sequencing. Intracellular MGL obtained from K. oxytoca BLM-1 by sonication was purified by Octyl-Sepharose and Sephadex G-200 column chromatography. MALDI-TOF-MS analysis of protein band (Mr ~ 63 kDa) confirmed the PLP-dependence and structural similarity with MGL enzyme. Purified MGL (1.1 μg) exhibited the maximum activity in potassium phosphate buffer (80 mM; with L-met 20 mM pH 7.0) at 37 °C. That further enhanced in the presence of NaCl (2 mM), Tween-80 (1.0 %; v/v) and EDTA (5 mM). Km and Vmax for purified MGL by using L-met as substrate was found to be 5.32 mM and 0.386 U/mL/min. The purified MGL showed PLP dependence and the half-life was 365.59 min. The MGL was effective against breast cancer (MCF7), gastric adenocarcinoma and human glioblastoma (U87MG) cancer cell lines with IC50 values of purified MGL 0.041 U/mL, 0.008 U/mL and 0.009 U/mL, respectively. The U87MG, greatly affected by MGL treatment, when cultured in DMEM medium (10 mL) with PLP, homocysteine and 10 % FCS as compared to control/untransformed mouse spleen cells.

Chemical Structure and Immune Activation of a Glucan From Rhizoma Acori Tatarinowii

Rhizoma Acori Tatarinowii is a traditional Chinese herb used to treat depression and coronary heart disease. Studies on its active components mainly focus on small molecular compounds such as asarone and other essential oil components, while the large molecular active components such as polysaccharides are ignored. In this study, we aimed to study the chemical structure and immune activation of polysaccharides from Rhizoma Acori Tatarinowii. In this study, a polysaccharide (RATAPW) was isolated and purified by DEAE-52 cellulose and Sephadex G-100 column chromatography from alkali extraction polysaccharide of Rhizoma Acori Tatarinowii. The average molecular weight of RATAPW was 2.51 × 104 Da, and the total carbohydrate contents of RATAPW were 98.23 ± 0.29%. The monosaccharide composition, methylation, and nuclear magnetic resonance (NMR) analysis results displayed that the polysaccharide was α-1,4-glucan with short α-1,6 branches. Immunofluorescence assay and inhibitor neutralization assay indicated that RATAPW could promote the TNF-α production of RAW264.7 macrophage through the nuclear factor kappa B (NF-κB) molecular signaling pathway. Treatment with 200 μg/ml of RATAPW enhanced a 38.77% rise in the proliferation rate of spleen lymphocytes. RATAPW also enhances ConA-induced T cells and lipopolysaccharide (LPS)-induced B cell proliferation in a dose-dependent effect. Our study lays a foundation for the discovery of natural polysaccharide immune modulators or functional food from Rhizoma Acori Tatarinowii.

Antioxidant Activities of Aqueous Extracts and Protein Hydrolysates from Marine Worm Hechong (Tylorrhynchus heterochaeta).

Hechong (Tylorrhynchus heterochaeta) is an edible marine worm widely distributed in the estuary area. The objective of this study is to determine the antioxidant activities of extracts and protein hydrolysates from Hechong. Results showed that the aqueous extracts of steamed Hechong had the highest antioxidant values using the methods of DPPH, ABTS, and FRAP testing (76.29 μmol TE/g, 181.04 μmol TE/g, and 10.40 mmol Fe2+/100 g, respectively). Furthermore, protein hydrolysates of Hechong were observed significant antioxidant activities when compared to crude Hechong. The purification was carried out by DEAE-52 cellulose and Sephadex G-100 column chromatography. The microspatial structure of glycoprotein showed fibrous shapes and cracks with uniform distribution. The study has concluded that the extract and protein hydrolysates of Hechong have significant antioxidant activities, which is merited to be further investigated in the food and pharmaceutical fields.

Enzyme mediated transformation of CO2 into calcium carbonate using purified microbial carbonic anhydrase

In this study, a bacterial carbonic anhydrase (CA) was purified from Corynebacterium flavescens for the CO2 conversion into CaCO3. The synthesized CaCO3 can be utilized in the papermaking industry as filler material, construction material and in steel industry. Herein, the CA was purified by using a Sephadex G-100 column chromatography having 29.00 kDa molecular mass in SDS-PAGE analysis. The purified CA showed an optimal temperature of 35 °C and pH 7.5. In addition, a kinetic study of CA using p-NPA as substrate showed Vmax (166.66 μmoL/mL/min), Km (5.12 mM), and Kcat (80.56 sec−1) using Lineweaver Burk plot. The major inhibitors of CA activity were Na2+, K+, Mn2+, and Al3+, whereas Zn2+ and Fe2+ slightly enhanced it. The purified CA showed a good efficacy to convert the CO2 into CaCO3 with a total conversion rate of 65.05 mg CaCO3/mg of protein. In silico analysis suggested that the purified CA has conserved Zn2+ coordinating residues such as His 111, His 113, and His 130 in the active site center. Further analysis of the CO2 binding site showed conserved residues such as Val 132, Val 142, Leu 196, Thr 197, and Val 205. However, a substitution has been observed where Trp 208 of its closest structural homolog T. ammonificans CA is replaced with Arg 207 of C. flavescens. The presence of a hydrophilic mutation in the CO2 binding hydrophobic region is a further subject of investigation.

Optimization of alkali extraction, structure, and antioxidant activity of protein-bound polysaccharide from seeds of Plantago ovata Forssk

In this study, the effects of different solvents on the viscosity of the seeds of Plantago ovata Forssk (POF) were compared to select the extraction solvent; then, a three-level and four-variable Box–Behnken design (BBD) was applied to determine the optimum extraction conditions for the maximum yields of polysaccharides from POF. Two novel protein-bound heteropolysaccharides (POFG-1a and POFG-2a) with molecular weights of 118 kDa and 113 kDa were isolated and purified by ethanol fractionation precipitation, DEAE-650 M anion exchange column chromatography, and Sephadex G-100 column chromatography. POFG-1a and POFG-2a contain 49.6% and 17.2% of polysaccharide, 26.9% and 56.4% of protein, 9.1% and 5.0% of uronic acid, respectively; they are rich in monosaccharides, Ara, Gal, and Rha, and amino acids, Ser, Pro, Ile, and Phe. Based on FT-IR, β-elimination, methylation analysis, NMR spectroscopy, and some literature data, it was found that POFG-1a is a homogeneous acidic protein-bound heteropolysaccharide with O-glycosidic linkage. The main chain of POFG-1a is connected by 1→4 Araf and 1→5 Araf with a high degree of branching, connected to the branch at the O-2 and O-3 positions of 1→5 Araf. The POFGs exhibited strong scavenging activity on DPPH, ABTS, and NO free radicals, which is rarely observed in other plant polysaccharides. These results indicate that POFGs might have potential applications in the medical and food industries as novel natural antioxidants.

Purification of wild type and recombinant xylanases and evaluation of their arabinoxylan hydrolysis ability

Arabinoxylan hydrolyzing enzymes are very rich and diversified groups, among these, endo-1,4-β-xylanase is the most important group which catalyzes the cleavage of xylan main skeleton to release arabinoxylan oligosaccharides such as D-xylose, xylobiose, L-arabinose, xylotetraose, xylopentose. This group of enzyme was efficiently produced from natural microorganism including bacteria, yeast and fungi and recombinant strains. In this study, we have purified wild-type and recombinant xylanase to compare the difference in hydrolysis products of these two enzymes. Using sephadex G-100 and DEAE-sephadex column chromatography, we have purified xylanase from wild-type Aspergillus niger DSM1957 with molecular weight of 25 kDa, specific activity of 3240 IU/mL, the purity increases 1.68 folds compare to crude extract, and a recovery rate of 36%. The recombinant enzyme from Pichia pastoris GS115/pPXlnA was purified by ProbondTM affinity chromatography column, with a molecular weight of 36 kDa; the purity increased 3.2 folds than the crude extract, and with the recovery efficiency of 21.1%. The activity of commercial wild-type xylanase was better than that of our recombinant and wild-type enzymes. Our wild-type xylanase could hydrolyze arabinoxylan into L-arabinose in 50 mM CH3COONa pH 5.0. Our results showed that xylanases purified from different sources were stable and highly specific to xylan. Xylanase has potential application in the production of high quality biotechnological products.

Structural characteristics and immune-enhancing activity of fractionated polysaccharides from Athyrium Multidentatum (Doll.) Ching.

Polysaccharides coded as CP were extracted from Athyrium Multidentatum (Doll.) Ching and then fractionated into five fractions (FP-1, FP-2, FP-3, FP-4 and FP-5). A purified polysaccharide designated as FP-3-4 was prepared from FP-3 by Sephadex G-100 column chromatography. Chemical analysis disclosed that CP and these fractions were heteropolysaccharides and mainly composed of glucose, galactose, arabinose, mannose, rhamnose, xylose, fucose, ribose and uronic acid with different molar ratios. They presented different images of SEM. FP-3-4 was highly branched polymers with sixteen types of linkages. The in vitro immunomodulatory results stated that CP and these fractions could promote macrophage proliferation, enhance macrophage phagocytosis and increase the production of NO, TNF-α, IFN-γ, IL-1β, IL-6, IL-10 and IL-2, indicating remarkable immune enhancement activities. RNA sequencing analysis revealed that CP and FP-3 induced macrophage activation mainly through MAPK and alternative NF-κΒ signaling pathways via CD14/TLR4 and Dectin-2 receptors, which were verified by RT-qPCR and western blot.

PURIFICATION AND CHARACTERIZATION OF L-GLUTAMINASE ENZYME FROM SYMBIONT ENDOPHYTIC BACTERIA OF MARINE PLANT Sargassum sp. AS ANTIMICROBIAL ADJUVANTS

Endophytic bacterial symbiont of marine plant seaweed is a potential source of various bioactive compounds as a raw material for drugs. This research aimed to study the antimicrobial potential of L-glutaminase enzyme isolated from seaweed bacterial symbiont collected from Cikoang Island, South Sulawesi Indonesia. The production of L-glutaminase of bacterial symbiont marine plant Sargassum sp was optimum in nutrient broth using a shaker at 37 °C for 3 d. L-glutaminase was obtained from extraction and fractionation with ammonium sulphate at increased saturation levels of 0-20%, 20-40%, 40-60% and 60-80%, continued by dialysis and column chromatography. Antimicrobial activity assessment was conducted by an agar diffusion method using paper discs. Enzyme fractions were produced by purification using DEAE-Cellulose and Sephadex G-100 column chromatography with a purity of 12.23 and 16.95 times, respectively. The enzyme characterization showed that the optimum condition was at pH 8.0 and 37oC, where at this condition, enzyme activity showed a slight increase after the addition of K+ and Na+ ions and however, significant decrease in enzyme activity after the addition of Cu2+ and Fe2+ ions. The results of the antimicrobial activity evaluation revealed that the L-glutaminase of sephadex G-100 fraction had the highest activity against Escherchia coli and Staphylococcus aureus with the inhibition zone of 12.1 ± 0.29 and 17.4 ± 0.21mm, respectively. These results demonstrated that the environmentally friendly bacterial symbiont of the marine plant Sargassum binderi could be a new alternative as a source to produce the antimicrobial L-glutaminase enzyme. This work probably provides an essential finding for further research on the antimicrobial action of protein fraction from bacterial symbiont of marine plant Sargassum sp. or the other species.

Structural characterization and immunomodulatory activity of exopolysaccharide from Aureobasidium pullulans CGMCC 23063

Exopolysaccharide (EPS-1) was isolated from Aureobasidium pullulans CGMCC 23063 and purified with DEAE 650M and Sephadex G-100 column chromatography. The structural characteristics and immunomodulatory activity of EPS-1 were investigated. The results showed EPS-1 (294.9 KDa) was β-glucan consisting of the backbone (1→3)-linked β-Glcp and β-(1,6)-branches by Ion chromatography, gas chromatography–mass spectrometry, and 1D/2D NMR spectroscopy. The SEM and AFM showed that EPS-1 composed spheres joined by triple-helix conformation into chains and circles. In vitro cell experiments, EPS-1 displayed immunological activity on the RAW264.7 cells. After being treated with EPS-1 (400 μg/mL), the concentration of NO, TNF-α, and IL-6 could reach 17.05 μM, 448.46 pg/mL, and 15.18 pg/mL, respectively. In addition, the cytokines mRNA levels also demonstrated the above results at the molecular level. The western-blot results demonstrated that the expression of phosphorylated p65 and p-erk associated with cytokine release, was increased with the dose of EPS-1.

Structure characterization, antioxidant and hypoglycemic activity of an arabinogalactoglucan from Scutellaria baicalensis Georgi

An arabinogalactoglucan SBP-1 was purified from Scutellaria baicalensis by DEAE-52 and Sephadex G-100 column chromatography. The structure of SBP-1 was characterized using HPLC, IR, GC–MS, 1-D and 2-D NMR. The antioxidant and hypoglycemic activity of SBP-1 was investigated by vitro evaluation. The results showed that SBP-1 was composed of arabinose, glucose and galactose in a molar ratio of 1.0:5.9:1.1 and its Mw were 91,156. The backbone of SBP-1 was mainly composed of repeating →1)-α-D-Glcp-(4 → 1)-α-D-Glcp-(3 → 1)-α-D-Galp-(4→. The braches were composed of →2)-α-L-Araf-(1→, →3)-β-D-Glcp-(1→ and α-D-Glcp-(1→, which mainly substituted at O-6 of Glc, while terminal residue was α-L-Araf-(1→ and α-D-Glcp-(1→. Vitro bioactivity showed that SBP-1 had dose-dependent antioxidant and hypoglycemic activity. The scavenging rate on ABTS, DPPH, hydroxyl and superoxide radicals was all beyond 60% as SBP-1 concentration reached 4 mg/mL, and the inhibition rate on α-glucosidase and α-amylase was both more than 80%, which was closely to that of acarbose.

Acid-Catalyzed Water Extraction of Two Polysaccharides from Artemisia argyi and Their Physicochemical Properties and Antioxidant Activities.

In this study, two purified polysaccharide fractions, Artp1 and Artp2, were obtained using acid-catalyzed water extraction, and then purified by DEAE-52 cellulose and Sephadex G-200 column chromatography from the crude polysaccharides of Artemisia argyi. Their physicochemical properties were investigated by gel permeation chromatography (GPC), high-performance anion exchange chromatography (HPAEC), Fourier transform infrared (FT-IR), scanning electron microscope (SEM), thermal analysis, and methylation analysis. The average molecular weight (Mw) of Artp1 and Artp2 were estimated to be 42.17 kDa and 175.22 kDa, respectively. Monosaccharide composition analysis revealed that the Rha, Gal, and GalA occupied main proportion in Artp1 with the molar ratio of 25.1:24.7:40.4, while the Rha, Gal, Xly, and GalA occupied the main proportion in Artp2 with the molar ratio of 16.7:13.5:12.8:38.7. Due to the high yield and the relatively high carbohydrate content, the Artp1 was determined by the methylation analysis and NMR. The results of Artp1 indicated that 1,4-GalpA and 1,2,4-Rhap formed the backbone with some 1,2-Rhap, 1,3-Galp, and 1,6-Galp in the backbone or the side chains. Artp1 and Artp2 exhibited effective antioxidant activities by DPPH radical scavenging assay and hydroxyl radical scavenging assay in a dose-dependent manner. These investigations of the polysaccharides from A. argyi. provide a scientific basis for the uses of Artp1 and Artp2 as ingredients in functional foods and medicines.

Characterization of an Acidic Polysaccharides from Carrot and Its Hepatoprotective Effect on Alcoholic Liver Injury in Mice.

The characteristics of acidic polysaccharides extracted from Daucus carota L. var. sativa Hoffm were investigated and its hepatoprotective effects on alcoholic liver injury were determined in the mice model. A carrot polysaccharide (CPS-I: Carrot polysaccharide-I) with the molecular weight of 3.40×104 kDa was isolated from Daucus carota L. and purified by diethylaminoethyl-52 and Sephadex G-150 column chromatography. The components were analyzed by HPLC, which revealed that CPS-I consisted of galacturonic acid, rhamnose, xylose, arabinose, fructose, and galactose at a relative ratio of 1 : 3.16 : 1.13 : 5.53 : 3.45 : 7.76. Structural characterization analysis suggested that CPS-I was mainly composed of →6)-β-D-Galp-(1→ and →5)-α-L-Araf-(1→. The hepatoprotective effect of CPS-I was evaluated by alcoholic liver injury mice model. The results showed that the administration of CPS-I (300 mg/kg/day) alleviated the alcoholic liver injury in mice by increasing the levels of ADH and ALDH and reducing oxidative stress. CPS-I ameliorated the pathological changes of liver characterized by lipid accumulation, and reduced the number of lipid droplets.

Structural characterization of novel arabinoxylan and galactoarabinan from citron with potential antitumor and immunostimulatory activities

This study aimed to extract polysaccharides from citron and analyze their structures and potential bioactivities. Two novel polysaccharides CM-1 and CM-2 were purified from citron by DEAE-Sepharose Fast Flow and Sephadex G-100 column chromatography. Monosaccharide composition, linkage and NMR data were used to infer their sugar chains composition. The anti-breast cancer cells and immunoregulatory activities of CM-1 and CM-2 were investigated. Results indicated that CM-1 (Mw = 21,520 Da), composed of arabinose, xylose, mannose and glucose in a molar ratio of 10.78:11.53:1.00:1.70, was arabinoxylan (AX) with (1 → 4)-linked β-d-Xylp skeleton monosubstituted with α-l-Araf units at O-3 position. While CM-2 (Mw = 22,303 Da), composed of arabinose, mannose, glucose and galactose in a molar ratio of 25.46:1.45:1.00:6.57, was galactoarabinan (GA) with (1 → 5)-linked α-l-Araf backbone substituted by β-d-Galp units at O-2 and/or O-3 positions. Both polysaccharides exhibited potential inhibiting cancer and immunostimulatory activities in vitro, especially CM-1. These results provide a basis for further research on citron polysaccharides.

Purification of high molecular weight thermotolerant esterase from Serratia sp. and its characterization.

In the present study, an extracellular esterase from Serratia sp. was purified 24.46 fold using an initial ammonium sulphate precipitation step (optimized concentration of 30-40%), followed by Diethylaminoethyl cellulose (DEAE-cellulose) chromatography and size exclusion Sephadex G-200 column chromatography steps. The molecular weight of the esterase using native polyacrylamide gel electrophoresis (PAGE) was determined to be 236kDa and by using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) was found to be 60kDa suggesting that the enzyme was a tetramer of 4 subunits. The purified esterase was able to catalyze the hydrolysis of p-nitrophenyl esters, especially p-nitrophenyl acetate. Maximum esterase activity was achieved in 0.15M Tris-HCl buffer of pH 8.5 at 50°C after 10min. The enzyme was stable for at least 8h at 4 and 35°C but the half-life was determined to be 4.5h at 50°C and 3h at 60°C. The esterase activity was inhibited by detergents (1mM) (Triton X-100, Tween 60, Tween 80, ethylenediamine tetraacetic acid and SDS) except Tween 20. The esterase activity was inhibited by organic solvents (1mM) such as ethanol, methanol, acetone, acetonitrile and was stable in the presence of glycerol, isopropanol but the organic solvent dimethyl sulfoxide (DMSO) significantly (p < 0.05) enhanced esterase activity. The matrix-assisted laser desorption ionization-time of flight mass spectrometry showed that the enzyme exhibited similarity with the pimeloyl-[acyl carrier protein] methyl ester esterase of Serratia marcescens.

Ultrasound-assisted extraction of boulardii yeast cell wall polysaccharides: Characterization and its biological functions on early-weaned lambs.

Firstly, this study was designed to determine the optimal ultrasound‐assisted extraction parameters of Saccharomyces boulardii yeast wall polysaccharides (BYWP). Besides, the molecular weight and the ratio of mannose to glucose in compositions of BYWP were determined. Also, the effects of BYWP on the gain feed ratio, diarrhea frequency, intestinal morphology, intestinal immunity, and intestinal microbial flora of early‐weaned lambs were investigated. Single‐factor tests and Response surface optimization analysis (RSA) were used to obtain the optimal ultrasound‐assisted extraction conditions. Sephadex G‐100 column chromatography and liquid chromatography were used to analyze the molecular weight and ratio of mannose to glucose. The feeding trial was used to observe the biological functions of BYWP on early‐weaned lambs. A total of 72 36‐day‐old crossbred early‐weaned lambs were randomly divided into 4 groups with 3 replicates per group and 6 lambs per replicate. Lambs in the four treatments were fed basal milk replacer without supplement (Group I), basal milk replacer+0.1% BYWP (Group II), basal milk replacer+0.3% BYWP (Group III), and basal milk replacer+0.5% BYWP (Group IV), respectively. The optimal ultrasound‐assisted extraction parameters were as follows: NaOH addition: 52.63%, ultrasonic power: 143.15 W, ultrasonic time: 86.20 min, and the optimized extraction yield reached 37.54%. The molecular weights of main components BLC‐1 and BLC‐2 were 164.68 KDa and 13.21 KDa, and their proportions in BYWP were 24.57% and 66.08%, respectively. The proportions of glucose, mannose in BLC‐1 and BLC‐2 were 47.68%, 39.18%, and 76.59%, 6.86%, respectively. The addition of 0.3% and 0.5% BYWP in basal milk replacer significantly increased the average daily gain and feed conversion rate, and decreased the average fecal index and diarrhea rate of early‐weaned lambs. The addition of 0.3% and 0.5% BYWP significantly enhanced the intestinal morphology (villus height, crypt depth, and V/C value) of jejunum, ileum (p < .05). The addition of 0.3% and 0.5% BYWP significantly improved the levels of SIgA and IL‐10, but significantly decreased the level of IL‐1 in the ileum (p < .05). The addition of 0.3% and 0.5% BYWP significantly increased the number of Lactobacillus, but significantly suppressed the growth of Salmonella and Clostridium perfringens (p < .05). The results of the present study suggest that the supplementation of BYWP in the diet of early‐weaned lambs could increase feed utilization rate, and enhance intestinal morphology, immunological competence, microbial flora balance, and decrease the rate of diarrhea occurrence.

Purification and Structural Characterization of Dendrobium officinale Polysaccharides and Its Activities.

In this study, Dendrobium officinale polysaccharide (named DOPS-1) was isolated from the stems of Dendrobium officinale by hot-water extraction and purified by using Sephadex G-150 column chromatography. The structural characterization, antioxidant and cytotoxic activity were carried out. Based on the results of HPLC, GC, Congo red experiment, together with periodate oxidation, Smith degradation, SEM, FT-IR, and NMR spectral analysis, it expressed that DOPS-1 was largely composed of mannose, glucose and galacturonic acid in a molar ratio of 3.2 : 1.3 : 1. The molecular weight of DOPS-1 was 1530 kDa and the main chain was composed of (1→4)-β-D-Glcp, (1→4)-β-D-Manp and 2-O-acetyl-(1→4)-β-D-Manp. The measurement results of antioxidant activity showed that DOPS-1 had the strong scavenging activities on hydroxyl radicals, DPPH radicals and superoxide radicals and the high reducing ability in vitro. Moreover, DOPS-1 was cytotoxic to all three human cancer cells of MDA-MB-231, A549 and HepG2.