The simultaneous saccharification and fermentation of pretreated woody crops to ethanol

Four promising woody crops (Populusmaximowiczii x nigra (NE388), P.trichocarpa x deltoides (Nll), P.tremuloides, and SweetgumLiquidambar styraciflua) were pretreated by dilute sulfuric acid and evaluated in the simultaneous saccharification and fermentation (SSF) process for ethanol production. The yeastSaccharomyces cerevisiae was used in the fermentations alone, and in mixed cultures with β -glucosidase producingBrettanomyces dausenii. Commercial Genencor 150L cellulase enyme was either employed alone or supplemented with β- glucosidase. All SSFs were run at 37 …C for 8 d and compared to saccharifications at 45…C under the same enzyme loadings.S. cerevisiae alone achieved the highest ethanol yields and rates of hydrolysis at the higher enzyme loadings, whereas the mixed culture performed better at the lower enzyme loadings without β -glucosidase supplementation. The best overall rates of fermentation (3 d) and final theoretical ethanol yields (86–90%) were achieved with P.maximowiczii x nigra (NE388) and SweetgumLiquidambar styraciflua, followed by P.tremuloides and P.trichocarpa xdeltoides (N1l) with slightly slower rates and lower yields. Although there were some differences in SSF performance, all these pretreated woody crops show promise as substrates for ethanol production.     

Acid Pullulanase from Bacillus polymyxa MIR-23

Within the frame of a screening program aimed at the isolation of amylolytic sporeformers, one strain with high amylolytic activity designated MIR-23 was selected. The microbial characterization was carried out by morphological and biochemical tests and, by means of statistical treatment, was identified asBacillus polymyxa. The organism could grow in acidic conditions (pH 5.0) on a starch medium and produce α-amylase, pullulanase, and α-glucosidase. Batch cultures showed the highest enzyme activities in the stationary phase. Pullulanase activity exhibited an optimal temperature of 52–57°C at pH 4.5–5.5. These properties would allow its use in the saccharification processes in the starch industries.     

Purification and Characterization of an Intracellular β-Glucosidase from Lactobacillus casei ATCC 393

The lactic acid bacterium,Lactobacillus casei, produces an intracellular β-glucosidase when grown on Man-Rogosa-Sharpe (MRS) medium with cellobiose as carbon source. The β-glucosidase activity is produced intracellulary, and no extracellulary activity was detected. The enzyme was purified by ion-exchange chromatography and gel filtration. The molecular mass of the purified intracellular β-glucosidase as estimated by gel filtration was 480 kDa, consisting of six probably identical subunits. The enzyme exhibited optimum activity at 35°C and pH 6.3 with citrate-phosphate buffer. The enzyme was active against soluble glycosides with (1→4)-β configuration and from Lineweaver Burk plots, K_m value of 16 mmol/L was found for β-pNPG. The β-glucosidase was competitively inhibited by glucose, and no glycosyl transferase activity was observed in the presence of ethanol.     

Hydrolysis of cellobiose by immobilized β-glucosidase entrapped in maintenance-free gel spheres

A crude preparation of Aspergillus niger β-glucosidase (27.5 cello-biase U/mg protein at 40°C, pH 5.0) was immobilized on concanavalin A-Sepharose (CAS). The cellobiase activity of the immobilized enzyme was 1334 U/mg dried CAS or 108 U/mL CAS gel. The β-glucosidase-CAS complex was entrapped within crosslinked propylene glycol alginate/bone-geletin gel spheres that possessed between 0.67 and 2.35 cellobiase U/mL spheres, depending on their size. The effect of cellobiose concentration (10–300 mM) on the activity of native, immobilized, and gel-entrapped enzyme was determined. It was shown that concentrations of cellobiose between 10 and 180 mM were not inhibitory to the entrapped enzyme, although inhibition was found to occur with the native and immobilized enzyme. Exogenous ion addition was not necessary to maintain the structural integrity of the spheres, which were stable for 4 d at 40°C.     

Anti-Inflammatory,Antidiabetic Properties and In Silico Modeling of Cucurbitane-Type Triterpene Glycosides from Fruits of an Indian Cultivar of Momordica charantia L.

Diabetes mellitus is a chronic disease and one of the fastest-growing health challenges of the last decades. Studies have shown that chronic low-grade inflammation and activation of the innate immune system are intimately involved in type 2 diabetes pathogenesis. Momordica charantia L. fruits are used in traditional medicine to manage diabetes. Herein, we report the purification of a new 23-O-β-d-allopyranosyl-5β,19-epoxycucurbitane-6,24-diene triterpene (charantoside XV, 6) along with 25ξ-isopropenylchole-5(6)-ene-3-O-β-d-glucopyranoside (1), karaviloside VI (2), karaviloside VIII (3), momordicoside L (4), momordicoside A (5) and kuguaglycoside C (7) from an Indian cultivar of Momordica charantia. At 50 µM compounds, 2–6 differentially affected the expression of pro-inflammatory markers IL-6, TNF-α, and iNOS, and mitochondrial marker COX-2. Compounds tested for the inhibition of α-amylase and α-glucosidase enzymes at 0.87 mM and 1.33 mM, respectively. Compounds showed similar α-amylase inhibitory activity than acarbose (0.13 mM) of control (68.0–76.6%). Karaviloside VIII (56.5%) was the most active compound in the α-glucosidase assay, followed by karaviloside VI (40.3%), while momordicoside L (23.7%), A (33.5%), and charantoside XV (23.9%) were the least active compounds. To better understand the mode of binding of cucurbitane-triterpenes to these enzymes, in silico docking of the isolated compounds was evaluated with α-amylase and α-glucosidase.     

Cellulose filter paper immobilized α-glucosidase and its application to screening inhibitors from traditional Chinese medicine

In this study, α-glucosidase was successfully immobilized on cellulose filter paper and further applied to screening inhibitors from traditional Chinese medicines combined with capillary electrophoresis analysis. For α-glucosidase immobilization, a cellulose filter paper was used as the carrier and grafted with amino groups by coating chitosan, then α-glucosidase was covalently bonded on the amino-modified carrier via epoxy ring-opening reaction using polyethylene glycol diglycidyl ether as the crosslinker. Several parameters influencing the enzyme immobilization were optimized and the optimal values were enzyme concentration of 4 U/mL, polyethylene glycol diglycidyl ether concentration of 1.25%, chitosan concentration of 7.5 mg/mL, immobilization pH 7.0, crosslinking time of 4 h and immobilization time of 2 h. The immobilized α-glucosidase exhibited good batch-to-batch reproducibility (RSD = 2.1%, n = 5), excellent storage stability (73.5% of its initial activity after being stored at 4°C for 15 days), and reusability (75% of its initial activity after 10 repeated cycles). The Michaelis constant of immobilized α-glucosidase and half-maximal inhibitory concentration of acarbose were calculated to be 1.12 mM and 0.38 μM, respectively. Finally, the immobilized α-glucosidase was used for screening inhibitors from 14 kinds of Traditional Chinese Medicine extracts, and Sanguisorbae Radix showed the strongest inhibitory effect on α-glucosidase.     

UHPLC-DAD Characterization of Origanum vulgare L. from Atacama Desert Andean Region and Antioxidant,Antibacterial and Enzyme Inhibition Activities

The Lamiaceae family is an important source of species among medicinal plants highly valued for their biological properties and numerous uses in folk medicine. Origanum is one of the main genera that belong to this family. The purpose of the study was to determine the phenolic composition of the Origanum vulgare extract and evaluate the antimicrobial, antioxidant, and inhibitory activities of this species that grows in the Andean region of the Atacama Desert. High-performance liquid chromatography was performed to determine the main phenols. Rosmarinic acid was identified as the predominant phenolic compound in this species (76.01 mg/100 g DW), followed by protocatechuic acid, which to our knowledge, no previous study reported similar concentrations in O. vulgare. The oregano extract exhibited a content of total phenolic (3948 mg GAE/100 g DW) and total flavonoid (593 mg QE/100 g DW) with a higher DPPH antioxidant activity (IC₅₀ = 40.58 µg/mL), compared to the same species grown under other conditions. Furthermore, it was found to inhibit α-glucosidase activity with an IC₅₀ value (7.11 mg/mL) lower than acarbose (129.32 mg/mL). Pseudomonas syringae and Pantoea agglomerans (both MIC 0.313 mg/mL and MBC 1.25 mg/mL) were the bacteria most susceptible to oregano extract with the lowest concentration necessary to inhibit bacterial growth. These results open the door for the potential use of this plant to manage chronic diseases, and they expand the knowledge of the species cultivated in arid environmental conditions.     

Four Novel Phenanthrene Derivatives with α-Glucosidase Inhibitory Activity from Gastrochilus bellinus

Four new phenanthrene derivatives, gastrobellinols A-D (1–4), were isolated from the methanolic extract of Gastrochilus bellinus (Rchb.f.) Kuntze, along with eleven known phenolic compounds including agrostophyllin (5), agrostophyllidin (6), coniferyl aldehyde (7), 4-hydroxybenzaldehyde (8), agrostophyllone (9), gigantol (10), 4-(methoxylmethyl)phenol (11), syringaldehyde (12), 1-(4′-hydroxybenzyl)-imbricartin (13), 6-methoxycoelonin (14), and imbricatin (15). Their structures were determined by spectroscopic methods. Each isolate was evaluated for α-glucosidase inhibitory activity. Compounds 1, 2, 3, 7, 9, 13, and 15 showed higher activity than the drug acarbose. Gastrobellinol C (3) exhibited the strongest α-glucosidase inhibition with an IC₅₀ value of 45.92 μM. A kinetic study of 3 showed competitive inhibition on the α-glucosidase enzyme. This is the first report on the phytochemical constituents and α-glucosidase inhibitory activity of G. bellinus.     

哈茨木霉CGMCC 2979生物转化栀子中的京尼平苷制备京尼平

采用微生物直接转化药材的方法,将栀子中的京尼平苷转化为京尼平,无需糖苷酶和京尼平苷的制备. 在培养温度为30 ℃,pH 6.1以及栀子载量为80 g/L的条件下,48 h京尼平苷的转化率为97.8%. 转化后的京尼平通过XAD-16N大孔树脂偶联硅胶层析的方法,制备得到纯度大于95%的京尼平,收率为62.3%. 在催化、转化机制研究中,从哈茨木霉CGMCC2979的发酵液中分离得到了分子量为74.4 kDa的京尼平苷β-葡萄糖苷酶,该酶最优催化条件为50 ℃和pH 4.0-5.0. K_m和V_max分别为3.6 mmol/L和775 μmol/h/mg蛋白. 本文提供了一种简便、高效制备京尼平的新方法.