Purification,characterisation and application of α‐l‐rhamnosidase from Penicillium citrinum MTCC‐8897

An α‐l ‐rhamnosidase secreted by Penicillium citrinum MTCC‐8897 has been purified to homogeneity from the culture filtrate of the fungal strain using ammonium sulphate precipitation and cation‐exchange chromatography on carboxymethyl cellulose. The sodium dodecyl sulphate/polyacrylamide gel electrophoresis analysis of the purified enzyme gave a single protein band corresponding to the molecular mass 51.0 kDa. The native polyacrylamide gel electrophoresis also gave a single protein band confirming the enzyme purity. The K_m and V_max values of the enzyme for p‐nitrophenyl α‐l ‐rhamnopyranoside were 0.36 mm and 22.54 μmole min^?1 mg^?1, respectively, and k_cat value was 17.1 s^?1 giving k_cat/K_m value of 4.75 × 10⁴ m ^?1 s^?1. The pH and temperature optima of the enzyme were 7.0 and 60 °C, respectively. The purified enzyme liberated l ‐rhamnose from naringin, rutin, hesperidin and wine, indicating that it has biotechnological application potential for the preparation of l ‐rhamnose and other pharmaceutically important compounds from natural glycosides containing terminal α‐l ‐rhamnose and also in the enhancement of wine aroma.     

Purification and functional characterisation of an α‐l‐rhamnosidase from Penicillium citrinum MTCC‐3565

An extracellular α‐l ‐rhamnosidase from Penicillium citrinum MTCC‐3565 has purified to homogeneity from its culture filtrate using ethanol precipitation and cation‐exchange chromatography on carboxymethyl cellulose. The purified enzyme gave a single protein band corresponding to molecular mass of 45.0 kDa in SDS‐PAGE analysis showing the purity of the enzyme preparation. The native PAGE analysis showed the monomeric nature of the purified enzyme. Using p‐nitrophenyl α‐l ‐rhamnopyranoside as substrate, K_m and V_max values of the enzyme were 0.30 mm and 27.0 μm min mg^?1, respectively. The k_cat value was 20.1 s giving k_cat/K_m value of 67.0 mm s^?1 for the same substrate. The pH and temperature optima of the enzyme were 8.5 and 50 °C, respectively. The activation energy for the thermal denaturation of the enzyme was 29.9 KJ mol^?1. The α‐l ‐rhamnosidase was able to hydrolyse naringin, rutin and hesperidin and liberated l ‐rhamnose, indicating that the purified enzyme can be used for the preparation of α‐l ‐rhamnose and pharmaceutically important compounds by derhamnosylation of natural glycosides containing terminal α‐l ‐rhamnose. The α‐l ‐rhamnosidase was active at the level of ethanol concentration present in wine, indicating that it can be used for improving wine aroma.     

α‐l‐Rhamnosidase from Aspergillus flavus MTCC‐9606 isolated from lemon fruit peel

An α‐l ‐rhamnosidase producing fungal strain has been isolated from decaying lemon fruit. The fungal strain has been identified as Aspergillus flavus. The α‐l ‐rhamnosidase has been purified from the culture filtrate of the fungal strain using ultra filtration and cation exchange chromatography on carboxy methyl (CM) cellulose. The molecular mass of the purified enzyme determined by SDS–PAGE analysis was 41 kDa. The K_m values of the enzyme using p‐nitrophenyl‐α‐l ‐rhamnopyranoside and naringin as the substrates were 1.89 and 1.6 mm respectively. The pH and temperature optima of the enzyme were 11.0 and 50 °C respectively. The effects of various chemical species present in grape fruit juice and wine on the activity of the enzyme have been determined.     

l‐Citrulline Supplementation‐Increased Skeletal Muscle PGC‐1α Expression Is Associated with Exercise Performance and Increased Skeletal Muscle Weight

Scope

l ‐citrulline has recently been reported as a more effective supplement for promoting intracellular nitric oxide (NO) production compared to l ‐arginine. Here, the effect of l ‐citrulline on skeletal muscle and its influence on exercise performance were investigated. The underlying mechanism of its effect, specifically on the expression of skeletal muscle peroxisome proliferator‐activated receptor‐gamma coactivator‐1α (PGC‐1α), was also elucidated.

Methods and results

Six‐week‐old ICR mice were orally supplemented with l ‐citrulline (250 mg kg^?1) daily, and their performance in weight‐loaded swimming exercise every other day for 15 days, was evaluated. In addition, mice muscles were weighed and evaluated for the expression of PGC‐1α and PGC‐1α‐regulated genes. Mice orally supplemented with l ‐citrulline had significantly higher gastrocnemius and biceps femoris muscle mass. Although not statistically significant, l ‐citrulline prolonged the swimming time to exhaustion. PGC‐1α upregulation was associated with vascular endothelial growth factor α (VEGFα) and insulin‐like growth factor 1 (IGF‐1) upregulation. VEGFα and IGF‐1 are important for angiogenesis and muscle growth, respectively, and are regulated by PGC‐1α. Treatment with NG‐nitro‐l ‐arginine methyl ester hydrochloride (l ‐NAME), a nitric oxide synthesis inhibitor, suppressed the l ‐citrulline‐induced PGC‐1α upregulation in vitro.

Conclusion

Supplementation with l ‐citrulline upregulates skeletal muscle PGC‐1α levels resulting in higher skeletal muscle weight that improves time to exhaustion during exercise.

     

An α‐l‐rhamnosidase from Aspergillus awamori MTCC‐2879 and its role in debittering of orange juice

The extracellular α‐l ‐rhamnosidase has been purified by growing a new fungal strain Aspergillus awamori MTCC‐2879 in the liquid culture growth medium containing orange peel. The purification procedure involved ultrafiltration using PM‐10 membrane and anion‐exchange chromatography on diethyl amino ethyl cellulose. The purified enzyme gave single protein band in SDS‐PAGE analysis corresponding to molecular mass 75.0 kDa. The native PAGE analysis of the purified enzyme also gave a single protein band, confirming the purity of the enzyme. The K_m and V_max values of the enzyme for p‐nitrophenyl‐α‐l ‐rhamnopyranoside were 0.62 mm and 27.06 μmole min^?1 mg^?1, respectively, yielding k_cat and k_cat/k_m values 39.90 s^?1 and 54.70 mm ^?1 s^?1, respectively. The enzyme had an optimum pH of 7.0 and optimum temperature of 60 °C. The activation energy for the thermal denaturation of the enzyme was 35.65 kJ^?1 mol^?1 K^?1. The purified enzyme can be used for specifically cleaving terminal α‐l ‐rhamnose from the natural glycosides, thereby contributing to the preparation of pharmaceutically important compounds like prunin and l ‐rhamnose.     

3‐Hydroxypyridinone‐l‐phenylalanine conjugates with antimicrobial and tyrosinase inhibitory activities as potential shrimp preservatives

To explore the potential of novel tyrosinase inhibitors, 3‐hydroxypyridinone‐l ‐phenylalanine conjugates, as shrimp preservatives, compounds 1 and 2 were evaluated for the antimicrobial activity, and compound 2 was investigated for the shrimp preservative efficacy. It was found that they both possess a stronger antibacterial effect than kojic acid against two Gram‐positive bacteria and three Gram‐negative bacteria. The minimum inhibitory concentrations (MICs) of compound 2 against Escherichia coli, Staphylococcous aureus, Salmonella gallinarum, Vibrio parahaemolyticus and Bacillus subtilis were determined as 18.5, 37, 148, 37 and 295 μg mL^?1, respectively, whereas MICs of kojic acid against the same 5 bacterial strains were determined to be 355, 178, 1420, 1420 and 355 μg mL^?1, respectively. It has also been demonstrated that treatment with compound 2 improves the sensory properties, retards the growth of spoilage bacteria, decreases the total volatile basic nitrogen (TVB‐N) and increases the pH of Penaeus vannamei Boone, thereby extending the shelf life to 10 days. In contrast, the shelf life of shrimp treated with kojic acid and the control group was 7 and 6 days, respectively. Clearly, 3‐hydroxypyridinone‐l ‐phenylalanine conjugates could find application as shrimp preservatives by inhibiting melanosis and by preventing the growth of bacteria during the storage.     

Characterization of the Supermolecular Structure of Polydatin/6‐O‐α‐Maltosyl‐β‐cyclodextrin Inclusion Complex

Polydatin is the main bioactive ingredient in many medicinal plants, such as Hu‐zhang (Polygonum cuspidatum), with many bioactivities. However, its poor aqueous solubility restricts its application in functional food. In this work, 6‐O‐α‐Maltosyl‐β‐cyclodextrin (Malt‐β‐CD), a new kind of β‐CD derivative was used to enhance the aqueous solubility and stability of polydatin by forming the inclusion complex. The phase solubility study showed that polydatin and Malt‐β‐CD could form the complex with the stoichiometric ratio of 1:1. The supermolecular structure of the polydatin/Malt‐β‐CD complex was characterized by ultraviolet–visible spectroscopy (UV), Fourier transform infrared spectroscopy (FT‐IR), X‐ray diffractometry (XRD), thermogravimetric/differential scanning calorimetry (TG/DSC), and proton nuclear magnetic resonance (¹H‐NMR) spectroscopy. The changes of the characteristic spectral and thermal properties of polydatin suggested that polydatin could entrap inside the cavity of Malt‐β‐CD. Furthermore, to reasonably understand the complexation mode, the supermolecular structure of polydatin/Malt‐β‐CD inclusion complex was postulated by a molecular docking method based on Autodock 4.2.3. It was clearly observed that the ring B of polydatin oriented toward the narrow rim of Malt‐β‐CD with ring A and glucosyl group practically exposed to the wide rim by hydrogen bonding, which was in a good agreement with the spectral data.     

Investigation of the interaction between (−)‐epigallocatechin‐3‐gallate with trypsin and α‐chymotrypsin

Tea polyphenol (TP) inhibits digestive enzymes and reduces food digestibility. To explore the interaction between TP with digestive enzymes, bindings of ‐epigallocatechin‐3‐gallate (EGCG) to trypsin and α‐chymotrypsin were studied in detail using fluorescence, resonance light‐scattering, circular dichroism, fourier transform infrared spectroscopy methods and protein‐ligand docking. The binding parameters were calculated according to Stern–Volmer equation, and the thermodynamic parameters were determined by the van't Hoff equation. The results indicated that EGCG was capable of binding trypsin and α‐chymotrypsin with high affinity, resulting in a change of native conformation of these enzymes. EGCG had a greater influence on the structure of α‐chymotrypsin than trypsin. This study can be used to explain the binding interaction mechanism between TP and digestive enzymes.