Characterization of alpha-Galactosidase from Cucumber Leaves

Two forms of alpha-galactosidase (alpha-d-galactoside galactohydrolase, E.C. 3.2.1.22) which differed in molecular weight were resolved from Cucumis sativus L. leaves. The enzymes were partially purified using ammonium sulfate fractionation, Sephadex gel filtration, and diethylaminoethyl-Sephadex chromatography. The molecular weights of the two forms, by gel filtration, were 50,000 and 25,000. The 50,000-dalton form comprised approximately 84% of the total alpha-galactosidase activity in crude extracts from mature leaves and was purified 132-fold. The partially purified 25,000-molecular weight form rapidly lost activity unless stabilized with 0.2% albumin and accounted for 16% of the total alpha-galactosidase activity in the crude extract. The smaller molecular weight form was not found in older leaves.The two forms were similar in several ways including their pH optima which were 5.2 and 5.5 for the 50,000- and 25,000-dalton form, respectively, and activation energies, which were 15.4 and 18.9 kilocalories per mole for the larger and smaller forms. Both enzymes were inhibited by galactose as well as by excess concentrations of p-nitrophenyl-alpha-d-galactoside sub-strate. K(m) values with this substrate and with raffinose and melibiose were different for each substrate, but similar for both forms of the enzyme. With stachyose, K(m) values were 10 and 30 millimolar for the 50,000- and 25,000- molecular weight forms, respectively.     

Identification of a cerulenin resistance gene from Monascus pilosus.

Detoxification is essential for the fungal growth in the drug stress environments, and the multidrug transporters play an important role in this process. Here a cerulenin transporter gene (MpMdt, AB206476) was identified from Monascus pilosus. MpMdt mRNA contains 1951 bp and encodes a protein of 559 amino acid residues with 11 trans-membrane domains; and there is no difference in the sequence of MpMdt mRNA between the wild type M. pilosus IFO4520 and its cerulenin resistant mutant MK-1. Up-expression of MpMdt renders the cerulenin resistance of the mutant MK-1. Over-expression of MpMdt could also increase the cerulenin tolerance in the transgenic M. pilosus IFO4520. These results suggested that MpMdt is able to efflux-transport the anti-fungal antibiotic cerulenin and increase the cerulenin resistance of M. pilosus.     

Production, Partial Purification and Characterization of Extracelluar Xylanase from Chaetomium globosum

Culture conditions for efficient production of extracellular xylanase by fungus, Chaetomium globosum isolate Cg2, have been standardized. Further, xylanase has been partially purified and characterized. Xylanase activity was maximum after 9 days of incubation when amended in medium with 1.5 % xylan as carbon source and 0.6% NH₄H₂PO₄ as nitrogen source. Partial purification of the xylanase was accomplished by ammonium sulphate precipitation, followed by further purification by anion exchange chromatography on DEAE-Sephadex A-50 column. The partially purified enzyme was electrophoresed on SDS-PAGE and a single band produced corresponded to molecular weight, 32 kD. The optimum temperature and pH for maximum activity of purified xylanase were 30°C and 5.5, respectively. Both the purified xylanase and culture filtrate have shown the antifungal activity against Bipolaris sorokiniana, a causal organism of spot blotch of wheat. Purified xylanase at 100 μg ml^?1 concentration caused 100 per cent inhibition of conidia germination of B. sorokiniana, whereas the culture filtrate was able to inhibit germination up to 67.5 per cent.     

Effects of the principal nutrients on lovastatin production by Monascus pilosus

Lovastatin production is dependent on the substrates provided. We investigated how several carbon and nitrogen sources in the medium affect lovastatin production by Monascus pilosus. M. pilosus required a suitable concentration of organic nitrogen peptone for high lovastatin production. As sole carbon source with peptone, although glucose strongly repressed lovastatin production, maltose was responsible for high production. Interestingly, glycerol combined with maltose enhanced lovastatin production, up to 444 mg/l in the most effective case. Moreover, an isolated mutant, in which glucose repression might be relieved, easily produced the highest level of lovastatin, 725 mg/l on glucose-glycerol-peptone medium. These observations indicate that lovastatin production by M. pilosus is regulated by strict glucose repression and that an appropriate release from this repression by optimizing medium composition and/or by a mutation(s) is required for high lovastatin production.     

Antimicrobial Activity of Monascus pilosus IFO 4520 against Contaminant of Koji

Antimicrobial activity of Monascus pilosus IFO 4520 was examined to prevent contamination during beni-koji making in the open air. The antibacterial effect of the beni-koji prepared with this strain occured with 30 mg/ml of beni-koji extract in combination with 0.5% lactic acid against two contaminants of koji, Micrococcus varians and Bacillus subtilis. There were two compounds, antibacterial and antiyeast substances, in the beni-koji extract. These results suggest a possibility of inhibiting the growths of contaminants during beni-koji making using beni-koji extract and lactic acid.     

Secondary metabolites produced by the fungus Monascus pilosus and their anti-inflammatory activity

A detailed phytochemical study of the n-BuOH-soluble fraction of the 95% EtOH extract of red yeast rice fermented with the fungus Monascus pilosus BCRC 38072 (Eurotiaceae) revealed a new azaphilone derivative, namely monascusazaphilol (1) (rel-(3S,3aR,9aR)-3-(1-hydroxydecyl)-9a-methyl-6-((E)-propenyl)-3a,4,8,9a-tetrahydro-3H-furo[3,2-g]isochromene-2,9-dione), along with ten known compounds (2–11). Their structures were established by direct interpretation of their spectral data, mainly high resolution electrospray ionization mass spectrometry (HR-ESI-MS), 1D and 2D NMR (COSY, ROESY, HSQC and HMBC), and comparison of the spectroscopic data with those reported for structurally related compounds. We also evaluated the inhibitory effects of some isolates on the production of tumor necrosis factor (TNF-α) induced by lipopolysaccharide (LPS). Among the isolates, compounds 1–3 inhibited TNF-α production in U937 cells in vitro, and the IC₅₀ values were 1.24, 2.35 and 3.75 μg/ml, respectively.     

Partial Purification and Properties of an Alkaline alpha-Galactosidase from Mature Leaves of Cucurbita pepo

A fourth molecular from of α-galactosidase, designated L_IV, an alkaline α-galactosidase, was isolated from leaves of Cucurbita pepo and purified 165-fold. It was active over a narrow pH range with optimal hydrolysis of p-nitrophenyl-α-d-galactoside and stachyose at pH 7.5. The rate of stachyose hydrolysis was 10 times that of raffinose. K_m determinations in McIlvaine buffer (200 millimolar Na₂-phosphate, 100 millimolar citric acid, pH 7.5) for p-nitrophenyl-α-d-galactoside, stachyose, and raffinose were 1.40, 4.5, and 36.4 millimolar, respectively. L_IV was partially inhibited by Ca²⁺, Mg²⁺, and Mn²⁺, more so by Ni²⁺, Zn²⁺, and Co²⁺, and highly so by Cu²⁺, Ag²⁺, Hg²⁺ and by p-chloromercuribenzoate. It was not inhibited by high concentrations of the substrate p-nitrophenyl-α-d-galactoside or by myo-inositol, but α-d-galactose was a strong inhibitor. As observed for most other forms of α-galactosidase, L_IV only catalyzed the hydrolysis of glycosides possessing the α-d-galactose configuration at C₁, C₂, and C₄, and did not hydrolyze p-nitrophenyl-α-d-fucoside (α-d-galactose substituted at C₆). The enzyme was highly sensitive to buffers and chelating agents. Maximum hydrolytic activity for p-nitrophenyl-α-d-galactoside was obtained in McIlvaine buffer (pH 7.5). In 10 millimolar triethanolaminehydrochloride-NaOH (pH 7.5) or 10 millimolar Hepes-NaOH (pH 7.5), hydrolytic activity was virtually eliminated, but the addition of low concentrations of either ethylenediaminetetraacetate or citrate to these buffers restored activity almost completely. Partial restoration of activity was also observed, but at higher concentrations, with pyruvate and malate. Similar effects were found for stachyose hydrolysis, but in addition some inhibition of L_IV in McIlvaine buffer, possibly due to the high phosphate concentration, was observed with this substrate. It is questionable whether the organic acid anions possess any regulatory control of L_IVin vivo. It was possible that the results reflected the ability of these anions, and ethylene-diaminetetraacetate, to restore L_IV activity through coordination with some toxic cation introduced as a buffer contaminant.     

Production,Rapid Purification and Catalytic Characterization of Extracellular Phytase from Aspergillus Ficuum

Abstract

A rapid purification scheme utilizing three chromatographic steps resulted in 6 fold purification of Aspergillus ficuum phytase (myo-inositol-hexakis-phosphate 3-phosphohydrolase, EC 3.1. 3.8). At pH 5.0 and 60°C the enzyme performed acceptably for 2.0 hr with only 30% diminished catalytic rate at the end. Substrate concentration exceeding 2nM was inhibitory. The inorganic orthophosphate, the product and a weak inhibitor, exhibited a Ki of 1.9 × 10^?3M. The extracellular phytase has the potential for industrial use since it can be over produced, easily purified, remain catalytically active for a longer period and is not subjected to severe product inhibition.     

Purification and characterization of heterogeneous glucoamylases from Monascus purpureus

Two forms of an extracellular glucoamylase, MpuGA-I and MpuGA-II, were purified to homogeneity from Monascus purpureus RY3410. The molecular weights of these enzymes were estimated to be 60,000 (MpuGA-I) and 89,000 (MpuGA-II). These enzymes were glycoproteins with a carbohydrate content of 15.0% (MpuGA-I) and 16.2% (MpuGA-II) respectively. The pH optima were 5.0 for both enzymes, and the optimal temperatures were 50 degrees C (MpuGA-I) and 65 degrees C (MpuGA-II). The Km values for soluble starch were calculated to be 4.0+/-0.8 mg/ml (MpuGA-I) and 1.1+/-0.2 mg/ml (MpuGA-II) respectively.     

Antimicrobial activity of Monascus pilosus IFO 4520 against contaminant of Koji.

Antimicrobial activity of Monascus pilosus IFO 4520 was examined to prevent contamination during beni-koji making in the open air. The antibacterial effect of the beni-koji prepared with this strain occured with 30 mg/ml of beni-koji extract in combination with 0.5% lactic acid against two contaminants of koji, Micrococcus varians and Bacillus subtilis. There were two compounds, antibacterial and antiyeast substances, in the beni-koji extract. These results suggest a possibility of inhibiting the growths of contaminants during beni-koji making using beni-koji extract and lactic acid.     

Purification and characterization of an extracellular beta-glucosidase from Monascus purpureus

An extracellular beta-glucosidase produced by Monascus purpureus NRRL1992 in submerged cultivation was purified by acetone precipitation, gel filtration, and hydrophobic interaction chromatography, resulting in a purification factor of 92-fold. A 22 central-composite design (CCD) was performed to find the best temperature and pH conditions for enzyme activity. Maximum activity was observed in a wide range of temperature and pH values, with optimal conditions set at 50 degrees and pH 5.5. The beta-glucosidase showed moderate thermostability, was inhibited by HgCl2, K2CrO4, and K2Cr2O7, whereas other reagents including beta- mercaptoethanol, SDS, and EDTA showed no effect. Activity was slightly stimulated by low concentrations of ethanol and methanol. Hydrolysis of p-nitrophenyl-beta-D-glucopyranoside (pNPG), cellobiose, salicin, n-octyl-beta-D-glucopyranoside, and maltose indicates that the beta-glucosidase has broad substrate specificity. Apparently, glucosyl residues were removed from the nonreducing end of p-nitrophenyl-beta-Dcellobiose. beta-Glucosidase affinity and hydrolytic efficiency were higher for pNPG, followed by maltose and cellobiose. Glucose and cellobiose competitively inhibited pNPG hydrolysis.     

Anti-tumor-initiating effects of monascin, an azaphilonoid pigment from the extract of Monascus pilosus fermented rice (red-mold rice)

Monascin (1) constitutes one of the azaphilonoid pigments in the extracts of Monascus pilosus-fermented rice (red-mold rice). Compound 1 was evaluated for its anti-tumor-initiating activity via oral administration on the two-stage carcinogenesis of mouse skin tumor induced by peroxynitrite (ONOO-; PN) or by ultraviolet light B (UVB) as an initiator and 12-O-tetradecanoylphorbol-13-acetate (TPA) as a promoter. Compound 1 exhibited marked inhibitory activity on both PN- and UVB-induced mouse skin carcinogenesis tests. These findings suggest that compound 1 may be valuable as potential cancer chemopreventive agent in chemical and environmental carcinogenesis.     

Production,Purification, and Characterization of D-Aminoacylase from Alcaligenes xylosoxydans subsp. xylosoxydans A-6

The best inducers for D-aminoacylase from Alcaligenes xylosoxydans subsp. xylosoxydans A-6 (Alcaligenes A-6) were a poor substrate, N-acetyl-;-methyl-D-leucine, and an inhibitor, N-acetyl-D-alloisoleucine. The enzyme has been homogeneously purified. The molecular weight of the native enzyme was estimated to be 58,000 by gel filtration. A subunit molecular weight of 52,000 was measured by SD8–PAGE, indicating that the native protein is a monomer. The isoelectric point was 5.2. The enzyme was specific to the D-isomer and hydrolyzed N-acetyl derivatives of D-leucine, D-phenylalanine, D-norleucine, D-methionine, and D-valine, and also N-formyl, N-butyryl, and N-propionyl derivatives of D-leucine. The K_m for N-acetyl-D-leucine was 9.8mM. The optimum pH and temperature were 7.0 and 50°C, respectively. The stabilities of pH and temperature were 8.1 and 40°C. D-Aminoacylases from three species of the genus Alcaligenes differ in inducer and substrate specificities, but are similar with respect to molecular weight and N-terminal amino acid sequence.     

Characterization, Production, and Purification of Leucocin H, a Two-Peptide Bacteriocin from Leuconostoc MF215B

Leuconostoc MF215B was found to produce a two-peptide bacteriocin referred to as leucocin H. The two peptides were termed leucocin Hα and leucocin Hβ. When acting together, they inhibit, among others, Listeria monocytogenes, Bacillus cereus, and Clostridium perfringens. Production of leucocin H in growth medium takes place at temperatures down to 6°C and at pH below 7. The highest activity of leucocin H in growth medium was demonstrated in the late exponential growth phase. The bacteriocin was purified by precipitation with ammonium sulfate, ion-exchange (SP Sepharose) and reverse phase chromatography. Upon purification, specific activity increased 10⁵-fold, and the final specific activity was 2 × 10⁷ BU/OD₂₈₀. Amino acid composition analyses of leucocin Hα and leucocin Hβ indicated that both peptides consisted of around 40 amino acid residues. Their N-termini were blocked for Edman degradation, and the methionin residues of leucocin Hβ did not respond to Cyanogen Bromide (CNBr) cleavage. Absorbance at 280 nm indicated the presence of tryptophan residues and tryptophan-fracturing opened for partial sequencing by Edman degradation. From leucocin Hα, the sequence of 20 amino acids was obtained; from leucocin Hβ the sequence of 28 amino acid residues was obtained. No sequence homology to other known bacteriocins could be demonstrated. It also appeared that the two peptides themselves shared little or no sequence homology. The presence of soy oil did not affect the activity of leucocin H in agar. Received: 10 February 1999 / Accepted: 15 March 1999     

Selection of an effective red-pigment producing Monascus pilosus by efficient transformation with aurintricarboxylic acid

The filamentous fungus Monascus pilosus was genetically transformed with a reporter plasmid, pMS-1.5hp, by aurintricarboxylic acid (ATA) treatment to obtain an efficient red-pigment producing mutant. The transformation efficiency of Monascus pilosus was higher with the ATA-treatment than with either a non-restriction-enzyme-mediated integration (REMI) or a REMI method. This valid and convenient random mutagenesis method shows that ATA can be applied in fungi for efficient genetic transformation.     

Characterization of dehydromonacolin-MV2 from Monascus purpureus mutant

AIM: Characterization of dehydromonacolin-MV2, a bioactive metabolite isolated from Monascus purpureus mutant (CFR 410-11). METHODS AND RESULTS: Chloroform extract of rice, fermented with a hyperpigment-producing mutant of M. purpureus (CFR 410-11) was found to contain metabolites that inhibited the growth of Bacillus, Pseudomonas and Streptococcus in agar gel diffusion assays. The extract inhibited lipid peroxidation and scavenged 2,2-diphenyl-1-pycrylhydrazyl and hydroxyl radicals. The active compound purified by silica gel column chromatography was characterized by NMR. The carbon, proton and 2D HSQCT assignments identified dehydromonacolin-MV2 as the bioactive metabolite. CONCLUSIONS: Dehydromonacolin-MV2 apparently originated in the mutant by hydroxylation and oxidation of monacolin-J, an intermediate of monacolin biosynthetic pathway. SIGNIFICANCE AND IMPACT OF THE STUDY: Identification of the production of dehydromonacolin-MV2 by M. purpureus mutant (CFR 410-11) is new to literature. Bioactive properties of the compound suggested its pharmaceutical applications.     

Production, Purification, and Characterization of a Xylooligosaccharides-forming Xylanase from High-butanol-producing Strain Clostridium sp. BOH3

An endo-acting xylanase is isolated from the culture medium of Clostridium sp. BOH3 when xylan, glucose, xylose, or sugarcane bagasse hydrolysate (SBH) is used as a carbon source. Crude xylanase is purified by using an anionic Q-column with a yield of 39 %. The pure xylanase has a molecular weight of 35.8 kDa, and it shows optimal activity at pH 5 and 60 °C. When beechwood xylan is used as a substrate, this xylanase liberates short oligosaccharides (XOS) predominantly, ranging from xylobiose (X2) to xylopentaose (X5). However, no xylose can be detected, suggesting that this is an endo-β-1,4-xylanase. Kinetic study of this xylanase reveals that K _m and V _max are 1.36 mg/ml and 212 μmol/(min. mg protein), respectively. On the basis of amino acid sequence, this enzyme shows homology to xylanase (xynb) from Clostridium acetobutylicum ATCC 824, but this enzyme has several distinctive characteristics. For example, its activity can be enhanced with the addition of divalent metal ions, and it produces XOS exclusively when xylan is used as a substrate. These unique characteristics suggest that this is a new enzyme.     

Simultaneous enrichment of deglycosylated ginsenosides and monacolin K in red ginseng by fermentation with Monascus pilosus

To improve its bioavailability and pharmacological effects in humans, red ginseng was fermented with a newly isolated fungus, Monascus pilosus KMU103. Most of the ginsenosides were converted to deglycosylated ginsenocides, such as Rh(1), Rh(2), and Rg(3). The total amount of ginsenosides Rh(1), Rh(2), and Rg(3) was 838.7 mg/kg in the red ginseng, and increased to 4,117 mg/kg after 50 L fermentation in 13% red ginseng and 2% glucose. In addition, the Monascus-fermented red ginseng contained 3,089 mg/kg of monacolin K, one of the metabolites produced by Monascus known to reduce cholesterol in the blood. This newly developed Monascus-fermented red ginseng should result in improved health effects, not only by biotransforming gisenosides to deglycosylated ones but also by creating additional bioactive compounds.     

Simultaneous Enrichment of Deglycosylated Ginsenosides and Monacolin K in Red Ginseng by Fermentation with Monascus pilosus

To improve its bioavailability and pharmacological effects in humans, red ginseng was fermented with a newly isolated fungus, Monascus pilosus KMU103. Most of the ginsenosides were converted to deglycosylated ginsenocides, such as Rh₁, Rh₂, and Rg₃. The total amount of ginsenosides Rh₁, Rh₂, and Rg₃ was 838.7 mg/kg in the red ginseng, and increased to 4,117 mg/kg after 50 L fermentation in 13% red ginseng and 2% glucose. In addition, the Monascus-fermented red ginseng contained 3,089 mg/kg of monacolin K, one of the metabolites produced by Monascus known to reduce cholesterol in the blood. This newly developed Monascus-fermented red ginseng should result in improved health effects, not only by biotransforming gisenosides to deglycosylated ones but also by creating additional bioactive compounds.     

Development of monacolin K-enriched ganghwayakssuk (Artemisia princeps Pamp.) by fermentation with Monascus pilosus

Monacolin K-enriched ganghwayakssuk (Artemisia princeps Pamp.) was developed by fermentation with Monascus sp. Among the 15 Monascus spp. isolated previously from Monascus fermentation products, Monascus pilosus KMU108 produced 2,219 mg/kg of monacolin K during ganghwayakssuk fermentation with no detectable citrinin. The optimum concentrations of ganghwayakssuk and glucose determined from the response surface methodology (RSM) design were 2.2% and 3.8%, respectively. By applying these conditions, the monacolin K productivity was increased to 3,007 mg/kg after 15 days of fermentation. On the other hand, other characteristics such as the total content of flavonoids and phenolic compounds, and the antioxidant activity were relatively unchanged. Therefore, Monascusfermented ganghwayakssuk is an excellent biomaterial for the development of functional foods because of its high level of monacolin K, known to lower cholesterol levels.