Purification and characterization of a feruloyl esterase from the fungus Penicilliumexpansum

An extracellular phenolic acid esterase produced by the fungus Penicillium expansum in solid state culture released ferulic and ρ-coumaric acid from methyl esters of theacids, and from the phenolic-carbohydrate esters O-[5-O-(trans-feruloyl)-α- l -arabinofuranosyl]-(1 → 3)-O-β- d -xylopyranosyl-(1 → 4)- d -xylopyranose (FAXX) and O-[5-O-((E)-ρ-coumaroyl)-α- l -arabinofuranosyl]-(1 → 3)-O-β- d -xylopyranosyl-(1 → 4)- d -xylopyranose(PAXX). The esterase was purified 360-fold in successive stepsinvolving ultrafiltration and column chromatography by gel filtration, anion exchange andhydrophobic interaction. These chromatographic methods separated the phenolic acid esterasefrom α- l -arabinofuranosidase, pectate and pectin lyase, polygalacturonase,xylanase and β- d -xylosidase activities. The phenolic acid esterase had an apparentmass of 65 kDa under non-denaturing conditions and a mass of 57·5 kDa underdenaturing conditions. Optimal pH and temperature were 5·6 and 37 °C,respectively and the metal ions Cu2+ and Fe³+ atconcentrations of 5 mmol l⁻¹ inhibited feruloyl esterase activity by 95% and44%, respectively, at the optimum pH and temperature. The apparent K_m and V_max of the purified feruloyl esterase for methyl ferulate at pH 5·6 and 37 °Cwere 2·6 mmol l⁻¹ and 27·1 μmol min⁻¹ mg⁻¹. The corresponding constants of ρ-coumaroylesterase for methyl coumarate were 2·9 mmol l⁻¹ and 18·6μmol min−1 mg⁻¹.     

Comparative study of extracellular and intracellular β-glucosidases of a new strain of Zygosaccharomyces bailii isolated from fermenting agave juice

A yeast strain isolated in the laboratory was studied and classified as a Zygosaccharomyces bailii. Both intracellular and extracellular β-glucosidases of this yeast were purified by ion-exchange chromatography, gel filtration and hydroxylapatite (only for the intracellular enzyme). The tetrameric structure of the two β-glucosidases was determined following treatment of the purified enzyme with dodecyl sulphate. The intracellular β-glucosidase exhibited optimum activity at 65°C and pH 5.5. The extracellular enzyme exhibited optimum catalytic activity at 55°C and pH 5. The molecular mass of purified intracellular and extracellular β-glucosidases, estimated by gel filtration, was 440 and 360 kDa, respectively. Both enzymes are active against glycosides with (1 → 4)-β, (1 → 6)-β and (1 → 4)-α linkage configuration. The intracellular enzyme possesses (1 → 6)-α-arabinofuranosidase activity and extracellular enzyme (1 → 6)-α-rhamno-pyranosidase activity. The two β-glucosidases are competitively inhibited by glucose and by D-gluconic-acid-lactone and a slight glucosyl transferase activity is observed in the presence of ethanol. Since the glycosides present in wine and fruit juices represent a potential source of aromatic flavour, the possible use of the yeast β-glucosidases for the liberation of the bound aroma is discussed.     

Production and properties of ααα-glucosidase from the thermotolerant bacteriumThermomonospora curvata

Thermomonospora curvata contains α-1,4-glucosidase that is induced duringgrowth on maltose and starch. Maltose acts as an inducer of α-glucosidase even in thepresence of glucose. An intracellular thermostable α-glucosidase from T. curvata wasdetected in the crude extract on SDS-PAGE by means of modified colour reaction afterrenaturation of the enzyme. The enzyme was purified 59-fold to homogeneity with a yield of17·7% by a combination of ion-exchange and hydrophobic interaction chromatography andgel filtration. The enzyme has an apparent molecular mass of 60±1 kDa and isoelectric point4·1. The α-glucosidase exhibits optimum activity at pH 7·0–7·5 and54°C. The activity is inhibited by heavy metals and is positively affected by Ca²+ andMg²+. The enzyme hydrolyses maltose, sucrose, p-nitrophenyl-α- d -glucopyranoside and maltodextrins from maltotriose up to maltoheptaose with a decreasingefficiency. The K_m for maltose and p-NPG are 12 and 2·3 mmol l⁻¹,respectively.     

Purification and characterization of ββ-glucosidase from Aspergillus niger strain 322

An extracellular β-glucosidase enzyme was purified from the fungus Aspergillus niger strain 322 . The molecular mass of the enzyme was estimated to be 64 kDa by SDS gel electrophoresis. Optimal pH and temperature for β-glucosidase were 5·5 and 50 °C, respectively. Purified enzyme was stable up to 50 °C and pH between 2·0 and 5·5. The K_m was 0·1 mmol l⁻¹ for cellobiose. Enzyme activity was inhibited by several divalent metal ions.     

Isolation and characterization of an intracellular esterase from Lactobacillus casei subsp. casei IFPL731

An intracellular esterase from Lactobacillus casei subsp. casei IFPL731 was purified 1000-fold by ion exchange chromatography and gel filtration chromatography. The relative molecular mass of the native enzyme was 105 kDa, while the subunit molecular mass was estimated to be 38 kDa. The esterase hydrolysed tributyrin and had a preference for esters of short-chain fatty acids (butyrate, caproate and caprylate), while it did not hydrolyse palmitate and sterate esters. The apparent Michaelis-Menten constant of the enzyme on p -nitrophenyl butyrate was 0·3 mmol l⁻¹ while on p -nitrophenyl caprylate, it was 0·04 mmol l⁻¹. The esterase was active over a broad range of pH and temperature values, and retained about 50% of maximal activity at pH 5·0 and 12 °C. Activity was strongly inhibited by 5 mmol l⁻¹ phenylmethylsulphonyl fluoride, β-mercaptoethanol and N -ethylmaleimide, and was stimulated by Zn²⁺ at 1 mmol l⁻¹.     

Glucosyltransferase activity of commercial juice-processing enzyme preparations

Of various commercial enzyme preparations examined, Cytolase M102 was found to contain the highest glucosyltransferase activity (55 U ml⁻¹). It rapidly converted maltose to panose (Glcα1 → 6Glcα1 → 4Glc) with a V _max value of 5·8 mmol l⁻¹ min⁻¹ at 50°C in 0·05 mol l⁻¹ sodium acetate buffer (pH 4·4). The K _m value of the enzyme for maltose was 750 mmol l⁻¹. Yields of panose and glucose after 45 min of reaction, for example, were 47·2% and 52·8%, respectively, on the basis of the amount of maltose consumed.     

The linamarase of Mucor circinelloides LU M40 and its detoxifying activity on cassava

Mucor circinelloides LU M40 produced 12·2 mU ml⁻¹ of linamarase activity when grown in a 3 l fermenter in the following optimized medium (g l⁻¹ deionized water): pectin, 10·0; (NH₄)₂SO₄,
1·0; KH₂PO₄, 2·0; Na₂HPO₄, 0·7; MgSO₄.7H₂O, 0·5; yeast extract, 1·0; Tween-80,
1·0, added after 48 h of fermentation. The purified linamarase was a dimeric protein with a molecular mass of 210 kDa; the enzyme showed optimum catalytic activity at pH 5·5 and 40 °C and had a wide range (3·0–7·0) of pH stability. The enzyme substrate specificity on plant cyanogenic glycosides was wide; the K_m value for linamarin was 2·93 mmol l⁻¹. The addition, before processing, of the fungal crude enzyme to cassava roots facilitated and shortened detoxification; after 24 h of fermentation, all cyanogenic glycosides were hydrolysed.     

Histidine carboxylase of Leuconostoc œnos 9204: purification, kinetic properties, cloning and nucleotide sequence of the hdc gene

Histidine decarboxylase (HDC) was purified to homogeneity from Leuconostoc ' nos 9204, a wine lactic acid bacterium. Histidine decarboxylase comprised two subunits, respectively α and β. The hdc gene was cloned and sequenced. The gene encodes a single polypeptide of 315 amino acids, demonstrating that Leuc. ' nos 9204 HDC was synthesized as a precursor proHDC π₆ (Mr 205 000). A cleavage between Ser-81 and Ser-82 generated the α (Mr 25 380) and β (Mr 8840) chains, which suggested that the holoenzyme exists as a hexameric structure (αβ)₆. At the optimal pH of 4·8, the HDC activity exhibited a simple Michaelis-Menten kinetic ( K _m = 0·33 mmol l⁻¹, V _max = 17·8 μmol CO₂ min⁻¹ mg⁻¹), while at pH 7·6 it was sigmoidal (cooperativity index of 2). Histamine acted as a competitive inhibitor ( K _i = 32 mmol l⁻¹). The similarities of these results with those described for other bacterial HDC support the assumption that the pyruvoyl enzymes evolved from a common ancestral protein and have similar catalytic mechanisms. These results also confirmed that the main lactic acid bacterial species responsible for malolactic fermentation in red wine is able to produce histamine. Bacteria carrying the HDC activity must be avoided during selection of strains for the production of malolactic starters.     

Note: Purification of amylase secreted from Bifidobacterium adolescentis

Bifidobacterium adolescentis Int-57 isolated from human faeces produced extracellular amylase. The enzyme was purified from the culture supernatant fluids by ammonium sulphate precipitation, gel-filtration chromatography (Sephadex-G-75), ion-exchange chromatography (CM-cellulose) and FPLC. SDS-PAGE of the purified enzyme revealed a major band with an apparent molecular weight of 66 kDa. The pI was 5·2. Enzyme activity was optimal at 50°C, and at pH 5·5. The enzyme was stable at 20–40°C, and at pH 5–6 with a K _m value of 2·4 g l⁻¹ soluble starch. The activation energy was 42·3 kJ mol⁻¹. The enzyme was significantly inhibited by maltose (10%), glucose (10%), Cu²+ (5 mmol l⁻¹), Zn²+ (5 mmol l⁻¹), N- bromosuccinimide (5 mmol l⁻¹), EDTA (5 mmol l⁻¹), I₂ (1 mmol l⁻¹) and activated by β-mercaptoethanol (10 mmol l⁻¹).     

Purification and characterization of galacto-oligosaccharide-producing β-galactosidase from Sirobasidium magnum

Galacto-oligosaccharide-producing β-galactosidase from Sirobasidium magnum CBS6803 was purified to homogeneity with a yield of 60% by DEAE–toyopearl, butyl–toyopearl, p -aminobenzyl 1-thio-β- d -galactopyranoside–agarose and concanavalin A–agarose columns, from a solubilized cell wall preparation. The isoelectric point (pI) of purified β-galactosidase was 3·8, and the relative molecular mass was 67 000 as estimated by SDS gel electrophoresis, and 135 000 as estimated by gel filtration. Optimal β-galactosidase activity was observed at a temperature and pH of 65°C and pH 4·5–5·5, respectively. The K _m values for o -nitrophenyl-β- d -galactopyranoside and lactose were 14·3 and 5·5 mmol l⁻¹, respectively, and the V _max values for these substrates were 33·4 and 94·5 μmol min⁻¹ mg of protein⁻¹, respectively. In addition this enzyme possessed a high level of transgalactosylation activity, and 72 mg ml⁻¹ galacto-oligosaccharide was produced from 200 mg ml⁻¹ lactose.     

Glycosides from Roots of Cyathula officinalis Kuan

Abstract: To search for new and bioactive compounds from traditional Chinese medicines, a new glycoside, 3-O-[α- L -rhamnopyranosyl-(1→3)-( n -butyl-β- D -glucopyranosiduronate)]-28-O-β- D -glucopyranosyloleanolic acid ( 1 ), was isolated from the roots of Cyathula officinalis Kuan, along with 3-O-(methyl-β- D -glucopyranosiduronate)-28-O-β- D -glucopyranosyl oleanolic acid ( 2 ), 3-O-β- D -glucopyranosyl oleanolic acid ( 3 ), 3-O-β- D -glucuronopyranosyl oleanolic acid ( 4 ), 3-O-[β- L -rhamnopyranosyl-(1→3)-(β- D -glucuronopyranosyl)] oleanolic acid ( 5 ), 3-O-(β- D -glucuronopyranosyl)-28-O-β- D -glucopyranosyl oleanolic acid ( 6 ), 28-O-β- D -glucuronopyranosyl-(1→4)-β- D -glucopyranosyl hederagenin ( 7 ), 3-O-[β- L -rhamnopyranosyl-(1→3)-β- D -glucuronopyranosyl]-28-O-β- D -glucopyranosyl oleanolic acid ( 8 ), and 3-O-[β- D -glucopyranosyl-(1→2)-α- L -rhamnopyranosyl-(1→3)-β- D -glucuronopyranosyl]-28-O-β- D -glucopyranosyl oleanolic acid ( 9 ). The structures of these compounds were determined based on spectral and chemical evidence. The 50 per cent growth-inhibiting (GI₅₀) of compounds 1 and 5 against MDA-MB-231 (a human breast cancer cell line) was 3.44 × 10^-4 and 4.66 × 10^-4 mol/L, respectively.
(Managing editor: Wei WANG)     

Effect of chlorination on β-D-galactosidase activity of sewage bacteria and Escherichia coli

The effect of chlorine on β- D- galactosidase activity of sewage bacteria and Escherichia coli was studied. β- D- galactosidase activity of sewage was more resistant to chlorine than faecal coliform cultivability. At low initial dosage (0·05 mg Cl₂ l⁻¹) neither cultivability (colony-forming units (cfu)), nor enzyme activity of E. coli suspensions were severely impaired. When initial chlorine concentration was increased to 0·1 mg Cl₂ l⁻¹, the cfu number decreased whereas enzyme activity remained high, i.e. the enzyme activity calculated cfu⁻¹ increased. At higher chlorine doses both cfu and enzyme activity were reduced, but non-cultivable cells retained assayable activity after chlorination. Mean values of the enzyme activity calculated cfu⁻¹ decreased when the chlorine dosage was increased from 0·1 to 0·5 mg Cl₂ l⁻¹, but were not significantly different ( P > 0·05) for dosages of 0·2–0·7 mg Cl₂ l⁻¹. After chlorination, β- D- galactosidase activity of E. coli was less reduced than cfu and direct viable count numbers, but more reduced than 5-cyano-2-3, ditolyl tetrazolium chloride and total cell counts, and the enzyme activity represented an alternative activity parameter of chlorinated samples.     

A New Steroidal Glycoside from Ophiopogon japonicus （Thunb.） Ker-Gawl.

To study the chemical constituents from traditional Chinese herb Ophiopogon japonicus （Thunb.） Ker-Gawl., a new steroidal glycoside, named ophiopojaponin C （1）, together with two known ones, was isolated by column chromatography. Spectroscopic and chemical evidence revealed the structures to be ophiopogenin 3-O-[α-L-rhamnopyranosyl（1→2）]-β-D-xylopyranosyl（1→4）-β-D-glucopyranoside （1）, diosgenin 3-O-[2-O-acetyl-α-L-rhamnopyranosyl（1→2）]-β-D-xylopyranosyl（1→3）-β-D-glucopyranoside （2）, and ruscogenin 1-O-[2-O-acetyl-α-L-rhamnopyranosyl（1→2）]-β-D-xylopyranosyl（1→3）-β-D-fucopyranoside （3）.     

Partial purification, characterization and regulation of cellulolytic enzymes from Trichoderma longibrachiatum

A net purification of 9·46-, 18·6- and 16·7-fold for filter paper (FP) hydrolytic activity, carboxymethyl (CM) cellulase and β-glucosidase, respectively was achieved through ion exchange and gel chromatographies. The purified enzyme preparation showed an optimal pH of 5·0 for CM cellulase and 5·5 for the other two components. The enzyme activities increased up to 60°–65°C for the three enzyme components and they were stable at 30° or 40°C and pH 4·5 to 5·0 after 20–30 min treatment. The four enzyme components, that is, two FP activities (unadsorbed and adsorbed), a CM cellulase and a β-glucosidase, had K_m values of 47·6 mg, 33·3 mg, 4·0 mg and 0·18 mmol/l with V _max of 4, 1·28, 66·5 and 1·28 units per mg protein. The molecular weights as determined with SDS-PAGE were found to be 44000, 38000, 55000 and 63000 for the above four enzyme components in the same sequence. A distinct type of synergistic action was observed between these components by their action on dewaxed cotton. Glycerol at 1% strongly repressed the formation of all the cellulolytic enzymes. The role of proteolytic enzymes in in vitro inactivation of cellulases was not apparent.     

Purification and further characterization of a glycoprotein from the skin mucus of Japanese eels

A glycoprotein (EGP) was purified from the skin mucus of Japanese eel Anguilla japonica . Apparent average molecular mass of the EGP was estimated to be 500 000. The EGP was found to contain 30·8% NeuAc, 26·4% GalNAc, 6·4% Gal, 0·4% NeuGc and 25·1% Thr‐rich protein. EGP was treated with alkaline borohydride for the release of carbohydrate chains (oligosaccharide alditols). Three carbohydrate chains were isolated from the released carbohydrate chains by Sephadex G‐25 (superfine) gel filtration and HPLC. Using ¹H‐NMR spectroscopy, methylation analysis and glycosidase digestion, the structures of the three carbohydrate chains were determined to be NeuAcα2→6GalNAc‐ ol , NeuAcα2→3GalNAc‐ ol and NeuAcα2→6(GalNAcα1→3)GalNAc‐ ol . An overall structure for the sialoglycoprotein from the skin mucus is proposed: the molecule is considered to consist of highly glycosylated 10 glycopeptide units (containing >40 carbohydrate chains) that are linked to the hydroxyl amino acids and spaced on average two amino acids apart.     

Characteristics of glucoamylase from Aspergillus terreus

Glucose was the only product of starch hydrolysis liberated by glucoamylase. The enzyme was a glycoprotein with an isoelectric point at pH 3·4 and was optimally active at pH 4·0 and 60°C. It was remarkably stable over a wide range of pH and at elevated temperatures. Divalent Mg²⁺'and Ca²⁺ slightly stimulated glucoamylase activity. The enzyme exhibited specificity for substrates containing α(1 → 4) glucosidic linkages and the Km for starch hydrolysis was 4·0 g/l.     

Purification and characterization of a β-glucosidase from the phytopathogenic fungus Fusarium oxysporum f. sp. melonis

T.M. ALCONADA AND M.J. MARTÍNEZ. 1996. Fusarium oxysporum f. sp. melonis produces cellulase and β-glucosidase activities in a medium with glucose and avicel as carbon source. A β-glucosidase from this crude material was purified by gel filtration and ion exchange chromatography successively. This enzyme is a unique band of protein in SDS-PAGE and isoelectric focussing. It had a molecular weight of 66000 and a pI of 5. Using p -nitrophenyl-β-D-glucopyranoside as substrate β-glucosidase shows a K _m of 210 μmol 1^-1, an optimum pH of 5.5 and an optimum reaction temperature of 60°C, being stable in a pH range of 5–7 for 48 h at room temperature.     

An effective method for isolating alginate lyase-producing Bacillus sp. ATB-1015 strain and purification and characterization of the lyase

A new alginate lyase-producing micro-organism, designated as Bacillus sp. strain ATB-1015, was effectively isolated from soil samples pretreated for 3 months with a substrate of the enzyme, sodium alginate. Alginate lyase activity was assayed by the degrading activity of biofilm on Teflon sheet discs, which was formed by a mucoid strain of Pseudomonas aeruginosa PAM3 selected from clinical isolates. The extracellular alginate lyase was precipitated with ammonium sulphate from the culture broth, and purified by gel filtration and anion exchange chromatography. The molecular weight of the lyase was estimated to be 41 kDa by SDS polyacrylamide gel electrophoresis and Sephacryl S-200 HR column chromatography. The optimum pH and temperature for the enzyme activity were around 7·5 and 37 °C, respectively, and the Km value was 0·17% with the substrate, sodium alginate. The lyase activity was completely inhibited by treatment with 1 mmol l⁻¹ of EDTA and the decreased activity was almost completely recovered by the addition of 2 mmol l⁻¹ of CaCl₂. The activity was not affected by treatment with the protein denaturants, 0·01 mol l⁻¹ of SDS or 1 mmol l⁻¹ of urea. The lyase had substrate specificity for both the poly-guluronate and poly-mannuronate units in the alginate molecule.     

Variations in faecal bacterial enzyme activities and associations with bowel function and diet in elderly subjects

Daily and inter-individual variations of faecal bacterial β-glucuronidase and β-glucosidase activities and their associations with parameters of bowel function were studied in 10 residents of an old people's home during two 1-week periods 2 weeks apart. The effect of sampling method (a spot sample vs an aliquot of the homogenized sample from a total daily collection) on the activities of these enzymes and that of urease was also assessed. Intestinal transit time was determined using the radio-opaque Sitzmark^®; capsules, and questionnaires on bowel function and intakes of fluids and fibre-containing foods were completed. The mean (95% confidence interval) β-glucuronidase and β-glucosidase levels were 3·08 (2·75–3·41) and 11·53 (10·79–12·26) nmol min⁻¹ mg protein⁻¹. Daily variations in enzyme activities within individuals were not significant ( P = 0·277 and 0·990, respectively), whilst those between individuals were highly significant ( P = 0·000). Faecal frequency correlated negatively with β-glucuronidase and urease, but no other associations of the enzymic activities with parameters of bowel function and diet were observed. β-Glucuronidase and β-glucosidase were not affected by the sampling method, while significantly higher urease was obtained by spot sampling as compared with the aliquot representing the total daily collection. Large inter-individual variations in faecal enzyme activities should be taken into consideration when planning experiments and interpreting results on these faecal parameters.