Purification and Properties of Endo-polygalacturonase from Aspergillus japonicus

An endo-polygalacturonase from culture extracts of Aspergillus japonicus was purified about 34-fold by ammonium sulfate fractionation, SE-Sephadex column chromatography and gel filtration. The purified enzyme was homogeneous on ultracentrifugation and disc electrophoresis. Using gel filtration a molecular weight of 35,500 was estimated for the enzyme. The enzyme rapidly reduced the viscosity of pectic acid and released reducing groups in a random manner, yielding a mixture of mono-, di- and trigalacturonic acids as end products. The pH optimum of the enzyme for viscosity-reducing activity was 4.5 with pectin and pectic acid as substrates, and that for releasing reducing groups was also 4.5 with various pectic substances. The purified enzyme was able to macerate various kinds of plant tissues by itself.     

甘油氧化酶发酵条件及酶学性质的研究

日本曲霉 (AspergillusjaponicusAj113)发酵生产甘油氧化酶 (GlycerolOxidaseEC 1 1 3 - )的最适产酶条件 :初始pH 6 0- 6 5 ,温度 2 9± 1℃ ,培养时间 36h ,5 0 0ml三角瓶发酵液的装量为 10 0ml;酶的最适作用pH为 5 0 ,该酶在pH9 5 ,温度 30℃以下时稳定性较好 ;0 0 5mol L的硼砂 -碳酸钠缓冲液 (pH9 5 )对酶有较好的保存效果 ,Zn2 + 、Cu2 + 、Fe3+ 、Ca2 + 离子对酶有激活作用 ,Hg2 +离子对酶有强烈的抑制作用     

A New Enzyme,Agmatine Oxidase,from Fungi

A new enzyme, agmatine oxidase, was found in Penicillium chrysogenum. The oxidation products of agmatine with the enzyme were identified as γ-guanidinobutyraldehyde, NH₃ and H₂O₂. The enzyme rapidly oxidized agmatine, and slightly oxidized histamine, putrescine, 1,3-diaminopropane and cadaverine. Monoamines, polyamines and guanidyl derivatives were not oxidized by the enzyme. Maximal formation of the enzyme of P. chrysogenum was observed in the early stationary phase of growth, and thereafter the enzyme disappeared with consumption of substrate. In addition to agmatine, spermine, spermidine and putrescine were also effective as nitrogen sources. Agmatine oxidase was found in mycelia of fungi belonging to the genera of Aspergillus, Penicillium, Absidia, Fusarium, Mucor, Gibberella, Cylindrocarpon and Monascus when they were grown in agmatine-containing medium.     

Purification and Properties of Hydroxycinnamic Acid Ester Hydrolase from Aspergillus japonicus

Hydroxycinnamic acid ester hydrolase from the wheat bran culture medium of Aspergillus japonicus was purified 255-fold by ammonium sulfate fractionation, DEAE-Sephadex treatment and column chromatographies on DEAE-Sephadex, CM-Sephadex and various other Sephadexes. The purified enzyme was free from tannase and found to be homogeneous on polyacrylamide disc gel electrophoresis. Its molecular weight was estimated to be 150,000 by gel filtration and 142,000 by SDS-gel electrophoresis. The isoelectric point of the enzyme was pH 4.80. As to its amino acid composition, aspartic acid and glycine were abundant. The optimum pH and temperature for the enzyme reaction were, respectively, 6.5 and 55°C when chlorogenic acid was used as a substrate. The enzyme was stable between pH 3.0 to 7.5 and inactivated completely by heat treatment at 70°C for 10 min.

All metal ions examined did not activate the enzyme, while Hg⁺⁺ reduced its activity. The enzyme was markedly inhibited by diisopropylfluorophosphate and an oxidizing reagent, iodine, although it was not affected so much by metal chelating or reducing reagents. The purified enzyme hydrolyzed not only esters of hydroxycinnamic acids such as chlorogenic acid, caffeoyl tartaric acid and p-coumaroyl tartaric acid, but also ethyl and benzyl esters of cinnamic acid. However, the enzyme did not act on ethyl esters of crotonic acid and acrylic acid or esters of hydroxybenzoic acids.     

Purification and Properties of a New Enzyme,Glutathione Oxidase from Penicillium sp. K-6-5

A new flavoprotein enzyme, GSH oxidase, was found in the aqueous extract of a wheat bran culture of Penicillium sp. K-6-5. The oxidase is also produced extra and intracellularly in the liquid culture, although the production is much lower than that in the wheat bran culture.

The enzyme has been purified to homogeneity. It shows absorption maxima at 270, 350 and 444 nm and a shoulder around 465 nm and contains 2 mol of FAD per mol of enzyme. The enzyme has a molecular weight of approximately 95,000 and consists of two subunits identical in molecular weight (about 47,000). Balance studies show that 2 mol of GSH are converted to 1 mol of GSSG and hydrogen peroxide with the consumption of 1 mol of oxygen. In addition to GSH, several sulfhydryl compounds are oxidized by the enzyme to a lesser extent. The Michaelis constants are as follows: 0.69 mm for GSH, 3.6 mm for l-cysteine and 6.7 mm for dithiothreitol at pH 7.4. The oxidase scarcely acts on reduced RNase A in contrast to the known sulfhydryl oxidases. The isoelectric point and the optimal pH are 4.2 and 7.4, respectively. The enzyme activity is completely inhibited by addition of 1 mm ZnSO₄.     

Cellulase Enzyme from Aspergillus niger

Cellulase enzyme was produced by a selected strain of Aspergillus niger isolated from deteriorated wood and grown on different carbon sources. Filter paper gave the highest yield, followed by carboxymethyl cellulose (CMC). Cellobiose as well as glucose gave a low yield, while the yield from lactose was negligible. The concentration of filter paper cellulose that induced the maximum yield of the enzyme was 1%. Both soluble cellulose (CMC) and cotton cellulose treated with phosphoric acid (swollen) were easily hydrolyzed by cellulase; an increase in cellulase concentration lead to more hydrolysis of CMC and gave linearity in the reaction velocity. At certain concentrations of the enzyme, increase in CMC concentration, (up to 1%) resulted in more reducing sugar. Beyond this point no more hydrolysis occur.     

Crystallization and Characterization of Polyamine Oxidase from Aspergillus terreus

A thin-layer flow cell system for the determination of l-ascorbic acid by an ascorbate electrode was constructed and several components of this system were investigated. The most preferable conditions for optimum operation of the system were as follows: injection volume 150 μ1, delay coil length 60 cm, flow rate 1 ml/min, temperature 20°C, cell spacer thickness 0.2 mm. The linear response region was 0.2-3.0 mm and 0.02-0.5 mm (original l-ascorbic acid concentration) in the cases of pure oxygen and atmospheric oxygen bubbling, respectively. The relative standard variation at 1.5 mm l-ascorbic acid was 3.1 % for 20 successive assays. The measuring time was 2–3 min for each of these assays.     

Isolation and properties of pectinases from the fungus Aspergillus japonicus

Using anion-exchange chromatography on different carriers and phenyl-Sepharose hydrophobic chromatography, five pectolytic enzymes were isolated from the culture liquid of a mutant strain of Aspergillus japonicus: two endo-polygalacturonases (I and II, 38 and 65 kD, pI5.6 and 3.3), pectin lyase (50 kD, pI3.8), and two pectinesterases (I and II) with similar molecular weights (46 and 47 kD) and the same pI(3.8). The pectinesterases apparently represent two isoforms of the same enzyme. All purified enzymes were homogenous according to SDS-PAGE and polyacrylamide gel-IEF, except for endo-polygalacturonase II that gave two bands on isoelectric focusing, but one band on electrophoresis. All enzymes had maximal activity in an acid medium (at pH 4.0-5.5). The pectin lyase and pectinesterase were stable at 40-50°C. The thermal stability of both endo-polygalacturonases was much lower (after 3 h of incubation at 30°C, endo-polygalacturonases I and II lost 40 and 10% of the activity, respectively). The activity of endo-polygalacturonases I and II towards polygalacturonic acid strongly depended on NaCl concentration (optimal concentration of the salt was 0.1-0.2 M); the enzymes were also capable of reducing the viscosity of pectin solution, but rather slowly. The pectin lyase had no activity towards polygalacturonic acid. The activity of the pectin lyase increased with increasing degree of methylation of pectins. Both endo-polygalacturonases demonstrated synergism with the pectinesterase during the hydrolysis of highly methylated pectin. On the contrary, in the mixture of pectin lyase and pectinesterase an antagonism between the two enzymes was observed.     

New paralytic alkaloids, asperparalines A, B and C, from Aspergillus japonicus JV-23

New paralytic alkaloids, asperparalines A (1), B (2) and C (3), were isolated from okara (the insoluble residue of whole soybean) that had been fermented with Aspergillus japonicus JV-23. Their structures were elucidated by spectroscopic methods and X-ray crystallography. These asperparalines showed paralytic activity against silk worms.     

Isolation of Cycloclavine from the Culture Broth of Aspergillus japonicus SAITO

Changes of protein components (LA, LB, LC, and X) in mouse serum after administration of an antitumor agent, LC 9018 (lyophilized preparation of Lactobacillus casei, YIT 9018), were investigated. The intraperitoneal injection of LC 9018 caused an increase in these proteins one or two days after administration. The LC component was purified from mouse serum and was identified as haptoglobin. In addition, LA was identified as an intermediate of a haptoglobin-hemoglobin complex [Hp-Hb(α₁β₁)]and X as a haptoglobin-hemoglobin complex [Hp-Hb(α₂β₂)] because addition of increasing amount of hemoglobin to purified haptoglobin formed X via LA and all of these components were immunoprecipitated with anti-haptoglobin IgG.     

Crystal structure of the copper-containing quercetin 2,3-dioxygenase from Aspergillus japonicus

Quercetin 2,3-dioxygenase is a copper-containing enzyme that catalyzes the insertion of molecular oxygen into polyphenolic flavonols. Dioxygenation catalyzed by iron-containing enzymes has been studied extensively, but dioxygenases employing other metal cofactors are poorly understood. We determined the crystal structure of quercetin 2,3-dioxygenase at 1.6 A resolution. The enzyme forms homodimers, which are stabilized by an N-linked heptasaccharide at the dimer interface. The mononuclear type 2 copper center displays two distinct geometries: a distorted tetrahedral coordination, formed by His66, His68, His112, and a water molecule, and a distorted trigonal bipyramidal environment, which additionally comprises Glu73. Manual docking of the substrate quercetin into the active site showed that the different geometries of the copper site might be of catalytic importance.     

Properties of Arylamidase from Aspergillus oryzae

α-Amylase-like proteins were synthesized in a heterologous cell-free protein synthesizing system prepared from Escherichia coli. The proteins were precipitable with anti-α-amylase serum and detected only when RNA extracted from α-amylase producing Bacillus subtilis cells was used as messenger. The in vitro α-amylase-like products seemed to consist of two components having molecular weights of 30,000 and 13,000.     

Enzyme productivity from the protoplast regenerants of Aspergillus awamori

Protoplasts from the hydroiase enzyme producer Aspergillus awamori were isolated using Bulgarian enzyme preparations—Trichocease-SU and Xylanase. Among the regenerated colonies from protoplasts two were selected with enhanced productivity of xylanase, endoglucanase and β-glucosidase. The endoglucanase produced by the parent and one of the regenerants differ in their absorptivity, sensitivity to product inhibition and heat stability.     

Properties of cellobiase from Aspergillus niger

Summary Cellobiase enzyme was partially purified from the culture filtrate of Aspergillus niger AS-101 and the general and kinetic properties of the enzyme were examined. The enzyme was unstable on storage. However, it was protected by the addition of BSA, glycerol or sodium azide. Addition of glycerol also protected the enzyme from denaturation due to freezing and thawing. Effect of thiol group reagents revealed the presence of — SH groups at the active site of the enzyme. Different modulators such as metal ions and macroionic compounds illustrated varying effects on the purified cellobiase. Offprint requests to: A. Singh     

川麦冬中的新C_(27)甾体甙(英文)

从百合科植物川麦冬 (OphiopogonjaponicusKer_Gawl.)块根中分离得到 4个C2 7甾体甙类化合物 ,其中两个为新化合物 :ophiopojaponinA ( 1)和B ( 2 )。它们的结构通过波谱和化学方法鉴定为 :pennogenin 3_O_[2′_O_acetyl_α_L_rhamnopyranosyl ( 1→ 2 ) ]_β_D_xylopyranosyl ( 1→ 3)_β_D_glucopyranoside ( 1) ,2 6_O_β_D_glucopyranosyl_( 2 2 ξ,2 5R)_3β ,14α ,2 2 ξ ,2 6_tetrahydroxyfurost_5_ene 3_O_α_L_rhamnopyranosyl ( 1→ 2 )_β_D_glucopyranoside ( 2 ) ,diosgenin 3_O_[α_L_rhamnopyra nosyl( 1→ 2 ) ]_β_D_xylopyrano_syl ( 1→ 3)_β_D_glucopyranoside ( 3)andruscogenin 1_O_[α_L_rhamnopyranosyl( 1→ 2 ) ]_β_D_xy lopyranosyl ( 1→ 3)_β_D_fucopyranoside ( 4)。     

New Steroidal Glycosides from Ophiopogon japonicus

Two new C 27 steroidal glycosides, named ophiopojaponin A (1) and B (2), together with two known ones, were isolated from the tubers of the famous traditional Chinese herb Ophiopogon japonicus Ker-Gawl. The spectroscopic and chemical evidence revealed their structures to be pennogenin 3-O-［2′-O-acetyl-α-L-rhamnopyranosyl (1→2)］-β-D-xylopyranosyl (1→3)-β-D-glucopyranoside (1), 26-O-β-D-glucopyranosyl-(22ξ, 25R)-3β,14α,22ξ, 26-tetrahydroxyfurost-5-ene 3-O-α-L-rhamnopyranosyl (1→2)-β-D-glucopyranoside(2), diosgenin 3-O-［α-L-rhamnopyranosyl (1→2)］-β-D-xylopyrano-syl (1→3)-β-D-glucopyranoside (3) and ruscogenin 1-O-［α-L-rhamnopyranosyl (1→2)］-β-D-xylopyranosyl (1→3)-β-D-fucopyranoside (4).     

Bilirubin Oxidase from Trachyderma tsunodae K-2593, a Multi-copper Enzyme

The gel-setting and gel-melting temperatures of aqueous gelatin solutions (0.5 and 1.0 wt%) were measured by a steady state hot-wire method during treatment under high hydrostatic pressure up to 200 MPa. The high-pressure treatment caused both the gel-setting and gel-melting temperatures to increase with increasing pressure. The hot-wire method was proved to be effective for assessing the coagulation or gelation of food materials under pressurized conditions.     

Glycerol oxidase, a novel copper hemoprotein from Aspergillus japonicus. Molecular and catalytic properties of the enzyme and its application to the analysis of serum triglycerides

Glycerol oxidase purified from Aspergillus japonicus AT 008 had Mr = 400,000 and contained 1 mol of protoheme IX and 2 g atoms of copper/mol of enzyme protein. The absorption maxima of the oxidized form were found at 557, 530, 420, 280, and 238 nm, and those of the reduced form at 557 and 430 nm. Anaerobic addition of glycerol to the enzyme produced both a shift of the Soret band from 420 to 410 nm and bleaching of the alpha and beta bands at 557 and 530 nm. The ESR spectrum of glycerol oxidase showed three major signals at g = 1.99, g = 2.00, and g = 2.02. The signals at g = 1.99 and g = 2.02 were diminished by the anaerobic addition of glycerol, and the three signals completely disappeared after the addition of either dithionite or diethyldithiocarbamate. Exposure of glycerol oxidase to a borate buffer of pH 10.0 resulted in activation of the enzyme with concomitant enhancement of the ESR signals at g = 1.99 and g = 2.02. Since glycerol oxidase acts predominantly on glycerol, the enzyme can be employed in a specific colorimetric assay for serum triglycerides in combination with lipoprotein lipase.     

Glycerol uptake mutants of the hyphal fungus Aspergillus nidulans

A new class of glycerol non-utilizing mutants, designated glcC, has been isolated. The glcC gene was mapped in linkage group VI and mutants were found to complement the reference strains glcA1 (linkage group V) and glcB33 (linkage group I) in diploids. The new mutants were unable to grow on glycerol. However, in contrast to the glcA and glcB phenotype these mutants did grow well on dihydroxyacetone and D-galacturonate. By in vivo 13C NMR spectroscopy it was shown that the glcC mutant did not take up glycerol but did take up dihydroxyacetone. The latter substrate was converted intracellularly into glycerol which was then catabolized as normal.     

植物细胞离析酶的制备和应用

用 Aspergillus sp．A-19菌经固体发酵研制成一种新的植物细胞离析酶(SeparatasezA—P)。其离析单细胞的酶活力平均为70 767u／g,有效作用的pH在3．0—7．0,温度为20—45℃。发酵培养基配方是麸皮：桔皮粉：(NH₄)₂SO₄(w／w)为100:100:O．63,料水比为1 :2．0,培养适宜条件为25℃、60小时。