Release of protoplasts from Penicillium paxilli by Penicillium purpurogenum enzymes

Spherical, osmotically fragile protoplasts were obtained from the mycelium of Penicillium paxilli. The lytic enzymes were derived from the culture filtrate of P. purpurogenum grown on disintegrated mycelium of P. paxilli. Maximum numbers of protoplasts were released from 36 h mycelium, using 1 M sorbitol as an osmotic stabilizer, at pH 5.8, after 2 h incubation with lytic enzymes at 30 degrees C.     

Production of Rubratoxin B by Penicillium purpurogenum Stoll

Culture filtrates and chloroform extracts from Penicillium purpurogenum isolated from foodstuffs were subjected to preliminary survey for toxic effects on HeLa cells and mice. The toxic metabolite was isolated in a crystalline form from P. purpurogenum and was identified as rubratoxin B. Pathological findings observed in HeLa cells and mice treated with the metabolite are briefly described.     

产紫青霉EL-02的分离鉴定及其絮凝活性

摘要：【目的】分离鉴定有絮凝活性真菌,同时对其絮凝活性进行初步研究。【方法】采用梯度稀释、平板划线、18S rDNA检测等方法分离鉴定絮凝活性菌株。通过高速离心、超声破碎、乙醇沉淀、定性试验等方法确定絮凝活性物质性质。【结果】从渤海湾海岸土壤样品中分离筛选出一株有较高絮凝活性的真菌,经鉴定为产紫青霉（Penicillium purpurogenum）,命名为产紫青霉EL-02（P. purpurogenum EL-02）。超声破碎试验证实其絮凝活性主要存在于发酵上清液。根据絮凝活性曲线,确定4 d为积累絮     

Glucose-induced production of a Penicillium purpurogenum xylanase by Aspergillus nidulans

The heterologous secretion of xylanase B from Penicillium purpurogenum using glucose as inducer was performed in Aspergillus nidulans. For this purpose, plasmid pEVXB, containing the xylanase B cDNA (including its own signal peptide) under the control of the glyceraldehyde-3-phosphate dehydrogenase promoter, was constructed and used to transform A. nidulans. Analysis of transformed clones showed that several of them secreted extracellular xylanase activity when grown in a medium containing glucose. The clone showing the highest xylanase activity was chosen for further work. When this clone was grown on glucose, xylanase activity (0.72 U/ml), was detected in the culture supernatant. This was confirmed by a zymogram analysis and by the amplification of xynB cDNA from this clone. To our knowledge, this is the first example of the production of a xylanase from Penicillium in heterologous fungal hosts using glucose as inducer.     

Cellulase production by Penicillium echinulatum on lactose

The inducer effect of lactose on cellulase activity in Penicillium echinulatum 9A02S1 was studied. Submerged cultivation was performed using different concentrations of lactose and cellulose, in which the pH, mycelial mass, soluble proteins, filter paper activity (FPA), and activity of β-glucosidases were measured. The cultures containing lactose only presented low FPAs (0.1 FPU/ml). The cultures with associated cellulose and lactose and those containing cellulose only presented similar enzymatic activities (1.5 FPU/ml), suggesting the possibility of up to 75% reduction in the cellulose concentration. In relation to the β-glucosidases, increasing the lactose/cellulose ratio results in a proportional increase of enzymatic activity. In the cultures using both inducers, there is a longer duration of the acid phase in relation to treatments using only cellulose or lactose, indicating diauxia and catabolic repression.     

Beta-glucosidase from Penicillium purpurogenum: purification and properties.

beta-Glucosidase was purified from the culture supernatant of Penicillium purpurogenum. The purified enzyme was homogeneous on both nondenaturing and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The enzyme is a monomeric glycoprotein with M(r) of 90,000 as determined by gel filtration on Bio-Gel P-300 and SDS-polyacrylamide gels. Two enzyme forms were resolved by chromatofocusing and isoelectric focusing, and the pI values obtained with both methods were 4.2 (major form) and 6.0. The major form was characterised further. Enzyme activity was optimal at pH 3.5 and at 60 degrees C. The enzyme was stable in the pH range 2.5-9.5 for 24 h at 4 degrees C. Kinetic analysis gave Kms of 0.8 mM for cellobiose and 85 microM for p-nitrophenyl-beta-D-glucopyranoside. The enzyme hydrolyses a wide range of substrates including aryl-beta-glucosides, cellobiose, and amygdalin. Glucose inhibits competitively and glucono-delta-lactone is a mixed inhibitor of the enzyme.     

Improvement in Inulinase Production by Simultaneous Action of Physical and Chemical Mutagenesis in Penicillium purpurogenum

Summary An extracellular inulinase (fructanase)-producing strain of Penicillium purpurogenum was isolated from the rhizosphere soil of chicory. Conidia of this selected strain were subjected to simultaneous treatment with NTG–UV (N-methyl-N′-nitro-N-nitrosoguanidine and ultraviolet radiation) and EtBr–UV (Ethidium bromide–ultraviolet radiation). After mutagenesis, colonies were screened and among them a few were selected to carry out the inulinase study, which showed a significantly higher inulinase activity with higher I/S (inulin/sucrose) ratio in all the selected colonies, indicating enhancement of inulinase production after mutagenic treatments in all the selected mutants.     

Characterization of dextranase from Penicillium purpurogenum (Ftoll)]

An extracellular dextranase (E. C. 3.2.1.11) was purified from cell-free culture filtrates of Penicillium purpurogenum (Ftoll). The enzyme was most active at pH 5,5. The dextranase was endo-type, it split quickly isomaltotetraose into two isomaltose molecules, slowly degraded isomaltotriose, and did not act on isomaltose. The rate of isomaltooligosaccharides hydrolysis was increased with the increase of the polymerization degree. Polyols obtained from isomaltooligosaccharides were split more slowly than the respective sugars. The isomaltopentaitol was split at two glucosidic linkages, 38% of hydrolyzed linkages being the second linkage from the sorbitol end of the molecule and 62% being the third one. The degree of degradation of dextrans depended on amount of 1,6 linkages. Isomaltose and tetrasaccharides of two types, 2(2)-alpha-D-glucosylmaltotriose and linear tetrasaccharide(s), are the lowest molecular weight products of exhaustive hydrolysis of branched dextrans.     

Culture conditions for enhanced cellulase production by a native strain of Penicillium purpurogenum

A cellulolytic wild-type strain of Penicillium purpurogenum was isolated from a soil sample in southern Chile. It grew best at 28°C from an inoculum of 4×10⁷ spores/100 ml medium. Highest endoglucanase activity was with Sigmacell as carbon source and corn steep liquor as nitrogen source. Wheat bran enhanced the production of endoglucanase and -glucosidase. The enzymes in the crude supernatants were stable up to 50°C and between pH 4.4 and 5.6 for 48 h.J.Steiner and C. Socha are with the Facultad de Ciencias Quimicas y Farmacéuticas, Universidad de Chile, Casilla 233, Santiago 1, Chile; J. Eyzaguirre is with the Laboratorio de Bioquímica, Pontificia Universidad Católica de Chile, Casilla 114-D, Santiago, Chile.     

The role of ethanol in preventing biofilm formation of Penicillium purpurogenum

The use of fungi in biotechnology requires that no cell loss takes place; a maximal level of cell–nutrient interaction is required to achieve efficient performance. The occurrence of high cell densities or loss of biomass through cell–surface interaction prevents the desired result. The main purpose of adding ethanol was to manipulate the cell–cell and cell–surface adhesion through manipulating cell surface properties. Scanning electron microscopy indicated that the type of surface and its treatment with ethanol controls the adhesion and biofilm formation of Penicillium purpurogenum. Gamma irradiation slightly affected the wettability of polystyrene strips at 0.5 and 1 kGy, thus slightly decreasing the adhesion, but was not as effective as using ethanol to control the adhesion. The presence of ethanol in the media caused a decrease in surface-bound proteins from 0.348 to 0.133 mg/ml, while surface exopolysaccharides showed a minimal decrease. Ethanol induced oxidative stress which reached its peak when 2.5 % v/v ethanol was added to the media; this was represented by both intracellular and extracellular catalase and lipid peroxidation. On the other hand, fungal biomass and pigment showed a decrease as the ethanol concentrations increased. Therefore, ethanol could be employed to control the surface properties of a fungus, and to inhibit biofilm formation to obtain a high surface area for the fungus to be employed in any biotechnological process.     

絮凝活性产紫青霉EL-02的分离鉴定及其絮凝活性初探

【目的】分离鉴定有絮凝活性真菌,同时对其絮凝活性进行初步研究。【方法】采用梯度稀释、平板划线、18SrDNA检测等方法分离鉴定絮凝活性菌株。通过高速离心、超声破碎、乙醇沉淀、定性试验等方法确定絮凝活性物质性质。【结果】从渤海湾海岸土壤样品中分离筛选出一株有较高絮凝活性的真菌,经鉴定为产紫青霉(Penicillium purpurogenum),命名为产紫青霉EL-02(P.purpurogenum EL-02)。超声破碎试验证实其絮凝活性主要存在于发酵上清液。根据絮凝活性曲线,确定4d为积累絮凝活性产物的最佳发酵时间。乙醇沉淀法获得该菌株絮凝活性物质。经鉴定该菌株所产絮凝活性物质为糖类,且其活性在pH2-11,温度-70℃-100℃范围内保持稳定。【结论】分离筛选到一株有絮凝活性的产紫青霉EL-02,经鉴定其产生糖类絮凝活性物质。     

Isolation of mutants of Penicillium purpurogenum resistant to catabolite repression

 The strain Penicillium purpurogenum P-26 was subjected to UV irradiation and N-methyl-N′-nitro-N-nitrosoguanidine treatment and mutants were isolated capable of synthesizing cellulase under the conditions of a high concentration of glucose. Initially mutants resistant to catabolite repression by 2-deoxy-D-glucose were isolated on Walseth’s cellulose/agar plates containing 15–45 mM 2-deoxy-D-glucose. These mutants were again screened for resistance to catabolite repression by glycerol or glucose on Walseth’s cellulose/agar plates containing 50 g/l glycerol or 50 g/l glucose respectively. Four mutants with different sizes of clearing zone on Walseth’s cellulose/agar plates containing 50 g/l glucose were selected for flask culture. Among them, the mutant NTUV-45-4 showed better carboxymethylcellulase activity in flask culture containing 1% Avicel plus 3% glucose than did the parental strain. Received: 9 October 1995/Received revision: 27 November 1995/Accepted: 8 January 1996     

An alpha-L-arabinofuranosidase from Penicillium purpurogenum: production, purification and properties

Penicillium purpurogenum secretes arabinofuranosidase to the growth medium. Highest levels of enzyme (1.0 U ml(-1)) are obtained when L-arabitol is used as carbon source, while 0.85 and 0.7 U ml(-1) are produced with sugar beet pulp and oat spelts xylan, respectively. By means of a zymogram, three bands with arabinofuranosidase activity have been detected in the supernatant of a culture grown in oat spelts xylan. One of the enzymes was purified to homogeneity from this supernatant using gel filtration (BioGel P-100), cation exchange chromatography (CM-Sephadex C-50), hydrophobic interaction chromatography (phenyl agarose) and a second BioGel P-100 column. The enzyme is a monomer of 58 kDa with a pI of 6.5. Optimum pH is 4.0 and optimal temperature 50 degrees C. The arabinofuranosidase is highly specific for alpha-L-arabinofuranosides and liberates arabinose from arabinoxylan. The enzyme shows hyperbolic kinetics towards p-nitrophenyl-alpha-L-arabinofuranoside with a K(M) of 1.23 mM. A 36-residue N-terminal sequence is over 70% identical to that of fungal arabinofuranosidases belonging to family 54 of the glycosyl hydrolases. Based on the sequence similarity and other biochemical properties it is proposed that the purified enzyme from P. purpurogenum belongs to family 54.     

Heterologous expression of a Penicillium purpurogenum pectin lyase in Pichia pastoris and its characterization

Lignocellulose is the major component of plant cell walls and it represents a great source of renewable organic matter. One of lignocellulose constituents is pectin. Pectin is composed of two basic structures: a ‘smooth’ region and a ‘hairy’ region. The ‘smooth’ region (homogalacturonan) is a linear polymer of galacturonic acid residues with α-(1→4) linkages, substituted by methyl and acetyl residues. The ‘hairy’ region is more complex, containing xylogalacturonan and rhamnogalacturonans I and II. Among the enzymes which degrade pectin (pectinases) is pectin lyase (E.C. 4.2.2.10). This enzyme acts on highly esterified homogalacturonan, catalysing the cleavage of α-(1→4) glycosidic bonds between methoxylated residues of galacturonic acid by means of β-elimination, with the formation of 4,5-unsaturated products. In this work, the gene and cDNA of a pectin lyase from Penicillium purpurogenum have been sequenced, and the cDNA has been expressed in Pichia pastoris. The gene is 1334 pb long, has three introns and codes for a protein of 376 amino acid residues. The recombinant enzyme was purified to homogeneity and characterized. Pectin lyase has a molecular mass of 45 kDa as determined by SDS-PAGE. It is active on highly esterified pectin, and decreases 40 % the viscosity of pectin with a degree of esterification ≥85 %. The enzyme showed no activity on polygalacturonic acid and pectin from citrus fruit 8 % esterified. The optimum pH and temperature for the recombinant enzyme are 6.0 and 50 °C, respectively, and it is stable up to 50 °C when exposed for 3 h. A purified pectin lyase may be useful in biotechnological applications such as the food industry where the liberation of toxic methanol in pectin degradation should be avoided.     

Removal of Chromium(VI) Ions from Synthetic Solutions by the Fungus Penicillium purpurogenum

The ability of Penicillium purpurogenum to bind high amounts of chromium(VI) from aqueous solutions is demonstrated. Cr(VI) adsorption capacity increases with time during the first four hours and then leveled off toward the equilibrium adsorption capacity. Biosorption of Cr(VI) ions reached equilibrium in four hours. Binding of Cr(VI) ions with Penicillium purpurogenum biomass was clearly pH dependent. Cr(VI) loading capacity increased with increasing pH. The adsorption of Cr(VI) ions reached a plateau value at a pH of approx. 6.0. The maximum capacity of adsorption of Cr(VI) ions onto the fungal biomass was 36.5 mg/g. Adsorption behavior of Cr(VI) ions can be approximately described with the Langmuir equation. When applying the Langmuir model, the maximum adsorption capacity (Q_max) and the Langmuir constant were found to be 40 mg/g and 3.9 × 10^–3 mg/L. Elution of Cr(VI) ions was performed by means of 0.5 M HCl. It was possible to use the biomass of Penicillium purpurogenum for six cycles for biosorption.     

斜卧青霉纤维素酶和木聚糖酶高产菌株的选育

以纤维素酶高产菌株斜卧青霉A50为出发菌株,通过紫外诱变原生质体获得1株木聚糖酶活力提高80%而纤维素酶活力没有改变的6号菌。蛋白质电泳和酶谱检测结果显示,纤维素酶谱基本无差别,而木聚糖酶谱显示6号菌比A50多了一条带。6号菌优化后的产酶培养基组成为:麸皮7%、葡萄糖0.1%,该条件下,纤维素酶活为19.7IU/mL,木聚糖酶活力为215.4IU/mL。     

大豆连作障碍研究Ⅰ．大豆连作土壤紫青霉菌的毒素作用研究

研究分析了大豆连作、轮作土壤微生物区系,发现连作大豆根际土壤真菌富集,以其优势真菌回接大豆．紫青霉菌（Ｐｅｎｉｃｉｌｌｉｕｍｐｕｒｐｕｒｏｇｅｎｕｍ）能强烈抑制大豆生长发育．在实验室条件下分离获得该菌产生的毒素粗结晶,５μｇ·ｍｌ－１水培液中即可观察到大豆根系受害,根毛很少生长;３０μｇ·ｍｌ－１水培液中大豆主根褐变严重,侧根几乎不再生长;２００μｇ·ｍｌ－１导致一些大豆品种幼苗在２周内死亡这些结果表明,连作大豆土壤中该菌的大量存在及其产生的毒素是大豆连作障碍产生的主要因素．