Viscometric method for assaying of total endodepolymerase activity of pectinases

An improved method for assaying of the total endodepolymerase activity of pectinases has been developed. The method is based on the determination of the viscosity of a citrus pectin solution in the presence of the enzyme using an Ostwald viscometer. The depolymerizing activity of different pectinases can be detected including polygalacturonase, polymethylgalacturonase, pectin lyase, and pectate lyase. One unit of the endodepolymerase activity corresponds to the activity resulting in 50% decrease in the relative viscosity of 0.5% citrus pectin solution for 5 min at 40°C and the appropriate pH. Depending on the pH-optima of the enzymes, two modifications of the method are described: 1) for acid pectinases at pH 5.0, and 2) for neutral (mildly alkaline) pectinases at pH 8.0. The modifications differed in the control and in the calculation of the activity. Six enzyme preparations were used to demonstrate the applicability of the method. The parameter used for the calculation of the enzymatic activity was directly proportional to the enzyme concentration (the dependence was linear in the range of at least 10-fold change in the enzyme concentration). The relative error of the method did not exceed 10%.     

Sequential production of pectinases byPenicillium frequentans

The pectinases produced byPenicillium frequentans were subjected to pectin- or sodium polypectate-PAGE. The fungus secreted one endo- and one exo-polygalacturonase during the first 10 h of incubation, either in media supplemented with pectin or in the absence of carbohydrate source. After 17 h of cultivation, another two exo-and three endo-polygalacturonases were detected in cultures supplemented with pectin. The results indicate that two constitutive polygalacturonases are secreted initially, followed by the synthesis of other inducible pectinases, in a sequential production of the pectinolytic complex. Monogalacturonic acid and sodium polypectate were better inducers for exo- than for endo-polygalacturonases, indicating that these enzymes are independently controlled.The authors are with the Faculdade de Ciências Farmacêuticas de Ribeirão Preto, USP, Avenida do Café s/n^o, 14040-903 Ribeirão Preto, São Paulo, Brazil     

Partial purification and properties of pectin lyase from Penicillium expansum

A pectin lyase, poly(methoxygalacturonide) lyase, EC 4.2.2.10, from a culture filtrate of Penicillium expansum was partially purified 33-fold with 7.3% yield. The enzyme was monomeric with a molecular mass of 36.5 kDa. The enzyme did not contain pectate lyase activity and degraded citrus and apple pectin best at pH 7.0 and 40 to 45°C. The K _m for citrus pectin was 9 mg ml^-1.     

Purification and characterization of pectinesterase and polygalacturonase from Trichoderma reesei

Abstract Exopolygalacturonase, endopolygalacturonase and pectinesterase were separated from culture filtrates of Trichoderma reesei QM9414 by Sephadex chromatography. Exopolygalacturonase was characterized by specific cleavage of pectic acid to form d -galactopyranuronic acid, and by the hydrolysis of oligomers (highest reaction rate at pentamer). Polygalacturonase exhibited 2 pH-optima peaks (at 4.8 and 5.1) and 10 bands with enzyme activity by isoelectric focusing (IEF) (p I 4.6–8.5). Pectinesterase showed a pH-optimum at 7.6, and 6 enzyme-activity bands on an IEF zymogram which seemed identical with those of higher plants (tomato, alfalfa).     

Expression of Pectinase Activity Among Aspergillus Flavus Isolates from Southwestern and Southeastern United States

Aspergillus flavus is a widely distributed filamentous fungus that contaminates crops with the potent carcinogen aflatoxin. This species can be divided into S and L strains on the basis of sclerotial morphology. During crop infection, A. flavus can secrete a large array of hydrolytic enzymes. These include pectinase, which aids fungal spread through plant tissues. A survey of pectinase expression by soil isolates derived from different regions of the United States revealed geographic polymorphisms. Strain L isolates from Arizona produced moderate to high levels of a specific pectinase P2c, while S strain isolates produced variable amounts of P2c. In contrast, L strain isolates from southeastern U.S. yielded variable P2c production, while S strain isolates consistently expressed high P2c levels. These results were corroborated by pectinase surveys of additional collections of A. flavus from soil and cottonseed. Expression patterns for P2c and pectinmethylesterase were evaluated for a select number of isolates using an isoelectric focusing technique. Clear zone reactions from the pectinase plate assay corresponded to the presence of P2c, while red ring reactions corresponded to the lack of P2c. Commercial cottonseed infected by S strain isolates frequently contained aflatoxin, even when infected by S strain isolates that did not produce pectinase P2c. Thus, although P2c-lacking isolates have reduced invasiveness, these isolates still have sufficient pathogenicity to cause aflatoxin contamination.     

The heterologous expression of polysaccharidase-encoding genes with oenological relevance in Saccharomyces cerevisiae

AIMS: The main objective of this study was to develop polysaccharide-degrading wine strains of Saccharomyces cerevisiae, which are able to improve aspects of wine processing and clarification, as well as colour extraction and stabilization during winemaking. METHODS AND RESULTS: Two yeast expression/secretion gene cassettes were constructed, namely (i) a pectinase gene cassette (pPPK) consisting of the endo-polygalacturonase gene (pelE) from Erwinia chrysanthemi and the pectate lyase gene (peh1) from Erwinia carotovora and (ii) a glucanase/xylanase gene cassette (pEXS) containing the endo-beta-1,4-glucanase gene (end1) from Butyrivibrio fibrisolvens and the endo-beta-1,4-xylanase gene (xynC) from Aspergillus niger. The commercial wine yeast strain, VIN13, was transformed separately with these two gene cassettes and checked for the production of pectinase, glucanase and xylanase activities. Pinot Noir, Cinsaut and Muscat d'Alexandria grape juices were fermented using the VIN13[pPPK] pectinase- and the VIN13[pEXS] glucanase/xylanase-producing transformants. Chemical analyses of the resultant wines indicated that (i) the pectinase-producing strain caused a decrease in the concentration of phenolic compounds in Pinot Noir whereas the glucanase/xylanase-producing strain caused an increase in phenolic compounds presumably because of the degradation of the grape skins; (ii) the glucanase/xylanase-producing strain caused a decrease in wine turbidity, especially in Pinot Noir wine, as well as a clear increase in colour intensity and (iii) in the Muscat d'Alexandria and Cinsaut wines, the differences between the control wines (fermented with the untransformed VIN3 strain) and the wines produced by the two transformed strains were less prominent showing that the effect of these polysaccharide-degrading enzymes is cultivar-dependent. CONCLUSIONS: The recombinant wine yeasts producing pectinase, glucanase and xylanase activities during the fermentation of Pinot Noir, Cinsaut and Muscat d'Alexandria grape juice altered the chemical composition of the resultant wines in a way that such yeasts could potentially be used to improve the clarity, colour intensity and stability and aroma of wine. SIGNIFICANCE AND IMPACT OF THE STUDY: Aspects of commercial-scale wine processing and clarification, colour extraction and stabilization, and aroma enhancement could potentially be improved by the use of polysaccharide-degrading wine yeasts without the addition of expensive commercial enzyme preparations. This offers the potential to further improve the price:quality ratio of wine according to consumer expectations.     

Isolation and characterization of extracellular pectin lyase from Penicillium canescens

Pectin lyase A (molecular weight 38 kD by SDS-PAGE, pI 6.7) was purified to homogeneity from culture broth of the mycelial fungus Penicillium canescens using chromatographic techniques. During genomic library screening, the gene encoding pectin lyase A from P. canescens (pelA) was isolated and sequenced, and the amino acid sequence was generated by applying the multiple alignment procedure (360 residues). A theoretical model for the three dimensional structure of the protein molecule was also proposed. Different properties of pectin lyase A were investigated: substrate specificity, pH- and temperature optimum of activity, stability under different pH and temperature conditions, and the effect of Ca2+ on enzyme activity. In the course of the laboratory trials, it was demonstrated that pectin lyase A from P. canescens could be successfully applied to production and clarification of juice.     

Characterization of galactose-induced extracellular and intracellular pectolytic activities from the exo-1 mutant strain of Neurospora crassa

Pectolytic enzymes from the hyperproducer exo-1 mutant of Neurospora crassa are induced either by pectin or galactose. Galactose-induced pectinases, in contrast with pectin-induced enzymes, are not affected by glucose repression. Here, the pectolytic enzymes induced by galactose were purified and characterized. Extracellular pectolytic activities were separated into two main fractions. Pool I contained lyases, and a polygalacturonase (PG) copurifying as a complex of about 80 kDa (gel filtration). Pool II contained PG only. Under urea-SDS-PAGE the lyases and polygalacturonase from pool I migrated with an apparent MW of 56.2 kDa, and 34.3 kDa, respectively. PG from pool II exhibited an apparent MW of 44.7 kDa. Cell extracts contained PG free of lyase activities. Purified intracellular PG migrated (SDS-PAGE) as a single band of apparent MW of 31.5 kDa. All pectinases were glycoproteins (18.5–39% carbohydrate), with stability and optimum pH at 5–6 and 9–10 for PG and lyases, respectively. Temperature optima were 40–50°C, respectively. All enzymes were inactivated at 60°C, with a half-life from 1.5 to 5 min. Activation energy (Ea) values for extracellular and intracellular PG varied between 0.45 and 2.0 Kcal mol⁻¹. Pool II and intracellular PG and lyases, exhibited a random mechanism of hydrolysis. Pool I PG exhibited an exo character. Received 20 October 1997/ Accepted in revised form 28 February 1998     

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.     

Production of pectinases by Aspergillus niger in solid state fermentation at high initial glucose concentrations

Exopectinase (exo-p) and endopectinase (endo-p) production by Aspergillus niger CH4 in solid state culture was studied at initial glucose concentrations of 100, 250, 350 and 450 g/l. The highest activity of exo-p (35 U/g) was produced at 72 and 120 h in the medium containing 100 and 250 g glucose/l, respectively. The maximum endo-p activity (9 U/g) was produced at 72 h in the medium with 250 g glucose/l. The reduction in pectinase production at 350 and 450 g/l initial glucose concentration was due neither to repression of the synthesis of the enzyme nor to the glucose consumption rate of the strain but due to a drastic drop in pH of the medium.S. Solis-Pereyra, E. Favela-Torres, M. Gutiérrez-Rojas, G. Saucedo-Castañeda and G. Viniegra-González are with the Departamento de Biotecnologia, Universidad Autónoma Metropolitana, A.P. 55-535, C.P. 09340, México D.F., México; S. Roussos is with the Laboratoire de Biotechnologie, ORSTOM, B.P. 5045, 34032, Montpellier Cedex, France, and P. Gunasekaran is with the Department of Microbial Technology, School of Biological Sciences, Madurai Kamaraj University, Madurai, 625-021, India.     

Polygalacturonase, Pectate Lyase and Pectin Methylesterase Activity in Pathogenic Strains of Phytophthora capsici Incubated under Different Conditions

Pectolytic enzymes are found mainly in fungi and bacteria. The most widely occurring enzymes are polygalacturonase (PGs), pectin methylesterase (PMEs) and pectate lyase (PLs) produced during the infection process and during culturing. The secretion of these enzymes results in the disorganization of the plant cell walls, which is responsible for the pathogenicity of the pathogens. These enzymes degrade the pectin of plants causing maceration of plant tissues and the enzyme activity increases under favourable environmental conditions. We have found that Phytophthora capsici , a pathogenic oomycete, produces levels of these three enzymes equal to those produced by soft-rotting Erwinia chrysanthemi . The activity of PGs, PLs and PMEs was investigated at the optimum temperature, pH and ionic strength in highly pathogenic P. capsici strains cultivated in two kinds of liquid medium containing either crude pepper extracts plus pectin or pectin as the carbon source. Virulence tests and enzymes activity showed that there was a high correlation between the enzyme activity and the pathogenicity of P. capsici . The effects of different carbon sources on the enzyme activity showed that pepper extract plus pectin was the best source for the carbon source.     

黑曲霉果胶裂解酶基因毕赤酵母在Pichia pastoris GS115中的表达

利用RT-PCR技术从黑曲霉(EIM-6)中扩增得到去除信号肽的果胶裂解酶基因A,将其插入到毕赤酵母表达载体pPIC9k上,构建重组表达质粒pPIC9K-pelA,电击转化毕赤酵母GS115,得到了表达成功的工程菌株。用终浓度为1.5%的甲醇对其进行诱导,将发酵上清液浓缩后,用盐酸法测定其酶活可以达到2.3U/mL。通过对重组毕赤酵母诱导表达产物进行SDS-PAGE鉴定,发现重组毕赤酵母分泌了1个约38kD的蛋白,与该酶基因产物的理论值相符,并通过水解圈法测定验证,均说明果胶裂解酶得到正确的分泌表达.     

Cell-wall lysing enzymes and products of cell-wall digestion elicit ethylene in citrus

Ethylene production was induced in Valencia oranges [ Citrus sinensis (L.) Osbcck] by injection of the fungal enzyme mixture Pectolyase ( Aspergillus japonicus ) which contains pectolytic enzymes into the peel. The mixture also stimulated production of 1-aminocyclopropane-1-carboxylic acid (ACC). Cycloheximide partially inhibited the Pectolyase-induced ethylene response. Pectin fragments, resulting from partial acid hydrolysis or Pectolyase digestion, caused an increase in ethylene production when injected into the peel of intact orange fruits. Pectic fragments produced by fungal enzymes are known to be elicitors of phytoalexins and in this study are shown to elicit ethylene in citurs.     

Isolation, Structure elucidation, and antimycobacterial properties of dimeric naphtho-gamma-pyrones from the marine-derived fungus Aspergillus carbonarius

Two new dimeric naphtho-gamma-pyrones, compounds 1 and 2, were isolated from the AcOEt extract of the fungal strain WZ-4-11 of Aspergillus carbonarius, together with eight known analogues, including 10,10'-bifonsecin B (3), 6'-O-demethylnigerone (4), nigerone (5), isonigerone (6), fonsecin (7), rubrofusarin B (8), TMC 256A1 (9), and flavasperone (10). Their structures were elucidated by means of UV, CD, IR, and 1D- and 2D-NMR spectroscopy, in combination with HR-MS analysis. The fully assigned (1)H- and (13)C-NMR data of 3, and the (13)C-NMR data of 6 are reported for the first time. Compounds 1 and 2 showed weak antimycobacterial activities against Mycobacterium tuberculosis H37Rv, with MIC values of 43.0 and 21.5 microM, resp.     

Determination of the enzymatic activity of pectinases from different microorganisms

The decrease in viscosity is widely used to estimate the activity of pectinolytic enzymes. This method is shown to be influenced by the production strain and this prevents an accurate comparison between the activities of different microorganisms, especially under different conditions.A.E. Maiorano and Y. Ogaki are with the Divisão de Quimica, with the Agrupamento de Biotecnologia, Instituto de Pesquisas Tecnológicas do Estado de São Paulo S/A.-IPT, Cidade Universitária, Caixa Postal 7141, CEP 01064-970, São Paulo, SP, Brazil; W. Schmidell is with the Departamento de Engenharia Quimica, Escola Politécnia, Universidade de São Paulo, Caixa Postal 61548, CEP 05424-970, São Paulo, SP, Brazil.     

Isolation and characterization of a cellulase-free pectinolytic and hemicellulolytic thermophilic fungus

A thermophilic fungus belonging to the Deuteromyces, having pectinase and xylanase activities, was grown at its optimum temperature of 55°C. It grew over a wide pH range of 4 to 10, being optimal at 6. The fungus grew well on modified Mandels' medium in which cellulose was substituted either with hemicellulose or pectin. With citrus pectin as carbon source, 121 units/ml of pectinase activity were obtained and with larch wood xylan as carbon source, 83 units/ml of xylanase activity were obtained.