Starch-degrading enzymes of two species ofFusarium

Starch supported production of maximum α-amylases (dextrinizing and saccharifying) byFusarium oxysporum andF. scirpi. Addition of gibberellic acid resulted in an increased production of α-amylase. Presence of glucose depressed the enzyme production. pH 4.5 and 4.0 was found to be optimum for the dextrinizing enzyme secreted by both species. The temperature of 25 and 40 °C was optimum for the dextrinizing enzyme secreted byF. oxysporum andF. scirpi, respectively. Saccharifying enzymes of both species showed their optimum at pH 6.9. The optimum temperature for the activity of the saccharifying enzyme was 30 and 40 °C, respectively.     

Comparative studies on the effect of vitamins on sporulation in Fusarium oxysporum schlecht. ex. Fr. and Fusarium moniliforme V. subglutinans Wr. & Rg.

On the basis of sporulation (total output of all the three spore forms taken together) and fungal mat production bothF. oxysporum Schlecht ex.Fr. andF. moniliforme v.subglutinans Wr. &Rg. are auxoheterotrophic for thiamine, biotin, inositol, riboflavin and pyridoxine. The first three vitamins are selective in accelerating macro-conidial production also inF. moniliforme, which otherwise shows decrease with advance in days of incubation.F. moniliforme is an auxo-autotroph for nicotinic acid, Ca-pantothenate and folic acid and auxoheterotroph for ascorbic acid. Auxoautotrophy for Ca-pantothenate, folic acid, l-ascorbic acid and p-aminobenzoic acid cannot be suggested forF. oxysporum. Whereas nicotinic acid is a depressent of sporulation inF. oxysporum, inF. moniliforme another vitamin p-aminobenzoic acid depresses sporulation. As the two species ofFusarium show differences in preference as well as inhibition to at least five of the vitamins studied and also varied trends of pH changes exists there is full justification for their separate taxonomic placements.     

Influence of various nitrogen and carbon sources on the production of pectolytic,cellulolytic and proteolytic enzymes byAspergillus niger

Three isolates ofAspergillus niger produced polygalacturonase (PG) and pectin methyl galacturonase (PMG) in the presence of organic and inorganic nitrogen sources. Complete inhibition of PG PMG cellulase (C_x) and proteinase synthesis was found in the presence of cystine in all isolates. Maximum biomass was found in sodium nitrate whereas no isolate could grow in the presence of cystine. A correlation between biomass and enzyme production could be obtained when sodium nitrate and cystine were added to the medium separately. All isolates produced pectic cellulolytic and proteolytic enzymes in the presence of various native carbon sources. Sodium polypectate was found to be the best carbon source for the production of PG and PMG; pectin inhibited completely the production of PG and PMG. Maximum cellulase production was brought about by cotton in all three isolates. Maximum proteinase production was observed with gelatin which served as poor substrate for fungal growth. Sucrose supported maximum fungal growth in comparison with all other native carbon sources. The increased production of pectolytic cellulolytic and proteolytic enzymes in the presence of sodium polypectate reflected a stimulation rather than an induction of synthesis of these enzymes.     

Antibiotic inhibition of pectolytic and cellulolytic enzyme activity in twoFusarium species

Among all antibiotics tested, amoxycillin (500 ppm) completely inhibited the polygalacturonase and pectinmethylgalacturonase enzyme activity inF. oxysporum; none of the antibiotics did so inF. moniliforme. No antibiotic completely inhibited the cellulase activity in both test organisms, however, amoxycillin was better than other antibiotics in inhibiting the cellulase activity in both the organisms.     

Cellulose digestion in a leaf eating insect,the mexican bean beetle,Epilachna varivestis

Adult Mexican bean beetles, Epilachna varivestis (Mulsant) (Coleoptera: Coccinellidae), are able to digest cellulose. Females digest nearly three times as much cellulose and retain food in their guts three times longer than do males. There is no sexual dimorphism in enzyme activity although pH exerts a profound effect. The approximate pH optima are 4.5 for C₁-cellulase, 5.5 for C_x-cellulase and 4.5–4.8 for β-glucosidase. The acidic gut of beetles (pH 4.9–5.8) should permit maximum cellulase activity.     

Tissue maceration during pathogenesis ofFusarium oxysporum andF.moniliforme

Tissue extracts of fruits infected withF. oxysporum orF. moniliforme did not cause tissue maceration at any pH tested. WithF. oxysporum insignificant tissue maceration was observed at pH 8. Pectic enzymes were not responsible for tissue maceration.     

Purification and characterization of a chitinase from Serratia proteamaculans

A chitinase gene from Serratia proteamaculans 18A1 was cloned, sequenced, and expressed in Escherichia coli M15. Recombinant enzyme (ChiA) was purified by Ni-NTA affinity column chromatography. The ChiA gene contains an open reading frame (ORF), encoding an endochitinase with a deduced molecular weight 60 kDa and predicted isoelectric point of 6.35. Comparison of ChiA with other chitinases revealed a modular structure containing an N-terminal PKD-domain, a family 18 catalytic domain and a C-terminal putative chitin-binding domain. Turn over rate (K _cat) of the enzyme was determined using colloidal chitin (49.71 ± 1.15 S^?1) and crystalline β-chitin (17.20 ± 0.83 S^?1) as substrates. The purified enzyme was active over a broad range of pH (pH 4.5–9.0) and temperature (4–70°C) with a peak activity at pH 5.5 and 55°C. However, enzyme activity was found to be stable up to 45°C for longer incubation periods. Purified enzyme was shown to inhibit fungal spore germination and hyphal growth of pathogenic fungi Fusarium oxysporum and Aspergillus niger.     

Screening of zearalenone-producing Fusarium species in Egypt and chemically defined medium for production of the toxin

296 isolates of Fusarium spp. from 100 samples of cereale grains were examined for their ability to produce zearalenone on liquid culture medium. Thin layer Chromatographic analysis revealed that the mycotoxin was detected from 45 isolates, (F. oxysporum, 36; F. moniliforme, 8; and F. equiseti, one isolate). A suitable liquid medium and some optimal conditions for the biosynthesis of zearalenone were reported. Glutamine and riboflavin stimulated the production of the toxin. The maximum amount of zearalenone appeared at pH 7, after 12 days of incubation at 20 °C.     

Stability of phycocyanin extracted from Spirulina sp.: Influence of temperature,pH and preservatives

Temperature and pH play an important role in the stability of phycocyanin, a natural blue colorant. Systematic investigations showed the maximum stability of phycocyanin was in the pH range 5.5–6.0. Incubation at temperatures between 47 and 64 °C caused the concentration (C_R) and half-life of phycocyanin in solution to decrease rapidly. The C_R value remained at approximately 50% after incubating for 30 min at 59 °C. After heating at 60 °C for 15 min, the C_R value of phycocyanin at pH 7.0 was maintained at around 62–70% when 20–40% glucose or sucrose was added, and the half-life increased from 19 min to 30–44 min. 2.5% sodium chloride was found to be an effective preservative for phycocyanin at pH 7.0 as a C_R value of 76% was maintained and the half-life of 67 min was increased.     

Purification and characterization of a novel solvent-tolerant lipase from Fusarium heterosporum

A microorganism producing a solvent-tolerant lipase was identified as Fusarium (F.) heterosporum. The lipase was purified from the culture filtrate to homogeneity as judged by disc-PAGE and SDS-PAGE. The purification included SP-Sephadex chromatography, gel filtration and isoelectric focusing, and the recovery yield was 38%. The lipase was a monomeric protein with a molecular weight of 31 kDa estimated by SDS-PAGE, and a pI of 7.0. The optimum pH at 40°C and optimum temperature at pH 5.6 were 5.5–6.0 and 45–50°C, respectively, when olive oil was used as the substrate. The lipase was stable over a pH range of 4–10 at 30°C for 4 h, and up to 40°C at pH 5.6 for 30 min. Furthermore, the enzyme was not inactivated even after incubation at 30°C in 50% solvent such as dimethylsulfoxide (DMSO), hexane, benzene and ether for 20 h. The activity did not decrease in a reaction with stirring in a mixture containing 50% DMSO or dimethylformamide. The lipase preferably reacted on middle-chain fatty acid triglycerides (6≤C≤12), and cleaved only 1,3-ester bonds of triolein. The enzyme had an N-terminal sequence of Ala-Val-Thr-Val-Thr-Thr-Gln-Asp-Leu-Ser, which has not previously been found in any other protein. We compared the properties of lipases from F. heterosporum and another strain F. oxysporum.     

Isomerization of glucose to fructose: production and some properties of glucose isomerase from Streptomyces sp. strain C7

Nine strains of actinomycetes isolated from Iraqi soils were investigated for glucose isomerase production. Only one strain, Streptomyces sp., C₇, was active. The maximum conversion ratio of the enzyme for the cells grown in d-xylose medium after 24 h incubation at 70°C and pH 6.9, was 64 and 48% for crude extract and cell-bound enzyme, respectively. The optimum pH value and temperatures for both enzymes were 8.0 and 70°C.     

Production of ethanol from sugars in wood hydrolysate byFusarium oxysporum

Wood hydrolysate used for ethanol production by two strains ofFusarium oxysporum contained 2.3% (w/v) reducing sugars (xylose and glucose). Ethanol production at the optimum reducing sugar concentration of 54.8 g/l medium, at pH 5.5, and 30°C was 12.3 g/l and 11.7 g/l byF. oxysporum D-140 and NCIM-1072, respectively in shake flasks during 96 h fermentation. The maximum production of ethanol under optimum cultural conditions, and in the presence of yeast extract plus minerals, was 13.2 g/l medium byF. oxysporum D-140 over 108 h fermentation.

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Résumé L'hydrolysat de bois utilisé pour la production d'éthanol par deux souches deFusarium oxysporum contenait 2.3% (poids/vol.) de sucres réducteurs (xylose et glucose). La production d'éthanol, à la concentration optimum en sucres réducleurs de 54.8 g par litre de milieu à pH 5.5 et à 30°C était de 12.3 g/l et 11.7 g/l respectivement chezF. oxysporum D-140 et NCIM-1072, en flacons agités pendant 96 h de fermentation. La production maximum d'éthanol, dans les conditions optimum de culture, et en prosence d'extrait de levure et de minéraux a mit de 13.2 g par litre de milieu chezF. oxysporum D-140 en 108 h de lermentation.

     

Cellulase activity of some phytopathogenic fungi isolated from diseased leaves of broad bean

Forty-five fungal species and two varieties belonging to the 23 genera were collected from 50 samples of diseased leaves of broad bean collected from Qena Governorate in Egypt on glucose-Czapek’s, dichloran-chloramphenicol-malt extract and dichloran-chloramphenicol-peptone agar at 28 °C. The most common genera were Alternaria, Aspergillus, Cladosporium, Fusarium, Mycosphaerella and Penicillium. The most prevalent species were Alternaria alternata, Aspergillus flavus, A. fumigatus, A. niger, Cladosporium cladosporioides, Fusarium merismoides, Mycosphaerella tassiana and Penicillium chrysogenum. Among the eight dematiaceous Hyphomycetes phytopathogenic fungi screened for their abilities to produce both exo- and endo-β-1,4-glucanase enzymes (C₁ and C_x), five species had high C₁ enzyme production and two species had high C_x enzyme production. However, two and three species were moderately active in producing C₁ and C_x enzymes, respectively. The remaining species had low production of both C₁ and C_x enzymes. The highest cellulase activity was recorded by Alternaria citri and Cochliobolus spicifer for C₁ and by A alternata and A. citri for C_x enzyme. Maximum production of C₁ enzyme by A. citri and C. spicifer was obtained after six days of incubation at 30 °C with an initial pH of 6 in a culture medium containing lactose and calcium nitrate as the carbon and nitrogen sources, respectively. The highest C_x enzyme production was by A. alternata and A. citri,which was recorded after eight days of incubation at 30 °C with an initial pH of 6 in a culture medium containing carboxymethyl cellulose and sodium or calcium nitrate as the carbon and nitrogen sources, respectively.     

Resilience of rice (Oryza spp.) pollen germination and tube growth to temperature stress

Resilience of rice cropping systems to potential global climate change will partly depend on the temperature tolerance of pollen germination (PG) and tube growth (PTG). Pollen germination of high temperature‐susceptible Oryza glaberrima Steud. (cv. CG14) and Oryza sativa L. ssp. indica (cv. IR64) and high temperature‐tolerant O. sativa ssp. aus (cv. N22), was assessed on a 5.6–45.4 °C temperature gradient system. Mean maximum PG was 85% at 27 °C with 1488 μm PTG at 25 °C. The hypothesis that in each pollen grain, the minimum temperature requirements (T_n) and maximum temperature limits (T_x) for germination operate independently was accepted by comparing multiplicative and subtractive probability models. The maximum temperature limit for PG in 50% of grains (T_x(50)) was the lowest (29.8 °C) in IR64 compared with CG14 (34.3 °C) and N22 (35.6 °C). Standard deviation (s_x) of T_x was also low in IR64 (2.3 °C) suggesting that the mechanism of IR64's susceptibility to high temperatures may relate to PG. Optimum germination temperatures and thermal times for 1 mm PTG were not linked to tolerating high temperatures at anthesis. However, the parameters T_x(50) and s_x in the germination model define new pragmatic criteria for successful and resilient PG, preferable to the more traditional cardinal (maximum and minimum) temperatures.     

Purification and Characterization of a Polygalacturonase-Inhibiting Protein from Phaseolus vulgaris L

Homogeneous endo-polygalacturonase (PG) was covalently bound to cyanogen-bromide-activated Sepharose, and the resulting PG-Sepharose conjugate was utilized to purify, by affinity chromatography, a protein from Phaseolus vulgaris hypocotyls that binds to and inhibits PG. Isoelectric focusing of the purified PG-inhibiting protein (PGIP) showed a major protein band that coincided with PG-inhibiting activity. PGIP formed a complex with PG at pH 5.0 and at low salt concentrations. The complex dissociated in 0.5 m Na-acetate and pH values lower than 4.5 or higher than 6.0. Formation of the PG-PGIP complex resulted in complete inhibition of PG activity. PG activity was restored upon dissociation of the complex. The protein exhibited inhibitory activity toward PGs from Colletotrichum lindemuthianum, Fusarium moniliforme and Aspergillus niger. The possible role of PGIP in regulating the activity of fungal PG's and their ability to elicit plant defense reactions are discussed.     

Statistical optimization of solid state fermentation conditions for the enhanced production of thermoactive chitinases by mesophilic soil fungi using response surface methodology and their application in the reclamation of shrimp processing by-products

The mesophilic strains Aspergillus flavus CFR 10 and Fusarium oxysporum CFR 8 are potent producers of extracellular thermoactive chitinases (endo-chitinase and β-N-acetylhexosaminidase). Chitinases have a wide range of applications in many areas including reclamation of seafood processing chitinous by-products. In the present study, the interactive effects of four fermentation conditions on thermoactive chitinase production by solid state fermentation (SSF) using commercial wheat bran (CWB) was investigated employing response surface methodology (RSM). Further, these chitinases were applied for the preparation of N-acetyl chitooligosaccharides from shrimp chitin. Statistical optimization resulted in the production (unit/g initial dry substrate, U/g IDS) of 19.8 endo-chitinase and 649.0 β-N-acetylhexosaminidase activity by A. flavus CFR 10, and 17.5 endo-chitinase and 319.9 β-N-acetylhexosaminidase activity by F. oxysporum CFR 8. Activity of crude endo-chitinase and β-N-acetylhexosaminidase were found to be optimum at 62?±?1 °C in a wide pH range. Hydrolysis of colloidal chitin with crude chitinases produced the maximum N-acetyl chitooligosaccharides yield (mmol/l) of 10.4?±?0.28 at 6 h and 10.2?±?0.01 at 30 h post-reaction initiation, respectively, by the enzymes of A. flavus CFR 10 and F. oxysporum CFR 8. HPLC analysis revealed the presence of N-acetyl chitooligosaccharides with N-acetyl chitotriose as the main end product of the colloidal chitin hydrolysis. These results indicate the potential of mesophilic A. flavus CFR 10 and F. oxysporum CFR 8 in the production of thermoactive chitinases employing the economical SSF process using CWB as an ideal substrate, as well as the potential of these chitinases for the reclamation of abundant shrimp processing by-products and production of defined N-acetyl chitooligosaccharides.     

Preparation,characterization and laboratory-scale application of an immobilized glucoamylase

Glucoamylase (1,4-α-d-glucan glucohydrolase, EC 3.2.1.3) has been covalently immobilized on a polyacrylamide-type support containing carboxylic groups activated by water-soluble carbodiimide. The activity was 5.5– 6.0 units g^?1solid. The optimum pH for catalytic activity was pH 3.8. The apparent optimum temperature was found at 60°C. With soluble starch as substrate the K_m value was 14 mg ml^?1. The pH for maximum stability was pH 4.0–4.5. In the presence of 8 m urea the immobilized glucoamylase retained most of its catalytic activity but it was more susceptible to guanidinium hydrochloride than the soluble enzyme. The practical applicability of immobilized glucoamylase was tested in batch process and continuous operation.     

Kinetic characterization of a crude β-d-glucosidase from Aspergillus wentii Pt 2804

The kinetic characteristics of β-d-glucosidase (cellobiase, β-d-glucosidase glucohydrolase, EC 3.2.1.21) from the filtered broth of a well grown culture of Aspergillus wentii have been studied. Both cellobiose and 4-nitrophenyl-β-d-glucoside (4NPG) were used as substrates and values of K_m, V_max for both the substrates were determined. Activity was maximum over a pH range of 4.5–5.5 but declined sharply beyond 5.5 for both substrates. The optimum temperature was between 60 and 65°C. Half-life of the cellobiase was ～38.0 h at 60°C and ～6.3 h at 65°C. However, the enzyme was found to be quite stable at 50°C. The activation and deactivation energies for 4NPG hydrolysis were 33.2 and 111.3 kJ mol^?1 K^?1, and 43.6 and 63.7 kJ mol K^?1 for cellobiose hydrolysis. Product inhibition was found to be of the competitive type. Preliminary experiments showed that marked synergistic activity exists between Trichoderma reesei and A. wentii cellulases [see 1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] for cellulose hydrolysis.     

Characterization and expression of glucosamine-6-phosphate synthase from Saccharomyces cerevisiae in Pichia pastoris

Glucosamine-6-phosphate (GlcN-6-P) synthase from Saccharomyces cerevisiae was expressed in Pichia pastoris SMD1168 GIVING maximum activity of 96 U ml^?1 for the enzyme in the culture medium. By SDS-PAGE, the enzyme, a glycosylated protein, had an apparent molecular mass of 90 kDa. The enzyme was purified by gel exclusion chromatography to near homogeneity, with a 90 % yield and its properties were characterized. Optimal activities were at pH 5.5 and 55 °C, respectively, at which the highest specific activity was 6.8 U mg protein ^?1. The enzyme was stable from pH 4.5 to 5.5 and from 45 to 60 °C. The K_m and V_max of the GlcN-6-P synthase towards d-fructose 6-phosphate were 2.8 mM and 6.9 μmol min^?1 mg^?1, respectively.     

Incidence of Fusarium spp. and Levels of Fumonisin B1 in Maize in Western Kenya

Maize kernel samples were collected in 1996 from smallholder farm storages in the districts of Bomet, Bungoma, Kakamega, Kericho, Kisii, Nandi, Siaya, Trans Nzoia, and Vihiga in the tropical highlands of western Kenya. Two-thirds of the samples were good-quality maize, and one-third were poor-quality maize with a high incidence of visibly diseased kernels. One hundred fifty-three maize samples were assessed for Fusarium infection by culturing kernels on a selective medium. The isolates obtained were identified to the species level based on morphology and on formation of the sexual stage in Gibberella fujikuroi mating population tests. Fusarium moniliforme (G. fujikuroi mating population A) was isolated most frequently, but F. subglutinans (G. fujikuroi mating population E), F. graminearum, F. oxysporum, F. solani, and other Fusarium species were also isolated. The high incidence of kernel infection with the fumonisin-producing species F. moniliforme indicated a potential for fumonisin contamination of Kenyan maize. However, analysis of 197 maize kernel samples by high-performance liquid chromatography found little fumonisin B₁ in most of the samples. Forty-seven percent of the samples contained fumonisin B₁ at levels above the detection limit (100 ng/g), but only 5% were above 1,000 ng/g, a proposed level of concern for human consumption. The four most-contaminated samples, with fumonisin B₁ levels ranging from 3,600 to 11,600 ng/g, were from poor-quality maize collected in the Kisii district. Many samples with a high incidence of visibly diseased kernels contained little or no fumonisin B₁, despite the presence of F. moniliforme. This result may be attributable to the inability of F. moniliforme isolates present in Kenyan maize to produce fumonisins, to the presence of other ear rot fungi, and/or to environmental conditions unfavorable for fumonisin production.