Dextranase production from Penicillium funiculosum

Twenty-eight Penicillium cultures were screened for dextranase activity. Dextranase yield of about 2000 DU/ml was obtained with Penicillium funiculosum SH-5. Maximum dextranase concentration was attained only when cell mass decreased. The kinetics of the dextranase production was correlated with the cell mass by a two-parameter model. The optimum pH and temperature for dextranase were 5.0-5.5 and 55°C, respectively. Crude dextranase preparation was inhibitory to insoluble glucan formation by streptococcus mutans 6715 in vitro.     

Glucohydrolase from Penicillium funiculosum

Summary A 1,4--d-glucan glucohydrolase (EC 3.2.1.74) was isolated from culture filtrates of Penicillum funiculosum and purified by isoelectric focussing. The purified enzyme was homogeneous as indicated by electrophoresis on sodium dodecyl sulphate-polyacrylamide gels. The enzyme had a molecular weight of 20 000 and the pI was 4.45. The hydrolysis of Avicel by the purified enzyme and culture broth using equal amounts of Walseth units were comparable. The glucohydrolase did not act in synergism with endoglucanase or cellobiohydrolase from the same culture. The enzyme had little ability to attack carboxymethyl cellulose. It showed activity towards Avicel, Walseth cellulose and cellooligosaccharides (G₃-G₅), producing glucose as the end product, indicating that the enzyme is a -1–4 glucan glucohydrolase. The enzyme exhibited transglucosidase activity, producing higher oligosaccharides from cellobiose.NCL Communication no. 3899     

Effect of pretreatment on the hydrolysis of cellulose by Penicillium funiculosum cellulase and recovery of enzyme

Penicillium funiculosum produces a complete cellulase which brings about 97% hydrolysis of cotton and has high beta-glucosidase, xylanase, laminarinase, and lichenase activities. This article deals with the effect of different pretreatments on the hydrolysis of sugarcane bagasse by P. funiculosum enzymes and the recovery of enzyme from the insoluble residues. Enzymic saccharification of bagasse pretreated with hot 1N NaOH followed by neutralization with HCI and steam treated under pressure (7 kg/cm(2)) gave 63 and 59% saccharification, respectively, in 48 h. Hemicellulose is not lost in these pretreatments. With a 30% slurry of steam-treated bagasse, a semisolid mass containing 14% sugar was obtained. A 90% recovery of CMCase, beta-glucosidase, and filter paper activity from the hydrolysates was obtained under the following conditions: (1) maintaining the ratio of enzyme to substrate high by stepwise addition of substrate, (2) brief grinding of the residual substrate with glass powder, and (3) addition of 0.4% Tween-80 to the eluting buffer. The high recovery of cellulolytic enzymes indicates that the adsorption of these enzymes on cellulose is not irreversible.     

Hydrolysis of cellulose materials during successive treatment with cellulase from Penicillium funiculosum

Cellulase from Penicillium funiculosum exhibited different hydrolysis tendencies when acting on cellulose materials. Successive addition of fresh cellulase to enzymatic pre-treated substrates showed foolscap paper to be the most susceptible for enzymatic hydrolysis followed by filter paper, newsprint and microcrystalline cellulose.     

Thermostability of cellulase from Penicillium funiculosum

When cellulase from Penicillium funiculosum was held at between 25°C and 60°C prior to incubation with CM-cellulose and filter paper as cellulosic substrates, it then had a higher thermostability towards soluble CM-cellulose than insoluble filter paper.     

Strain selection criteria for Penicillium funiculosum in enzymic hydrolysis of lignocellulosics

Summary The mutants ofPenicillium funiculosum viz. N-4, BU-36 and Cu-1 producing different proportions of cellulase components have been isolated. Results of saccharification experiments using various lignocellulose substrates with culture filtrates of these mutants suggested criteria for their selection.     

Cellulases from two Penicillium sp. strains isolated from subtropical forest soil: production and characterization

AIMS: To isolate new fungal strains from subtropical soils and to identify those that produce high cellulase activity. To select microbial strains producing thermostable cellulases with potential application in industry. METHODS AND RESULTS: The new strains Penicillium sp. CR-316 and Penicillium sp. CR-313 have been identified and selected because they secreted a high level of cellulase in media supplemented with rice straw. Analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis, isoelectric focussing and zymography showed that the studied strains secreted multiple enzymes that hydrolyse cellulose. Cellulase activity of Penicillium sp. CR-316, the strain showing higher production, was analysed. Optimum temperature and pH of carboxymethyl cellulase activity were 65 degrees C and pH 4.5, respectively. Activity remained stable after incubation at 60 degrees C and pH 4.5 for 3 h. CONCLUSIONS: Fungal strains that secrete high levels of cellulase activity have been characterized and selected from soil. The isolated strains have complex sets of enzymes for cellulose degradation. Crude cellulase produced by Penicillium sp. CR-316 showed activity and stability at high temperature. SIGNIFICANCE AND IMPACT OF THE STUDY: Two fungal strains with biotechnological potential have been isolated. The strains secrete high levels of cellulase, and one of them, Penicillium sp. CR-316, produces a thermostable cellulase, that makes it a good candidate for industrial applications.     

Intracellular glucose oxidase from Penicillium funiculosum 46.1

A method for isolating extracellular glucose oxidase from the fungus Penicillium funiculosum 46.1, using ultrafiltration membranes, was developed. Two samples of the enzyme with a specific activity of 914-956 IU were obtained. The enzyme exhibited a high catalytic activity at pH above 6.0. The effective rate constant of glucose oxidase inactivation at pH 2.6 and 16 degrees C was 2.74 x 10(-6) s-1. This constant decreased significantly as pH of the medium increased (4.0-10.0). The temperature optimum for glucose oxidase-catalyzed beta-D-glucose oxidation was in the range 30-65 degrees C. At temperatures below 30 degrees C, the activation energy for beta-D-glucose oxidation was 6.42 kcal/mol; at higher temperatures, this parameter was equal to 0.61 kcal/mol. Kinetic parameters of glucose oxidase-catalyzed delta-D-glucose oxidation depended on the initial concentration of the enzyme in the solution. Glucose oxidase also catalyzed the oxidation of 2-deoxy-D-glucose, maltose, and galactose.     

Growth characteristics and glucose oxidase production by mutant Penicillium funiculosum strains

The main parameters of growth and glucose oxidase production by the mutant Penicillium funiculosum strains BIM F-15.3, NMM95.132, and 46.1 were studied. The synthesis of extracellular glucose oxidase by these strains was constitutive and occurred following the phase of exponential growth. The mutant strains also synthesized extracellular invertase and cell-associated catalase and glucose oxidase. The syntheses of invertase, the cell-associated enzymes, and extracellular glucose oxidase were found to be maximum between 14 and 18 h, between 48 and 52 h, and by the 96th h of cultivation, respectively. Among the mutants studied, P. funiculosum 46.1 showed the maximal rates of growth and glucose oxidase synthesis.     

Isolation of a Pure Dextranase from Penicillium funiculosum

A dextranase, produced by Penicillium funiculosum, was purified 1,000-fold to yield the enzyme which was demonstrated by gel electrophoresis and electrofocusing to be a homogeneous protein. The purification method included acetone partition, ammonium sulfate fractionation, gel filtration, iron defecation and precipitation, and diethylaminoethyl-cellulose chromatography. The pure enzyme was also obtained by preparative gel electrophoresis. Gel-permeation chromatography indicates a molecular weight of 41,000. An isoelectric pH of 4.6 was established by electrofocusing. A 1-mg amount of the enzyme hydrolyzes a dextran substrate to yield 27,000 isomaltose reducing units in 2 hr.     

Preparation and some properties of immobilized Penicillium funiculosum 258 dextranase

The production of dextranase was investigated in static cultures of Penicillium funiculosum 258. Maximal enzyme productivity was attained at pH 8.0, with 3.5% (w/v) dextran (MW, 260 000) as carbon source, NaNO₃ (1%, w/v) and yeast extract (0.2%, w/v) as nitrogen source, 0.4% (w/v) K₂HPO₄ and 0.06% (w/v) MgSO₄. It was possible to increase the productivity of dextranase to 41.8 units ml⁻¹ in the modified medium. The enzyme was immobilized on different carriers by different techniques of immobilization. The enzyme prepared by covalent binding on chitosan using glutaraldehyde had the highest activity, the immobilized enzyme retaining 63% of its original specific activity. Compared with the free dextranase, the immobilized enzyme exhibited: a higher pH optimum, a higher optimal reaction temperature and energy of activation, a higher Michaelis constant, improved thermal stability and higher values of deactivation rate constant. The immobilized enzyme retained about 80% of the initial catalytic activity even after being used for 12 cycles.     

Production and properties of alpha-amylase from Penicillium chrysogenum and its application in starch hydrolysis

Fungi were screened for their ability to produce alpha-amylase by a plate culture method. Penicillium chrysogenum showed high enzymatic activity. Alpha-amylase production by P. chrysogenum cultivated in liquid media containing maltose (2%) reached its maximum at 6-8 days, at 30 degrees C, with a level of 155 U ml(-1). Some general properties of the enzyme were investigated. The optimum reaction pH and temperature were 5.0 and 30-40 degrees C, respectively. The enzyme was stable at a pH range from 5.0-6.0 and at 30 degrees C for 20 min and the enzyme's 92.1% activity's was retained at 40 degrees C for 20 min without substrate. Hydrolysis products of the enzyme were maltose, unidefined oligosaccharides, and a trace amount of glucose. Alpha-amylase of P. chrysogenum hydrolysed starches from different sources. The best hydrolysis was determined (98.69%) in soluble starch for 15 minute at 30 degrees C.     

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.     

Comparative production of cellulases by mutants of Penicillium janthinellum NCIM 1171 and its application in hydrolysis of Avicel and cellulose

Mutants of Penicillium janthinellum NCIM 1171 were evaluated for cellulase production using both submerged fermentation (SmF) and solid state fermentation (SSF). Mutant EU2D-21 gave highest yields of cellulases in both SmF and SSF. Hydrolysis of Avicel and cellulose were compared using SmF and SSF derived enzyme preparations obtained from EU2D-21. Surprisingly, the use of SSF derived preparation gave less hydrolysis compared to SmF derived enzymes. This may be due to inactivation of β-glucosidase at 50 °C in SSF derived enzyme preparations. SmF derived enzyme preparations contained both thermostable and thermosensitive β-glucosidases where as SSF derived enzyme preparations contained predominantly thermosensitive β-glucosidase. This is the first report on less thermostability of SSF derived β-glucosidase which is the main reason for getting less hydrolysis.     

Saccharification of wastepaper mixtures with cellulase from Penicillium funiculosum

Different used paper materials and mixtures thereof were saccharified with Penicillium funiculosum cellulase. Non-similar biodegradation patterns were concluded to be operating as well as declining bioconversion efficiencies with increasing biodegradation. Biowaste mixtures were less effectively biodegraded indicating the importance of separating biowaste into distinctive materials prior to developing it as a resource of bioproduct synthesis.     

The isolation, purification and properties of the cellobiohydrolase component of Penicillium funiculosum cellulase.

1. A cellobiohydrolase component was isolated from a Penicillium funiculosum cellulase preparation by chromatography on DEAE-Sephadex, and purified by isoelectric focusing. 2. Purified in this way, the enzyme was homogeneous as judged by electrophoresis on sodium dodecyl sulphate/polyacrylamide gels and isoelectric focusing in polyacrylamide gels. 3. Acting in isolation, the enzyme had little hydrolytic activity to highly ordered celluloses such as cotton fibre, but, when recombined in the original proportions with the other components [endo-(1 leads to 4)-beta-D-glucanase and beta-D-glucosidase] of the complex, 98% of the original activity was recovered. 4. Synergistic effects were also observed when the enzyme was acting in concert with endo-(1 leads to 4)-beta-D-glucanase from other fungal sources. 5. Less-well-ordered celluloses, such as that swollen in H3PO4, were extensively hydrolysed, the principal product being cellobiose. 6. Attack on carboxymethyl-cellulose (CM-cellulose), which is the substrate normally used to assay for endo-(1 leads to 4)-beta-D-glucanase activity, was minimal. 7. The enzyme was associated with 9% of neutral sugar, 88% of which was mannose. It was isoelectric at pH 4.36 (4 degrees C) and had a mol.wt. of 46 300 (determined by gel chromatography on a calibrated column of Ultrogel). 8. The enzyme was specific for the beta-(1 leads to 4)-linkage.