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     

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.     

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.     

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.     

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.     

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.     

Purification and properties of a cellobiohydrolase from Penicillium pinophilum

Cellobiohydrolase II, isolated from the extracellular cellulase system of Penicillium pinophilum by chromatography on DEAE-Sephadex and DEAE-Sepharose followed by chromatofocusing, gave a single homogeneous band in SDS-gel electrophoresis and gel electrophoresis and gel electrofocusing. It had a molecular weight of 50,700 and of pI of 5.0, and was associated with 19% of carbohydrate. Cellobiose was the sole product of hydrolysis of the cellulosic materials, Avicel and H₃PO₄-swollen cellulose. No cross reaction was observed with antiserum prepared with another purified cellobiohydrolase (I) isolated from the same cellulase system. Cellobiohydrolase II showed no capacity for producing short fibres from filter paper. Avicel was hydrolysed extensively, but little or no hydrolysis of cotton fibre was apparent. However, cotton fibre was hydrolysed with a reconstituted mixture of the purified cellobiohydrolase II and the four major endo-(1→4)-β-d-glucanases isolated during fractionation. The action of cellobiohydrolase II on H₃PO₄-swollen cellulose was stimulated by high concentrations of cellobiose, but inhibited by high concentrations of d-glucose. Other notable inhibitors were Mn²⁺ and carbodi-imide. The properties of cellobiohydrolase II and the immunologically unrelated cellobiohydrolase I are compared.     

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.     

Immobilization of Penicillium funiculosum cellulase on a soluble polymer

The three cellulase [see 1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] components of Penicillium funiculosum have been immobilized on a soluble, high molecular weight polymer, poly(vinyl alcohol), using carbodiimide. The immobilized enzyme retained over 90% of cellulase [1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4], and exo-β-d-glucanase [1,4-β-d-glucan cellobiohydrolase, EC 3.2.1.91] and β-d-glucosidase [β-d-glucoside glucohydrolase, EC 3.2.1.21] activities. The bound enzyme catalysed the hydrolysis of alkali-treated bagasse with a greater efficiency than the free cellulase. The potential for reuse of the immobilized system was studied using membrane filters and the system was found to be active for three cycles.     

A Highly Regioselective Esterase from Penicillium Frequentans IMI 92265

An esterase isolated from Penicillium frequentans IMI 92265 selectively hydrolyses the acetoxyethyl ester in preference to the acetoxymethyl esters in the triacetate substrate 1,4-diacetoxy-2-acetoxymethylbutane (1) which was converted to 4-acetoxy-3-acetoxymethylbutan-l-ol (2) in good yield. The enzyme gave no detectable hydrolysis of l,3-diacetoxy-2-acetoxymethylpropane (4). When immobilised to a cyanogen bromide activated Sepharose resin the enzyme was highly stable and showed no loss of regioselectivity in the hydrolysis of (1). A method is described for the elective isolation of microorganisms which have the ability to hydrolyse (1).     

Quartz sand as an adsorbent for purification of extracellular glucose oxidase from Penicillium funiculosum 46.1

The procedure of purification of extracellular glucose oxidase (GO, EC 1.1.3.4) from culture-liquid filtrate (CLF) of the fungus Penicillum funiculosum 46.1 using alluvial quartz sand as an adsorbent has been developed. The modification of sand by changing the charge and polarity did not lead to a significant increase in its adsorption capacity towards GO. The effectiveness of sand and aluminum oxide, used as sorbents for isolation of GO from CLF, was compared. Glucose oxidase, isolated from CLF by adsorption on sand, exhibited a greater catalytic activity compared to the enzyme specimens obtained by column chromatography on CLF. Sand adsorbed GO from P. funiculosum 46.1 more effectively than aluminum oxide. It is concluded that sand may be used for fractionation of partly purified GO.     

Quartz Sand as an Adsorbent for Purification of Extracellular Glucose Oxidase from Penicillium funiculosum 46.1

A procedure for purification of extracellular glucose oxidase (GO, EC 1.1.3.4) from a filtrate of culture liquid (CLF) of the fungus Penicillium funiculosum 46.1 has been developed using alluvial quartz sand as an adsorbent. Modifying the sand by changing the charge and polarity did not lead to a significant increase in its adsorption capacity towards GO. The effectiveness of sand and aluminum oxide used as adsorbents for GO isolation from CLF has been compared. Glucose oxidase isolated from CLF by adsorption on sand exhibited a greater catalytic activity than enzyme preparations obtained by column chromatography on CLF. Glucose oxidase from P. funiculosum 46.1 was adsorbed on sand more effectively than on aluminum oxide. It is concluded that sand may be used for fractionation of partially purified GO.     

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.     

Laminarinase from Penicillium funiculosum and its role in release of beta-glucosidase

An extracellular laminarinase (1----3)-beta-glucan glucohydrolase (EC 3.2.1.6) was purified from culture filtrates of Penicillium funiculosum. It was homogeneous on polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate. It had a Mr of 14,000 and isoelectric point of pH 4.2. The apparent Km value for lamimarinase was 8.3 mg/ml and Vmax was 8 mumol/min/mg. The distribution of beta-glucosidase activity in two different species of Penicillium showed that P. funiculosum had a higher ratio of extracellular to cell wall bound activity than Penicillium janthinellum. Treatment of mycelia of both species with NaCl, EDTA, Triton X-100, or proteolytic enzymes did not release the cell wall bound beta-glucosidase. Incubation of the mycelia with the laminarinase released 2-4 times more beta-glucosidase than the estimated cell bound activity in P. janthinellum and P. funiculosum.     

A bacterial GH6 cellobiohydrolase with a novel modular structure

Cel6D from Paenibacillus barcinonensis is a modular cellobiohydrolase with a novel molecular architecture among glycosyl hydrolases of family 6. It contains an N-terminal catalytic domain (family 6 of glycosyl hydrolases (GH6)), followed by a fibronectin III-like domain repeat (Fn3_1,2) and a C-terminal family 3b cellulose-binding domain (CBM3b). The enzyme has been identified and purified showing catalytic activity on cellulosic substrates and cellodextrins, with a marked preference for phosphoric acid swollen cellulose (PASC). Analysis of mode of action of Cel6D shows that it releases cellobiose as the only hydrolysis product from cellulose. Kinetic parameters were determined on PASC showing a K _m of 68.73 mg/ml and a V _max of 1.73 U/mg. A series of truncated derivatives of Cel6D have been constructed and characterized. Deletion of CBM3b caused a notable reduction in hydrolytic activity, while deletion of the Fn3 domain abolished activity, as the isolated GH6 domain was not active on any of the substrates tested. Mutant enzymes Cel6D-D146A and Cel6D-D97A were constructed in the residues corresponding to the putative acid catalyst and to the network for the nucleophilic attack. The lack of activity of the mutant enzymes indicates the important role of these residues in catalysis. Analysis of cooperative activity of Cel6D with cellulases from the same producing P. barcinonensis strain reveals high synergistic activity with processive endoglucanase Cel9B on hydrolysis of crystalline substrates. The characterized cellobiohydrolase can be a good contribution for depolymerization of cellulosic substrates and for the deconstruction of native cellulose.

     

Properties and applications of Penicillium funiculosum cellulase immobilized on a soluble polymer

Summary The cellobiase and xylanase activities of Penicillium funiculosum were immobilized on a soluble polymer poly(vinyl alcohol) (PVA). The kinetic parameters and the adsorption characteristics of the bound and free enzymes were compared. The Km value of the immobilized preparation was the same as the free enzyme. The hydrolysis of different cellulosic substrates by the bound enzyme is investigated.     

Stabilization of dextranase from Penicillium funiculosum and Fusarium solani during heating and freeze-drying

Freeze-drying of highly purified dextranse from Penicillium funiculosum and Fusarium solani was accompanied by 90% losses of enzyme activity and solubility. Many carbohydrates were tested as stabilizers, e.g. glucose, maltose, lactose, polyglucine, dextranase hydrolyzate of polyglucine as well as mannitol and ammonium sulfate. Polyglucine, its hydrolyzate, and glucose proved most effective stabilizers. The stabilizing effect of polyglucine hydrolyzate of dextranase during its heating and freeze-drying was compared. The effective concentration of the stabilizer during freeze-drying was 10 times lower than during heating.