Production of xylanolytic enzymes in association with the cellulolytic activities of Penicillium funiculosum

Penicillium funiculosum produced 16 and 0.4 units ml^?1 of d-xylanase (1,4-β-d-xylan xylanohydrolase, EC 3.2.1.8) and β-d-xylosidase (1,4-β-d-xylan xylohydrolase, EC 3.2.1.37), respectively, in shake flasks. Both enzymes were 100% stable when heated at 50°C for 30 min and on prolonged heating d-xylanase and β-d-xylosidase showed 46 and 20% loss, respectively. Maximum hydrolysis (75%) of d-xylan was obtained when the end products were removed. The addition of β-d-xylosidase markedly influenced the degree of hydrolysis of d-xylan. End-product analysis of the d-xylan hydrolysate showed the presence of d-xylose, d-xylobiose, d-xylotriose, d-xylotetraose, d-xylopentose and l-arabinose. The fractionation of culture filtrate of Penicillium funiculosum grown on cellulose powder or in a combination of cellulose powder and wheat bran indicated the presence of two d-xylanases. The role of cellulase [see 1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] and d-xylanase on the overall hydrolysis of pure cellulose and lignocellulosic substrates is discussed.     

Occurrence of a procellulase in the culture filtrates of Penicillium janthinellum

The endo-1,4-β-d-glucanase [cellulase, 1,4-(1,3:1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] activity of two-day old culture filtrates of Penicillium janthinellum has been enhanced four-fold by incubating with a 10-day old culture filtrate of Penicillium funiculosum grown on the same medium. An inactive protein isolated by fractionation of two-day old culture filtrate of P. janthinellum using preparative isoelectric focusing, showed 30- to 50-fold enhancement of endo-1,4-β-d-glucanase activity. This fraction has been designated the ‘procellulase’ in the present paper. The purity of the procellulase was confirmed by analytical isoelectric focusing and sodium dodecyl sulphate-polyacrylamide gel electrophoresis. It had a molecular weight of 68 000 and an isoelectric point of pH 3.7.     

Optimization of the composition of the nutrient medium for cellulase and protein biosynthesis by thermophilic Aspergillus fumigatus NRC 272

An active strain of Aspergillus spp. has been selected for the production of cellulolytic enzymes and proteins when grown on peracetic acid-treated wheat straw. This strain produced a considerable amount of cellulase [see 1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] in the extracellular supernatant and exhibited good overall cellulolytic activity, as measured using filter paper and Avicel as substrates. Also, under the same conditions the strain showed high activities of β-d-glucosidase (β-d-glucoside glucohydrolase, EC 3.2.1.21) and β-d-xylosidase (1,4-β-d-xylan xylohydrolase, EC 3.2.1.37). The maximum enzyme yields (carboxymethylcellulose activity 26.4 units ml^?1, filter paper activity 2.26 units ml^?1 and Avicel activity 16.82 units ml^?1; β-d-glucosidase 9.09 units ml^?1 and β-d-xylosidase 1.92 units ml^?1) were obtained after 96 h incubation at 45°C.     

Immobilization of cellulase and d-xylanase complexes from Aspergillus terreus F-413 on controlled porosity glasses

The major components of cellulase [see 1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] and d-xylanase (see 1,4-β-d-xylan xylanohydrolase, EC 3.2.1.8) complexes have been immobilized on glass beads activated by 3-aminopropyltriethoxysilane or 3-glycidoxypropyltrimethoxysilane. The final preparations contained over 20 mg protein g^?1 glass beads. The activity retained was 71.6–98.1% for cellulase complexes and 81–100% for d-xylanase complexes. The immobilization of the enzymes spread their optimum pH range. Cellulose and d-xylan were quantitatively hydrolysed by the immobilized enzymes. The major reaction products were identified as a d-glucose and d-xylose respectively.     

Effect of attached carbohydrates on the activity of Trichoderma viride cellulases

Trichoderma viride 1,4-β-d-glucan cellobiohydrolase (exo-cellobiohydrolase, 1,4-β-d-glucan cellobiohydrolase, EC 3.2.1.91) purified from a commercial product to electrophoretic homogeneity by a procedure including affinity and DEAE-Sephadex chromatography, has attached carbohydrates in addition to the glycoprotein constituents. These carbohydrates are lost by consecutive gel filtration steps in Sephadex G-25 columns, whereupon there is a rapid increase in enzymatic activity. A single gel filtration step can eliminate d-glucose or cellobiose added to a solution of this enzyme, but not the carbohydrates attached during incubation with Avicel.After free carbohydrate elimination from crude cellulase complexes by Sephadex G-25 chromatography, liberation of d-glucose following incubation at 50°C and pH 4.8 was observed. This indicates that some carbohydrates remain bound after gel filtration. The elimination of carbohydrate from whole cellulase complex [see 1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] was favoured by a yeast treatment, with a simultaneous increase in activity, but the process is not reproducible, as a secondary inactivation process exists.     

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.     

Purification and properties of cellulase enzymes from Robillarda sp. Y-20

One endo-β-1,4-glucanase (EC 3.2.1.4) and two unique β-glucosidases (EC 3.2.1.21) have been isolated from culture filtrates Robillarda sp. Y-20 by combinations of DEAE A-50 column chromatography and isoelectric focusing. These enzymes were homogeneous on gel filtration, isoelectric focusing and polyacrylamide gel electrophoresis with and without sodium dodecyl sulphate (SDS). The molecular weights of endoglucanase, and the two β-glucosidases, I and II by SDS-polyacrylamide gel electrophoresis were 59000, 76000 and 54000, respectively. The pI values were 3.5, 7.5, and 3.8 for endoglucanase, β-glucosidase I and II, respectively. The major β-glucosidase I was a glycoprotein, but the endoglucanase and β-glucosidase II were not. The endoglucanase rapidly reduced the viscosity of carboxymethyl (CM) cellulose with concomitant production of reducing sugar. The enzyme had very low activity with crystalline cellulose such as insoluble acid treated cellulose, Avicel and filter paper. The endoglucanase attacked celloheptaose to cellotetraose more readily than cellotriose, but did not hydrolyze cellobiose. Both β-glucosidases attacked celloheptaose to cellotetraose more readily than cellotriose and cellobiose, but did not hydrolyze CM-cellulose and insoluble acid treated cellulose. Strong synergism was observed for hydrolysis of CM-cellulose by the endoglucanase and β-glucosidases.     

The cellulase of Penicillium pinophilum. Synergism between enzyme components in solubilizing cellulose with special reference to the involvement of two immunologically distinct cellobiohydrolases.

Two immunologically unrelated cellobiohydrolases (I and II), isolated from the extracellular cellulase system elaborated by the fungus Penicillum pinophilum, acted in synergism to solubilize the microcrystalline cellulose Avicel; the ratio of the two enzymes for maximum rate of attack was approx. 1:1. A hypothesis to explain the phenomenon of synergism between two endwise-acting cellobiohydrolases is presented. It is suggested that the cellobiohydrolases may be two stereospecific enzymes concerned with the hydrolysis of the two different configurations of non-reducing end groups that would exist in cellulose. Only one type of cellobiohydrolase has been isolated so far from the cellulases of the fungi Fusarium solani and Trichoderma koningii. Only cellobiohydrolase II of P. pinophilum acted synergistically with the cellobiohydrolase of the fungi T. koningii or F. solani to solubilize Avicel. Cellobiohydrolase II showed no capacity for co-operating with the endo-1,4-beta-glucanase of T. koningii or F. solani to solubilize crystalline cellulose, but cellobiohydrolase I did. These results are discussed in the context of the hypothesis presented.     

Purification and characterization of two d-xylanases from Trichoderma harzianum

Two endo-1,4-β-d-xylanases (1,4-β-d-xylan xylanohydrolase, EC 3.2.1.8) from Trichoderma harzianum E58 have been purified by ultrafiltration and chromatography on carboxymethyl-Sepharose, phenyl-Sepharose and Sephadex G-75. The d-xylanases were shown to be homogeneous by the criteria of dodecyl sulphate polyacrylamide gel electrophoresis and isoelectric focusing. The molecular weights were estimated to be 20 000 and 29 000, with pl values of 9.4 and 9.5, respectively. Typically, 456 mg of the 20 000 dalton and 1.9 mg of the 29 000 dalton d-xylanases were purified from 4.2 litre of culture filtrate with specific activities of 370 and 75 U mg^?1, respectively. Optimum d-xylanase activities were obtained when the enzymes were incubated at pH 5, 50°C, for the 20 000 dalton protein and pH 5, 60°C for the 29 000 dalton protein.     

A rapid and simplified procedure for purification of a cellulase from Fusarium lini

An endo-beta-1,4-glucanase (EC 3.2.1.4) was obtained in high yields in purified form a culture filtrate of Fusarium lini by an extremely simple method. The method consists of precipitation of the culture filtrate with ammonium sulphate (290 g/L), followed by chromatography of the precipitated fraction on Biogel P-150. The purification is based on the unusual property of the enzyme being eluted after cytochrome C, even though it molecular weight is 2.8 x 10(4) (by SDS PAGE). The yield of pure enzyme was 6.8 mg/L culture broth. The homogeneity of the enzyme was established by ultracentrifugation, isoelectric focusing, and electrophoresis in polyacrylamide gels containing SDS. The enzyme was isoelectric at pH 8.3 and contained 2.9% carbohydrate. The K(m) value for carboxymethyl (CM) cellulose was 11.6 mg/mL. The enzyme showed high viscosity reducing activity towards CM cellulose but very low activity with Walseth cellulose and crystalline celluloses such as Avicel and cotton. The purified enzyme has activity towards xylan. The amino acid analysis showed a predominance of acidic and neutral amino acids and low contents of histidine, arginine, and methionine. One-half of the cysteine content was 11 residues/mol enzyme, and no free-SH group was detectable.     

Functional characteristics of two d-xylanases purified from Trichoderma harzianum

Two endo-1,4-β-d-xylanases (1,4-β-d-xylan xylanohydrolase, EC 3.2.1.8) were purified from Trichoderma harzianum culture filtrates. From kinetic analyses, apparent V_max and K_m values of 580 U mg^?1 protein and 0.16% d-xylan were obtained for the 20 000 dalton endo-1,4-β-d-xylanase, while values of 100 U mg^?1 protein and 0.066% d-xylan were obtained for the 29 000 dalton endo-1,4-β-d-xylanase. Substrate levels >1% (w/v) d-xylan were found to be inhibitory to both enzymes. Both d-xylanases were highly active against d-xylans obtained from various sources. Of the polymeric sugars tested, carboxymethyl cellulose was the only substrate which was hydrolysed to any extent. Little or no activity was observed against cellulose. Analyses by h.p.l.c. demonstrated the absence of hydrolytic activity by both d-xylanases on d-xylobiose. d-Xylotriose was cleaved to a limited extent by the 29 000 dalton d-xylanase only, while d-xylotetraose was hydrolysed by both. In the presence of d-xylotetraose, the 20 000 dalton d-xylanase had an associated transxylosidase activity which was not observed with the 29 000 dalton enzyme. When the solubilization assay was used, neither of the d-xylanases was inhibited by high concentrations of d-xylose and xylobiose.     

Lignocellulose biotransformation with immobilized cellulase,d-glucose oxidase and fungal peroxidases

Three enzymes, cellulase [see 1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4], d-glucose oxidase (β-d-glucose: oxygen 1-oxidoreductase, EC 1.1.3.4) and peroxidase (donor:hydrogen peroxide oxidoreductase, EC 1.11.1.7) immobilized on glass beads, have been incubated with lignocellulose. Fungal peroxidases from Trametes versicolor and Inonotus radiatus when mixed with cellulase and d-glucose oxidase were able to liberate phenolic compounds and d-glucose from lignocellulose. Three lignin monomers were identified. When the immobilized enzymes were incubated individually with lignocellulose they did not degrade lignin.     

An endo-(1→6-β-D-glucanase from Mucor hiemalis

An endo-(1→6)-β-D-glucanase (EC 3.2.1), isolated from the culture filtrate of Mucor hiemalis, was purified by ammonium sulphate fractionation and gel filtration. The homogeneity of the enzyme was confirmed by disc electrophoresis. The enzyme had a wide range of temperature and pH stability, high substrate specificity, and an action pattern of the endo-type.     

Application of cellulase and pectinase from fungal origin for the liquefaction and saccharification of biomass

Commercial cellulase [see 1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] from Trichoderma viride and pectinase [poly(1,4-α-d-galacturonide) glycanohydrolase, EC 3.2.1.15] from Aspergillus niger have been applied to produce fermentation syrups from sugar-beet pulp and potato fibre. Cellulosic, hemicellulosic and pectic polysaccharides of these substrates were hydrolysed extensively. Recovery of enzymes has been investigated in a packed-column reactor, connected with a hollow-fibre ultrafiltration unit. Enzymes appeared to be stable in this type of reactor, although part of the enzyme activity was lost, especially by adsorption onto the substrate residue.     

Production and Characteristics of Avicel-Digesting and Non-Avicel-Digesting Cellobiohydrolases from Aspergillus ficum

Two immunologically related cellobiohydrolases, cellobiohydrolase I (CBH I) and cellobiohydrolase II (CBH II), were purified from Aspergillus ficum. The Avicel-adsorbable CBH I (molecular weight, 128,000) digested Avicel, cotton, and cellulose powder to cellobiose, but the Avicel-unadsorbable CBH II (molecular weight, 50,000) could not digest those substrates. Both enzymes hydrolyzed insoluble cellooligosaccharides ( 25) to cellobiose. High-pressure liquid chromatographic analysis of soluble cellooligosaccharide hydrolysates revealed that both enzymes split off strictly cellobiose units from the nonreducing end of the cellulose chain with an exowise mechanism. CBH I showed glucosyltransferase activity, but CBH II did not. The N-bromosuccinimideoxidized CBH I was completely inactive but retained the ability to adsorb to Avicel. This suggested that CBH I has separate sites for binding to cellulose and for catalyzing cleavage of glycosidic linkages. Cellobiohydrolases were of two types, CBH I and CBH II. The former can adsorb to and digest Avicel, while the latter can do neither.     

Mutants of the white-rot fungus Sporotrichum pulverulentum with increased cellulase and β-d-glucosidase production

This paper reports the isolation of mutants of the white-rot fungus Sporotrichum pulverulentum and the results of a survey of enzymic activity among these mutants. The strains were screened for extracellular cellulase [see 1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] and β-d-glucosidase (β-d-glucoside glucohydrolase, EC 3.2.1.21) production in shake flask experiments. Apart from strain 63-2, strains 6, 63, 9, L5, E-1 and UV-18 showed equal or higher endo-1,4-β-d-glucanase (cellulase), filter paper-degrading and β-d-glucosidase activities than S. pulverulentum. The cellulase activity obtained, measured as filter paper activity, was comparable to that reported for Trichoderma reesei QM9414. However, the β-d-glucosidase activity was about six times higher than for the QM9414 strain. The pH and temperature-activity profiles of crude β-d-glucosidase preparations from the various strains were determined and were found to be identical. The thermal stability at pH 4.5 and 40°C was 5 days for all these preparations.     

Cellobiohydrolase from Trichoderma reesei.

A 1,4-beta-D-glucan cellobiohydrolase (EC 3.2.1.91) was purified from the culture liquid of Trichoderma reesei by using biospecific sorption on amorphous cellulose and immunoaffinity chromatography. A single protein band in polyacrylamide-gel electrophoresis and one arc in immunoelectrophoresis corresponded to the enzyme activity. The Mr was 65 000. The pI was 4.2-3.6. The purified enzyme contained about 10% hexose. The enzyme differs from previously described cellobiohydrolases in being more effective in the hydrolysis of cellulose.     

Studies on enzymic methods for extraction of inulin from Jerusalem artichokes

The release of inulin, d-fructose and protein from Jerusalem artichokes has been studied under diffusion and maceration conditions. The effects on release of added inulinase (2,1-β-d-fructan fructanohydrolase, EC 3.2.1.7), protease and cellulase [see 1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] enzymes have been investigated. The results indicate that added enzymes do not improve the efficiency of inulin or d-fructose release and that mechanical methods represent the most efficient means of carbohydrate solubilization. Treatment of plant extracts with inulinase is shown to have the disadvantage of increasing peptide solubilization. The potential for improved inulin solubilization by use of endo-inulinases is discussed.     

Substrate specificities and kinetic properties of two (1→3), (1→4)-β-d-glucan endo-hydrolases from germinating barley (Hordeum vulgare)

Two β-d-glucan endo-hydrolases purified from germinating barley (Hordeum vulgare) hydrolyse (1→4)-β linkages in (1→3),(1→4)-β-d-glucans where the d-glucosyl residue is substituted at O-3, but will not hydrolyse (1→3)-β-d-glucans or (1→4)-β-d-glucans. Methylation analysis of hydrolytic products released from barley (1→3),(1→4)-β-d-glucan indicates that 3-O-β-cellobiosyl-d-glucose and 3-O-β-cellotriosyl-d-glucose are the major oligomers formed. The enzymes exhibit characteristic endo-hydrolase action-patterns on this substrate. Both enzyme can therefore be classified as (1→3),(1→4)-β-d-glucan 4-glucanohydrolases (EC 3.2.1.73). The reduced, pneumococcal polysaccharide RS III, which consists of alternating (1→3)- and (1→4)-linked β-d-glucosyl residues, is hydrolysed by the enzymes to release laminaribiose as a major oligomeric product. Although the kinetic parameters of the two enzymes are similar, one hydrolyses barley (1→3),(1→4)-β-d-glucan at a significantly higher rate than the other and is more stable at elevated temperatures.     

Purification and properties of pig brain guanine deaminase.

Guanine deaminase (guanine aminohydrolase, EC 3.5.4.3) from pig brain was purified to homogeneity by column chromatography and ammonium sulphate fractionation. Homogeneity was established by polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulphate (SDS). The molecular weight of 110 000 was determined by gel filtration and sucrose density gradient centrifugation. SDS polyacrylamide gel electrophoresis indicated subunits of a molecular weight of 50 000. The amino acid composition, the isoelectric point and the number of -SH groups were determined. 5.5'-Dithiobis-(2-nitrobenzoic acid) reacts with about seven -SH groups in the native enzyme, but upon denaturation with SDS, 10 -SH groups react with this former reagent. Using electrolytic reduction, 44 half-cystines were determined in accordance with the number of cysteic acid residues determined by amino acid analysis after performic acid oxidation. The Km values determined for substrates of the enzyme were 1.1 . 10(-5) M for guanine in 0.1 M Tris. HCl buffer (pH 8.0) and 3.3 . 10(-4) M for 8-azaguanine in 0.1 M phosphate buffer, pH 6.4. The pKa values determined for ionizable groups of the active site of the enzyme were near pH 6.2 and pH 8.2. The chemical and kinetic evidence suggests that cysteine and histidine may be essential for the catalysis.