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.     

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.     

Purification and properties of two exo-cellobiohydrolases from a cellulolytic culture of Fusarium lini

Two distinct exo-cellobiohydrolases (1,4-β-d-glucan cellobiohydrolase, EC 3.2.1.91) have been isolated from culture filtrates of Fusarium lini by repeated ammonium sulphate fractionation and isoelectric focusing. The purified enzymes were evaluated for physical properties, kinetics and the mechanism of their action. The results of this work were as follows. (1) A two-step enzyme purification procedure was developed, involving isoelectric focusing and ammonium sulphate fractionation. (2) Yields of pure cellobiohydrolases I and II were 45 and 36 mg l^?1 of culture broth, respectively. (3) Both enzymes were found to be homogeneous, as determined by ultracentrifugation, isoelectric focusing, electrophoresis in polyacrylamide gels containing SDS and chromatography on Sephadex. (4) The molecular weights of the two cellobiohydrolases, as determined by gel filtration and SDS gel electrophoresis, were 50 000–57 000. (5) Both cellobiohydrolases had low viscosity-reducing and reducing sugar activity from carboxymethyl cellulose and high activity with Walseth cellulose and Avicel. (6) The enzymes produced only cellobiose as the end product from filter paper and Avicel, indicating that they are true cellobiohydrolases. (7) Cellobiohydrolase I hydrolysed d-xylan whereas cellobiohydrolase II was inactive towards d-xylan. (8) There was a striking synergism in filter paper activity when cellobiohydrolase was supplemented with endo-1,4-β-d-glucanase [cellulase, 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).     

Synthesis and properties of some O-(2,2-dialkoxyethyl)glycolaldehydes

β-d-Mannosidase (β-d-mannoside mannohydrolase EC 3.2.1.25) was purified 160-fold from crude gut-solution of Helix pomatia by three chromatographic steps and then gave a single protein band (mol. wt. 94,000) on SDS-gel electrophoresis, and three protein bands (of almost identical isoelectric points) on thin-layer iso-electric focusing. Each of these protein bands had enzyme activity. The specific activity of the purified enzyme on p-nitrophenyl β-d-mannopyranoside was 1694 nkat/mg at 40° and it was devoid of α-d-mannosidase, β-d-galactosidase, 2-acet-amido-2-deoxy-d-glucosidase, (1→4)-β-d-mannanase, and (1→4)-β-d-glucanase activities, almost devoid of α-d-galactosidase activity, and contaminated with <0.02% of β-d-glucosidase activity. The purified enzyme had the same K_m for borohydride-reduced β-d-manno-oligosaccharides of d.p. 3–5 (12.5mm). The initial rate of hydrolysis of (1→4)-linked β-d-manno-oligosaccharides of d.p. 2–5 and of reduced β-d-manno-oligosaccharides of d.p. 3–5 was the same, and o-nitrophenyl, methylumbelliferyl, and naphthyl β-d-mannopyranosides were readily hydrolysed. β-d-Mannobiose was hydrolysed at a rate ～25 times that of 6¹-α-d-galactosyl-β-d-mannobiose and 6³-α-d-galactosyl-β-d-mannotetraose, and at ～90 times the rate for β-d-mannobi-itol.     

Some kinetic properties of the β-d-glucosidase (cellobiase) in a commercial cellulase product from Penicillium funiculosum and its relevance in the hydrolysis of cellulose

Some kinetic parameters of the β-d-glucosidase (cellobiase, β-d-glucoside glucohydrolase, EC 3.2.1.21) component of Sturge Enzymes CP cellulase [see 1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] from Penicillium funiculosum have been determined. The Michaelis constants (K_m) for 4-nitrophenyl β-d-glucopyranoside (4NPG) and cellobiose are 0.4 and 2.1 mM, respectively, at pH 4.0 and 50°C. d-Glucose is shown to be a competitive inhibitor with inhibitor constants (K_i) of 1.7 mM when 4NPG is the substrate and 1 mM when cellobiose is the substrate. Cellobiose, at high concentrations, exhibits a substrate inhibition effect on the enzyme. d-Glucono-1,5-lactone is shown to be a potent inhibitor (K_i = 8 μM; 4NPG as substrate) while d-fructose exhibits little inhibition. Cellulose hydrolysis progress curves using Avicel or Solka Floc as substrates and a range of commercial cellulase preparations show that CP cellulase gives the best performance, which can be attributed to the activity of the β-d-glucosidase in this preparation in maintaining the cellobiose at low concentrations during cellulose hydrolysis.     

Purification and some properties of a (1→4)-β-d-glucan glucohydrolase associated with the cellulase from the fungus Penicillium funiculosum

The (1→4)-β-d-glucan glucohydrolase from Penicillium funiculosum cellulase was purified to homogeneity by chromatography on DEAE-Sephadex and by iso-electric focusing. The purified component, which had a molecular weight of 65,000 and a pI of 4.65, showed activity on H₃PO₄-swollen cellulose, o-nitrophenyl β-d-glucopyranoside, cellobiose, cellotriose, cellotetraose, and cellopentaose, the K_m values being 172 mg/mL, and 0.77, 10.0, 0.44, 0.77, and 0.37 mm, respectively. d-Glucono-1,5-lactone was a powerful inhibitor of the action of the enzyme on o-nitrophenyl β-d-glucopyranoside (K_i 2.1 μm), cellobiose (K_i 1.95 μm), and cellotriose (K_i 7.9 μm) [cf.d-glucose (K_i 1756 μm)]. On the basis of a Dixon plot, the hydrolysis of o-nitrophenyl β-d-glucopyranoside appeared to be competitively inhibited by d-glucono-1,5-lactone. However, inhibition of hydrolysis by d-glucose was non-competitive, as was that for the gluconolactone-cellobiose and gluconolactone-cellotriose systems. Sophorose, laminaribiose, and gentiobiose were attacked at different rates, but the action on soluble O-(carboxymethyl)cellulose was minimal. The enzyme did not act in synergism with the endo-(1→4)-β-d-glucanase component to solubilise highly ordered cotton cellulose, a behaviour which contrasts with that of the other exo-(1→4)-β-d-glucanase found in the same cellulase, namely, the (1→4)-β-d-glucan cellobiohydrolase.     

Simultaneous saccharification and fermentation of cellulose: effect of β-d-glucosidase activity and ethanol inhibition of cellulases

Compared with saccharification in the absence of yeast, simultaneous saccharification and fermentation (SSF) using Trichoderma cellulases and Saccharomyces cerevisiae enhanced cellulose hydrolysis rates by 13–30%. The optimum temperature for SSF was 35°C. The requirement for β-d-glucosidase (β-d-glucoside glucohydrolase, EC 3.2.1.21) in SSF was lower than for saccharification: maximal ethanol production was attained when the ratio of the activity of β-d-glucosidase to filter paper activity was ～1.0. Ethanol inhibited cellulases uncompetitively, with an inhibition constant of 30.5 gl ^?1, but its effect was less severe than that of an equivalent concentration of cellobiose or glucose. No irreversible denaturation of cellulases [1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] by ethanol was observed.     

Cellulase and ethanol production from cellulose by Neurospora crassa

Two strains of Neurospora crassa have been identified which utilize cellulase and produce 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]. The activities were detected as early as 48 h in the culture broth. These cultures also fermented d-glucose, d-xylose and cellulosic materials to ethanol as the major product of fermentation. The conversion of cellulose to ethanol was >60%, indicating the potential of using Neurospora for the direct conversion of cellulose to ethanol.     

Production of Cellulase byTrichoderma reesei on Waste Cellophane

Utilization of waste cellophane for production of extracellular cellulose-degrading enzymes byTrichoderma reesei was studied on laboratory-scale experiments using liquid media containing varnished or non-varnished cellophane disintegrated physically to different degrees. Enzyme activities were directly proportional to the degree of cellophane disintegration,i.e. its specific surface area. Hydrolytic activities of cellulase on 1 % milled cellophane reached approximately 50 % yield of the activities acquired on 1 % microcrystalline cellulose. Addition of Xylocel, concentrated prehydrolyzate of beech wood, added to the media in amounts corresponding to 0.2 to 1.0 % concentration of reducing groups, did not improve the yield of cellulase on milled cellophane. However, β-_D-xylanase activity was increased several times due to the presence of lower xylooligosaccharides serving as specific β-_D-xylanase inducers. No differences in occurrence of multiple forms of endo-l,4-β-_D-glueanases and endo-l,4-β-_D-xylanases and their isoelectric points were observed.     

Properties of Trichoderma reesei cells immobilized by the irradiation technique

Aerobic cells of Trichoderma reesei have been immobilized by the radiation polymerization technique using fibrous substances and hydroxyethyl methacrylate. The enzyme [cellulase, 1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] productivity and growth of the cells in the immobilized growing cells have been studied. The enzyme (filter paper) activity in the immobilized cells was comparable to that of the intact cells, showing that the cells immobilized with fibrous materials grow and become adhered to the surface of the fibrils. The filter paper activity of the immobilized cells was affected mainly by monomer concentration and the content of the fibrous materials, as well as the irradiation dose. It was demonstrated that in repeated batch culture of the immobilized cells the filter paper activity gave a constant value, and leakage of the cells was not observed.     

Purification and properties of a β-N-acetylaminoglucohydrolase from malted barley

β-N-Acetylaminoglucohydrolase (β-2-acetylamino-2-deoxy-D-glucoside acetylaminodeoxyglucohydrolase, EC 3.2.1.30) was extracted from malted barley and purified. The partially purified preparation was free from α-and β-glucosidase, α- and β-galactosidase, α-mannosidase and β-mannosidase. This preparation was free from α-mannosidase only after affinity chromatography with p-amino-N-acetyl-β-D-glucosaminidine coupled to Sepharose. The enzyme was active between pH 3 and 6.5 and had a pH optimum at pH 5. A MW of 92000 was obtained by sodium dodecyl sulfate-acrylamide gel electrophoresis and a sedimentation coefficient of 4.65 was obtained from sedimentation velocity experiments. β-N-Acetylaminoglucohydrolase had a K_m of 2.5 × 10^?4 M using the p-nitrophenyl N-acetyl β-D-glucosaminidine as the substrate.     

1,3-β-Glucanase from Vigna aconitifolia and its possible use in enzyme bioreactor fabrication

Endo-1,3(4)-β-glucanase (EC 3.2.1.6) from Vigna aconitifolia sprouts was purified to 14.5 fold by gel filtration and ion-exchange chromatography. The enzyme was found to be a glycoprotein, its activity was Ca²⁺ dependent and specific for β-1,3 linkages in different polysaccharides. The K_m value of the enzyme was estimated to be 3.0 mg ml⁻¹ for β-d-glucan as substrate. Circular dichroism studies revealed 8% α-helix, 48% β-pleated and 44% random coil in its secondary structure. Purified β-glucanase was then successfully co-immobilized with glucose oxidase in agarose-chitosan beads, showing better immobilization yield, operational range and stability as compared with the crude β-glucanase beads. The immobilized β-glucanase was successfully used for mini-bioreactor fabrication.     

Purification and properties of neutral β-galactosidases from human liver

Two neutral β-galactosidase isozymes were purified from human liver. The initial step of purification was removal of the acidic β-galactosidases by adsorption on concanavalin A-Sepharose 4B conjugate. Subsequent purification steps included ammonium sulfate precipitation, diethylaminoethyl cellulose column chromatography, Sephadex G-100 gel filtration, and preparative polyacrylamide-gel isoelectric focusing. The final step of purification was affinity chromatography of the separated isoelectric forms on ?-aminocaproyl-β-d-galactosylamine-Sepharose 4B conjugate. The purified β-galactosidase isozymes had activity toward both β-d-galactoside and β-d-glucoside derivatives of 4-methylumbelliferone and p-nitrophenol with a pH optimum around 6.2. These enzyme forms were also found to possess lactosylceramidase II activity with a pH optimum in the range of 5.4 to 5.6, but not lactosylceramidase I activity and no activity toward galactosylceramide or GM₁-ganglioside. The molecular weight was found to be in the range of 37,500–39,500 for the two neutral isozymes and they had similar K_m and V values; the more acidic form (designated β-galactosidase N₁) was more heat stable than the other form (designated β-galactosidase N₂). Antibodies evoked against the N₁ and N₂ β-galactosidases gave identical precipitin lines retaining enzymatic activity. No cross-reactivity was observed between the neutral and the acidic isozymes when examined with the respective antisera.     

Cellulose digestion in termites and cockroaches: What role do symbionts play?

• 1.1. Termites and cockroaches are excellent models for studying the role of symbionts in cellulose digestion in insects: they eat cellulose in a variety of forms and may or may not have symbionts.
• 2.2. The wood-eating cockroach, Panesthia cribrata, can be maintained indefinitely, free of microorganisms, on a diet of crystalline cellulose. Under these conditions the RQ is 1, indicating that the cockroach is surviving on glucose produced by endogenous cellulase.
• 3.3. The in vitro rate at which glucose is produced from crystalline cellulose by gut extracts from P. cribrata and Nasutitermes walkeri is comparable to the in vivo production of CO₂ in these insects, clearly indicating that the rate of glucose production from crystalline cellulose is sufficient for their needs.
• 4.4. In all termites and cockroaches examined, cellulase activity was found in the salivary glands and predominantly in the foregut and midgut. These regions are the normal sites of secretion of digestive enzymes and are either devoid of microorganisms (salivary glands) or have very low numbers.
• 5.5. Endogeneous cellulases from termites and cockroaches consist of multiple endo-β-1,4-glucanase (EC 3.2.1.4) and β-1,4-glucosidase (EC 3.2.1.21) components. There is no evidence that an exo-β-1,4-glucanase (cellobiohydrolase) (EC 3.2.1.91) is involved in, or needed for, the production of glucose from crystalline cellulose in termites or cockroaches as the endo-β-1,4-glucanase components are active against both crystalline cellulose and carboxymethylcellulose.
• 6.6. There is no evidence that bacteria are involved in cellulose digestion in termites and cockroaches. The cellulase associated with the fungus garden of M. michaelseni is distinct from that in the midgut; there is little indication that the fungal enzymes are acquired or needed. Lower termites such as Coptotermes lacteus have Protozoa in their hindgut which produce a cellulase(s) quite distinct from that in the foregut and midgut.

     

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.     

Major characteristics of the cellulolytic system of Clostridium thermocellum coincide with those of the purified cellulosome

The properties of the cellulosome (a cellulose-binding, multiple cellulase-containing protein complex isolated from Clostridium thermocellum) have been compared with the previously reported characteristics for crude cellulase [see 1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] preparations. Similar to the crude enzyme system, true cellulolytic activity was demonstrated for the purified cellulosome on the basis of extensive solubilization of microcrystalline cellulose. The cellulolytic activity of the purified cellulosome was enhanced both by calcium ions and by thiols, and was inhibited by cellobiose (the major end product of the cellulosome-mediated cellulose degradation). In addition, at low ionic strength, cellulose-adsorbed cellulosome was detached intact from the cellulose matrix. Using controlled conditions, maximum enzymatic activity was shown to correspond to suboptimal conditions of cellulosome adsorption to cellulose. The results suggest that previous data accumulated for the crude cellulase system in C. thermocellum essentially reflect the contribution of the cellulosome.     

Expression of β1,4-galactosyltransferase in the development of mouse brain

β_1,4-Galactosyltransferase (GalTase, EC 2.4.1.38) transfers galactose to the terminal N-acetylglucosamine of complex-type N-glycans, which have great importance for cell-cell interactions during fertilization and early embryogenesis. In this study, the activity of β_1,4-galactosyltransferase in mouse brain during development was measured with the method of reverse HPLC using a fluorescence-labeled biantenary sugar chain, GlcNAcβ1-2Manα1-6(GlcNAcβ1-2Manα1-3) Manβ1-4GlcNAcβ1-4GlcNAc-PA. The level of messenger RNA of this enzyme during the development of mouse brain was also investigated with Northern blot analysis. The results showed that: (1) β_1,4-galactosyltransferase showed similar branch specificity and kinetics for the biantenary substrate during development; (2) GalTase activity in fetal mouse brain was four times higher than that in adult mouse brain and decreased gradually in the course of development; (3) messenger RNA level was highest in fetal mouse and decreased dramatically after birth. However, the contents of mRNA were not parallel to the enzyme activity.     

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.     

β-d-Glucosidase: multiplicity of activities and significance to enzymic saccharification of cellulose

Activity measurement of protein components resolved by polyacrylamide gel electrophoresis has indicated that the β-d-glucosidase (β-d-glucoside glucohydrolase, EC 3.2.1.21) system is composed of multiple enzymes, each differing in substrate specificity. The proportions of these enzymes varied in preparations obtained from Aspergillus phoenicis, almond emulsion and Trichoderma reesei. The enzyme component showing the highest cellobiase activity was most useful in improving cellulose saccharification by Trichoderma cellulases. The optimum ratio between filter paper and cellobiase activities, expressed in the appropriate units, was 1.0:0.9. The results indicate that for saccharification purposes, the β-d-glucosidase activity should be measured using cellobiose as a substrate, rather than salicin, esculin or 4-nitrophenyl-β-d-glucopyranoside.     

Thermostable alkaline cellulase from an alkaliphilic isolate, Bacillus sp. KSM-S237

Thermostable alkaline cellulase (endo-1,4-β-glucanase, EC 3.2.1.4) activity was detected in the culture medium of a strictly alkaliphilic strain of Bacillus, designated KSM-S237. This novel enzyme was purified to homogeneity by a two-step column-chromatographic procedure with high yield. The N-terminal amino acid sequence of the purified enzyme was Glu-Gly-Asn-Thr-Arg-Glu-Asp-Asn-Phe-Lys-His-Leu-Leu-Gly-Asn-Asp-Asn-Val-Lys-Arg. The enzyme had a molecular mass of approximately 86 kDa and an isoelectric point of pH 3.8. The enzyme had a pH optimum of 8.6–9.0 and displayed maximum activity at 45°C. The alkaline enzyme was stable up to 50°C and more than 30% of the original activity was detectable after heating at 100°C and at pH 9.0 for 10 min. The enzyme hydrolyzed carboxymethylcellulose, lichenan (β-1,3;1,4-linkage), and p-nitrophenyl derivatives of cellotriose and cellotetraose. Crystalline forms of cellulose (Avicel and filter paper), H₃PO₄-swollen cellulose, NaOH-swollen cellulose, curdlan (β-1,3-linkage), laminarin (β-1,3;1,6-linkage), and xylan were barely hydrolyzed at all. Received: April 28, 1997 / Accepted: May 24, 1997