Cellobiose metabolism and cellobiohydrolase I biosynthesis by Trichoderma reesei

The relationship between β-linked disaccharide (cellobiose, sophorose) utilization and cellulase, particularly cellobiohydrolase I (CBH I) synthesis by Trichoderma reesei, was investigated. During growth on cellobiose and sophorose as carbon sources in batch as well as resting-cell culture, only sophorose induced cellulase formation. In the latter experiments, sophorose was utilized at a much lower rate than cellobiose, and the more cellulase produced, the lower its rate of utilization. Cellobiose and sophorose were utilized by the fungus mainly via hydrolysis by the cell wall- and cell membrane-bound β-glucosidase. Addition of sophorose to T. reesei growing on cellulose did not further stimulate cellulase synthesis, and addition of cellobiose was inhibitory. Cellobiose, however, promoted cellulase formation in both batch and resting cell cultures, when its hydrolysis by β-glucosidase was inhibited by nojirimycin. No cellulase formation was observed when the uptake of glucose (produced from cellobiose by β-glucosidase) was inhibited by 3-O-methylglucoside. Cellodextrins (C₂ to C₆) promoted formation of low levels of cellobiohydrolase I in indirect proportion to their rate of hydrolysis by β-glucosidase. Studies on the uptake of [³H]cellobiose, [³H]sophorose, and [¹⁴C]glucose in the presence of inhibitors of β-glucosidase (nojirimycin) and glucose transport (3-O-methylglucoside) show that glucose transport occurs at a much higher rate than disaccharide hydrolysis. Extracellular disaccharide hydrolysis accounts for at least 95% of their metabolism. The presence of an uptake system for cellobiose was established by demonstrating the presence of intracellular labeled [³H]cellobiose in T. reesei after its extracellular supply. The data are consistent with induction of cellulase and particularly CBH I formation in T. reesei by β-linked disaccharides under conditions where their uptake is favored at the expense of extracellular hydrolysis.     

Induction and regulation of cellulase synthesis in Trichoderma pseudokoningii mutants EA3-867 and N2-78

Two mutants, EA₃-867 and N₂-78, with high cellulase yields were obtained from wild strains of Trichoderma pseudokoningii Rifai, 1096 and Mo₃, respectively, by mutagenic treatments with a linear accelerator, ⁶⁰Co, u.v., nitrosoguanidine (NTG) and diethylsulphate (DTS). The mutants grew slowly to produce small colonies on agar plates with synthetic medium. On agar plates of peptone-yeast extract, the small colonies were as large as those of wild strains. The cellulase activities of these mutants in Koji extracts, shake flask culture filtrates, and enzyme preparations were markedly higher than those of their parents. The mutant N₂-78 reached quite high cellulase activity level when cultured for 60 h in shake flasks in a simple medium containing milled straw, wheat bran, mineral salts plus waste glucose molasses. The cellulase saccharifying activities on CMC, filter paper and cotton, were 255, 8.2 and 13.4 mg glucose/ml enzyme, respectively, or 11, 4.3 and 6 times more than those of its parent Mo₃.The cellulase synthesis of EA₃-867 and N₂-78 was strongly induced by sophorose, isolated from pods of Sophora japonica L., and was inhibited by glucose, sugar phosphates, glycerol and organic acids. We conclude that cellulase synthesis of the mutants is regulated by catabolite repression as well as by induction. The increase in cellulase production by both mutants results from changes in the regulatory systems for cellulase synthesis, i.e. the mutants showed higher sensitivity to inducer and lower susceptibility to catabolite repression than did the wild types.A cellulase preparation of Trichoderma pseudokoningii Rifai N₂-78 induced by sophorose was fractionated by DEAE-Sephadex A-50 and Sephadex G-100 column chromatography, selective inactivation and polyacrylamide gel electrophoresis. The components C₁(exo-β1,4-glucanase), C_x(endo-β1,4-glucanase) and β-glucosidase were separated, and their molecular weights were estimated to be 67 000, 62 000 and 42 000 respectively. The homogeneity of C₁ was verified by polyacrylamide gel electrophoresis, immunoelectrophoresis and ultracentrifugal analysis. It is a glycoprotein and is rich in glycine, aspartic acid, threonine, serine and glutamic acid. The C₁ showed a strong synergistic action with C_x in the degradation of cotton, Avicel and Walseth cellulose.A poly(A)-RNA, induced by sophorose in N₂-78 mycelium, was isolated by oligo(dT)-cellulose affinity chromatography.     

Cellulase induction by lactose in Trichoderma reesei PC-3-7

In an attempt to clarify the function of lactose in cellulase induction, experiments were carried out on cellulase formation by lactose along with other sugars in a resting cell system of Trichoderma reesei PC-3-7, a hypercellulase-producing mutant. Although lactose alone induces little cellulase under the conditions used, a synergistic effect on cellulase formation was observed following the respective addition of sophorose, cellobiose or galactose to lactose. The lactose consumption was more rapid when these sugars were added than in their absence. Furthermore, following lactose addition 10 h after the beginning of cultivation in the presence of cellobiose, cellulase formation was initiated with only a little lag, and lactose consumption started immediately, being complete in 14 h. \-Galactosidase induction experiments suggested that the rapid consumption of lactose is possibly not dependent on lactose degradation by the enzyme. From these results, it is suggested that lactose may function as an inducer for cellulase formation if it is taken up in the mycelium of T. reesei PC-3-7, and that sophorose, cellobiose or galactose may induce a putative lactose permease. *** DIRECT SUPPORT *** AG903066 00005     

Sophorose induction of an intracellular b-glucosidase in Trichoderma

The disaccharide sophorose induces Trichoderma to increase a solube intracellular b-glucosidase that hydrolyses cellobiose, sophorose, and p-nitrophenyl-b-D-glucopyranoside. Simultaneously, it depresses the activity of a similar insoluble enzyme that is associated with the mycelium. Gel electrophoresis indicates that a single enzyme is responsible for all the soluble intracellular b-glucosidase activity. Cycloheximide severely inhibits sophorose induction of this enzyme indicating that the increase in activity normally obtained with sophorose is due to the de novo formation of the enzyme. The same sugars that promote the formation and release of cellulase by Trichoderma induce an increase in the soluble intracellular b-glucosidase. A function of the soluble intracellular enzyme appears to be the hydrolysis of cellobiose, which would otherwise accumulate during cellulose degradation, and thus to prevent cellobiose inhibition of cellulase.     

Induction of cellulolytic enzymes in Trichoderma reesei by sophorose.

Sophorose (2-O-beta-glucopyranosyl-D-glucose) induces carboxymethyl cellulase in Trichoderma reesei QM6a mycelium with 1.5 to 2 h. The induction response to sophorose concentration, although complicated by the metabolism of sophorose, shows saturation kinetics. Most of the cellulase appears after most of the sophorose has been taken up, but the presence of an inducer is required to maintain cellulase synthesis because enzyme production ceases after separation of the mycelium from the induction medium. Cellulase appears simultaneously in the medium and in the mycelium, and no appreciable levels accumulate in the mycelium. Response to pH suggest either that synthesis and secretion of the enzyme are closely associated or concurrent events affected by surface interactions with the medium. Effects of temperature and pH on cellulase induction by sophorose are similar to those reported for induction by cellulose. The kinetics of absorption by mycelium differs from that of other beta-linked saccharides and glucose, the uptake of sophorose being much slower. Under our cultural conditions, sophorose appears to induce an incomplete array of cellulase enzymes, as indicated by enzymatic and electrophoretic studies.     

Induction of cellulase formation in Trichoderma reesei by cellobiono-1,5-lacton

Induction of synthesis of cellulolytic enzymes in Trichoderma reesei QM 9414 by cellobiono-1,5-lactone (CBL) has been investigated in a replacement system lacking additional carbon source. CBL induced cellulase secretion optimally at pH 5 and a concentration of 70 g/ml. Higher concentrations lead to lower induction. De novo induction of cellulases was proven by the inhibitory effect of cycloheximide addition. Induction by CBL was shown to act synergistically on induction by sophorose, as it decreased the concentration of sophorose required for maximal induction. Maximal endo--1,4-glucanase activities induced by either sophorose or CBL were comparable. The CBL-induced cellulase system contained all the major cellulolytic enzymes of T. reesei, i.e. cellobiohydrolase I and II, and endoglucanase I, as shown by SDS-PAGE, Western blotting and detection with specific mono- and polyclonal antibodies. No differences were seen in the types of individual enzymes formed upon induction by either sophorose or CBL. No other hydrolytic enzymes appear to be induced by CBL (i.e. amylase, laminarinase, xylanase).Abbreviations SDS-PAGE polyacrylamide gel electrophoresis in the presence of sodium-dodecylsulfate - CBL cellobiono-1,5-lacton - CBH cellobiohydrolase - EG endoglucanase - IgG immunoglobulin G     

Biochemical properties of xylanases from a thermophilic fungus,Melanocarpus albomyces, and their action on plant cell walls

Melanocarpus albomyces, a thermophilic fungus isolated from compost by enrichment culture in a liquid medium containing sugarcane bagasse, produced cellulase-free xylanase in culture medium. The fungus was unusual in that xylanase activity was inducible not only by hemicellulosic material but also by the monomeric pentosan unit of xylan but not by glucose. Concentration of bagasse-grown culture filtrate protein followed by size-exclusion and anion-exchange chromatography separated four xylanase activities. Under identical conditions of protein purification, xylanase I was absent in the xylose-grown culture filtrate. Two xylanase activities, a minor xylanase IA and a major xylanase IIIA, were purified to apparent homogeneity from bagasse-grown cultures. Both xylanases were specific forβ-1,4 xylose-rich polymer, optimally active, respectively, at pH 6.6 and 5.6, and at 65°C. The xylanases were stable between pH 5 to 10 at 50°C for 24 h. Xylanases released xylobiose, xylotriose and higher oligomers from xylans from different sources. Xylanase IA had a M_r of 38 kDa and contained 7% carbohydrate whereas xylanase IIIA had a M_r of 24 kDa and no detectable carbohydrate. The K_m for larchwood xylan (mg ml⁻¹) and V_max (μmol xylose min⁻¹ mg⁻¹ protein) of xylanase IA were 0.33 and 311, and of xylanase IIIA 1.69 and 500, respectively. Xylanases IA, II and IIIA showed no synergism in the hydrolysis of larchwood glucuronoxylan or oat spelt and sugarcane bagasse arabinoxylans. They had different reactivity on untreated and delignified bagasse. The xylanases were more reactive than cellulase on delignified bagasse. Simultaneous treatment of delignified bagasse by xylanase and cellulase released more sugar than individual enzyme treatments. By contrast, the primary cell walls of a plant, particularly from the region of elongation, were more susceptible to the action of cellulase than xylanase. The effects of xylanase and cellulase on plant cell walls were consistent with the view that hemicellulose surrounds cellulose in plant cell walls.     

Turgor regulation in a novel Halomonas species

The mechanisms by which a novel eubacterium, identified as belonging to the genus Halomonas, adapted to increases in the extracellular osmotic potential were investigated. It was shown that the ability of the bacterium to grow after hyperosmotic shock was dependent on the presence of potassium ions. Growth of the bacterium in 2 M NaCl medium could be limited by low concentrations of K⁺ and this enabled the affinity for K⁺ to be determined (K _s=21.5 M). Rubidium salts could be substituted for those of potassium, but the lowest concentration of Rb⁺ that allowed growth in 2 M NaCl medium was 50-fold greater than the minimum concentration of K⁺. ¹³C-NMR spectroscopy and HPLC analysis were used to demonstrate the accumulation of organic solutes in the cytoplasm after exposure to high salinities. The major osmolyte was ectoine, but glutamate and ectoine hydroxide were also present. Addition of exogenous glycine betaine to 3.25 M NaCl medium resulted in the accumulation of high intracellular concentrations of glycine betaine in the bacterium. This reduced the level of ectoine accumulation but did not fully inhibit the synthesis of this compound in the cytoplasm.Abbreviation Specific growth rate (generations/h)     

Effects of thyroid hormones and aldosterone on mineralocorticoid binding sites in the toad bladder

Summary In the urinary bladder of the toadBufo marinus triiodothyronine selectively inhibits the late effect of aldosterone on Na⁺ transport. We have investigated whether T₃ might mediate its antimineralocorticoid action by controlling: i) the level of aldosterone binding sites in the soluble (cytosolic) pool isolated from tissues treated with T₃ (60nm) for up to 20 hr of incubation; ii) the kinetics of uptake of³H-aldosterone into cytoplasmic and nuclear fractions after 2 or 20 hr of exposure to T₃. The number and the affinity of Type I (high affinity, low capacity) and Type II (low affinity, high capacity) cytosolic binding sites (measured at 0°C) did not vary significantly after 18 hr of exposure to T₃, while aldosterone-dependent Na⁺ transport was significantly inhibited. In addition, T₃ did not modify the kinetics of uptake (90 min) of³H-aldosterone into cytoplasmic and nuclear fractions of toad bladder incubatedin vitro at 25°C. By contrast, aldosterone itself was able to down-regulate its cytosolic and nuclear binding sites after an 18-hr exposure to the steroid hormone (10 or 80nm). T₃ slightly (20%) but significantly potentiated the down regulation of nuclear binding sites. In conclusion, T₃ does not appear to have major effects on the regulation of the aldosterone receptor, which could explain in a simple manner its antimineralocorticoid action.     

Preparation and properties of an immobilized cellulase on the reversibly soluble matrix Eudragit L-100

Abstract

To prepare a smart biocatalyst, cellulase was immobilized on the reversibly soluble matrix Eudragit L-100 by non-covalent and covalent methods. Covalent immobilization using carbodiimide coupling exhibited superior enzyme loading and reusability compared with non-covalent immobilization, and the covalent loading was increased by almost 20% through the addition of N-hydroxysuccinimide. The temperature optimum of the cellulase was not improved apparently by immobilization but the pH optimum increased from 4.75 to 5.25. Immobilized cellulase was more active than free cellulase above pH 5.0. Immobilized cellulase was more stable than free cellulase during storage at 4°C, room temperature and 50°C. K_m values of immobilized and free cellulase were 85.55 and 73.84 g L^?1, respectively. About 50% productivity was retained after five cycles for hydrolysis of steam-exploded straw.     

Cellulase production and activity in a species ofCladosporium

ACladosporium species produced large amounts of cellulase enzyme components when grown in shake-culture with medium containing carboxymethylcellulose. There was significantly less activity when Avicel, filter paper or cotton were used as substrates. KNO₃ was better than NH₄Cl or urea for the production of cellulase. Tween 80 at 0.1% (w/v) increased the production of cellulase by 1.5 to 4.5-fold. All the cellulase components were optimally active in the assay at pH 5.0 and 60°C.     

Cadmium biosorption by a cadmium resistant strain of Bacillus thuringiensis: regulation and optimization of cell surface affinity for metal cations

A marine bacterial strain putatively identified asBacillus thuringiensis strain DM55, showed multiple heavy metal resistance and biosorption phenotypes. Electron microscopic studies revealed that DM55 cells are encased in anionic cell wall polymers that can immobilize discrete aggregates of cations. Factors affecting cell surface affinity for metal cations, monitored by means of Cd²⁺ binding capability, are investigated. The mechanisms of cadmium resistance and Cd²⁺ biosorption by the bacterium appeared to be inducible and coincident. Medium components affecting metal removal under cadmium-stressed growth conditions were explored based on the application of two sequential multi-factorial statistical designs. Concentrations of potassium phosphates and peptone were the most significant variables. Optimized culture conditions allowed DM55 cells grown in the presence of 0.25 mM CdCl₂ to remove about 79% of the metal ions within 24 h with a specific biosorption capacity of 21.57 mg g^–1 of biomass. Both fresh and dry cells of DM55 prepared under cadmium-free optimal nutrient condition were also able to biosorb Cd²⁺. In addition to the concentration of phosphate in the medium, KinA, a major phosphate provider in the phosphorelay of Bacillus cells, was also demonstrated to regulate the magnitude of cell surface affinity for cadmium ions.     

Kinetics of lactose transport inKluyveromyces fragilis grown in a chemostat on diluted whey permeate

Lactose transport was studied inKluyveromyces fragilis grown in lactose-limited chemostat cultures. Kinetic parameters were determined using a method based on genetic population evolution. Lactose transport was carried out via three carriers characterized respectively byK _m of 0.1 mM, 3 mM and 15.5 mM. The synthesis of these lactose carriers and their capacity (V _max) are dependent on the dilution rate (D). At D=0.12 h^–1, the high affinity transporter is prominent. For intermediate dilution rate, only the high and the medium affinity systems are present. In cells growing at D=0.4 h^–1, these carriers are absent but instead, the low affinity transporter is present. The effect on lactose transport of such metabolic inhibitors as CCCP, a proton ionophore, and Antimycin A, an energy inhibitor, were also investigated. The high affinity system is the most sensitive to the effect of these inhibitors. Lactose transport through this carrier is probably a mechanism dependent on the proton motive force.     

Purification and characterization of a novel halostable cellulase from Salinivibrio sp. strain NTU-05

A halostable cellulase with a molecular mass of 29 kDa was purified from culture supernatants of the halophilic bacterium Salinivibrio sp. NTU-05 by way of the Fast Protein Liquid Chromatography method and the biochemical properties of the halostable cellulase was studied. The enzyme was active over a range of 0–25% sodium chloride examined in culture broth. The optimum cellulase activity was observed at 5% sodium chloride. Results from the salinity stability test indicated 24% of enzyme activity was retained at 25% sodium chloride for 4 h. The enzyme was also shown to be slightly thermostable with 40% residual activity under 60 °C for 4 h. The enzyme has a K_m of 3.03 mg/ml and a V_max of 142.86 mol/min/mg when tested using carboxymethyl-cellulose (CMC). The enzyme activity increased in the presence of K⁺, Mg²⁺, Na⁺ ions and decreased when Hg²⁺ ions were present. The deduced internal amino acid sequence of the Salinivibrio sp. NTU-05 cellulase showed similarity to the sequence of the glycoside hydrolase family protein. These are some of the novel characteristics that make this enzyme have potential applications in cellulose biodegradation.     

N2-fixation in Erwinia herbicola

Four from 18 strains of Erwinia herbicola tested had nitrogenase activity and grew with N₂ as sole source of nitrogen under strict anaerobic conditions with a doubling time of 20–24 h. Nitrogenase activity started only 96–120 h after transfer to a special medium maintained under anaerobic conditions. A ten fold increase in protein per culture found after the maximum nitrogenase activity of 80–130 nmol C₂H₄. mg protein^-1·min^-1 was accompanied by a fall in pH of the medium (20 mM phosphate buffer and in 125 mM Tris-buffer) from pH 7.2 to 5.4 or less, but only to 6.8 in 100 mM phosphate buffer. In all cases we found a sharp curtailing of nitrogenase activity 48 h after the maximum. The bacteria utilized only 35–50% of the nitrogen fixed for growth. Erwinia herbicola strains differed from two strains of Enterobacter agglomerans in being unable to fix nitrogen on agar surfaces exposed to air. Specific nitrogenase activity in Erwinia herbicola is compared with data reported for other Enterobacteriaceae and is found to be higher than that reported for Klebsiella pneumoniae, Enterobacter cloacae or Citrobacter freundii.     

Purification and characterization of a novel endo-β-1,4-glucanase from the thermoacidophilic <Emphasis Type="Italic">Aspergillus terreus</Emphasis>

An endo-β-1,4-glucanase from a thermoacidophilic fungus, Aspergillus terreus M11, was purified 18-fold with 14% yield and a specific activity of 67 U mg⁻¹ protein. The optimal pH was 2 and the cellulase was stable from pH 2 to 5. The cellulase had a temperature optimum of 60°C measured over 30 min and retained more than 60% of its activity after heating at 70°C for 1 h. The molecular mass of the cellulase was about 25 kDa. Its activity was inhibited by 77% by Hg²⁺ (2 mM) and by 59% by Cu²⁺ (2 mM).     

An improved method of measuring photosynthetic electron transport in Euglena under in vivo conditions

A method is described to measure photochemical activity in intact cells of Euglena under in vivo conditions. The method employs a cell wall digesting enzyme (cellulysin) to induce enough permeability in the cell walls and membranes in order to allow dyes, commonly used to investigate light-dependent electron transport reactions to enter, but without inducing a concomittant efflux of metabolites. Between 1 and 2 h of incubation in 5% (w/v) cellulysin provided conditions which allowed measurement of photosystem I-, II- and I+II-dependent electron transport with rates up to 600% higher than in control cells; whereas other cell wall degrading enzymes (cellulase and pectinase) still did not increase the entry of the dyes. Cellulysin up to 2 h of incubation had little or no effect on whole cell respiration, photosynthetic O₂ evolution, or the export of potassium and (¹⁴C) labeled compounds out of cells; therefore cellulysin obviously did not change the normal habit or physiology of Euglena. Cellulysin (4 h digestion), cellulase and pectinase (2–4 h of incubation) on the other hand led to a lowering of respiration and light-dependent O₂ evolution, and increased the efflux of K⁺, but apparently decreased that of (¹⁴C)labeled fixation products.Abbreviations DBMIB dibromothymoquinone - DCPIP 2,6-dichlorophenol-indophenol - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DMMIB 2,3-dimethyl-5,6-methylenedioxy-p-benzoquinone - MV methylviologen - PSI photosystem I - PS II photosystem II     

柚子皮黑曲霉的分离鉴定及产酶特性研究

对柚子皮上自然生长的黑曲霉进行分离鉴定,并探讨其产酶特性。以平板稀释法从柚子皮上分离出一株霉菌菌株,通过观察其形态特征和培养特征,对照《真菌鉴定手册》判定该菌株的种属;采用鉴定培养基法对其产酶特性进行分析。根据柚子皮的成分特性,以干柚子皮为主要原料,该菌为生产菌株,采用固态发酵法探究培养基的成分、柚子皮含量、培养基初始含水量及发酵时间4个因素对纤维素酶活力的影响。结果表明,该菌株为黑曲霉(Aspergillus nige),可产淀粉酶、蛋白酶、纤维素酶、果胶酶;固态发酵培养基中添加柚子皮12g,麸皮0.5 g和(NH_4)_2SO_40.5 g,培养基初始含水量保持在68.5 mL/100 g,培养时间控制在60 h左右时纤维素酶产量较高。     

Energetic aspects of anaerobic growth of Aerobacter aerogenes in complex medium

Molar growth yields for anaerobic growth of Aerobacter aerogenes in complex medium were much higher than for growth in minimal medium. In batch cultures the molar growth yield for glucose varied from 44 to 50 and Y _ATP from 17.1 to 18.8. For glucose-limited chemostat cultures a value of 17.5 g/mole was found for Y _ATP ^max and a value of 2.3 mmoles ATP/g dry weight h for the maintenance coeficient. Growth dependent pH changes were used to control the addition of fresh medium, containing excess of glucose to a continuous culture. The specific growth rate and the population density were dependent on the pH difference between the inflowing medium and the culture. At a value of 1.44 h^-1 the molar growth yield for glucose was about 70 and Y _ATP about 28.5. An-equation is presented, which gives the relation between theoretical and experimental Y _ATP ^max values.