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.     

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.     

Effect of enzyme supplementation at moderate cellulase loadings on initial glucose and xylose release from corn stover solids pretreated by leading technologies

Moderate loadings of cellulase enzyme supplemented with beta-glucosidase were applied to solids produced by ammonia fiber expansion (AFEX), ammonia recycle (ARP), controlled pH, dilute sulfuric acid, lime, and sulfur dioxide pretreatments to better understand factors that control glucose and xylose release following 24, 48, and 72 h of hydrolysis and define promising routes to reducing enzyme demands. Glucose removal was higher from all pretreatments than from Avicel cellulose at lower enzyme loadings, but sugar release was a bit lower for solids prepared by dilute sulfuric acid in the Sunds system and by controlled pH pretreatment than from Avicel at higher protein loadings. Inhibition by cellobiose was observed to depend on the type of substrate and pretreatment and hydrolysis times, with a corresponding impact of beta-glucosidase supplementation. Furthermore, for the first time, xylobiose and higher xylooligomers were shown to inhibit enzymatic hydrolysis of pure glucan, pure xylan, and pretreated corn stover, and xylose, xylobiose, and xylotriose were shown to have progressively greater effects on hydrolysis rates. Consistent with this, addition of xylanase and beta-xylosidase improved performance significantly. For a combined mass loading of cellulase and beta-glucosidase of 16.1 mg/g original glucan (about 7.5 FPU/g), glucose release from pretreated solids ranged from 50% to75% of the theoretical maximum and was greater for all pretreatments at all protein loadings compared to pure Avicel cellulose except for solids from controlled pH pretreatment and from dilute acid pretreatment by the Sunds pilot unit. The fraction of xylose released from pretreated solids was always less than for glucose, with the upper limit being about 60% of the maximum for ARP and the Sunds dilute acid pretreatments at a very high protein mass loading of 116 mg/g glucan (about 60 FPU).     

UDPGLUCOSE PYROPHOSPHORYLASE ACTIVITY IN THE DIATOM CYCLOTELLA CRYPTICA. PATHWAY OF CHRYSOLAMINARIN BIOSYNTHESIS 1

UDPglucose pyrophosphorylase activity was detected in cell-free extracts of the diatom Cyclotella cryptica TI3L Reimann, Lewin and Guillard. When assayed in the direction of UDPglucose formation, the enzyme had maximal activity at pH 7.8 and was stimulated by Mg²⁺and Mn²⁺ions. 3-Phosphoglycerate and inorganic phosphate had little effect on enzymatic activity, and the enzyme was relatively insensitive to feedback inhibition from UDPglucose (K, > I millimolar). A glucan was formed from UDP-[¹⁴C]glucose in cell-free extracts of C. cryptica. This glucan had a median molecular weight of 4600 (as determined by gel filtration chromatograbhy) and could be hydrolyzed by laminarinase. Partial acid hydrolysis of the glucan resulted in the formation of glucose and laminaribiose. but not cellobiose. These results suggest that the synthesis of chrysolaminarin (the major storage carbohydrate of diatoms) occurs via the activity of UDPglucose pyrophosphorylase. followed by glucosyl transfer from UDPglucose to the growing β-(1–3)-linked glucan.     

Derepressed synthesis of cellulase by Cellulomonas.

A Cellulomonas sp. was isolated from the soil which hydrolyzed cellulose, as shown by clear-zone formation on cellulose agar medium. Catabolite repression of cellulase synthesis occurred when moderate levels of glucose were added to the medium. A stable mutant that no longer exhibits catabolite repression was produced through treatment of the wild-type organism with N-methyl-N'-nitro-N-nitrosoguanidine. Both enzyme concentration and specific activity, as determined by the rate of hydrolysis of carboxymethylcellulose, were greater with the mutant than with the wild-type organism under various test conditions. The wild type had no measurable cellulase activity when grown in the presence of either 1.0% glucose or cellobiose. Cellobiose, but not glucose, inhibited enzyme activity towards both cellulose and carboxymethylcellulose. Cellobiose, cellulose, and sophorose at low concentrations induced cellulase synthesis in both the wild-type and the mutant organism. Cellulase regulation appears to depend upon a complex relationship involving catabolite repression, inhibition, and induction.     

玉米秸秆不同部位的组成及其对预处理的影响

不同玉米秸秆部位的成分组成及分布对预处理和酶解影响显著。研究表明:韧皮部与髓芯的成分相近,但叶子的差异较大,其木聚糖和总糖的质量分数最高,分别为29.48%和66.15%,而木质素的质量分数最低,因而叶子更容易预处理。玉米秸秆在稀酸预处理过程中可回收96.9%葡聚糖和50.0%～70.0%木聚糖,其中50.0%～60.0%木聚糖水解成木糖溶出;不同部位的木聚糖损失率与初始的木聚糖含量正相关;经稀酸预处理后,叶子中葡聚糖的质量分数最高,达72.40%,叶子和髓芯易于被纤维素酶水解生成葡萄糖,而韧皮部困难。不同部位的酶解得率与自身的葡聚糖含量正相关,与酸不溶木质素含量负相关,同时受原料的物理结构、葡聚糖和木质素大分子的化学组成等影响。     

An intermediate in cyclic beta 1-2 glucan biosynthesis

Incubation of UDP-[14C]Glc with the inner membranes of Agrobacterium tumefaciens leads to the formation of cyclic beta 1-2 glucan and trichloroacetic acid-insoluble compounds. The proteolysis products of the latter show a positive charge in acid and a negative charge in alkaline buffers. The cyclic beta 1-2 glucan and the trichloroacetic acid insoluble compounds yield the same products on partial acid hydrolysis. Addition of excess non-radioactive UDP-Glc to the reaction mixture nearly stops the formation of radioactive beta 1-2 glucan and leads to a rapid fall of radioactivity in the trichloroacetic acid precipitate. Alkaline treatment of the insoluble compounds under conditions of beta-elimination leads to the partial release of free saccharides (about 30%). It is concluded that beta 1-2 glucan chains are built up joined to a protein and then released as free cyclic beta 1-2 glucan.     

Evidence for a glycosidic linkage between chitin and glucan in the cell wall of Candida albicans

The alkali-insoluble glucan was isolated from regenerating spheroplasts and intact cells of Candida albicans. Sequential enzymic hydrolysis of this fraction by Zymolyase 100T and purified chitinase and subsequent gel filtration produced a fraction which was enriched in glycosaminoglycans. This fraction was analysed by partial acid hydrolysis, TLC and GLC-MS. The GLC-MS peaks identified included 2,3,4,6-tetra-O-methylglucitol acetate and 2,3,4-tri-O-methylglucitol acetate of beta-1,6-glucan and the 3,6-di-O-methyl-2-N-methylglucosaminitol acetate of chitin. In addition, 3-O-methyl-2-N-methylglucosaminitol acetate was identified, which indicated a branch point in chitin. These data provide evidence for a covalent linkage between chitin and beta-(1,6)-glucan through a glycosidic linkage at position 6 of N-acetylglucosamine and position 1 of the glucose in the glucan.     

beta-1, 3-Glucan Synthase from Lilium longiflorum Pollen

A particulate fraction from pollen tubes and ungerminated pollen of Lilium longiflorum incorporated ¹⁴C-glucose from UDP-glucose-¹⁴C into a lipid fraction and into β-1, 3-glucan. Partial hydrolysis of the glucan yielded laminaribiose as the only radioactive disaccharide. The preferred substrate was UDP-glucose, and enzyme activity was stimulated by glucose and by β-linked di- and trisaccharides. Enzyme from growing pollen tubes synthesized β-1, 3-glucan more rapidly and produced a higher proportion of alkali-insoluble glucan than did enzyme from ungerminated pollen. The onset of pollen tube growth may be dependent on altered activity of β-1, 3-glucan synthase.     

Characteristics of the beta-glucan receptor of murine macrophages

Phagocytosis of heat-killed yeast (HK-yeast), zymosan, and glucan particles by thioglycollate-elicited mouse peritoneal macrophages (Tg-macrophages) was inhibited by soluble glucan polymers/oligomers. The inhibitory capacity of soluble glucans decreased steeply with the decrease in the degree of polymerization (DPn); i.e., the concentration at which 50% inhibition of phagocytosis was attained was 0.23 microgram/ml for glucan 1 (DPn 24.8), 0.8 microgram/ml for glucan 2 (DPn 21.9), and greater than 40 micrograms/ml for glucan 3 (DPn 13.8). The glucan polymers were obtained by partial hydrolysis of glucan particles with formic acid (90%, 95 degrees C, 20 min) and fractionation according to solubility in ethanol water mixtures. A short preincubation (5 min, 4 or 37 degrees C) of Tg-macrophages with glucan 1 led to a subsequent inhibition of HK-yeast phagocytosis. Recovery of the phagocytic function was slow (27% in 3 h; 68% in 5 h) and required protein synthesis. beta-Glucan receptor expression was also suppressed by dexamethasone treatment. Mannan exerted at high concentrations (5 mg/ml) a partial inhibitory activity which was totally abrogated by beta-glucanase treatment. Treatment of macrophages with glucan together with mannan did not enhance the inhibitory capacity of glucan beyond the component abrogated by enzyme treatment. Contribution of local opsonization of HK-yeast to the phagocytic response (involvement of complement receptors) was indirectly negated; (a) glucan 1 which inhibits HK-yeast phagocytosis by up to 95% is not an activator of complement and therefore could not compete for the opsonizing proteins; (b) cycloheximide treatment in itself inhibited only partially HK-yeast phagocytosis whereas it inhibited the reexpression of the glucan receptors; (c) glucan 1 did not affect the phagocytosis of serum opsonized HK-yeast. Thus under the experimental conditions described, phagocytosis of HK-yeast by murine macrophages is mediated by and large by the beta-glucan receptors, while the mannose receptors and complement receptors do not contribute to the process.     

Interaction of the β-Transglycosylase of Trichoderma longibrachiatum with Cellulose

The effects of cellulose on the production and stimulation of β-transglycosylase were studied. The β-transglycosylase of Trichoderma longibrachiatum was produced specifically in the presence of cellulose in Czapeck-Dox medium containing sucrose as a sole carbon source. The enzyme activity was stimulated by the addition of cellulose in the reaction mixture, where the transfer reaction product (a water-insoluble glucan) was apparently synthesized on the surface of the added cellulose fibers.

The hyphal wall fraction of the fungus had the same stimulatory effect on β-transglycosylase as the cellulose fibers. A cellulose-like material in this fraction was found by partial acid hydrolysis and gas chromatography. Cellotriose was the smallest substrate effective for the synthesis of a water-insoluble glucan in the presence of cellulose by the β-transglycosylase, though a significant amount of glucan could not be synthesized without the addition of cellulose.     

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.     

UDP-glucose: Glucan Synthetase in Developing Cotton Fibers: II. Structure of the Reaction Product

The solubility properties, composition, and structure of the radioactive product synthesized from UDP-[¹⁴C]glucose by a highly active cotton fiber glucan synthetase have been determined. Product obtained under the following three different conditions was analyzed: at high and low substrate concentrations by detached fibers, and at high substrate concentrations with an isolated particulate preparation. The results of acetic and nitric acid digestion, enzyme digestion, total acid hydrolyses, periodate oxidation, partial acid hydrolyses, and methylation analyses all support the conclusion that the product of the glucan synthetase produced under all three assay conditions is a linear β-(1→3)-glucan.     

(1–3)-β-Glucan synthesis ofNeurospora crassa

(1–3)--d-Glucan synthase activity ofNeurospora crassa was localized to the plasma membrane by autoradiography of colloidal gold-labeled plasma membranes. The active site of glucan synthase for substrate hydrolysis was determined to be cytoplasmic facing. However, glucan synthase activity present in intact protoplasts was partially sensitive to Novozym 234 and to glutaraldehyde treatments, suggestive that enzyme activity is transmembrane. Enzyme activity also directed the formation of microfibrils in vitro. Taken together, these and previous results support the following scheme for glucan synthesis: 1. The sequential addition of glucose residues from UDP-glucose to glucan chains occurs on the cytoplasmically facing portion of glucan synthase. 2. As each glucan chain is synthesized, it is extruded to the extracytoplasmic side of the plasma membrane. 3. As each chain is extruded, it forms interchain hydrogen bonds with adjacent chains, resulting in glucan microfibril assembly.     

Short‐term lime pretreatment of poplar wood

Short‐term lime pretreatment uses lime and high‐pressure oxygen to significantly increase the digestibility of poplar wood. When the treated poplar wood was enzymatically hydrolyzed, glucan and xylan were converted to glucose and xylose, respectively. To calculate product yields from raw biomass, these sugars were expressed as equivalent glucan and xylan. To recommend pretreatment conditions, the single criterion was the maximum overall glucan and xylan yields using a cellulase loading of 15 FPU/g glucan in raw biomass. On this basis, the recommended conditions for short‐term lime pretreatment of poplar wood follow: (1) 2 h, 140°C, 21.7 bar absolute and (2) 2 h, 160°C, and 14.8 bar absolute. In these two cases, the reactivity was nearly identical, thus the selected condition depends on the economic trade off between pressure and temperature. Considering glucose and xylose and their oligomers produced during 72 h of enzymatic hydrolysis, the overall yields attained under these recommended conditions follow: (1) 95.5 g glucan/100 g of glucan in raw biomass and 73.1 g xylan/100 g xylan in raw biomass and (2) 94.2 g glucan/100 g glucan in raw biomass and 73.2 g xylan/100 g xylan in raw biomass. The yields improved by increasing the enzyme loading. An optimal enzyme cocktail was identified as 67% cellulase, 12% β‐glucosidase, and 24% xylanase (mass of protein basis) with cellulase activity of 15 FPU/g glucan in raw biomass and total enzyme loading of 51 mg protein/g glucan in raw biomass. Ball milling the lime‐treated poplar wood allowed for 100% conversion of glucan in 120 h with a cellulase loading of only 10 FPU/g glucan in raw biomass. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Identification of amino acid residues in Streptococcus mutans glucosyltransferases influencing the structure of the glucan product.

The glucosyltransferases (GTFs) of mutans streptococci are important virulence factors in the sucrose-dependent colonization of tooth surfaces by these organisms. To investigate the structure-function relationship of the GTFs, an approach was initiated to identify amino acid residues of the GTFs which affect the incorporation of glucose residues into the glucan polymer. Conserved amino acid residues were identified in the GTF-S and GTF-I enzymes of the mutans streptococci and were selected for site-directed mutagenesis in the corresponding enzymes from Streptococcus mutans GS5. Conversion of six amino acid residues of the GTF-I enzyme to those present at the corresponding positions in GTF-S, either singly or in multiple combinations, resulted in enzymes synthesizing increased levels of soluble glucans. The enzyme containing six alterations synthesized 73% water-soluble glucan in the absence of acceptor dextran T10, while parental enzyme GTF-I synthesized no such glucan product. Conversely, when residue 589 of the GTF-S enzyme was converted from Thr to either Asp or Glu, the resulting enzyme synthesized primarily water-insoluble glucan in the absence of the acceptor. Therefore, this approach has identified several amino acid positions which influence the nature of the glucan product synthesized by GTFs.     

A water-insoluble (1-->3)-beta-D-glucan from the alkaline extract of an edible mushroom Termitomyces eurhizus

A water-insoluble glucan, TEINS has been isolated from the hot alkaline extract of an edible mushroom Termitomyces eurhizus. The total carbohydrate content of the polysaccharide fraction was found to be 98.4%, and it was found to contain only glucose as the monosaccharide constituent. On the basis of total acid hydrolysis, a methylation experiment, periodate oxidation and (13)C NMR experiment, the repeating unit of the polysaccharide was established as: -->3)-beta-D-Glcp-(1-->.     

An alkali-soluble polysaccharide from the cell walls of Coprinus lagopus

An alkali-soluble polysaccharide was isolated from the purified mycelial walls of Coprinus lagopus. The hydrolysis products, optical rotation, and infrared spectrum indicate a -glucan. Hydrolysis of the glucan after permethylation gave only 2,3,4,6-tetra-, 2,4,6-tri-, and 2,4-di-O-methyl-d-glucose. These methylated sugars and their relative quantities reveal that the glucan is a polysaccharide containing -1,3-linked glucose units with about 14% of the sugars having 1,6-linked branch points. Partial hydrolysis of the product derived from Smith degradation of the glucan released laminaribiose and gentiobiose suggesting that the branches are generally longer than a single glucose unit.Adapted from a portion of a dissertation submitted in partial fullfillment of the requirements for the Ph.D. degree in botany at the University of Connecticut under the direction of Dr. Ralph P. Collins     

Surface carbohydrates of Rhizobium I. β-1, 2-Glucans

Because of increased interest in surface carbohydrates of Rhizobium in relation to host specificity, phenol — water extractions were carried out of whole cells of Rhizobium strains of the species R. leguminosarum, R. phaseoli, R. trifolii and R. meliloti.Fractionation of the crude extracts with cetavlon afforded polysaccharide mixtures, which were essentially free of RNA and acidic exopolysaccharide (EPS). They could be separated into a high molecular weight heteropolysaccharide fraction of lipopolysaccharide (LPS) nature and a low molecular weight glucan fraction. Glucan turned out to be the principal polysaccharide component of the cells (up to 10% of the dry cell weight), when cultivated in carbohydrate-rich media, and to be present as firmly attached capsular material.Glucan (mol wt 3000) structure was elucidated by methylation and periodate oxidation techniques. Methylation yielded 3, 4, 6-tri-O-methyl-d-glucose, characterized by GLC-MS, as the only product of hydrolysis of the fully methylated glucan. The glucan consumed 1 mole of periodate per mole anhydroglucose unit and gave sophorose on partial hydrolysis. From these data a linear -1,2-linked glucan structure was deduced. The occurrence of -1,2-glucan and the implications for the specific binding properties of Rhizobium cells are discussed.