Induction of α-amylase by maltooligosaccharides in Thermomonospora curvata

The action pattern of the α-amylase produced by Thermomonospora curvata is unique. Maltooligosaccharides (maltose to maltopentaose) were tested individually for their ability to induce α-amylase in this thermophilic actinomycete. Maltotetraose was the most inductive followed by maltotriose. Maltose was a good inducer of amylase production when used as sole carbon source, but had relatively little inductive capacity in the presence of either glucose or cellobiose. When cellobiose was added during exponential growth on maltose, maltose utilization and extracellular α-amylase accumulation were transiently inhibited. With maltotriose as the initial carbon source, addition of cellobiose did not inhibit the utilization of the trisaccharide; however, cellobiose, whether added during exponential growth or stationary phase, resulted in the rapid degradation of amylase when maltotriose was depleted from the medium. This inactivation did not appear to be a growth phase-induced phenomenon because stationary phase cells in the absence of cellobiose maintained their peak extracellular amylase level. This cellobiose-mediated α-amylase inactivation would be particularly important during production of the enzyme on a complex lignocellulosic substrate.     

Degradation of cellulosome-produced cello-oligosaccharides by an extracellular non-cellulosomal beta-glucan glucohydrolase, BglA, from Clostridium cellulovorans

Clostridium cellulovorans degrades cellulose efficiently to small oligosaccharides, which are used as an energy source. To characterize enzymes related to degrading small oligosaccharides, a gene was cloned for an extracellular non-cellulosomal beta-glucan glucohydrolase (BglA) classified as a family-1 glycosyl hydrolase in C. cellulovorans. Recombinant BglA (rBglA) had higher activity on long glucooligomers than on cellobiose. When cellulosomes and rBglA were incubated with cellulose, the oligosaccharides produced were degraded more effectively to cellobiose and glucose, than with cellulosomes alone, indicating that BglA facilitated the degradation of accessible cello-oligosaccharides produced from cellulose by C. cellulovorans cellulosomes. Thus, this is an example of an extracellular non-cellulosomal enzyme working in a cooperative manner with cellulosomes to degrade cellulose to sugars.     

Cellulase enzyme production during continuous culture growth of Sporotrichum (Chrysosporium) thermophile

Summary The cellulolytic fungus Sporotrichum (Chrysosporium) thermophile produces an extracellular cellobiose dehydrogenase during batch culture on cellulose or cellobiose. In chemostat culture at pH 5.6 on cellobiose this enzyme was produced in parallel with endo-cellulase. At pH 5.0 in continuous or fed-batch culture such a pattern was not evident. At constant growth rate in a chemostat with varying pH, activity of these enzymes was found to be poorly correlated. Thus the induction of cellobiose dehydrogenase shows a dependence on pH and cellobiose concentration which is different to that for endo-cellulase. The natural inducer of these enzymes and the role of cellobiose dehydrogenase remain to be elucidated.     

Phanerochaete chrysosporium beta-Glucosidases: Induction, Cellular Localization, and Physical Characterization

Phanerochaete chrysosporium produces intracellular soluble and particulate beta-glucosidases and an extracellular beta-glucosidase. The extracellular enzyme is induced by cellulose but repressed in the presence of glucose. The molecular weight of this enzyme is 90,000. The K(m) for p-nitrophenyl-beta-glucoside is 1.6 x 10 M; the K(i) for glucose, a competitive inhibitor, is 5.0 x 10 M. The K(m) for cellobiose is 5.3 x 10 M. The intracellular soluble enzyme is induced by cellobiose; this induction is prevented by cycloheximide. The presence of 300 mM glucose in the medium, however, had no effect on induction. The K(m) for p-nitrophenyl-beta-glucoside is 1.1 x 10 M. The molecular weight of this enzyme is approximately 410,000. Both enzymes have an optimal temperature of 45 degrees C and an E(act) of 9.15 kcal (ca. 3.83 x 10 J). The pH optima, however, were approximately 7.0 and 5.5 for the intracellular and extracellular enzymes, respectively.     

Component-specific stimulation of cellulase secretion in Thermomonospora curvata by the surfactant Tween 80

The non-ionic surfactant, Tween 80, stimulated the secretion of extracellular proteins by 35–140% in Thermomonospora curvata during growth on a variety of substrates. Cellulase secretion was also stimulated but fractionation of extracellular proteins by ion-exchange high performance liquid chromatography showed that this stimulation was largely confined to a single enzymatic component (or group of closely related components) active against crystalline cellulose. The surfactant's effect was more pronounced during growth on cellobiose octaacetate than on the soluble sugar, cellobiose, or on crystalline cellulose.     

Preferential Utilization of Cellobiose by Thermomonospora curvata

Thermomonospora curvata was cultivated on mineral salts medium containing glucose and cellobiose under conditions that increasingly favored the uptake of glucose. In each case cellobiose was utilized in preference to glucose and induced β-glucosidase and endoglucanase activity. [¹⁴C]glucose metabolism studies indicated that cellobiose was not cleaved by extracellular β-glucosidase and transported as glucose. No evidence of cellobiose phosphorylase or a cellobiose-specific phosphoenolpyruvate-phosphotransferase system was observed.     

Effect of extracellular pH on growth and proton motive force of Bacteroides succinogenes, a cellulolytic ruminal bacterium.

The utilization of cellulose or cellobiose by Bacteroides succinogenes S85 was severely inhibited at pH values of less than 5.7. Since low pH inhibited the utilization of both cellobiose and cellulose, changes in cellulase activity could not explain the effect. At an extracellular pH of 6.9, the pH gradient (delta pH) across the cell membrane was only 0.07 U. As extracellular pH declined from 6.9 to 5.7, intracellular pH decreased to a smaller extent than extracellular pH and delta pH increased. Below pH 5.7, there was a linear and nearly proportional decrease in intracellular pH. B. succinogenes took up the lipophilic cation tetraphenylphosphonium ion (TPP+) in the presence of cellobiose, and uptake was sensitive to the ionophore valinomycin. As pH was decreased with phosphoric acid, the cells lost TPP+ and electrical potential, delta psi, decreased. From extracellular pH 6.9 to 5.7, the decrease in delta psi was compensated for by an increase in delta pH, and the proton motive force ranged from 152 to 158 mV. At a pH of less than 5.7, there was a large decrease in proton motive force, and this decrease corresponded to the inhibition of cellobiose utilization.     

Do the extracellular enzymes cellobiose dehydrogenase and manganese peroxidase form a pathway in lignin biodegradation?

The extracellular enzyme manganese peroxidase is believed to degrade lignin by a hydrogen peroxide-dependent oxidation of Mn(II) to the reactive species Mn(III) that attacks the lignin. However, Mn(III) is not able to directly oxidise the non-phenolic lignin structures that predominate in native lignin. We show here that pretreatment of a non-phenolic lignin model compound with another extracellular fungal enzyme, cellobiose dehydrogenase, allows the manganese peroxidase system to oxidise this molecule. The mechanism behind this effect is demethoxylation and/or hydroxylation, i.e. conversion of a non-phenolic structure to a phenolic one, mediated by hydroxyl radicals generated by cellobiose dehydrogenase. This suggests that cellobiose dehydrogenase and manganese peroxidase may act in an extracellular pathway in fungal lignin biodegradation. Analytical techniques used in this paper are reverse-phase high-pressure liquid chromatography, gas chromatography connected to mass spectroscopy and UV-visible spectroscopy.     

Purification and characterization of a chloride-stimulated cellobiosidase from Bacteroides succinogenes S85.

A cellobiosidase with unique characteristics from the extracellular culture fluid of the anaerobic gram-negative cellulolytic rumen bacterium Bacteroides succinogenes grown on microcrystalline cellulose (Avicel) in a continuous culture system was purified to homogeneity by column chromatography. The enzyme was a glycoprotein with a molecular weight of approximately 75,000 and an isoelectric point of 6.7. When assayed at 39 degrees C and pH 6.5, the activity of the enzyme with p-nitrophenyl-beta-D-cellobioside as the substrate was stimulated by chloride, bromide, fluoride, iodide, nitrate, and nitrite, with maximum activation (approximately sevenfold) occurring at concentrations ranging from 1.0 mM (Cl-) to greater than 0.75 M (F-). The presence of chloride (0.2 M) did not affect the Km but doubled the Vmax. In the presence of chloride (0.2 M), the pH optimum of the enzyme was broadened, and the temperature optimum was increased from 39 to 45 degrees C. The enzyme released terminal cellobiose from cellotriose and cellobiose and cellotriose from longer-chain-length cellooligosaccharrides and acid-swollen cellulose, but it had no activity on cellobiose. The enzyme showed affinity for cellulose (Avicel) but did not hydrolyze it. It also had a low activity on carboxymethyl cellulose.     

Effect of extracellular pH on growth and proton motive force of Bacteroides succinogenes, a cellulolytic ruminal bacterium

The utilization of cellulose or cellobiose by Bacteroides succinogenes S85 was severely inhibited at pH values of less than 5.7. Since low pH inhibited the utilization of both cellobiose and cellulose, changes in cellulase activity could not explain the effect. At an extracellular pH of 6.9, the pH gradient (delta pH) across the cell membrane was only 0.07 U. As extracellular pH declined from 6.9 to 5.7, intracellular pH decreased to a smaller extent than extracellular pH and delta pH increased. Below pH 5.7, there was a linear and nearly proportional decrease in intracellular pH. B. succinogenes took up the lipophilic cation tetraphenylphosphonium ion (TPP+) in the presence of cellobiose, and uptake was sensitive to the ionophore valinomycin. As pH was decreased with phosphoric acid, the cells lost TPP+ and electrical potential, delta psi, decreased. From extracellular pH 6.9 to 5.7, the decrease in delta psi was compensated for by an increase in delta pH, and the proton motive force ranged from 152 to 158 mV. At a pH of less than 5.7, there was a large decrease in proton motive force, and this decrease corresponded to the inhibition of cellobiose utilization.     

Study of cellulases from a newly isolated thermophilic and cellulolytic <Emphasis Type="Italic">Brevibacillus</Emphasis> sp. strain JXL

A potentially novel aerobic, thermophilic, and cellulolytic bacterium designated as Brevibacillus sp. strain JXL was isolated from swine waste. Strain JXL can utilize a broad range of carbohydrates including: cellulose, carboxymethylcellulose (CMC), xylan, cellobiose, glucose, and xylose. In two different media supplemented with crystalline cellulose and CMC at 57°C under aeration, strain JXL produced a basal level of cellulases as FPU of 0.02 IU/ml in the crude culture supernatant. When glucose or cellobiose was used besides cellulose, cellulase activities were enhanced ten times during the first 24 h, but with no significant difference between these two simple sugars. After that time, however, culture with glucose demonstrated higher cellulase activities compared with that from cellobiose. Similar trend and effect on cellulase activities were also obtained when glucose or cellobiose served as a single substrate. The optimal doses of cellobiose and glucose for cellulase induction were 0.5 and 1%. These inducing effects were further confirmed by scanning electron microscopy (SEM) images, which indicated the presence of extracellular protuberant structures. These cellulosome-resembling structures were most abundant in culture with glucose, followed by cellobiose and without sugar addition. With respect to cellulase activity assay, crude cellulases had an optimal temperature of 50°C and a broad optimal pH range of 6–8. These cellulases also had high thermotolerance as evidenced by retaining more than 50% activity at 100°C after 1 h. In summary, this is the first study to show that the genus Brevibacillus may have strains that can degrade cellulose.     

Induction of Cellulases in chaetomium cellulolyticum by cellobiose

The production of extracellular cellulases by Chaetomium cellulolyticum could be induced by slow feeding of cellobiose to the cultures. Both the rate of production and the amount of activity were comparable to that obtained in batch cultivation on cellulose. The specific filter paper activity of 2.06 U per mg protein was almost two times higher than that obtained in cellulose medium. Cellulases were not induced when glucose was slowly fed to the cultures. Changing the feed stream from glucose to cellobiose resulted in a rapid accumulation of cellulases. Thus cellobiose has a similar role in cellulase induction in C. cellulolyticum, as earlier shown for Trichoderma reesei.     

Formation and release of cellulolytic enzymes during growth of Trichoderma reesei on cellobiose and glycerol

Summary Production and release of cellulolytic enzymes by Trichoderma reesei QM 9414 were studied under induced and non-induced conditions. For that purpose, a method was developmed to produce cellulases by Trichoderma reesei QM 9414 using the soluble inducer, cellobiose, as the only carbon source. The production was based on continuous feeding of cellobiose to a batch culture. For optimum production, the cellobiose supply had to be adjusted according to the consumption so that cellobiose was not accumulated in the culture. With a proper feeding program the repression and/or inactivation by cellobiose could be avoided and the cellulase production by Trichoderma reesei QM 9414 was at least equally as high as with cellulose as the carbon source.During the cultivation, specific activities against filter paper, carboxymethyl cellulose (CMC) and p-nitrophenyl glucoside were analyzed from the culture medium as well as from the cytosol and the cell debris fractions. There was a base level of cell debris bound hydrolytic activity against filter paper and p-nitrophenyl glucoside even in T. reesei grown non-induced on glycerol. T. reesei grown on cellobiose was induced to produce large amounts of extracellular filter paper and CMC hydrolyzing enzymes, which were actively released into the medium even in the early stages of cultivation. -Glucosidase was mainly detected in the cell debris and was not released unless the cells were autolyzing.     

Synthesis of cello-oligosaccharides from cellobiose with β-glucosidase II from Aspergillus niger

An extracellular -glucosidase II of Aspergillus niger catalysed the synthesis of cello-oligosaccharides from cellobiose (15%, w/v). The enzyme was stable at and below 4°C for at least 230 days and also stable at 30°C with the presence of 2.0% (w/v) cellobiose. The maximum yield of cello-oligosaccharides was about 30% (mol/mol), based on cellobiose (130 mg/mL) consumed. © Rapid Science Ltd. 1998     

哈氏噬纤维菌吸附纤维素的影响因素

[目的]探索哈氏噬纤维菌(Cytophaga hutchinsonii)吸附纤维素的作用机制.[方法]通过比较不同因素对哈氏噬纤维菌吸附纤维素的影响,包括:菌龄、pH、温度、表面电荷、细胞活力、细胞表面蛋白、细胞表面多糖以及纤维素类似物等,寻找在吸附过程中起重要作用的细胞成分.[结果]菌体经蛋白酶及热处理,对纤维素的吸附能力完全丧失;叠氮化钠、甲醛和戊二醛处理对菌体吸附能力影响不明显;菌体经刚果红和高碘酸钠处理,吸附能力变化不大;菌体对纤维素底物的吸附具有特异性,吸附作用不受纤维二糖和羧甲基纤维素的抑制.[结论]实验表明,哈氏噬纤维菌吸附纤维素的能力与菌体表面蛋白密切相关,而受细胞的代谢活性和胞外多糖影响较小,推测细胞表面可能存在特异性的纤维素结合蛋白.     

Formation, Location, and Regulation of Endo-1,4-beta-Glucanases and beta-Glucosidases from Cellulomonas uda

The formation and location of endo-1,4-beta-glucanases and beta-glucosidases were studied in cultures of Cellulomonas uda grown on microcrystalline cellulose, carboxymethyl cellulose, printed newspaper, and some mono- or disaccharides. Endo-1,4-Glucanases were found to be extracellular, but a very small amount of cell-bound endo-1,4-beta-glucanase was considered to be the basal endoglucanase level of the cells. The formation of extracellular endo-1,4-beta-glucanases was induced by cellobiose and repressed by glucose. Extracellular endoglucanase activity was inhibited by cellobiose but not by glucose. beta-Glucosidases, on the other hand, were formed constitutively and found to be cell bound. beta-Glucosidase activity was inhibited noncompetitively by glucose. Some characteristics such as the optimal pH for and the thermostability of the endoglucanases and beta-glucosidases and the end products of cellulose degradation were determined.     

An adhesion-defective mutant of Ruminococcus albus SY3 is impaired in its capability to degrade cellulose

An adhesion-defective mutant of Ruminococcus albus SY3 was isolated by a subtractive enrichment procedure, which involved repetitive adsorption of cellobiose-grown cells to cellulose. The growth characteristics of the mutant were compared with those of the wild type. Like the wild-type cells, the mutant was capable of growing on soluble substrates, i.e. cellobiose and xylan. However, in contrast to the wild type strain, the mutant was impaired in its capacity to utilize insoluble substrates, e.g. crystalline cellulose, acid-swollen cellulose or alfalfa cell walls. Scanning electron microscopy revealed protuberance-like surface structures on the wild-type strain which were absent on the mutant. The levels of endoglucanase and xylanase enzymatic activities released into the extracellular culture fluid were higher in the wild type compared to the mutant. However, Avicelase activity was not detected in the extracellular culture fluid of either strains when grown on cellobiose.     

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.     

Cellobiose dehydrogenase production by Sclerotium species pathogenic to plants

AIMS: Evaluation of various Sclerotium spp. as producers of the biotechnologically attractive enzyme cellobiose dehydrogenase. METHODS AND RESULTS: All isolates of S. coffeicola, S. delphinii and S. rolfsii grown in shaken flasks on a cellulose-based medium produced appreciable amounts of the extracellular enzyme cellobiose dehydrogenase. CONCLUSIONS: Cellobiose dehydrogenase seems to play an important role in phytopathogenic Sclerotium spp.; a possible function could be either in the degradation of rigid lignocellulose or as a protective mechanism against toxic quinones. SIGNIFICANCE AND IMPACT OF THE STUDY: S. coffeicola and S. delphinii were identified as potent, not-yet-described producers of cellobiose dehydrogenase (CDH). The high levels of intact CDH produced by the different Sclerotium species should make them attractive producers for further studies and applications.