Column cellulose hydrolysis reactor: Cellulase adsorption profile

Summary A column cellulose hydrolysis reactor was set up using a single passage of cellulase enzyme which was followed with a continuous percolation of buffer. Hydrolysis rates were found to decline precipitously upon the removal of the non-adsorbed cellulase components. By comparing specific activities of the cellulase before and after adsorption on the cellulose column, it was concluded that the adsorption efficiencies for the cellulase components decreased from exoglucanase (1,4--d-glucan cellobiohydrolase EC 3.2.1.91) to endoglucanase [1,4-(1,3;1,4)--d-glucan 4-glucanohydrolase, EC 3.2.1.4] to -glucosidase (-d-glucoside glucohydrolase, EC 3.2.1.21). Of the adsorbed cellulase components, the rate of endoglucanase leaching from the cellulose column was 20 times that for the exoglucanase despite the greater adsorption efficiency of the latter. By analysing the cellulase components which were bound and not bound by the cellulose column and comparing them with a purified exoglucanase enzyme on sodium dodecyl sulfate polyacrylamide gels, it was confirmed that the major cellulase component adsorbed to the cellulose column was an exoglucanase component. The resultant loss of other cellulase components from the reactor was probably the cause for the much reduced rate of cellulose hydrolysis when these components were flushed out of the column.     

Shear deactivation of cellulase, exoglucanase, endoglucanase, and beta-glucosidase in a mechanically agitated reactor.

Shear deactivation of cellulase and its major component enzymes, viz., exoglucanase (exo-1,4-beta-D-glucan-4-cellobiohydrolase), endoglucanase (endo-1,4-beta-D-glucanhydrolase), and 1,4-beta-glucosidase, was carried out by exposing cellulase to shear in a mechanically agitated reactor in the presence as well as in the absence of the substrate cellulose. Cellulase was found to undergo deactivation when subjected to shear, and the extent of deactivation increased with increasing speed of agitation. Among the three major component enzymes of cellulase, exoglucanase showed rapid deactivation and contributed the most to cellulase deactivation. The presence of a substrate did not affect the deactivation of cellulase.     

Adsorption of cellulase from Trichoderma viride on cellulose

The adsorption of cellulase from Trichoderma viride (Meicelase CEP) on the surface of pure cellulose was studied. The adsorption was found to obey apparently the Langmuir isotherm. From the data concering the effects of temperature and the crystallinity of cellulose on the Langmuir adsorption parameters, the characteristics of the adsorption of the individual cellulase components, namely CMCase (endoglucanase) and Avicelase (exoglucanase), were discussed. While beta-glucosidase also adsorbed on the surface of cellulose at 5 degrees C, it did not at 50 degrees C.     

Enhanced cellulase production using Solka Floc in a fed-batch fermentation

Summary Use of a fed-batch mode of cultivation of T. reesei has permitted high concentrations of substrate to be consumed. This has resulted in the production of high titre cellulase preparations around 30 FPU/ml at high volumetric productivities (177 IU/L.hr).Perhaps the most obvious area for major improvement in the process of cellulose utilization is the production of cellulase enzyme for hydrolysis of wood and agricultural residues. It has been estimated that some 50% of the cost of producing glucose from cellulosic material is attributable to enzyme production alone (Perez, et al., 1980). Improvements in the area would therefore have a dramatic impact, and are of paramount importance if economical hydrolysis processes are to be realized. The first major thrust in the area has been the development of improved mutant strains of T. reesei, free from catabolite repression and capable of constitutive cellulase production (Montenecourt and Eveleigh, 1977; Gallo, 1982).While this effort continues to develop further high yielding mutants, improvement must also come from developments in fermentation techniques. A major advance is the use of fed-batch cultivation, which provides a means of avoiding the agitation and aeration difficulties, as well as repression effects encountered with high substrate concentration batch fermentation. This report briefly compares batch and fed-batch operation over a range of substrate concentrations.     

Purification and characterization of endoglucanase and exoglucanase components from Trichoderma viride

Major cellulase components—four endoglucanases (Endo I, II, III and IV) and one exoglucanase (Exo II)—were isolated from a commercial cellulase preparation derived from Trichoderma viride by a series of chromatographic procedures. The average molecular weights were determined by SDS-polyacrylamide gel electrophoresis. Endos I, III and IV, with M_rs of 52,000, 42,000 and 38,000, respectively, exhibited a more random hydrolytic mode on carboxymethylcellulose (CMC) than Endo II, which has an M_r of 60,000. Endo II showed low activity towards CMC, but out of the four purified endoglucanases this enzyme had the highest specific activity against Avicel. In the hydrolysis of H₃PO₄-swollen cellulose by Endos I, III and IV, cellobiose was the major product, but equimolar amounts of glucose and cellobiose were formed by Endo II. Exo II, with an M_r of 62,000, released cellobiose as the main product in the hydrolysis of H₃PO₄-swollen cellulose, but glucose was negligible. The combination of Endo I, II, III or IV with Exo II resulted in a synergistic effect in the degradation of Avicel at various combination ratios of these enzymes; the specific optimum ratio of endoglucanase to exoglucanase was largely dependent upon the random hydrolytic mode of the endoglucanase. On the other hand, adsorption of cellulase components was found apparently to obey the Langmuir isotherm, and the thermodynamic parameter (ΔH) was calculated from the adsorption equilibrium constant (K). The enthalpies of adsorption of the endoglucanases were in the range of −2.6–−7.2 KJmol⁻¹, much smaller than that of Exo II (−19.4 KJmol⁻¹). This suggest that Exo II shows stronger preferential adsorption than endoglucanases, and that the enthalpy of adsorption will be effective in distinguishing endoglucanase from exoglucanase.     

Sporotrichum thermophile Growth, Cellulose Degradation, and Cellulase Activity

The activity of components of the extracellular cellulase system of the thermophilic fungus Sporotrichum thermophile showed appreciable differences between strains; β-glucosidase (EC 3.2.1.21) was the most variable component. Although its endoglucanase (EC 3.2.1.4) and exoglucanase (EC 3.2.1.91) activities were markedly lower, S. thermophile degraded cellulose faster than Trichoderma reesei. The production of β-glucosidase lagged behind that of endoglucanase and exoglucanase. The latter activities were produced during active growth. When growth was inhibited by cycloheximide treatment, the hydrolysis of cellulose was lower than in the control in spite of the presence of both endoglucanase and exoglucanase activities in the culture medium. Degradation of cellulose was a growth-associated process, with cellulase preparations hydrolyzing cellulose only to a limited extent. The growth rate and cell density of S. thermophile were similar in media containing cellulose or glucose. A distinctive feature of fungal development in media incorporating cellulose or lactose (inducers of cellulase activity) was the rapid differentiation of reproductive units and autolysis of hyphal cells to liberate propagules which were capable of renewing growth immediately.     

Studies on the mechanism of enzymatic hydrolysis of cellulosic substances.

Most cellulosic substances contain appreciable amounts of cellulose and hemicellulose, which on enzymatic hydrolysis mainly yield a mixture of glucose, cellobiose, and xylose. In this paper, studies on the mechanisms of hydrolysis of bagasse (a complex native cellulosic waste left after extraction of juice from cane sugar) by the cellulase enzyme components are described in light of their adsorption characteristics. Simultaneous adsorption of exo- and endoglucanases on hydrolyzable cellulosics is the causative factor of the hydrolysis that follows immediately after. It supports the postulate of synergistic enzyme action proposed by Eriksson. Xylanase pretreatment enhanced the hydrolysis of bagasse owing to the creation of more accessible cellulosic regions that are readily acted upon by exo- and endoglucanases. The synergistic action of the purified exoglucanase, endoglucanase, and xylanse has been found to be most effective for hydrolysis of bagasse but not for pure cellulose. Significant quantities of glucose are produced in beta-glucosidase-free cellulase action on bagasse. Individual and combined action of the purified cellulase components on hydrolysis of native and delignified bagasse are discussed in respect to the release of sugars in the hydrolysate.     

Expression of a bifunctional cellulase with exoglucanase and endoglucanase activities to enhance the hydrolysis ability of cellulase from a marine Aspergillus niger

Low exoglucanase and endoglucanase activities of marine Aspergillus niger cellulase decreased the hydrolyzing ability of cellulase. To increase the activity of halostable cellulase obtained from a marine A. niger, a cellulase with endoglucanase and exoglucanase activity was efficiently expressed by constructing a vector with promoter glaA. Exoglucanase and endoglucanase activities increased from 0.21 and 4.51 U/ml of the original strain to 0.89 U/ml and 15.12 U/ml of the transformant, respectively. Filter paper activity (FPA) increased by 7.1 folds from 0.63 to 4.47 U/ml. The release of glucose by hydrolysis of wheat straw with cellulase from the transformant was 1.37 folds higher than that with cellulase from the original strain under high salinity condition. Cellulase with endoglucanase and exoglucanase activities could be well expressed in marine A. niger. The cellulase from the transformant not only showed higher activity, but also retained halostability. An appreciate proportion of β-glucosidase, exoglucanase, endgolucanasein cellulase was important for hydrolyzing cellulose.     

Cellulolytic and physiological properties of Clostridium thermocellum

Three strains of Clostridium thermocellum obtained from various sources were found to have nearly identical deoxyribonucleic acid guanosine plus cytosine contents that ranged from 38.1–39.5 mole-%. All strain examined fermented only cellulose and cellulose derivatives, but not glucose, or xylose or other sugars. The principal cellulose fermentation products were ethanol, lactate, acetate, hydrogen and carbon dioxide. Growth of C. thermocellum on cellulose resulted in the production of extracellular cellulase that was non-oxygen labile, was thermally stable at 70° C for 45 min and adsorbed strongly on cellulose. Production of cellulase during fermentation correlated linearly with growth and cellulose degradation. Both the yield and specific activity of crude cellulase varied considerably with the specific growth substrates. Highest cellulase yield was obtained when grown on native cellulose, -cellulose and low degree of polymerization cellulose but not carboxymethylcellulose or other carbohydrate sources. Cellulase activity was not detected when cells were grown on cellobiose. Crude extracellular protein preparations lacked proteolytic and cellobiase activity. The pH and temperafure optima for endoglucanase activity were 5.2 and 65° C, respectively, while that of the exoglucanase activity were 5.4 and 64° C, respectively. The specific activity at 60° c for exoglucanase and endoglucanase of crude cellulase obtained from cells grown on cellulose (MN 300) was 3.6 moles reducing sugar equivalents released per h (unit)/mg of protein and 1.5 mole reducing sugar equivalent released per min (unit)/mg of protein, respectively. The yield of endoglucanase was 125 units per g of cellulose MN 300 degraded and that of exoglucanase was 300 units per g of cellulose MN 300 degraded. Glucose and cellobiose were the hydrolytic end products of crude cellulase action on cellulose, cellotraose and cellotriose in vitro.     

Some characteristics of the cellulolytic enzyme system of Chaetomium cellulolyticum

Summary The effects of pH and temperature on the activities of endoglucanase, exoglucanase and -glucosidase of C. cellulolyticum were studied. Thermal stability of these enzymes was characterized. Enzymatic hydrolyses of cellulose were performed yielding predominantly glucose and cellobiose. Glucose was shown to be a potent inhibitor of its own formation in cellulose saccharification.     

Adsorption of cellulase on cellulose: Effect of physicochemical properties of cellulose on adsorption and rate of hydrolysis

In the cellulase-cellulose reaction system, the adsorption of cellulase on the solid cellulose substrate was found to be one of the important parameters that govern the enzymatic hydrolysis rate of cellulose. The adsorption of cellulase usually parallels the rate of hydrolysis of cellulose. The affinity for cellulase varies depending on the structural properties of cellulose. Adsorption parameters such as the half-saturation constant, the maximum adsorption constant, and the distribution coefficient for both the cellulase and cellulsoe have been experimentally determined for several substrates. These adsorption parameters vary with the source of cellulose and the pretreatment methods and are correlated with the crystallinity and the specific surface area of cellulose substrates. The changing pattern of adsorption profile of cellulase during the hydrolysis reaction has also been elucidated. For practical utilization of cellulosic materials, the cellulose structural properties and their effects on cellulase adsorption, and the rate of hydrolysis must be taken into consideration.     

Enhancement of enzymatic hydrolysis of cellulose by surfactant

Effects of surfactants on enzymatic saccharification of cellulose have been studied. Nonionic, amphoteric, and cationic surfactants enhanced the saccharification, while anionic surfactant did not. Cationic and anionic surfactants denatured cellulase in their relatively low concentrations, namely, more than 0.008 and 0.001%, respectively. Using nonionic surfactant Tween 20, which is most effective to the enhancement (e.g., the fractional conversion attained by 72 h saccharification of 5 wt % Avicel in the presence of 0.05 wt % Tween 20 is increased by 35%), actions of surfactant have been examined. As the results, it was suggested that Tween 20 plays an important role in the hydrolysis of crystalline cellulose and that Tween 20 disturbs the adsorption of endoglucanase on cellulose, i.e., varies the adsorption balance of endo- and exoglucanase, resulting in enhancing the reaction. The influence of Tween 20 to the saccharification was found to remain in simultaneous saccharification and fermentation of Avicel.     

Effects of agitation level on the adsorption, desorption, and activities on cotton fabrics of full length and core domains of EGV (Humicola insolens) and CenA (Cellulomonas fimi)

The activities (at pH 7 and 50 degrees C) of purified EGV (Humicola insolens) and CenA (Cellulomonas fimi) were determined on cotton fabrics at high and low levels of mechanical agitation. Similar activity measurements were also made by using the core domains of these cellulases. Activity experiments suggested that the presence of cellulose binding domains (CBDs) is not essential for cellulase performance in the textile processes, where high levels of mechanical agitation are applied. The binding reversibilities of these cellulases and their cores were studied by dilution of the treatment liquor after equilibrium adsorption. EGV showed low percentage of adsorption under both levels of agitation. It was observed that the adsorption/desorption processes of cellulases are enhanced by higher mechanical agitation levels and that the binding of cellulase with CBD of family I (EGV) is more reversible than that of CBD of the cellulase of family II (CenA).     

Production and characterization of monoclonal antibodies to the cellulases produced by Talaromyces emersonii CBS 814.70

Talaromyces emersonii CBS 814.70 produces a cellulase complex during growth on cellulose and lactose. The cellulase system consists of endoglucanase, exoglucanase and ß-glucosidase, and each type of enzyme exists as isoforms. In order to probe this complex system with respect to studying structure-function relationships of the components and also to pursue our studies on the genetic control of expression of the various components in the T. emersonii system, we decided to produce highly specific monoclonal antibodies to those cellulase components. Here we report on the production of the first three monoclonal antibodies to be made to the T. emersonii cellulase system. One of those antibodies is specific for endoglucanase and has been useful in demonstrating differences in the isoforms of endoglucanase produced during growth of the organism on lactose- and cellulose-containing media. The antibody is highly sensitive, being capable of detective less than 50 ng of endoglucanase in an ELISA system. The antibody also cross reacts with one major endoglucanase produced by Trichoderma reesei.     

Extracellular cellulase production by tropical isolates of Aureobasidium pullulans

Cellulase production by Aureobasidium pullulans from the temperate regions has remained speculative, with most studies reporting no activity at all. In the current study, tropical isolates from diverse sources were screened for cellulase production. Isolates were grown on a synthetic medium containing cell walls of Msasa tree (Brachystegia sp.) as the sole carbon source, and their cellulolytic activities were measured using carboxymethyl cellulose and alpha-cellulose as substrates. All isolates studied produced carboxymethyl cellulase (endoglucanase) and alpha-cellulase (exoglucanase) activity. Endoglucanase-specific activities of ten selected isolates ranged from 2.375 to 12.884 micromol glucose.(mg protein)-1.h-1, while activities on alpha-cellulose (exoglucanase activity) ranged from 0.293 to 22.442 micromol glucose.(mg protein)-1.day-1. Carboxymethyl cellulose induced the highest cellulase activity in the selected isolates, while the isolates showed variable responses to nitrogen sources. The current study indicates that some isolates of A. pullulans of tropical origin produce significant extracellular cellulolytic activity and that crude cell walls may be good inducers of cellulolytic activity in A. pullulans.     

Simultaneous saccharification and fermentation of cellulose: effect of ethanol on enzymatic saccharification of cellulose

It was confirmed that simultaneous saccharification and fermentation are effective for accelerating enzymatic saccharification of cellulose. In this work, the effects of ethanol on the saccharification of tissue paper by Trichoderma cellulase (Meicelase CEPB) have been investigated. The following results were obtained. (1) Saccharification was inhibited by at least 0.2M ethanol. (2) Less than 4M ethanol did not affect the enzymatic activities of beta-glucosidase and endoglucanase (C(x)) at all. The thermal stability of endoglucanase was not also varied by ethanol. (3) It is suggested that ethanol depresses the adsorption of exoglucanase on cellulose. (4) The rate expression of saccharification of cellulose in the presense of ethanol is proposed. (5) The inhibititory effect of ethanol was found to become more significant in the later stages of the reaction than just the initial stage.     

Factors Involved in Hydrolysis of Microcrystalline Cellulose by Acetivibrio cellulolyticus

Acetivibrio cellulolyticus cellulase obtained by the water elution of residual cellulose from the growth medium was compared with the cellulase activity present in culture supernatants. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis indicated that water elution released most of the protein bands which adhered to undigested cellulose from the culture medium. The enzyme in the culture supernatant and that eluted from residual cellulose had specific activities for Avicel hydrolysis that were 20- to 40-fold greater than that of Trichoderma reesei cellulase. However, Ca²⁺ and a reducing agent such as dithiothreitol were required for maximum Avicel hydrolysis rates by these A. cellulolyticus enzyme preparations. The effect of these agents on p-nitrophenyl lactopyranoside hydrolysis suggested that they were required by an exoglucanase component. Supernatant enzyme preparations contained large amounts of carbohydrate which was separated from most of the cellulase protein by phenyl-Sepharose chromatography. Removal of this carbohydrate, which interfered with protein fractionations, allowed for an activity stain analysis of the supernatant enzyme.     

Cellulase adsorption and relationship to features of corn stover solids produced by leading pretreatments

Although essential to enzymatic hydrolysis of cellulosic biomass to sugars for fermentation to ethanol or other products, enzyme adsorption and its relationship to substrate features has received limited attention, and little data and insight have been developed on cellulase adsorption for promising pretreatment options, with almost no data available to facilitate comparisons. Therefore, adsorption of cellulase on Avicel, and of cellulase and xylanase on corn stover solids resulting from ammonia fiber expansion (AFEX), ammonia recycled percolation (ARP), controlled pH, dilute acid, lime, and sulfur dioxide (SO₂) pretreatments were measured at 4°C. Langmuir adsorption parameters were then estimated by non‐linear regression using Polymath software, and cellulase accessibility to cellulose was estimated based on adsorption data for pretreated solids and lignin left after carbohydrate digestion. To determine the impact of delignification and deacetylation on cellulose accessibility, purified CBHI (Cel7A) adsorption at 4°C and hydrolysis with whole cellulase were followed for untreated (UT) corn stover. In all cases, cellulase attained equilibrium in less than 2 h, and upon dilution, solids pretreated by controlled pH technology showed the greatest desorption followed by solids from dilute acid and SO₂ pretreatments. Surprisingly, the lowest desorption was measured for Avicel glucan followed by solids from AFEX pretreatment. The higher cellulose accessibility for AFEX and lime pretreated solids could account for the good digestion reported in the literature for these approaches. Lime pretreated solids had the greatest xylanase capacity and AFEX solids the least, showing pretreatment pH did not seem to be controlling. The 24 h glucan hydrolysis rate data had a strong relationship to cellulase adsorption capacities, while 24 h xylan hydrolysis rate data showed no relationship to xylanase adsorption capacities. Furthermore, delignification greatly enhanced enzyme effectiveness but had a limited effect on cellulose accessibility. And because delignification enhanced release of xylose more than glucose, it appears that lignin did not directly control cellulose accessibility but restricted xylan accessibility which in turn controlled access to cellulose. Reducing the acetyl content in corn stover solids significantly improved both cellulose accessibility and enzyme effectiveness. Biotechnol. Bioeng. 2009;103: 252–267. © 2009 Wiley Periodicals, Inc.     

Cellulase and hemicellulase production by Cellulomonas flavigena NIAB 441

Summary Cellulomonas flavigena (strain NIAB 441) produced cellulase and hemicellulase activities when grown on Leptochloa fusca L. Kunth (Kallar grass), found to be the best inducer for enzyme production. The enzyme possessed the potential to saccharify bagasse, Kallar grass straw, wheat straw, carboxymethyl cellulose (CMC) and xylan to reducing sugars.     

Isolation, characterization and manipulation of cellulase genes

The complete hydrolysis of cellulose requires a number of different enzymes including endoglucanase, exoglucanase and beta-glucosidase. These enzymes function in concert as part of a 'cellulase'complex called a cellulosome. In order (i) to develop a better understanding of the biochemical nature of the cellulase complex as well as the genetic regulation of its integral components and (ii) to utilize cellulases either as purified enzymes or as part of an engineered organism for a variety of purposes, researchers have, as a first step, used recombinant DNA technology to isolate the genes for these enzymes from a variety of organisms. This review provides some perspective on the current status of the isolation, characterization and manipulation of cellulase genes and specifically discusses (i) strategies for the isolation of endoglucanase, exoglucanase and beta-glucosidase genes; (ii) DNA sequence characterization of the cellulase genes and their accompanying regulatory elements; (iii) the expression of cellulase genes in heterologous host organisms and (iv) some of the proposed uses for isolated cellulase genes.