Subcellulosome preparation with high cellulase activity from Clostridium thermocellum.

We have prepared a much simpler cellulase preparation than that of cellulosomes from the extracellular broth of Clostridium thermocellum. This "subcellulosome" preparation from C. thermocellum was obtained by column chromatography on CM-Bio-Gel A and then on a lectin-affinity material (Jacalin). The subcellulosome preparation is a macromolecular complex, composed of six main protein subunits (molecular weight, 210,000 to 58,000) revealed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The specific activities of carboxymethylcellulase (CMCase) and Avicelase are 15- and 8-fold-higher, respectively, than those of crude extracellular cellulase. We could not further fractionate this preparation without denaturing it. The optimum pH and temperature of the subcellulosome preparation are 5.5 to 7.0 and 70 degrees C for CMCase and 5.5 to 7.0 and 65 degrees C for Avicelase. The subcellulosome preparation acted on various types of carboxymethyl cellulose, cellulose, and p-nitrophenyl-beta-D-cellobioside but not on p-nitrophenyl-beta-D-glucoside. Sulfhydryl reagents and N-bromosuccinimide inhibited both CMCase and Avicelase activities, whereas EDTA and o-phenanthroline inhibited Avicelase activity only.     

Streptomyces flavogriseus cellulase: Evaluation under various hydrolysis conditions

Streptomyces flavogriseus CMCase and Avicelase were very stable at 30 degrees C but not at 40 degrees C or higher. beta-Glucosidase was less stable at all temperatures tested. Stabilities were similar at pH values between 5.5 and 7, the optimal range for enzyme activity. Cellulose solubilizing activity was reduced by 40% at a cellobiose concentration of 150mM but glucose inhibited activity by only 10% at this concentration. beta-Glucosidase was inhibited by 40% at a glucose concentration of 10mM (ten times the substrate concentration). Relatively dilute S. flavogriseus cellulase extensively hydrolysed acid-swollen cellulose at concentrations as high as 10%. More highly crystalline forms of cellulose were more resistant to attack.     

Identification of Mycelium-Associated Cellulase from Streptomyces reticuli

Among 180 Streptomyces strains tested, 25 were capable of hydrolyzing microcrystalline cellulose (Avicel) at 30°C. Streptomyces reticuli was selected for further studies because of its ability to grow at between 30 and 50°C on Avicel. Enzymatic activities degrading Avicel, carboxymethyl cellulose, and cellobiose were found both in the culture supernatant and in association with the mycelium and crystalline substrate. The bound enzymes were efficiently solubilized by repeated washes with buffer of low ionic strength (50 mM Tris hydrochloride [pH 7.5]) and further purified by fast protein liquid chromatography. A high-molecular-weight Avicelase of >300 kilodaltons could be separated from carboxymethyl cellulase (CMCase) and β-glucosidase activities (molecular mass, 40 to 50 kilodaltons) by gel filtration on Superose 12. The CMCase fraction was resolved by Mono Q anion-exchange chromatography into two enzymes designated CMCase 1 and CMCase 2. The β-glucosidase activity was found to copurify with CMCase 2. The purified cellulase components showed optimal activity at around pH 7.0 and temperatures of between 45 and 50°C. Avicelase (but not CMCase) activity was stimulated significantly by the addition of CaCl₂.     

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.     

Carboxymethylcellulase and avicelase activities from a cellulolytic Clostridium strain A11

The extracellular cellulase enzyme system of Clostridium A11 was fractionated by affinity chromatography on Avicel: 80% of the initial carboxymethylcellulase (CMCase) activity was adhered. This cellulase system was a multicomponent aggregate. Several CMCase activities were detected, but the major protein P1 had no detectable activity. Adhered and unadhered cellulases showed CMCase activity with the highest specific activity in Avicel-adhered fraction. However, only afhered fractions could degrade Avicel. Thus, efficiency of the enzymatic hydrolysis of Avicel was related to the cellulase-adhesion capacity. Carboxymethylcellulase and Avicelase activities were studied with the extracellular enzyme system and cloned cellulases. Genomic libraries from Clostridium A11 were constructed with DNA from this Clostridium, and a new gene cel1 was isolated. The gene(s) product(s) from cel1 exhibited CMCase and p-nitrophenylcellobiosidase (pNPCbase) activities. This cloned cellulase adhered to cellulose. Synergism between adhered enzyme system and cloned endoglucanases was observed on Avicel degradation. Conversely, no synergism was observed on CMC hydrolysis. Addition of cloned endoglucanase to cellulase complex led to increase of the V_max without significant K _m variation. Cloned endoglucanases can be added to cellulase complexes to efficiently hydrolyze cellulose.     

Simultaneous production and induction of cellulolytic and xylanolytic enzymes in aStreptomyces sp.

Extracellular cellulolytic and xylanolytic enzymes ofStreptomyces sp. EC22 were produced during submerged fermentation. The cell-free culture supernatant of the streptomycete grown on microcrystalline cellulose contained enzymes able to depolymerize both crystalline and soluble celluloses and xylans. Higher cellulase and xylanase activities were found in the cell-free culture supernatant of the strain when grown on microcrystalline cellulose than when grown on xylan. Total cellulase and endoglucanase [carboxymethyl-cellulase (CMCase)] activities reached maxima after 72 h and xylanase activity was maximal after 60h. Temperature and pH optima were 55°C and 5.0 for CMCase activity and 60°C and 5.5 for total crystalline cellulase and xylanase activities. At 80°C, approximate half-lives of the enzymes were 37, 81 and 51 min for CMCase, crystalline cellulose depolymerization and xylanase, respectively.     

Production of cellulase enzymes during the solid-state fermentation of empty palm fruit bunch fiber

Penicillium verruculosum COKE4E is a fungal strain isolated from bituminous coal. The microorganism cultivated in a minimal medium supplemented with Avicel, carboxymethylcellulose, and oat spelt xylan produced cellulase enzymes as exhibiting carboxymethylcellulase (CMCase), Avicelase, xylanase, and cellobiosidase activities. In this study, the productivity of the extracellular enzymes in the strain was evaluated by using empty palm fruit bunch fiber (EPFBF), a lignocellulosic biomass, as a substrate for solid-state bioconversion. The highest cellulase activities were observed after 6 days of fermentation at pH 6.0 and 30 °C. The enzymes were secreted as cellulosomes for the degradation of EPFBF as a sole carbon source. Focused ion beam analysis showed that P. verruculosum COKE4E produced cellulolytic enzymes that were able to effectively biodegrade EPFBF during solid-state fermentation. In this process, 6.5 U of CMCase, 6.8 U of Avicelase, and 8.8 U of xylanase per gram of dry solid EPFBF were produced. These results demonstrate that EPFBF may be a potential raw material in solid-state fermentation for the production of cellulase enzymes to be used for biofuel production.     

New isolate of Streptomyces sp. with novel thermoalkalotolerant cellulases

A Streptomyces sp. was isolated that produced novel thermoalkalotolerant cellulase activity after growth on crystalline cellulose at 50°C. Three major components of the cellulases (CMCase, Avicelase and cellobiase) were produced with maximal activities (11.8, 7.8 and 3.9 IU/ml) and maximum specific activities 357, 276 and 118 IU/mg protein, respectively, after 120 h growth. Maximum CMCase activity was between 50 and 60°C measured over 3 h. The enzyme also retained 88% of its maximum activity at 70°C and pH 5, and 80% of the activity at pH 10 and 50°C when assayed after 1 h. After incubation at 40°C for 1 h with commercial detergent (Tide) at pH 11, 95% activity was retained. The enzyme mixture produced glucose from crystalline cellulose.     

Comparison of Cellulolytic Activities in Clostridium thermocellum and Three Thermophilic, Cellulolytic Anaerobes

Avicelase, carboxymethyl cellulase (CMCase), and β-glucosidase activities have been compared between Clostridium thermocellum and three extremely thermophilic, cellulolytic anaerobes, isolates TP8, TP11, and KT8. The three isolates were all small, gram-negative staining, oval-ended rods which occurred singly and, at exponential phase, in long chains. They were nonflagellated and no spores were visible. The KT8 and TP11 isolates caused clumping of the cellulose during growth. In all four organisms the CMCase activity paralleled cell growth; however, in C. thermocellum and TP8 the avicelase activity did not increase until early stationary phase. Total CMCase activity in C. thermocellum was significantly higher than in the three isolates; however, avicelase activities were much more comparable among the four organisms. C. thermocellum produced higher levels of ethanol, and all four organisms produced similar concentrations of acetate. The amounts of free and bound CMCase and avicelase activities were investigated. In C. thermocellum and TP8 most of the CMCase and avicelase activities were bound to the cellulose in the medium. In contrast, most of the CMCase activity in TP11 and KT8 was free in the culture supernatant; a significant percentage of avicelase activity was also free. The TP8 isolate was also grown on a defined medium with urea as sole nitrogen source and cellulose serving as the carbon source. Under these conditions the pattern of enzyme production was the same as that in the enriched medium, although the level of that production was considerably reduced.     

Saccharification of cellolulose by the cellulolytic enzyme system of Thermonospora sp. I. Stability of cellulolytic activities with respect to time,temperature, and pH

The stabilities and optima with respect to temperature and pH of the β-glucosidase, Avicelase, and carboxymethylcellulase (CMCase) activity of Thermomonospora sp., in the culture filtrate, culture whole broth, and filtrate after sonication of culture solids, are reported. The β-glucosidase is cell associated and has an optimal activity at about pH 6.5 and 55°C. In the whole culture broth, it has a half-life of about 8 hr at 55°C and less than 1 hr at 60°C, while the half-life of the activity in the sonicated, cell-free filtrate is less than 1 hr at 55°C. The Avicelase and CMCase activities occur in the extracellular culture fluid and have optima at about pH 7.0 and 5.9, and 65 and 70°C, respectively. The CMCase activity is stable over 24 hr at 60°C, but declines by 50% in the same period at 65°C. The Avicelase activity declines by 15% over 24 hr at 55°C, and by 50% at 60°C. The highest pH studied (pH 7.3) was the most destabilizing for all three activities. The thermostable characteristics of the cellulases from Themomonospora appear to make them suitable for commercial saccharification processes operating at elevated temperatures.     

Cellulase system of a free-living, mesophilic clostridium (strain C7).

The enzymatic activity responsible for crystalline cellulose degradation (Avicelase activity) by a mesophilic clostridium (strain C7) was present in culture supernatant fluid but was not detected in significant amounts in association with whole cells or in disrupted cells. Cells of the mesophilic clostridium lacked cellulosome clusters on their surface and did not adhere to cellulose fibers. The extracellular cellulase system of the mesophilic clostridium was fractionated by Sephracryl S-300 gel filtration, and the fractions were assayed for Avicelase and carboxymethylcellulase activities. The Avicelase activity coincided with an A280 peak that eluted in the 700,000-Mr region. Nondenaturing polyacrylamide gel electrophoresis and sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of the 700,000-Mr fractions showed that Avicelase was present as a multiprotein aggregate that lost the ability to hydrolyze crystalline cellulose when partially dissociated by sodium dodecyl sulfate treatment. Proteins resulting from the partial dissociation of the aggregate retained carboxymethylcellulase activity. An Avicelase-deficient mutant of strain C7 (strain LS), which was not capable of degrading crystalline cellulose, lacked the Avicelase-active 700,000-Mr peak. The results indicated that an extracellular 700,000-Mr multiprotein complex, consisting of at least 15 proteins, is utilized by the mesophilic clostridium for the hydrolysis of crystalline cellulose. At least six different endo-1,4-beta-glucanases may be part of the cellulase system of strain C7. Sephacryl S-300 column fractions, corresponding to an A280 peak in the 130,000-Mr region, contained carboxymethylcellulase-active proteins that may serve as precursors for the assembly of the Avicelase-active complex by the mesophilic clostridium.     

Adsorption kinetics and behaviors of cellulase components on microcrystalline cellulose

In order to investigate the interactive adsorption behaviors between each cellulase component purified from Trichoderma viride cellulase on microcrystalline cellulose, the adsorption of CMCase, Avicelase, and various compositions of CMCase and Avicelase was performed at 25–45°C. All adsorptions were found to apparently obey the Langmuir isotherm and the thermodynamic parameters, ΔH_a, ΔS_a, and ΔG_a were calculated from the adsorption equilibrium constant, K_ad. The adsorption process was found to be endothermic and an adsorption entropy-controlled reaction. The amount of adsorption of cellulase components decreased with increasing temperature and varied with a change in composition of the cellulase components. The maximum synergistic degradation occurred at the specific mass ratio of the cellulase components at which the maximum affinity of cellulase components occurred.     

Cellulases and xylanase of an anaerobic rumen fungus grown on wheat straw, wheat straw holocellulose, cellulose, and xylan.

The activities of cellulolytic and xylanolytic enzymes produced by an anaerobic fungus (R1) which resembled Neocallimastix sp. were investigated. Carboxymethylcellulase (CMCase), cellobiase, and filter paper (FPase) activities had pH optima of 6.0, 5.5, and 6.0, respectively. CMCase and cellobiase activities both had a temperature optimum of 50 degrees C, whereas FPase had an optimum of 45 degrees C. The pH and temperature optima for xylanase activity were pH 6.0 and 50 degrees C, respectively. Growth of the fungus on wheat straw, wheat straw holocellulose, or cellulose resulted in substantial colonization, with at least 43 to 58% losses in substrate dry matter and accumulation of comparable amounts of formate. This end product was correlated to apparent loss of substrate dry weight and could be used as an indicator of fungal growth. Milling of wheat straw did not enhance the rate or extent of substrate degradation. Growth of the R1 isolate on the above substrates or xylan also resulted in accumulation of high levels of xylanase activity and lower cellulase activities. Of the cellulases, CMCase was the most active and was associated with either low or trace amounts of cellobiase and FPase activities. During growth on xylan, reducing sugars, including arabinose and xylose, rapidly accumulated in the medium. Xylose and other reducing sugars, but not arabinose, were subsequently used for growth. Reducing sugars also accumulated, but not as rapidly, when the fungus was grown on wheat straw, wheat straw holocellulose, or cellulose. Xylanase activities detected during growth of R1 on media containing glucose, xylose, or cellobiose suggested that enzyme production was constitutive.(ABSTRACT TRUNCATED AT 250 WORDS)     

Properties of a genetically reconstructed Prevotella ruminicola endoglucanase.

A pUC19-derived plasmid was constructed that coded for a hybrid cellulase with the Thermomonospora fusca E2 cellulose-binding domain at its C terminus joined to the Prevotella ruminicola 40.5-kDa carboxymethyl cellulase (CMCase). The hybrid enzyme was purified and characterized enzymatically. It bound tightly to cellulose, and its specific activities on carboxymethyl cellulose, amorphous cellulose, and ball-milled cellulose were 1.5, 10, and 8 times that of the 40.5-kDa CMCase, respectively. Furthermore, the modified enzyme gave synergism with an exocellulase in the degradation of filter paper, while the 40.5-kDa CMCase did not.     

Cellulases and xylanase of an anaerobic rumen fungus grown on wheat straw, wheat straw holocellulose, cellulose, and xylan

The activities of cellulolytic and xylanolytic enzymes produced by an anaerobic fungus (R1) which resembled Neocallimastix sp. were investigated. Carboxymethylcellulase (CMCase), cellobiase, and filter paper (FPase) activities had pH optima of 6.0, 5.5, and 6.0, respectively. CMCase and cellobiase activities both had a temperature optimum of 50 degrees C, whereas FPase had an optimum of 45 degrees C. The pH and temperature optima for xylanase activity were pH 6.0 and 50 degrees C, respectively. Growth of the fungus on wheat straw, wheat straw holocellulose, or cellulose resulted in substantial colonization, with at least 43 to 58% losses in substrate dry matter and accumulation of comparable amounts of formate. This end product was correlated to apparent loss of substrate dry weight and could be used as an indicator of fungal growth. Milling of wheat straw did not enhance the rate or extent of substrate degradation. Growth of the R1 isolate on the above substrates or xylan also resulted in accumulation of high levels of xylanase activity and lower cellulase activities. Of the cellulases, CMCase was the most active and was associated with either low or trace amounts of cellobiase and FPase activities. During growth on xylan, reducing sugars, including arabinose and xylose, rapidly accumulated in the medium. Xylose and other reducing sugars, but not arabinose, were subsequently used for growth. Reducing sugars also accumulated, but not as rapidly, when the fungus was grown on wheat straw, wheat straw holocellulose, or cellulose. Xylanase activities detected during growth of R1 on media containing glucose, xylose, or cellobiose suggested that enzyme production was constitutive.(ABSTRACT TRUNCATED AT 250 WORDS)     

Properties of a genetically reconstructed Prevotella ruminicola endoglucanase.

A pUC19-derived plasmid was constructed that coded for a hybrid cellulase with the Thermomonospora fusca E2 cellulose-binding domain at its C terminus joined to the Prevotella ruminicola 40.5-kDa carboxymethyl cellulase (CMCase). The hybrid enzyme was purified and characterized enzymatically. It bound tightly to cellulose, and its specific activities on carboxymethyl cellulose, amorphous cellulose, and ball-milled cellulose were 1.5, 10, and 8 times that of the 40.5-kDa CMCase, respectively. Furthermore, the modified enzyme gave synergism with an exocellulase in the degradation of filter paper, while the 40.5-kDa CMCase did not.     

Characteristics of Clostridium thermocellum strain SS8: A broad saccharolytic thermophile

Clostridium thermocellum SS8 produced both carboxymethylcellulase (CMCase) and Avicelase when grown on cellulose. CMCase activity was unaffected by Ca²⁺, Mg²⁺, dithionate or dithiothreitol (DTT). Avicelase activity increased 2-fold with 5 mM DTT and 10 mM Ca²⁺. Cellulase and amylase were produced when a celluloseadapted culture was grown on starch. The mould grew best on sucrose and was inhibited by NaCl above 10 g/l.     

稻草秸秆预处理方法对烟曲霉产纤维素酶的影响

采用机械粉碎、高温、酸碱处理等方法对稻草秸秆进行预处理,以烟曲霉为实验菌株,研究预处理方法对菌株产纤维素酶的影响。结果表明,取机械粉碎后的稻草（30~120目）进行121℃高压蒸汽处理20min（即灭菌处理）,有利于菌株的生长与纤维素酶的产生;与未粉碎的稻草秸秆相比,烟曲霉羧甲基纤维素钠（CMC）酶、微晶纤维素酶、β-葡萄糖苷酶和滤纸（FPA）酶的活力分别提高了63.2%、164.0%、10.2%和14.1%。而采用不同种类、不同浓度的酸碱常温处理稻草秸秆4d或100℃高温处理30min,纤维素酶活力均出现了不同程度的下降。     

Effect of dithiocarbamates on cellulase activity in culture filtrates of Trichoderma reesei

Different concentrations of tetramethylthiuram disulfide (TMTD), sodium dimethyldithiocarbamate (NaDDC), and zinc dimethyldithiocarbamate (ZnDDC) affected the amount of cellulase(s) activity in the culture of Trichoderma reesei. After eight days incubation at 28 degrees C the greatest increase in Avicelase, CMCase, and beta-glucosidase over the control were observed at 0.1 ppm (TMTD) and 0.4 ppm (NaDDC and ZnDCC). There was decrease in the growth in the ZnDDC, but beta-glucosidase activity was reduced considerably. Total protein in the culture filtrate increased with the increase in cellulase(s) activity. No change in pH was observed at eight days incubation but pH increased (not exceeding 5.9) at 12 days incubation.