Recovery of cellulase activity after ethanol stripping in a novel pilot-scale unit

Recycling of enzymes has a potential interest during cellulosic bioethanol production as purchasing enzymes is one of the largest expenses in the process. By recycling enzymes after distillation, loss of sugars and ethanol are avoided, but depending on the distillation temperature, there is a potential risk of enzyme degradation. Studies of the rate of enzyme denaturation based on estimation of the denaturation constant K _D was performed using a novel distillation setup allowing stripping of ethanol at 50–65 °C. Experiments were performed in a pilot-scale stripper, where the effect of temperature (55–65 °C) and exposure to gas–liquid and liquid–heat transmission interfaces were tested on a mesophilic and thermostable enzyme mixture in fiber beer and buffer. Lab-scale tests were included in addition to the pilot-scale experiments to study the effect of shear, ethanol concentration, and PEG on enzyme stability. When increasing the temperature (up to 65 °C) or ethanol content (up to 7.5 % w/v), the denaturation rate of the enzymes increased. Enzyme denaturation occurred slower when the experiments were performed in fiber beer compared to buffer only, which could be due to PEG or other stabilizing substances in fiber beer. However, at extreme conditions with high temperature (65 °C) and ethanol content (7.5 % w/v), PEG had no enzyme stabilizing effect. The novel distillation setup proved to be useful for maintaining enzyme activity during ethanol extraction.     

Biosynthesis of nicotinic acid from 3-cyanopyridine by a newly isolated Fusarium proliferatum ZJB-09150

In this study, nitriles were used as sole sources of nitrogen in the enrichments to isolate nitrile-converting microorganisms. A novel fungus named ZJB-09150 possessing nitrile-converting enzymes was obtained with 3-cyanopyridine as sole source of nitrogen, which was identified by morphology, biology and 18S rDNA gene sequence as Fusarium proliferatum. It was found that F. proliferatum had ability to convert nitriles to corresponding acids or amides and showed wide substrate specificity to aliphatic nitriles, aromatic nitriles and ortho-substituted heterocyclic nitriles. The nitrile converting enzymes including nitrilase and nitrile hydratase in ZJB-09150 were induced by ε-caprolactam. Nitrilase obtained in this study showed high activity toward 3-cyanopyridine. It was active within pH 3.0–12.0 and temperature ranging from 25 to 65 °C with optimal at pH 9.0 and temperature 50–55 °C. The enzyme was thermostable and its half-life was 12.5 and 6 h at 45 and 55 °C, respectively. Under optimized reaction conditions, 60 mM 3-cyanopyridine was converted to nicotinic acid in 15 min, which indicated ZJB-09150 has potentials of application in large scale production of nicotinic acid.     

Chitinase production by a newly isolated thermotolerant Paenibacillus sp. BISR-047

Chitinase is one of the important mycolytic enzymes with industrial significance, and is produced by a number of organisms, including bacteria. In this study, we describe isolation, characterization and media optimization for chitinase production from a newly isolated thermotolerant bacterial strain, BISR-047, isolated from desert soil and later identified as Paenibacillus sp. The production of extracellularly secreted chitinase by this strain was optimized by varying pH, temperature, incubation period, substrate concentrations, carbon and nitrogen source,etc. The maximum chitinase production was achieved at 45 °C with media containing (in g/l) chitin 2.0, yeast extract 1.5, glycerol 1.0, and ammonium sulphate 0.2 % (media pH 7.0). A three-fold increase in the chitinase production (712 IU/ml) was found at the optimized media conditions at 6 days of incubation. The enzyme showed activity at broad pH (3–10) and temperature (35–100 °C) ranges, with optimal activity displayed at pH 5.0 and 55 °C, respectively. The produced enzyme was found to be highly thermostable at higher temperatures, with a half-life of 4 h at 100 °C.     

Molecular basis for thermal properties of <Emphasis Type="Italic">Streptomyces thermovulgaris</Emphasis> fumarase C hinge at hydrophilic amino acids R163, E170 and S347

Industrially, the use of high temperatures (40–60°C) in the l-malate production process could result in rapid inactivation of the mesophilic fumarases, warranting constant replenishment of the biocatalyst. Thus, a thermostable fumarase C that is active and stable at high temperatures would be ideal. Biochemical studies using recombinant fumarase C from thermophilic Streptomyces thermovulgaris (stFUMC) indicated that it was optimally active at 50°C and highly stable even after 24 h of incubation at 40°C. The same gene from mesophilic Streptomyces coelicolor (scfumC) was also cloned and expressed as soluble proteins for comparison in thermal properties of both enzymes. In contrast to stFUMC, scFUMC exhibited a lower temperature optima of 30°C and was rapidly denatured at 50°C. The specific activity of stFUMC was also higher than that of scFUMC by 20-fold. After primary sequence comparison, three hydrophilic amino acid residues, R163, E170 and S347, were forged into the thermolabile scFUMC either singly or in combination for the investigation of their contributions in the thermal properties of the mutant enzymes. Of the mutants studied, the A347S scFUMC mutant resulted in the highest increase in optimum temperature of 10°C and a fourfold enhancement in specific activity. G163R/G170E and G163R/G170E/A347S scFUMC mutants are more thermostable than wild-type scFUMC. These findings support stFUMC as a highly efficient, thermostable fumarase C with industrial potential and suggest that R163, E170 and S347 are involved in the enhancement of thermal properties in fumarase C.     

Purification and properties of an acidic β-glucosidase from Acidobacterium capsulatum

Acidobacterium capsulatum, an acidophilic, mesophilic and chemoorganotrophic bacterium, produced an inducible, acidic β-glucosidase in the cellobiose medium. The enzyme was successively purified 109 times by CM-Sepharose, Sephacryl S-200 chromatography and preparative discontinuous polyacrylamide gel electrophoresis. Polyacrylamide gel electrophoresis of the purified enzyme gave a single band at pH 4.3. The enzyme had an optimum pH of 3.0 and optimum reaction temperature of 55°C, being stable from pH 1.5 to 6.0 and at temperatures from 20 to 45°C. No activity was detected above pH 6.5 or above 65°C. The molecular weight of 90,000 was estimated by gel filtration and the enzyme had an isoelectric point of 7.0. The enzyme hydrolyzed aryl-β-glycosides and β-linked disaccharides.     

Increased salinity improves the thermotolerance of mesophilic nitrification

Nitrification is a well-studied and established process to treat ammonia in wastewater. Although thermophilic nitrification could avoid cooling costs for the treatment of warm wastewaters, applications above 40 °C remain a significant challenge. This study tested the effect of salinity on the thermotolerance of mesophilic nitrifying sludge (34 °C). In batch tests, 5 g NaCl L^?1 increased the activity of aerobic ammonia-oxidizing bacteria (AerAOB) by 20–21 % at 40 and 45 °C. For nitrite-oxidizing bacteria (NOB), the activity remained unaltered at 40 °C, yet decreased by 83 % at 45 °C. In a subsequent long-term continuous reactor test, temperature was increased from 34 to 40, 42.5, 45, 47.5 and 50 °C. The AerAOB activity showed 65 and 37 % higher immediate resilience in the salt reactor (7.5 g NaCl L^?1) for the first two temperature transitions and lost activity from 45 °C onwards. NOB activity, in contrast to the batch tests, was 37 and 21 % more resilient in the salt reactor for the first two transitions, while no difference was observed for the third temperature transition. The control reactor lost NOB activity at 47.5 °C, while the salt reactor only lost activity at 50 °C. Overall, this study demonstrates salt amendment as a tool for a more efficient temperature transition for mesophilic sludge (34 °C) and eventually higher nitrification temperatures.     

Impact of cofactor-binding loop mutations on thermotolerance and activity of E. coli transketolase

Improvement of thermostability in engineered enzymes can allow biocatalysis on substrates with poor aqueous solubility. Denaturation of the cofactor-binding loops of Escherichia coli transketolase (TK) was previously linked to the loss of enzyme activity under conditions of high pH or urea. Incubation at temperatures just below the thermal melting transition, above which the protein aggregates, was also found to anneal the enzyme to give an increased specific activity. The potential role of cofactor-binding loop instability in this process remained unclear. In this work, the two cofactor-binding loops (residues 185–192 and 382–392) were progressively mutated towards the equivalent sequence from the thermostable Thermus thermophilus TK and variants assessed for their impact on both thermostability and activity. Cofactor-binding loop 2 variants had detrimental effects on specific activity at elevated temperatures, whereas the H192P mutation in cofactor-binding loop 1 resulted in a two-fold improved stability to inactivation at elevated temperatures, and increased the critical onset temperature for aggregation. The specific activity of H192P was 3-fold and 19-fold higher than that for wild-type at 60 °C and 65 °C respectively, and also remained 2.7-4 fold higher after re-cooling from pre-incubations at either 55 °C or 60 °C for 1 h. Interestingly, H192P was also 2-times more active than wild-type TK at 25 °C. Optimal activity was achieved at 60 °C for H192P compared to 55 °C for wild type. These results show that cofactor-binding loop 1, plays a pivotal role in partial denaturation and aggregation at elevated temperatures. Furthermore, a single rigidifying mutation within this loop can significantly improve the enzyme specific activity, as well as the stability to thermal denaturation and aggregation, to give an increased temperature optimum for activity.     

Activity of supplemental enzymes and their effect on nutrient utilization and growth performance of growing chickens as affected by pelleting temperature

Activity of supplemental enzymes in a barley‐soybean‐maize based diet at 60, 75 and 90°C pelleting temperatures was studied using feed viscosity, in‐vitro enzyme activity and broiler performance data.

High pelleting temperatures increased feed viscosity but supplemented enzymes reduced the viscosity at all three temperatures levels by 11, 14 and 17%, respectively. Water intake and losses in excreta of birds were found to be affected by feed viscosity. Activity of cellulase enzyme, measured using the radial diffusion method, was unaffected at 60 and 75°C, but reduced by 73% in feed processed at 90°C. Enzymes increased the weight gain of broilers by 11.1% at 90°C, but no effect could be seen at low pelleting temperatures possibly due to high dietary protein and energy contents. Feed intake was unaffected by enzymes. Birds consumed 6% more feed and grew 9% faster when the pelleting temperature was increased from 60 to 75°C. Reduced feed intake and daily weight gain observed at 90° C could be fully compensated by the enzyme supplementation. High pelleting temperature reduced energy metabolizability (3.2%) and nitrogen utilization (4%) but enzyme almost compensated them (by 3.3% and 2.6%, respectively). No interaction could be detected between the pelleting temperatures and enzymes.

It is concluded that pelleting temperatures as high as 90°C drastically reduce cellulase activity, energy and nitrogen utilization thus lowering broiler performance. Either the remaining activity of cellulase or other thermostable enzymes can prevent the losses.     

Engineering chimeric thermostable GH7 cellobiohydrolases in Saccharomyces cerevisiae

We report here the effect of adding different types of carbohydrate-binding modules (CBM) to a single-module GH7 family cellobiohydrolase Cel7A from a thermophilic fungus Talaromyces emersonii (TeCel7A). Both bacterial and fungal CBMs derived from families 1, 2 and 3, all reported to bind to crystalline cellulose, were used. Chimeric cellobiohydrolases with an additional S–S bridge in the catalytic module of TeCel7A were also made. All the fusion proteins were secreted in active form and in good yields by Saccharomyces cerevisiae. The purified chimeric enzymes bound to cellulose clearly better than the catalytic module alone and demonstrated high thermal stability, having unfolding temperatures (T _m) ranging from 72 °C to 77 °C. The highest activity enhancement on microcrystalline cellulose could be gained by a fusion with a bacterial CBM3 derived from Clostridium thermocellum cellulosomal-scaffolding protein CipA. The two CBM3 fusion enzymes tested were more active than the reference enzyme Trichoderma reesei Cel7A both at moderate (45 °C and 55 °C) and at high temperatures (60 °C and 65 °C), the hydrolysis yields being two- to three-fold better at 60 °C, and six- to seven-fold better at 65 °C. The best enzyme variant was also tested on a lignocellulosic feedstock hydrolysis, which demonstrated its potency in biomass hydrolysis even at 70 °C.     

Thermostable Artificial Enzyme Isolated by In Vitro Selection

Artificial enzymes hold the potential to catalyze valuable reactions not observed in nature. One approach to build artificial enzymes introduces mutations into an existing protein scaffold to enable a new catalytic activity. This process commonly results in a simultaneous reduction of protein stability as an undesired side effect. While protein stability can be increased through techniques like directed evolution, care needs to be taken that added stability, conversely, does not sacrifice the desired activity of the enzyme. Ideally, enzymatic activity and protein stability are engineered simultaneously to ensure that stable enzymes with the desired catalytic properties are isolated. Here, we present the use of the in vitro selection technique mRNA display to isolate enzymes with improved stability and activity in a single step. Starting with a library of artificial RNA ligase enzymes that were previously isolated at ambient temperature and were therefore mostly mesophilic, we selected for thermostable active enzyme variants by performing the selection step at 65°C. The most efficient enzyme, ligase 10C, was not only active at 65°C, but was also an order of magnitude more active at room temperature compared to related enzymes previously isolated at ambient temperature. Concurrently, the melting temperature of ligase 10C increased by 35 degrees compared to these related enzymes. While low stability and solubility of the previously selected enzymes prevented a structural characterization, the improved properties of the heat-stable ligase 10C finally allowed us to solve the three-dimensional structure by NMR. This artificial enzyme adopted an entirely novel fold that has not been seen in nature, which was published elsewhere. These results highlight the versatility of the in vitro selection technique mRNA display as a powerful method for the isolation of thermostable novel enzymes.     

Biochemical characterization of psychrophilic Mn-superoxide dismutase from newly isolated Exiguobacterium sp. OS-77

Many types of superoxide dismutases have been purified and characterized from various bacteria, however, a psychrophilic Mn-superoxide dismutase (MnSOD) has not yet been reported. Here, we describe the purification and the biochemical characterization of the psychrophilic MnSOD from Exiguobacterium sp. strain OS-77 (EgMnSOD). According to 16S rRNA sequence analysis, a newly isolated bacterium strain OS-77 belongs to the genus Exiguobacterium. The optimum growth temperature of the strain OS-77 is 20 °C. The EgMnSOD is a homodimer of 23.5 kDa polypeptides determined by SDS-PAGE and gel filtration analysis. UV-Vis spectrum and ICP-MS analysis clearly indicated that the homogeneously purified enzyme contains only a Mn ion as a metal cofactor. The optimal reaction pH and temperature of the enzyme were pH 9.0 and 5 °C, respectively. Notably, the purified EgMnSOD was thermostable up to 45 °C and retained 50 % activity after 21.2 min at 60 °C. The differential scanning calorimetry also indicated that the EgMnSOD is thermostable, exhibiting two protein denaturation peaks at 65 and 84 °C. The statistical analysis of amino acid sequence and composition of the EgMnSOD suggests that the enzyme retains psychrophilic characteristics.     

Activity and stability of a thermostable alpha-amylase compared to its mesophilic homologue: mechanisms of thermal adaptation

To elucidate how enzymes adapt to extreme environmental conditions, a comparative study with a thermostable alpha-amylase from Bacillus licheniformis (BLA) and its mesophilic homologue from Bacillus amyloliquefaciens (BAA) was performed. We measured conformational stability, catalytic activity, and conformational fluctuations on the picosecond time scale for both enzymes as a function of temperature. The objective of this study is to analyze how these properties are related to each other. BLA shows its maximal catalytic activity at about 90-95 degrees C and a strongly reduced activity (only 20% of the maximum) at room temperature. Although B. licheniformis itself is a mesophilic organism, BLA shows an activity profile typical for a thermophilic enzyme. In contrast to this, BAA exhibits its maximal activity at about 80 degrees C but with a level of about 60% activity at room temperature. In both cases the unfolding temperatures T(m) are only 6 degrees C (BAA, T(m) = 86 degrees C) and 10 degrees C (BLA, T(m) = 103 degrees C), respectively, higher than the temperatures for maximal activity. In contrast to many previous studies on other thermophilic-mesophilic pairs, in this study a higher structural flexibility of the thermostable BLA was measured as compared to the mesophilic BAA. The findings of this study neither indicate a proportionality between the observed dynamics and the catalytic activity nor support the idea of more "rigid" thermostable proteins, as often proposed in the concept of "corresponding states".     

Ectopic expression of bacterial amylopullulanase enhances bioethanol production from maize grain

Key message

Heterologous expression of amylopullulanase in maize seeds leads to partial starch degradation into fermentable sugars, which enhances direct bioethanol production from maize grain.

Abstract

Utilization of maize in bioethanol industry in the United States reached ±13.3 billion gallons in 2012, most of which was derived from maize grain. Starch hydrolysis for bioethanol industry requires the addition of thermostable alpha amylase and amyloglucosidase (AMG) enzymes to break down the α-1,4 and α-1,6 glucosidic bonds of starch that limits the cost effectiveness of the process on an industrial scale due to its high cost. Transgenic plants expressing a thermostable starch-degrading enzyme can overcome this problem by omitting the addition of exogenous enzymes during the starch hydrolysis process. In this study, we generated transgenic maize plants expressing an amylopullulanase (APU) enzyme from the bacterium Thermoanaerobacter thermohydrosulfuricus. A truncated version of the dual functional APU (TrAPU) that possesses both alpha amylase and pullulanase activities was produced in maize endosperm tissue using a seed-specific promoter of 27-kD gamma zein. A number of analyses were performed at 85 °C, a temperature typically used for starch processing. Firstly, enzymatic assay and thin layer chromatography analysis showed direct starch hydrolysis into glucose. In addition, scanning electron microscopy illustrated porous and broken granules, suggesting starch autohydrolysis. Finally, bioethanol assay demonstrated that a 40.2 ± 2.63 % (14.7 ± 0.90 g ethanol per 100 g seed) maize starch to ethanol conversion was achieved from the TrAPU seeds. Conversion efficiency was improved to reach 90.5 % (33.1 ± 0.66 g ethanol per 100 g seed) when commercial amyloglucosidase was added after direct hydrolysis of TrAPU maize seeds. Our results provide evidence that enzymes for starch hydrolysis can be produced in maize seeds to enhance bioethanol production.     

Differences in the catalytic mechanisms of mesophilic and thermophilic indole-3-glycerol phosphate synthase enzymes at their adaptive temperatures

Thermophilic enzymes tend to be less catalytically-active at lower temperatures relative to their mesophilic counterparts, despite having very similar crystal structures. An often cited hypothesis for this general observation is that thermostable enzymes have evolved a more rigid tertiary structure in order to cope with their more extreme, natural environment, but they are also less flexible at lower temperatures, leading to their lower catalytic activity under mesophilic conditions. An alternative hypothesis, however, is that complementary thermophilic-mesophilic enzyme pairs simply operate through different evolutionary-optimized catalytic mechanisms. In this communication, we present evidence that while the steps of the catalytic mechanisms for mesophilic and thermophilic indole-3-glycerol phosphate synthase (IGPS) enzymes are fundamentally similar, the identity of the rate-determining step changes as a function of temperature. Our findings indicate that while product release is rate-determining at 25°C for thermophilic IGPS, near its adaptive temperature (75°C), a proton transfer event, involving a general acid, becomes rate-determining. The rate-determining steps for thermophilic and mesophilic IGPS enzymes are also different at their respective, adaptive temperatures with the mesophilic IGPS-catalyzed reaction being rate-limited before irreversible CO2 release, and the thermophilic IGPS-catalyzed reaction being rate limited afterwards.     

EFFECT OF TEMPERATURE ON TWO ENZYMES FROM A PSYCHROPHILIC CHLOROMONAS (CHLOROPHYTA)1

A psychrophilic green alga belonging to the Chloromonas genus and here named ANT1 was collected in Antarctica. The activities of two enzymes, nitrate reductase and argininosuccinate lyase, were measured at various temperatures and compared to the corresponding enzyme activities in the mesophilic species Chlamydomonas reinhardtii Dangeard. For both enzymes, the temperature for apparent optimal activity was about 20°C lower in ANT1 than in C. reinhardtii. The enzymes were also submitted to various heat treatments before measuring their activities. Both psychrophilic enzymes were more sensitive to heat than the corresponding mesophilic enzymes. It is worth stressing, however, that in both species nitrate reductase was much more sensitive to heat than argininosuccinate lyase, which probably indicates that the peculiar structure of each protein primarily determines its dependence to temperature. Secondary adaptations to low temperatures should then occur to confer the psychrophilic character.     

Continuous lipase-catalyzed esterification of soybean fatty acids under ultrasound irradiation

This work investigates the continuous production of alkyl esters from soybean fatty acid (FA) charges using immobilized Novozym 435 as catalyst. The experiments were performed in a packed-bed bioreactor evaluating the effects of FA charge to alcohol (methanol and ethanol) molar ratio, from 1:1 to 1:6, substrate flow rate in the range of 0.5–2.5 mL/min and output irradiation power up to 154 W, at fixed temperature of 65 °C, on the reaction conversion. Results showed that almost complete conversions to fatty acids ethyl esters were achieved at mild ultrasonic power (61.6 W), FA to ethanol molar ratio of 1:6, operating temperature (65 °C) and remained nearly constant for long-term reactions without negligible enzyme activity losses.     

Production of a thermostable ATPase by the facultative thermophile,Bacillus coagulans

The properties of the ATPase in the facultative thermophile, Bacillus coagulans, grown at thermophilic or mesophilic temperatures were similar. Arrhenius plots did not show discontinuities indicative of thermoadaptation. Magnesium stimulation of the enzyme was dependant on the assay temperature but independant of the growth temperature. The ATPase in cells grown at 35°C or 55°C was equally thermostable at 65°C. In contrast, the ATPase from the mesophile, Bacillus megaterium (T _max=42°C) was completely inactivated at 55°C in 5 min.     

Thermostability enhancement of polyethylene terephthalate degrading PETase using self- and nonself-ligating protein scaffolding approaches

Polyethylene terephthalate (PET) hydrolase enzymes show promise for enzymatic PET degradation and green recycling of single-use PET vessels representing a major source of global pollution. Their full potential can be unlocked with enzyme engineering to render activities on recalcitrant PET substrates commensurate with cost-effective recycling at scale. Thermostability is a highly desirable property in industrial enzymes, often imparting increased robustness and significantly reducing quantities required. To date, most engineered PET hydrolases show improved thermostability over their parental enzymes. Here, we report engineered thermostable variants of Ideonella sakaiensis PET hydrolase enzyme (IsPETase) developed using two scaffolding strategies. The first employed SpyCatcher-SpyTag technology to covalently cyclize IsPETase, resulting in increased thermostability that was concomitant with reduced turnover of PET substrates compared to native IsPETase. The second approach using a GFP-nanobody fusion protein (vGFP) as a scaffold yielded a construct with a melting temperature of 80°C. This was further increased to 85°C when a thermostable PETase variant (FAST PETase) was scaffolded into vGFP, the highest reported so far for an engineered PET hydrolase derived from IsPETase. Thermostability enhancement using the vGFP scaffold did not compromise activity on PET compared to IsPETase. These contrasting results highlight potential topological and dynamic constraints imposed by scaffold choice as determinants of enzyme activity.     

Purification and enzymatic properties of α-galactosidase from Penicillium janthinellum

α-Galactosidase (E.C.3.2.1.22) from Penicillium janthinellum was purified by precipitation and fractionation with ammonium sulphate, cold acetone or ethanol, calcium phosphate gel, and column chromatographies on Sephadex G-100 and G-200. The enzyme was purified about 110.39-fold when Sephadex G-100 was used. α-Galactosidase exhibited the optimum pH and temperature at 4.5 and 60°C, respectively. The optimum enzyme stability was obtained at pH 3.5 for 24 h (at room temperature). The enzyme was found to be thermostable below 65°C up to 40 minutes and was gradually inactivated by increasing the temperature above this degree. The MICHAELIS constant was 0.55 mM for p-nitrophenyl-α-D-galactoside. The α-galactosidase activity was strongly inhibited by Hg⁺⁺ and slightly activated by Mn⁺⁺. The results show the possibility of producing a thermostable enzyme from a low-priced agricultural product, for instance, lupine.     

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.