Cellulase Protein Research Articles

The adsorptive and hydrolytic performance of cellulase on cationised cotton

In this research, the cotton fabrics were cationised by a cationic agent to change their surface electric properties. The cationised cotton fabrics were then bio-polished by cellulase to explore the influence of cationisation on the adsorptive and hydrolytic performance of cellulase. The experimental results from cellulase adsorption reveal the cationisation of cotton can obviously improve the binding efficiency of cellulase protein mainly by the improved electrostatic attraction between oppositely charged cellulase and cationised cotton. The adsorption parameters calculated can further prove this improvement trend. Through measuring the concentration of reducing sugars released and weight loss of cotton during the bio-polishing, it is found the hydrolytic activity toward cotton is partially damaged by the cationisation. The reason can be attributed to the movement restriction and dysfunction of cellulase protein by the cationisation.

The effects of carbonates removal on adsorption, immobilization and activity of cellulase in a calcareous soil

Enzymes are able to be sorbed and immobilized by clay minerals and humic colloids in soil environments. The present study was done to elucidate the effects of soil innate carbonates on the sorption, immobilization and activity of cellulase in a calcareous soil by treating it with different levels of acetic acid. The study of adsorption isotherm revealed that soil carbonates removal with acetic acid increased the intensity of cellulase adsorption (1/n) estimated by the Freundlich model. Based on the Freundlich isotherm model it is predicted that cellulase molecules will be increasingly adsorbed on acetic acid treated soil with increasing cellulase concentration. However, practically the amounts of cellulase protein adsorbed and immobilized on untreated soil with high levels of ECC were significantly higher than those on acetic acid treated soils. The immobilized cellulase activity and particularly its specific activity decreased considerably with increasing carbonate contents in soil.

Application of ultrafiltration and nanofiltration for recycling cellulase and concentrating glucose from enzymatic hydrolyzate of steam exploded wheat straw

Application of combined ultrafiltration (UF) and nanofiltration (NF) was examined to recycle cellulase and concentrate glucose present in lignocellulosic hydrolyzate. With PES10 membrane operated at 25.6l/m2h, 73.9% of cellulase protein present in the hydrolyzate suspension could be recovered while allowing free transmission of glucose. The permeate obtained from UF was then concentrated by NF. With NF270 membrane operated at 13.3l/m2h, the glucose concentration in the ultrafiltered hydrolyzate increased from 30.2 to 110.2g/l. Recycling cellulase by UF could reduce the hydrolysis cost of lignocellulosic feedstock, while concentrating glucose by NF could improve the fermentation efficiency of lignocellulosic hydrolyzate and lower the separation and purification cost of fermentative product. Therefore, the use of UF and NF for treating lignocellulosic hydrolyzate could be a promising approach in fermentative production of bioproducts and biofuels using lignocellulosic feedstock as substrate.

The Enzymatic Action of Cellulase on Pigment-Dyed Cotton Fabrics

In this research, cotton fabrics were colored with a pigment suspension, and then treated with an acid cellulase to investigate the influences of pre-existing pigment on cellulase action. It was found that the pigment on cotton surface could increase the affinity between cotton and cellulase protein due to the mutual attraction between pigment molecule and non-polar residues in the enzyme. However, it was observed that the existing pigment would suppress enzymatic activity by testing the soluble reducing sugars liberated during the treatment. The main reason is that the pigment could reduce the accessibility of cellulase protein to the cotton molecular chains by steric hindrance.

Improvement of cellulose conversion caused by the protection of Tween-80 on the adsorbed cellulase

For the hydrolysis of pure cellulose, the positive effect of Tween-80 was controversial. In this study, it was found that Tween-80 had little effect on pure cellulose conversion under the shaking speed of 0 and 100 rpm. However, when the shaking speed increased to 180 rpm, Tween-80 resulted in an obvious improvement of cellulose conversion. The mechanism of Tween-80 effect was investigated from three aspects: protecting free cellulase from deactivation, decreasing cellulase proteins adsorption and reducing the cellulase deactivation during the interaction between enzyme and substrate. Tween-80 did not show more positive effects at high speed in protecting free cellulase against deactivation and decreasing cellulase proteins adsorption. From the studies of endoglucanase and exoglucanase relative activities in the supernatant of hydrolysis, it was found that the improved conversion of cellulose with Tween-80 at high shaking speed was mainly due to the reduction of the adsorbed cellulase deactivation resulting from enzyme–substrate interaction.

Fabrication of cellulase protein fibers through concentric electrospinning

. ► Crosslinked cellulase fibers were prepared for the first time on glass paper by electrospinning. ► Core–shell electrospinning techniques were employed for the preparation of enzyme fibers. ► The supported cellulase membrane was active for the conversion of cellulose. Cellulase catalyzes the hydrolysis of cellulose to glucose and is of great interest for biofuel production. Cellulase fibers with diameters ranging from 100 to 500 nm were fabricated for the first time by core–shell electrospinning. The enzymes in the core were cross-linked using glutaraldehyde and the PEO outer shell washed away using water and methanol to produce a free standing nonwoven enzyme fiber mat. The activity of the cross-linked protein fibers was determined to be 24% that of the free enzyme for hydrolysis of an insoluble paper substrate. Also, the cellulase was complexed with Ca 2+ and electrospun for EDX analysis. Elemental mapping of Ca 2+ showed fibers that cross-linked cellulase fibers maintained the fibrous morphology after washing away the PEO shell.

The effects of CaCO3 on adsorption, immobilization and activity of cellulase in a decarbonated soil

The interaction of organic molecules with mineral surfaces is a subject of interest in a variety of disciplines. The present study was done to elucidate some aspects of sorption and immobilization of cellulase on soil components by analysis of the sorption, desorption, immobilization and activity of cellulase on a decarbonated soil treated with different levels of CaCO3 (0, 2.5, 10 and 20 %). Applied concentrations of cellulase protein on Ca-homoionized soil suspensions were 0, 0.014, 0.028, 0.070, 0.140, 0.280, 0.701, 0.981 and 1.402 mg mL-1. After shaking for 1 h in sterile conditions, they were centrifuged and the amount of the cellulase protein remaining in solution was determined. The adsorbed cellulase protein was calculated. The immobilized and desorbed cellulase proteins were calculated after washing soil suspensions thrice with distilled water. Analysis of variance showed that the effects of the enzyme concentration, CaCO3 level and their interaction on cellulase protein adsorption and activity were statistically significant. The adsorption and immobilization capacities of the decarbonated soil increased by application of CaCO3. However, these effects of CaCO3 were only significant when high concentrations of cellulase protein were added to the soil. The desorption of cellulase protein from the decarbonated soil did not depend on the amount of cellulase adsorbed on the soil and the CaCO3 level in the soil. The immobilized cellulase activity, and particularly its specific activity, decreased considerably by increasing CaCO3 levels in the soil. This negative effect of CaCO3 on the cellulase specific activity in the decarbonated soil was significant even for low levels.

Identification of genes that enhance cellulase protein production in yeast

In order to enhance heterologous cellulase protein production in yeast, a plasmid harboring the endoglucanase gene from Clostridium thermocellum ( Ctcel8A) was used to systematically transform a homozygous diploid yeast deletion strain collection. We identified 55 deletion strains that exhibited enhanced endoglucanase activity compared with that of the wild-type strain. Genes disrupted in these strains were classified into the categories of transcription, translation, phospholipid synthesis, endosome/vacuole function, ER/Golgi function, nitrogen starvation response, and cytoskeleton. The vps3Δ and vps16Δ strains, which have deletion in genes encoding components of the class C core vacuole/endosome tethering (CORVET) complex, also exhibited enhanced β-glucosidase activity when Ctcel8A was heterologously expressed. Moreover, multiple gene deletion strains were constructed by using the vps3Δ strain. Endoglucanase activity of the resulting rav1Δ vps3Δ double deletion strain was exhibited higher than that of the rav1Δ or vps3Δ strains. Our genome-wide analyses using the yeast deletion strain collection identified useful genes that allow efficient expression of cellulase.

In Situ, Rapid, and Temporally Resolved Measurements of Cellulase Adsorption onto Lignocellulosic Substrates by UV−vis Spectrophotometry

This study demonstrated two in situ UV-vis spectrophotometric methods for rapid and temporally resolved measurements of cellulase adsorption onto cellulosic and lignocellulosic substrates during enzymatic hydrolysis. The cellulase protein absorption peak at 280 nm was used for quantification. The spectral interferences from light scattering by small fibers (fines) and particulates and from absorptions by lignin leached from lignocelluloses were corrected using a dual-wavelength technique. Wavelengths of 500 and 255 nm were used as secondary wavelengths for correcting spectral interferences from light scattering and absorption of leached lignin. Spectral interferences can also be eliminated by taking the second derivative of the measured spectra of enzymatic hydrolysate of cellulose or lignocelluloses. The in situ measured cellulase adsorptions in cellulose and lignocellulose suspensions by these two spectrophotometric methods showed general agreement with batch sampling assayed by the Bradford method. The in situ methods not only eliminated tedious batch sampling but also can resolve the kinetics of the initial adsorption process. The measured time-dependent cellulase adsorptions were found to follow pseudo-second-order kinetics.

Monitoring Cellulase Protein Adsorption and Recovery Using SDS-PAGE

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was employed to study the sorption behaviors of cellulases on microcrystalline celluloses and hardwood pulp. The adsorption and recovery of cellulases from Aspergillus niger and Trichoderma reesei were investigated at 25 °C. Cellulase recovery was conducted by rinsing adsorbed enzymes with sodium acetate buffer, Milli-Q water, and sodium hydroxide solution. The initial, equilibrium, and recovered enzymes were analyzed using SDS-PAGE gels. Gels were scanned and analyzed using ImagePro software. The molecular weights of cellulase proteins were determined using a protein marker having seven known proteins. The cellulase system from Trichoderma reesei had a higher adsorption on all substrates studied than the cellulase system from Aspergillius niger, and higher pH favored desorption from the substrates studied. Experimental results also demonstrated that adsorption and desorption amounts determined by SDS-PAGE were proportional to protein concentrations in their crude mixtures.

The activity of a wall-bound cellulase is required for and is coupled to cell cycle progression in the dinoflagellate Crypthecodinium cohnii.

Cellulose synthesis, but not its degradation, is generally thought to be required for plant cell growth. In this work, we cloned a dinoflagellate cellulase gene, dCel1, whose activities increased significantly in G(2)/M phase, in agreement with the significant drop of cellulose content reported previously. Cellulase inhibitors not only caused a delay in cell cycle progression at both the G(1) and G(2)/M phases in the dinoflagellate Crypthecodinium cohnii, but also induced a higher level of dCel1p expression. Immunostaining results revealed that dCel1p was mainly localized at the cell wall. Accordingly, the possible role of cellulase activity in cell cycle progression was tested by treating synchronized cells with exogenous dCelp and purified antibody, in experiments analogous to overexpression and knockdown analyses, respectively. Cell cycle advancement was observed in cells treated with exogenous dCel1p, whereas the addition of purified antibody resulted in a cell cycle delay. Furthermore, delaying the G(2)/M phase independently with antimicrotubule inhibitors caused an abrupt and reversible drop in cellulase protein level. Our results provide a conceptual framework for the coordination of cell wall degradation and reconstruction with cell cycle progression in organisms with cell walls. Since cellulase activity has a direct bearing on the cell size, the coupling between cellulase expression and cell cycle progression can also be considered as a feedback mechanism that regulates cell size.

Expression of recombinant sea urchin cellulase SnEG54 using mammalian cell lines

We previously identified the cellulase SnEG54 from Japanese purple sea urchin Strongylocentrotus nudus, the molecular mass of which is about 54 kDa on SDS–PAGE. It is difficult to express and purify a recombinant cellulase protein using bacteria such as Escherichia coli or yeast. In this study, we generated mammalian expression vectors encoding SnEG54 to transiently express SnEG54 in mammalian cells. Both SnEG54 expressed in mammalian cells and SnEG54 released into the culture supernatant showed hydrolytic activity toward carboxymethyl cellulose. By using a retroviral expression system, we also established a mammalian cell line that constitutively produces SnEG54. Unexpectedly, SnEG54 released into the culture medium was not stable, and the peak time showing the highest concentration was approximately 1–2 days after seeding into fresh culture media. These findings suggest that non-mammalian sea urchin cellulase can be generated in human cell lines but that recombinant SnEG54 is unstable in culture medium due to an unidentified mechanism.

Effect of xylanase supplementation of cellulase on digestion of corn stover solids prepared by leading pretreatment technologies

Solids resulting from pretreatment of corn stover by ammonia fiber expansion (AFEX), ammonia recycled percolation (ARP), controlled pH, dilute acid, lime, and sulfur dioxide (SO 2) technologies were hydrolyzed by enzyme cocktails based on cellulase supplemented with β-glucosidase at an activity ratio of 1:2, respectively, and augmented with up to 11.0 g xylanase protein/g cellulase protein for combined cellulase and β-glucosidase mass loadings of 14.5 and 29.0 mg protein (about 7.5 and 15 FPU, respectively)/g of original potential glucose. It was found that glucose release increased nearly linearly with residual xylose removal by enzymes for all pretreatments despite substantial differences in their relative yields. The ratio of the fraction of glucan removed by enzymes to that for xylose was defined as leverage and correlated statistically at two combined cellulase and β-glucosidase mass loadings with pretreatment type. However, no direct relationship was found between leverage and solid features following different pretreatments such as residual xylan or acetyl content. However, acetyl content not only affected how xylanase impacted cellulase action but also enhanced accessibility of cellulose and/or cellulase effectiveness, as determined by hydrolysis with purified CBHI (Cel7A). Statistical modeling showed that cellulose crystallinity, among the main substrate features, played a vital role in cellulase–xylanase interactions, and a mechanism is suggested to explain the incremental increase in glucose release with xylanase supplementation.

Effects of cellulase and xylanase enzymes on the deconstruction of solids from pretreatment of poplar by leading technologies

Comparative data is presented on glucose and xylose release for enzymatic hydrolysis of solids produced by pretreatment of poplar wood by ammonia fiber expansion (AFEX), ammonia recycled percolation (ARP), controlled pH, dilute acid, flowthrough (FT), lime, and sulfur dioxide (SO(2)) technologies. Sugar solubilization was measured for times of up to 72 h using cellulase supplemented with beta-glucosidase at an activity ratio of 1:2, respectively, at combined protein mass loadings of 5.8-116 mg/g of glucan in poplar wood prior to pretreatment. In addition, the enzyme cocktail was augmented with up to 11.0 g of xylanase protein per gram of cellulase protein at combined cellulase and beta-glucosidase mass loadings of 14.5 and 29.0 mg protein (about 7.5 and 15 FPU, respectively)/g of original potential glucose to evaluate cellulase-xylanase interactions. All pretreated poplar solids required high protein loadings to realize good sugar yields via enzymatic hydrolysis, and performance tended to be better for low pH pretreatments by dilute sulfuric acid and sulfur dioxide, possibly due to higher xylose removal. Glucose release increased nearly linearly with residual xylose removal by enzymes for all pretreatments, xylanase leverage on glucan removal decreased at high cellulase loadings. Washing the solids improved digestion for all pretreatments and was particularly beneficial for controlled pH pretreatment. Furthermore, incubation of pretreated solids with BSA, Tween 20, or PEG6000 prior to adding enzymes enhanced yields, but the effectiveness of these additives varied with the type of pretreatment.

Subcellular targeting is a key condition for high‐level accumulation of cellulase protein in transgenic maize seed

Ethanol from lignocellulosic biomass is being pursued as an alternative to petroleum-based transportation fuels. To succeed in this endeavour, efficient digestion of cellulose into monomeric sugar streams is a key step. Current production systems for cellulase enzymes, i.e. fungi and bacteria, cannot meet the cost and huge volume requirements of this commodity-based industry. Transgenic maize (Zea mays L.) seed containing cellulase protein in embryo tissue, with protein localized to the endoplasmic reticulum, cell wall or vacuole, allows the recovery of commercial amounts of enzyme. E1 cellulase, an endo-beta-1,4-glucanase from Acidothermus cellulolyticus, was recovered at levels greater than 16% total soluble protein (TSP) in single seed. More significantly, cellobiohydrolase I (CBH I), an exocellulase from Trichoderma reesei, also accumulated to levels greater than 16% TSP in single seed, nearly 1000-fold higher than the expression in any other plant reported in the literature. The catalytic domain was the dominant form of E1 that was detected in the endoplasmic reticulum and vacuole, whereas CBH I holoenzyme was present in the cell wall. With one exception, individual transgenic events contained single inserts. Recovery of high levels of enzyme in T2 ears demonstrated that expression is likely to be stable over multiple generations. The enzymes were active in cleaving soluble substrate.

A Novel Stepwise Recovery Strategy of Cellulase Adsorbed to the Residual Substrate after Hydrolysis of Steam Exploded Wheat Straw

Cellulase distribution between residual substrate and supernatant in the process of enzymatic hydrolysis of steam-exploded wheat straw was investigated. Subsequently, a novel stepwise recovery strategy with three successive steps was adopted to recover cellulase adsorbed to the residual substrate. The results showed that cellulase protein in the supernatant increased as the hydrolysis time increased. When hydrolysis ended, the cellulase remaining on the residual substrate accounted for 33-42% of the original added cellulase according to the different cellulase loading. To obtain the maximum cellulase recovery rate, the residual substrate was dealt with in three successive steps: washed with sodium acetate buffer (step 1), shaken with sodium acetate buffer (step 2), and then treated with 0.0015 mol/L, pH 10 Ca(OH)2 (step 3). The total cellulase protein recovered by the three steps reached 96.70-98.14%. The enzyme activity of cellulase recovered by the first two steps was kept well. The ratios of the specific activity between the recovered cellulase and the original were 89-96%, which was by far higher than that using step 3 (the value was 48% approximately 56%).

Correlation of cellulase gene expression and cellulolytic activity throughout the gut of the termite Reticulitermes flavipes

Termites have developed cellulose digestion capabilities that allow them to obtain energy and nutrition from nutritionally poor food sources, such as lignocellulosic plant material and residues derived from it ( e.g., wood and humus). Lower termites, which are equipped with both endogenous ( i.e., of termite origin) and symbiotic cellulases, feed primarily on wood and wood-related materials. This study investigated cellulase gene diversity, structure, and activity in the lower termite, Reticulitermes flavipes (Kollar). We initially used a metagenomics approach to identify four genes encoding one endogenous and three symbiotic cellulases, which we refer to as Cell-1, -2, -3 and -4. These four genes encode proteins that share significant sequence similarity with known endoglucanases, exoglucanases and xylanases. Phylogenetic analyses further supported these inferred relationships by showing that each of the four cellulase proteins clusters tightly with respective termite, protozoan or fungal cellulases. Gene structure studies revealed that Cell-1, -3 and -4 are intron-free, while Cell-2 contains the first intron sequence to be identified from a termite symbiont cellulase. Quantitative real-time PCR (qRT-PCR) revealed that the endogenous Cell-1 gene is expressed exclusively in the salivary gland/foregut, whereas symbiotic Cell-2, -3, and -4 are highly expressed in the hindgut (where cellulolytic protists are harbored). Cellulase activity assays mapped the distribution pattern of endoglucanase, exoglucanase and xylanase activity throughout the R. flavipes digestive tract. Cellulase gene expression correlated well with the specific types of cellulolytic activities observed in each gut region (foregut + salivary gland, midgut and hindgut). These results suggest the presence of a single unified cellulose digestion system, whereby endogenous and symbiotic cellulases work sequentially and collaboratively across the entire digestive tract of R. flavipes.

Optimization and modeling of cellulase protein from Trichoderma reesei Rut C30 using mixed substrate

Bioethanol from cellulosic raw material has proved to be the best alternative renewable energy source. Cellulase is a multienzyme complex catalyses the bioconversion of cellulose to glucose, which can be used for ethanol production. The objective of this research is to reduce the cost of cellulase production by optimization of fermentation conditions and modeling of the fermentation process. Research surface methodology was suggested for optimization of process conditions of cellulase biosynthesis. Logistic kinetic model was the best model for the mixed substrates. A conceptual Artificial Neural Network (ANN) model was well incorporated in the fermentative production of cellulase. By adopting these models high yield of cellulase was obtained. Keywords: Cellulose, Lactose, Cellulase, Optimization, Modeling, Response surface methodology, Logistic model, Artificial Neural Network

Optimization and modeling of cellulase protein from Trichoderma reesei Rut C30 using mixed substrate

Bioethanol from cellulosic raw material has proved to be the best alternative renewable energy source. Cellulase is a multienzyme complex catalyses the bioconversion of cellulose to glucose, which can be used for ethanol production. The objective of this research is to reduce the cost of cellulase production by optimization of fermentation conditions and modeling of the fermentation process. Research surface methodology was suggested for optimization of process conditions of cellulase biosynthesis. Logistic kinetic model was the best model for the mixed substrates. A conceptual Artificial Neural Network (ANN) model was well incorporated in the fermentative production of cellulase. By adopting these models high yield of cellulase was obtained.

Fermentative production and kinetics of cellulase protein on Trichoderma reesei using sugarcane bagasse and rice straw

Cellulase a multienzyme made up of several proteins finds extensive applications in food, fermentation and textile industries. Trichoderma reesei is an efficient producer of cellulase protein. The comparative study was made on various carbon sources on the production of cellulase using strains of T. reesei QM 9414, 97.177 and Tm3. Pretreatment of sugarcane bagasse and rice straw offers very digestible cellulose and potentially less inhibition. Cellulase production was enhanced by multiple carbon sources because of diauxic pattern of utilization of substrates. This is the first attempt of combining the synthetic substrate (xylose, lactose) with natural substrate (sugarcane bagasse, rice straw). The mixture of substrates produced the highest maximal enzyme activity on cellulose with xylose, cellulose with lactose, bagasse with xylose, bagasse with lactose, rice straw with xylose and rice straw with lactose. In addition Monod growth kinetics and Leudeking piret product formation kinetics were studied using T. reesei with optimized medium under optimized conditions of inoculum concentration, D.O. level, agitator speed, temperature and pH.