A mechanistic model for rational design of optimal cellulase mixtures

A model‐based framework is described that permits the optimal composition of cellulase enzyme mixtures to be found for lignocellulose hydrolysis. The rates of hydrolysis are shown to be dependent on the nature of the substrate. For bacterial microcrystalline cellulose (BMCC) hydrolyzed by a ternary cellulase mixture of EG2, CBHI, and CBHII, the optimal predicted mixture was 1:0:1 EG2:CBHI:CBHII at 24 h and 1:1:0 at 72 h, at loadings of 10 mg enzyme per g substrate. The model was validated with measurements of soluble cello‐oligosaccharide production from BMCC during both single enzyme and mixed enzyme hydrolysis. Three‐dimensional diagrams illustrating cellulose conversion were developed for mixtures of EG2, CBHI, CBHII acting on BMCC and predicted for other substrates with a range of substrate properties. Model predictions agreed well with experimental values of conversion after 24 h for a variety of enzyme mixtures. The predicted mixture performances for substrates with varying properties demonstrated the effects of initial degree of polymerization (DP) and surface area on the performance of cellulase mixtures. For substrates with a higher initial DP, endoglucanase enzymes accounted for a larger fraction of the optimal mixture. Substrates with low surface areas showed significantly reduced hydrolysis rates regardless of mixture composition. These insights, along with the quantitative predictions, demonstrate the utility of this model‐based framework for optimizing cellulase mixtures. Biotechnol. Bioeng. 2011;108: 2561–2570. © 2011 Wiley Periodicals, Inc.     

Competitive inhibition of cellobiohydrolase I by manno-oligosaccharides

In the hydrolysis of softwood, significant amounts of manno-oligosaccharides (MOS) are released from mannan, the major hemicelluloses in softwood. However, the impact of MOS on the performance of cellulases is not yet clear. In this work, the effect of mannan and MOS in cellulose hydrolysis by cellulases, especially cellobiohydrolase I (CBHI) from Thermoascus aurantiacus (Ta Cel7A), was studied. The glucose yield of Avicel decreased with an increasing amount of added mannan. Commercial cellulases contained mannan hydrolysing enzymes, and β-glucosidase played an important role in mannan hydrolysis. Addition of 10 mg/ml mannan reduced the glucose yield of Avicel (at 20 g/l) from 40.1 to 24.3%. No inhibition of β-glucosidase by mannan was observed. The negative effects of mannan and MOS on the hydrolytic action of cellulases indicated that the inhibitory effect was at least partly attributed to the inhibition of Ta Cel7A (CBHI), but not on β-glucosidase. Kinetic experiments showed that MOS were competitive inhibitors of the CBHI from T. aurantiacus, and mannobiose had a stronger inhibitory effect on CBHI than mannotriose or mannotetraose. For efficient hydrolysis of softwood, it was necessary to add supplementary enzymes to hydrolyze both mannan and MOS to less inhibitory product, mannose.     

Improvement of cellulose-degrading ability of a yeast strain displaying Trichoderma reesei endoglucanase II by recombination of cellulose-binding domains

To improve the cellulolytic activity of a yeast strain displaying endoglucanase II (EGII) from the filamentous fungus Trichoderma reesei QM9414, the genes encoding the cellulose-binding domain (CBD) of EGII, cellobiohydrolase I (CBHI) and cellobiohydrolase II (CBHII) from T. reesei QM9414, were fused with the catalytic domain of EGII and expressed in Saccharomyces cerevisiae. Display of each of the recombinant EGIIs was confirmed using immunofluorescence microscopy. In the case of EGII-displaying yeast strains in which the CBD of EGII was replaced with the CBD of CBHI or CBHII, the binding affinity to Avicel and hydrolytic activity toward phosphoric acid swollen Avicel were similar to that of a yeast strain displaying wild-type EGII. On the other hand, the three yeast strains displaying EGII with two or three tandemly aligned CBDs showed binding affinity and hydrolytic activity higher than that of the yeast strain displaying wild-type EGII. This result indicates that the hydrolytic activity of yeast strains displaying recombinant EGII increases with increased binding ability to cellulose.     

Evaluation of minimal Trichoderma reesei cellulase mixtures on differently pretreated Barley straw substrates

The commercial cellulase product Celluclast 1.5, derived from Trichoderma reesei (Novozymes A/S, Bagsvaerd, Denmark), is widely employed for hydrolysis of lignocellulosic biomass feedstocks. This enzyme preparation contains a broad spectrum of cellulolytic enzyme activities, most notably cellobiohydrolases (CBHs) and endo-1,4-beta-glucanases (EGs). Since the original T. reesei strain was isolated from decaying canvas, the T. reesei CBH and EG activities might be present in suboptimal ratios for hydrolysis of pretreated lignocellulosic substrates. We employed statistically designed combinations of the four main activities of Celluclast 1.5, CBHI, CBHII, EGI, and EGII, to identify the optimal glucose-releasing combination of these four enzymes to degrade barley straw substrates subjected to three different pretreatments. The data signified that EGII activity is not required for efficient lignocellulose hydrolysis when addition of this activity occurs at the expense of the remaining three activities. The optimal ratios of the remaining three enzymes were similar for the two pretreated barley samples that had been subjeced to different hot water pretreatments, but the relative levels of EGI and CBHII activities required in the enzyme mixture for optimal hydrolysis of the acid-impregnated, steam-exploded barley straw substrate were somewhat different from those required for the other two substrates. The optimal ratios of the cellulolytic activities in all cases differed from that of the cellulases secreted by T. reesei. Hence, the data indicate the feasibility of designing minimal enzyme mixtures for pretreated lignocellulosic biomass by careful combination of monocomponent enzymes. This strategy can promote both a more efficient enzymatic hydrolysis of (ligno)cellulose and a more rational utilization of enzymes.     

Cloning of two cellobiohydrolase genes from <Emphasis Type="Italic">Trichoderma</Emphasis><Emphasis Type="Italic">viride</Emphasis> and heterogenous expression in yeast <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Cellobiohydrolase genes cbhI and cbhII were isolated from Trichoderma viride AS3.3711 and T. viride CICC 13038, respectively, using RT-PCR technique. The cbhI gene from T. viride AS3.3711 contains 1,542 nucleotides and encodes a 514-amino acid protein with a molecular weight of approximately 53.96 kDa. The cbhII gene from T. viride CICC 13038 was 1,413 bp in length encoding 471 amino acid residues with a molecular weight of approximately 49.55 kDa. The CBHI protein showed high homology with enzymes belonging to glycoside hydrolase family 7 and CBHII is a member of Glycoside hydrolase family 6. CBHI and CBHII play a role in the conversion of cellulose to glucose by cutting the disaccharide cellobiose from the non-reducing end of the cellulose polymer chain. The two cellobiohydrolase (CBHI, CBHII) genes were successfully expressed in Saccharomyces cerevisiae H158. Maximal activities of transformants Sc-cbhI and Sc-cbhII were 0.03 and 0.089 units ml⁻¹ under galactose induction, respectively. The optimal temperatures of the recombinant enzymes (CBHI, CBHII) were 60 and 70°C, respectively. The optimal pHs of recombinant enzymes CBHI and CBHII were at pH 5.8 and 5.0, respectively.     

Widely different off rates of two closely related cellulose-binding domains from Trichoderma reesei.

The filamentous fungus Trichoderma reesei produces two cellobiohydrolases (CBHI and CBHII). These, like most other cellulose-degrading enzymes, have a modular structure consisting of a catalytic domain linked to a cellulose-binding domain (CBD). The isolated catalytic domains bind poorly to cellulose and have a much lower activity towards cellulose than the intact enzymes. For the CBDs, no function other than binding to cellulose has been found. We have previously described the reversibility and exchange rate for the binding of the CBD of CBHI to cellulose. In this work, we studied the binding of the CBD of CBHII and showed that it differs markedly from the behaviour of that of CBHI. The apparent binding affinities were similar, but the CBD of CBHII could not be dissociated from cellulose by buffer dilution and did not show a measurable exchange rate. However, desorption could be triggered by shifting the temperature. The CBD of CBHII bound reversibly to chitin. Two variants of the CBHII CBD were made, in which point mutations increased its similarity to the CBD of CBHI. Both variants were found to bind reversibly to cellulose.     

Utilization of agro-industrial orange peel and sugar beet pulp wastes for fungal endo- polygalacturonase production

The pectinase enzymes are involved in several industrial applications, and industrial waste is one of the largest environmental pollutants, so this study aims to Endo-polygalacturonase (endo-PG) producing using Aspergillus niger AUMC 4156, Penicillium oxalicum AUMC 4153 and P. variotii AUMC 4149 by using some agro-industrial wastes (dried orange peel and sugar beet pulp) as a sole raw carbon source for degradation these waste in the process of urban wastes disposal. The fermentation process was carried out as a submerged culture technique under both shaken and static culture conditions. A. niger AUMC 4156 was the most promising producer of endo-PG under static conditions while P. oxalicum AUMC 4153 was the highest producer of endo-PG under shaken conditions. Sugar beet pulp proved to be the most preferable to orange peel as the only source of carbon in both shaken and static cultures. The medium that encompassing orange peel as a single carbon source afforded the highest protein content with all tested fungal strains in stirred and static cultures in comparison with sugar beet pulp. The highest activity of endo-polygalacuronase that produced using A. niger AUMC 4156 and P. oxalicum AUMC 4153 was achieved by using sugar beet pulp at 3% concentration under static cultures, meanwhile maximal enzyme activity produced by both fungal strains required 2% sugar beet pulp under shaken cultures. Sugar beet pulp showed promised potential as a good inducer for endo-polygalacturoase production, and enzymes production depended on fungal strains, culture medium, and submerged fermentation conditions.     

The 1,4-beta-D-glucan cellobiohydrolases from Phanerochaete chrysosporium. I. A system of synergistically acting enzymes homologous to Trichoderma reesei.

A physico-chemical and structural characterization of three 1,4-beta-D-glucan cellobiohydrolases (EC. 3.2.1.91), isolated from a culture filtrate of the white-rot fungus Phanerochaete chrysosporium, reveals that the cellulolytic enzyme secretion pattern and thus the general degradation strategy for P. chrysosporium is similar to that of Trichoderma reesei. Partial sequence data show that two of the isolated enzymes, i.e., CBHI, pI 3.82 and CBH62, pI 4.85, are homologous with CBHI and EGI from T. reesei; while, the third, i.e., CBH50, pI 4.87, is homologous to T. reesei CBHII. Limited proteolysis with papain cleaved each of the three enzymes into two domains: a core protein which retained full catalytic activity against low molecular weight substrates and a peptide fragment corresponding to the cellulose binding domain, in striking similarity to the structural organization of T. reesei. CBHI and CBH62 have their binding domain located at the C-terminus, whereas in CBH50 it is located at the N-terminus. It is evident that synergistically acting cellobiohydrolases is a general requirement for efficient hydrolysis of crystalline cellulose by cellulolytic fungi.     

Biodegradation of chestnut shell and lignin-modifying enzymes production by the white-rot fungi Dichomitus squalens,Phlebia radiata

As a discarded lignocellulosic biomass, chestnut shell is of great potential economic value, thus a sustainable strategy is needed and valuable for utilization of this resource. Herein, the feasibility of biological processes of chestnut shell with Dichomitus squalens, Phlebia radiata and their co-cultivation for lignin-modifying enzymes (LMEs) production and biodegradation of this lignocellulosic biomass was investigated under submerged cultivation. The treatment with D. squalens alone at 12 days gained the highest laccase activity (9.42 ± 0.73 U mg^?1). Combined with the data of laccase and manganese peroxidase, oxalate and H₂O₂ were found to participate in chestnut shell degradation, accompanied by a rapid consumption of reducing sugar. Furthermore, specific surface area of chestnut shell was increased by 77.6–114.1 % with the selected fungi, and total pore volume was improved by 90.2 % with D. squalens. Meanwhile, the surface morphology was observably modified by this fungus. Overall, D. squalens was considered as a suitable fungus for degradation of chestnut shell and laccase production. The presence of LMEs, H₂O₂ and oxalate provided more understanding for decomposition of chestnut shell by the white-rot fungi.     

Efficient secretion of two fungal cellobiohydrolases by Saccharomyces cerevisiae

Two different cellobiohydrolases, CBHI and CBHII, of the filamentous fungus Trichoderma reesei both hydrolyse highly crystalline cellulose. Cellulolytic strains of the yeast Saccharomyces cerevisiae were constructed by transferring cDNAs coding for these enzymes into yeast on an expression plasmid. These cellulolytic yeasts were able to secrete efficiently the large, heterologous proteins to the culture medium. The recombinant cellulases were observed to be heterogeneous in Mr due, at least partly, to variable N-glycosylation. Recombinant CBHII was able to bind to crystalline cellulose, although slightly less efficiently than the native enzyme. Both of the two recombinant cellulases were able to degrade amorphous cellulose. In a fermenter cultivation, around 100 micrograms/ml of CBHII was secreted into the yeast growth medium.     

Isolation and purification of isoenzymes of cellobiohydrolase I and II of trichoderma reesei using LPLC methods

Five cellulases were fractionated from a commercial cellulase preparation (Celluclast^TM) Two isoenzymes of cellobiohydrolase I (CBHI)(pI = 4.1) could be proved to be real exo-glucanases due to their activity towards MU (=methylumbelliferyl)-lactoside being inhibited by cellobiose (5 mM) and due to production of cellobiose from carboxymethylcellulose (CMC) as the sole final product.Two isoenzymes of CBHII (pI=6.15, 6.0) were shown to act as endo-glucanases because they produced glucose, cellobiose and cellotetraose from CMC and because they were not inhibited by cellobiose when decomposing MU-lactoside. Results confirm recent reports in the literature classifying CBHI and CBHII as exo-type and endo-type cellulases, respectively. Both the CBHI and the CBHII isoenzymes were shown to be active towards CMC and amorphous cellulose.CBHI and CBHII reactions could be differentiated from one another by the velocities of decomposition of CMC: CBHI acts slowly and linearly whereas CBHII acts strongly and exponentially.The fifth of the purified enzymes must be classed as a conventional endoglucanase which exhibits activity towards CMC but fails to be active towards MU-lactoside and amorphous cellulose.     

Method for characterization of the enzyme profile and the determination of CBH I (Cel 7a) core protein in Trichoderma reesei cellulase preparations

Summary Fast protein liquid chromatography (FPLC) was used to characterize a commercial cellulase preparation (Celluclast 1.5L, Novozymes) in relation to its protein profile and activity against hydroxyethylcellulose (HEC) and other substrates. Co-elution of CBHII (Cel 6A) with other enzyme components of the cellulase system was characterized by immunochemical assays using monoclonal antibodies, whereas the occurrence of EGII (Cel 5A) was assessed based on its ability to cleave the heterosidic bond of 4-methylumbellyferyl-β-d-cellotrioside (MUmbG₃). The main cellulase constituents of Celluclast 1.5L were pooled into six fractions containing EGII (Cel 5A) and EGIII (Cel 12A) (F1), EGII and CBHII (Cel 6A) (F2), CBHII and EGI (Cel 7B) (F3), EGI (F4), and CBHI (Cel 7A) (F5). The occurrence of CBHI core protein within the CBHI fraction of the FPLC profile was determined by hydrophobic interaction chromatography. Using this method, we were able to demonstrate that the batch of Celluclast 1.5L used in this study contained 10.9–18.8% of CBHI as its corresponding free core protein.     

Fractionation of sugar beet pulp into pectin, cellulose, and arabinose by arabinases combined with ultrafiltration.

Incubation of beet pulp with two arabinases (alpha-L-arabinofuranosidase and endo-arabinase), used singularly or in combination at different units of activity per gram of beet pulp, caused the hydrolysis of arabinan, which produced a hydrolyzate consisting mainly of arabinose. Pectin and a residue enriched with cellulose were subsequently separated from the incubation mixture. The best enzymatic hydrolysis results were obtained when 100 U/g of beet pulp of each enzyme worked synergistically with yields of 100% arabinose and 91.7% pectin. These yields were higher than those obtained with traditional chemical hydrolysis. The pectin fraction showed a low content of neutral sugar content and the cellulose residue contained only a small amount of pentoses. Semicontinuous hydrolysis with enzyme recycling in an ultrafiltration unit was also carried out to separate arabinose, pectin, and cellulose from beet pulp in 7 cycles of hydrolysis followed by ultrafiltration. The yields of separation were similar to those obtained in batch experiments, with an enzyme consumption reduced by 3.5 times and some significant advantages over batch processes.     

Effects of non-ionic surfactants on the interactions between cellulases and tannic acid: a model system for cellulase-poly-phenol interactions

Addition of non-ionic surfactants (NIS) is known to accelerate enzymatic lignocellulose hydrolysis. The mechanism behind this accelerating effect is still not elucidated but has been hypothesized to originate from favorable NIS-lignin interactions which alleviate non-productive adsorption of cellulases to lignin. In the current work we address this hypothesis using tannic acid (TAN) as a general poly-phenolic model compound (for lignin and soluble phenolics) and measure the mutual interactions of cellulases (CBHI, CBHII, EGI, EGII and BG), TAN and NIS (Triton X-100) using isothermal titration calorimetry (ITC). The experimental results suggest rather strong enzyme-specific interactions with TAN in reasonable agreement with enzyme specific lignin inhibition found in the literature. Enzyme-TAN interactions were disrupted by the presence of NIS by a mechanism of strong TAN-NIS interaction. The presence of NIS also alleviated the inhibitory effect of TAN on cellulase activity. All together the current work provides strong indications that favorable NIS-poly-phenol interactions alleviate non-productive cellulase-poly-phenol interactions and hence may provide a mechanism for the accelerating effect of NIS on lignocellulose hydrolysis.     

Thermal Stabilization of Erwinia chrysanthemi Pectin Methylesterase A for Application in a Sugar Beet Pulp Biorefinery

Directed evolution approaches were used to construct a thermally stabilized variant of Erwinia chrysanthemi pectin methylesterase A. The final evolved enzyme has four amino acid substitutions that together confer a T_m value that is approximately 11°C greater than that of the wild-type enzyme, while maintaining near-wild-type kinetic properties. The specific activity, with saturating substrate, of the thermally stabilized enzyme is greater than that of the wild-type enzyme when both are operating at their respective optimal temperatures, 60°C and 50°C. The engineered enzyme may be useful for saccharification of biomass, such as sugar beet pulp, with relatively high pectin content. In particular, the engineered enzyme is able to function in biomass up to temperatures of 65°C without significant loss of activity. Specifically, the thermally stabilized enzyme facilitates the saccharification of sugar beet pulp by the commercial pectinase preparation Pectinex Ultra SPL. Added pectin methylesterase increases the initial rate of sugar production by approximately 50%.Pectin is a heterogenous structural polysaccharide found in plant primary cell walls. Pectin helps to connect and cross-link other cell wall polysaccharides, such as cellulose and hemicellulose, to contribute to cell wall rigidity. The pectin backbone is comprised of α-(1,4)-linked galacturonic acid (GalA) subunits. In addition to the “smooth” regions of homogalacturonans, there are variable proportions of “hairy” regions consisting of α-(1,5)-linked arabinans and/or β-(1,4)-linked galactans as well as other neutral sugars. Some of the GalA subunits carry methoxyl and acetyl esters at C-6 and C-2/C-3, respectively. The degree of C-6 methylesterification, in particular, influences the rheological properties of the polymer (18, 19, 24).Pectin methylesterases (PMEs; EC 3.1.1.11) catalyze the demethylesterification of GalA C-6 producing methanol, protons, and polygalacturonate. This reaction is significant in a number of contexts. In muro, the activity of plant PMEs helps control cell wall rigidity and plays a major role in pectin remodeling related to cell wall growth and processes such as fruit ripening (15). In the case of bacterial and fungal phytopathogens, PMEs are virulence factors that are necessary for pathogen invasion and spread through plant tissues (2, 3, 23). PMEs along with other pectinolytic enzymes are widely used in the food and beverage industries and paper and fiber industries, among others (9).PMEs work in concert with other pectinolytic enzymes, pectate lyases (EC 4.2.2.2), and pectate glycohydrolases (EC 3.2.1.15), among others, to depolymerize pectin. Highly esterified pectin is largely resistant to depolymerization (1). Shevchik and colleagues demonstrated that pretreatment of purified sugar beet pectin with the Erwinia chrysanthemi PMEA resulted in a 10- to 20-fold enhancement in the catalytic rate of E. chrysanthemi pectate lyases PELA, PELB, PELC, PELD, and PELL relative to that of the untreated substrate (21). Similarly, Christgau and colleagues found that depolymerization of purified apple pectin by pectate glycohydrolase from Aspergillus aculeatus was dependent on added PME (5).E. chrysanthemi produces at least two PMEs. PMEA is a 342-amino-acid secreted protein, and the 433-amino-acid PMEB is bound to the outer membrane (12, 20). PMEA is a novel aspartate-esterase that folds into a right-handed parallel β-helix, similar to other pectinolytic enzymes, such as pectate lyases and polygalacturonases (8, 10). The enzyme is active over a broad pH range (5-9) and has optimal activity around 50°C (11, 16).Sugar beet pulp is the by-product of sucrose production from the tap root of Beta vulgaris. Sugar beet pulp is rich in pectin, hemicellulose, and cellulose and relatively low in lignin content. It exits the sugar refinery as heated (ca. 60°C) thin slices approximately 75% water by weight. These and other considerations make sugar beet pulp an attractive biomass target for enzymatic saccharification and subsequent conversion of sugars to value-added products.In order to create a PME suited to the saccharification of sugar beet pulp, we employed directed evolution approaches to engineer a variant of E. chrysanthemi PMEA that would function at 60°C in sugar beet pulp. Here, we report the development of a thermostabilized PMEA variant with four amino acid substitutions that demonstrates efficacy in the saccharification of sugar beet pulp.     

Comparative analyses of Podospora anserina secretomes reveal a large array of lignocellulose-active enzymes

The genome of the coprophilous fungus Podospora anserina harbors a large and highly diverse set of putative lignocellulose-acting enzymes. In this study, we investigated the enzymatic diversity of a broad range of P. anserina secretomes induced by various carbon sources (dextrin, glucose, xylose, arabinose, lactose, cellobiose, saccharose, Avicel, Solka-floc, birchwood xylan, wheat straw, maize bran, and sugar beet pulp (SBP)). Compared with the Trichoderma reesei enzymatic cocktail, P. anserina secretomes displayed similar cellulase, xylanase, and pectinase activities and greater arabinofuranosidase, arabinanase, and galactanase activities. The secretomes were further tested for their capacity to supplement a T. reesei cocktail. Four of them improved significantly the saccharification yield of steam-exploded wheat straw up to 48 %. Fine analysis of the P. anserina secretomes produced with Avicel and SBP using proteomics revealed a large array of CAZymes with a high number of GH6 and GH7 cellulases, CE1 esterases, GH43 arabinofuranosidases, and AA1 laccase-like multicopper oxidases. Moreover, a preponderance of AA9 (formerly GH61) was exclusively produced in the SBP condition. This study brings additional insights into the P. anserina enzymatic machinery and will facilitate the selection of promising targets for the development of future biorefineries.     

Purification and partial characterisation of three endo-glucanases from dichomitus squalens

Three endo-glucanases (En I-III) were obtained by chromatofocusing fractionation of a culture supernatant from the white-rot fungus Dichomitus squalens. They were purified further on Phenyl-Sepharose CL-4B, DEAE-Trisacryl, and Ultrogel AcA 54; ∼21-, ∼16-, and ∼9-fold purifications were obtained for En I-III, respectively. The enzymes appeared as homogeneous proteins on disc gel electrophoresis with and without SDS (sodium dodecyl sulphate), and on isoelectric focusing; the respective mol. wts. were 42,000, 56,000, and 47,000, and the isoelectric points 4.8, 4.3, and 4.1. Optimum conditions for the hydrolysis of CM-cellulose were pH 4.8 and 55° for each enzyme, and each was stable over the pH range 4.0–8.5 but inactivated completely within 30 min at 70°. None of the purified enzymes exhibited β-d-glucosidase or cellobiohydrolase activity, but En II was weakly active towards laminaran and xylan. En I and En II acted more randomly on CM-cellulose than did En III. Cellotetraose was degraded by each endo-glucanase, whereas only En III could hydrolyse cellotriose.     

Biodegradation of lignocellulosic waste by Aspergillus terreus

Biodegradation of lignocellulosic waste by Aspergillus terreus is reported for the first time. This isolate produced 250 CMCase (carboxymethyl cellulase or endoglucanase) U.ml^-1 and biodegraded hay and straw during 3 days and the biomass production on straw was 5g.L^-1dry weight from 0.25 cm² inoculated mycellium. This strain secreted endocellulases and exocellulases in the culture medium, but some of the enzymes produced, remained cell membrane bound. Cell bound enzymes were released by various treatments. The highest amount of endoglucanase and exoglucanase was released when the cells were treated with sonication. Aspergillus terreus was added to two tanks containing sugar wastewater and pulp manufacturing waste, as a seed for COD removal. This fungus reduced the COD by 40–80 percent, also, ammonia was reduced from 14.5 mM to 5.6 mM in sugar beet wastewater. The effects of crude enzyme of this fungus for COD removal was studied.     

Enhanced production of cellobiohydrolases in Trichoderma reesei and evaluation of the new preparations in biofinishing of cotton

In the search for suitable cellulase combinations for industrial biofinishing of cotton, five different types of Trichoderma reesei strains were constructed for elevated cellobiohydrolase production: CBHI overproducers with and without endoglucanase I (EGI), CBHII overproducers with and without endoglucanase II (EGII) and strains overproducing both CBHI and CBHII without the major endoglucanases I and II. One additional copy of cbh1 gene increased production of CBHI protein 1.3-fold, and two copies 1.5-fold according to ELISA (enzyme-linked immunosorbent assay). The level of total secreted proteins was increased in CBHI transformants as compared to the host strain. One copy of the cbh2 expression cassette in which the cbh2 was expressed from the cbh1 promoter increased production of CBHII protein three- to four-fold when compared to the host strain. T. reesei strains producing elevated amounts of both CBHI and CBHII without EGI and EGII were constructed by replacing the egl1 locus with the coding region of the cbh1 gene and the egl2 locus with the coding region of cbh2. The cbh1 was expressed from its own promoter and the cbh2 gene using either the cbh1 or cbh2 promoter. Production of CBHI by the CBH-transformants was increased up to 1.6-fold and production of CBHII up to 3.4-fold as compared with the host strain. Approximately similar amounts of CBHII protein were produced by using cbh1 or cbh2 promoters. When the enzyme preparation with elevated CBHII content was used in biofinishing of cotton, better depilling and visual appearance were achieved than with the wild type preparation; however, the improvement was not as pronounced as with preparations with elevated levels of endoglucanases (EG).     

Dynamic Interaction of Trichoderma reesei Cellobiohydrolases Cel6A and Cel7A and Cellulose at Equilibrium and during Hydrolysis

The binding of cellobiohydrolases to cellulose is a crucial initial step in cellulose hydrolysis. In the search for a detailed understanding of the function of cellobiohydrolases, much information concerning how the enzymes and their constituent catalytic and cellulose-binding domains interact with cellulose and with each other and how binding changes during hydrolysis is still needed. In this study we used tritium labeling by reductive methylation to monitor binding of the two Trichoderma reesei cellobiohydrolases, Cel6A and Cel7A (formerly CBHII and CBHI), and their catalytic domains. Measuring hydrolysis by high-performance liquid chromatography and measuring binding by scintillation counting allowed us to correlate activity and binding as a function of the extent of degradation. These experiments showed that the density of bound protein increased with both Cel6A and Cel7A as hydrolysis proceeded, in such a way that the adsorption points moved off the initial binding isotherms. We also compared the affinities of the cellulose-binding domains and the catalytic domains to the affinities of the intact proteins and found that in each case the affinity of the enzyme was determined by the linkage between the catalytic and cellulose-binding domains. Desorption of Cel6A by dilution of the sample showed hysteresis (60 to 70% reversible); in contrast, desorption of Cel7A did not show hysteresis and was more than 90% reversible. These findings showed that the two enzymes differ with respect to the reversibility of binding.