Effect of pH on cellulase production of Trichoderma ressei RUT C30

Currently, the high market price of cellulases prohibits commercialization of the lignocellulosics-to-fuel ethanol process, which utilizes enzymes for saccharification of cellulose. For this reason research aimed at understanding and improving cellulase production is still a hot topic in cellulase research. Trichoderma reesei RUT C30 is known to be one of the best hyper producing cellulolytic fungi, which makes it an ideal test organism for research. New findings could be adopted for industrial strains in the hope of improving enzyme yields, which in turn may result in lower market price of cellulases, thus making fuel ethanol more cost competitive with fossil fuels. Being one of the factors affecting the growth and cellulase production of T. reesei, the pH of cultivation is of major interest. In the present work, numerous pH-controlling strategies were compared both in shake-flask cultures and in a fermentor. Application of various buffer systems in shake-flask experiments was also tested. Although application of buffers resulted in slightly lower cellulase activity than that obtained in non-buffered medium, β-glucosidase production was increased greatly.     

Evidence for a novel mechanism of microbial cellulose degradation

There are two well studied mechanisms that are used by cellulolytic microorganisms to degrade the cellulose present in plant cell walls and a third less well studied oxidative mechanism used by brown rot fungi. The well studied mechanisms use cellulases to hydrolyze the β-1,4 linkages present in cellulose, however the way in which cellulases are presented to the environment are quite different for each mechanism. Most aerobic microorganisms secrete a set of cellulases outside the cell (free cellulase mechanism) while most anaerobic microorganisms produce large multi enzyme complexes on their outer surface (cellulosomal mechanism). Their genomic sequences suggest that the aerobic bacterium, Cytophaga hutchinsonii and the anaerobic bacterium, Fibrobacter succinogenes, do not use either of these mechanisms for degrading cellulose, as these organisms only code for normal endocellulases not for processive cellulases like exocellulases and processive endocellulases which are used in both of the well studied mechanisms.     

β-Glucosidase production by Trichoderma reesei

The hydrolysis of cellulose to the water-soluble products cellobiose and glucose is achieved via synergistic action of cellulolytic proteins. The three types of enzymes involved in this process are endoglucanases, cellobiohydrolases, and β-glucosidases. One of the best fungal cellulase producers is Trichoderma reesei RUT C30. However, the amount of β-glucosidases secreted by this fungus is insufficient for effective cellulose conversion. We investigated the production of cellulases and β-glucosidases in shake-flask cultures by applying three pH-controlling strategies: (1) the pH of the production medium was adjusted to 5.8 after the addition of seed culture with no additional pH adjustment performed, (2) the pH was adjusted to 6.0 daily, and (3) the pH was maintained at 6.0 by the addition of Tris-maleate buffer to the growth medium. Different carbon sources—Solka Floc 200, glucose, lactose, and sorbitol—were added to standard Mandels nutrients. The lowest β-glucosidase activities were obtained when no pH adjustment was done regardless of the carbon source employed. Somewhat higher levels of β-glucosidase were measured in the culture filtrates when daily pH adjustment was carried out. The effect of buffering the culture medium on β-glucosidase liberation was most prominent when a carbon source inducing the production of other cellulases was applied.     

Maximum Saccharification of Cellulose Complex by an Enzyme Cocktail Supplemented with Cellulase from Newly Isolated <Emphasis Type="BoldItalic">Aspergillus fumigatus</Emphasis> ECU0811

Either the natural biodegradation process or the industrial hydrolytic process requires synergistic interactions between various cellulases. However, it is sometimes impeded by low hydrolytic rate of existing cellulases and the lack of accessory enzymes. Herein, the ability of a commercial cellulase (Spezyme CP, from Genencor) to degrade steam explosion-pretreated corn stover was significantly improved. Firstly, a fungal cellulase producer, Aspergillus fumigatus ECU0811, was isolated from hundreds of soil samples. A 96-deep-well microscale-based platform was developed here to reduce the labor-intensive screening work and proved to be consistent with macroscale screening work. After optimization of fermentation, 3% corn cob could induce A. fumigatus ECU0811 to yield the highest cellulase production. Based on the high activities of β-glucosidase and xylanase by A. fumigatus ECU0811, 0.91 and 125 U/mg protein, respectively, an enzyme cocktail was composed with a fixed dosage of Spezyme CP (CPCel) at 14.2 filter paper units (FPU)/g glucan and varied dosages of A. fumigatus cellulase (AFCel). Consequently, the glucan-to-glucose conversion of corn stover was increased from 25.6% in the presence of CPCel at a dosage of 14.2 FPU/g glucan to 99.5% in the presence of the enzyme cocktail (14.2 FPU CPCel plus 1.21 FPU AFCel per gram of glucan). On the other side, it reduced the total protein amount of CPCel by as much as tenfold, which extremely improved the hydrolytic rate of Spezyme CP and reduced its dosage.     

A Simplified Filter Paper Assay Method of Cellulase Enzymes Based on HPLC Analysis

A simplified filter paper assay (FPA) method of cellulase enzymes was proposed based on high-performance liquid chromatography (HPLC) measurement. The method was according to the sum of glucose and cellobiose concentrations measured by HPLC that was able to be correlated with filter paper units (FPU) of the cellulase enzymes assayed by the traditional FPA method, regardless of the differences in the sources, activities, and components of the cellulases. This simple and quick assay method for the cellulase enzymes provided another parameter of the ratio of glucose to cellobiose (G/C ratio) representing the capacity of cellulase enzymes degrading cellulose into fermentable monomeric sugars.     

Xylanolytic enzyme production by anAspergillus niger isolate

Production of xylanolytic enzymes by anAspergillus niger CCMI 850 isolate was investigated in batch cultures. The effect of the composition of a fermentation medium that did not include chemical inducers, on β-xylanase, β-xylosidase, α-l-arabinofuranosidase, and total cellulase activity was studied. With 4% xylan as the carbon source, about 65 U/mL of β-xylanase was obtained, whereas the total cellulase activity was undetectable, under the specified conditions. This β-xylanase activity represents the highest reported for a wild-type strain ofA. niger. The effect of pH and temperature on the activity of β-xylanase was studied. Partial characterization of the β-xylanase showed that with insoluble birchwood as substrate theK _m andV _max were 0.3 mM and 19 μmol/min, respectively. Aspects of using the crude β-xylanase preparation for applications in the pulp and paper industry were discussed.     

Effects of ammonia fiber explosion treatment on activity of endoglucanase from Acidothermus cellulolyticus in transgenic plant

A critical parameter affecting the economic feasibility of lignocellulosic bioconversion is the production of inexpensive and highly active cellulase enzymes in bulk quantity. A promising approach to reduce enzyme costs is to genetically transform plants with the genes of these enzymes, thereby producing the desired cellulases in the plants themselves. Extraction and recovery of active proteins or release of active cellulase from the plants during bioconversion could have a significant positive impact on overall lignocellulose conversion economics. The effects of ammonia fiber explosion (AFEX) pretreatment variables (treatment temperature, moisture content, and ammonia loading) on the activity of plant-produced heterologous cellulase enzyme were individually investigated via heat treatmett or ammonia treatment. Finally, we studied the effects of all these variables in concert through the AFEX process. The plant materials included transgenic tobacco plants expressing E1 (endoglucanase from Acidothermus cellulolyticus). The E1 activity was measured in untreated and AFEX-treated tobacco leaves to investigate the effects of the treatment on the activity of this enzyme. The maximum observed activity retention in AFEX-treated transgenic tobacco samples compared with untreated samples was approx 35% (at 60°C, 0.5∶1 ammonia loading, and 40% moisture). Based on these findings, it is our opinion that AFEX pretreatment is not a suitable option for releasing cellulase enzyme from transgenic plants.     

Probing substrate binding to Metallo-β-Lactamase L1 from Stenotrophomonas maltophilia by using site-directed mutagenesis

Background  

The metallo-β-lactamases are Zn(II)-containing enzymes that hydrolyze the β-lactam bond in penicillins, cephalosporins, and carbapenems and are involved in bacterial antibiotic resistance. There are at least 20 distinct organisms that produce a metallo-β-lactamase, and these enzymes have been extensively studied using X-ray crystallographic, computational, kinetic, and inhibition studies; however, much is still unknown about how substrates bind and the catalytic mechanism. In an effort to probe substrate binding to metallo-β-lactamase L1 from Stenotrophomonas maltophilia, nine site-directed mutants of L1 were prepared and characterized using metal analyses, CD spectroscopy, and pre-steady state and steady state kinetics.     

Effect of media composition and growth conditions on production of β-glucosidase by Aspergillus niger C-6

The hydrolytic activity of fungal originated β-glucosidase is exploited in several biotechnological processes to increase the rate and extent of saccharification of several cellulosic materials by hydrolyzing the cellobiose which inhibits cellulases. In a previous presentation, we reported the screening and liquid fermentation with Aspergillus niger, strain C-6 for β-glucosidase production at shake flask cultures in a basal culture medium with mineral salts, corn syrup liquor, and different waste lignocellulosic materials as the sole carbon source obtaining the maximum enzymatic activity after 5–6 d of 8.5 IU/mL using native sugar cane bagasse. In this work we describe the evaluation of fermentation conditions: growth temperature, medium composition, and pH, also the agitation and aeration effects for β-glucosidase production under submerged culture using a culture media with corn syrup liquor (CSL) and native sugar cane bagasse pith as the sole carbon source in a laboratory fermenter. The maximum enzyme titer of 7.2 IU/mL was obtained within 3 d of fermentation. This indicates that β-glucosidase productivity by Aspergillus niger C-6 is function of culture conditions, principally temperature, pH, culture medium conditions, and the oxygen supply given in the bioreactor. Results obtained suggest that this strain is a potential microorganism that can reach a major level of enzyme production and also for enzyme characterization.     

Production of cellulase/β-glucosidase by the mixed fungi culture of Trichoderma reesei and Aspergillus phoenicis on dairy manure

A cellulase production process was developed by growing the fungi Trichoderma reesei and Aspergillus phoenicis on dairy manure. T. reesei produced a high total cellulase titer (1.7 filter paper units [FPU]/mL, filter paper activity) in medium containing 10 g/L of manure (dry basis [w/w]), 2 g/L KH₂PO₄, 2 mL/L of Tween-80, and 2mg/L of CoCl₂. However, β-glucosidase activity in the T. reesei-enzyme system was very low. T. reesei was then cocultured with A. phoenicis to enhance the β-glucosidase level. The mixed culture resulted in a relatively high level of total cellulase (1.54 FPU/mL) and β-glucosidase (0.64 IU/mL). The ratio of β-glucosidase activity to filter paper activity was 0.41, suitable for hydrolyzing manure cellulose. The crude enzyme broth from the mixed culture was used for hydrolyzing the manure cellulose, and the produced glucose was significantly (p<0.01) higher than levels obtained by using the commercial enzyme or the enzyme broth of the pure culture T. reesei.     

Cellulase production of Trichoderma reesei rut C 30 using steam-pretreated spruce

Various techniques are available for the conversion of lignocellulosics to fuel ethanol. During the last decade processes based on enzymatic hydrolysis of cellulose have been investigated more extensively, showing good yield on both hardwood and softwood. The cellulase production of a filamentous fungi, Trichoderma reesei Rut C30, was examined on carbon sources obtained after steam pretreatment of spruce. These materials were washed fibrous steam-pretreated spruce (SPS), and hem icellulose hydrolysate. The hemicellulose hydrolysate contained, besides water-soluble carbohydrates, lignin and sugar degradation products, which were formed during the pretreatment and proved to be inhibitory to microorganisms. Experiments were performed in a 4-L laboratory fermentor. The hydrolytic capacity of the produced enzyme solutions was compared with two commercially available enzyme preparations, Celluclast and logen Cellulase, on SPS, washed SPS, and Solka Floc cellulose powder. There was no significant difference among the different enzymes produced by T. reesei Rut C30. However, the conversion of cellulose using these enzymes was higher than that obtained with logen or Celluclast cellulases using steam-pretreated spruce as substrate.     

Effect of acetic acid and furfural on cellulase production of Trichoderma reesei RUT C30

Because of the high temperature applied in the steam pretreatment of lignocellulosic materials, different types of inhibiting degradation products of saccharides and lignin, such as acetic acid and furfural, are formed. The main objective of the present study was to examine the effect of acetic acid and furfural on the cellulase production of a filamentous fungus Trichoderma reesei RUT C30, which is known to be one of the best cellulase-producing strains. Mandels’s mineral medium, supplemented with steam-pretreated willow as the carbon source at a concentration corresponding to 10 g/L of carbohydrate, was used. Four different concentration levels of acetic acid (0–3.0 g/L) and furfural (0–1.2 g/L) were applied alone as well as in certain combinations. Two enzyme activities, cellulase and β-glucosidase, were measured. The highest cellulase activity obtained after a 7-d incubation was 1.55 FPU/mL with 1.0 g/L of acetic acid and 0.8 g/L of furfural added to the medium. This was 17% higher than that obtained without acetic acid and furfural. Furthermore, the results showed that acetic acid alone did not influence the cellulase activity even at the highest concentration. However, β-glucosidase activity was increased with increasing acetic acid concentration. Furfural proved to be an inhibiting agent causing a significant decrease in both cellulase and β-glucosidase production.     

Purification and properties of a β-galactosidase with potential application as a digestive supplement

Functional-based screening of crude β-galactosidase activities from 42 yeast strains resulted in the selection of a single enzyme of potential interest as a digestive supplement. β-Galactosidase produced by Kluyveromyces marxianus DSM5418 was purified to homogeneity by a combination of gel filtration, ion-exchange, and hydroxylapatite chromatographies. The denatured (123 kDa) and native molecular masses (251 kDa) suggest that the enzyme is a homodimer. The optimum pH and temperature of the purified enzyme were 6.8 and 37°C, respectively. The unpurified β-galactosidase in particular displayed a high level of stability when exposed to simulated intestinal conditions in vitro for 4 h. Matrix-assisted laser desorption ionization mass sectrometry analysis revealed that the enzyme's trypsin-generated peptide mass fingerprint shares several peptide fragment hits with β-galactosidases from Kluyveromyces lactis. This confirms the enzyme's identity and indicates that significant sequence homology exists between these enzymes.     

Production of Cellulolytic Enzymes by <Emphasis Type="Italic">Aspergillus phoenicis</Emphasis> in Grape Waste using Response Surface Methodology

The production of cellulolytic enzymes by the fungus Aspergillus phoenicis was investigated. Grape waste from the winemaking industry was chosen as the growth substrate among several agro-industrial byproducts. A 2 × 2 central composite design was performed, utilizing the amount of grape waste and peptone as independent variables. The fungus was cultivated in submerged fermentation at 30 °C and 120 rpm for 120 h, and the activities of total cellulases, endoglucanases, and β-glucosidases were measured. Total cellulases were positively influenced by the linear increase of peptone concentration and decrease at axial concentrations of grape waste and peptone. Maximum activity of endoglucanase was observed by a linear increase of both grape waste and peptone concentrations. Concentrations of grape waste between 5 and 15 g/L had a positive effect on the production of β-glucosidase; peptone had no significant effects. The optimum production of the three cellulolytic activities was observed at values near the central point. A. phoenicis has the potential for the production of cellulases utilizing grape waste as the growth substrate.     

Hydrolytic Enzyme of Cellulose for Complex Formulation Applied Research

To improve the enzymatic hydrolytic efficiency and reduce the supplementation of enzymes, the mixture designed experimental approach was used to optimize the composition of enzyme mixture and promote the hydrolysis of ball-milled corn stover. From the experimental results, a synergistic effect was found when combinations of the three enzymes, two kinds of cellulases and a kind of xylanase, were used. The optimal hydrolysis of pretreated corn stover accorded with enzymes activity ration of FPU/CMCase/β-glucosidase/xylanase = 4.4:1:75:829, and the hydrolysis efficiency of corn stover increased significantly compared with using individual enzyme. The results indicated that the mixture design experiment could be an effective tool for optimized enzyme mixture for lignocellulose hydrolysis.     

Weak lignin-binding enzymes

Economic barriers preventing commercialization of lignocellulose-to-ethanol bioconversion processes include the high cost of hydrolytic enzymes. One strategy for cost reduction is to improve the specific activities of cellulases by genetic engineering. However, screening for improved activity typically uses “ideal” cellulosic substrates, and results are not necessarily applicable to more realistic substrates such as pretreated hardwoods and softwoods. For lignocellulosic substrates, nonproductive binding and inactivation of enzymes by the lignin component appear to be important factors limiting catalytic efficiency. A better understanding of these factors could allow engineering of cellulases with improved activity based on reduced enzyme-lignin interaction (“weak lignin-binding cellulases”). To prove this concept, we have shown that naturally occurring cellulases with similar catalytic activity on a model cellulosic substrate can differ significantly in their affinities for lignin. Moreover, although cellulose-binding domains (CBDs) are hydrophobic and probably participate in lignin binding, we show that cellulases lacking CBDs also have a high affinity for lignin, indicating the presence of lignin-binding sites on the catalytic domain.     

Cellulase retention and sugar removal by membrane ultrafiltration during lignocellulosic biomass hydrolysis

Technologies suitable for the separation and reuse of cellulase enzymes during the enzymatic saccharification of pretreated corn stover are investigated to examine the economic and technical viability of processes that promote cellulase reuse while removing inhibitory reaction products such as glucose and cellobiose. The simplest and most suitable separation is a filter with relatively large pores on the order of 20–25 mm that retains residual corn stover solids while passing reaction products such as glucose and cellobiose to form a sugar stream for a variety of end uses. Such a simple separation is effective because cellulase remains bound to the residual solids. Ultrafiltration using 50-kDa polyethersulfone membranes to recover cellulase enzymes in solution was shown not to enhance further the saccharification rate or overall conversion. Instead, it appears that the necessary cellulase enzymes, including β-glucosidase, are tightly bound to the substrate; when fresh corn stover is contacted with highly washed residual solids, without the addition of fresh enzymes, glucose is generated at a high rate. When filtration was applied multiple times, the concentration of inhibitory reaction products such as glucose and cellobiose was reduced from 70 to 10 g/L. However, an enhanced saccharification performance was not observed, most likely because the concentration of the inhibitory products remained too high. Further reduction in the product concentration was not investigated, because it would make the reaction unnecessarily complex and result in a product stream that is much too dilute to be useful. Finally, an economic analysis shows that reuse of cellulase can reduce glucose production costs, especially when the enzyme price is high. The most economic performance is shown to occur when the cellulase enzyme is reused and a small amount of fresh enzyme is added after each separation step to replace lost or deactivated enzyme.     

Improved Cellulase Production by <Emphasis Type="Italic">Trichoderma reesei </Emphasis>RUT C30 under SSF Through Process Optimization

The major constraint in the enzymatic saccharification of biomass for ethanol production is the cost of cellulase enzymes. Production cost of cellulases may be brought down by multifaceted approaches which includes the use of cheap lignocellulosic substrates for fermentation production of the enzyme, and the use of cost efficient fermentation strategies like solid state fermentation (SSF). The current study investigated the production of cellulase by Trichoderma reesei RUT C30 on wheat bran under SSF. Process parameters important in cellulase production were identified by a Plackett and Burman design and the parameters with significant effects on enzyme production were optimized for maximal yield using a central composite rotary design (CCD). Higher initial moisture content of the medium had a negative effect on production whereas incubation temperature influenced cellulase production positively in the tested range. Optimization of the levels of incubation temperature and initial moisture content of the medium resulted in a 6.2 fold increase in production from 0.605 to 3.8 U/gds of cellulase. The optimal combination of moisture and temperature was found to be 37.56% and 30 °C, respectively, for maximal cellulase production by the fungus on wheat bran.     

Mixed submerged fermentation with two filamentous fungi for cellulolytic and xylanolytic enzyme production

The efficient saccharification of lignocellulosic materials requires the cooperative actions of different cellulase enzyme activities: exoglucanase, endoglucanase, β-glucosidase, and xylanase. Previous studies with the fungi strains Aureobasidium sp. CHTE-18, Penicillium sp. CH-TE-001, and Aspergillus terreus CH-TE-013, selected mainly because of their different cellulolytic and xylanolytic activities, have demonstrated the capacity of culture filtrates of cross-synergistic action in the saccharification of native sugarcane bagasse pith. In an attempt to improve the enzymatic hydrolysis of different cellulosic materials, we investigated a coculture fermentation with two of these strains to enhance the production of cellulases and xylanases. The 48-h batch experimental results showed that the mixed culture of Penicillium sp. CH-TE-001 and A. terreus CH-TE-013 produced culture filtrates with high protein content, cellulase (mainly β-glucosidase), and xylanase activities compared with the individual culture of each strain. The same culture conditions were used in a simple medium with mineral salts, corn syrup liquor, and sugarcane bagasse pith as the sole carbon source with moderate shaking at 29°C. Finally, we compared the effect of the cell-free culture filtrates obtained from the mixed and single fermentations on the saccharification of different kinds of cellulosic materials.