Detrimental effect of cellulose oxidation on cellulose hydrolysis by cellulase

To effectively convert complex and recalcitrant biomass carbohydrates to simple platform sugars useful for fuel and chemicals production, mechanical or chemical pre-treatments are often required to make the carbohydrates more accessible for enzymatic hydrolysis. Due to their harsh conditions, some pre-treatments might negatively affect enzymatic hydrolysis because of events such as cellulose oxidation. To study how oxidative modification may impact cellulose's reactivity toward hydrolysis by cellulases, we prepared three cellulose substrates by cupric ion and hypochlorite oxidations, and subjected the derived celluloses to hydrolysis by various cellobiohydrolases from glycoside hydrolase families 6 and 7, and one cellulolytic Hypocrea jecorina extracellular enzyme mixture. We observed a profound decrease of enzymatic hydrolysis on the oxidized celluloses. The effect was attributed to the interference, from oxidized functional groups in cellulose, on its binding/activation in the active pocket/tunnel of cellobiohydrolases. Potential implication of the observed effect from cellulose oxidation on pre-treatment optimization and cellulase improvement was discussed.     

Alternatives to Trichoderma reesei in biofuel production

Mutant strains of Trichoderma reesei are considered indisputable champions in cellulase production among biomass-degrading fungi. So, it is not surprising that most R&D projects on bioethanol production from lignocellulosics have been based on using T. reesei cellulases. The present review focuses on whether any serious alternatives to T. reesei enzymes in cellulose hydrolysis exist. Although not widely accepted, more and more data have been accumulated that demonstrate that fungi belonging to the genera Penicillium, Acremonium and Chrysosporium might represent such alternatives because they are competitive to T. reesei on some important parameters, such as protein production level, cellulase hydrolytic performance per unit of activity or milligram of protein.     

里氏木霉产纤维素酶研究进展

木质纤维素类生物质被认为是重要且可持续的可再生能源,其主要组成部分是纤维素.纤维素酶是一种能将纤维素分解为葡萄糖的复合酶,能有效地降解木质纤维素生物质.真菌、细菌、放线菌、酵母等多种微生物均可以产生纤维素酶,其中里氏木霉具有完整的纤维素酶系结构,常作为生物技术领域中一个重要菌株,广泛应用于纤维素酶的商业生产.介绍了纤维...     

Inhibitory Effects of Methylcellulose on Cellulose Degradation by Ruminococcus flavefaciens

Highly methylated, long-chain celluloses strongly inhibited cellulose degradation by several species of cellulolytic bacteria of ruminal origin. Specifically, the inhibitory effects of methylcellulose on the growth of Ruminococcus flavefaciens FD1 were concentration dependent, with complete inhibition at 0.1% (wt/vol). However, methylcellulose did not inhibit growth on cellobiose or cellulooligosaccharides. Mixtures of methylated cellulooligosaccharides having an average degree of polymerization of 6.7 to 9.5 inhibited cellulose degradation, but those with an average degree of polymerization of 1.0 to 4.5 did not. Similar inhibitory effects by methylcellulose and, to a lesser extent, by methyl cellulooligosaccharides were observed on cellulase activity, as measured by hydrolysis of p-nitrophenyl-beta-d-cellobioside. R. flavefaciens cultures hydrolyzed cellulooligosaccharides to cellobiose and cellotriose as final end products. Cellopentaose and cellohexaose were cleaved to these end products, but cellotetraose was also formed from cellohexaose. Methylcellulose did not inhibit hydrolysis of cellulooligosaccharides. These data are consistent with the presence of separate cellulase (beta-1,4-glucanase) and cellulodextrinase activities in R. flavefaciens.     

Cellulase production by Trichoderma reesei when cultured on xylose-based media supplemented with sorbose

The ability of L-sorbose to stimulate cellulase production In shake flask culture of Trichoderma reesei was examined in mineral salts media (initial pH 5.0) containing either 1.0% D-xylose, 1.0% cellulose, and/or 0.1, 0.3, or 0.5% L-sorbose. When sorbose was the only carbon source, growth was limited, little substrate was utilized, pH increased, and cellulase activity was not apparent. The other carbon sources promoted good growth, pH dropped sharply to 2.5-3.0, substrate was utilized rapidly, and cellulase activity was detected. After three weeks of fermentation, twice as much cellulase activity was detected in the medium containing only cellulose as the carbon source, as compared to xylose as the carbon source. Cellulase activity was higher when media contained xylose supplemented with sorbose compared to xylose as the only carbon source. At 0.3 and 0.5% levels of sorbose supplementation of xylose-based media, cellulase activity was similar to that in cellulose-based media.     

An effective method for bioconversion of delignified waste-cellulose fibers from the paper industry with a cellulase complex

An effective method for production of glucose was developed using enzymatic hydrolysis of waste-cellulose fibers by the cellulase complex from Trichoderma reesei. However, these cellulosic materials are strongly resistant to direct enzymatic hydrolysis (the degree of degradation after 48 h of enzyme treatment did not exceed 14%) apparently due to the presence of various chemical substances used in the process of paper production. This adhesive “envelope” around the cellulose fibers was effectively pretreated with 0.25% H₃PO₄ and after that the free cellulose mass was extensively hydrolyzed by the cellulase complex (degree of degradation more than 80%). The HPLC analyses of the enzyme hydrolysates revealed glucose as the main component as well as some cellobiose and xylose.     

The Cellulases Endoglucanase I and Cellobiohydrolase II of Trichoderma reesei Act Synergistically To Solubilize Native Cotton Cellulose but Not To Decrease Its Molecular Size

Degradation of cotton cellulose by Trichoderma reesei endoglucanase I (EGI) and cellobiohydrolase II (CBHII) was investigated by analyzing the insoluble cellulose fragments remaining after enzymatic hydrolysis. Changes in the molecular-size distribution of cellulose after attack by EGI, alone and in combination with CBHII, were determined by size exclusion chromatography of the tricarbanilate derivatives. Cotton cellulose incubated with EGI exhibited a single major peak, which with time shifted to progressively lower degrees of polymerization (DP; number of glucosyl residues per cellulose chain). In the later stages of degradation (8 days), this peak was eventually centered over a DP of 200 to 300 and was accompanied by a second peak (DP, (apprx=)15); a final weight loss of 34% was observed. Although CBHII solubilized approximately 40% of bacterial microcrystalline cellulose, the cellobiohydrolase did not depolymerize or significantly hydrolyze native cotton cellulose. Furthermore, molecular-size distributions of cellulose incubated with EGI together with CBHII did not differ from those attacked solely by EGI. However, a synergistic effect was observed in the reducing-sugar production by the cellulase mixture. From these results we conclude that EGI of T. reesei degrades cotton cellulose by selectively cleaving through the microfibrils at the amorphous sites, whereas CBHII releases soluble sugars from the EGI-degraded cotton cellulose and from the more crystalline bacterial microcrystalline cellulose.     

Distribution of carboxylate groups introduced into cotton linters by the TEMPO-mediated oxidation

The 2,2,6,6-tetramethylpiperidine-1-oxy radial (TEMPO)-mediated oxidation was applied to aqueous slurries of cotton linters. The water-insoluble fibrous fractions thus obtained in the yields of more than 78% were characterized by solid-state ¹³C-NMR, X-ray diffraction and scanning electron microscopic analyses for evaluation of distribution of carboxylate groups formed in the TEMPO-oxidized celluloses. The patterns of solid-state ¹³C-NMR spectra revealed that the oxidation occurred at the C6 primary hydroxyl groups of cellulose. X-ray diffraction and scanning electron microscopic analyses showed that such C6 oxidation took place at the surfaces of cellulose I crystallites without any oxidation at the C6 of inside cellulose I crystallites. Thus, carboxylate and aldehyde groups introduced into the TEMPO-oxidized celluloses are densely present on the surfaces of cellulose I crystallites. In addition, the obtained results revealed that the shoulder signal due to non-crystalline C6 carbons at about 63 ppm in solid-state ¹³C-NMR spectra of native celluloses is ascribed to those of surfaces of cellulose I crystallites or those of cellulose microfibrils.     

Correlation between X-ray diffraction measurements of cellulose crystalline structure and the susceptibility to microbial cellulase

Chemical and physical treatments of cotton cellulose have been studied in order to elucidate the relationship between the degree of crystallinity of cellulose and the susceptibility of cellulose to cellulase. Cotton cellulose powder was treated with the following solvents: 60% H₂SO₄, Cadoxen, and DMSO-p -formaldehyde. The dissolved celluloses were recovered at high yield of over 97% by addition of nine volumes of cold acetone. X-ray diffraction for measurements of relative crystallinity showed that the crystalline structure of cellulose declined in quantity and perfection by the dissolving treatment and changed to an amorphous form that is highly susceptible to enzymatic hydrolysis. These reprecipitated celluloses were hydrolyzed almost completely within 48 hr by Aspergillus niger cellulase containing mainly 1,4-β-glucan glucanohydrolase (EC 3.2.1.4), without action of 1,4-β-glucan cellobiohydrolase (EC 3.2.1. 91). On the other hand, cryo-milled cellulose (below 250 mesh) still had a crystalline structure, was resistant to cellulase, and gave a low percentage of saccharification. These results indicate that in pure cellulose there are good correlations between x-ray diffractograms and susceptibility to microbial cellulase.     

Cellulases: Biosynthesis and applications

Strains of Trichoderma, particularly T. reesei and its mutants, are good sources of extracellular cellulase suitable for practical saccharification. They secrete a complete cellulase complex containing endo- and exo-glucanases plus β-glucosidase (cellobiase) which act syngergistically to degrade totally even highly resistant crystalline cellulose to soluble sugars. All strains investigated to date are inducible by cellulose, lactose, or sophorose, and all are repressible by glucose. Induction, synthesis and secretion of the β-glucanase enzymes appear to be closely associated. The composition and properties of the enzyme complex are similar regardless of the strain or inducing substrate although quantities of the enzyme secreted by the mutants are higher. Enzyme yields are proportional to initial cellulose concentration. Up to 15 filter paper cellulase units (20 mg of cellulase protein) per ml and productivities up to 80 cellulase units (130 mg cellulase protein) per litre per hour have been attained on 6% cellulose. The economics of glucose production are not yet competitive due to the low specific activity of cellulase (0.6 filter paper cellulase units/mg protein) and poor efficiency (about 15% of predicted sugar levels in 24 h hydrolyses of 10–25% substrate). As hydrolysis proceeds, the enzyme reaction slows due to increasing resistance of the residue, product inhibition, and enzyme inactivation. These problems are being attacked by use of pretreatments to increase the quantity of amorphous cellulose, addition of β-glucosidase to reduce cellobiose inhibition, and studies of means to overcome instability and increase efficiency of the cellulases. Indications are that carbon compounds derived from enzymatic hydrolysis of cellulose will be used as fermentation and chemical feedstocks as soon as the process economics are favourable for such application.     

Specific quantification of trichoderma reesei cellulases in reconstituted mixtures and its application to cellulase-cellulose binding studies

Specific quantifications of the major cellulolytic components of the Trichoderma reesei enzyme complex, i.e., endoglucanases I and III and cellobiohydrolases I and II, are described and, employing a defined mixture of these four cellulases reconstituted according to the composition of the native Trichoderma cellulase complex, used to determine the binding of each individual component onto filter paper. During substrate degradation by this enzyme mixture, the specific adsorption of each individual cellulase gradually increases and no preferential binding of one enzyme component in any particular phase of cellulose hydrolysis is found. T. reesei cellobiohydrolases I and II admixed with endoglucanases I and III represent a "full-value" cellulase system that is capable of degrading semicrystalline cellulose efficiently. In comparison with the crude Trichoderma enzyme complex, almost identical adsorption properties and similar hydrolytic efficiency are found for the reconstituted mixture. (c) 1994 John Wiley & Sons, Inc.     

Characterization of the cellulolytic activity of a Bacillus isolate

A group I Bacillus strain, DLG, was isolated and characterized as being most closely related to Bacillus subtilis. When grown on any of a variety of sugars, the culture supernatant of this isolate was found to possess cellulolytic activity, as demonstrated by degradation of trinitrophenyl-carboxymethyl cellulose. Growth in medium containing cellobiose or glucose resulted in the greatest production of cellulolytic activity. The cellulolytic activity was not produced until the stationary phase of growth, and the addition of glucose or cellobiose to a culture in this phase had no apparent effect on enzyme production. Fractionation of the culture supernatant showed that the molecular weight of the enzymatic activity was less than 100,000. Maximum cellulolytic activity in assays was observed at pH 4.8 and at 58C, although maximum thermal stability of the activity. Kinetic experiments suggested that more than one enzyme was acting upon trinitrophenyl-carboxymethyl cellulose. Exocellular protein produced by this Bacillus isolate showed roughly one-fifth the cellulolytic activity displayed by Trichoderma reesei C30 on noncrystalline, cellulosic substrates. In contrast to T. reesei cellulase, the Bacillus enzymatic activity showed no ability to degrade crystalline forms of cellulose, nor was cellobiase activity detectable.     

Characterization of the cellulolytic activity of a Bacillus isolate.

A group I Bacillus strain, DLG, was isolated and characterized as being most closely related to Bacillus subtilis. When grown on any of a variety of sugars, the culture supernatant of this isolate was found to possess cellulolytic activity, as demonstrated by degradation of trinitrophenyl-carboxymethyl cellulose. Growth in medium containing cellobiose or glucose resulted in the greatest production of cellulolytic activity. The cellulolytic activity was not produced until the stationary phase of growth, and the addition of glucose or cellobiose to a culture in this phase had no apparent effect on enzyme production. Fractionation of the culture supernatant showed that the molecular weight of the enzymatic activity was less than 100,000. Maximum cellulolytic activity in assays was observed at pH 4.8 and at 58C, although maximum thermal stability of the activity. Kinetic experiments suggested that more than one enzyme was acting upon trinitrophenyl-carboxymethyl cellulose. Exocellular protein produced by this Bacillus isolate showed roughly one-fifth the cellulolytic activity displayed by Trichoderma reesei C30 on noncrystalline, cellulosic substrates. In contrast to T. reesei cellulase, the Bacillus enzymatic activity showed no ability to degrade crystalline forms of cellulose, nor was cellobiase activity detectable.     

Determination of the number-average degree of polymerization of cellodextrins and cellulose with application to enzymatic hydrolysis

A rapid and accurate method for determining the number-average degree of polymerization (DP(n)) was established for insoluble cellulose and soluble cellodextrins as the ratio of glucosyl monomer concentration determined by the phenol-sulfuric acid method divided by the reducing-end concentration determined by a modified 2,2'-bicinchoninate (BCA) method. The modified BCA method, featuring incubation at 75 degrees C for 30 min, did not result in beta-glucosidic bond cleavage, whereas substantial cleavage was observed at higher temperature. Solubilization of insoluble cellulose in cold phosphoric acid prior to measurement of the reducing-end concentration by the BCA method was found not to be necessary for several model celluloses such as microcrystalline cellulose, but such solubilization was required for large fibers of cellulose such as Whatman No. 1 filter paper. The phenol-sulfuric acid method can be used for measuring the glucosyl monomer concentration of soluble cellodextrins, and also for insoluble cellulose if preceded by a liquefaction step. Standard deviations of < or =2% were obtained for both reducing and glucosyl monomer determination and of < or =3% for overall determination of DP. By use of the reported method, hydrolysis of phosphoric acid-swollen cellulose (PASC) by the Trichoderma reesei cellulase system was shown to result in a rapid decrease in DP as hydrolysis proceeded. By contrast, the DP of Avicel remained nearly constant during hydrolysis. The specific enzymatic cellulose hydrolysis rate is 100-fold higher for PASC as compared to Avicel.     

High-yield cellulase production by Trichoderma reesei ZU-02 on corn cob residue

Cellulase production using corn cob residue from xylose manufacture as substrate was carried out by Trichoderma reesei ZU-02. It was found that on the same cellulose basis, the cellulase activity and yield produced on corn cob residue were comparable with that on purified cellulose. Under batch process, the optimum concentration of substrate was 40 g/l and the optimum C/N ratio was 8.0. In 500 ml flasks, cellulase activity reached 5.25 IU/ml (213.4 IU/g cellulose) after seven days' cultivation. In a 30 m(3) stirred fermenter for large scale production, cellulase and cellobiase activity were 5.48 IU/ml (222.8 IU/g cellulase) and 0.25 IU/ml (10.2 IU/g cellulose), respectively, after four days' submerged fermentation. The produced cellulase could effectively hydrolyze the corn cob residue, and the yield of enzymatic hydrolysis reached 90.4% on 10% corn cob residue (w/v) when the cellulase dosage was 20 IU/g substrate.     

Enzymic saccharification of sugarcane bagasse pretreated by autohydrolysis-steam explosion

Pretreatment of bagasse by autohydrolysis at 200 degrees C for 4 min and explosive defibration resulted in the solubilization of 90% of the hemicellulose (a heteroxylan) and in the production of a pulp that was highly susceptible to hydrolysis by cellulases from Trichoderma reesei C-30 and QM 9414, and by a comercial preparation, Meicelase. Saccharification yields of 50% resulted after 24 h at 50 degrees C (pH 5.0) in enzymic digests containing 10% (w/v) bagasse pulps and 20 filter paper cellulase units (FPU). Saccharifications could be increased to more than 80% at 24 h by the addition of exogenous beta-glucosidase from Aspergillus niger. The crystallinity of cellulose in bagasse remained unchanged following autohydrolysis-explosion and did not appear to hinder the rate or extent of hydrolysis of cellulose. Autohydrolysis-exploded pulps extracted with alkali or ethanol to remove lignin resulted in lowere conversions of cellulose (28-36% after 25 h) than unextracted pulps. Alkali extracted pulps arising from autohydrolysis times of more than 10 min at 200 degrees C were less susceptible to enzymic hydrolysis than unextracted pulps and alkali-extracted pulps arising from short autohydrolysis times (e.g., 2 min at 200 degrees C). Autohydrolysis-explosion was as effective a pretreatment method as 0.25M NaOH (70 degrees C/2 h) both yielded pulps that resulted in high cellulose conversions with T. reesei cellulase preparations and Meicelase. Supplementation of T. reesei C-30 cellulose preparations with A. niger beta-glucosidases was effective in promoting the conversion of cellulose into glucose. A ration of FPU to beta-glucosidase of 1:1.25 was the minimum requirement to achieve more than 80% conversion of cellulose into glucose within 24 h. Other factors which influenced the extent of saccharification of autohydrolysis-exploded bagasse pulps were the enzyme-substrate ratio, the substrate concentration, and the saccharification mode.     

Formation of cross-fractures in cellulose microfibril structure by an endoglucanase-cellobiohydrolase complex from Trichoderma reesei

Abstract An endoglucanase-cellobiohydrolase from Trichoderma reesei culture fluids was purified by means of preparative isoelectric focusing. The cellulase complex had a common apparent isoelectric point (p I ) of 3.8. Beyond this p I , the electrophoretic mobilities of endoglucanase and cellobiohydrolase were different under conditions of titration curves. The effect of this endoglucanase-cellobiohydronalase complex on Sinapis cellulose microfibril ultranstructure was observed by transmission electron microscopy after metal shadowing of the specimen. By the action of this cellulase complex, the microfibril structure was converted into an amorphous form of cellulose. Moreover, the hydrolase complex induced visible cross-fractures within the cellulose microfibril structure. The mean cellulose microfibril lenght of 1.2 μm was reduced to 0.9 μm in the presence (12 h) of this cellulase complex by the formation of shorter microfibril fragments.     

The enzymatic hydrolysis and fermentation of pretreated wood substrates

Aspenwood chips were pretreated by steam explosion. The various wood fractions obtained were assayed for their ability to act as substrates for growth and cellulase production of different Trichoderma and Clostridium thermocellum species. Steam exploded aspenwood was as efficiently utilized as solka floc and correspondingly high cellulase activities were detected in the various culture filtrates. When T. harzianum E58 was grown on increasing concentrations of solka floc, highest cellulase and xylanase activities were detected at 1% substrate concentrations while high substrate concentrations (10-20%) inhibited growth and enzyme production. When the cellulosic substrates were supplemented with increasing amounts of glucose, cellulase and xylanase production were inhibited when the glucose concentration exceeded 0.1%. Highest xylanase activities were detected after growth of T. reesei C30 and T. harianum E58 on xylan and solka floc respectively. All of the steam exploded fractions were at least partially hydrolyzed by the T. harzianum E58 cellulase system. The extent of the pretreatment also influenced the ability of Zymomonas mobilis and Saccharomyces cerevisiae to ferment the liberated sugars to ethanol. About 85% of the theoretical yield of ethanol from cellulose could be obtained from the combined hydrolysis and fermentation of pretreated aspenwood.     

Regenerating cellulose from ionic liquids for an accelerated enzymatic hydrolysis

The efficient conversion of lignocellulosic materials into fuel ethanol has become a research priority in producing affordable and renewable energy. The pretreatment of lignocelluloses is known to be key to the fast enzymatic hydrolysis of cellulose. Recently, certain ionic liquids (ILs) were found capable of dissolving more than 10wt% cellulose. Preliminary investigations [Dadi, A.P., Varanasi, S., Schall, C.A., 2006. Enhancement of cellulose saccharification kinetics using an ionic liquid pretreatment step. Biotechnol. Bioeng. 95, 904-910; Liu, L., Chen, H., 2006. Enzymatic hydrolysis of cellulose materials treated with ionic liquid [BMIM]Cl. Chin. Sci. Bull. 51, 2432-2436; Dadi, A.P., Schall, C.A., Varanasi, S., 2007. Mitigation of cellulose recalcitrance to enzymatic hydrolysis by ionic liquid pretreatment. Appl. Biochem. Biotechnol. 137-140, 407-421] suggest that celluloses regenerated from IL solutions are subject to faster saccharification than untreated substrates. These encouraging results offer the possibility of using ILs as alternative and non-volatile solvents for cellulose pretreatment. However, these studies are limited to two chloride-based ILs: (a) 1-butyl-3-methylimidazolium chloride ([BMIM]Cl), which is a corrosive, toxic and extremely hygroscopic solid (m.p. approximately 70 degrees C), and (b) 1-allyl-3-methylimidazolium chloride ([AMIM]Cl), which is viscous and has a reactive side-chain. Therefore, more in-depth research involving other ILs is much needed to explore this promising pretreatment route. For this reason, we studied a number of chloride- and acetate-based ILs for cellulose regeneration, including several ILs newly developed in our laboratory. This will enable us to select inexpensive, efficient and environmentally benign solvents for processing cellulosic biomass. Our data confirm that all regenerated celluloses are less crystalline (58-75% lower) and more accessible to cellulase (>2 times) than untreated substrates. As a result, regenerated Avicel((R)) cellulose, filter paper and cotton were hydrolyzed 2-10 times faster than the respective untreated celluloses. A complete hydrolysis of Avicel((R)) cellulose could be achieved in 6h given the Trichoderma reesei cellulase/substrate ratio (w/w) of 3:20 at 50 degrees C. In addition, we observed that cellulase is more thermally stable (up to 60 degrees C) in the presence of regenerated cellulose. Furthermore, our systematic studies suggest that the presence of various ILs during the hydrolysis induced different degrees of cellulase inactivation. Therefore, a thorough removal of IL residues after cellulose regeneration is highly recommended, and a systematic investigation on this subject is much needed.     

Cellulase production by a solid state culture system

Production of cellulase using solid culture systems of Trichoderma reesei QM9414 and Sporotrichum cellulophilum on wheat bran was studied. By using moisture-controlled solid culture equipment, the effect of water content of wheat bran on cell growth and cellulase production was investigated. Cellular biomass grown on solid substrate was estimated by measuring oxygen consumption rate and glucosamine content of the cells. These parameters were shown to have a good linear correlation with the specific growth rate. This reliable method of estimating the cell growth rate enabled us to simulate the enzyme production in a solid culture system by means of multiple linear regression analysis which takes into account of the water content, cell mass, and the oxygen consumption rate as variables. The cell growth and cellulase production were maximized at different water content of the medium. A high water content, 57% for T. reesei and 70% for S. cellulophilum, favored mycelial growth, while the maximum cellulase activity was obtained at a lower water content such as 50% for both fungi. It was observed that cellulase production by T. reesei depended on the culture conditions that support the optimal growth rate for the maximum enzyme production.