The effect of oxidative pretreatment on cellulose degradation by Poria placenta and Trichoderma reesei cellulases

The possible role of hydrogen peroxide in brown-rot decay was investigated by studying the effects of pretreatment of spruce wood and microcrystalline Avicel cellulose with H₂O₂ and Fe²⁺ (Fenton's reagent) on the subsequent enzymatic hydrolysis of the substrates. A crude endoglucanase preparation from the brown-rot fungus Poria placenta, a purified endoglucanase from Trichoderma reesei and a commercial Trichoderma cellulase were used as enzymes. Avicel cellulose and spruce dust were depolymerized in the H₂O₂/Fe²⁺ treatment. Mainly hemicelluloses were lost in the treatment of spruce dust. The effect of the pretreatment on subsequent enzymatic hydrolysis was found to depend on the nature of the substrate and the enzyme preparation used. Pretreatment with H₂O₂/Fe²⁺ clearly increased the amount of enzymatic hydrolysis of spruce dust with both the endoglucanases and the commercial cellulase. In all cases the amount of hydrolysis was increased about threefold. The hydrolysis of Avicel with the endoglucanases was also enhanced, whereas the hydrolysis with the commercial cellulase was decreased. Received: 23 December 1996 / Received revision: 17 April 1997 / Accepted: 19 April 1997     

Starch hydrolysis using enzyme in supercritical carbon dioxide

Summary The enzymatic hydrolysis reaction with supercritical carbon dioxide(SC-CO₂) as a reaction medium to make glucose from starch was investigated. The reaction rate was enhanced at higher temperature and pressure, especially near the critical point of the CO₂. The -amylase and glucoamylase were found to be active in a SC-CO₂.     

The effect of bovine serum albumin on batch and continuous enzymatic cellulose hydrolysis mixed by stirring or shaking

Bovine serum albumin (BSA) was applied as a model non-catalytic protein to enzymatic hydrolysis of Avicel and dilute acid pretreated corn stover at different reaction conditions to improve the understanding of its ability to enhance cellulose hydrolysis. Addition of BSA improved the 72 h hydrolysis yields in shake flasks by up to 26% for both substrates by reducing de-activation of the exoglucanases and by facilitating reductions in particle size and crystallinity during a magnetically stirred pre-incubation step. The enzyme stabilizing effect of BSA addition was most striking for batch hydrolysis in a stirred tank reactor, with glucose yields increasing by 76% after 72 h for Avicel and by 40% after 145 h for corn stover. Application of BSA to continuous hydrolysis for a mean residence time of 24h gave 33% and 40% higher glucose yields for corn stover and Avicel compared to the controls.     

Triterpeneglycosides as Inhibitors of Fungal Growth and Metabolism

Aescin in phosphate buffer reduced, to some extent, the production of ¹⁴CO₂ from uniformly labelled glucose in mycelia of Ophiobolus graminis and Neurospora crassa, whereas aescin in succinate buffer had no effect. The enzymatic hydrolysis of sucrose was, however, severely affected, no production of ¹⁴CO₂ from labelled sucrose being found after treatment of mycelia of O. graminis with 100 mg/l of aescin and N. crassa with 300 mg/l for 90 and 300 min, respectively. In Aspergillus niger the production of ¹⁴CO₂ from glucose or sucrose was not affected. The ATPase in whole cells and isolated plasma membranes was not inhibited by the aescin treatment, on the contrary, the ATPase in whole cells of N. crassa was somewhat stimulated.     

The isolation, characterization and effect of lignin isolated from steam pretreated Douglas-fir on the enzymatic hydrolysis of cellulose

Douglas-fir was SO₂-steam pretreated at different severities (190, 200, and 210 °C) to assess the possible negative effect of the residual and isolated lignins on the enzymatic hydrolysis of the steam pretreated substrates. When various isolated lignins were added to the Avicel hydrolysis reactions, the decrease in glucose yields ranged from 15.2% to 29.0% after 72 h. It was apparent that the better hydrolysis yields obtained at higher pretreatment severities were more a result of the greater accessibly of the cellulose rather than any specific change in the non-productive binding of the lignin to the enzymes. FTIR and ¹³C NMR characterization indicated that the lignin in the steam pretreated substrates became more condensed with increasing severity, suggesting that the cellulases were adsorbed to the lignin by hydrophobic interactions. Electrostatic interactions were also involved as the positively charged cellulase components were preferentially adsorbed to the lignins.     

Effect of agitation on growth and enzyme production of Trichoderma reesei in batch fermentation

Growth, enzyme-producing activity and respiratory properties of Trichoderma reesei QM 9414 were examined under various agitation intensities. Two substrates were compared: lactose and Avicel. Pellet formation occurred at all agitation intensities for both substrates. Oxygen dependence at the lower agitation rate varied with the substrate type. With lactose as the carbon source, linear growth was observed, despite a regulation of the dissolved oxygen concentration at 30% saturation. The enzyme production was strongly affected by the agitation. At the higher agitation rates the enzyme production dropped. With Avicel as the carbon source, the production of enzymes surged as soon as the growth was limited by the hydrolysis of Avicel.Growth on Avicel, in the conditions we used, was limited by Avicel hydrolysis. Cubic growth was observed when lactose was the carbon source. A new derivation for a model of the observed cubic growth is proposed and is used to correlate growth, CO₂ production and oxygen consumption in a consistent way, impossible with exponential growth models.     

Enzymatische Hydrolyse und Ethanolgärung von Lignocellulosen

The kinetics of enzymatic hydrolysis of different lignocellulosic materials (wheat straw, newspaper and microcrystalline cellulose Avicel PH 101) was studied using the cellulase complexes from Trichoderma reesei QM 9414 and its mutants M 5, M 6, MHC 15 and MHC 22. The maximum yields of hydrolysis were obtained with wheat straw partially delignified with 1% NaOH as substrate, and using the enzyme from the mutants T. reesei M 6 and MHC 22. The possibility of simultaneous enzymatic hydrolysis and ethanol fermentation of wheat straw using the enzyme complex from M 6 and yeasts of the genus Candida and Torulopsis was also investigated. A good conversion of liberated glucose and cellobiose to ethanol was obtained, however, xylose was not fermented.     

A continuous spectrophotometric assay for the determination of cellulase solubilizing activity

A cellulase assay was developed for the continuous measurement of colored cellulose oligosaccharides (total carbohydrates) released during enzymatic hydrolysis of dyed crystal-line cellulose. Several cellulosic substrates were uniformly dyed by Remalzol brilliant blue R salt without altering their physical properties. Dyed Avicel (6.5%, w/w) was selected as the most representative substrate for the assay procedure. The assay was performed continuously in a simple, thermally controlled apparatus designed for filtration of the reaction mixture via a 5-μm-pore-size nylon filter to retain the crystalline dyed cellulose while spectrophotometrically monitoring the absorbance at 595 nm of the reaction filtrate. Crude supernatant cellulase of Trichoderma viride QM9414 was used to test the assay procedure. The activity of cellulase on dyed Avicel as measured by ΔA_595nm correlated directly with the total carbohydrates formed. The initial reaction rate of cellulase solubilizing activity was readily determined with high sensitivity. The continuous assay has utility for the study of cellulase kinetics and for the comparison of activities from different microorganisms.     

CO2 enrichment and its relationship to bioconversion of cellulosic biomass of sweet potato (Ipomoea batatas L.) into fermentable sugars

Sweet potatoes (Ipomoea batatas L. (Lam) ‘Georgia-Jet’) were grown in open field plots and open top chambers at CO₂ concentrations of 354, 431, 506, and 659 μl liter⁻¹ for 90 days. The leaves and stems after the harvest were used as substrates for the production of fermentable sugars. Elevated CO₂ concentrations increased the cellulose content of stems, being most pronounced at 506 μl liter⁻¹. Hemicellulose content of leaves and stems as well as lignin content of stems decreased as a result of CO₂ enriched environment resulted in increased conversion of cellulose into fermentable sugars. The saccharification was greater in stems than in leaves. It was also found that chemical pretreatment of stems and leaves enhanced the enzymatic hydrolysis and the yields of glucose were higher than those from untreated stems and leaves.     

Access of cellulase to cellulose and lignin for poplar solids produced by leading pretreatment technologies

Adsorption of cellulase on solids resulting from pretreatment of poplar wood by ammonia fiber expansion (AFEX), ammonia recycled percolation (ARP), controlled pH, dilute acid (DA), flowthrough (FT), lime, and sulfur dioxide (SO₂) and pure Avicel glucan was measured at 4°C, as were adsorption and desorption of cellulase and adsorption of β‐glucosidase for lignin left after enzymatic digestion of the solids from these pretreatments. From this, Langmuir adsorption parameters, cellulose accessibility to cellulase, and the effectiveness of cellulase adsorbed on poplar solids were estimated, and the effect of delignification on cellulase effectiveness was determined. Furthermore, Avicel hydrolysis inhibition by enzymatic and acid lignin of poplar solids was studied. Flowthrough pretreated solids showed the highest maximum cellulase adsorption capacity (σ_solids = 195 mg/g solid) followed by dilute acid (σ_solids = 170.0 mg/g solid) and lime pretreated solids (σ_solids = 150.8 mg/g solid), whereas controlled pH pretreated solids had the lowest (σ_solids = 56 mg/g solid). Lime pretreated solids also had the highest cellulose accessibility (σ_cellulose = 241 mg/g cellulose) followed by FT and DA. AFEX lignin had the lowest cellulase adsorption capacity (σ_lignin = 57 mg/g lignin) followed by dilute acid lignin (σ_lignin = 74 mg/g lignin). AFEX lignin also had the lowest β‐glucosidase capacity (σ_lignin = 66.6 mg/g lignin), while lignin from SO₂ (σ_lignin = 320 mg/g lignin) followed by dilute acid had the highest (301 mg/g lignin). Furthermore, SO₂ followed by dilute acid pretreated solids gave the highest cellulase effectiveness, but delignification enhanced cellulase effectiveness more for high pH than low pH pretreatments, suggesting that lignin impedes access of enzymes to xylan more than to glucan, which in turn affects glucan accessibility. In addition, lignin from enzymatic digestion of AFEX and dilute acid pretreated solids inhibited Avicel hydrolysis less than ARP and flowthrough lignin, whereas acid lignin from unpretreated poplar inhibited enzymes the most. Irreversible binding of cellulase to lignin varied with pretreatment type and desorption method. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Characterization of a Symbiotic Coculture of Clostridium thermohydrosulfuricum YM3 and Clostridium thermocellum YM4

Clostridium thermohydrosulfuricum YM3 and C. thermocellum YM4 were isolated from a coculture which was obtained from an enrichment culture inoculated with volcanic soil in Izu Peninsula, Japan. Strain YM3 had advantages over reported C. thermohydrosulfuricum strains in that it fermented inulin and could accumulate ethanol up to 1.3% (wt/vol). The highest ethanol yield obtained was 1.96 mol/mol of anhydroglucose unit in cellobiose. Strain YM4 had features different from those reported in C. thermocellum strains: it formed spores rarely (at a frequency of <10^-5), it required CO₂ and Na₂CO₃ for growth, and it fermented sucrose. Strain YM4 completely decomposed 1% Avicel within 25 h when the inoculum constituted 2% of the culture medium volume, and it produced 0.22 U of Avicelase and 2.21 U of carboxymethylcellulase per ml of the medium. The doubling times on Avicel, cellobiose, and glucose were 2.7, 1.1, and 1.6 h, respectively. Reconstructed cocultures of strains YM3 and YM4 were very stable and degraded Avicel more rapidly than did strain YM4 monoculture. Without yeast extract, neither microorganism was able to grow. However, the coculture grew on cellulose without yeast extract and produced ethanol in high yield. Moreover, cell-free spent culture broth of strain YM3 could replace yeast extract in supporting the growth of strain YM4. The symbiotic relationship of the two bacteria in cellulose fermentation is probably a case of mutualism.     

Enzymatic fractionation of carbon isotopes by phosphoenolpyruvate carboxylase from c(4) plants

The carbon atoms of glucose and malate in C₄ plants are 2 to 3‰ enriched in ¹²C with respect to atmospheric CO₂; whereas these intermediates in C₃ plants are 15 to 18‰ enriched with ¹²C with respect to atmospheric CO₂. The enzymatic synthesis of malate from phosphoenolpyruvate and bicarbonate in preparations of leaves of Sorghum bicolor, Haygrazer result in a carbon isotope fractionation of about 3‰. The enzymatic synthesis of phosphoglyceric acid from ribulose 1,5-diP and CO₂ in these preparations (contaminated with carbonic anhydrase) at 24 C and 37 C result in a carbon isotope fractionation of 33.7‰ and 18.3‰, respectively. These data are consistent with the conclusion that the small enrichment of ¹²C in the carbon atoms of malate and glucose (with respect to atmospheric CO₂) in leaves of Sorghum bicolor, Haygrazer occurs at the phosphoenolpyruvate carboxylase step.     

Supercritical carbon dioxide explosion as a pretreatment for cellulose hydrolysis

Summary Cellulosic material Avicel was treated with supercritical carbon dioxide to increase the reactivity of cellulose, thereby to enhance the rate and the extent of cellulose hydrolysis. Upon an explosive release of the carbon dioxide pressure, the disruption of the cellulosic structure increases the accessible surface area of the cellulosic substrate to enzymatic hydrolysis. This explosion pretreatment enhances the rate of the Avicel hydrolysis as well as increases glucose yield by as much as 50%.     

Hydrolysis of dilute acid pretreated mixed hardwood and purified microcrystalline cellulose by cell-free broth from Clostridium thermocellum

The cellulase activity in cell-free broths from the thermophilic, ethanol-producing anaerobic bacterium Clostridium thermocellum is examined on both dilute-acid-pretreated mixed hardwood (90% maple, 10% birch) and Avicel. Experiments were conducted in vitro in order to distinguish properties of the cellulase from properties of the organism and to evaluate the effectiveness of C. thermocellum cellulase in the hydrolysis of a naturally occurring, lignin-containing substrate. The results obtained establish that essentially quantitative hydrolysis of cellulose from pretreated mixed hardwood is possible using this enzyme system. Pretreatment with 1% H(2)SO(4) and a 9-s residence time at 220, 210, 200, and 180 degrees C allowed yields after enzymatic hydrolysis (percentage of glucan solubilized/ glucan potentially solubilized) of 97.8, 86.1, 82.0, and 34.6%, respectively. Enzymatic hydrolysis of mixed hardwood with no pretreatment resulted in a yield of 10.1%. Hydrolysis yields of >95% were obtained from approximately 0.6 g/L mixed hardwood pretreated at 220 degrees C in 7 h at broth strengths of 60 and 80% (v/v) and in approximately 48 h with 33% broth. Hydrolysis of pretreated mixed hardwood is compared to hydrolysis of Avicel, a pure microcrystalline cellulose studied previously. The initial rate of Avicel hydrolysis saturates with respect to enzyme, whereas the initial rate of hydrolysis of pretreated wood is proportional to the amount of enzyme present. Initial hydrolysis rates for pretreated wood and Avicel at 0.6 g/L are greater for wood at low broth dilutions (1.25: 1 to 5 :1) by up to 2.7-fold and greater for Avicel at high broth dilutions (5 : 1 to 50 : 1) by up to 4.3-fold. Maximum rates of hydrolysis are achieved at <2 g substrate/L for both pretreated wood and Avicel. The substrate concentration at one-half the maximum observed rate for C. thermocellum broths is smaller for pretreated mixed hardwood than for Avicel and decreases with increasing broth dilution for both substrates. An initial activity per volume broth of approximately 11 mumol soluble glucose equivalent produced/L broth/min is observed for mixed hardwood pretreated at 220 degrees C and for Avicel at high broth dilutions; the initial activity per volume broth for Avicel is lower at low broth dilutions. The results indicate that pretreated wood is hydrolyzed at rates comparable to Avicel under many conditions and at rates significantly faster than Avicel under several conditions.     

Carboxymethylcellulase and avicelase activities from a cellulolytic Clostridium strain A11

The extracellular cellulase enzyme system of Clostridium A11 was fractionated by affinity chromatography on Avicel: 80% of the initial carboxymethylcellulase (CMCase) activity was adhered. This cellulase system was a multicomponent aggregate. Several CMCase activities were detected, but the major protein P1 had no detectable activity. Adhered and unadhered cellulases showed CMCase activity with the highest specific activity in Avicel-adhered fraction. However, only afhered fractions could degrade Avicel. Thus, efficiency of the enzymatic hydrolysis of Avicel was related to the cellulase-adhesion capacity. Carboxymethylcellulase and Avicelase activities were studied with the extracellular enzyme system and cloned cellulases. Genomic libraries from Clostridium A11 were constructed with DNA from this Clostridium, and a new gene cel1 was isolated. The gene(s) product(s) from cel1 exhibited CMCase and p-nitrophenylcellobiosidase (pNPCbase) activities. This cloned cellulase adhered to cellulose. Synergism between adhered enzyme system and cloned endoglucanases was observed on Avicel degradation. Conversely, no synergism was observed on CMC hydrolysis. Addition of cloned endoglucanase to cellulase complex led to increase of the V_max without significant K _m variation. Cloned endoglucanases can be added to cellulase complexes to efficiently hydrolyze cellulose.     

A novel green enzymatic synthetic route of 2, 3-dihydroxybenzoic acid from glucose and CO2 fixation

The enzymatic carbon fixation is a promising approach to deal with greenhouse gas emission and is usually accompanied with energy consumption during the reduction of CO₂. As a very important route, the carboxylation can convert CO₂ to organic carbon without extra requirement of reduction power and is hoped as a greener solution, especially for some non-bulk chemicals, such as medical intermediates. Here, a concept-proof trail of green enzymatic process of conversing both of CO₂ and benzene to produce 2,3-dihydroxybenzoic acid (2,3-DHBA), which is the intermediate for fine chemicals, is introduced with O₂ from air and glucose. The results showed that the conversion catechol by 2, 3-dihydroxybenzoic acid decarboxylase (2,3-DHBD) alone was around 30 %, with an overall conversion from phenol of 2.4 %, which was limited by the in-situ production of catechol. This trail contributed a green enzymatic route for the production of 2,3-DHBA.     

The cellulase activity of an extreme thermophile

Summary The carboxymethylcellulase activity concentrated from the extremely thermophilic anaerobe H173 was found to have a pH optimum of 6.5–7.0. The enzyme activity was stabilised by the addition of dithiothreitol and CaCl₂·2H₂O and was very stable at 80° C, retaining 77% of the initial activity after 120 min incubtation. At min and after 120 min only 3% of the initial activity remained. With the enzyme dissolved in buffer, glucose and cellobiose were formed from the hydrolosis of Avicel. In culture medium the Avicel-solubilising activity was insensitive to the presence of up tp 50 mm glucose and showed linear glucose accumulation over a period of days at 70° C. HPLC analysis established that glucose was the major end-product of hydrolysis in the culture broths.Offprint requests to: H. W. Morgan     

Cellulase immobilized by sol–gel entrapment for efficient hydrolysis of cellulose

Cellulase from Trichoderma reesei (Celluclast 1.5 L, Novozyme) was immobilized by sol–gel encapsulation, using binary or ternary mixtures of tetramethoxysilane (TMOS) with alkyl- or aryl-substituted trimethoxysilanes as precursors. Optimization of immobilization conditions resulted in 92 % recovery of total enzymatic activity in the best immobilized preparate. The immobilized cellulase exhibiting the highest activity, obtained from tetramethoxysilane and methyltrimethoxysilane precursors at 3:1 molar ratio, was investigated in the hydrolysis reaction of microcrystalline cellulose (Avicel PH101). Although the optimal values did not change significantly, both temperature and pH stabilities of the sol–gel entrapped cellulase improved compared to the native enzyme. Immobilization also conferred superior resistance against the inactivation effect of glucose. Reuse of the sol–gel entrapped cellulase showed 40 % retention of the initial activity after five batch hydrolysis cycles, demonstrating the potential of this biocatalyst for large-scale application.     

A mutant β-glucosidase increases the rate of the cellulose enzymatic hydrolysis

The enzymatic hydrolysis of cellulose and lignocellulosic materials is marked by a rate decrease along the reaction time. Cellobiohydrolase slow dissociation from the substrate and its inhibition by the cellobiose produced are relevant factors associated to the rate decrease. In that sense, addition of β-glucosidases to the enzyme cocktails employed in cellulose enzymatic hydrolysis not only produces glucose as final product but also reduces the cellobiohydrolase inhibition by cellobiose. The digestive β-glucosidase GH1 from the fall armyworm Spodoptera frugiperda, hereafter called Sfβgly, containing the mutation L428V showed an increased k_cat for cellobiose hydrolysis. In comparison to assays conducted with the wild-type Sfβgly and cellobiohydrolase TrCel7A, the presence of the mutant L428V increased in 5 fold the initial rate of crystalline cellulose hydrolysis and reduced to one quarter the time needed to TrCel7A produce the maximum glucose yield. As our results show that mutant L428V complement the action of TrCel7A, the introduction of the equivalent replacement in β-glucosidases is a promising strategy to reduce costs in the enzymatic hydrolysis of lignocellulosic materials.     

Purification and properties of a cellobiohydrolase from Penicillium pinophilum

Cellobiohydrolase II, isolated from the extracellular cellulase system of Penicillium pinophilum by chromatography on DEAE-Sephadex and DEAE-Sepharose followed by chromatofocusing, gave a single homogeneous band in SDS-gel electrophoresis and gel electrophoresis and gel electrofocusing. It had a molecular weight of 50,700 and of pI of 5.0, and was associated with 19% of carbohydrate. Cellobiose was the sole product of hydrolysis of the cellulosic materials, Avicel and H₃PO₄-swollen cellulose. No cross reaction was observed with antiserum prepared with another purified cellobiohydrolase (I) isolated from the same cellulase system. Cellobiohydrolase II showed no capacity for producing short fibres from filter paper. Avicel was hydrolysed extensively, but little or no hydrolysis of cotton fibre was apparent. However, cotton fibre was hydrolysed with a reconstituted mixture of the purified cellobiohydrolase II and the four major endo-(1→4)-β-d-glucanases isolated during fractionation. The action of cellobiohydrolase II on H₃PO₄-swollen cellulose was stimulated by high concentrations of cellobiose, but inhibited by high concentrations of d-glucose. Other notable inhibitors were Mn²⁺ and carbodi-imide. The properties of cellobiohydrolase II and the immunologically unrelated cellobiohydrolase I are compared.