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.     

Enzymatic degradation of steam-pretreated Lespedeza stalk (Lespedeza crytobotrya) by cellulosic-substrate induced cellulases

The cellulase production by Trichoderma viride, cultivated on different substrates, namely steam-pretreated Lespedeza, filter paper, microcrystalline cellulose (MCC) or carboxymethyl cellulose (CMC), was studied. Different cellulase systems were secreted when cultivated on different substrates. The cellulolytic enzyme from steam-pretreated Lespedeza medium performed the highest filter paper activity, exoglucanase and endoglucanase activities, while the highest β-glucosidase activity was obtained from the enzyme produced on filter paper medium. The hydrolytic potential of the enzymes produced from different media was evaluated on steam-pretreated Lespedeza. The cellulase from steam-pretreated Lespedeza was found to have the most efficient hydrolysis capability to this specific substrate. The molecular weights of the cellulases produced on steam-pretreated Lespedeza, filter paper and MCC media were 33, 37 and 40 kDa, respectively, and the cellulase from CMC medium had molecular weights of 20 and 43 kDa. The degree of polymerization, crystallinity index and micro structure scanned by the scanning electron microscopy of degraded steam-pretreated Lespedeza residues were also studied.     

Cultivation of anaerobic fungi in a 10-l fermenter system for the production of (hemi-)cellulolytic enzymes

 Two species of anaerobic fungi, i.e. Piromyces strain E2 and Neocallimastix patriciarum strain N2, were cultivated in a 10-l batch fermenter with filter- paper cellulose as the carbon source. The accumulation of fermentation products, production of extracellular protein and (hemi-)cellulolytic enzymes were monitored during growth. Growth of Piromyces E2 in the fermenter resulted in a shift in the fermentation pattern to more acetate and formate and less ethanol, lactate, succinate and malate, possibly because of removal of hydrogen. The specific activities of Avicelase, endoglucanase, β-glucosidase and xylanase were up to threefold higher compared to small batch cultures. Enzyme activities produced per gram of cellulose were up to four times the values reported for Piromyces E2 grown in a semi-continuous coculture with the methanogen Methanobacterium formicicum. The performance of fermenter enzyme preparations from the anaerobic fungi with respect to hydrolysis of Avicel compared well to that of preparations of Trichoderma reesei. However, addition of exogenous β-glucosidase was indispensible with the latter preparation for the complete conversion to glucose. Received: 14 December 1995/Received revision: 19 March 1996/Accepted: 25 March 1996     

Mutants ofXanthomonas campestris defective in secretion of extracellular enzymes

Summary Mutants ofXanthomonas campestris B 1459 were isolated that are defective in secretion of both cellulase and amylase. Both enzymes accumulated in the periplasmic space. The defects in secretion of cellulase or amylase were partly overcome by introducing into the mutants specific multiple copies of DNA cloned fromX. campestris, and presumed to code for cellulase or amylase enzymes. The mutant strains also showed reduced amounts of extracellular pectinase and protease activities, as if the mutants were generally defective for secretion of extracellular enzymes. The mutants showed reduced pathogenesis for turnip seedlings. The secretion-defective mutants may allow production of xanthan gum with reduced cellulose, pectin, protein and starch-degrading enzyme activities, thereby allowing more widespread mixing of microbially produced xanthan gum with these commercially important water-soluble polymers.     

Use of 2-deoxyglucose in liquid media for the selection of mutant strains of Penicillium echinulatum producing increased cellulase and β-glucosidase activities

Mutagenesis and selection were applied to a strain of Penicillium echinulatum by treating conidia with hydrogen peroxide or 1,2,7,8-diepoxyoctane and then by incubating the conidia for 48 h in broth containing microcrystalline cellulose washed in 0.5% (w/v) aqueous 2-deoxyglucose before plating them onto cellulose agar containing 1.5% (w/v) glucose from which colonies showing the fastest production of halos of cellulose hydrolysis were selected. This process resulted in the isolation of two new cellulase-secreting P. echinulatum mutants: strain 9A02S1 showing increased cellulase secretion (2 IU ml⁻¹, measured as filter paper activity) in submerged culture in agitated flasks containing a mineral salts medium and 1% of cellulose, and strain 9A02D1, which proved more suitable for the production of cellulases in semisolid bran culture where it produced 23 IU of β-glucosidase per gram of wheat bran.     

β-Glucanase expression by Ruminococcus flavefaciens FD-1

Abstract Ruminococcus flavefaciens has been hypothesized to produce cellulase constitutively. We have studied the effect of carbon source, either cellobiose or cellulose, on the production of cellulase in batch cultures of R. flavefaciens FD-1. Total CMCase and ¹⁴C-cellulase activity was approximately 2-fold higher in cellobiose grown cells than in cellulose grown cells, whereas p-nitrophenyl-β- d -cellobiosidase (PNPCase) activity was not affected by culture conditions. The addition of cellulose to cells growing on cellobiose did not alter the amount or rate of PNPCase and ¹⁴C-cellulase production. Northern blot analysis of mRNAs produced by R. flavefaciens FD-1 grown using either cellobiose or cellulose as the substrate indicated that two of the four β-glucanase genes cloned from R. flavefaciens FD-1 were only expressed in cells grown with cellulose as the substrate. Although the adherence of cells and cellulase enzyme to native cellulose can complicate interpretations of these data, the results indicate that cellulase synthesis by R. flavefaciens is differentially regulated by carbon source.     

Enhanced cellulose degradation using cellulase-nanosphere complexes

Enzyme catalyzed conversion of plant biomass to sugars is an inherently inefficient process, and one of the major factors limiting economical biofuel production. This is due to the physical barrier presented by polymers in plant cell walls, including semi-crystalline cellulose, to soluble enzyme accessibility. In contrast to the enzymes currently used in industry, bacterial cellulosomes organize cellulases and other proteins in a scaffold structure, and are highly efficient in degrading cellulose. To mimic this clustered assembly of enzymes, we conjugated cellulase obtained from Trichoderma viride to polystyrene nanospheres (cellulase:NS) and tested the hydrolytic activity of this complex on cellulose substrates from purified and natural sources. Cellulase:NS and free cellulase were equally active on soluble carboxymethyl cellulose (CMC); however, the complexed enzyme displayed a higher affinity in its action on microcrystalline cellulose. Similarly, we found that the cellulase:NS complex was more efficient in degrading natural cellulose structures in the thickened walls of cultured wood cells. These results suggest that nanoparticle-bound enzymes can improve catalytic efficiency on physically intractable substrates. We discuss the potential for further enhancement of cellulose degradation by physically clustering combinations of different glycosyl hydrolase enzymes, and applications for using cellulase:NS complexes in biofuel production.     

Effect of different carbon sources on cellulase production by Hypocrea jecorina (Trichoderma reesei) strains

The ascomycete Hypocrea jecorina, an industrial (hemi)cellulase producer, can efficiently degrade plant polysaccharides. At present, the biology underlying cellulase hyperproduction of T. reesei, and the conditions for the enzyme induction, are not completely understood. In the current study, three different strains of T. reesei, including QM6a (wild-type), and mutants QM9414 and RUT-C30, were grown on 7 soluble and 7 insoluble carbon sources, with the later group including 4 pure polysaccharides and 3 lignocelluloses. Time course experiments showed that maximum cellulase activity of QM6a and QM9414 strains, for the majority of tested carbon sources, occurred at 120 hrs, while RUT-C30 had the greatest cellulase activity around 72 hrs. Maximum cellulase production was observed to be 0.035, 0.42 and 0.33 µmol glucose equivalents using microcrystalline celluloses for QM6a, QM9414, and RUTC-30, respectively. Increased cellulase production was positively correlated in QM9414 and negatively correlated in RUT-C30 with ability to grow on microcrystalline cellulose.     

Production of cellulases from alkali-treated bagasse in Trichoderma reesei

Summary Most of the mutants of Trichoderma reesei had good cellulase productivity on Avicel but this was low on alkali-treated bagasse, which could be a most promising cellulosic biomass to use as an inexpensive carbon source for cellulase production. Two T. reesei mutants, PC-3-7 and X-31, in which strong cellulase activity is inducible by l-sorbose, were, however, found to produce cellulase on alkali-treated bagasse. They produced about 100 units of CMCase per ml in 5-1 jar fermentor culture with 4% alkali-treated bagasse as carbon source. They also showed higher cellulase productivity than other mutants on other easily saccharified substrates, such as alkali-treated rice straw and Walseth's cellulose.Production of Ethanol from Biomasses Part IV.Production of Ethanol from Biomasses Part IV.     

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.     

Enhanced production of cellulase,hemicellulase, and β-glucosidase by Trichoderma reesei (Rut C-30)

The Production of cellulases and Hemicellulases was studied with Trichoderma reesei Rut C-30, This organism produced, together with high cellulase activities, considerable amounts of xylanases and β-glucosidase. Three cellulose concentration (1, 2.5, and 5.0%) were examined to determined the maximum levels of cellulase activity obtainable in submerged culture. Temperature and pH profiling was used to increase cell mass to maximum levels within two days and thereby enhancing fermentor productivity at higher substrate levels. The effect of temperature, pH, Tween-80 concentration, carbon sources, and substrate concentration on the ration of mycelial growth and extracellulose enzyme production are described.     

Effect of cellulose physical characteristics, especially the water sorption value, on the efficiency of its hydrolysis catalyzed by free or immobilized cellulase

Cellulase, an enzymatic complex that synergically promotes the degradation of cellulose to glucose and cellobiose, free or adsorbed onto Si/SiO₂ wafers at 60 °C has been employed as catalyst in the hydrolysis of microcrystalline cellulose (Avicel), microcrystalline cellulose pre-treated with hot phosphoric acid (CP), cotton cellulose (CC) and eucalyptus cellulose (EC). The physical characteristics such as index of crystallinity (I_C), degree of polymerization (DP) and water sorption values were determined for all samples. The largest conversion rates of cellulose into the above-mentioned products using free cellulase were observed for samples with the largest water sorption values; conversion rates showed no correlation with either I_C or DP of the biopolymer. Cellulose with large water sorption value possesses large pore volumes, hence higher accessibility. The catalytic efficiency of immobilized cellulase could not be correlated with the physical characteristics of cellulose samples. The hydrolysis rates of the same cellulose samples with immobilized cellulase were lower than those by the free enzyme, due to the diffusion barrier (biopolymer chains approaching to the immobilized enzyme) and less effective contact between the enzyme active site and its substrate. Immobilized cellulase, unlike its free counterpart, can be recycled at least six times without loss of catalytic activity, leading to higher overall cellulose conversion.     

Sorbose mediated enhancement of cellulase biosynthesis inTrichoderma reesei

Addition of L-sorbose, a non-metabolizable non-inducing ketohexose, toTrichoderma reesei cultures growing on cellobiose or Avicel-cellulose lead to increased cellulase activities. Addition of sorbose resulted in a 6-fold increase in cellodextrins (cellotriose, cellotetraose, cellopentaose) concentration on day 3 in cellobiose cultures and 1.3-fold increase in cellodextrins concentrations on day 4 in Avicel cellulose cultures. This increase in intracellular cellodextrins concentration matched closely with the increase in endoglucanase activity at these time points. Treatment of the cell-free extracts with cellulase preparation led to disappearance of the cellodextrins and increase of glucose. These observations suggested a more direct involvement of cellodextrins in cellulase induction process. The cellulases produced in sorbose-supplemented cellobiose medium hydrolyzed microcrystalline cellulose as effectively as the ones produced on Avicel cellulose medium.     

Cellulase production on glucose-based media by the UV-irradiated mutants of Trichoderma reesei

From 22,791 mutants of a cellulase hyper-producing strain of Trichoderma reesei (Hypocrea jecorina), ATCC66589, as the parent, we selected two mutants, M2-1 and M3-1, that produce cellulases in media containing both cellulose and glucose. The mutation enabled the mutants to produce cellulases, which were measured as p-nitrophenyl β-d-lactopyranoside-hydrolyzing activities, in media with glucose as a sole carbon source, although M2-1 exhibited different sensitivities to glucose from M3-1. When the mutants were grown for 8 days on a medium with cellulose as a sole carbon source, the filter-paper-degrading activities (FPAs) per gram of cellulose were 257 and 281 U for M2-1 and M3-1, respectively, values that were 1.1–1.2 times higher than that of the parental strain. Cellulase production by M2-1 and M3-1 on a medium with a continuously fed mixture of glucose and cellobiose resulted in 214 and 210 U of FPA/gram carbon sources, respectively, whereas less efficient production (140 U of FPA/gram carbon source) was achieved by the parental strain. The improved cellulase productivity of the mutants allows us to use glucose as a carbon source for efficient on-site production of cellulases with quality/quantity-controlled feeding of soluble carbon sources and inducers.     

Simultaneous production and induction of cellulolytic and xylanolytic enzymes in aStreptomyces sp.

Extracellular cellulolytic and xylanolytic enzymes ofStreptomyces sp. EC22 were produced during submerged fermentation. The cell-free culture supernatant of the streptomycete grown on microcrystalline cellulose contained enzymes able to depolymerize both crystalline and soluble celluloses and xylans. Higher cellulase and xylanase activities were found in the cell-free culture supernatant of the strain when grown on microcrystalline cellulose than when grown on xylan. Total cellulase and endoglucanase [carboxymethyl-cellulase (CMCase)] activities reached maxima after 72 h and xylanase activity was maximal after 60h. Temperature and pH optima were 55°C and 5.0 for CMCase activity and 60°C and 5.5 for total crystalline cellulase and xylanase activities. At 80°C, approximate half-lives of the enzymes were 37, 81 and 51 min for CMCase, crystalline cellulose depolymerization and xylanase, respectively.     

Cellulose degradation and carboxymethylcellulase production by Sporocytophaga myxococcoides grown in an air-lift fermenter

Summary Sporocytophaga myxcoccoides was grown in a 31 air-lift fermenter using a medium containing 2% w/v insoluble cellulose. The cellulose content of the medium reduced the k_La of the fermenter but during growth the dissolved oxygen concentration did not fall below 75% saturation. Rates of cellulose degradation and extracellular enzyme production were similar to those reported for a stirred-tank fermenter.     

Automatic cellulase assay in computer coupled pilot fermentation

Equipment for the automatic measurement of cellulase activity was connected to a computer-coupled pilot fermenter. The measurement was based on the use of dyed Avicel cellulose as substrate. An intermittent sampling device was used to take a sample from the fermenter at intervals of one hour. The computer was programmed to calculate the cellulase activity during fermentation. The measured activity values were corrected against a standard sample of known activity by means of a mathematical model for the calibration curve which was stored in the computer. On-line measurement of enzyme activity was successfully performed.     

Production and Characteristics of Avicel-Disintegrating Endoglucanase from a Protease-Negative Humicola grisea var. thermoidea Mutant

Mutational experiments were performed to decrease the protease productivity of Humicola grisea var. thermoidea YH-78 using UV light and N-methyl-N′-nitro-N-nitrosoguanidine. A protease-negative mutant, no. 140, exhibited higher endoglucanase activity than the parent strain in mold bran culture at 50°C for 4 days. The culture extract rapidly disintegrated filter paper but produced a small amount of reducing sugar. About 30% of total endoglucanase activity in the extract was adsorbed onto Avicel. The electrophoretically homogeneous preparation of Avicel-adsorbable endoglucanase (molecular weight, 128,000) showed intensive filter-paper-disintegrating activity but did not release reducing sugar. The preparation also exhibited a highly synergistic effect with the cellulase preparation from Trichoderma reesei in the hydrolysis of microcrystalline cellulose. This endoglucanase was observed via scanning electron microscopy to disintegrate Avicel fibrils layer by layer from the surface, yielding thin sections with exposed chain ends. A mutant, no. 191, producing higher protease activity and an Avicel-unadsorbable, Avicel-nondisintegrating endoglucanase was isolated. The purified enzyme (molecular weight, 63,000) showed no disintegrating activity on filter paper and Avicel and a less synergistic effect with the T. reesei cellulase in hydrolyzing microcrystalline cellulose than did the former enzyme. Endoglucanase was therefore divided into two types, Avicel disintegrating and Avicel nondisintegrating.     

Enzymatic hydrolysis of cellulose: Evaluation of cellulase culture filtrates under use conditions

Culture filtrates from three mutant strains of Trichoderma reesei grown on lactose and on cellulose were compared under use conditions on four cellulose substrates. Cellulose culture filtrates contained five to six times as much cellulase as lactose culture filtrates. Unconcentrated cellulose culture filtrates produced up to 10% sugar solutions from 15% cellulose in 24 h. Specific activity in enzyme assays and efficiency in saccharification tests were low for enzymes from all the mutants. Over a wide range the percent saccharification of a substrate in a given times was directly proportional to the logarithm of the ratio of initial concentrations of enzyme and substrate. As a result of this, dilute enzyme is more efficient than concentrated enzyme, but if high sugar concentrations are desired, very large quantities of enzyme are required. Since the slopes of these plots varied, the relative activity of cellulase on different substrates may be affected by enzyme concentration.     

Evaluation of nanoparticle-immobilized cellulase for improved ethanol yield in simultaneous saccharification and fermentation reactions

Ethanol yields were 2.1 (P = 0.06) to 2.3 (P = 0.01) times higher in simultaneous saccharification and fermentation (SSF) reactions of microcrystalline cellulose when cellulase was physisorbed on silica nanoparticles compared to enzyme in solution. In SSF reactions, cellulose is hydrolyzed to glucose by cellulase while yeast simultaneously ferments glucose to ethanol. The 35°C temperature and the presence of ethanol in SSF reactions are not optimal conditions for cellulase. Immobilization onto solid supports can stabilize the enzyme and promote activity at non-optimum reaction conditions. Mock SSF reactions that did not contain yeast were used to measure saccharification products and identify the mechanism for the improved ethanol yield using immobilized cellulase. Cellulase adsorbed to 40 nm silica nanoparticles produced 1.6 times (P = 0.01) more glucose than cellulase in solution in 96 h at pH 4.8 and 35°C. There was no significant accumulation (<250 μg) of soluble cellooligomers in either the solution or immobilized enzyme reactions. This suggests that the mechanism for the immobilized enzyme's improved glucose yield compared to solution enzyme is the increased conversion of insoluble cellulose hydrolysis products to soluble cellooligomers at 35°C and in the presence of ethanol. The results show that silica-immobilized cellulase can be used to produce increased ethanol yields in the conversion of lignocellulosic materials by SSF.