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.     

Proteomic analysis of pH and strains dependent protein secretion of Trichoderma reesei

Bioenergy, particularly biofuel, from lignocellulosic biomass has been considered as one of the most promising renewable and sustainable energies. The industrial productivity and efficiency of microbial lignocellulolytic enzymes for cellulosic biofuel applications are significantly affected by pH of culture condition. This study established and compared hydrolytic protein expression profiles of Trichoderma reesei QM6a, QM9414, RUT C30 and QM9414MG5 strains at different pH in cellulosic culture media. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of secretome of T. reesei cultured from pH 3.0-9.0 revealed significantly higher hydrolytic protein expressions at acidic pH. The Bray-Curtis similarity indices, clustering, and Shannon diversity index elucidated differences in protein secretion at different pHs in individuals and among the strains. This study demonstrated a comparative lignocellulolytic enzyme secretion profile of T. reesei and its mutants at different pHs and provides pH sensitive and resistance enzyme targets for industrial lignocellulose hydrolysis.     

Production of thermotolerant and alkalotolerant cellulolytic enzymes by isolated <Emphasis Type="Italic">Nocardiopsis</Emphasis> sp. KNU

A novel cellulolytic bacterium was isolated from the forest soil of KNU University campus. Through 16S rRNA sequence matching and morphological observation it was identified as Nocardiopsis sp. KNU. This strain can utilize a broad range of cellulosic substrates including: carboxymethyl cellulose (CMC), avicel, xylan, cellobiose, filter paper and rice straw by producing a large amount of thermoalkalotolerant endoglucanase, exoglucanase, xylanase and glucoamylase. Optimal culture conditions (Dubos medium, 37°C, pH 6.5 and static condition) for the maximal production of the cellulolytic enzymes were determined. The activity of cellulolytic and hemicelluloytic enzymes produced by this strain was mainly present extracellularly and the enzyme production was dependent on the cellulosic substrates used for the growth. Effect of physicochemical conditions and metal additives on the cellulolytic enzymes production were systematically investigated. The cellulases produced by Nocardiopsis sp. KNU have an optimal temperature of 40°C and pH of 5.0. These cellulases also have high thermotolerance as evidenced by retaining 55–70% activity at 80°C and pH of 5.0 and alkalotolerance by retaining >55% of the activity at pH 10 and 40°C after 1 h. The efficiency of fermentative conversion of the hydrolyzed rice straw by Saccharomyces cerevisiae (KCTC-7296) resulted in 64% of theoretical ethanol yield.     

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.     

Chemical and gamma-ray-modified bagasse as substrates for bioproduction of cellulases and protein

Production of enzymes in the cellulolytic complex was determined in culture filtrates of six fungal isolates grown on chemically treated or gamma-irradiated bagasse. The enzymatic activities of the filtrates were determined by measurement of glucose release from cotton, filter paper, carboxymethylcellulose, cellobiose, and cellobiose octaacetate. Cultures grown on base-treated and gamma-irradiated plus acid-treated bagasse provided culture filtrates with the highest enzymatic activities whereas alpha-cellulose, untreated, and acid-treated bagasse were the poorest substrates for enzyme production. Filtrates of Trichoderma reesei QM 9414 yielded the highest cellulolytic activity in all test media. The largest accumulation of fungal-derived, extracellular protein was observed in media containing gamma-irradiated bagasse as the carbon substrate.     

Swollenin, a Trichoderma reesei protein with sequence similarity to the plant expansins, exhibits disruption activity on cellulosic materials.

Plant cell wall proteins called expansins are thought to disrupt hydrogen bonding between cell wall polysaccharides without hydrolyzing them. We describe here a novel gene with sequence similarity to plant expansins, isolated from the cellulolytic fungus Trichoderma reesei. The protein named swollenin has an N-terminal fungal type cellulose binding domain connected by a linker region to the expansin-like domain. The protein also contains regions similar to mammalian fibronectin type III repeats, found for the first time in a fungal protein. The swollenin gene is regulated in a largely similar manner as the T. reesei cellulase genes. The biological role of SWOI was studied by disrupting the swo1 gene from T. reesei. The disruption had no apparent effect on the growth rate on glucose or on different cellulosic carbon sources. Non-stringent Southern hybridization of Trichoderma genomic DNA with swo1 showed the presence of other swollenin-like genes, which could substitute for the loss of SWOI in the disruptant. The swollenin gene was expressed in yeast and Aspergillus niger var. awamori. Activity assays on cotton fibers and filter paper were performed with concentrated SWOI-containing yeast supernatant that disrupted the structure of the cotton fibers without detectable formation of reducing sugars. It also weakened filter paper as assayed by an extensometer. The SWOI protein was purified from A. niger var. awamori culture supernatant and used in an activity assay with Valonia cell walls. It disrupted the structure of the cell walls without producing detectable amounts of reducing sugars.     

Comparative study of the growth of Trichoderma reesei and Penicillium occitanie

During the last years, a great deal of research on the production of energetic substances was directed to the use of cellulosic by-products. A technique of special interest is the production of fermentissible sugars by the enzymatic hydrolysis of cellulose. Mandels and Reese (1960) showed that a fungi strain of Trichoderma reesei is the best performant microorganism in the production of cellulolytic enzymes. Nevertheless, recent investigation indicated that the rate and the yield of conversion of cellulose to glucose of this strain are limited by its poor beta-glucosidase activity. In order to increase the efficiency of the hydrolytic power of the cellulasic complex two approaches can be considered. Beta-glucosidase enrichment of Trichoderma reesei enzymes. The selection and use of strains with a better performance. In our laboratory, we chose the second approach using Penicillium occitanie comparing it to Trichoderma reesei.     

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.     

Cellulolytic Enzyme System of Thermoactinomyces sp. Grown on Microcrystalline Cellulose

The carboxymethyl-cellulase and Avicelase activities of Thermoactinomyces sp., strain YX, were produced simultaneously with cell growth. Throughout the growth phase these activities were primarily extracellular, with up to 50% adsorbed to residual cellulosic substrate at any one time. On the other hand, the β-glucosidase activity was associated with the culture solids throughout the entire fermentation and appears to be intracellular. Preparative isoelectric focusing of the culture filtrate, in the pH range 3 to 5, separated three major fractions with cellulolytic activities towards both carboxymethyl-cellulose and Avicel. Based on the carboxymethyl-cellulase and Avicelase activities of these separated fractions, it was not possible to discriminate between endo- and exoglucanases produced by Thermoactinomyces sp. Analytical isoelectric focusing of culture filtrates obtained throughout the growth phase of Thermoactinomyces indicated that all the extracellular cellulolytic enzymes are produced and released into the culture filtrate simultaneously, with no evidence of a sequential appearance of the various enzymes or isoenzymes.     

Production and properties of a cellobiose-oxidizing enzyme from a newly isolatedcellulolytic bacterium Cytophaga sp. LX-7

A cell-bound cellobiose-oxidizing enzyme was produced by cellulolytic Cytophaga sp. LX-7. It was found that both the cellulosic substrates and the soluble carbohydrate substrates tested promoted the production of the cellobiose-oxidizing enzyme, and the highest specific activities were obtained with cellulose powder MN300, carboxy- methylcellulose CM22, maltose and cellobiose. Among the nitrogen sources examined, peptone gave the best cellobiose-oxidizing enzyme production, whereas inorganic nitrogen sources gave very poor growth. The medium buffered with Tris/HCl, pH 7.1, yielded the highest levels of cellobiose-oxidizing enzyme activity and the temperature optimum for crude enzyme activity was 40°C.     

Stimulatory effect of oxidized cellulose on cellulase formation by Trichoderma reesei

Crystalline cellulase has been electrochemically oxidized to yield preparations containing various different percentages of oxidized end-groups. These celluloses have been used as carbon sources for growth and cellulase production by Trichoderma reesei . A low content of oxidized end groups in the celluloses (0.1–0.65%) stimulated cellulase production but not growth, whereas higher contents (> 1%) where inhibitory to both. The cellulolytic enzyme system secreted under stimulated conditions contained the same proportion of individual cellulase enzymes (cellobiohydrolase I and II, endoglucanase I) as the control, indicating a general stimulatory effect of oxidized cellulose. Activity of cellulases against oxidized celluloses in vitro was not stimulated, and only slightly inhibitory at high degrees of oxidation. The data support a potential role of cellulose oxidation in regulating cellulase formation by T. reesei .     

Evaluation of culture conditions for cellulase production by two Trichoderma reesei mutants under solid-state fermentation conditions

Response surface methodology (RSM) was used to evaluate the effects of fermentation parameters for cellulase production by Trichoderma reesei QM9414 and T. reesei MCG77 in solid-state fermentation using rice bran as substrate. Initial pH, moisture content and temperature were optimized using filter paper activity (FPA) as response. Statistical analysis of the results for T. reesei QM9414 showed that only moisture content had significant effect on cellulase activity and had a linear effect on enzyme activity (maximum enzyme activities were obtained at 70% moisture content). The results for T. reesei MCG77 showed that temperature and moisture content were the most significant parameters for cellulase activity. The optimum cellulase production was in the temperature range of 25-30 degrees C and moisture content between 55% and 70%. After the optimization, the FPA in T. reesei MCG77 was increased by 2.5 folds compared to that of T. reesei QM9414.     

Tamarind kernel powder co-induces xylanase and cellulase production during submerged fermentation of <Emphasis Type="Italic">Termitomyces clypeatus</Emphasis>

Tamarind kernel powder (TKP), a soluble agro-residue, was used to examine the production of both cellulolytic and xylanolytic enzymes in a submerged culture of Termitomyces clypeatus, an edible mushroom. Soluble TKP containing xyloglucan as the major polysaccharide induced all cellulolytic and xylanolytic enzymes, and enzyme production increased up to 3% (w/v) TKP with culture filtrate consisting of xylanase and CMCase at a ratio of 4: 1 app. Strong catabolic repression of enzyme production was also observed with the soluble substrate, although fed-batch addition of soluble substrate at late growth phase modified the enzyme kinetics by improving the yield by 30%. The results indicate that inducers were possibly released from TKP by cellulose and xylan fractions of the lignocellulosic polymer. Therefore, the present study reports the successful economic utilization of TKP, an abundantly available soluble agro-residue, for the production of both cellulolytic and xylanolytic enzymes in a single fermentation method.     

Development of a culture medium for large-scale production of cellulolytic enzymes by Trichoderma reesei

Culture filtrates of CL-847 strain of Trichoderma reesei grown on different carbon sources have been compared. The highest enzyme production is obtained with Whatman C 41 cellulose: 17.9 mg/mL of soluble proteins and 13.7 units of filter paper (FP) activity. Wood pulps gave lower production values and more viscous culture media. About one-third of maximal enzyme production is obtained on lactose as the sole carbon source. Addition of 0.5% cellulosic inducer to 6% lactose media enhances enzyme production up to the following levels: 14.1 mg/mL of soluble proteins and 8.4 units of FP activity.     

Optimization of ellagic acid production from ellagitannins by co-culture and correlation between its yield and activities of relevant enzymes

Aspergillus oryzae was co-cultured with Trichoderma reesei using acorn cups extract containing up to 62% ellagitannins as substrate to produce ellagic acid with relatively high levels of ellagitannin acyl hydrolase, cellulase and xylanase. Ellagitannins concentration, initial pH, T. reesei and A. oryzae during the fermentation were identified as important process parameters effecting ellagic acid accumulation and the enzymes syntheses. These parameters were optimized by uniformity design to determine the optimum condition for ellagic acid production. Under optimum operational condition, ellagic acid yield could be arrived at 24%, when the fermentation run lasted 96h with an initial pH of 4.5, an ellagitannins concentration of 4gl(-1), T. reesei of 3ml and A. oryzae of 3ml. Meanwhile, it was found that the three enzymes activities correlated very well with ellagic acid yield, resulting in model with high coefficient of determination (R(2)=0.98). The results indicate that the mixed culture of T. reesei and A. oryzae is an effective approach to produce an enzyme system of degrading ellagitannins for ellagic acid production.     

Kinetic properties and contribution to cellulose saccharification of a cloned Pseudomonas beta-glucosidase

The plasmid pND71, which encodes beta-glucosidase (cellobiase) activity, cloned from the cellulolytic Pseudomonad, PS2-2, was mobilized by conjugation into 10 Pseudomonas strains. The highest specific activity was produced by 17498 (pND71) and the properties of the enzyme produced from this transconjugant were studied. The enzyme was shown to be cell associated, to have a temperature optimum of 37 degrees C, a pH optimum of 7.0 and Km values of 1.33 and 2.94 mM for pNPG and cellobiose respectively. It was competitively inhibited by glucose, with a Ki of 30 mM. Evidence was obtained which suggested that the enzyme was produced constitutively and that synthesis was not repressed by glucose. When culture preparations were used in combination with Trichoderma reesei QM9414 and C30 enzyme preparations to saccharify cellulose, 17498 (pND71) was more effective than preparations of PS2-2 in acting synergistically with T. reesei to solubilize more carbohydrate and produce more glucose.     

Extractive fermentation for improved production of endoglucanase by an intergeneric fusant of Trichoderma reesei/Saccharomyces cerevisiae using aqueous two-phase system

Extractive aqueous two-phase fermentation of endoglucanase, a key enzyme for the conversion of cellulosic substances to fermentable sugars, from an intergeneric fusant of Trichoderma reesei/Saccharomyces cerevisiae is a meaningful approach for better production and simple recovery of this enzyme. A phase composition of 6.5% (w/w) dextran and 7.5% (w/w) polyethylene glycol 6000, having a partition coefficient of 2.89 and 1.31 for endoglucanase from an intergeneric fusant of T. reesei/S. cerevisiae and T. reesei (WT) (being a control in this study), respectively, was chosen for extractive fermentation of the enzyme. Endoglucanase production is higher in medium containing polyethylene glycol (PEG) 6000 than in medium without PEG 6000. Comparative analysis of endoglucanase fermentation by fusant and T. reesei was carried out in shake culture and environment-controlled bioreactor conditions. The fusant produced 0.43U of endoglucanase (overall production: 0.34U) in the top phase of an aqueous two-phase system (ATPS), compared to 0.3U in medium without the phase system in shake culture. In a batch reactor, the endoglucanase level for the fusant in the top phase of ATPS was 0.49U (overall production: 0.40U), compared to 0.38U produced in medium without aqueous two-phase components. To corroborate this study, T. reesei produced 8.41U of endoglucanase (overall production: 5.96U) in the top phase of ATPS, compared to 7.18U in the medium without the phase system in shake culture. On the other hand, in a batch bioreactor, T. reesei produced 10.13U of endoglucanase (overall production: 6.90U) in the top phase of ATPS, compared to 8.56U of the enzyme in medium without aqueous two-phase components. The lower overall enzyme production by T. reesei in the two-phase system might be due to limitation in oxygen transfer to the dispersed phase where the enzyme is produced. A higher cell concentration and a reduced lag phase was obtained in ATPS, compared to a similar medium without phase forming polymers for both the intergeneric fusant of T. reesei/S. cerevisiae and T. reesei.     

Characterization of cellulolytic enzymes and bioH2 production from anaerobic thermophilic Clostridium sp. TCW1

A thermophilic anaerobic bacterium Clostridium sp. TCW1 was isolated from dairy cow dung and was used to produce hydrogen from cellulosic feedstock. Extracellular cellulolytic enzymes produced from TCW1 strain were identified as endoglucanases (45, 53 and 70 kDa), exoglucanase (70 kDa), xylanases (53 and 60 kDa), and β-glucosidase (45 kDa). The endoglucanase and xylanase were more abundant. The optimal conditions for H₂ production and enzyme production of the TCW1 strain were the same (60 °C, initial pH 7, agitation rate of 200 rpm). Ten cellulosic feedstock, including pure or natural cellulosic materials, were used as feedstock for hydrogen production by Clostridium strain TCW1 under optimal culture conditions. Using filter paper at 5.0 g/L resulted in the most effective hydrogen production performance, achieving a H₂ production rate and yield of 57.7 ml/h/L and 2.03 mol H₂/mol hexose, respectively. Production of cellulolytic enzyme activities was positively correlated with the efficiency of dark-H₂ fermentation.