Levels Of Cellulase Research Articles

Effect of Natural and Pretreated Soybean Hulls on Enzyme Production by Trichoderma reesei

AbstractThe enzyme induction utility of soybean hulls (SBH), consisting in excess of 50 wt% non‐starch polysaccharides (NSP) cellulose, hemicellulose, and pectin, was studied through cultivation of the carbohydrase‐producing fungus Trichoderma reesei Rut C‐30. Shake flask systems of T. reesei were grown in a medium consisting of defatted soybean flour as a nitrogen source and SBH, some of which were untreated and others pretreated by liquid hot water, alkaline, and supercritical carbon dioxide, as carbon source. Cellulase, xylanase, and polygalacturonase activities were measured for the systems, and the natural hull systems were found to yield optimum enzyme production. Controlled batch fermentation experiments were carried out to compare enzyme production resulting from media with Avicel® (FMC BioPolymer, Philadelphia, PA, USA) versus natural SBH with and without soybean flour as the nitrogen source. Soybean hull fermentations were also performed at several pH levels to observe the effects on enzyme production. Soybean hulls induced comparable levels of cellulase, and higher levels of xylanase and polygalacturonase, than Avicel®. With SBH, cellulase and xylanase production were enhanced at higher pH levels (6.0), and polygalacturonase was enhanced at lower pH levels (4.0–4.5). Enzyme production was largely unaffected by the presence of soybean flour as the nitrogen source.

High Level of Cellulase and β-Glucosidase Production by T.reesei and T.koningii Combinations on Corn Straws of Alkali Pretreatment

In order to obtain high level of β-glucosidase and filter paper activity production, the screening of strain combinations and the characteristic of enzymes from co-culture were investigated. The results showed that both the β-glucosidase (0.347 U/ml) and FPA (3.21 U/ml) reached the highest level via co-culture of T.reesei and T.koningii with the corn straw of alkali pretreatment as fermentation substrate. After 72 h of fermentation, both two enzyme activities reached the highest level. And the optimal temperature and pH value were 55°C and 5.5, respectively. These two enzyme stability decreased rapidly when the temperatures exceeded 90°C. The conversion efficiency reached 0.652 g reducing sugar per g pretreated corn straw using the enzymes from these two fungi, which was 1.49-fold and 1.64-fold compared with those from T.reesei (0.439 g/g) and T.koningii (0.398 g/g). The present work demonstrated an effective way to develop an efficient system for the bioconversion of agricultural waste straws to sugar.

Biomass hydrolyzing enzymes from plant pathogen Xanthomonas axonopodis pv. punicae: optimizing production and characterization

Xanthomonas axonopodis pv. punicae strain—a potent plant pathogen that causes blight disease in pomegranate—was screened for cellulolytic and xylanolytic enzyme production. This strain produced endo-β-1,4-glucanase, filter paper lyase activity (FPA), β-glucosidase and xylanase activities. Enzyme production was optimized with respect to major nutrient sources like carbon and nitrogen. Carboxy methyl cellulose (CMC) was a better inducer for FPA, CMCase and xylanase production, while starch was found to be best for cellobiase. Soybean meal/yeast extract at 0.5 % were better nitrogen sources for both cellulolytic and xylanolytic enzyme production while cellobiase and xylanase production was higher with peptone. Surfactants had no significant effect on levels of extracellular cellulases and xylanases. A temperature of 28 °C and pH 6–8 were optimum for production of enzyme activities. Growth under optimized conditions resulted in increases in different enzyme activities of around 1.72- to 5-fold. Physico-chemical characterization of enzymes showed that they were active over broad range of pH 4–8 with an optimum at 8. Cellulolytic enzymes showed a temperature optimum at around 55 °C while xylanase had highest activity at 45 °C. Heat treatment of enzyme extract at 75 °C for 1 h showed that xylanase activity was more stable than cellulolytic activities. Xanthomonas enzyme extracts were able to act on biologically pretreated paddy straw to release reducing sugars, and the amount of reducing sugars increased with incubation time. Thus, the enzymes produced by X. axonopodis pv. punicae are more versatile and resilient with respect to their activity at different pH and temperature. These enzymes can be overproduced and find application in different industries including food, pulp and paper and biorefineries for conversion of lignocellulosic biomass.

Evaluating biopulping as an alternative application on oil palm trunk using the white-rot fungus Trametes versicolor

The objective of the research was to investigate the suitability of the white-rot fungus Trametes versicolor for biopulping using oil palm biomass as the substrate. Fungi are grown on solid-state cultures Kirk's enrichment media to determine the lignocellulolytic activities. Samples subjected to fungal pretreatment for periods of 1, 2, 3, and 4 weeks were investigated and compared to the untreated control. The crude enzyme extracts were assayed using specific substrates and enzyme activities were calculated. The highest level of laccase activity was 218.66 U/L; the peak activity of manganese peroxidase was 162.10 U/L, and lignin peroxidase is 42.56 U/L. The activity levels of cellulase and hemicellulase were insignificant in all extracts (53.30 and 1.50 U/L, respectively). When the chips were pulped mechanically the Kappa number, pulp yield, and screened pulp yield decreased significantly and paper strength increased marginally with the exposure time. Hand sheet properties were also improved significantly by fungal treatment. Weight loss, lignin loss, cellulose, and holocellulose loss were 8.45%, 9.35%, 4.58%, and 7.2%, respectively. Images from SEM seem to indicate a simultaneous type of decay pattern involving cell wall breakdown combined with lignin modification. Considering all its pulping and papermaking properties, the performance of T. versicolor is good and has potential for use in large-scale biotechnological processes.

Environmentally safe treatment of black liquor with Comamonas sp. B‐9 under high‐alkaline conditions

The strain Comamonas sp. B-9 was isolated from steeping fluid of erosive bamboo slips derived from Kingdom Wu during the Three-Kingdoms Dynasty of ancient China (A.D. 220-280). It could be used to treat black liquor (BL) with high-alkaline pH and with an initial chemical oxygen demand (COD) of 18,000-25,000 mg L(-1) , without the addition of other carbon and nitrogen sources. The results revealed that Comamonas sp. B-9 was capable of reducing the COD, color, and lignin content of BL by up to 56.8, 35.3, and 43.5%, respectively. High levels of laccase, manganese peroxidase, cellulase, and xylanase enzymatic activities were also observed, and these enzymes could play an important role in the biotreatment of BL. Further, GC-MS analysis showed that most of the compounds detected in BL after biotreatment with Comamonas sp. B-9 were diminished, while 4-methyl benzaldehyde, 3,4,5-trihydroxybenzoic acid ethyl ester, and 4-hydroxy-3,5-dimethoxy benzaldehyde were produced as metabolites. The presented results indicate that Comamonas sp. B-9 has potential application for the treatment of wastewaters from pulp and paper processing with high COD load under high-alkaline conditions.

Simplifying cellulase production by using environmental selection pressures and recycling substrate

The production of crude cellulase in solid-state fermentation was simplified by using environmental selection pressures as an alternative to specialized strains and by re-using cellulose-to-ethanol fermentation substrate for cellulase production. The performance of wild strains of Trichoderma viride isolated from wheat and bark were found not to differ significantly from a ‘cellulase-enhanced’ strain of Trichoderma reesei. The filter paper activity of a strain of Aspergillus niger isolated in the laboratory was more than two times higher than the specialized T. reesei at 10 days. Activities of enrichment cultures obtained from compost and horse manure were more than double T. reesei after 10 days. Using the solid residue of previous simultaneous saccharification and fermentations of wheat straw to ethanol as substrate for cellulase production brought forward the onset of cellulase production and increased cellulase levels by 50%. The findings may help to simplify the on-site manufacture of cellulase for cellulose-to-ethanol conversion and reduce the amount of substrate required for the overall process.

Characterization of the newly isolated Geobacillus sp. T1, the efficient cellulase-producer on untreated barley and wheat straws

A thermophile cellulase-producing bacterium was isolated and identified as closely related to Geobacillus subterraneus. The strain, named Geobacillus sp. T1, was able to grow and produce cellulase on cellobiose, microcrystalline cellulose, carboxymethylcellulose (CMC), barley straw, wheat straw and Whatman No. 1 filter paper. However, barley and wheat straws were significantly better substrates for cellulase production. When Geobacillus sp. T1 was cultivated in the presence of 0.5% barley straw, 0.1% Tween 80 and pH 6.5 at 50°C, the maximum level of free cellulase up to 143.50U/mL was produced after 24h. This cellulase (∼54kDa) was most active at pH 6.5 and 70°C. The enzyme in citrate phosphate buffer (10mM) was stable at 60°C for at least 1h. Geobacillus sp. T1 with efficient growth and cellulase production on straws seems a potential candidate for conversion of agricultural biomass to fuels.

Deciphering the metabolic capabilities of a lipase producing Pseudomonas aeruginosa SL-72 strain

Pseudomonads have been reported for their metabolic, nutritional and ecological versatility, which motivated us to prospect the metabolic profile of a lipolytic strain Pseudomonas aeruginosa SL-72. The strain SL-72 was found to produce high levels of lipase and pectinase (1,555.62IU/mL and 1,490.33IU/mL, respectively), esterase and amylase, besides low levels of xylanase, proteinase and cellulase. The strain also tested positive for different plant growth-promoting traits-production of ammonia, hydrogen cyanide, siderophores, phosphate solubilization, nitrate reduction and antifungal activity. The high levels of activity of aryl sulphatase, alkaline phosphatase and fluorescein diacetate hydrolase makes it a useful strain for enhanced nutrient cycling in soil. The strain SL-72 produced rhamnolipids, a biosurfactant and its production was enhanced when starch was used as carbon source (0.256g/L) and utilized polycyclic hydrocarbon compounds viz. anthracene, phenanthrene, pyrene, fluorene and its mixture. The multifaceted nature of the culture illustrates its promise in bioremediation, industry, besides its use as an inoculant.

Production of a xylose-stimulated β-glucosidase and a cellulase-free thermostable xylanase by the thermophilic fungus Humicola brevis var. thermoidea under solid state fermentation

Humicola brevis var. thermoidea cultivated under solid state fermentation in wheat bran and water (1:2 w/v) was a good producer of β-glucosidase and xylanase. After optimization using response surface methodology the level of xylanase reached 5,791.2 ± 411.2 U g(-1), while β-glucosidase production was increased about 2.6-fold, reaching 20.7 ± 1.5 U g(-1). Cellulase levels were negligible. Biochemical characterization of H. brevis β-glucosidase and xylanase activities showed that they were stable in a wide pH range. Optimum pH for β-glucosidase and xylanase activities were 5.0 and 5.5, respectively, but the xylanase showed 80 % of maximal activity when assayed at pH 8.0. Both enzymes presented high thermal stability. The β-glucosidase maintained about 95 % of its activity after 26 h in water at 55 °C, with half-lives of 15.7 h at 60 °C and 5.1 h at 65 °C. The presence of xylose during heat treatment at 65 °C protected β-glucosidase against thermal inactivation. Xylanase maintained about 80 % of its activity after 200 h in water at 60 °C. Xylose stimulated β-glucosidase activity up to 1.7-fold, at 200 mmol L(-1). The notable features of both xylanase and β-glucosidase suggest that H. brevis crude culture extract may be useful to compose efficient enzymatic cocktails for lignocellulosic materials treatment or paper pulp biobleaching.

Production of cellulases from Aspergillus niger NS-2 in solid state fermentation on agricultural and kitchen waste residues

Various agricultural and kitchen waste residues were assessed for their ability to support the production of a complete cellulase system by Aspergillus niger NS-2 in solid state fermentation. Untreated as well as acid and base-pretreated substrates including corn cobs, carrot peelings, composite, grass, leaves, orange peelings, pineapple peelings, potato peelings, rice husk, sugarcane bagasse, saw dust, wheat bran, wheat straw, simply moistened with water, were found to be well suited for the organism’s growth, producing good amounts of cellulases after 96h without the supplementation of additional nutritional sources. Yields of cellulases were higher in alkali treated substrates as compared to acid treated and untreated substrates except in wheat bran. Of all the substrates tested, wheat bran appeared to be the best suited substrate producing appreciable yields of CMCase, FPase and β-glucosidase at the levels of 310, 17 and 33U/g dry substrate respectively. An evaluation of various environmental parameters demonstrated that appreciable levels of cellulases could be produced over a wide range of temperatures (20–50°C) and pH levels (3.0–8.0) with a 1:1.5 to 1:1.75 substrate to moisture ratio.

Function of meiobenthos and microorganisms in cellulose breakdown in sediments of wetlands with different origins in Hokkaido

To validate the mechanism of cellulose breakdown in cold climate wetlands, we investigated cellulase activity in sediments collected from 17 wetland sites in Hokkaido, the northern area of Japan. We evaluated cellulase activity by quantitative analysis of glucose released from carboxymethyl cellulose and found that sediments from peat fens demonstrated high activity, followed by sediments from lagoons and estuaries. Sediments from peat fens also contained greater amounts of organic matter, followed by lagoons and estuaries, thereby suggesting a strong positive correlation between organic matter content and cellulase activity. Evaluation of cellulase activity by qualitative cellulose zymographic analysis showed that various cellulases with different molecular sizes were implicated in cellulose breakdown in wetlands. Among them, cellulose breakdown in Meguma Pond (peat fen), Notsuke Gulf (peat fen), and Lake Utonai (lagoon) was potentially due to microorganism cellulase, while that in Lake Chobushi (lagoon) was ascribed to meiobenthos (Oligochaeta species) cellulase. The findings presented herein suggest that the origin and activity level of cellulase vary depending on the type of cold climate wetland.

Characterization of hemicellulases from thermophilic fungi

The thermophilic fungi Thermomyces lanuginosus, Malbranchea cinnamomea, Myceliophthora fergusii and the thermotolerant Aspergillus terreus were cultivated on various carbon sources, and hemicellulolytic and cellulolytic enzyme profiles were evaluated. All fungi could grow on locust bean galactomannan (LBG), Solka floc, wheat bran and pectin, except T. lanuginosus, which failed to utilize LBG for growth. Different levels of cellulase and hemicellulase activities were produced by these fungal strains. Depending on the carbon source, variable ratios of thermostable hydrolytic enzymes were obtained, which may be useful in various applications. All strains were found to secrete xylanolytic and mannanolytic enzymes. Generally, LBG was the most efficient carbon source to induce mannanase activities, although T. lanuginosus was able to produce mannanase only on wheat bran as a carbon source. Xylanolytic activities were usually highest on wheat bran medium, but in contrast to other investigated fungi, xylanase production by M. fergusii was enhanced on pectin medium. Preliminary thermostability screening indicated that among the investigated species, thermotolerant glycosidases can be found. Some of the accessory activities, including the α-arabinosidase activity, were surprisingly high. The capability of the produced enzymes to improve the hydrolysis of lignocellulosic pretreated substrate was evaluated and revealed potential for these enzymes.

Sediment-complex-binding cellulose breakdown in wetlands of rivers

We have been assessing the activity level of cellulase in wetland sediments to clarify the significance of cellulase for the turnover of plant cellulose in wetlands. In this study, we investigated the cellulose degrading function of sediment in wetlands to clarify the biochemical breakdown mechanism of cellulose. Specifically, we measured cellulase activities of sediments collected from wetlands of the Chikugo River (Fukuoka Prefecture), Midori River (Kumamoto Prefecture), Hamado River (Kumamoto Prefecture), Yodo River (Osaka Prefecture), and Tanaka River (Mie Prefecture). The results revealed that the activity levels differed significantly among rivers. Additionally, the cellulase activities of the sediment were not completely suppressed in the presence of chloramphenicol. These findings suggested that a portion of the cellulase activities were derived from cellulases binding to the components of sediments. Actually, sediments also showed the ability to bind fungal cellulase. Comparison of the binding ability of clay and plant residues, the main components of sediments, revealed that plant residues had significantly higher abilities to bind cellulase. This finding was supported by the fact that there was a strong correlation between the organic matter content in the sediment and the cellulase-binding ability (R = 0.66). Results of this study show that sediment complexes harboring cellulases might function as a bioreactor to degrade cellulose in wetlands.

Production of cellulases and β-glucosidase in Trichoderma reesei mutated by proton beam irradiation

To obtain mutant strains producing high levels of cellulases (FPase and CMCase) and β-glucosidase, Trichoderma reesei KCTC 6950 was mutated by proton beam irradiation. Five mutants were selected out of 1,000 mutants of T.reesei treated with proton beam irradiation, based on their ability for enzyme production on a plate screening medium. In submerged cultures containing Mandel’s fermentation medium, the mutant strain T-2 (MT-2) demonstrated a 165% increase in the activity of FPase, a 146% increase in the activity of CMCase, and a 313% increase in the activity of β-glucosidase, compared with the wild type strain. Additionally, the properties of high level β-glucosidase produced by MT-2 were the same as those of the wild type strain, e.g., an optimum pH of 4.8, and an optimum temperature of 65 °C. Moreover, the protein concentrations of β-glucosidase produced by the wild type strain and MT-2 were measured by SDS-PAGE, and then β-glucosidase activities were detected by the MUG-zymogram assay.

Enhanced Microbial Utilization of Recalcitrant Cellulose by an Ex Vivo Cellulosome-Microbe Complex

A cellulosome-microbe complex was assembled ex vivo on the surface of Bacillus subtilis displaying a miniscaffoldin that can bind with three dockerin-containing cellulase components: the endoglucanase Cel5, the processive endoglucanase Cel9, and the cellobiohydrolase Cel48. The hydrolysis performances of the synthetic cellulosome bound to living cells, the synthetic cellulosome, a noncomplexed cellulase mixture with the same catalytic components, and a commercial fungal enzyme mixture were investigated on low-accessibility recalcitrant Avicel and high-accessibility regenerated amorphous cellulose (RAC). The cell-bound cellulosome exhibited 4.5- and 2.3-fold-higher hydrolysis ability than cell-free cellulosome on Avicel and RAC, respectively. The cellulosome-microbe synergy was not completely explained by the removal of hydrolysis products from the bulk fermentation broth by free-living cells and appeared to be due to substrate channeling of long-chain hydrolysis products assimilated by the adjacent cells located in the boundary layer. Our results implied that long-chain hydrolysis products in the boundary layer may inhibit cellulosome activity to a greater extent than the short-chain products in bulk phase. The findings that cell-bound cellulosome expedited the microbial cellulose utilization rate by 2.3- to 4.5-fold would help in the development of better consolidated bioprocessing microorganisms (e.g., B. subtilis) that can hydrolyze recalcitrant cellulose rapidly at low secretory cellulase levels.

Role of alkaline-tolerant fungal cellulases in release of total antioxidants from agro-wastes under solid state fermentation

The alkaline-tolerant marine-derived fungus Chaetomium globosum was tested for the production of enhanced levels of cellulases and free phenolics under highly alkaline conditions using agro wastes (cotton seed, sugar cane bagasse) as substrates under solid state fermentation (SSF) processes. In both the agro wastes used, an increase in cellulases (β-endoglucanase, β-Glucosidase, and β-exoglucanase) production was observed with increase in pH. This enhanced carbohydrate-hydrolyzing enzymes (β-endoglucanase, β-Glucosidase and β-exoglucanase) and thereby enriched the total phenolic release from agro-wastes under SSF conditions of higher pH. A linear correlation was observed between released total phenolic contents of agro-wastes and total antioxidant property. The increased antioxidant activity on free radical scavenging was also observed with the increase in pH. Thus, the present study makes it possible to produce nutraceutical ingredients cost-effectively from agricultural wastes.

Differential Regulation of the Cellulase Transcription Factors XYR1, ACE2, and ACE1 in Trichoderma reesei Strains Producing High and Low Levels of Cellulase

Due to its capacity to produce large amounts of cellulases, Trichoderma reesei is increasingly being investigated for second-generation biofuel production from lignocellulosic biomass. The induction mechanisms of T. reesei cellulases have been described recently, but the regulation of the genes involved in their transcription has not been studied thoroughly. Here we report the regulation of expression of the two activator genes xyr1 and ace2, and the corepressor gene ace1, during the induction of cellulase biosynthesis by the inducer lactose in T. reesei QM 9414, a strain producing low levels of cellulase (low producer). We show that all three genes are induced by lactose. xyr1 was also induced by d-galactose, but this induction was independent of d-galactose metabolism. Moreover, ace1 was carbon catabolite repressed, whereas full induction of xyr1 and ace2 in fact required CRE1. Significant differences in these regulatory patterns were observed in the high-producer strain RUT C30 and the hyperproducer strain T. reesei CL847. These observations suggest that a strongly elevated basal transcription level of xyr1 and reduced upregulation of ace1 by lactose may have been important for generating the hyperproducer strain and that thus, these genes are major control elements of cellulase production.

New isolate of Trichoderma viride strain for enhanced cellulolytic enzyme complex production

A new Trichoderma viride stain was isolated from Singapore soil samples. Its mutants were developed by using ethyl methyl sulfonate (EMS) treatment and UV-irradiation followed by a semi-quantitative plate clearing assay on phosphoric-acid-swollen cellulose plates. Mutant EU2-77 proved to be the most promising extracellular cellulase producer among 20 mutants in a screening program performed in shake flask fermentation after plate screening. Soluble protein content, filter paper cellulase (FPase) activity, β-glucosidase activity and endoglucanase (CMCase) activity of the fermentation broths of the mutant strain were increased to 1.67, 2.49, 2.16, and 2.61 folds, respectively, compared with the wild strain. This enzyme complex produced by mutant EU2-77 contained FPase (2.19 IU/ml), CMCase (16.46 IU/ml), β-glucosidase (4.04 IU/ml), xylanase (42.37 IU/ml), and β-xylosidase (0.12 IU/ml). The soluble protein concentration in the enzyme complex was 1.69 mg/ml. The hydrolytic capacities of fermentation supernatants of T. reesei Rut-C30, the wild strain T. viride NP13a and mutant T. viride EU2-77 were compared with the commercial enzymes on the hydrolysis of waste newspaper. The crude enzymes prepared by T. viride EU2-77 showed much higher hydrolysis performance than that from the commercial strain Rut-C30 and demonstrated much comparable hydrolytic performances with the commercial enzyme mixtures. T. viride mutant EU2-77 produced high levels of extracellular cellulases as well as β-glucosidase, rendering the supplementation of β-glucosidase unnecessary in waste newspaper hydrolysis.

Isolation and characterization of a Bacillus licheniformis strain capable of degrading zearalenone

The worldwide contamination of cereals, oilseeds, and other crops by mycotoxin-producing moulds is a significant problem. Mycotoxins have adverse effects on humans and animals that result in illnesses and economic losses. Reduction or elimination of mycotoxin contamination in food and feed is an important issue. This study aimed to screen soil bacteria for degradation of zearalenone (ZEN). A pure culture of strain CK1 isolated from soil samples showed most capable of degradation of ZEN. Using physiological, biochemical, and 16S rRNA gene sequence analysis methods, CK1 was identified as Bacillus licheniformis. Addition of 2 ppm of ZEN in Luria–Bertani (LB) medium, B. licheniformis CK1 decreased 95.8% of ZEN after 36 h of incubation. In ZEN-contaminated corn meal medium, B. licheniformis CK1 decreased more than 98% of ZEN after 36 h of incubation. In addition, B. licheniformis CK1 was non-hemolytic, non-enterotoxin producing, and displayed high levels of extracellular xylanase, cellulase, and protease activities. These findings suggest that B. licheniformis CK1 could be used to reduce the concentrations of ZEN and improve the digestibility of nutrients in feedstuffs simultaneously.

Xylanase from a soil isolate, Bacillus pumilus: gene isolation, enzyme production, purification, characterization and one-step separation by aqueous-two-phase system

A xylanase producer, Bacillus pumilus SB-M13, was isolated from soil and identified using various tests based on carbohydrate fermentation preferences and fatty acid analysis. Xylanase gene, isolated using PCR amplification, was partially sequenced and it showed 89–94% sequence similarity to the xylanase genes of other B. pumilus strains. Xylanase with very low level of cellulase was produced on agricultural byproducts. The enzyme has been purified 186-fold by hydrophobic interaction chromatography and biochemically characterized. It has a molecular weight of 24.8 kDa and pI of 9.2. Xylanolytic activity is stable at alkaline pH and highest activity is observed at 60 °C and pH 7.5. Enzyme K m and k cat values were determined as 1.9 mg/mL and 42,600 U/mg, respectively. In aqueous-two-phase system, xylanase always partitioned to the top phase. Basic pH, low PEG concentration, salt addition, and presence of microbial cells enhanced xylanase partitioning. A maximum sevenfold purification, 10-fold concentration and 100% xylanase recovery were obtained, separately, by adjusting system parameters. A fourfold concentrated xylanase was obtained with 70% enzyme recovery only in one step ATPS process without cell harvesting.