Enhance of Cellulase Production and Biomass Degradation by Transformation of the Trichoderma reesei RUT-C30∆ zface1 Strain

Abstract The second-generation bioethanol employs lignocellulosic materials degraded by microbial cellulases in their production. The fungus Trichoderma reesei is one of the main microorganisms producing cellulases, and its genetic modification can lead to the optimization in obtaining hydrolytic enzymes. This work carried out the deletion of the sequence that encodes the zinc finger motif of the transcription factor ACE1 (cellulase expression repressor I) of the fungus T. reesei RUT-C30. The transformation of the RUT-C30 lineage was confirmed by amplification of the 989 bp fragment relative to the selection marker, and by the absence of the zinc finger region amplification in mutants, named T. reesei RUT-C30Δzface1. The production of cellulases by mutants was compared to RUT-C30 and measured with substrates carboxymethylcellulose (CMC), microcrystalline cellulose (Avicel®) and Whatman filter paper (PF). The results demonstrated that RUT-C30Δzface1 has cellulolytic activity increased 3.2-fold in Avicel and 2.1-fold in CMC and PF. The mutants presented 1.4-fold higher sugar released in the hydrolysis of the biomass assays. These results suggest that the partial deletion of ace1 gene is an important strategy in achieving bioethanol production on an industrial scale at a competitive price in the fuel market.

Production of thermostable ß-glucosidase and CMCase by Penicillium sp: LMI01 isolated from the Amazon region

Background: Cellulolytic enzymes of microbial origin have great industrial importance because of their wide application in various industrial sectors. Fungi are considered the most efficient producers of these enzymes. Bioprospecting survey to identify fungal sources of biomass-hydrolyzing enzymes from a high-diversity environment is an important approach to discover interesting strains for bioprocess uses. In this study, we evaluated the production of endoglucanase (CMCase) and ß-glucosidase, enzymes from the lignocellulolytic complex, produced by a native fungus. Penicillium sp. LMI01 was isolated from decaying plant material in the Amazon region, and its performance was compared with that of the standard isolate Trichoderma reesei QM9414 under submerged fermentation conditions. Results: The effectiveness of LMI01 was similar to that of QM9414 in volumetric enzyme activity (U/mL); however, the specific enzyme activity (U/mg) of the former was higher, corresponding to 24.170 U/mg of CMCase and 1.345 U/mg of ß-glucosidase. The enzymes produced by LMI01 had the following physicochemical properties: CMCase activity was optimal at pH 4.2 and the ß-glucosidase activity was optimal at pH 6.0. Both CMCase and ß-glucosidase had an optimum temperature at 60°C and were thermostable between 50 and 60°C. The electrophoretic profile of the proteins secreted by LMI01 indicated that this isolate produced at least two enzymes with CMCase activity, with approximate molecular masses of 50 and 35 kDa, and ß-glucosidases with molecular masses between 70 and 100 kDa. Conclusions: The effectiveness and characteristics of these enzymes indicate that LMI01 can be an alternative for the hydrolysis of lignocellulosic materials and should be tested in commercial formulations.

Improving the thermostability of Trichoderma reesei xylanase 2 by introducing disulfide bonds

Background: Xylanases are considered one of the most important enzymes in many industries. However, their low thermostability hampers their applications in feed pelleting, pulp bleaching, and so on. The main aim of this work was to improve the thermostability of Trichoderma ressei xylanase 2 (Xyn2) by introducing disulfide bonds between the N-terminal and α-helix and the ß-sheet core. Results: In this work, two disulfide bonds were separately introduced in the Xyn2 to connect the N-terminal and α-helix to the ß-sheet core of Xyn2. The two disulfide bonds were introduced by site-directed mutagenesis of the corresponding residues. The half-life of the mutants Xyn2C14­52 (disulfide bond between ß-sheets B2 and B3) and Xyn2C59­149 (disulfide bond between ß-sheets A5 and A6) at 60°C was improved by approximately 2.5- and 1.8-fold compared to that of the wild type Xyn2. In addition, the enzyme's resistance to alkali and acid was enhanced. Conclusion: Our results indicated that the connection of the N-terminal and α-helix to the ß-sheet core is due to the stable structure of the entire protein.

Isolation and expression of two polyketide synthase genes from Trichoderma harzianum 88 during mycoparasitism

Abstract Metabolites of mycoparasitic fungal species such as Trichoderma harzianum 88 have important biological roles. In this study, two new ketoacyl synthase (KS) fragments were isolated from cultured Trichoderma harzianum 88 mycelia using degenerate primers and analysed using a phylogenetic tree. The gene fragments were determined to be present as single copies in Trichoderma harzianum 88 through southern blot analysis using digoxigenin-labelled KS gene fragments as probes. The complete sequence analysis in formation of pksT-1 (5669 bp) and pksT-2 (7901 bp) suggests that pksT-1 exhibited features of a non-reducing type I fungal PKS, whereas pksT-2 exhibited features of a highly reducing type I fungal PKS. Reverse transcription polymerase chain reaction indicated that the isolated genes are differentially regulated in Trichoderma harzianum 88 during challenge with three fungal plant pathogens, which suggests that they participate in the response of Trichoderma harzianum 88 to fungal plant pathogens. Furthermore, disruption of the pksT-2 encoding ketosynthase–acyltransferase domains through Agrobacterium -mediated gene transformation indicated that pksT-2 is a key factor for conidial pigmentation in Trichoderma harzianum 88.

The improvement of competitive saprophytic capabilities of Trichoderma species through the use of chemical mutagens

Abstract The antagonistic potential of Trichoderma strains was assayed by studying the effect of their culture filtrate on the radial growth of Sclerotium rolfsii, the causal agent of chickpea collar rot. Trichoderma harzianum-1432 (42.2%) and Trichoderma atroviride (40.3%) were found to be strong antagonists. To enhance their antagonistic potential, mutagenesis of these two selected strains was performed. Two mutants, Th-m1 and T. atroviride m1, were found to be more effective than their parent strains. The enzymatic activities of the selected parent and mutant strains were assayed, and although both mutants were found to have enhanced enzymatic activities compared to their respective parent strains, Th-m1 possessed the maximum cellulase (5.69 U/mL) and β-1,3-glucanase activity (61.9 U/mL). Th-m1 also showed high competitive saprophytic ability (CSA) among all of the selected parent and mutant strains, and during field experiments, Th-m1 was found to successfully possess enhanced disease control (82.9%).

Cultural conditions on the production of extracellular enzymes by Trichoderma isolates from tobacco rhizosphere

Abstract Twelve isolates of Trichoderma spp. isolated from tobacco rhizosphere were evaluated for their ability to produce chitinase and β-1,3-glucanase extracellular hydrolytic enzymes. Isolates ThJt1 and TvHt2, out of 12 isolates, produced maximum activities of chitinase and β-1,3-glucanase, respectively. In vitro production of chitinase and β-1,3-glucanase by isolates ThJt1 and TvHt2 was tested under different cultural conditions. The enzyme activities were significantly influenced by acidic pH and the optimum temperature was 30 °C. The chitin and cell walls of Sclerotium rolfsii, as carbon sources, supported the maximum and significantly higher chitinase activity by both isolates. The chitinase activity of isolate ThJt1 was suppressed significantly by fructose (80.28%), followed by glucose (77.42%), whereas the β-1,3-glucanase activity of ThJt1 and both enzymes of isolate TvHt2 were significantly suppressed by fructose, followed by sucrose. Ammonium nitrate as nitrogen source supported the maximum activity of chitinase in both isolates, whereas urea was a poor nitrogen source. Production of both enzymes by the isolates was significantly influenced by the cultural conditions. Thus, the isolates ThJt1 and TvHt2 showed higher levels of chitinase and β-1,3-glucanase activities and were capable of hydrolyzing the mycelium of S. rolfsii infecting tobacco. These organisms can be used therefore for assessment of their synergism in biomass production and biocontrol efficacy and for their field biocontrol ability against S. rolfsii and Pythium aphanidermatum infecting tobacco.

Determination of lytic enzyme activities of indigenous Trichoderma isolates from Pakistan

Abstract This study investigated lytic enzyme activities in three indigenous Trichoderma strains namely, Trichoderma asperellum, Trichoderma harzianum and Trichoderma sp. Native Trichoderma strains and a virulent strain of Rhizoctonia solani isolated from infected bean plants were also included in the study. Enzyme activities were determined by measuring sugar reduction by dinitrosalicylic acid (DNS) method using suitable substrates. The antagonists were cultured in minimal salt medium with the following modifications: medium A (1 g of glucose), medium B (0.5 g of glucose + 0.5 g of deactivated R. solani mycelia), medium C (1.0 g of deactivated respective antagonist mycelium) and medium D (1 g of deactivated R. solani mycelia). T asperellum showed presence of higher amounts of chitinases, β-1, 3-glucanases and xylanases in extracellular protein extracts from medium D as compared to medium A. While, the higher activities of glucosidases and endoglucanses were shown in medium D extracts by T. harzianum. β-glucosidase activities were lower compared with other enzymes; however, activities of the extracts of medium D were significantly different. T. asperellum exhibited maximum inhibition (97.7%). On the other hand, Trichoderma sp. did not show any effect on mycelia growth of R. solani on crude extract.

Purification and characterization of xylanases from Trichoderma inhamatum

Background Two xylanases, Xyl I and Xyl II, were purified from the crude extracellular extract of a Trichoderma inhamatum strain cultivated in liquid medium with oat spelts xylan. Results The molecular masses of the purified enzymes estimated by SDS-PAGE and gel filtration were, respectively, 19 and 14 kDa for Xyl I and 21 and 14.6 kDa for Xyl II. The enzymes are glycoproteins with optimum activity at 50°C in pH 5.0-5.5 for Xyl I and 5.5 for Xyl II. The xylanases were very stable at 40°C and in the pH ranges from 4.5-6.5 for Xyl I and 4.0-8.0 for Xyl II. The ion Hg2+ and the detergent SDS strongly reduced the activity while 1,4-dithiothreitol stimulated both enzymes. The xylanases showed specificity for xylan, Km and Vmax of 14.5, 1.6 mg·mL-1 and 2680.2 and 462.2 U·mg of protein-1 (Xyl I) and 10.7, 4.0 mg·mL-1 and 4553.7 and 1972.7 U·mg of protein-1 (Xyl II) on oat spelts and birchwood xylan, respectively. The hydrolysis of oat spelts xylan released xylobiose, xylotriose, xylotetrose and larger xylooligosaccharides. Conclusions The enzymes present potential for application in industrial processes that require activity in acid conditions, wide-ranging pH stability, such as for animal feed, or juice and wine industries.

Molecular characterization of a Xylanase-producing fungus isolated from fouled soil

Xylanase (EC 3. 2. 1. 8), hydrolyzes xylo-oligosaccharides into D-xylose and required for complete hydrolysis of native cellulose and biomass conversion. It has broad range of applications in the pulp and paper, pharmaceutical and Agri-food industries. Fifty fungal species were isolated from the fouled soil around an oil refinery and screened for the production of xylanase enzyme by enrichment culture techniques. The isolated fungal strain was identified as Hypocrea lixii SS1 based on the results of biochemical tests and 18s rRNA sequencing. The phylogenetic tree was constructed using the MEGA 5 software. Further, Hypocrea lixii SS1 was tested for the ability to utilize the sunflower oil sludge (waste from the oil industry) as the sole carbon source for xylanase production. The growth characteristics of Hypocrea lixii SS1 were also studied and maximum growth was found on the 7th day of incubation. The fungus showed a remarkable xylanase production of 38.9 U/mL. Xylanase was purified using a combination of 0-50% NH4SO2 precipitation, DEAE-sepharose and Sephacryl S-200 chromatography. Single peak obtained in RP-HPLC confirms the purity of xylanase. Further the enzyme produced was affirmed as xylanase with its molecular weight (29 kDa) using SDS-PAGE.

Filamentous fungi and media for cellulase production in solid state cultures

Cellulase production was evaluated in two reference strains (T. reesei Rut-C30 and T. reesei QM9414), two strains isolated from a sugarcane cultivation area (Trichoderma sp. IPT778 and T. harzianum rifai IPT821) and one strain isolated in a program for biodiversity preservation in São Paulo state (Myceliophthora thermophila M77). Solid state cultures were performed using sugarcane bagasse (C), wheat bran (W) and/or soybean bran (S). The highest FPA was 10.6 U/gdm for M77 in SC (10:90) at 80% moisture, which was 4.4 times higher than production in pure W. C was a strong inducer of cellulase production, given that the production level of 6.1 U/gdm in WC (40:60) was 2.5 times higher than in pure W for strain M77; T. reesei Rut-C30 did not respond as strongly with about 1.6-fold surplus production. S advantageously replaced W, as the surplus production on SC (20:80) was 2.3 times relative to WC (20:80) for M77.

Sugarcane bagasse as feedstock for cellulase production by Trichoderma harzianum in optimized culture medium

This work aimed at the production of cellulases from pretreated sugarcane bagasse by the filamentous fungus Trichoderma harzianum IOC 3844 and their application in the hydrolysis of this same substrate, for a future use in second-generation ethanol production. The production of cellulases was optimized, which resulted in high enzymatic activities after 42 hrs of process in an instrumented bioreactor (CMCase 27,017 U x L-1; FPase 1,225 U x L-1; and β-glucosidase 609 U x L-1). The enzymatic extract was concentrated by using a hollow fiber membrane filtration system. The concentrated extract was applied in the hydrolysis of pretreated sugarcane bagasse, after 28 hrs of enzymatic reaction, displaying a similar catalytic performance of that attained with a commercial enzymatic preparation (hydrolysis efficiency of roughly 50%). Finally, the enzymatic extract was partially characterized in terms of the molecular weights of the main activities of the enzymatic pool. Electrophoretic analysis identified eleven protein bands; six of them were related to CMCase activity and revealing molecular weights that varied from 48 to 78 kDa, and two bands were associated with β-glucosidase activity and having molecular weights of 75 and 85 kDa.

Bioethanol production from rice straw residues

A rice straw -cellulose utilizing mold was isolated from rotted rice straw residues. The efficient rice straw degrading microorganism was identified as Trichoderma reesei. The results showed that different carbon sources in liquid culture such as rice straw, carboxymethyl cellulose, filter paper, sugar cane bagasse, cotton stalk and banana stalk induced T. reesei cellulase production whereas glucose or Potato Dextrose repressed the synthesis of cellulase. T. reesei cellulase was produced by the solid state culture on rice straw medium. The optimal pH and temperature for T. reesei cellulase production were 6 and 25 ºC, respectively. Rice straw exhibited different susceptibilities towards cellulase to their conversion to reducing sugars. The present study showed also that, the general trend of rice straw bioconversion with cellulase was more than the general trend by T. reesei. This enzyme effectively led to enzymatic conversion of acid, alkali and ultrasonic pretreated cellulose from rice straw into glucose, followed by fermentation into ethanol. The combined method of acid pretreatment with ultrasound and subsequent enzyme treatment resulted the highest conversion of lignocellulose in rice straw to sugar and consequently, highest ethanol concentration after 7 days fermentation with S. cerevisae yeast. The ethanol yield in this study was about 10 and 11 g.L-¹.

Effect of acid hydrolysis and fungal biotreatment on agro-industrial wastes for obtainment of free sugars for bioethanol production

This study was designed to evaluate selected chemical and microbiological treatments for the conversion of certain local agro-industrial wastes (rice straw, corn stalks, sawdust, sugar beet waste and sugarcane bagasse) to ethanol. The chemical composition of these feedstocks was determined. Conversion of wastes to free sugars by acid hydrolysis varied from one treatment to another. In single-stage dilute acid hydrolysis, increasing acid concentration from 1 % (v/v) to 5 % (v/v) decreased the conversion percentage of almost all treated agro-industrial wastes. Lower conversion percentages for some treatments were obtained when increasing the residence time from 90 to 120min. The two-stage dilute acid hydrolysis by phosphoric acid (1.0 % v/v) followed by sulphuric acid (1.0 % v/v) resulted in the highest conversion percentage (41.3 % w/w) on treated sugar beet waste. This treatment when neutralized, amended with some nutrients and inoculated with baker's yeast, achieved the highest ethanol concentration (1.0 % v/v). Formation of furfural and hydroxymethylfurfural (HMF) were functions of type of acid hydrolysis, acid concentration, residence time and feedstock type. The highest bioconversion of 5 % wastes (37.8 % w/w) was recorded on sugar beet waste by Trichoderma viride EMCC 107. This treatment when followed by baker's yeast fermentation, 0.41 % (v/v) ethanol and 8.2 % (v/w) conversion coefficient were obtained.

The hydrolysis of agro-industrial residues by holocellulose-degrading enzymes

Holocellulose structures from agro-industrial residues rely on main and side chain attacking enzymes with different specificities for complete hydrolysis. Combinations of crude enzymatic extracts from different fungal species, including Aspergillus terreus, Aspergillus oryzae, Aspergillus niger and Trichoderma longibrachiatum, were applied to sugar cane bagasse, banana stem and dirty cotton residue to investigate the hydrolysis of holocellulose structures. A. terreus and A. oryzae were the best producers of FPase and xylanase activities. A combination of A. terreus and A. oryzae extracts in a 50% proportion provided optimal hydrolysis of dirty cotton residue and banana stem. For the hydrolysis of sugar cane bagasse, the best results were obtained with samples only containing A. terreus crude extract.

Comparative study for the kinetics of extracellular xylanases from Trichoderma harzianum and chaetomium thermophilum

Xylanases assume special importance in the paper and pulp industry as they replace toxic chemicals such as chlorine and chlorine dioxide for developing eco-friendly processes. This study evaluated xylanases produced by two fungi, the mesophilic fungus Trichoderma harzianum and a thermophilic fungus Chaetomium thermophilum. Among the polymeric substrates studied for xylanase production by both the fungi, birch wood xylan was found to be the best inducer of xylanases. Xylanases induction was subject to glucose repression. Partially purified xylanases preparation from T. harzianum and C. thermophilum exhibited optimal activities at pH 5 and 6 and at 60ºC and 70ºC, respectively. The apparent Km and Vmax values for the partially purified xylanase from T. harzianum using oat spelt xylan as a substrate were 4.8 mg mL-1 and 0.526 µmol min-1 mg-1, respectively. Whereas values of the partially purified xylanase from C. thermophilum were 2.96 mg mL-1 and 0.25 µmol min-1 mg-1, respectively. These findings in this study have great implications for the future applications of xylanases.

Production of lytic enzymes by Trichoderma isolates during in vitro antagonism with Aspergillus niger, the causal agent of collar rot of peanut

Twelve isolates of Trichoderma (six of T. harzianum, five of T. viride, one of T. virens), which reduced variably the incidence of collar rot disease caused in peanut by Aspergillus niger Van Tieghem, were evaluated for their potential to produce lytic enzymes during in vitro antagonism. T. viride 60 inhibited highest (86.2%) growth of test fungus followed by T. harzianum 2J (80.4%) at 6 days after inoculation (DAI) on PDA media. The specific activities of chitinase, â-1,3-glucanase and protease were 11, 3.46 and 9 folds higher in T6 antagonist (T. viride 60 and A. niger interactions) followed by 8.72, 2.85 and 9 folds in T8 antagonist (T. harzianum 2J and A. niger interactions), respectively, compared to the activity produced by control petri plate T13 (A. niger alone) at 6 DAI. Activity of these lytic enzymes induced in antagonists' plates comprises the growth of Trichoderma isolates. However, cellulase and poly galacturonase were found least amount in these antagonists treatment. A significant positive correlation (p=0.01) between percentage growth inhibition of test fungus and lytic enzymes - (chitinase, â-1,3-glucanase and protease) in the culture medium of antagonist treatment established a relationship to inhibit growth of fungal pathogen by increasing the levels of these enzymes. Among the Trichoderma isolates, T. viride 60 was found best strain to be used in biological control of plant pathogen A. niger.

Biocontrol capacity of wild and mutant Trichoderma harzianum (Rifai) strains on Rhizoctonia solani 618: effect of temperature and soil type during storage

Wild (Th11, Th12 and Th650) and mutant (Th11A80.1, Th12A40.1, Th12C40.1 and Th650-NG7) Trichoderma harzianum strains were stored for 180 days at 5ºC or at 22ºC, in two types of soils. Strains recovered at 90, 120 and 180 days from the two types of soils, retained their full capacity to biocontrol Rhizoctonia solani 618, that produces crown and root rot of tomatoes. Recovery, estimated as colony forming units (cfu) of the wild and mutant strains, showed that all increased their cfu after storage independently of the type of soil and temperature, although kinetic behavior differed among strains. Ratios of recovery after storage in type B soil/ type A soil or at 22ºC/5ºC, higher or lower than one respectively, allowed to establish that Th11 and Th12 were the most appropriate strains for the biocontrol of R. solani in conditions where growth of the phytopathogen is optimal.

Influence of buffer systems on Trichoderma reesei Rut C-30 morphology and cellulase production

The cellulase enzyme production is a key issue in the enzymatic hydrolysis of lignocellulosic materials. Since fungal morphology influences the productivity of fungal fermentations, it is of major importance to well know the fungal behavior during culture for cellulase production. In this work, the influence of medium supplementation, with different buffer systems at two different concentrations and pH conditions, on the morphology of T. reesei Rut C-30 and cellulase production, was investigated. A medium without buffer was used as control. The results suggest that fungal morphology is significantly dependent on the addition of different buffer systems to the nutrient broth. The mycelial morphology shows a clear transition from clumped to pelleted forms in cultures with variation of buffer systems and concentration. The higher filter paper activity was obtained using 100 mM succinate buffer, at pH 4.8, in the medium supplementation, corresponding to a dispersed mycelial morphology.

Hydrolysis of xylans by enzyme systems from solid cultures of Trichoderma harzianum strains

Xylanase activity was isolated from crude extracts of Trichoderma harzianum strains C and 4 grown at 28oC in a solid medium containing wheat bran as the carbon source. Enzyme activity was demonstrable in the permeate after ultrafiltration of the crude extracts using an Amicon system. The hydrolysis patterns of different xylans and paper pulps by xylanase activity ranged from xylose, xylobiose and xylotriose to higher xylooligosaccharides. A purified ß-xylosidase from the Trichoderma harzianum strain released xylose, xylobiose and xylotriose from seaweed, deacetylated, oat spelt and birchwood xylans. The purified enzyme was not active against acetylated xylan and catalyzed the hydrolysis of xylooligosaccharides, including xylotriose, xylotetraose and xylopentaose. However, the enzyme was not able to degrade xylohexaose. Xylanase pretreatment was effective for hardwood kraft pulp bleaching. Hardwood kraft pulp bleached in the XEOP sequence had its kappa number reduced from 13.2 to 8.9 and a viscosity of 20.45 cp. The efficiency of delignification was 33

"Região cis-atuante reguladora da transcrição do gene que codifica celobiohidrolase I em Trichoderma reesei: expressão basal e induzida / Cis regulatory region of gene transcription which codify cellobiohydrolase I in Trichoderma reesei: induced and basal expression\"

A enzima celobiohidrolase I (CBHI) do fungo multicelular Trichoderma reesei catalisa a liberação de celobiose a partir das extremidades redutoras das cadeias de celulose. Esta enzima é um dos membros do sistema de celulases extracelular necessário para a hidrólise completa da celulose até glicose. A expressão do gene cbhl é controlada pela fonte de carbono energético usado no meio de cultura. O crescimento em presença de celulose - não de glicose ou glicerol - resulta na indução do transcrito de cbhl em pelo menos 1200 vezes. Esta indução parece requerer a expressão basal do sistema das celulases, as quais são necessárias para catalisar a formação do indutor solúvel a partir da celulose. A expressão do transcrito de cbhl também é controlada pelo estado metabólico da mitocôndria; os transcritos das celulases são regulados sob condições tidas como repressoras da respiração mitocondrial...