Fungus Trichoderma Reesei Research Articles

The effects of deletion of cellobiohydrolase genes on carbon source-dependent growth and enzymatic lignocellulose hydrolysis in Trichoderma reesei.

The saprophytic fungus Trichoderma reesei has long been used as a model to study microbial degradation of lignocellulosic biomass. The major cellulolytic enzymes of T. reesei are the cellobiohydrolases CBH1 and CBH2, which constitute more than 70% of total proteins secreted by the fungus. However, their physiological functions and effects on enzymatic hydrolysis of cellulose substrates are not sufficiently elucidated. Here, the cellobiohydrolase-encoding genes cbh1 and cbh2 were deleted, individually or combinatively, by using an auxotrophic marker-recycling technique in T. reesei. When cultured on media with different soluble carbon sources, all three deletion strains (Δcbh1, Δcbh2, and Δcbh1Δcbh2) exhibited no dramatic variation in morphological phenotypes, but their growth rates increased apparently when cultured on soluble cellulase-inducing carbon sources. In addition, Δcbh1 showed dramatically reduced growth and Δcbh1Δcbh2 could hardly grew on microcrystalline cellulose (MCC), whereas all strains grew equally on sodium carboxymethyl cellulose (CMC-Na), suggesting that the influence of the CBHs on growth was carbon source-dependent. Moreover, five representative cellulose substrates were used to analyse the influence of the absence of CBHs on saccharification efficiency. CBH1 deficiency significantly affected the enzymatic hydrolysis rates of various cellulose substrates, where acid pre-treated corn stover (PCS) was influenced the least. CBH2 deficiency reduced the hydrolysis of MCC, PCS, and acid pre-treated and delignified corncob but improved the hydrolysis ability of filter paper. These results demonstrate the specific contributions of CBHs to the hydrolysis of different types of biomass, which could facilitate the development of tailor-made strains with highly efficient hydrolysis enzymes for certain biomass types in the biofuel industry.

A simple approach to mediate genome editing in the filamentous fungus Trichoderma reesei by CRISPR/Cas9-coupled in vivo gRNA transcription.

To simplify CRISPR/Cas9 genome editing in the industrial filamentous fungus Trichoderma reesei based on in vivo guide RNA (gRNA) transcription. Two putative RNA polymerase III U6 snRNA genes were identified in the genome of T. reesei QM6a by BLASTN using Myceliophthora. thermophila U6 snRNA gene as the template. The regions approximately 500bp upstream of two U6 genes were efficient promoters for the in vivo expression of gRNA. The CRISPR system consisting of Cas9 and in vivo synthesized gRNA under control of the T. reesei U6 snRNA promoters was sufficient to cause a frameshift mutation in the ura5 gene via non-homologous end-joining-mediated events. We report a simple gene editing method using a CRISPR/Cas9-coupled in vivo gRNA transcription system in T. reesei.

Engineering of Trichoderma reesei for enhanced degradation of lignocellulosic biomass by truncation of the cellulase activator ACE3

BackgroundThe filamentous fungus Trichoderma reesei is a major workhorse employed to produce cellulase, which hydrolyzes lignocellulosic biomass for the production of cellulosic ethanol and bio-based products. However, the economic efficiency of biorefineries is still low.ResultsIn this study, the truncation of cellulase activator ACE3 was identified and characterized in T. reesei classical mutant NG14 and its direct descendants for the first time. We demonstrated that the truncated ACE3 is the crucial cause of cellulase hyper-production in T. reesei NG14 branch. Replacing the native ACE3 with truncated ACE3 in other T. reesei strains remarkably improves cellulase production. By truncating ACE3, we engineered a T. reesei mutant, PC-3-7-A723, capable of producing more cellulase than other strains. In a 30-L fermenter, fed-batch fermentation with PC-3-7-A723 drastically increased the maximum cellulase titer (FPase) to 102.63 IU/mL at 240 h, which constitutes a 20–30% improvement to that of the parental strain PC-3-7.ConclusionsThis work characterized the function of truncated ACE3 and demonstrated that analysis of classical mutants allows rational engineering of mutant strains with improved cellulase production necessary to process lignocellulosic biomass. Our rational engineering strategy might be useful for enhancing the production of other bio-based products.

Optimised red- and green-fluorescent proteins for live cell imaging in the industrial enzyme-producing fungus Trichoderma reesei

The filamentous fungus Trichoderma reesei is a major source of cellulolytic enzymes in biofuel production. Despite its economic relevance, our understanding of its secretory pathways is fragmentary. A major challenge is to visualise the dynamic behaviour of secretory vesicles in living cells. To this end, we establish a location juxtaposing the succinate dehydrogenase locus as a “soft-landing” site for controlled expression of 4 green-fluorescent and 5 red-fluorescent protein-encoding genes (GFPs, RFPs). Quantitative and comparative analysis of their fluorescent signals in living cells demonstrates that codon-optimised monomeric superfolder GFP (TrmsGFP) and codon-optimised mCherry (TrmCherry) combine highest signal intensity with significantly improved signal-to-noise ratios. Finally, we show that integration of plasmid near the sdi1 locus does not affect secretion of cellulase activity in RUT-C30. The molecular and live cell imaging tools generated in this study will help our understanding the secretory pathway in the industrial fungus T. reesei.

One New Terphenyl Glycoside From a Sponge-Derived Fungus Trichoderma reesei (HN-2016-018)

One new terphenyl glycoside (1), gliocladinin D, together with 4 known compounds (2-5) were isolated from the sponge-derived fungus Trichoderma reesei (HN-2016-018). The structure of the new compound was elucidated by the comprehensive spectroscopic analysis, including 1-dimensional and 2-dimensional nuclear magnetic resonance, and high-resolution electrospray ionization mass spectrometry. Compound 3 exhibited moderate Topo I inhibitory activity.

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

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.

Biotechnological Impacts of Different Fungal and Bacterial Strains Treatment on Nutritive Value of Some Straws

The current work was carried out to investigate the biotechnological impact of different strains of fungi (Pleurotus Ostreatus , Trichoderma viride and Trichoderma reesei) and bacterial treatment by LAB (Lactobacillus Plantarum with or without addition of molasses) on the nutritive value of agricultural residues for production of good quality straw silage. Four types of straws namely rice straw (RS), bean straw (BS), mixture of rice and bean straw (RBS) and wheat straw (WS) were subjected to biological treatment under solid state fermentation) with each Microorganism. The experimental groups include, control group of each substrate which did not inoculated with any microorganism (CON) , and five treatments groups , T1 (straws treated by Lactobacillus plantarum with molasses), T2 (straws treated with lactobacillus plantarum without molasses), T3 (straws treated by Trichoderma viride ),T4 (Straws treated by pleurotus ostreatus ) and T5 (Straw treated by Trichoderma reesei). Each treatment was carried out for four types of straws. The results of proximate analysis showed increased crude protein, ether extract and ash contents in all treatment compared to the un treated straws .The biotechnological treatment also led to reduction in dry matter, organic matter, crude fiber and fiber fractions contents in all treatments relative to the control group. So it is recommended to treat agricultural residues by cellulosic fungi and bacteria especially Lactobacillus plantarum for improving its nutritive value for animal feeding. Further investigations are required in vivo to study the effect of feeding of the produced biomass on the performance of ruminants.

Enhancement of Cellulase Production in Trichoderma reesei via Disruption of Multiple Protease Genes Identified by Comparative Secretomics.

The filamentous fungus Trichoderma reesei is one of the most studied cellulolytic organisms and the major producer of cellulases for industrial applications. However, undesired degradation of cellulases often happens in culture filtrates and commercial enzyme preparations. Even studies have been reported about describing proteolytic degradation of heterologous proteins in T. reesei, there are few systematic explorations concerning the extracellular proteases responsible for degradation of cellulases. In this study, the cellulase activity was observed to rapidly decrease at late cultivation stages using corn steep liquor (CSL) as the nitrogen source in T. reesei. It was discovered that this decrease may be caused by proteases. To identify the proteases, comparative secretomics was performed to analyze the concomitant proteases during the cellulase production. 12 candidate proteases from the secretome of T. reesei were identified and their encoding genes were individually deleted via homologous recombination. Furthermore, three target proteases (tre81070, tre120998, and tre123234) were simultaneously deleted by one-step genetic transformation. The triple deletion strain ΔP70 showed a 78% decrease in protease activity and a six-fold increase in cellulase activity at late fermentation stages. These results demonstrated the feasibility of improvement of cellulase production by genetically disrupting the potential protease genes to construct the T. reesei strains with low extracellular protease secretion. This dataset also provides an efficient approach for strain improvement by precise genetic engineering combined with “omics” strategy for high-production of industrial enzymes to reduce the cost of lignocellulose bioconversion.

CLP1, a Novel Plant Homeo Domain Protein, Participates in Regulating Cellulase Gene Expression in the Filamentous Fungus Trichoderma reesei.

The stringent regulatory network of cellulase gene expression in the filamentous fungus Trichoderma reesei involves multiple transcriptional regulators. However, identification and mechanistic investigation of these regulators are still insufficient. Here, we identified a novel transcriptional regulator, CLP1, a plant homeo domain (PHD) Protein that participates in regulating T. reesei cellulase gene expression. Phylogenetic analyses demonstrated that CLP1 homologs are widely distributed in filamentous fungi including Trichoderma, Penicillium, Fusarium, Neurospora, and Aspergillus species. We demonstrated that CLP1 is a nuclear protein and lack of CLP1 significantly impaired the induced expression of cellulase genes. ChIP experiments showed CLP1 binding to the cellulase gene promoters specifically under cellulose conditions and compromised XYR1 occupancy on the same promoters in the absence of CLP1 at the early induction stage. XYR1 overexpression fully rescued the defect in cellulase production but not the defect in conidia formation in the clp1 null mutant. Further analysis showed that the PHD is required for the CLP1 appropriate subcellular localization as well as the induced cellulase gene expression and conidiation. Taken together, these data demonstrated an important role of CLP1 in the regulation of cellulase and xylanase gene expression in T. reesei.

Microbial industrial enzymes: An annotated selection of World Wide Web sites relevant to the topics in microbial biotechnology.

https://link.springer.com/chapter/10.1007/978-3-319-52287-6_28 This review article in an e-book provides an excellent overview to the field of industrial enzymes. In addition to the current commonly used enzymes, it contains information of emerging enzymes of interest. http://www.novozymes.com/-/media/Project/Novozymes/.../Enzymes_at_work.pdf This web brochure produced by Novozymes provides many examples of industrial enzymes, their origins and applications. http://www1.lsbu.ac.uk/water/enztech/detergent.html This web article is a little dated but it nevertheless provides a good overview of how enzymes are used on washing formulations. https://www.aasv.org/shap/issues/v18n2/v18n2p90.html Phytase is an enzyme that releases phosphate from phytate, a hexaphosphate ester of inositol. Because much phosphate in grain and seeds is stored in phytate, phytase enzyme is added to animal feed to help release phosphate from the feed. https://www.ocl-journal.org/articles/ocl/pdf/2017/04/ocl170015.pdf Lipases, mostly obtained from fungi, is used extensively in industry. Major uses are in detergents and in food oil processing. https://www.heraproject.com/files/22-F-07_PROTEASE_HERA_Final%20Edition%20(unsecured%20-%20PDFA-1b).pdf Subtilisin is a protease, originally obtained from strains of Bacillus. It is used in laundry, dishwashing and contact lens cleaning applications. The current document contains much information relevant to the industrial use of subtilisin. https://www.chemistryworld.com/podcasts/chymosin/3007682.article Chymosin is an important enzyme used in the conversion of milk to cheese. It was originally obtained from the digestive tracts of young ruminant animals. More recently, it is produced in recombinant form by microbial fermentation processes. https://microbialcellfactories.biomedcentral.com/articles/10.1186/s12934-016-0507-6 Cellulases are important industrial enzymes in biomass conversion. A major organism long studied for it enzymes for degrading cellulose is the fungus Trichoderma reesei. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3769773/ This review article discusses the uses of amylases, mostly in converting various food products such as starches in corn, wheat, rice, and tapioca. A very large process currently is the conversion of corn starch to glucose by amylase coupled to the isomerization of glucose to fructose. https://www.researchgate.net/publication/315803185_Xylanases_and_their_industrial_applications_A_review This Research Gate website contains an abstract on xylanases and a link to the full review article on the topic. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3347563/ This review article covers enzymes used in the production of semi-synthetic beta-lactam antibiotics. The major focus is on protein expression and engineering. https://www.creative-enzymes.com/similar/glucose-isomerase_319.html Glucose isomerase, also known as xylose isomerase, is widely used in the production of high-fructose corn syrup. https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/pectinase Pectinases are widely used in producing clarified fruit juices. The pectinases hydrolyze linkages in the pectic substance found in the cell walls of plants. https://www.novozymes.com/en Novozymes is a major enzyme producing company, headquartered in Denmark. http://biosciences.dupont.com DuPont is a very large producer of enzymes with currently 900 commercial products on the market. https://www.dupont.com/products-and-services/industrial-biotechnology/industrial-enzymes-bioactives/primagreen.html Many enzymes are used in textile processing and clothing finishing. This website addresses are group of enzymes used in those applications. http://animalnutrition.dupont.com/productsservices/feed-enzymes/ Animal nutrition enzymes are largely derived from bacteria and aid in the animal digestion of their foods. The enzymes are principally phytase, proteases, and carbohydrate hydrolyzing enzymes. https://www.basf.com/global/en/products/cross-industry-solutions/enzymes.html The German chemical company BASF made a large entry into industrial enzymes with the the purchase of the Verenium Corporation. https://www.amano-enzyme.com/uk/about-company/ Amano produces microbial enzymes for use in food, supplement, and medical diagnostic industries.

EFFECT OF BACTERIA <em>BACILLUS PSEUDOFIRMUS</em> AND FUNGUS <em>TRICHODERMA REESEI</em> ON SELF-HEALING ABILITY OF CONCRETE

There are usually used inorganic substances for the strengthening of self-healing ability of concrete but some specific types of bacteria or fungi may also be considered for use. The bacteria’s ability to fill pores and micro-cracks was investigated on cylindrical concrete specimens with the diameter 55 mm and height 5 mm. Bacteria Bacillus pseudofirmus and fungus Trichoderma reesei was used for the experiment. The main objective of the experiment was to investigate, if it is possible to use Bacillus pseudofirmus and Trichoderma reesei for self-healing concrete. The results show, that it is more advantageous to use Bacillus pseudofirmus than Trichoderma reesei in alkaline environment. It is desirable to create the most ideal conditions for microorganism’s growth, as possible. Bacteria should have positive effect on self-healing ability of concrete. Any effect of fungus on self-healing ability of concrete wasn’t confirmed.

N,N-dimethylformamide induces cellulase production in the filamentous fungus Trichoderma reesei

BackgroundThe filamentous fungus Trichoderma reesei produces cellulase enzymes that are widely studied for lignocellulose bioconversion to biofuel. N,N-dimethylformamide (DMF) is a versatile organic solvent used in large quantities in industries.ResultsIn this study, we serendipitously found that biologically relevant concentrations of extracellular DMF-induced cellulase production in the T. reesei hyper-cellulolytic mutant Rut-C30 and wild-type strain QM6a. Next, by transcriptome analysis, we determined that plc-e encoding phospholipase C was activated by DMF and revealed that cytosolic Ca2+ plays a vital role in the response of T. reesei to DMF. Using EGTA (a putative extracellular Ca2+ chelator) and LaCl3 (a plasma membrane Ca2+ channel blocker), we demonstrated that DMF induced a cytosolic Ca2+ burst via extracellular Ca2+ and Ca2+ channels in T. reesei, and that the cytosolic Ca2+ burst induced by DMF-mediated overexpression of cellulase through calcium signaling. Deletion of crz1 confirmed that calcium signaling plays a dominant role in DMF-induced cellulase production. Additionally, 0.5–2% DMF increases the permeability of T. reesei mycelia for cellulase release. Simultaneous supplementation with 1% DMF and 10 mM Mn2+ to T. reesei Rut-C30 increased cellulase activity approximately fourfold compared to that without treatment and was also more than that observed in response to either treatment alone.ConclusionsOur results reveal that DMF-induced cellulase production via calcium signaling and permeabilization. Our results also provide insight into the role of calcium signaling in enzyme production for enhanced cellulase production and the development of novel inducers of cellulase.

Systematic deletions in the cellobiohydrolase (CBH) Cel7A from the fungus Trichoderma reesei reveal flexible loops critical for CBH activity

Glycoside hydrolase family 7 (GH7) cellulases are some of the most efficient degraders of cellulose, making them particularly relevant for industries seeking to produce renewable fuels from lignocellulosic biomass. The secretome of the cellulolytic model fungus Trichoderma reesei contains two GH7s, termed TrCel7A and TrCel7B. Despite having high structural and sequence similarities, the two enzymes are functionally quite different. TrCel7A is an exolytic, processive cellobiohydrolase (CBH), with high activity on crystalline cellulose, whereas TrCel7B is an endoglucanase (EG) with a preference for more amorphous cellulose. At the structural level, these functional differences are usually ascribed to the flexible loops that cover the substrate-binding areas. TrCel7A has an extensive tunnel created by eight peripheral loops, and the absence of four of these loops in TrCel7B makes its catalytic domain a more open cleft. To investigate the structure-function relationships of these loops, here we produced and kinetically characterized several variants in which four loops unique to TrCel7A were individually deleted to resemble the arrangement in the TrCel7B structure. Analysis of a range of kinetic parameters consistently indicated that the B2 loop, covering the substrate-binding subsites -3 and -4 in TrCel7A, was a key determinant for the difference in CBH- or EG-like behavior between TrCel7A and TrCel7B. Conversely, the B3 and B4 loops, located closer to the catalytic site in TrCel7A, were less important for these activities. We surmise that these results could be useful both in further mechanistic investigations and for guiding engineering efforts of this industrially important enzyme family.

Lignocellulolytic characterization and comparative secretome analysis of a Trichoderma erinaceum strain isolated from decaying sugarcane straw

The fungus Trichoderma reesei is employed in the production of most enzyme cocktails used by the lignocellulosic biofuels industry today. Despite significant improvements, the cost of the required enzyme preparations remains high, representing a major obstacle for the industrial production of these alternative fuels. In this study, a new Trichoderma erinaceum strain was isolated from decaying sugarcane straw. The enzyme cocktail secreted by the new isolate during growth in pretreated sugarcane straw-containing medium presented higher specific activities of β-glucosidase, endoxylanase, β-xylosidase and α-galactosidase than the cocktail of a wild T. reesei strain and yielded more glucose in the hydrolysis of pretreated sugarcane straw. A proteomic analysis of the two strains' secretomes identified a total of 86 proteins, of which 48 were exclusive to T. erinaceum, 35 were exclusive to T. reesei and only 3 were common to both strains. The secretome of T. erinaceum also displayed a higher number of carbohydrate-active enzymes than that of T. reesei (37 and 27 enzymes, respectively). Altogether, these results reveal the significant potential of the T. erinaceum species for the production of lignocellulases, both as a possible source of enzymes for the supplementation of industrial cocktails and as a candidate chassis for enzyme production.

Trichoderma reesei improved the nutrition status of wheat crop under salt stress

ABSTRACT The use of plant endophytes to release salt stress is cheap and quick. In this respect, we have isolated an endophytic fungus (Trichoderma reesei) from Solanum surattense that promoted the wheat growth under salt stress. The results showed that the fungal inoculated wheat plants had higher chlorophyll a and b, carotenoids, reduced glutathione (GSH), ascorbate, peroxidase, catalase, weight of plant, flavonoids, total soluble protein, stomatal conductance, transpiration rate, and relative water contents. Also, T. reesei treated plants showed higher IAA, GA, Ca and K, while lower ABA, H2O2, phenol, sugar, proline, electrical conductivity, malondialdehyde (MDA), Na+ and Cl−. The results concluded that the integrative use of T. reesei might help the wheat plants to stand salt stress.

An efficient shortened genetic transformation strategy for filamentous fungus Trichoderma reesei.

The filamentous fungus Trichoderma reesei is one of the most important fungi for the production of cellulases and xylanases, which can be used for biofuel production from lignocellulose. We aimed to develop an effective selection marker system for more extensive functional genomic studies in the fungus T. reesei, and to construct better industrial transformants for producing cellulases. Here, we present a novel effective G418 selection marker to use a codon-optimized neomycin phosphotransferase II gene nptII to transform T. reesei. We developed an effective and erasable selection marker, lcNG, and a combined genetic transformation system for gene manipulation in T. reesei using a two-Agrobacterium-mediated transformation method. This transformation strategy combines two steps in the transformation protocol, which saves 15-30-day's time. The system could be a useful tool for the genetic engineering of T. reesei.

The Change in the Composition of Trichoderma reesei Carbohydrases Complex as a Result of Gamma Mutagenesis.

The filamentous fungus Trichoderma reesei is traditionally used as the main industrial producer of cellulases and hemicellulases. Recently, the relevance of carbohydrases hydrolyzing nonstarch polysaccharides of cereals has significantly increased in feed production. In processing of grain raw materials, endodepolymerases, mainly xylanases and endoglucanases, play a key role. Earlier, we carried out gamma mutagenesis of an industrial strain T. reesei BCM18.2/KK to increase the proportion of endodepolymerases in its enzyme complex. Endoglucanase activity of the strain was increased 5-fold, while xylanase activity increased more than 8-fold. It was interesting to determine the carbohydrases composition in enzyme preparations obtained from the original and mutant T. reesei strains. So, the strains were cultured in laboratory fermenters; concentrated preparations were obtained using freeze dryer. It was established that gamma mutagenesis resulted in significant changes in the carbohydrases complex of the strain. Cellobiohydrolase I being the major carbohydrase in the original strain was absent in the enzyme complex of the mutant. The share of xylanases and endoglucanases in the preparation from the mutant strain increased by 6% and 6.5%, respectively, compared with the preparation from the original strain. The obtained data show the ability of gamma irradiation to affect the component composition of T. reesei carbohydrase complex.

Bioremediation of Diesel Oil Spill by Filamentous Fungus Trichoderma reesei H002 in Aquatic Environment

Bioremediation of aquatic environment could be a response to the oil spills threats. In this paper, Trichoderma reesei H002, a filamentous ascomycete fungus isolated from a polluted site in an orchard garden, Johor, Malaysia, was experimented for its biodegradation ability to degrade diesel oil. Varying nitrogen and carbon sources, pH, agitation on diesel oil by Trichoderma reesei H002 in liquid media were examined to find their impacts on TPHs, alkane, aromatic and NSO fractions of diesel oil degradation. Glucose and yeast extract were the most suitable nutrients for the development of T. reesei H002 and increased the degradation of total petroleum hydrocarbons (TPHs up to 94.78% at the end of the study (40 days) at 25 0C. The degradation of TPHs were performed by gravimetric analysis and degradation of alkane and aromatic fractions were confirmed by GC-FID analysis. Based on the findings of T. reesei H002 for the biodegradation of diesel oil, it can be proposed that T. reesei H002 can be applied to bioremediate diesel oil spill in aquatic environment, therefore protect the ecosystem.

The putative \u03b2-glucosidase BGL3I regulates cellulase induction in Trichoderma reesei

BackgroundThe filamentous fungus Trichoderma reesei (anamorph of Hypocrea jecorina) displays increased cellulase expression while growing on inducers such as lactose or cellulose. However, the mechanism of cellulase induction in T. reesei is not yet completely characterized. Here, a protein annotated as β-glucosidase (BGL3I) was found to be involved in cellulase induction in T. reesei. The effects of BGL3I on cellulase production have not yet been fully understood.ResultsDeletion of the bgl3i gene had no influence on the growth of T. reesei, but significantly increased its cellulase activities. Deletion of bgl3i also resulted in decreased extracellular galactosidase activity, but significantly increased transcription of lactose permeases, which might be involved in lactose transport. Furthermore, deletion of bgl3i enhanced the transcription levels of intracellular β-glucosidases cel1a, cel1b and the regulator xyr1, which are all essential for lactose induction in T. reesei. BGL3I was found to have a relatively high ability to hydrolyze sophorose, which is proposed to be the strongest natural inducer of cellulase synthesis in T. reesei.ConclusionsBGL3I may take part in the complex regulating system of cellulase induction. The deletion of bgl3i offers a new strategy to improve T. reesei strain performance.

A chitinase with antifungal activity from naked oat (Avena chinensis) seeds.

A chitinase was purified from naked oat (Avena chinensis) seeds using simple chromatographic techniques. Its molecular weight and isoelectric point were determined as 35kDa and 8.9, respectively. The purified chitinase exhibited specific activity of 3.6 U/mg and 15.6% yield using colloidal chitin as substrate. Partial amino acid sequence analysis and homology search indicated that it probably belonged to Class I plant chitinase, glycosyl hydrolase family 19. With chitin as substrate, the optimum pH and temperature of the chitinase were pH 7.0 and 40°C, respectively. The chitinase was remarkably stable from 30°C up to 50°C, but was inactivated at high temperatures above 85°C. Antifungal activity in vitro tests demonstrated this purified chitinase had potent, dose-dependent inhibitory activity against the fungi Panus conchatus and Trichoderma reesei. PRACTICAL APPLICATIONS: Chitinase has broad applications in many fields including the food industry and is recognized as one of the antifungal substances with potential use in plant disease resistance or biological control in agriculture. This study developed cost-effective purification methods for producing chitinase from naked oat (Avena chinensis) seeds, which may favor large-scale production of the enzyme. The remarkable stability of the chitinase at moderate temperatures (30°C-50°C), makes it a potentially useful enzyme in bioprocessing to produce chitooligosaccharides for various applications in the food, health, and agriculture sectors.