Reesei Rut-C30 Research Articles

Improved production of Trichoderma harzianum endochitinase by expression in Trichoderma reesei.

The chromosomal endochitinase gene (ThEn-42) of the mycoparasite fungus Trichoderma harzianum P1 was isolated and overexpressed in the filamentous fungus Trichoderma reesei under the promoter of the major cellulase gene cbhl1. The host strain RutC-30 did not produce any endogenous endochitinase activity. The prepro region of the T harzianum endochitinase was correctly processed in T. reesei. No differences in expression were observed when the prepro region was replaced with the CBHI signal sequence. Shake flask cultivation yielded 130 mg of active enzyme per liter, which in terms of activity represents about a 20-fold increase over the endochitinase activity produced by T. harzianum. The presence of multiple copies of the expression cassette in the transformant resulted in limitation in transcription and/or regulation factors needed for full activity of the cbh1 promoter, although this was not the major limiting factor for higher expression of endochitinase. The endochitinase was very sensitive to an acidic protease at the late stages of T. reesei cultivation. T. reesei RutC-30 appeared to be tolerant of the endochitinase and can be used as a production host for this enzyme, which has antifungal activity toward plant pathogens.

Steady and dynamic shear characterization of cellulase-producingTrichoderma reesei suspensions

Suspension rheology of fungal fermentations is important in determination of mass transfer rates, as well as mixing quality. We have characterizedTrichorderma reesei RUT-C30 suspension rheology during growth on xylose (soluble) and cellulose (particulate) substrates, using both steady and dynamic shear measurements. Biomass growth was monophasic on xylose and biphasic on cellulose; the latter behavior is consistent with relatively rapid, early growth on soluble sugars derived from rapidly hydrolyzed material, followed by a second, slower growth phase owing to hydrolysis of more recalcitrant cellulose by increasing cellulase concentrations.

Modelling and simulation of the growth and enzyme production of Trichoderma reesei Rut C30

The growth of the fungus Trichoderma reesei Rut C30 on cellulosic materials, especially potato pulp, and the enzyme synthesis in batch and fed-batch cultures were investigated by Klingspohn (1990). Based on these experimental data, a comprehensive model was set up describing the uptake of substrate for growth, maintenance and enzyme synthesis including the enzymatic hydrolysis of the substrate (Bader, 1991). The model equations consist of non-linear systems of ordinary differential and algebraic equations. The model parameters were identified by means of the experimental results. The simulation results correspond well with the experimental data. Model computations for different operational conditions show the range of validity and the performance of the model. Possibilities of improvements of the yield and productivity can be deducted.

Production of enzymes from potato pulp using batch operation of a bioreactor

AbstractTrichoderma reesei strains MCG77 and Rut C30 were cultivated in batch operation in a stirred tank reactor on potato pulp (PP) consisting of starch, pectin, cellulose, and hemicellulose; potato pulp residue (PR) without starch and pectin; or enzymatic residue (ER), respectively, as substrate and potato protein liquor (PPL) as supplement. The enzyme mixtures produced by the fungi were characterized by four assays: with avicelase, CMCase, xylanase, and FP‐activities. Trichoderma reesei Rut C30 yielded higher activities than Trichoderma reesei MCG77. The enzyme activities and productivities obtained with PR are comparable to those obtained by other groups using pure cellulose and a synthetic nutrient solution in batch culture. An optimal operation strategy was derived to obtain maximum enzyme activity and productivity. After converting PR with the enzymes produced and using the residue, ER, as substrate for enzyme production, 85% (w/w) of the pulp was utilized.

Liquid fluidized bed starter culture ofTrichoderma reesei for cellulase production

A starter culture ofTrichoderma reesei (Rut-C30) prepared in a liquid fluidized bed reactor (LFBR) gave better growth and greater cellulase production in submerged fermentation than a conventional shake flask inoculum. The LFBR starter was prepared by first coatingT. reesei spores to 0.25 mm size corncob (1.0x108g-1) in a medium containing 1.0% corncob, 0.5 gL-1 xylose and 0.1 gL-1 lactose in a balanced salt solution, then fluidizing the particles in the LFBR for 36 h to allow germination of the spores, and covering the particles with an approx 30 μm thick biofilm. This biofilm that developed in constant adherence to the lignocellulosic carrier, apparently became well adapted to grow rapidly on insoluble cellulose substrates (Solca Floc), and had the enzymes of the cellulase complex induced for increased cellulase production.

Formation d'enzymes requises pour l'hydrolyse des polysaccharides de la paroi de plantes chez Trichoderma reesei

Enzyme preparations fromTrichoderma reesei RUT-C30, in addition to cellulase, contained various glucanase and glucosidase, acetylxylan esterase, glucuronidase and xylanase activities. These preparations were able to hydrolyse endosperm cell walls of corn and wheat and commercially-available xylans and plant gums having stright chains, but lacked the ability to hydrolyse branched or substituted hemicelluloses.

A method for online-estimation of cellulose concentration in a fermentation process

A method based on the elemental balance of the process has been developed to calculate the consumption of cellulose in cellulase production byTrichoderma reesei Rut-C30. Using a synthetic medium only the oxygen in effluent gas and consumption of base for pH-correction has to be measured.

Immunoelectron microscopic localization of endoglucanase in the frozen thin sections of a hypercellulolytic mutant of Trichoderma reesei

Endoglucanase (EG) is an enzyme of cellulase complex depolymerizing crystalline cellulose to cellulose microfibril fragments. T. reesei Rut-C30 is a catabolite repression resistant mutant which enhances endoplasmic reticulum (ER) and produces 15-20 times more endoglycanase than the wild type QM6a. About 90% of this enzyme is secreted.Purified secreted EG from three day old culture was used for polyclonal antibody production. The IgG was labeled with 15-18nm colloidal gold. EG secreting mycellia, grown in Vogel's medium with 1% Avicel PH101, were collected by centrifugation. This pellate was washed in sodium cacodylate buffer, fixed in tannic acid-glutaraldehyde mixture and cryoprotected with 30% glycerol. This material was placed on the specimen holders and frozen in liquid freon followed by liquid nitrogen. Frozen thin sections were prepared in a LKB Cryonova microtome using glass knives and collected on sucrose drops.

Cellulase and beta-glucosidase production by mexied culture of trichoderma reesei rut C30 and aspergillus phoenicis

When lignocellulosic materials are hydrolysed using cellulase fromTrichodermareesei, cellobiose accumulates due to a deficiency in β-glucosidase activity of the enzyme complex. Cellobiose decreases the rate and extent of hydrolysis through feedback inhibition of exo β-1,4-glucanase. Cellulase produced through mixed culture ofTrichoderma andAspergillus showed increased β-glucosidase activity and greatly improved hydrolytic potential.