RO126765A2 - Process for preparing a hydrolytic enzyme complex using a strain of trichoderma reesei - Google Patents

Abstract

The invention relates to a process for preparing a hydrolytic enzyme complex meant to be used in degradation of lignocellulosic materials, production of fuel from renewable sources etc., by submerged cultivation of 10% vegetative inoculum of Trichoderma reesei on a culture medium comprising 2% maize grit, 1% soya beans grit, 0.3% sodium nitrate, 0.2% acid potassium phosphate, 0.1% magnesium sulphate, 0.05% potassium chloride, 0.01% ferrous sulphate, at 30...31°C, while stirring at 220 rpm, after which the biomass is separated and the enzyme liquid is concentrated by ultrafiltration, on a module having an efficiency of the amylolytic activity of 80...82%, of the cellulosolytic activity of 88...90% and xylanasic activity of 86...88%, resulting in an enzyme concentrate which is precipitated with 70% ammonium sulphate to finally obtain a raw enzyme preparation.

Description

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The invention relates to a process for obtaining an enzymatic preparation with cellulosolytic activity from Tricoderma reesei cultures, capable of hydrolyzing cellulose, hemicellulose, starch, xylan and other polysaccharides from energy plants (different varieties of maize, wheat, cochineal straws, strains Miscanthus giganteus etc. used in anaerobic fermentation in biogas production.

At present, other processes for obtaining cellulosolytic preparations by culturing microorganisms are known, in a batch system on culture media with other carbon sources such as: glucose, sucrose, carboxymethylcellulose, etc. or nitrogen: corn extract, ammonium salts, etc., which induce the formation of a certain type of enzyme complex containing cellulases and hemicellulases.

It is known that the complete hydrolysis of cellulose residues from plants and wood is a difficult process, unresolved so far, especially due to the complex structure of cellulose, but also its association with other compounds (especially lignin). For their hydrolysis there are physical, chemical, enzymatic or combined processes. Currently, various enzymatic preparations are used which, depending on the component enzymes, achieve different degrees of hydrolysis of lignocellulosic materials.

The problem solved by the present invention is to establish the sequence of process steps, as well as the technical conditions for their realization so as to obtain a complex hydrolytic enzyme preparation, capable of degrading the existing components in energy plants, so that carbohydrates results from hydrolysis lead to increased yields in biogas, bioethanol.

The process, according to the invention, consists in that it comprises the following steps:

-submerged cultivation, in a discontinuous system of 10%, by volume, of the vegetative inoculum of Tricoderma reesei registered in the Collection of Microorganisms of the Center of Microbial Biotechnologies-Bucharest under no. 28 on a culture medium containing, 2.0% corn cob (corn cobs wholly chopped to a particle size of 0.5-1 mm), 1.0% soybean meal, 0.3% sodium nitrate , 0,2% potassium acid phosphate, 0,1% magnesium sulphate, 0,05% potassium chloride, 0,01% ferrous sulphate, percentage by volume, in temperature conditions: 30-3 l ° C, constant stirring at 220 rpm, variable air and pressure, so that the dissolved oxygen content in the medium does not fall below 50% for 96 hours after inoculation.

-processing of the culture medium which includes the separation of biomass and the concentration by ultrafiltration of the enzymatic liquid on a module with an amylolytic activity yield of 80-82%, cellulosolytic of 88-90% and xylanase of 86-88%, resulting in an enzymatic concentrate from which in the precipitation conditioning phase with ammonium sulphate, at 70% saturation, the crude enzyme preparation of Trichoderma reesei is obtained, at a yield of 33%, white-gray, non-toxic, with cellulase, amylase and xylanase activity as follows :

- specific cellulase activity of 2.1 U / mg protein

-specific xylanase activity of 17.35 U / mg protein

-specific amylase activity of 2.66 U / mg protein

The enzyme preparation obtained is active in the range of pH 5.5-6.5 and temperature: 35-50 ° C.

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By applying the invention, the advantage of synthesizing a complex enzymatic preparation is obtained, which contains active enzymatic fractions for the concomitant hydrolysis of cellulose, xylan and starch (up to maltose, glucose) from lignocellulosic materials (cocenes, whole corn, corn flour, straw, etc.). used in biogas production (before anaerobic fermentation).

The following is an embodiment of the invention.

Example

a) Submerged cultivation in a discontinuous system of the Tricoderma reesei strain, registered in the Collection of Microorganisms of the Center for Microbial Biotechnologies Bucharest, under no. 28.

In a bioreactor of 0.01 m, with 0.007 ... 0.008 m of culture medium containing, in percentage by volume, 2.0% corn cob (wholly chopped corn cobs up to a particle size of 0.5- 1 mm), 1.0% soybean meal, 0.3% sodium nitrate, 0.2% potassium acid phosphate, 0.1% magnesium sulphate, 0.05% potassium chloride, 0.01% sulphate of iron, with an initial pH of 5.5, a vegetative inoculum of Tricoderma reesei is sown, in a proportion of 10%, the inoculum was developed on PDB medium (Potato Dextrose Broth), for 48 hours, with stirring of 220 rpm at 28 ° C. Cultivation is carried out for 90-96 hours (stirring 220 rpm), variable aeration and pressure during the bioprocess, namely: 1.0-1.2 I air / min, so that its oxygen content do not fall below 50%.

The preinoculum consisted of a spore suspension of the fungal strain Tricoderma reesei used, well developed on PDA (Potato Dextrose Agar) medium, for 7 days at 28 ° C.

b) Processing the cultural environment

At the end of the bioprocess after 90-96 hours of cultivation of the producer, the processing of the culture medium is carried out in the following phases:

bl) Separation of biomass from Tricoderma reesei by filtration on Nutsche filter, with addition of 0.1% filtration aid (Perlite), average filtration rate 30 l / m ² / h b2) Ultrafiltration concentration of the culture liquid resulting from the phase biomass separation, using a Pellicon Standard module (Millipore Corp. equipped with a box of polysulfonic membranes type PTGC 00005 (exclusion limit NMWL10 kDa), achieving a concentration of up to 5 ... 10 times the filtrate with a yield of

82 ... 90%, resulting in liquid hydrolytic enzyme concentrate.

b3) Condidation of the liquid hydrolytic enzyme concentrate is performed by precipitation with ammonium sulfate, at 70% saturation; the precipitate is separated by centrifugation and dried under vacuum, obtaining the crude enzyme preparation, in the form of a white-gray powder, non-toxic, with specific cellulase activity (2, 1U / mg), specific amylase (2.66U / mg ) and specific xylanazine (17.35U / mg), active in the range of pH 5.5-6.5 and temperature: 35-50 ° C, non-toxic to tests performed on farm animals.

The product obtained by the process described in the invention allows to obtain polyenzymatic preparations containing mainly cellulases, amylases, xylanases, which can be used in the hydrolysis of energy plants (in order to obtain biofuels such as biogas or bioethanol) and in animal husbandry in the preparation of premix.

Claims (1)

Claim Process for the production of a hydrolytic enzyme preparation (cellulases, amylases, xylanases) from cultures of Tricoderma reesei, characterized in that it comprises the following steps: -submerged cultivation, in a discontinuous system of 10%, by volume, of the vegetative inoculum of Tricoderma reesei registered in the Collection of Microorganisms of the Center of Microbial Biotechnologies-Bucharest under no. 28 on a culture medium containing, 2.0% corn cob (corn cobs wholly chopped to a particle size of 0.5-1 mm), 1.0% soybean meal, 0.3% sodium nitrate , 0,2% potassium acid phosphate, 0,1% magnesium sulphate, 0,05% potassium chloride, 0,01% ferrous sulphate, percentage by volume, in temperature conditions: 30-31 ° C, stirring constant at 220 rpm, variable air and pressure, so that the dissolved oxygen content in the medium does not fall below 50% for 96 hours after inoculation. -processing of the culture medium which includes the separation of biomass and the concentration by ultrafiltration of the enzymatic liquid on a module with an amylolytic activity yield of 80-82%, cellulosolytic of 88-90% and xylanase of 86-88%, resulting in an enzymatic concentrate from which in the precipitation conditioning phase with ammonium sulphate, at 70% saturation, the crude enzyme preparation of Trichoderma reesei is obtained, at a yield of 33%, white-gray, non-toxic, with cellulase, amylase and xylanase activity as follows : - specific cellulase activity of 2.1 U / mg protein -specific xylanase activity of 17.35 U / mg protein -specific amylase activity of 2.66 U / mg protein The enzyme preparation obtained is active in the range of pH 5.5-6.5 and temperature: 35-50 ° C.