SU925108A1 - Method for preparing immobilized cellulose - Google Patents

Abstract

A method for producing an immobilized cellulose by: covalently binding cellulase to j: an immaterial carrier, characterized in that, in order to increase the enzymatic activity of the resulting product, chitin activated with p-benzoquinone is used as a polymeric carrier, and a piece is used. : carrier

Description

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The invention relates to a process for the preparation of dmobilized enzymes, in particular cellulase. Immobilized enzymes are used as biochemical reagents, medicinal preparations, as well as in the chemical, biological biological and food industries as heterogeneous biocatalysts.

Immunization of cellulase provides a more efficient hydrolysis agent.

The stabilization of cellulase in soluble form l and the immobilization of cellulase on insoluble carriers x, z,

The practical use of such enzymes as cellulase and 6 -glucosidase, which are part of the cellulolytic complex, requires the production of cellulase in an insoluble form.

A known method for producing immobilized cellulase by binding it to polyurethane foam. However, the process significantly inactivates enzyme 2,

There is also known a method for producing immobilized cellulase from Azreggillus niger by binding the enzyme to polymer 1 with a polyacrylonitrile resin carrier. Poo activity

mi

NH

As a result of the high reactivity of quinones, the liN reaction covalently binds the enzyme to the carrier quickly and with small losses of the enzyme. The duration of the reaction is 1 hour. Up to 72% of the taken amount of the enzyme is bound. This saves the enzyme. All components of the cellulolytic complex are well immobilized on this carrier. -1, 4-Endoglucan Cases are immobilized with retention of 80.9 from the initial activity (according to Na-CMC, PI, 4-glucanellobiohygrylase are immobilized with preservation of 60% of the initial activity (in cellobiose); Immobilized P-glucidase show 37% of activity from the activity of the initial p-glucosidases (according to p-nitrophenyl A, D-glucopyranoside J. Example 1. To 2.5 g of the carrier (containing 280 µg-eq, quinone groups per 1 (g of carrier)) add 1.0 g , cellulase drug in

immobilized cellulase. makes up 20% of the initial activity of the enzyme h.

The disadvantage of this method is the low activity of immobilized cellulase.

The aim of the invention is to increase the enzymatic activity of immobilized cellulase.

The goal is achieved by a method of producing immobilized cellulase, consisting in covalent bonding of cellulase to a polymer carrier, characterized by using chitin activated by p-benzoquinone as the polymer carrier and running the process at a weight ratio of cellulase: carrier (0.06%). , 0.4: 1).

Chitin, activated by p-benzoquinone, contains 0.2–0.28 µg eq of quinine groups per gram of carrier.

Electron acceptor carbonyl groups create a lack of electrons on the carbon ring, as a result of which quinones are electroacceptor compounds and easily react with nucleophilic reagents. The reaction scheme is as follows:

4- HZN-cellulase cellulose 10 ml 0.1 n, shdettnogo buffer (pH 4.8). A cellulose preparation from the fungus Geotrichum candidum is used. With a protein content of 50% and endoglucanase (ya-K1 1C activity 11.37 µmol glucose / mg protein, cellobiasis (cellobiose) activity 1.13 µmol glucose / mg protein and | 5-glucosidase (p-nitrophenyl-p-B-glucopyranoside) with an activity of 1.16 mg glucose / mg protein, kept at room temperature for 1 hour and filtered, washed with 3 x 15 ml of 1 M NaCl solution and 2 x 10 ml of acetate buffer. 72 % of taken amount of protein. The resulting preparation of immobilized cellulase contains 43.6 mg of be 1 g of carrier. Activity of immobilized cellulosis of lulase 1: endoglucanase 9.2 µmol glucose / mg protein (80.9%), cellobias H .afi 0.8 μmol glucose / mg belkd (69.8%, glucosidase 0/43 µmol glucose / mg protein (36.9%), Example 2. K1 g of the carrier sprinkle a solution of 0.2 g of the cellulase preparation from Geotrichum candidum in 3 ml of 0.05 n acetate buffer (pH 4.8). Hold at room temperature 1 h and filtered, Washed with 3 X 10 ml of 1 M NaCl solution and 2 X 5 ml of acetate buffer. A preparation of immobilized cellulase with the activity is obtained: endoglucanum 9.13 μmol glucose / mg protein (80.2%), cellobiasis 0.68 μmol glucose / mg-protein (60.0%, p-glucose dosage 0.42 μmol glucose / mg protein (37.1%). Example 3. A carrier solution of 0.3 g of cellulose preparation in 4 ml of 0.05 N, acetate buffer (pH 4.8) is added to the carrier. A cellulose preparation from the fungus Trichode is used. rm lignoEum with a protein content of 40% and endogluconase activity of 32 μmol glucose / mg protein, cellobiasis activity of 3.4 μmol glucose / mg protein, B - glucosidase activity of 1.9 μmol. glucose / mg of protein. Contained at 4 ° C. for 20 hours.Filtered and washed with 3 × 15 ml of M11-C1 and 1x10 ml of acetate buffer. A preparation of immobilized cellulase with a protein content of 43.2 mg per 1 g of carrier is obtained. 80.8%); cellobiasis at 2.0 glucose / mg protein (60.1%), p-glucosidase 0.7 μmol glucose / mg protein (37.0%). Determination of cellulase activities 1. Glucanase (by Ka-KMU . The activity is determined by the nd amount of reducing sugars formed by hydrolysis of 1 ml of 1% solution of carboxymethylcellulose sodium salt (Ka-CMC) for 1 hour at 50 ° C in 0.05 M acetate buffer (pH 4; 8) . The determination of reducing sugars is carried out with 3,5-dinitrosalicylic acid. The glucose calibration curve determines the activity in g glucose per 1 ml of the reaction mixture and is converted to µmol glucose per 1 mg of bound protein. 2. Cellobiasis fno cellobiose). Activity is determined by the amount of sucrose-reducing, formed by hydrolysis of 1 ml of a 0.2% cellobiose solution for 0.5 h with 0.25 M acetate buffer (pH 4.7). The determination of reducing sugars is carried out by the glucose oxidase method. 3.Aryl- (-glucosidase) (according to p-nitprophenyl-p-T-glucopyranoside. Activity is determined by the amount of p-nitrophenol formed during hydrolysis with 1 ml of 0.001 M p-nitrophenyl-P) - D -glucopyranoside over 10 min at 40s in 0.1 M acetate buffer (pH 4.7). After colorimetry (3 filter), the content of p-nitrophenol is determined from a calibration curve, converted to µmol per 1 mg of protein. The advantages of the proposed method are the saving of the enzyme preparation and the increase in the specific activity of the immobilized enzyme. High rates of all activities of immobilized cellulase are a prerequisite for the practical use of the immobilized preparation. Immobilized cellobiasis can be applied to the final hydrolysis of oligaxaxide Bf obtained in the process of enzymatic cleavage of the whole; 1ylose enzymes containing an insufficient amount of cellobiasis to glucose. Immobilized (Z-gluconase can be used for hydrolysis of -glucans. Immobilized D-glucosidase can be used to produce aglycones from 5-glucosides, which are used in the pharmaceutical industry.

Claims (1)

METHOD FOR PRODUCING IMMOBILIZED CELLULASE by covalently binding cellulase to a polymer carrier, characterized in that, in order to increase the enzymatic activity of the resulting product, chitin activated by p-benzoquinone is used as a polymer carrier, and the process is carried out at a weight ratio of cellulase: carrier (O, 06 -0.4): 1.