SU761477A1 - Method of preparing water-soluble cellulase preparate - Google Patents

Description

The invention relates to the production of a water-soluble cellulase preparation. Stabilized and immobilized enzyme preparations are widely used in the food and chemical industries, in medicine, in the practice of scientific research.

There are several ways to obtain immobilized cellulase. Described, for example, is a method for producing immobilized cellulase using polyurethane foam as a matrix [].

The preparations of immobilized cellulase obtained by a known method are stable, but insoluble and do not have any prospects from the point of view of their use for the biochemical processing of solid substrates - „

cellulase. ²⁰

A method of obtaining a water-soluble cellulase preparation by binding the enzyme with a polymeric stabilizing agent at the pH-optimum of the enzyme, followed by centrifugation, dialysis, and lyophilization is known. synthetic polymers are used as a polymeric stabilizing agent, in particular, copoly

2

ethylene-msleic anhydride or copolymer of vinyl acetate - maleic anhydride.

Cellulase preparations are obtained by treating cellulase with a 0.4% suspension of a copolymer in phosphate buffer (pH 7.48) with a polymer: enzyme ratio of 1: (3-4) at 4 ° C. This is followed by centrifugation, dialysis, and lyophilization. 18% of the soluble cellulase preparation is obtained with an activity yield of 32.7%, an insoluble 36% [2].

Along with ensuring the high activity of the final product, a known method. Gives a low yield of a water-soluble cellulase preparation (18%). It is due to the use of polymers, short are characterized by poor solubility in water and buffer. The use of polymer suspensions increases the proportion of the insoluble final complex. In addition, a large concentration of the enzyme with respect to the polymer is used, which contributes to the cross-linking of the polymer molecules with the enzyme. This causes the formation of end-products with a high molecular weight and, consequently, a flat * • "* · '' p-'U- ·; '·

761477

hoi solubility. This method is accompanied by large losses of enzyme and polymer. -

The purpose of the invention is to increase the yield of the water-soluble drug and the saving of the enzyme. ...... _ ....

This goal is achieved by binding the enzyme to the copolymer of vinylpyrrolidone and acrolein diethyl acetal at pH optimum of the enzyme, 1? C "3.0? C X enzyme: copolymer ratio (0.3-Ϊ): 1, and using a copolymer, pre-treated with 0.1 N -NS1 at 79-81®С within an hour, the resulting preparation is further subjected to centrifugation, "dialysis and lyophilization."

The process is usually carried out at a pH of 5.0. Preliminary activation of the copolymer with dilute hydrochloric acid leads to the hydrolysis of acetal

'“Grouping and the formation of aldehyde

groups. The latter are active ......

in relation to the amino groups of the enzyme and react with the formation of relatively stable azomethines.

The preparation of cellulase preparation can be represented by the following schemes:

1) hydrolysis of a copolymer of vinylpyrrolido'-a-diethyl acetal acrolein

ten

15

20

25

-rnn_n

I ^r

n ₅ s ₂ o

thirty

35

her

--СН - Снимно - CH - CH— = n, with OO Λ 1 -CH ₂ and -1 sh and

40

2) "the addition of the enzyme

45

Ch-sn, -sn ι sno

Sn -SI * ι

N

H – C ^ c = 0 ² · ι

+ H, MI, Elmo— "

vine

50

- * t

n "with • CH,

* H _g N (and, Elmo

“V p sn one n eleven MN is intact *. H, / С®0 * "". ⁿ g ^c - ^cN communication "

55

η "about

65

and pH 5-7.5) because the optimum action of cellulase at pH 4.8-7.5.

The use of a high molecular weight polymer (50,000) provides a good stabilizing effect without requiring additional cross-linking of polymer molecules with the enzyme, which saves the enzyme. The yield of the water-soluble cellulase preparation is 93-95%, the cellulase activity is 58-65% of the activity of the original cellulase.

Prev 1. To 0.20 g of a copolymer of vinylpyrrolidone-diethyl acetal acrolein add 4 ml of 0.1 N.HC1. Dissolve by stirring at 80 ° C for 1 hour. The resulting solution of vinylpyrrolidone-acrolein copolymer is neutralized with 0.1 n NaOH pH 7.5. 0.20 g of cellulase is added (polymer: enzyme = 1: 1). Stand 24 hours at 20 ° C. Centrifuged, separated_ insoluble portion (0.02 g). The soluble portion is dialyzed against water and lyophilized. The yield of water-soluble cellulase preparation is 0.37 g (93.8%), insoluble — 0.02 g (5%).

Cellulase activity of the soluble, my preparation 2.76 g glitch / g of farms, h / t. 60.6% of the activity of the original cellulase. '

Determination of cellulase activity.

Cellulase activity is determined by the Mandelra-Weber method _and by the amount of reducing sugars formed during the hydrolysis of 50 mg (0.5 · 0.5 cm) of Mlahtap No. 1 paper at 50 ° C in 0.05 M acetate buffer ( pH 4.8). After incubation, add 3,5-dinitrosalicylic acid and inactivate the enzyme by boiling for 15 minutes. Cool, filter and colorimeter (7 filter). According to the calibration curve of glucose, the activity in g of glucose per 1 mi of the reaction mixture is determined and the glucose is calculated as g per enzyme. ....... .....

In ^Example 2 to 0.19g copolymer ^Vini lpirrolidona acrolein-diethylacetal were added 4 ml of 0.1 N HC1. The hydrolysis is carried out analogously to example 1.

The copolymer solution obtained after hydrolysis is neutralized with 0.1. n Iahon up to ry 5. Add 0.064 g of cellulase (polymer: enzyme - 1: 0.3) ..

The preparation and isolation of a complex is similar to example1.

The yield of a water-soluble drug is 0.24 g (95%), an insoluble drug is 0.01 g (3.8%). Cellulase activity of soluble drug 255'G ^! Gl _YuK / _g farms.h ' ¹ (65% of the activity * of the original cellulose).

Pr and measures 3. To 0.30 g of the acetyl form of the copolymer add b ml of 0.1 n. HC1. The hydrolysis is carried out analogously to example 1. Obtained after

Vinylpyrrolidone-acrolein copolymer

completely soluble in water. %

The reaction can be carried out in soft

conditions (at room temperature

761477

hydrolysis of the copolymer solution is neutralized with 0.1 n. IOH to pH 6. Add 0.21 g of cellulase (polymer: enzyme 1: 0.7).

Production and isolation of complex 5 as in example 1.

The yield of the water-soluble drug is 0.48 g (94.7%) with cellulase activity of 2.92 g glitch / g of farm .’C * ’, i.e. 64.3% of the activity of the original cellula-C) PS.

Claims (2)

Claim 1. A method of obtaining a water-soluble cellulase preparation by binding the enzyme with a polymeric stabilizing agent at the pH-optimum of the enzyme, followed by centrifugation, dialysis and lyophilization, characterized in that, in order to increase the yield of the target product and save the enzyme, as a polymeric stabilizing agent a copolymer of vinylpyrrolidone and acrolein diethyl acetal, 0.1 pretreated ^with n.NSE at 79-81 ^C for one hour, and binding of enzyme copolymer is carried out at 18-20 ° C minutes .sootnoshen and accordingly the components (0,3-1): 1. 2. Method pop.1, characterized in that the process of binding the enzyme with the copolymer is carried out at a pH of 5.0-8.0.