CS220296B1 - Process for producing oligogalacturonic acids by enzymatic hydrolysis - Google Patents

Abstract

The invention relates to a method for the production of oligogalacturonic acids by enzyme hydrolysis, in which sodium pectan is treated with endo-D-galaturonanase from pectinase at pH 4.5 to 4.8, temperature 30 to 45°C for 60 to 90 minutes.

Description

220296 3

The present invention relates to a process for the preparation of oligo-galacturonic acids by enzyme hydrolysis.

Until now, oligogalacturonic acids have not been produced industrially. They have been prepared in some laboratories via chemical hydrolysis: Hatanaka C., Ozawa J .: J. Agr.Chem. Soc. Japan 40, p. 98 (1960); Rexová - Benková L .: Chem. Announcements 24, p. 59 (1970) or by enzymatic degradation by the effect of purified enzymes: Van HoundemhovenF .; Vitt P .; Vister J .: Carbohydr. Res. 34, p.233 (1974); H. Stutz E .: Advan. Enzymol. 20, p. 41 (1968); Nagel G. W., Wilson T.E .: J. Chromatogr. 41, p. 410 (1969). All the chemical hydrolysis procedures described above are multistage reactions that represent a complex isolation technique. The previously described enzymatic hydrolyses are based on the use of specific purified enzymes which are very time-consuming, equipment-intensive and especially chemical-intensive. These drawbacks are eliminated by the method for the production of oligogalacturonic acids by enzymatic hydrolysis, characterized in that sodium napectan is treated with pectinase endo-D-galacturonate at pH 4.5 to 4.8 at 30 to 45 ° C for 60 hours. up to 90 minutes. The advantage of this process is that endo-D-lactamuronases do not have to be isolated in purity for enzyme hydrolysis, but directly commercial preparations (Igrazym M 10, Pectofoethidine, Bulgarian Pectin and Leozyme R.) can be used without prior modification. used in the canning industry. Knowing the optimal conditions for the effect of these enzymes (pH, temperature, enzyme and substrate concentration) and their mode of action allows for targeted hydrolysis. The use of a particular preparation (from the heat) allows to obtain a certain composition of oligo-glacturonic acids as exemplified by 1, 2 and 3. Another great advantage of this method is that hydrolysis takes place in the form of a one-step reaction and no further substrate or conenzyme is required for the reaction to proceed.

Oligogalacturonic acids are substances important for the theoretical study of the anthocyte enzymes and pectin compounds and for the characterization of the industry used by the proteolytic enzymes. EXAMPLE 1 1 g of sodium pectane (average molecular weight 27,000, uronide content 90%) is dissolved in 100 µl of buffer solution (pH 4.8) successively, 30 mg of enzyme preparation (Igrazym M10) is dissolved in 100 ml of acetate buffer (pH 4.8). After filtering the solution of the enzyme, the two solutions are combined. The enzyme hydrolysis takes place at 40 ° C with agitation or shaking for 60 minutes. The course of enzyme hydrolysis is controlled by chromatography (degradation product) and spectrophotometric (degradation degree of polymer substrate). After hydrolysis for 60 minutes, the reaction was completed by boiling for 20 minutes. The cooling solution is filtered and the supernatant is freeze-dried. The products of D-galactopyranuronic acid, digalacturonic acid and tri-galacturonic acid de-gradation are dissolved in 0.05 M phosphate buffer solution into two well-connected Sephadex G-25 Fi-columns (molecular sieve) to separate them. nia. The effluents of the individual oligogalacturonic acids are concentrated by freeze-drying and desalted on a Sephadex G-10 column. The oligogalacturonic acid yield is up to 90%. Example 2

The procedure was as in Example 1, with the difference that 20 mg of the enzyme prepolymer (Pectofoetidine) was dissolved in the acetate buffer pH = 4.6 and the enzyme hydrolysis was carried out at 45 ° C for 80 minutes. Enzyme hydrolysis products include: D-galactopyranuronic acid, digalacturonic acid, trigalacturonic acid, tetragalacturonic acid, and pentagalacturonic acid. The yield was up to 70%. Example 3

The procedure was as in Example 1, with the difference that 25 mg of the enzyme prepaint (Bulgarian pectinase) was dissolved in acetate buffer pH 4.5 and the enzyme hydrolysis was carried out for 30 minutes at 30 ° C. The enzyme hydrolysis products were D-galactopyranuronic acid, digalacturonic acid, triga-lacturonic acid, tetragalacturonic acid, pentagalacturonic acid, hexagalacturonic acid, and heptagalacturonic acid. The yield of oligogalacturonic acids was 60 ° C. Example 4

Quantitative determination of individual oligogalacturonic acids was performed by thin layer chromatography on Silufol plates in a 1-butanol-formic-water (2: 3: 1) system by addition of aniline phthalate. Oligogalacturonic acids were identified based on

The Rf values are linearly dependent on the degree of oligogalacturonic acid polymerization. D-galactopyranuronic acid and single oligogalacturonic acid standards prepared by chemical hydrolysis were used as reference samples. The invention is based on the use of oligo-galacturonic acids for testing various enzymatic preparations used

Claims (1)

220296 S in the conservation and food industry as well as in the theoretical study of the properties and mechanism of action of pectolytic enzymes. OBJECT OF THE INVENTION A method for producing oligogalacturonic acids by enzymatic hydrolysis, characterized in that sodium pectate is treated with endo-D-inventive palacturonanase from pectinase at pH 4.5 to 4.8, at a temperature of 30 to 45 ° C for 60 to 90 minutes.