CS259204B1 - A method for preparing β-D-fructofuranosidase enzyme concentrates - Google Patents

Abstract

The solution relates to a method for preparing enzyme concentrates of β -D-fructofuranosidase from the yeast Saccharomyces cerevisiae, in particular from strains overproducing this enzyme. The procedure after releasing -D-fructofuranosidase from the cells into the solution includes the removal of ballast macromolecules by sedimentation after prior chemical or thermal treatment, selective concentration of the supernatant and dehydration under reduced pressure. The procedure can be used to obtain liquid or anhydrous preparations of β -D-fructofuranosidase with a high, specific activity, more than 7,500 U/ml, or 75,000 U/g dry matter, usable for food purposes.

Description

The invention relates to a process for the preparation of beta-D-fructofuranosidase enzyme concentrates.

Beta-D-fructofuranosidase (EC 3.2.1.26) is an enzyme used in various branches of the food industry for the hydrolysis of sucrose to glucose and fructose. It occurs in fungi, especially yeasts, capable of utilizing sucrose as the sole source of carbon and energy. In yeast cells, this enzyme occurs predominantly in the form of an external glycoprotein and, in small quantities, as cytoplasmic non-glycosylated beta-D-fructofuranosidase in isolated or immobilized form, as well as its activity in yeast, are important from a biotechnological point of view.

Its most important source is baker's yeast. The prior art processes for the preparation of beta-D-fructofuranosidase are considerably complicated and have only been of laboratory preparative importance. Utilization of alkyl glycol ethers and triethylene glycol for industrial enzyme production / Csl. pat. no. 101 971 (1960) has not yet taken place for economic and hygienic reasons.

Nowadays, in addition to the production of protein for food purposes from unicellular microorganisms, there is an endeavor to make comprehensive use of yeast biomass for the isolation of biologically active proteins as well as other valuable substances that we have not produced as microbial metabolism products in our country.

This is also the case with beta-D-fructofuranosidase, which is only imported to us for the food industry, especially confectionery.

An advantage of the process of preparing the beta-D-fructofuranosidase according to the invention is that the process takes into account the complex fractionation of yeast biomass and results in invertase with high specific activity, especially when applied to strains capable of significantly overproducing this enzyme. In addition, the ballast proteins, as an intermediate of the isolation process, can be used for food or feed purposes after treatment.

The principle of the invention is that beta-D-fructofuranosidase is first released from the yeast cells into solution, either by cell autolysis induced by electrolytes and organic solvents, or by enzymatic or enzymatic, respectively. mechanical disruption of cell walls.

More than 50% of the solubilized beta-D-fructofuranosidase remains in the cell-free extract after sedimentation of the particular cell structures. The specific activity of beta-D-fructofuranosidase in the extract is increased by hydrolysis and resp. sedimentation of ballast macromolecules after their acid denaturation and isoelectric precipitation. The resulting supernatant with beta-D-fructofuranosidase is freed of low molecular weight substances by ultrafiltration and the beta-D-fructofuranosidase content can be further concentrated by dehydration under reduced pressure or ethanol precipitation, respectively. aoetónom. In this way, beta-D-fructofuranosidase preparations having a specific activity of greater than 75,000 ϋ / g dry matter and 30-45% yield, respectively, can be obtained. 7 500 U / ml preparation.

The invention can be practiced according to one of the following examples without being limited thereto.

Example 1

R

The yeast cells of Saccharomyces serevisiae strain M2A-C 2 (3.5 g dry matter) were suspended in distilled water and disintegrated with an efficiency of more than 98% in a Braun homogenizer in the presence of glass beads (0.45 mm diameter) at 4 ° C for 2 min. The homogenates prepared from twice 3.5 g of cell dry matter were pooled, the beads were removed by filtration through a Sintr S1 frit, washed and the total volume of the homogenate was adjusted to 100 ml. Sedimentation of cell walls, nuclei and unbroken cells resulted in a cell-free beta-D-fructofuranosidase extract. Its pH was adjusted to pH 6 and the extract was incubated for 2 hours. at 50 ° C with occasional stirring.

After acidifying the basic extract with HCl to pH 3.6, the ballast macromolecules were separated by sedimentation. The resulting supernatant was subjected to ultrafiltration and diafiltration through a membrane, trapping 50,000 molecular weight particles and impervious to beta-D-fructofuranosidase. The resulting retentate having an activity of 8,360 U / ml was subjected to lyophilization to form an anhydrous beta-D-fructofuranosidase 0 preparation. U / g dry matter.

Example 2

ABOUT

100 g yeast from production strain s. cerevisiae M2A-C 2 was subjected to autolysis in the presence of NaCl (5 g / 100 ml) and ethanol (6 g / 100 ml) in a total volume of 120 ml at 40 ° C for 70 hours. The autolysed cell suspension was centrifuged, washed once with water and the supernatants were pooled. The resulting beta-D-fructofuranosidase extract was subjected to membrane ultrafiltration, trapping 50,000 molecular weight particles. The retentate was concentrated in vacuo to give a beta-D-fructofuranosidase preparation having a specific activity of 70,500 U / g.

Example 3

R

Yeast cells of Saccharomyces cerevisiae strain M2A-C 2 were subjected to enzymatic disintegration by glucanases prepared from the stomach of snails. Cells (10 g dry matter) were first exposed to a 2 minute 0.5 M mercaptoethanol at pH 9.3. They were then centrifuged, suspended in 100 ml of water containing 1 g of enzyme extract from the gastric snails and allowed to incubate at 30 ° C for 70 minutes. After sedimentation. enzymatically resistant cells, a cell-free beta-D-fructofuranosidase extract was formed, which was further subjected to thermal denaturation, isoelectric precipitation and ultrafiltration as described in Example 1.

The retentate after ultrafiltration had an activity of 7,750 U / ml. Upon dialysis of the retentate against water at 4 ° C and its lyophilization, an anhydrous preparation of beta-D-fructofuranosidase with a specific activity of 76,300 U / g dry matter was formed.

The invention finds wide application in the food industry in the industrial production of beta-B * fructofuranosidase and its application in the production of invert sugar, which is sweeter and more stable in solutions, respectively. more soluble than sucrose beet sugar.

Claims (1)

After acidifying the basic extract with HCl to pH 3.6, the ballast macromolecules are separated by sedimentation. The resulting supernatant was subjected to ultrafiltration and diafiltration through the membrane, capturing 50,000 molecular weight particles and impermeable to beta-D-fructofuranosidase. The resulting 8,360 U / ml retentate was subjected to lyophilization to form an anhydrous preparative of? g dry matter. Example 2 100 g of yeast from the production strain s. Cerevisiae M2A-C 2 was subjected to autolysis in the presence of NaCl / 5 g (100 ml) and ethanol (6 g / 100 ml) in a total volume of 120 ml at 40 ° C after The suspension of autolysed cells was centrifuged, washed once with water and the supernatants pooled. The resulting beta-D-fructofuranosidase extract was subjected to ultrafiltration through a membrane of 50,000 molecular weight. The retentate was vacuum concentrated to form a beta-D-fructofuranosidase preparation with a specific activity of 70,500 U / g. Example 3 R Yeast cells of Saccharomyces cerevisiae strain M2A-C 2 were subjected to enzymatic disintegration by glucanases prepared from the stomach of slugs. The cells (10 g of dry matter) were first exposed to a 0.5-minute effect of 0.5 M mercaptoethanol at pH 9.3. They were then centrifuged, suspended in 100 ml of water containing 1 g of slug stomach enzyme extract and incubated at 30 ° C for 70 minutes. After sedimentation. enzymatically resistant cells, a cell-free beta-D-fructofuranosidase extract was produced, which was further subjected to heat denaturation, isoelectric precipitation and ultrafiltration as described in Example 1. The retentate after ultrafiltration had an activity of 7,750 U / ml. After retentate dialysis with water at 4 ° C and lyophilization, an anhydrous preparation of beta-D-fructofuranosidase with a specific activity of 76,300 U / g dry weight was obtained. The invention finds wide application in the food industry for the industrial production of beta-B * fructofuranosidase and its application in the production of invert sugar, which is sweeter and more stable in solution, respectively. more soluble than beet sugar sucrose. BEFORE METHODS OF THE INVENTION A method of preparing enzyme concentrates of beta-D-fructofuranosidase from yeast cells of Saccharomyces cerevisiae, preferably strains conferring this enzyme, upon introduction of beta-D-fructofuranosidase into solution by autolysis, enzymatic or mechanical disintegration of cells, characterized in that the cytosol formed by after sedimentation of cellular structures, the ballast macromolecules are removed by further sedimentation after prior exposure to the cytosolution temperature of 20 ° C to 60 ° C, preferably 50 ° C, at pH 3 to 8, preferably pH 6, for 15 min 240 min preferably 120 min, without or after adjusting the pH to 3-4, the beta-D-fructofuranosidase is concentrated by ultrafiltration, on ultrafiltration trapping particles having a molecular weight of 50,000 to 500,000, and evaporation of water under reduced pressure at 4 ° C to 50 ° C.