SU1011056A3 - Process for preparing isomaltolose - Google Patents

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/ fanfye ayMr ftf ftuv / foi /% 2. The method according to claim.  1, with the use of Erwlnia rhapontic strains NCPPB 1578, NCPPB 139, NCPPB 1739 or ATCC 2928i as an isomaltu-forming microorganism.  3 The method according to claim.  1, that is, with the fact that the isomaltulose forming the microorganism is fixed in the form of whole cells.  The invention relates to the microbiological synthesis of substances and can be used for the enzymatic production of isomaltulose from sucrose, which can be used in the food industry.  A known method for producing isomal tulose from sucrose by the microbiological method during the fermentation of a solution containing from 15 to Q% by weight. / /weight.  sucrose.  The resulting isomaltulose is isolated by filtration and fl J crystallizes. Closest to the inventive and achievable design, a method for producing isomaltulose is obtained by enzymatically transforming sucrose solutions using isomaltulo-forming microorganisms, for example Protominobacter rubrum or Serratia plymuthica, using microorganisms that produce isomaltulose-forming microorganisms, for example, Protominobacter rubrum or Serratia plymuthica. .  For fermentation, a solution with a dry matter content of from 5 to 30%, preferably from 20 to 27% is used, with sucrose comprising from 90 to 98 solids.  In addition to Protominobacter rubrum and Serratfa plymuthlsa, Serratfa marscescens firwmia carotovora or Leuconostoc mesenterofdes can be used as an isomaltulose-forming microorganism 2.  The disadvantages of these methods are the need to use sucrose solutions with a concentration below tO i.  preferably about 25), the productivity of isomaltulose-forming microorganisms is not high enough, as well as the flow of 10-10-6 k.  The method according to claim.  1, that is, so that a soluble extract of the whole is used for fixation.  or crushed cell isomaltulose forming microorganism, 5.  The method according to claim.  1, which is based on the fact that an alginate gel is used as a gel.  side processes during aerobic fermentation of microorganisms that lead to the formation of substances that contaminate isomaltulose.  The purpose of the invention is to increase the stability and productivity of isomaltulose-forming microorganisms.  The goal is achieved according to the method of producing isomaltulose by enzymatically converting sucrose solutions using isomaltulose-cutting micro. organisms and the subsequent isolation and crystallization of isomaltulose, isomaltulose-forming microorganisms are fixed by incorporation into a gel and used to obtain isomaltulose from solutions containing 40–55% w / v.  sucrose.  In addition, Erwfnia rhaponticf of strain NCPpB 1578, NCPPB 139, NCPPB 1739 or ATCC 29284 is used as an isomal tuber-forming microorganism. The isomaltose-forming microorganism is fixed as whole cells.  For fixation, a soluble extract of whole or crushed cells of an isomaltulose-forming microorganism is used.  As the gel used alginate gel.  In the proposed method, immobilization is used to fix the enzyme or enzymes forming the enzyme system involved in the conversion of sucrose to isomaltulose.  You can use the enzyme system extracted. from an isomalulose-forming microorganism, for example or crushed microorganism cells, while the cells serve as a carrier for the enzyme system.  Some division of whole cells may occur after immobilization, but it is desirable that the immobilization method and other process parameters be chosen in such a way that the division is absent or is minimal.  Various methods can be used to immobilize the enzyme system, but gel retention is most convenient, since this method is suitable for immobilizing an enzymatically active cell extract, as well as whole or crushed cells.  Suitable gel materials include polyacrylate amide alginate, agar, a mixture of xanthan gum (lacquer gum resin, carrageenan kappa particles or a mixture of carrageenan capparticles), lacquer gum resin, collagen or cellulose acetate.  Of these materials, alginate gel, in particular calcium alginate gel, gives the best results.  Other alginate gels may be used, for example, those formed with other metals of the C group.  Calcium alginate gel is characterized by a high rate of penetrating diffusion of sucrose and a low rate of leakage of whole cells and protein to the outside.  .  An additional advantage is the exceptional stability of the isomaltulose-forming activity in fixed whole cells.  The half-time of the activity of cells retained in the calcium alginate gel is 10,000 hours, with 8500 hours being the typical value, while the longest half-period under comparable conditions for other methods of immobilization is less than 1000 hours.  In addition, immobilization in the algante gel does not result in a significant loss of activity.  To immobilize the enzyme system by itself, either in whole or in crushed cells, the enzyme system is mixed with an aqueous solution of soluble alginate, for example sodium alginate, in an algante gel.  The optimal procedure is to suspend whole cells with soluble alginate.  The cell concentration is 1-90% wet weight by volume, more optimally 10-40%, better still 20%.  The most acceptable concentrations of soluble alginate for use with whole cells or with other types of enzyme systems are 1-10% (wt. /about. ), more n | preferably 5% (.  weight. /about.  The resulting alginate mixture is dosed into a solution of a metal salt, with which soluble alginate forms a gel. If the gel is calcium alginate, suitable salts include calcium chloride with a concentration of 0.011, 0 M, preferably 0.05-0.5 M, or 0, 1M.  The temperature of the metal salt solution at dosing, preferably about, - the solution is stirred.  The stability of the fixed enzyme system is increased if the salt solution of the metal also contains some amount of dissolved sucrose, for example (weight. /about.  sucrose, preferably 20%.  When dosing a suspension or other alginate mixture in the form of individual drops, it is possible to obtain spherical granules of the gel that holds the enzymatic system.  The size of the granules may vary, but for ease of handling and to ensure high mass transfer efficiency when used, it is desirable to use granules with a diameter of 3-5 mm.  However, size and shape are of little value.  It is possible to fix the enzymatic system in a gel block (which is wound onto the mold for use or cut into pieces) or in microfiber particles (using high gradient conditions when dosing the alginate mixture).  Use other gels.  Kappa-carrageenan or a mixture of kappacaragenan xiolues are used in the same way: as used for alginate, with the exception that the solution in which the cell suspension was extruded is a mixture of IM CaCI and 1 M KCl at.  Agar is obtained by boiling 5% (wt. /about. ) agar in deionized water, cool the resulting gel until a cell suspension is added and the resulting suspension is extruded dropwise into ice water.  , 5 A mixture of xanthan / pitch of a false anguish half over with the noMoutbro method, as for agar, but the resin is heated to, and the gel is cooled to 60 ° C before adding the cells.  Polyacrylamide is used according to the method of Ch iba you (Chibate et a1.  (Method in Enzymology, 1976, p.  739).  Other methods of immobilization can be used to immobilize whole or fragmented cells of an isomaltulose-producing microorganism.  For example, cells can be physically adsorbed on an inert carrier;  bind them covalently to an inert carrier; to aggregate using a cross-linking reagent, with these methods, however, the preservation of activity and activity stability is lower.  However, these methods of immobilization have other advantages.  Thus, when cells are adsorbed on cellulose, the liquid obtained after transformation is crystal clear, which indicates that all impurities are adsorbed by DEAyocellulose.  Immobilization of crushed cells or dissolved extracts of the enzyme system allows you to completely avoid cell division occurring during their use, thereby eliminating the cracking of the granules and blocking the pores that sometimes occur when the division of fixed whole cells inside the granules of calcium alginate gel.  Moreover, when fixing crushed cells or dissolved extracts, the initial activity is higher, which indicates the absence of intact cell wall membranes that can act as a barrier to the diffusion of sucrose or isomaltulose.  Crushing can be done using a ball mill or other processing of whole cells.  The source of the enzyme system used for immobilization is isomaltulose producing microorganism of the genus Erwinla.  Bacteria of other genera, Servatia plymuthlca or Protaminobacter rubrum, may be used, but the first of these may be pathogenic to humans, and the latter tends to produce unwanted red pigment with a low molecular weight.  Bacteria of natural origin can be used.  From strains belonging to the genus Erwinla, E. strains are desirable. phapontici stored in the British National Collection of Plant Pathogenic Bacteria by registration numbers L / SRV 1578, N CPPB 139 and N CPPB 1739.  The SRPM 1578 code is stored in the American Type Culture Collection under the ATCC number 29283. ,  Strains N СРРВ 1578, 139 and 1739 ARE the most promising because they have a higher production specificity, greater initial activity and stability compared to other strains of Erwinla or with other strains of other genera.  The method is carried out as a continuous process by loading a fixed enzyme system into a column and passing sucrose substrate as a solution through a column or several columns installed in parallel, for example, on a carousel.  Column size greater than 20 ml.  When potassium sucrose is converted to isomaltulose, some amount of carbon dioxide is formed.  When using fixed whole cells, it is easier to remove this carbon dioxide if the liquid is passed up through the column.  Passing up the fluid promotes the fluidization of the bed of the fixed enzyme system and avoids the compaction that is possible by passing the fluid down.  As a substrate, a sucrose solution containing at least 301, preferably at least 40 | and, best of all, at least is used. about.  sucrose.  Stability (measured in (by the half life period of the enzymatic system for the synthesis of isomaltulose in fixed cells increases with increasing sucrose concentration, similarly the concentration of isomaltulose in the converted fluid increases.  FIG.  1 shows a plot of the stability of the fixed cells versus the concentration of sucrose solution; in fig.  2 — The effect of sucrose concentration on the productivity of fixed cells.  Data obtained using calcium alginate granules with a fixed strain E. rhapontici NCPPB 1739.  The flpH increase in sucrose concentration above 30-LO stability and productivity increase significantly, which is mainly due to the property of the fixed isomaltulose-forming enzyme system, and not the features of the fixation method. The immobilization method with the use of calcium alginate in this case gives better stability.  In addition, a high concentration of sucrose contributes to the inhibition of the division of fixed cells and prevents microbial contamination, facilitates the release of the product and reduces the volume of the treated fluid.  The upper viscosity limit of the solution is from 100 to 1000 cP, usually around 500 cP.  For the method using cells fixed in granules of a calcium alginate gel, the sucrose concentration in the starting material is about 55 (wt. /about. ).  The substrate does not necessarily have to be a solution of pure sucrose, instead of the latter can be included in the solution up to 15 (vol. /about. a) molasses or use the material obtained on.  sugar refinery, or a related syrup, and these two types of impurities are obtained with the usual refining of sugar. The conversion of sucrose to isomaltulose is carried out at 15 AOC, more, preferably at 20-35 ° C, a temperature of about 30 ° C is particularly acceptable; The pH of the sucrose solution is most preferably about 7.  Conversion to isomaltulose usually results in acidification of the medium, but in practice it is usually not necessary to include steps to maintain the pH in the range of 5 to 9.  In any case, the formation of acid usually decreases with increasing substrate concentration.  .  The method is carried out in such a way that sucrose is converted to isomaltulose and the accompanying products in a controlled manner.  With an increase in the residence time of sucrose in contact with a fixed enzymatic system, the degree of conversion increases to a practical maximum of 85 to 100% sucrose is converted into products.  However, maximum productivity is not necessarily achieved at the highest degree of conversion, because high throughput capacity and higher activity, obtained due to shorter residence time, can compensate for losses when operating with non-maximum conversion.  Thus, the maximum pro-.  The performance is achieved during operation.  not with the maximum transformation, but usually when turning into products from 70 to 95.  For calcium alginate granules with fixed Erwinia cells, a maximum productivity is usually observed when working in a conversion from 80 to 90.  FIG.  3 shows a plot of the productivity of the fixed cells versus the initial percent conversion (t. e.  the conversion rate is extracted back to zero time).  The data were obtained using calcium alginate granules with fixed E. cells. rhaponitici N CPPB 1739.  There is a noticeable maximum productivity near the value from 85 to 90% of the degree of sucrose to isomaltulose and related products.  The degree of conversion of sucrose to i products significantly affects the stability of the isomaltulose-forming enzyme system of fixed cells.  Stability may increase exponentially with increasing conversion of the concentrated sucrose solution.  FIG. A depicts a graph. the dependence of the half-life of the fixed cells on the initial percent conversion (at 55% conversion of the sucrose solution).  The data were obtained using calcium alginate granules with fixed E. cells. rhapontici N CPPB 1739.  Stability increases exponentially to a value of about 8500 hours at a 90% conversion.  After isomaltulose is obtained, it can be crystallized or otherwise purified.  For food and related products, crystallized isomaltulose can be used, which need not necessarily be clean; in some cases, an uncrystallized product can be used that contains impurities.  Example 1  Cells from Erwinia rhapontici culture N CPPB 1578, ATCC 29283) were diluted with 10 ml of phosphate buffer.  0.10 ml of the obtained suspension is used for seeding on 200 nutrient medium in sterilely conical flasks with 500 ml partition walls.  The composition of the medium is given in table.  one.  Table 1 Quantity Component (g / l distilled water) Sucrose Peptone (digested casein) Meat flask extract with crops three days with a speed of 120 oscillations per minute at 30 ° C for 70 hours, then biomass is collected.  Cells are harvested in the stationary growth phase, but can be harvested in the logarithmic growth phase or in the death phase.  The cells are centrifuged at 15,000 for 10 minutes at 30 ° C, about 1 g of packed wet cells per 100 ml of medium are taken into account (if large volumes of the medium to be injected are treated, a centrifuge rotor is used through which the medium is pumped at a rate of 100 ml / min) .  Collected tamped cells are suspended in a solution of sodium alginate 5% (dry weight. / v, J in deionized water in order to obtain 20% (wet.  weight. /about.  cell suspension (cell weight in grams is called wet weight (wet.  approximately converted to dry weight by dividing by 5.  Cell activities are expressed per gram of wet cells).  Cell suspension. extruded dropwise from a height of 10 cm into a stirred solution of calcium chloride C 0.1 M) with a temperature of 30 C, containing 20 (wt. /about.  sucrose, which serves to stabilize the isomaltulose-forming activity of cells.  The resulting granules are stirred for 1 h, then placed in a column (30 cm in height, 5 cm in diameter) equipped with a cooling jacket.  Then a sucrose solution of 55% is prepared (wt. /about. ) in deionized water and adjusted to pH 7.0 with 1.0 M NaOH.  The sugar solution is pumped up through the column at.  When the flow rate of sucrose is about 0.01 volumes of an empty column / h, the conversion of sucrose to isomaltulose and other products reaches equilibrium.  The equilibrium state is reached after 24 hours. By this time, the activity of the fixed cells is about 0.2 g of product / g. wet  cells / h  Cell stability, expressed as a half period, is about 1 year.  When the sucrose flow rate is increased so that the conversion is reduced to 80, the activity is about 0.325 g product / g wet. cells / h  Collect the eluate column, evaporated to about 70 (weight. /about. ), then, allowed to cool, or forcibly cooled with stirring, thus obtaining small white crystals.  The crystals can be isolated more quickly by priming the cooled solution with a small amount of dry isomaltulose.  The crystals are collected by filtration or centrifugation and dried at 700 mm Hg. Art.  in a vacuum oven.  The product is analyzed and then tested.  The crystals contain isomviltulose, with one molecule of water of crystallization per molecule of isomaltulose, the rest being other sugars.  Recrystallization three times gives pure isomaltulose.  If the substrate is converted to isomaltulose and related products by 80, 88 g of crystalline product per 100 g of sucrose substrate are obtained, and the productivity of the column is 0.30 tons of dry crystalline product / column volume / year.  During working use, the number of viable cells from the fixed cells quickly decreases: the number of viable cells reaches zero after about 500 hours of working use.  After this period, no viable cells remain and no involuntary cell forms are visible.  In spite of

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relatively rapid loss of viability, the isomaltyl forming activity of the fixed cells does not decrease: it is dependent and much slower with a half-life of 35 days.

This shows that viable cells are not needed for the formation of isomaltulose, and that there may be a single stable enzyme that converts sucrose to isomaltulose, for which rectifier cofactors or continuous regeneration of energy sources is not required. This is confirmed by the experience of using as a substrate 55% (w / v) sucrose 0.1 g / l chloramphenicol, which inhibits protein synthesis. The activity of isomaltulose synthesis does not change, but cell division stops, even with added nutrients.

It is possible to induce new growth of Erwinia cells in place by supplying nutrients in the form of a fresh, sterile medium, even when used in a column continuously for a long time; isomaltulose-forming activity falls about a small fraction of its original value. In these eight experiments, the medium was pumped up through the column at a rate of 0.01 from the empty column / h. Significant amounts of SP are released in this case by the cells fixed in the granules, the pH value of the spent medium washed out from the column drops to, an increase in the number of viable cells is observed, both in the granules and in the spent medium, and the analysis of nitrogen as the spent medium and granule cells, showed that cell growth occurs. After returning back to sucrose as a substrate, the isonutulose-forming activity of the granules increases in some cases to levels higher than the initial activity. There is no evidence of the formation of the enzyme in the original cells, but there is a fact that regeneration is caused solely by the growth of the k-let. The degree of restoration of the activity of the granules is proportional to the number of viable cells remaining immediately before the addition of nutrients, and no recovery of activity is observed in the granules that have been used for such long periods of time.

Time periods that all fixed cells became viable (500 h /:

After the working use of the regenerated cells during the subsequent period, the activity of the column can be activated in a similar way at least 3 times. The possibility of regeneration of columns with fixed cells provides a theoretically infinite stability of the column and suggests that the age of the fixed cells does not depend on the carrying capacity of the substrate.

Instead of supplying nutrients directly after using the pellets for a certain period of time, the nutrient medium can be fed to the column immediately after fixation. In these experiments, the slow growth of Erwinia cells occurs in situ. When 1 (wet. Weight, / about.) Cell material of granules is fixed, a 30-fold increase in the number of viable cells is observed, and cell growth occurs with a doubling time of 6 hours before expiration of the experiment, caused by a strong weakening of the structure of the alginate gel. When cells

used in this way, with frequent restoration of activity by providing conditions for cell growth, the internal stability of the enzyme system is less than when cell growth does not occur. Thus, the enzyme is much more stable when the cell is in a pseudo-latency or in a state of conservation, than when it is allowed to divide.

Example 2. The basic procedure of Example 1 is used with an additional stage in which the collected cells are crushed with a ball mill for 90 minutes with prior fixation. The initial activity of the granules is the same as when whole cells are used in Example 1, but the half life is approximately 170 days.

Example 3 The method is carried out as in example 1 using other strains of isomaltulose-forming microorganisms, in particular Erwinia rhapontici (N CPPB 1578, 139 and 1739 and ATCC 2928A), Erwinia caratovora var atroseptica N CPPB 139, Erwinia dissolvens and N CPPP. Prota .minobacter rubrum (CBS 57, 77) Servatia masscescens (N CIB 8285) and

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Serratia plymuthica (ATCC 15928). The specificity, activity and stability of the fixed strains are measured. Then, taking into account the pathogenicity of microorganisms and the tendency to produce a polymer or pigment, the strains under study are evaluated for suitability for their use in the method of Example 1.

In tab. Figure 2 shows the suitability of Yu strains for the production of isomaltulose. table 2

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With year-:

Strain

E.rhaponticI NCPPB 1578

F..rhapontlc NCPPB 139 E.rhapontlci NCPPB 1739

E.rhapontlci ATCC 2928 + -Y- +

E.carotovora var atroseptlca NCPPB 139

E.dissolvens NCPPB 1862 E.dissolvens NCPPB 2209 Serratia plymuthica

15928

ATCC

Serratia marscescens

NCIB8285 +

Protaminobacter rubrum CBS.57.77 +

An example. The method of Example 1 is carried out using a sucrose concentration of 12.5 to 60 (w / v). Activities and half-lives were measured at baseline percent conversion equal to%.

In tab. Figure 3 shows the effect of sucrose concentration on isomaltulose formations.

Table 3

- and

Continued table. 3

0 A change in sucrose concentration affects the performance of the fixing columns. The sucrose concentration affects the activity of the fixed cells, with the concentration

5 above about 0 (w / v. Has inhibitory effect. Stability (i.e. half life) of the fixed cells increases significantly with increasing sucrose concentration above those used in previous studies. Increasing the stability of fixed cells with increasing sucrose concentration outweighs the decrease in activity, so the productivity of the column increases with increasing sucrose concentration.

The equilibrium ratio of products to sucrose is strongly influenced by the concentration of sucrose in the substrate, with the highest equilibrium value obtained using sucrose (w / v). Thus, when sucrose is used, the equilibrium ratio of products to sucrose is 13.2: 1, which is BeotTTByeT 93% conversion to products.

Example 5. The method is carried out as in Example 1 using Erwinla rhapontic N CPPB 1578 cells fixed by other immobilization methods.

In tab. The effect of the method of fixing the activity of isomaltulose-forming microorganisms on the activity and half life is shown. 15101 Table k Calcium alginate 0.325 8500 EAE-cellulose 0.583 "00 Polyacrylamide 0.13 570 Cells aggregated with glutaraldehyde 0.153 O Kappa-carrageenan / pseudoactivity 0.2bZ 37.5 Bone char 0.01 25 Agar 0.3 27 Xanthan / pitch lacquer resin 0.10 8 Example 6. M-methyl-M-nitro-N-nitrosoguanidine (100 mg / ml) is dissolved in 25 ml of 0.10 M citrate buffer at pH 6.0 while incubating in a water bath at 37 ° C for 20 min. This solution is then added to 50 ml of the filtered nutrient broth containing approximately 1 g of freshly harvested E. rhapontici N CPPB 1578 cells. The cells are then incubated

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52.4 0.389 54.4 0.354 56.6 10 S 15 20 25 JQ

Table 5

59.52 52.53 47.77 616 30 min while in a water bath, then they are collected and suspended in a k% sucrose medium table. 1) overnight at C. Aliquots (0.1 ml) are placed on 1.5% (w / v) agar plates containing sucrose k% (w / v), tryptone 0.41 (weight 1% (wt./about., the pH of which was raised to 7.0, the incubation is carried out h. Separate colonies are then transplanted into shake flasks and cultured for 48 when shaken at 120 rpm, then the cells are harvested, subjected to Ticking and analysis. Mutant strains are less specific than the original cells, despite the fact that they produce isomaltulose. This is carried out as in Example 1 using an al-, gynat gel containing fixed cells CPBB 1578, which is either gelled in a block and cut into fragments, or gelled as a continuous cord and used in a form wound onto a rod or sliced segments When used in a column, these fixed cells do not show a noticeable difference in activity or stability compared to cells fixed in spherical granules. Example 8. Example 3 shows that strains of E.rhapontici are the most suitable. The subsequent comparison is carried out under standardized conditions using each of the three E. rhapontici strains fixed in calcium alginate. In tab. 5 shows the productivity of different strains of E.rhapontici. 17 Example 9. The enzyme system responsible for the conversion of sucrose to isomaltulose is extracted with a solvent from Erwlnia rhapontici N CPPB 1578 cells, using four different methods, such as Mickle shaking, osmotic shock, ultrasonic treatment or processing of detangent. To shake in mikle, 2 g of freshly harvested cells are shaken with 2 g of dry sand and 2 ml of distilled water for 20 minutes at room temperature, using a shaker of mikle. For the osmotic shock method, a portion of the cell suspension is suspended in 10 ml of deionized water for 30 minutes at. For ultrasound treatment, 2 g of the suspension of cells is suspended in 15 ml of deionized water and treated

0.63 0.567 0.094

0.70 0, 0.038

0.63 05T 0, 0.16 - 0, From the results given in table. 6, It can be seen that, upon shaking by Mikle, about 5% of the cell activity gets into the surface layer, and the remaining activity is associated with cell fragments. The soluble enzyme extract, i.e. surface layer, has an activity of 0.09 g of product / ml / h, specific activity of 0.0027, g of products / mg of protein / h and show twelve different protein bands when analyzed using polyacrylamide gel electrophoresis. The enzyme system can be more efficiently extracted from 0 ,, 60,0029

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0,00561,990,0028

0.92 618 ultrasound for 30 minutes using a 2.5 cm nozzle generator. For treating with a detergent, use 10 ml of an aqueous solution of Triton X-10P (0.15% o6./o6.J to treat 0.5 g After each appropriate treatment, the resulting suspensions are centrifuged at 17000 for 30 minutes with the cells or cellular fragments and the surface layer of the liquid is then individually fixed on the calcium alginate granules using the general method of Example 1. Then the granules are tested against 55% ( wt./about. sucrose to determine activity of isomaltulose synthesis. Table 6 shows the activity of isomaltulose-forming microorganisms with different methods of cell destruction. Table 6. Using osmotic shock of cells. About 9% of cell activity is extracted using this method, the extract has an activity of 0.253 g of products / ml / h and specific activity 0, g product / mg protein / h. Moreover, electrophoresis in a polyacrylamide gel shows only ten protein bands. The relatively high purity of such an enzyme extract is probably due to the fact that the cells do not rupture under the action of osmotic shock. The enzyme system is membrane bound and / or enclosed in a cell, and the periplasmic space is more likely to be located, especially since osmotic shock is the best method of extraction, and also because The periplasmic space of the enzymes, such as acid phosphatase, was also extracted by treatment with osmotic shock. From the data table. 6 it follows that crushed cells have the greatest activity. This is probably caused by the elimination of obstacles for diffusive transfer of the substrate created by the walls and membrane of an intact cell. However, there is no significant difference in the activities of cells subjected to osmotic shock, non-viable cells and fresh viable cells, which shows that the active transport system for sucrose influx into the cell does not work here, since such a system would be dependent on the integral metabolism of the whole cell and it could have been apparently lost in osmotic shock cells. ; Further investigation of the cell-free enzyme extract obtained by osmotic shock showed that it has an optimal pH value, equal to 7.0, optimal temperature and has a maximal activity for 35% Suspension / about sucrose solution. TOT FactJ is the optimum substrate concentration for soluble enzyme systems. topics lower than. for free cells 55) or for fixed β-cells, apparently, reflects a change in the intrinsic properties of the enzyme system, depending on localization. When the enzyme system, extracted by shaking by Mikle, is used on (w / v sucrose, the fixed enzyme has an initial activity of 0.0067 g of product / g of granules / h and has a half life of 620 h, ensuring an initial conversion rate of 81.5 Thus, the use of the proposed invention dramatically increases the stability and productivity of the isomaltulose-forming system, both the 3d through its immobilization in the gel, and through the use of new strains of microorganisms of the genus Erwinia, the use of more e, concentrated sucrose solutions and more complete conversion of sucrose to the desired product. Thus, immobilization of calcium alginate in the gel allows to increase the half-life of cells from several to 8500 hours, and the use of 5 and 55% solutions of sucrose results in a 1.510-fold increase in cell stability compared with experiments in which 35% solutions are used, without a significant change in the activity of the system. Furthermore, the invention allows the use of the isomaltulose-forming system of microorganisms without culturing and tim microorganisms themselves.

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Claims (5)

(577 1. METHOD FOR PRODUCING I30 MALTULOSE by enzymatically converting sucrose solutions using isomaltulose-forming microorganisms and subsequent isolation and crystallization of isomaltulose, characterized in that, in order to increase the stability and productivity of isomaltulose-forming microorganisms, the latter are used to fix the gel and use them to fix isomalt • vines from solutions containing 4055% (w / v) sucrose. 101 2. A method according to claim 1, characterized in that Erwinia rhapontici strains NCPPB 1578, NCPPB 139, NCPPB 1739 or ATCC 29284 are used as an isomalt-forming microorganism. 3. The method of claim 1, wherein the isomaltulose-forming microorganism is fixed as whole cells. 056 4. The method according to π. 1, which is characterized in that a soluble extract of the whole is used for fixation. or crushed cells of isomaltulose-forming microorganism, 5. A method according to claim 1, wherein the alginate gel is used as a gel.