Process for producing sucralose

专利摘要:
The novel chlorinated sugar O -a-D-6-chloro-6-deoxyg- alactopyranosyl-(1 → 6)-a-D-4-chloro-4-deoxygalactopyran- osyl-(1 - 2)-β-D-1, 6-dichloro-1, 6-dideoxyfructofuranoside (TCR) can be used to prepare sucralose by incubating the TCR in solution in the presence of an enzyme serving to remove the 6-chloro-6-deoxygalactosyl moiety from the 6-position, especially an enzyme derived from a strain of Mortierella vinacea, Circinella muscae or Aspergillus niger. TCR is prepared by treating raffinose with thionyl chloride in the presence of triphenylphosphine oxide.
公开号:SU1635905A3
申请号:SU864028456
申请日:1986-10-20
公开日:1991-03-15
发明作者:Брин Ратбоун Элнер；Султан Мафти Кхизар；Ахмед Кхан Риаз；Самюэл Джеймс Читхэм Петер；Джон Хэкинг Эндрю；Сег Дордик Джонатан
申请人:Тейт Энд Лайл Паблик Лимитед (Фирма)；
IPC主号:

专利说明:

The invention relates to biotechnology, is the production of a sugary product with a high. Sweetening effect — sucralose (, 4,1 -6-trichloro-4-1, b-trideoxyhalo-agaroza, known as TGS); 600 times sweeter than sucrose.
The purpose of the invention is to simplify the process by minimizing the formation of by-products.
The method consists in incubating chlorinated sucrose derivative TCR (0-o solution (-B-6-chloro-6-deoxygalactopyranosyl- (1-6) -c (-B-4-chloro-4-deoxylactopyranosyl) (1-2 ) - .ji-D-1,6-dichloro-1,6-dideoxyfructofuranoside) in the presence of an enzyme,
serving to remove the 6-chloro-deoxy-galactosyl residue from position 6.
Of particular interest are galactosidases. Melibioses are of much less interest due to the fact that although melibiosis (O-o-O-galak-tyranosyl- (1) -o-B-glucopyranose) is a component of raffinose, but chlorinated TXP has a reversed configuration. It has been found that many of these enzymes do not possess any beneficial activity against TXR even in cases where they are able to release sucrose from raffinose. About 20 enzymes of the row of 0-galactosidases derived from bacterial, fungal, and
about with
SP
WITH
R
01

cm
and plant sources, however, only 8 of them were active in the hydrolysis of TXR, all of them originated from fungal sources. Active enzymes were obtained from strains llortierella vinacea, Circinella muscae. The most active is Hortierella vinacea species rafiinoseutilizer (ATCC N 20034), supplied by the Hokkaido Sugar Company. The reaction rate with TCR in this case is only a few percent of the reaction rate with the participation of raffinose itself. However, taking into account the significant conformational and configurational differences between raffinose and TCR, even this rate (or activity) is unexpected and leads to almost complete hydrolysis of TCR in sucralose.
The granulated M.viracea cells of the raffinoseutilizer variety have been used in the production of beet sugar since 1968.
Enzymatic treatment is conveniently carried out in an aqueous solution. However, although the presence of water is required to carry out the reaction, it has been found that k / -galactosidase can intensively hydrolyze TXP to sucralose and in a number of organic solvents. The enzyme has been found to be the most stable and active in water-immiscible organic solvents, whereas TXR dissolves well only in hydrophilic solvents. Thus, the most suitable solvents for the hydrolysis of TCR are hydrophilic water-immiscible organic solvents, for example ethyl acetate, n-butanol and methyl isobutyl ketone. For example, the rate of hydrolysis of TXP, determined by the catalyst conversion number (Kko-) in ethyl acetate previously saturated with aqueous buffer (which gives enough water for hydrolysis), is similar to the rate of hydrolysis in aqueous buffer. It is assumed that the enzyme retains its full catalytic activity even when it is suspended in an organic solvent. Each of these organic solvents, being previously saturated with an aqueous buffer, remains as a single organic phase. The enzyme functions0
five
0
five
0
five
0
five
0
em in the organic phase. Hydrophilic water-immiscible solvents (e.g., ethyl acetate, n-butanol, and methyl isobutyl ketone) were able to maintain high catalytic activity and high solubility of TCR.
In tab. Table 1 shows the solubility data at 55 ° C TXR and Sucraloe in various solvents (all of them were pre-saturated with aqueous buffer) as compared with the aqueous buffer. It is clear that the solubility of TXP becomes maximal if hydrophilic water-immiscible solvents are used.
Other advantages of carrying out the process of hydrolysis in an organic medium also include an increase in thermal stability (the stability of bi-galactosidase at 55 s in ethyl acetate is about 50% higher than in an aqueous solution) and the solubility of the substrate (the solubility of TCR in the above solvents is over 50% (May / vol), whereas in an aqueous buffer, TXP solubility is only 15% (May / vol)).
In the case of using both aqueous and organic systems, the treatment should be carried out at optimum temperatures and pH values for this enzyme, which in the case of using 0 (, - M. caceacea galactosidase is approximately 55CC and pH 4.5-5.5. In organic solvents saturated with aqueous buffer, these pH values refer to the pH of the aqueous buffer. At the indicated temperature and pH 5.0 it was found that the resulting sucralose does not decompose.
The enzyme can be dissolved or dispersed in an aqueous system as an extract containing no cells, obtained, for example, by sonication of the granules, or it can be used in an immobilized form, for example, in the form of granules, placed in columns or arranged in layers. In organic systems, the enzyme remains in insoluble form and can be reused in layers or columns.
Sucralose obtained as a product is separated by any known method, for example, by evaporation and extraction in an organization.
solvent, chromatographic techniques, selective crystallization from aqueous or non-aqueous systems.
Example 1. Preparation of sucralose in aqueous solution (in micro-amounts).
An amount of 20 mg of tetrachlorine galactorafinosis is placed in a top-screwing vessel containing 2 ml of 0.1 M citrate-phosphate buffer (pH 5.0). Get a solution concentration of 1% (May / volume). The indicated amount of water is kept constant during the subsequent incubation by adding the required amount of water when required.
To the mixture, 10 mg of 0 mg (.- galaktoeidazy in the form of granulated Mortierella vinacea cells of raffinoseutilizer ATCC variety No. 20034 (supplied by Hokkaido Sugar Company, Japan) are added, and the resulting mixture is incubated for 100 hours at periodic monitoring. the degree of hydrolysis is carried out using thin layer chromatography, for which 250 μl aliquots are taken from the reaction mixture, which are eluted with ethyl acetate: ethanol: water (45: 5:: 1), or high pressure liquid chromatography on a column of Waters Dextropack C 18 with reverse fa this was applied for elution with 20% acetonitrile (v / v). The content of the products was measured with a detector based on the determination of the refractive index. The results are shown in Fig. 1. Thus, it was found that about 53% of the product was hydrolyzed after 48 hours and approximately 70% - after 90 hours. The products are separated by the chromatographic method, and in addition to sucralose, 6-chloro galactose and TXP are found. In this system, TXR can be used in amounts up to 15% (May / volume). In aqueous solution, this enzyme has a Michaelis constant (Kz) equal to 5.8 mm and a constant Кцдт equal to 52.5 mg per g enzyme per hour.
Example 2. Mortierella vinacea granules of raffinoseutylizer variety (ATCC No. 20034) in an amount of 3 g are hydrated in a 0.1 M solution of citrate-phosphate buffer (pH 5.0) containing 5% (May / vol) raffinose,
0
five
0
five

0
five
0
five
0
and fill the mixture with a column of 9 cm in size with a volume of a layer of 7 cm3 equipped with a water jacket. In this column, a temperature of 55PC is maintained using a circulating water pump. Using this column for hydro- lytic TXP to sucralose, it was found that the half-conversion time (up to 50% conversion) is 13 days.
Example 3. Microorganisms suitable for hydrolyzing TXP to sucralose.
.. Microorganisms possessing activity as o-galactosidases were obtained from the ATCC culture collection. They were grown in flasks with a capacity of 100 ml, containing medium based on mineral salts of 1 g / l of yeast extract and 5 g / l of rasinose, shaking at a temperature.
Cells are selected by centrifugation (5000 rpm by centrifuging for 10 minutes) in the late exponential growth stage, resuspended in five times the volume of citrate-phosphate buffer at pH 5.0 and kept at 0 ° C in an ice bath, and then destroyed by 3 runs of 20 s in an ultrasound device (Dave Soniprosb, type 1130A) at maximum power. The suspension thus obtained is centrifuged at 17,000 rpm for 30 minutes and examined for the presence of activity as t -galactosidase (both the precipitate and the upper layer), determined the indicated activity in units of 1 g dry weight of the cell residue and applied p-nitrophenyl- - D-galactopyranoside targets (Sigma). Virtually no enzymatic activity was found in the surface layer. For cultures that give positive results when tested for the hydrolysis of TXR according to the method described in example 1, the results obtained (see Table 2) are given in mg of sucralose obtained per 1 g dry weight of cell residue per hour (for 70% conversion rate).
Example 4. Obtaining Sucralose in an organic medium.
TXP in an amount of 23 mg is placed in a 5 ml tube with a screw cap and 2 ml of organic
the solvent previously saturated with aqueous buffer (sodium acetate 50 mM, pH 5.0), resulting in. 20 mM (1.15% May / volume) solution. To each of the tubes, 100 mg of β-galactoEdase in the form of granulated M. vinacea cells of raffinoseutilizer ATCC variety No. 20034 are added, and the reaction is initiated by introducing 1% (volume / volume) aqueous buffer, vigorously stirring for 30 seconds. and placing the tubes in a shaking water bath at 55 ° C and 100 rpm. Periodically, 25 µl samples are taken from the reaction mixture, which are introduced into the chromatographic analysis system at high pressure (system using reverse phase, Waters Dextropack C 18), used y eluant 20% aqueous acetonitrile, and the resulting measured quantity of residual sucralose and TXP. The effect of the presence of an organic solvent on obtaining sucralose during a 24-hour incubation is shown in Figure 2. The dependence of the degree of hydrolysis of TCR in sucralose on time is given in the form of curve 1 for an aqueous buffer and for three solvents: methyl isobutyl 5 - JQ N.C5. a 20 25 jg
6359058
tone (curve 2), ethyl acetate (curve 3) and n-butanol (curve 4). The reaction of hydrolysis in methyl isobutyl ketone and aqueous buffer is the same; in the case of using ethyl acetate and n-butanol, lower rates of reaction and conversion are recorded. In all cases, more than 50% conversion is observed over a period of 24 hours, with a conversion over the same period of 70% for the aqueous buffer and methyl isobutyl ketone.
In a separate experiment, the kinetic parameters of the Mihaelis-Menten enzyme for both water buffer and ethyl acetate, Kjn and K in ethyl acetate were 16.4 mM TXP and 56.3 mg sucralose / g, respectively. . The reason for the timing for the hydrolysis of TXP in ethyl acetate is lower than the corresponding schedule in the aqueous buffer (at 20 mM TXP) is a higher Ksc TCP value in ethyl acetate, and not a lower catalytic activity. When using 50 mM TXR, the rate and degree of conversion during the hydrolysis of TXP in ethyl acetate are similar to the corresponding values in aqueous buffer.
Example 5
Concentration TXR start of reaction, X 10
Enzyme Concentration, X5
Z cash sucralose through, h:
20.3
60.6
231.2
471.5
72
Example 6
The concentration of txp in the beginning of the reaction, X
Enzyme Concentration, X
X cash sucralose after, h:
2
five
22 3) 45 70
5 2.3 1 0.5 2.5 1.25 0.3 0.25
Example 7. Mortierel granules of vinacea var raffinoseutilizer ATCC 20034 in an amount of 12.5 g are hydrated in a solution of 0.1 M citrate-phosphate buffer, pH 5.0, containing 2.5% raffinose per volume. a 5.7 cm column with a nozzle volume (ecv) of 27 ml, maintaining the temperature at 55 ° C with a water jacket equipped with a circulation pump. A 2.5% aqueous solution of TCR was passed through the column at an initial flow rate of 0.11 ecv / h, which was reduced to 0.09 ecv / h after 13 days and to 0.038 ecv / h after 26 days, which gives about 19 n sucraloea in | 28 days (65% conversion). Half-decay of the enzyme is achieved between the 25th and 30th day.

权利要求:
Claims (2)
[1]
1. A method of producing sucralose, which involves chlorination of a derivative of sucrose, substituted in position 6, with the introduction of chlorine atoms into
Absidia grisiola
Aspergilla awamori
position 4, 1
61
sucrose and
five
0
five
The following removal of the 6-substituent, characterized in that, in order to simplify the process by minimizing the formation of by-products, raffinose is used as the 6-substituted sucrose derivative, and the removal of the 6-substituent is carried out by enzymatic hydrolysis of a 1-15% solution of O - -D-6-. chloro-5-deoxygalactopyranosyl- (1-6) - lb-B 4-chloro-4-deoxylactopyranosyl- (1-2), 6-dichloro-1,6-dideoxy-fructofuranoside in an aqueous system or in a hydrophobic solvent using oC β-galactosidase from Mortie-rella vinacea or Circinella muscae, showing a degree of hydrolysis of 1.6 to -4.3 mg sucralose per gram of dry cell residue in 1 hour at 30 to 50-70% conversion.
[2]
2. A process according to claim 1, characterized in that the solvent is methyl isobutyl ketone, ethyl acetate or n-butanol.
Table 1
table 2
20430 20431 22618 44733
fO 40 VO SO Yu
VRMP ikbibireLoni Fig1
YU
15 20 25 Lryp incubation
Fig 2

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法律状态:

优先权:

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申请号 | 申请日 | 专利标题

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GB858525871A|GB8525871D0|1985-10-21|1985-10-21|Chemical compound|LV920526A| LV5136A3|1985-10-21|1992-12-29|Saturation of sucralose|

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LTRP676A| LT2150B|1985-10-21|1993-06-22|THE SUCCESSFUL BUDGET|

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MD94-0113A| MD97C2|1985-10-21|1994-03-23|The method for preparation the sucralase|