JPS5982088A - Preparation of sorbitol by microorganism belonging to pichia genus - Google Patents

Abstract

PURPOSE:To prepare sorbitol in high efficiency, by culturing microorganisms belonging to Pichia genus in a pentose, and treating glucose with the living cell or cell extract of the cultured microorganisms in the presence of NADPH. CONSTITUTION:A microbial strain belonging to Pichia genus, e.g. Pichia quercuum IFO 0949, Pichia xylosa IFO 0950, etc. is cultured aerobically in a liquid medium containing 2-15% of pentose such as D-xylose, D-arabinose, D-ribose, etc. as a carbon source. The living cells, treated cells, disintegrated cells, cell extract, or their mixture is added to 1-60% glucose solution, and made to react with each other in the presence of 0.1mM-20mol of reduced nicotinamide adenine dinucleotide phosphate (NADPH) per 1mol of glucose.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing sorbitol R1 from glucose by a microbial method (from i:X<, t;
L Live bacterial cells, treated bacterial cells, and crushed bacterial cells of Pichia microorganisms J
Sorbitol can be efficiently converted from glucose in the presence of Bacterium nigra (using this extract, etc.) Concerning how to manufacture well.

Sorbitol has 11 unique tastes, is poor in reaction +I, is harmless, and has an orchid control effect, so it is used as it is in foods, toothpaste, and as an additive in 171 chemical products. It is widely used as an intermediate raw material for the production of vitamin C1 interfacial viscosity 1/1 agents.

Sorbi 1 is currently produced by catalytic reduction using an N1 catalyst such as glucose in a conduit, but such a method inevitably involves a reaction step. high pitch (160℃), high IT (170KO,/ar
n2), which requires a large amount of energy, requires a moon-resistant container, and has the inherent danger of explosion due to the handling of hydrogen.

The present inventors, having a different idea from the method mentioned above, attempted a method of producing sorbitol with a relaxation factor of 1 by microbiologically reducing η-lucose. −
1f) Reduction of dextrose to sorbyl 1~-l IXV 7
74 LSI animal cells (31, sheep seminal vesicles (H, G, l-1ers 111ocllell)
l.

, Biopt+ys, △cla 37 (Hlf
iO) 127 138) or in 1 nons (M,
L ou 131 oche IIl.

Biopbys, ΔCia 1 'I 1 (1
967)! ')4-/-5)5), but in microorganisms, the lees content is slightly lower than 11fl.
/l 5 2483 /I month and No. 46-23038, xylose was aerobically fermented in Kirin 1~-l and C'J!
ii) It merely discloses a method for reproducing original data. Furthermore, even if the strain disclosed in the above invention was cultured underground in Gurunisu 16, it was not possible to produce 1q of sorbyl.

There, the present inventors conducted tests on a wide variety of microorganisms. Freeze-dried bacterial cells N
We have now completed the present invention by devising the reduction of the substrate glucose to sorbyl 1-1 in high yield in the presence of P1-1.

Vinyl 1a and C used in this defense are Pichia gluccum (P 1cl)ia gucrcum) 1 "
00949. Be V A 4-Shiro (P 1chia)
xylosa) 1 [00950, among others, P+-"P, i-C11"JIFO0
95 (l is so frightening.

In growing the bacteria according to the present invention, -C contains 2 to 15% of I, iJ as a water source, Ij carbon sugars such as D-xylose, D-nononohinose, and D-ribose, and r-st extract or Culture at 25 to 35°C in a liquid medium supplemented with corn seed liquor or the like for 2 to 10 hours. As for the culture method, any aerobic conditions such as aerated culture, shaking culture, rotary i, ram d1, etc. are adopted.

In addition to the above ingredients, the culture medium also contains various vitamins and inorganic
, peptone, yeast-r, 4-s and other organic substances are added.
It is also possible to increase the proliferation rate.

The bacterial cells grown in this way were collected, washed, (jqk
Even if live bacteria are used as they are, 91 Song [, 1° is observed, but freeze-dried bacterial cells, freeze-thawed bacterial cells, 7' t1~
The LC effect is much more effective when using treated bacterial cells such as treated bacterial cells with organic solvents such as N, -Til, etc. In addition, it is also possible to use a microbial cell fragment d3 and microbial cell extract prepared by ultrasonication or using a crusher such as a Vibrogenrelmil.

So, what is the writing method for manufacturing sorbyl?
The treated microbial cells, crushed microbial cells, or the obtained microbial cells are preferably reacted at 25 to 35°C. In this case, as a supplementary M search, N
The coexistence of ADI) 1-1 dramatically improves the productivity of sorbitol.

The amount of NAD I) H to be added varies greatly depending on the preparation method of the bacterial cells used or the extraction procedure of the bacterial cells.
The amount per mole is 0.1 mmol to 20 mol, preferably 0.1 mol to 10 mol. The time required for the reaction also varies greatly depending on the conditions; for example, when using a bacterial cell extract,
The reaction is complete in 0 minutes to 14 days.

After the reaction is completed, sorbitol is separated from the mother liquor.

For the purification of sorbyl, centrifugation,
A combination of known methods such as ultrafiltration and ion exchange v1 is used.

Hereinafter, the present invention will be described in detail with reference to Examples.

Example 1 50 ml of a sterilized medium having the composition shown in Table 1 was placed in a 500 n+l digestion flask and Pichia kerculum 111r
o o9a9 strain and Pichia xylori l F
O, '0950 strain was inoculated into one platinum loop strain, and 3
The cells were cultured at 0°C for 0111 hours. The cultured bacterial cells were washed twice with phosphorus Mtj suspension, and the turbidity on the 6100 image was 25 (suspended in diphosphate buffer).This bacterial suspension was incubated for more than 20 minutes. Sonicate and 14,000 (+
The mixture was centrifuged for 20 minutes at N1, and the 1: oil fraction was used for the production of sorbitol.

Using both supernatants obtained in the above procedure, a reaction solution having the composition shown in Table 2 was prepared, the rlH was adjusted to 7.5, and the reaction solution was reacted at 30° C. for 6 hours. Pichia kerculum IF00949 strain has 21 μl lloles, Pichia xi[1sf F
For strain O0950, 81 μmoles of sorbyl was obtained.

D−×y10S0 8(1′:,”1ii K l−1:! l:).4 0
．． 1gMQ SO2・71-120 0.
05(]Ca C1n ・2+-1200,0I
C+Ma C1O,01(1 Casamino acid 0.11g Yase l-1kiss 0.1 (Table 2) 9 hearts-l', Wi it evening 5
1glucose 3, 6mmolesNΔ
IIPH0,2+n molcs Phosphorus M buffer Q, 7111 moles evaporation
Retained water 5) Mf recognition of 1 product [
Ic performed by J Thin Layer Chroma 1~Graphy, High Performance Liquid Chroma 1~Graphie and Gas Chromatographie
oAlso, I retired with a high-speed liquid pump L171゛ Granoy.

Example Me'1 Pichia = obtained by culturing in the same manner as in Example 1
Cells of IFO0950 were washed twice with phosphate buffer and then freeze-dried.

A reaction composition shown in Table 3 was prepared using the obtained freeze-dried cells, and 1 was reacted at 30° C. for 16 hours. 121 μmoles of sorbyl 1 was obtained from the reaction solution.

Table 3 Freeze-dried bacterial cells 0.5°glucose 10 m molesNADP
H2mmolcs phosphate buffer 3.5 mmoles evaporation
1 Example 3 20 ml of the reaction solution obtained by the method of Example 1 using the B1) 7. The sorbyl 1--ol was purified through The amount of sorbyl 1-1 obtained was 65 μmoles.

et al.1. 1 1. 1 1゜Hand Shadow Kenn't-Shoyo (White) Mr. Commissioner of the Patent Office 1, Indication of Matter A'1 Patent Application No. 1, 1982
90053 2-1, Title of invention 5, Date of amendment order 1 Voluntary action 6, Number of inventions increased by amendment None 7, Subject of amendment
[Detailed Description of the Invention Column j8 of the Specification, Contents of the Amendment As shown in the attached sheet, attached specification p (page 7, line 6, rvac+-+ in ``'' is l-N
h1 correct for a Cl j.

Claims (1)

[Scope of Claims] [1] Live microorganisms obtained by aerobically culturing Pichia microorganisms using a medium containing pentose as a main nutrient source,
1. A method for producing sorbitol using a microorganism, which comprises producing sorbitol by allowing at least one of a crushed bacterial cell material and a bacterial cell extract to act on glucose in the coexistence of reduced nicotinamide adenine dinucleotide phosphate. [2] The method for producing sorbitol using a microorganism according to claim 1, wherein the Pichia microorganism is Pichia kercum or Pichia xylosa. [Blade Pichia Kerkrum [F 0 0949. The method for producing sorbitol using a microorganism according to claim 2, wherein Pichia xylosa is IFO0950.