JPS5985296A - Production of diglycolic acid and beta-hydroxyethoxyacetic acid - Google Patents

Abstract

PURPOSE:To produce diglycolic acid and beta-hydroxyethoxyacetic acid from diethylene glycol, by using a yeast. CONSTITUTION:A yeast selected from the group consisting of the genera Candida, Torulopsis, Rhodotorula, Hansenula, Debaryomyces, Cryptococcus and Pichia, e.g. Candida albicans IFO 0601, is cultivated in a culture medium containing diethylene glycol or an immobilized microbial cell prepared by immobilizing the above-mentioned yeast by an optional method is brought into contact with diethylene glycol to separate and collect the resultant diglycolic acid and/or beta-hydroxyethoxyacetic acid.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing diglycolic acid and β-hydroxyethoxyacetic acid from diethylene glycol using yeast.

It has been known to culture #~ in a medium containing ethylene glycol and to separate and collect glycol (+'l) from the medium.
The issue discloses the use of a strain belonging to the genus Gyandeida.

However, using yeast-(diglycolic acid and β-
Regarding the production of hydroxyethoxy c:IH 7
v,1 Unknown1, The present invention uses diethylene glycol as a raw material to produce diglycolic acid and/-4k r,i,15-human [-7
Kishietogi/131 r'i of L1 Ketai that produces fermentation? ~
・It is based on the discovery of 41°
, A Yandeida spp., l-Ruloff'ns spp., Lord i. Le]) t-15, No. Nzenula spp., Debaryomyces spp., Chryso! Kotsukasu h1% ``producing and further separating and collecting f: 11 characterized diglycols'' 2 / 2 or β-hydrogyne[xy[ll 1'''I 1'' method.

According to the present invention, as a method of adding tIY mother to diethylene glycol to -f'+-J II, in diethylene glycol:'7-L well 41 medium, p; How to immobilize yeast using a method of immobilizing yeast (by immobilizing yeast cells with diethylene glycol (4 times a day)
One example is the method of self-service.

The medium used in the method of culturing yeast α1 in diethylene glycol sej medium is one that is generally used for culturing yeast, and the carbon source used in the medium is normal paraffin. Examples include hydrocarbons, ethanol, acetic acid, tuucrose, and glycerol/r. In 7, the source of g-X is 7, 1+ is ammonium chloride, 7 is inorganic nitrogen source such as ammonium phosphate, and ammonium chloride.
Examples of organic nitrogen sources include Peb I, Casamino I, urea or i', j Consude Epliquor, and inorganic salts include potassium phosphate, sodium phosphate, magnenow A sulfate, sulfur 112 mankan, Examples include iron sulfate, iron sulfate, and calcium chloride. In addition, yeast extract, meat extract, vitamins, etc. are circulated as nutritional sources that can be added accordingly, and an appropriate one is selected from these sources.

The addition of -4-lodiethylene glycol to the t'S base is usually about 1% to about 1%, preferably -41:L
<&: I about 2 to about 5%.

The yeast used in the present invention is yeast selected from the genus Campita, Torlobus 7us, Rhodotorula genus, Hansenula genus, Tevariomyces h≦, Cryp I cotscus or Pichia, among which are the following: The one shown in is preferable.

Campita albicans If”O[J
6U1 Gyandata Tropicalis 11 (
'O1, J5890-do1 Lula Minuta
IFO0,5870-$l Lula Rubra
IF"0 0709 Hansenula octosvorus IP'0 0415〃 ・Petersonii IF'0 1372 Hansenula sonnekii IFO0135〃 ・Winyi TFOLI so76〃 ・
Pino, Ncris IF “○ 1566 Teva IJ, l Mrs. Jabonicas IF’O
0L139 Chrysitococcus albitus 1
14"0U60'y'(Cant, raalburea
u voor SCkllmmeICuljul:'e
s(CBS)(7))'rs,L,HOof'lr1gu
Pichia Bini by eS de Mlrarlda
Valaive 1゛ Meribioni (Pbichia v
ini varlmelibiosi) and iF
On the second side of the ``佼'' was ``seven''. )1 note place (!kot;1
Selected from the genera mentioned above, it is inoculated, the temperature is about 20 to about 35 degrees Celsius, and the pH is about 6 to 9 degrees Fahrenheit.
It is cultured aerobically in the range of L < 5.57i to 8.

“t in pt medium 7) diethylicol concentration 6
"1, 通')・訃1゛J1 or I? +1 fJ%,
Preferably, in order to maintain a relationship of about 2 to about 5 degrees, the culture should be divided into several times during the culture, and added as needed:
) u-a can also be done.

Furthermore, in the present invention, since the pH in the medium is lowered by the generated diglycolic acid and/or t↓β-hydrohe-ethoxyacetic acid, an aqueous solution of Cal/Rum carbonate, Ryuno, etc. It is undesirable to neutralize it from time to time.

Another aspect of Ushidagi QJI is the method of contacting diethylene glycol to stimulate the fermented bacteria (δj).

rt-t as a material to standardize 1"1Y mother,
Cellulose with active groups, acarose, degistran, gichi/, alginate, agar, carrageenan,
Examples include tangerine endodermis pecnan and polyacrylamide. 7> The method of yi conversion includes a method of cultivating a polymeric substance 2 and adding a weight to it, a method of attaching a sol-like polymer (the proboscis 1i'jj to a bacterial cell (1) Here is an example of how 40V is converted into 1-J.

In the present invention, polyacrylamide gel is particularly suitable.

Various methods can be employed to bring the immobilized bacterial cells into contact with diethylene glycol. 1 way, how to bring 1 self-made body into contact with ``the above-mentioned diethylene glycol-containing'' ~ ground, 7g) jH. Zeru Power i
): can be mentioned. It is desirable that the identified specimens be cultured and grown in an immobilized trowel medium, heated, and then brought into contact with diethylene glycol.

and <(((immobilized bacterial cells: tyrene glycol-containing -1'
It is desirable to grow the immobilized bacterial cells in a J medium with aeration and agitation, and then contact them with a buffer solution containing diethylene glycol.

Diglycolic acid and/or β- produced according to the present invention
Hydroxyethoxy vinegar mk separation and collection method (・1, the general method can be applied. For example, the culture medium is sequentially t-
After removing the bacterial cells with a tg v sieve, diglycolic acid and/or β-hydroxyethoxyacetic acid is precipitated as Cal/Rum salt, and then the free diglycolic acid and/or β-Hydroquinoethoxyacetic acid can be used as a strain.

Infrared rays of a sample made from +η by recrystallization from acetone 1
The absorption spectrum matched that of the standard product.

The following Qζ'':, (indicates the service (J).

Example 1 10 g of glycerol, (I H,)2So, 4. !
9, KH21) 041g, K, HPo, I J'
, Mg5O, 7H, (,) Q, 5 Q, Jact' 0.1 g, CaCl2-2H2t, 'l
01. @, 1 g of yeast extract 1 mineral solution 1 me'tr・1 t t, i) l in water
``8-Understood.

°゛ζ；ζ；Tofurusouriyunoyon J1H3t-303
! 5 tJ U~ UuSO, 45Hz0 4 U〃)≦
)'Kl 11J
011ノ)1'F'eCz'3.6 )]□0
2 +, J +Jnv #111S (J4
・'20 4 fJ []]Il'fNa2
Mob, -2H, J nIJU”i7n, nSO
,・7 H,,04U IJm'i territory'I l (1)
Water 'r'('(+:+'+T L , +j 5 liters of this liquid) me V(, add 1 g of diethylene glycol U 0rrre into a shaking flask t1.1
21J℃IIJ division 11j.

This medium-\Gandita albinonons■Il”0
Connected L16tJ1, 60℃ 115rpm t:r
) 111<j culture was carried out on a shaker. Since the pH of the soap produced during the culture was low, the shaking culture was continued for 7 days while adjusting the pH to 7 twice a day with a 2N caustic soda aqueous solution.

After that, the culture was acidified and removed by centrifugation, and then passed through an ion exchange resin to separate organic acids.
diglycolic acid and β using gel chromatography
-Hydroquinoethoxy 11I'l' Hk was prepared in advance (2/ko).

As a result of methylating the product with diazomethane and placing it on a gas chromatograph, 1 ton of diglycolic acid was obtained from 1 ton of culture.
．． [J,! ? , 0.5g of β-hydroxyethoxyacetic acid
obtained at a rate of

Examples> to 4 The same procedures as in Example 1 were carried out except that the strains to be inoculated were as shown in Table 1.

Diglycolic acid 1 with culture gQ' snout 1t or C-, 1<j
... Yohi/Ma/ξQ: [Ratio of β-hydroxyethoxy vinegar is shown in Table 1.

1st Clothing Example Glycerol 10. ! ? , (liH4)28044&
XK1-12PO41〃, K) i21'o, 1.9X
MgSO4・7) 1200.5. ! V, f-st extract 1g, Fe50.・7H2011: Jmy, ZnSO
4,7f-12010rng, Mn5C)4.nH2O
10"W S: i*)'I'l was added to 1 ton of water to give a total phi of 6.5.

This liquid 50m/! and diegoulene glycol 1.5 &
? After sterilization in a 50Ume shake flask, torulozosis key% 77'-(da M', I"99
(?I佼)IlJIbacteriumWreJ'y1362Sita) was cultured in the same way as in Example 1.
,7.

'7: M, A, fgut'su〃・1゛〕times iR&(l
t, -C diglycolic acid and β-+'', t1:',
j,,7: f-1-K7 exit r separation 4 Seiyo Koshi C1
(I [shi/ζ佇; fruit, culture 1 1 t - diglycolic acid 1.1 g, β-human I + gear r-l-8 F11
I suggest, 1g's, 1Hl1 combination = 1:).

'No (2h 11 case 6 Apex Mj Using the same +1e medium as 11, 15, Pichia.
Bini Bala Idei Merihi’Oshi 4 MTl 1J5
7 (Ikai-1) 11 sho 1 廂 6 b U 6 yen) was cultured in the same aA at a pressure of 41.

Diglycolic acid and β-hydroxyethoxylic acid were purified in the same manner from J'i't 'J'E.

Guζ.

Culture (1 ton of diglycolic acid is 0.9 &,
β-Hyl-'C1=YShi]・2.2g of 1 acid in A
obtained at a rate of

Ability 1! i1 row 2 and 8 Torlovsis campita M'['99 (@ Engineering (iJ
t ft1'iG'T'31E1662) and Campita tropicalis x Po v58q, respectively.
Is it really the same 4 as the old I4 5? inoculated into the medium of 31
2.5) J culture at J℃.

Therefore, f;l'1. f') 111 pieces in y month J)
-Each+J, 1 tvi rin via shikai (r) H6,
5) So, is it revenge? I got '4'. Next 0,1M phosphorus f
'tAmugi(it)i (fl (1,)H6,5-)
1 (To this bacterial cell, h! It becomes cloudy, and then acrylic acid) 0. 5% 6-(dimethylamine) globio = 1-lyl U, bme and n-5% K2S20
Add 20.5ml'7, keep in water for 1 hour, and mix.
Da 4- Assimilated.

Meanwhile, add 1Qme'i of water to this assimilated/ζ gel-like '(, '+'+'r compound, and mix it with a homogenizer.
l < Crush, wash with water and tighten completely 7 times.

The above j・r rt:Yotsu C7!07g kl fixed Mukaguchibo was born.

(T)
2. Suspend 1 g of immobilized bacterial cells obtained by the method described in 2.
Shake for 1 U and 9 ml at °C.

Diglycolic acid and β-hydroxyethoxy alcohol were purified in the same manner from the qf3j 7&middot;

As a result, the values shown in Table 2 were obtained.

Example 19 Lyso and 1 (-1 ¥ Approximately 7 g of immobilized bacterial cells prepared by Q straining in the same manner as in Example 7 are shown in Example 1.
I put one in jθ〃le and made a 24 o'clock roll with ΔU'C. Ya'7) f immersion filtration 1~-C increase 1g
Collected bacteria.

20”g of shave 1/grill 1 tr 0
．． IM phosphate buffer (pH 7) 10r1/! inside,
The fixed footed carbohydrate 1y obtained by the method of Yu 1st was finely ground and shaken in a filter for 1 U at 0°C/ζ.

Thereafter, diglycolic acid and β-hydroglycolic acid were separated and purified using the buffer.

As a result, the value shown in the sixth figure was obtained.

Example 11 1 g of immobilized bacterial cells obtained in the same manner as in Examples 7 and 8 was suspended in 20 ml of the medium (pH 7) shown in Example 1 containing 200 mV of diethylene glycol, and incubated at 60°C for 2 hours.
It shows the time at 4 o'clock.

After that, diglycolic acid and β were extracted from the culture solution in the same manner.
- Separation and purification of human rogin etoggi/sacrifice.

As a result, the values shown in Table 7fJ4 VC were obtained.

Table 4) ↓Execution 12 to 18 Execution 1flJ Strain k to be inoculated in 1 5th fi”
': The following steps were performed in the same manner.

Similarly, the results are shown in Table 5 and Table VC.

Claims (2)

[Claims] (1) Yeast selected from the genera Giyandeida, Torulopsis, Rhodotorula, Hansenula, Debaryomyces, Cryptoconcus, and Pichia are reacted with diethylene glycol to produce diglycolic acid and/or β-hydroxyethoxyacetic acid. 1. A method for producing diglycolic acid and/or β-hydroxyethoquinoacetic acid, which comprises producing and then separating and collecting. (2) The production method according to claim 11, characterized in that immobilized bacterial cells are used as the yeast.