DE2108214A1 - Process for the production of 1 dairy farmer - Google Patents

Description

Andrejewsld & Honke patent attorneys Physicist Dr. Watter Andre je wskf

m. t. ~ * _A / r _A α. Graduate engineer

Anwalfsckte: 56 262 / H p £ ^

Essen, February 19, 1971 Kettwiger Str. 56

Patent application

KYOWA HAKKO KOGYO KABUSHIKI KAISHA

No. 6-1, 1-choae, Ohte-machi, Chiyoda-ku T ο k y ο - to / Japan

Method of Obtaining 1-Serine

The present invention relates to a method for the synthetic production of! -Serine. In particular concerns the present invention a process for the production of 1-Serln, which is indicated by the fact that one uses glycine and |

Formaldehyde in the presence of threonine aldolase (E.C.4-.1.2.5) » wolehö is derived from a microorganism, condenses.

1-serine is one of the proteins that make up amino acids. Its wiohtlclcelt as raw material for pharmaceuticals, additive on foods and cometics has been recognized.

Since X-SwIn is far from dead in natural protein sources, is it usually by the hydrolysis of silk yarn, Waste Kokondi Sericin and the like have been produced, wolehe one

10S838 / 1688

: BAD ORIGINAL

Patent attorneys Dr. W. Andrejewski, Dr. M. Honke, 43 Essen, Kettwiger Strasse

contain relatively large amounts of 1-serine. The 1-serine is then isolated from other amino acids and subjected to purification. This process has the disadvantage of a low yield and it is also not economical in view of the limited access to the raw material. Another method for the chemical synthesis of 1-serine is not known become. There is also the fact that the product made in this way has the disadvantage of producing the optically inactive dl foria, in which an optical cleavage must still be used to isolate ura 1-serine. These procedures are therefore sur commercial Obtaining 1-serine hardly suitable.

On the other hand, there is a method for producing 1-serine became known through the use of threonine aldolase, which dies from the mammalian liver. This method however, it has the disadvantages of requiring both relatively expensive tetrahydrofolates in addition to glyain and formaldehyde as well as a low yield *

It was found before kursea that the enzyme threonine aldolase can be obtained by microorganisms, e.g. Using a yeast such as Candida huralcola and a bacteriuras, like Bacterium cadaveris.

It is known * that threonin-aldolase-Iinsyra 1-tlireonin decomposes to glycine and acetaldehyde. However, it has unexpectedly been found that this ensym, if it is obtained by a microorganism, can produce 1-sorin from glycine and formaldehyde without requiring tetrahydrofolate. Furthermore is

ORIGfNAL 109838/1688

21082U

Patent attorneys Dr. W. Andreiewsici, Dr. M. Honke, 43 Essen, Kettwiger Strasse

In this reaction, which differs from the conviction of the Enzyme su threonine differs, the balance of the Reaction considerably towards the synthesis of! -Serine has been shifted so that in the reaction of glycine and Formaldehyde in the presence of this enzyme! -Serine in high Yield can be produced. According to the invention is Application- of tetrahydrofolate, which is normally used for the same Purpose is required, not required.

One object of the invention is a method for listening Rush ahead of l-Ssi'in without adding tetrahydrofolate need, under Verv / enduag of TIireonin-Älclolase, which by ei a microorganism

According to the invention, e:! N Verfata ⁱ cn sur Ge: v? Innunß von 1-Ssriii vergesshs ?! unfconr "Verwenduns of Threor-in-Aldolfs" derived from a njJcroorgariiLfc.us, valches glycine una Pormaldchyd einsclilieSfc condense dio in Ocf.onviert of threonine AldolQc "fciiteinandsr ·

_{Erfindur.:',sitsr:} SSS it is possible l-3erin in \ rorbusserter kind ή

even in Abrveaenhe.it to obtain from tetrsh / clrofolat, v. ^; What is required in other processes.

The for the or-findun ^ sgeinajSen Zvißck zu. using Threonine-Aidolrao irJ.rd obtained by cultivating various microorganisms, such as sB Candida Inmiicola, Candida iitilis, Ciindida ru / rosa, Bacterduai cadaveris * JEscherichia coil,

10 9 8 3 8/1688 | ^ AD OR1G1MAL

Patent attorneys Dr. W. Andrejewski, Dr, M. Honke, 43 Essen, Kettv / iger Strasse

Klebsieila, pneimiorüae, Arthrobacter simplex, Xanthomonas campestrie and the like. Preventive microorganisms for the ux'findunriSisetn & ßen purpose are Candida hraieola HjSk- (ATCC 20i6h), Candida huraicola 4586 (ATCC 20i? 6iJ), Candida utllie (./11 ¹ CC 20252), Candida rugosa (/.TCC 2026. ^ ), Bacterium cadaveriss (ATCC S> TbO), Eeohcriohia coll ES (ATCC 21252), Klebslella pnoureonlae (ATCC 21524), Arfchrobacter simpler 69> \ 6), and Xyithomonas oeiapeotxdr. (ATCC 7th

The microorganizations mentioned above are boi der / userlcen Tj-pe Culture Collection has been deposited and »teuon der Publickolfc free imi »available.

liciin erli; .idun; -r.ct ^< rr; ': /''. ri \ Vörfiihren hSiiners die ühlltihorweipe i'ür the ZL'Oiituirs of i';i!; roorG &iiisnj'jn voj ^ wronrietcjn carbon quf.; Q en, fit; ic Iratoffquöllen, anor ^ r -d, «eh - jn iial2 © urvi other iiHhrtitofrn ebonfalXs vcti ^ wenrJot Vferdcru tlo bo stand on the lioblcn-IXe-n \ ^ ri: -ir; _; r, tial?; u atjs Such, oiö cmrch uio Im provisi-

ki'Jn-nen, vrlc JJuckcrr.ioffe (z.3. Gluiio ;;; «, f'aecarou« ,. iiolai.se, rfcii.ri'xiiyi'ii.'Olysiii;), or ^ anißcliö traces (sB EssiccaU Kolileo-vr-cserstofiO, An Sticlcntoffouellca are cinc / ini-wnit ^ -iriulfitt, Ar'r.ionit & aiitr & t, IIarnrilOiTf, Arr: oninJc unci c ovi "~ i) isoha Stxc? ^ & Toffi: wiouollc". Iollcnal -antter, l ^ fooxtrakt, Pl'3iöcl "ie:; ti * rait. Ks3 iiraphonphafc, Iiatr3.i? r.: phoi; pbat, iinrnetiViL? -sulfate υ.-π, '. iLiinnen as cnorfraniiScJia ij ^ .lrifs are used »An & c-rB ^ ¹ U-PStOiTo \, lo natural irciimtofto, vitamins u.cU:l.

nt \: oi * don.

10983 8/1688

21082H

Patent attorneys Dr. W. Andrejewski, Dr. M. Honke, 43 Essen, Kettwiger Strasse

Potassium and ammonium ions can be added to the culture medium to increase the ensymatic activity, preferably in a concentration of 10%. The cultivation is carried out at temperatures between 25 and 27 ° C., preferably between 28 and 35 ° C., under aerobic conditions for 10 to 72 hours. The pH of the culture medium is kept between ^ 7 and Io during cultivation, lra special boi 6.5 in yeast and bsi 7, ο 'in bacteria

The crystalline enzyme test preparation obtained from the cultivation is yellowish and has a maximum absorption uptake of up to 420 ßiu and J&Q myx $ also at 280 ma. However, it has also been found that 1 col of this enzyme is coupled with 6 hols of pyridoxal phosphate. When the crystalline enzyme is treated with cysteine, the absorption spectrum passes from 420 nm to? J50 n «a.

Further, when the enzyme is treated with ammonium sulfate, J. an apo-3 enzyme arise. The apo-ensym is colorless and jsaigt no activity. However, its activity can be regained by adding pyridoxal phosphate. The Km value of the

fr

Apo-Enayms to Pyridoxalphoaphat is 2.5x 10 " ^{ M. Furthermore, there is a crystalline preparation of this enzyme evenly if it is there

ORIGINAL

109838/1 688

21082H

Patent attorneys Dr. W. Andrejewski, Dr. M. Honke, 43 Essen, Keüwiger Strasse 36

"Disc" is subjected to electrophoresis and is 11.6 S, and the diffusion index is 4.06x10 "^ cra ² / sec. From these data, a molecular weight of about 277,000 is determined.

Any type of enzyme can be used in the reaction Threonin aldolase used if only by one Microorganism originates and does not damage the reaction, e.g. Along with any I-purple robe corpses, braids perturbations ™ Ilicrobic bodies, broth, a fine crystal, a only partially fine product and the like.

According to another feature of the invention, better ergonomics can be obtained by reacting glycine and formaldehyde in a suitable solvent both in the presence of threonine aldocase, which is prepared before a KiJiroorcian ^. as well as from a SH-Re & rjcn / s. D: sr expression "SIi-Re ^. ^ BeÄölchnot one compound that contains one -CH-Grupp ^, such as SeB. Mercapfcoiithanol, Dithiothreitoi, Glutathione, 2, jS-Dithlogly-se or their Geraisöhe-. The suitable Kor.zcnt ration is depending on the BsakfcionobücUnr.ungen var5.iorcn.Bs is

• «'Ι- -1

jodoeh a consummation of approx. iixio bi3 5x10 ', bos.

ρ
Io Mol Bm most convenient.

The process of the present invention dor v «f ird; o \: Uhnlich in a solvent wäGßrigen voraugsT-ae foi at einsni p _{. of 7 Ms 11 executed , but you can get good results with a p _T1 of the above. 8.5 received. DiG Rciiktlcmafce is 20 to 50 C. Depending hülioi · the temperature, xxrrno Krgebnlsee.

109838/1688 [BAD ORIGINAL

21082U

Paientanv / ctlte Dr. VV. Andrejewski, Dr. M. Honke, 43 Essen, Keftwiger Strasse

After completion of the reaction, the 1-Serin -containing reaction product can be treated in the usual way by an ion-exchange resin process for the purpose of isolating and obtaining! "Serin z" B ". The treated material contains li? Erin in high concentration, so that it is irX ^; It is easy to obtain a considerable amount of the product as compared to the procedure for isolating! Serine according to the S horlcünsnlicbea sources.

When carrying out the reaction according to the invention, the use of equal moles of glysine and formaldehyde is preferable, but the same number of moles is not necessary. The KonEcatratioiion of glysine and formaldehyde varies depending on the activity usrs veri.xncloten Ew ^ na. The reaction can take place without causing serious damage, if

i. '. ^ en are used in glysine and formaldehyde.

'All runs the reaction ai «test at concentra- tion» of pipe as 0.1 \ ι / ν} 6 εη glycine or formaldehyde, too, is it? /; «£! i, advantageously one of the following Vo-bindingöii l- yridoiral ^ hoiiishat and potassium chloride ßen rieaktiont-iiOv-ipotieritea sur implementation of the invention

The naohfoli - / u; 3s Baispiele erlUutcrn 6ie invention · ^ 1 ° in the examples v-u? ^ onuetcxi HikrooivtraiiEkKen sirri at dor American Type Culture Cclleovion on uiiCinrceschri-V.-cter Er-sis, she th: Cx öor Üffeiitlichlrcit free vovftie.bar. All Px ¹ OECn! Tönc.ii'i-'On f'lnd Goviichtsproiicnte, bi.n.lor «e.nd on the Ksdium - viCTiii not taiclers angeloben ·

10 9 8 3 8/1688.

^r ORIGINAL BATHROOM

21082H

Potentanwälte Dr. W. Andrejewski, Dr. M, h'onke, 43 Essen, Kefiwiger Strasse 36

-X-

example 1

Candida hursicola 45 84 (ATCC 20 264) was inoculated on a medium containing a composition of 1-threonine (0.05 #), KU ₂ PO ₂ ,, (0.2 #), (Wlfy) ₂ SOj ^ (0.1 %), MeSO ₁ J.TH ₂ O (0.1Jg) and yeast extract (0.5 #). The resulting Naßzeil ca (0.2 g) were added to a solution containing 0.25 al of 1 Kiol.Glyzln - »0.25 ml of 1 mol. Fomialdeli3? D -, 0.2 ml of a 0.001 mol «fyridoxal phosphate solution, 0.20 ml of a 1 mol. KCl-1.0 ral a 0.2 mol. tris buffer solution (p "10). The reaction was carried out ^{at Jo 0 C in 24 hours.} The amount of 1-serine produced in the reaction mixture was 20 ing.

Example 2

A solution (0.2 g) containing Haßr.ftXlcn, which was obtained from the broths of Arthrobot Simplex (ATCC G $ k6) and from a medium of the composition according to Example 1; a solution of 1 mol of glysine (0 , 23 ml), 1 pint of formaldehyde (0.23 ml) * 0.001 Biol. Solution of pyridoxal phosphate (0.3.0 ml), 1 mol of KCl (0.20 earth) and 0.2 mol of a salic acidic PuiTorlcuivs (1.0 ml j p .. 10) zujpjesetst · This solution was 0.1 Hol Hero apt oethanol (0.2 el.) sugei'ti ^ t. The reaction proceeded at 150 ° C. After 16 stci. It ended and 21.4 kg of serine was obtained.

A reaction similar to that of Vörcenmintcn was added von tierep-pfcol: th: i.nol cußgeftUirt. They t; ar ended after 2 ^ hours, and 13.3 mg of 1-Ser.in were obtained

BATH ORIGINAL

109838/1 688

Patent attorneys Dr. W. Andrejewski, Dr. M. Honke, 43 Essen, Kettwiger Strasse 36

The invention just described, which is placed under protection is to be continued in the following claims

109838/1688 V ₉ * <*** »■

Claims (9)

Patent attorneys Dr. W. Andrejewski, Dr. M. Honke, 43 Essen, Kettwiger Strasse Yo Patent claims: 1 / Process for the production of 1-serine, characterized in that üim \ OXyzIn and formaldehyde condense in the presence of Thr-eonin-Aldolase, which originates from a microorganism. 2. The method of claim 1, characterized in that the Kondertsationsrß ,, & ction in a wiifsßrigen LÜ3ungi: mittol is carried out at p "from 7 to 11 and at a temperature of 20 to 50 ⁰ C. 3. The method according to claim 1, characterized eekeiinzc-icimwt that the concentration of glycine is greater than 0.1 Wv £ ■ . H, method according to claim 1, characterized in that more than 0.1 w / v % formaldehyde has been used. 5. The method according to claim 1, characterized in that the reaction is also countered by pyridoxal phosphate, Potassium chloride or mixtures thereof will, 6. Ancestors according to claim 1, characterized in that gckennzeiclmot the threonine aldolaao from a microorganism int, capable of threonine aldolai; «au pr and dor au der Gri'.p.je göhört that from Candida him J, cc la, Candida utllis, Candida rugona, Baoteriuni eadaverio, Eseherichia eoli, Klsbsiella pnsussjoniao, Arthrobacter simplex or Xanthomon-r.si ear: pöstris- betitaht. . · "■■ · '■ 109838/1688 [ BAD ORIGINAL 21082U Patent attorneys Dr. W. Andrejewski, Dr. M. Honke, 43 years old, Kettwiger Straßo 7. The method according to claim 6, characterized in that the micro-orgsnis-t-us suasevjöhlt is from Candida huirdcola ty5 84 (ATCC 20 Γ: 5'0 * Candida toimieola 4 5 C6 (ATCC 20 265), Candida utilis (ATCC 20 262), Candida rurjoea (A ^ OC 20 265), Bacterium ccd & verls (/.TCC 97 60 ) _s Ecchoricbia coii Ji 8 (ATCC 21 52j5), Klebsieila pn-.imioniae (ATCC 21 5P.-H), / « rtbr-obactor. «siraplox (AI ¹ CC 69 ^ 6) un-i Xanthoraonao ow;? 9ostrio (ATCC 73 81). 8. The method, according to claim 1, öaäurch cekeiuisüichnct, UnQ a Threcüin-ulclolaso ver ^ jendet vitd In Torrn a not 50-rcimlgtcn Prol.uzkoa, \ .- olchoa Bcn-tcwatGilo "or riikrobiellcn stage contains, by oils it produces liurue" , 9. Vor-fahron r.ich Anspi-uch 8, diMurch £; ei-: cnnnsichüDt _f that cli'.c icviin-Alcolano in For-: a dor Kiir ^ ohsnsfillcii veru-cr.det vrj.i;!. ' ^ ¥ erfßl; ri -) - i nach / in - ^ - ruch 1, ordurch ^ o ^ -i-nTizoichnot * i'aö cUo ICoüdcnnabäG'i In Gosenixrt einoi? Ycrl) Aiidu.nr * is thrown through, die cirto -Sli-irrupr 0 Civ :: lJi> lt. 11 «VorT« 'ih "-cJX ntiüb Claim ic, dahi-i-ch c: ^ci ' - ' ^{? I} ^ 2siehnet, tlaß das Sll ^ iicas ^ iii ;: ou -:." Cselects i ^ rt aisa I' ercap & oliU ^ -nal, DithiotrcitoJ., Glutathione, 2, > * Dt tlyl ο -Glycerin ei or tieren Ilir.chunr.en. P. Fehl- · η nach /, ncpruoh 3.0, uitiurcih colcännr-oichnofc, <if «f3 Uio Eorttration fies CH-K -.-:!: Sens 5χ1ΰ"^<1Γ to - ^ 1O * " ¹ Hol bütri ^ t. BAD ORIGINAt 109P38 / ie88