DK153571B - Process for the preparation of D-glucosone - Google Patents
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- DK153571B DK153571B DK040083A DK40083A DK153571B DK 153571 B DK153571 B DK 153571B DK 040083 A DK040083 A DK 040083A DK 40083 A DK40083 A DK 40083A DK 153571 B DK153571 B DK 153571B
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Description
DK 153571 BDK 153571 B
Den foreliggende opfindelse angår en fremgangsmåde til fremstilling af D-glucoson.The present invention relates to a process for the preparation of D-glucosone.
D-glucoson er et velegnet udgangsmateriale til fremstilling af 5 krystallinsk fructose. De enestående fysiske, kemiske og metaboliske egenskaber af krystallinsk fructose giver særlige anvendelsesmuligheder, som ikke findes for g 1ucoseisomerase-fremstillet majssirup med højt fructoseindhold (HFCS), som typisk indeholder 42% fructose, 50% dextrose og 8% polysacchari-der. Medens HFCS er omtrent så sød som saccharose, er krystallinsk fructose ca. 50% sødere og kan anvendes i lavere koncentrationer til frembringelse af den samme sødhed. HFCS konkurrerer primært på de direkte omkostninger med flydende sukkerarter i en lang række traditionelle næringsmiddelanvendelser i 15 enten tør eller flydende form, primært til farmaceutiske anvendelser og i næringsmidler, hvor der ønskes en reduktion af kalorieindholdet pr. sødhedsenhed. Krystallinsk fructose anvendes i diætslik og is og kan hensigtsmæssigt supplere eller erstatte glucose, saccharose eller HFCS i drikkevarer.D-glucosone is a suitable starting material for preparing crystalline fructose. The unique physical, chemical and metabolic properties of crystalline fructose provide special applications not available for g 1ucose isomerase-produced high fructose corn syrup (HFCS), typically containing 42% fructose, 50% dextrose and 8% polysaccharides. While HFCS is about as sweet as sucrose, crystalline fructose is approx. 50% sweeter and can be used at lower concentrations to produce the same sweetness. HFCS primarily competes on the direct cost of liquid sugars in a wide variety of traditional food uses in either dry or liquid form, primarily for pharmaceutical applications and in foods where a reduction in calorie content per day is desired. sødhedsenhed. Crystalline fructose is used in diet sweets and ice creams and can conveniently supplement or replace glucose, sucrose or HFCS in beverages.
2020
Fremstillingen af krystallinsk fructose i kommerciel målestok har stødt på en række alvorlige problemer.The production of crystalline fructose on a commercial scale has encountered a number of serious problems.
Et af de største problemer er fremstillingsprisen. Den eneste kommercielt acceptable produktionsmetode hidtil var baseret på 25 først at fremstille en glucose-fructose-sirupblanding ud fra glucose (under anvendelse af enten glucoseisomerase eller alkalisk isomerisering) eller saccharose (via invertsukker), derpå fysisk at separere de to sukkerarter (ionbytning eller selektiv calciumsaltudfældning) og endelig at udvinde fructo-30 sen fra den vandige opløsning i krystallinsk form (podning eller metanoludfældning).! Den fysiske separeringsbehandling af de to sukkerarter er nødvendig, da krystallinsk fructose i bedste fald vanskeligt og hyppigt umuligt kan udvindes fra en vandig opløsning, med mindre i alt væsentligt alle ioniske 35 stoffer, restglucose og andre forureninger fjernes.15 En sådan behandling er dyr og resulterer derfor i en høj markedspris for fructose - en pris, som ikke kan konkurrere med rørsukker.One of the biggest problems is the manufacturing cost. The only commercially acceptable production method so far was based on first preparing a glucose fructose syrup mixture from glucose (using either glucose isomerase or alkaline isomerization) or sucrose (via invert sugar), then physically separating the two sugars (ion exchange or selective calcium salt precipitation) and finally to recover the fructose from the aqueous solution in crystalline form (grafting or methanol precipitation). The physical separation treatment of the two sugars is necessary since crystalline fructose can be difficult and frequently impossible to recover from an aqueous solution at best, unless substantially all ionic substances, residual glucose and other contaminants are removed.15 Such treatment is expensive and therefore results in a high market price for fructose - a price that cannot compete with cane sugar.
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Litteraturen angiver, at lave koncentrationer af fructose kan fremstilles ud fra glucose. Så tidligt som i 1889 blev fructose dannet ved reduktion af D-glucoson med zinkpulver i vandig eddikesyre.6 Denne reduktion er blevet anvendt som en analytisk 5 test til bestemmelsen af D-glucoson i adskillige biologiske stoffer.8»'12 Reduktion af D-glucoson til D-fructose er også blevet gennemført med natriumborhydrid.I8 Omdannelse af D-glucoson til fructose er en mulig enzymatisk reaktion, da re-duktaser, som gennemfører reduktionen af -CHO--CH2OH, ken- 10 des.I*The literature indicates that low concentrations of fructose can be prepared from glucose. As early as 1889, fructose was formed by reduction of D-glucosone with zinc powder in aqueous acetic acid.6 This reduction has been used as an analytical test for the determination of D-glucosone in several biological substances.8 »'12 Reduction of D-glucosone Glucosone for D-fructose has also been carried out with sodium borohydride. Conversion of D-glucosone to fructose is a possible enzymatic reaction, as reductases which effect the reduction of -CHO - CH2OH are known.
Litteraturen angiver også, at lave koncentrationer af glucoson kan fremstilles ud fra glucose. Der er blevet beskrevet adskillige metoder til at oxidere glucose - med 1^024 eller med 15 kobberacetat.5 I hver af disse reaktioner er omdannelsesudbytterne lave (<30%), og der opstår mange biprodukter. Glucose er blevet omdannet til D-glucoson ved først at fremstille et derivat af glucosen (f.eks. ved omsætning med phenyl hydrazin til fremstilling af glucosazon6 eller ved omsætning med p-to-20 luidin7) og derpå kemisk behandle dette derivat til dannelse af D-glucoson. I disse reaktioner var udbytterne ikke over 50%, og de reagenser, der ikke genvindes, er for dyre til kommerciel brug. Anvendelsen af aromaholdige reagenser kunne desuden give problemer i forbindelse med opnåelse af sukkerarter 25 af rimelig kvalitet.The literature also indicates that low concentrations of glucosone can be prepared from glucose. Several methods have been described for oxidizing glucose - with 1 ^ 024 or with 15 copper acetate.5 In each of these reactions, the conversion yields are low (<30%) and many by-products occur. Glucose has been converted to D-glucosone by first preparing a derivative of the glucose (for example, by reaction with phenyl hydrazine to produce glucosazone 6 or by reaction with p-to-luidin7) and then chemically treating this derivative to form D-glucosone. In these reactions, the yields were not more than 50% and the non-recovered reagents are too expensive for commercial use. The use of flavoring reagents could additionally cause problems in obtaining sugars of reasonable quality.
Omdannelse af glucose til D-glucoson er også en kendt enzymatisk reaktion. Så tidligt som i 1932 blev det anført, at glucose blev oxideret til D-glucoson af den krystallinske form for en mollusk, Saxidomus giganteous.8 I 1937 blev det anført, 30 at D-glucoson dannes ved oxidationen af glucose, stivelse, maltose eller saccharose med plasmolyserede præparater af to skimmelsvampe, Aspergillus parasiticus og Aspergillus flavus-oryzae.Conversion of glucose to D-glucosone is also a known enzymatic reaction. As early as 1932, it was stated that glucose was oxidized to D-glucosone by the crystalline form of a mollusk, Saxidomus giganteous.8 In 1937, 30 was stated that D-glucosone is formed by the oxidation of glucose, starch, maltose or sucrose with plasmolyzed preparations of two molds, Aspergillus parasiticus and Aspergillus flavus oryzae.
35 i 1956 blev den enzymatiske oxidation af glucose til glucoson i en rød alge, Iridophycus flaccidum,10 beskrevet. En carbohy-dratoxydase blev isoleret fra mycelium af Basidiomyceten, Po-lyporus obtusus, som oxiderede glucose til D-glucoson.** Der 3In 1956, the enzymatic oxidation of glucose to glucosone in a red alga, Iridophycus flaccidum, 10 was described. A carbohydrate oxidase was isolated from the mycelium of the Basidiomycete, Polypyporus obtusus, which oxidized glucose to D-glucosone. ** There 3
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blev ikke angivet noget om udbytter. Endelig blev der i 1978 påvist glucose-2-oxidase-aktivitet i basidiomyceten, Oudeman-siella mucida, såvel som i andre træforrådnende svampe.oe bedste udbytter, der blev angivet, var ikke over 50%. I alle 5 disse tilfælde antages det, at D-glucosonproduktionen er ledsaget af dannelsen af hydrogenperoxid.nothing was stated about yields. Finally, in 1978, glucose-2-oxidase activity was detected in the basidiomycetine, Oudeman-siella mucida, as well as in other wood-decomposing fungi, and the best yields reported were not more than 50%. In all 5 of these cases, it is believed that D-glucosone production is accompanied by hydrogen peroxide formation.
Det har nu vist sig, at der kan fremstilles D-glucoson i højt udbytte ud fra glucose ved en fremgangsmåde, som er ejendom-1Q melig ved, at man tilvejebringer en vandig opløsning af glucose og omdanner ca. 95% af glucosen i opløsning til D-gluco-son ved enzymatisk oxidation med et oxidoreduktaseenzym i nærværelse af oxygen, mens man fjerner eller udnytter co-produce-ret hydrogenperoxid.It has now been found that in high yield, D-glucosone can be prepared from glucose by a process which is property-like by providing an aqueous solution of glucose and converting ca. 95% of the glucose in solution to D-gluconone by enzymatic oxidation with an oxidoreductase enzyme in the presence of oxygen while removing or utilizing co-produced hydrogen peroxide.
1515
Opfindelsen forklares nærmere under henvisning til de medfølgende tegninger, hvori fig. 1 er et skematisk diagram over et foretrukket eksempel på fremgangsmåden ifølge opfindelsen, 20 fig. 2 viser molekylstrukturen af D-glucose, fig. 3 viser molekylstrukturen af D-glucoson, og fig. 4 viser molekylstrukturen af D-fructose.The invention is explained in more detail with reference to the accompanying drawings, in which fig. 1 is a schematic diagram of a preferred example of the method of the invention; FIG. Figure 2 shows the molecular structure of D-glucose; 3 shows the molecular structure of D-glucosone; and FIG. 4 shows the molecular structure of D-fructose.
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Med hensyn til molekylstrukturen af D-glucoson vil fagfolk på dette område vide, at det er blevet hævdet, at der eksisterer adskillige andre former af molekylet i vandig opløsning. En række af disse er cykliske.2'3 30 I den foreliggende beskrivelse anvendes udtrykkene "glucose", "D-glucose" og "dextrose" skiftevis til at omfatte dette møno-saccharid i en eller anden form - opløsning eller tør. Figur 2 betegner glucose.As to the molecular structure of D-glucosone, those skilled in the art will know that it has been claimed that several other forms of the molecule exist in aqueous solution. A number of these are cyclic.23 In the present specification, the terms "glucose", "D-glucose" and "dextrose" are used interchangeably to include this monosaccharide in some form - solution or dry. Figure 2 represents glucose.
35 I den foreliggende beskrivelse anvendes udtrykkene "D-glucoson" og "D-arabino-2-hexosulose" i flæng. Figur 3 betegner D-glucoson.In the present specification the terms "D-glucosone" and "D-arabino-2-hexosulose" are used interchangeably. Figure 3 represents D-glucosone.
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I den foreliggende beskrivelse betegner udtrykkene "fructose", "D-fructose" og "levulose" ensbetydende den isomer af glucose, som er sødere end glucose. Udtrykket "krystallinsk fructose" anvendes i den foreliggende tekst til at betegne 5 dette monosaccharid i vandfri form. Figur 4 viser fructose.In the present description, the terms "fructose", "D-fructose" and "levulose" mean the isomer of glucose which is sweeter than glucose. The term "crystalline fructose" is used in the present text to denote this monosaccharide in anhydrous form. Figure 4 shows fructose.
Ifølge den foreliggende opfindelse omdannes generelt sagt glucose i vandig opløsning enzymatisk til D-glucoson med et passende enzym såsom carbohydratoxidase eller giucose-2-oxidase. j0 Denne omdannelse får lov til at forløbe spontant, hurtigt og i alt væsentligt fuldstændigt. D-glucoson kan så uden yderligere isolering omdannes til fructose ved passende kemisk hydrogenering.In general, according to the present invention, glucose in aqueous solution is enzymatically converted to D-glucosone with a suitable enzyme such as carbohydrate oxidase or giucose-2 oxidase. j0 This transformation is allowed to proceed spontaneously, quickly and substantially completely. Then, without further isolation, D-glucosone can be converted to fructose by appropriate chemical hydrogenation.
Som nævnt og henvist til ovenfor kendes 1avkoncentrationsom-1 5 dannelse af glucose til D-glucoson. Ifølge den foreliggende opfindelse omdannes glucose let og fuldstændigt til D-glucoson under anvendelse af et renset oxidoreduktaseenzym såsom carbohydratoxidase eller giucose-2-oxidase. Det ved den foreliggende opfindelse opnåede udbytte overstiger det i litteraturen 20 beskrevne for dette enzym. Denne høje omdannelse til D-glucoson fjerner behovet for fysisk at separere eventuelt reste rende uomdannet glucose.As mentioned and referred to above, the concentration of glucose to D-glucosone is known. According to the present invention, glucose is readily and completely converted to D-glucosone using a purified oxidoreductase enzyme such as carbohydrate oxidase or giucose-2 oxidase. The yield obtained by the present invention exceeds that described in literature 20 for this enzyme. This high conversion to D-glucosone eliminates the need to physically separate any residual unchanged glucose.
Visse oxidoreduktaser har katalytisk virkning med hensyn til 25 at oxidere hydroxylgruppen på det andet carbon af glucose, idet de fremmer oxidationen af denne hydroxylgruppe til en keto-gruppe, f.eks. ved omdannelse af strukturen i fig. 2 til strukturen i fig. 3. Disse specifikke oxidoreduktaseenzymer, der er beskrevet i eksemplerne heri, omtales varierende som 30 "glucose-2-oxidase", "pyranose-2-oxidase" og "carbohydratoxi dase", men opfindelsen er ikke nødvendigvis begrænset til således betegnede enzymer. Giucose-2-oxidase besidder en høj specificitet over for glucose som substrat, hvorimod carbohydratoxidase, selv om det har glucose som foretrukket substrat, 35 har en bredere substratspecificitet. Ethvert enzym, som er i stand til at omdanne hydroxylgruppen på det andet carbon af glucose til en ketogruppe og ikke ellers væsentligt påvirke resten af giucosemolekylet, falder inden for den foreliggende op- 5Certain oxidoreductases have a catalytic effect in oxidizing the hydroxyl group on the second carbon of glucose, promoting the oxidation of this hydroxyl group to a keto group, e.g. by converting the structure of FIG. 2 to the structure of FIG. 3. These specific oxidoreductase enzymes described in the Examples herein are variously referred to as "glucose-2-oxidase", "pyranose-2-oxidase" and "carbohydrate oxy-dase", but the invention is not necessarily limited to such enzymes. Giucose-2 oxidase has a high specificity to glucose as a substrate, whereas carbohydrate oxidase, although it has glucose as the preferred substrate, has a broader substrate specificity. Any enzyme capable of converting the hydroxyl group on the second carbon of glucose into a keto group and not otherwise significantly affecting the remainder of the giucose molecule falls within the present invention.
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findelses omfang. Et sådant enzym kan specificeres som et enzym, der har glucose-2-oxidaseaktivitet.scope of invention. Such an enzyme can be specified as an enzyme having glucose-2-oxidase activity.
Et foretrukket carbohydratoxidaseenzym er afledt af mikroorga-5 nismen Polyporus obtusus. Kilder for glucose-2-oxidase omfatter adskillige andre mikroorganismer, mollusker og røde alger, som refereret ovenfor. Disse enzymer og deres kilder er kun indikative og skal ikke betragtes som omfattende alle egnede enzymer og deres kilder inden for den foreliggende opfindelses omfang.A preferred carbohydrate oxidase enzyme is derived from the microorganism Polyporus obtusus. Sources of glucose-2 oxidase include several other microorganisms, mollusks and red algae, as referenced above. These enzymes and their sources are indicative only and are not to be considered as encompassing all suitable enzymes and their sources within the scope of the present invention.
1010
For at lette omtalen vil forskellige træk ved den foreliggende opfindelse blive beskrevet specielt, men ikke udelukkende i forbindelse med anvendelsen af den foretrukne carbohydratoxi- dase fra Polyporus obtusus og glucose-2-oxidasen fra Aspergil- 1 5 lus oryzae. Mikroorganismerne kan dyrkes i bevæget, neddyppet kultur ved stuetemperatur ved hjælp af sædvanlige metoder. Enzymet fremstilles ud fra mycelier af mikroorganismen, der dyrkes under betingelser for bevæget, neddyppet kultur.In order to facilitate the discussion, various features of the present invention will be described specifically but not exclusively in connection with the use of the preferred carbohydrate oxidase from Polyporus obtusus and the glucose-2 oxidase from Aspergil-15 lus oryzae. The microorganisms can be grown in stirred, immersed culture at room temperature by conventional methods. The enzyme is prepared from mycelia of the microorganism grown under conditions of moving, immersed culture.
20 Enzymet anvendes fortrinsvis i immobi1iseret form, selv om frit enzym også kan anvendes. Fremgangsmåderne til enzymimmobilisering er velkendte for fagfolk på dette område og består af omsætning af en opløsning af enzymet med et af et stort antal overfladebehandlede eller -ubehandlede organiske og uorga-25 niske støttematerialer. Indbefattet heri er polyacrylamid, ethylenmaleinsyrecopolymerer, methacrylbaserede polymerer, po-lypeptider, styrenbaserede polymerer, agarose, cellulose, dex-tran, silica, porøse plasperler, trækul eller carbon black, træ og savsmuld, hydroxyapatit og aluminium- eller 30 titanhydroxid. Enzymer i denne form har forøget stabilitet, forlænget levetid og anvendelighed og udvi ndelighéd. Reaktioner, hvori der anvendes immobi1iserede enzymer, kan gennemføres i søjler eller reaktionsbeholdere eller andre egnede reaktorer .The enzyme is preferably used in immobilized form, although free enzyme can also be used. The methods of enzyme immobilization are well known to those skilled in the art and consist of reacting a solution of the enzyme with one of a large number of surface-treated or untreated organic and inorganic support materials. Included herein are polyacrylamide, ethylene-maleic acid copolymers, methacrylic-based polymers, polypeptides, styrene-based polymers, agarose, cellulose, dextran, silica, porous beads, charcoal or carbon black, wood and sawdust, hydroxyapatite and aluminum or titanium hydroxide. Enzymes in this form have increased stability, extended life and usefulness and extensibility. Reactions using immobilized enzymes can be carried out in columns or reaction vessels or other suitable reactors.
3535
Foruden carbohydratoxidase- eller giucose-2-oxidaseenzymerne behøves en kilde for oxygen. En fremgangsmåde til hydrogenper-oxidfjernelse eller -udnyttelse kræves også i reaktionen til 6In addition to the carbohydrate oxidase or giucose-2 oxidase enzymes, a source of oxygen is needed. A process for hydrogen peroxide removal or utilization is also required in the reaction to 6
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omdannelse af glucose til D-glucoson på den mest effektive måde. Dette skyldes, at H2O2 oxiderer visse kritiske steder på enzymmolekylet, hvilket beskadiger dets funktion. Fremgangsmåder til hydrogenperoxidfjernelse omfatter (1) dekomponering 5 ved hjælp af enzymet katalase, (2) dekomponering ved hjælp af kendte kemiske midler og (3) dekomponering ved anvendelse af dekomponeringsmatrikser såsom manganoxid eller carbon black16'17 som det immobi1iserende støttemateriale for oxidoreduktaseenzymet. I en foretrukket alternativ metode kan 10 det dannede hydrogenperoxid, fremfor at blive dekomponeret, blive forbrugt til fremstilling af et værdifuldt co-produkt. Sammenkobling af D-glucosonfremst i 11 ing med propylenhalohy-drin- eller propylenoxidfremsti 11ing er et foretrukket eksempel som vist i fig. 1.conversion of glucose to D-glucosone in the most efficient way. This is because H2O2 oxidizes certain critical sites on the enzyme molecule, which damages its function. Methods for hydrogen peroxide removal include (1) decomposition 5 by the enzyme catalase, (2) decomposition by known chemical means and (3) decomposition using decomposition matrices such as manganese oxide or carbon black16'17 as the immobilizing support material for the oxidoreductase enzyme. In a preferred alternative method, the hydrogen peroxide formed, rather than being decomposed, can be consumed to produce a valuable co-product. Coupling D-glucosone production in 11 with propylene halohydrin or propylene oxide preparation is a preferred example as shown in FIG. First
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Den enzymatiske omdannelse af glucose til D-glucoson gennemføres fortrinsvis i vand ved omtrent neutral pH-værdi, men kan gennemføres inden for pH-værdiinterval let fra ca. tre til ca. otte ved anvendelse af passende puffere. Denne omdannelse gen-,0 nemføres fortrinsvis ved omgivelsernes temperatur, men kan gennemføres inden for temperatur interval1 et fra ca. 15°C til ca. 65°C. Trykket er fortrinsvis atmosfærisk tryk, men kan variere fra tryk under til tryk over atmosfæretryk. Ethvert carbohy-dratmateri ale, som ad kemisk eller enzymatisk vej giver glu-25 cose, er en egnet kilde for glucose til omdannelse til D-glucose. Disse stoffer omfatter, men er ikke begrænset til følgende: cellulose, stivelse, saccharose, majssirup, HFCS og andre sirupper indeholdende forskellige mængder af glucose og fructose.The enzymatic conversion of glucose to D-glucosone is preferably carried out in water at approximately neutral pH, but can be carried out within the pH value range easily from ca. three to approx. eight using appropriate buffers. This conversion is preferably carried out at ambient temperature, but can be carried out within the temperature range of from approx. 15 ° C to approx. 65 ° C. The pressure is preferably atmospheric pressure, but may vary from below pressure to atmospheric pressure. Any carbohydrate material that provides glucose or chemically through glucose is a suitable source of glucose for conversion to D-glucose. These substances include, but are not limited to, the following: cellulose, starch, sucrose, corn syrup, HFCS and other syrups containing various amounts of glucose and fructose.
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Prøverne, som anvendtes til at analysere sukkerarterne, er givet nedenfor: 1. Tyndt 1agskromatografi (TLC). Avicelbel agte glasplader fremkaldes med et 8:8:2:4 (efter volumen) isopropanol:pyridin:ed-35 dikesyre:vand-opløsningsmiddelsystem. Glucose har en Rf-værdi på 0,5-0,6. D-glucoson har en Rf på 0,4-0,5 streg. (Rf = den afstand, som stoffet vandrer fra begyndelsespunktet/opløs-ningsmiddelfrontafstanden fra begyndelsespunktet). Når pla- 7The samples used to analyze the sugars are given below: 1. Thin Layer Chromatography (TLC). Avicelbel like glass plates are developed with an 8: 8: 2: 4 (by volume) isopropanol: pyridine: acetic acid: water-solvent system. Glucose has an Rf value of 0.5-0.6. D-glucosone has an Rf of 0.4-0.5 bar. (Rf = the distance that the substance travels from the starting point / solvent front distance from the starting point). When pla- 7
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derne sprøjtes med tri phenyltetrazoliumchlorid (2% TTC i 0,5N NaOH), giver D-glucoson øjeblikkeligt røde pletter; glucose giver kun en rød plet efter opvarmning i 1Q min ved 100°C, Når pladerne sprøjtes med diphenylamin/ani1 in/phosphorsyre/ethy1-5 acetat-reagens (0,15 g/0,8 ml/11 ml/100 ml), giver glucose en brun plet, og D-glucoson giver en purpurfarvet streg.there is sprayed with tri phenyltetrazolium chloride (2% TTC in 0.5N NaOH), immediately giving D-glucosone red spots; glucose gives only a red spot after heating for 1Q min at 100 ° C, When the plates are sprayed with diphenylamine / ani1 in / phosphoric acid / ethyl 5 acetate reagent (0.15 g / 0.8 ml / 11 ml / 100 ml) , glucose gives a brown stain, and D-glucosone gives a purple streak.
2. Højeffektiv væskekromatografi (HPLC). En μ-Bondapak-carbo-hydratsøjle indkøbt hos Waters Associates køres med 15% vandig acetonitril indeholdende 0,001 M kaliumphosphatpuffer, pH-vær-di 7, ved en strømningshastighed på 2 ml/min. Glucose har en retentionstid R* på 11,5 og D-glucoson en R* på 14,0. Der gennemføres forsøg på et spektra physics SP8000-instrument under anvendelse af både en Waters Associates-brydningsindeksdetek-tor og en Schoeffel-UV-detektor med variabel bølgelængde indstillet til 192 nm.2. High performance liquid chromatography (HPLC). A μ-Bondapak carbohydrate column purchased from Waters Associates is run with 15% aqueous acetonitrile containing 0.001 M potassium phosphate buffer, pH 7, at a flow rate of 2 ml / min. Glucose has a retention time R * of 11.5 and D-glucosone an R * of 14.0. Experiments are performed on a spectra physics SP8000 instrument using both a Waters Associates refractive index detector and a Schoeffel UV variable wavelength detector set to 192 nm.
3. Massespektrometri (MS). Den følgende derivatiseringsproto-kol anvendes til at gøre de kemiske komponenter flygtige: 20 (a) Til ca. 100 mg af den lyofi 1 iserede prøve sættes 110 mg N,N-di phenylhydraziη (H2NN02) og 1 ml 75% vandig ethanol. Reaktionsblandingen omhvirvles og får derpå lov til at henstå natten over ved stuetemperatur.3. Mass Spectrometry (MS). The following derivatization protocol is used to make the chemical components volatile: 20 (a) 100 mg of the lyophilized 1 sample is added 110 mg of N, N-di phenylhydraziη (H2NNO2) and 1 ml of 75% aqueous ethanol. The reaction mixture is vortexed and then allowed to stand overnight at room temperature.
(b) 3 ml vand sættes til reaktionsblandingen, og det resulte- 2 5 rende bundfald skilles fra supernatanten ved centrifugering og dekantering. Til dette bundfald sættes 1 ml af en l:l-blanding af pyridin/eddikesyreanhydrid. Reaktionsblandingen anbringes i et 35-40°C vandbad i 15 min med lejlighedvis omrystning.(b) 3 ml of water is added to the reaction mixture and the resulting precipitate is separated from the supernatant by centrifugation and decanting. To this precipitate is added 1 ml of a 1: 1 mixture of pyridine / acetic anhydride. The reaction mixture is placed in a 35-40 ° C water bath for 15 minutes with occasional shaking.
30 (c) 2 ml vand tilsættes for at standse reaktionen, og derpå ekstraheres blandingen 2 gange med 3 ml portioner af ethyl-ether. Etheren tørres over en ringe mængde vandfrit natriumsulfat, og derpå afdrives etheren ved svag opvarmning (-40°C) og strømmende nitrogen.(C) 2 ml of water is added to quench the reaction and then the mixture is extracted twice with 3 ml portions of ethyl ether. The ether is dried over a small amount of anhydrous sodium sulfate and then the ether is stripped off by gentle heating (-40 ° C) and flowing nitrogen.
35 (d) Det resulterende faste stof eller den resulterende sirup er klar til massespektrometrisk analyse.(D) The resulting solid or resultant syrup is ready for mass spectrometric analysis.
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Forventede reaktioner: -CHO -CH=NNø2 >C=0 (1) h2NNø2-^ ^C=0 (upåvirket) 5 -OH (2) Ac20, pyridin -OAcExpected reactions: -CHO -CH = NNo2> C = O (1) h2NNo2- ^ C = O (unaffected) 5 -OH (2) Ac2O, pyridine -OAc
Glucose giver en molekylion ved masse 556 (C28H32N2°lo) °S en baseion ved masse 168 (Nø2-ionfragment); D-glucoson giver en molekylion ved masse 512 (C2gH28N20g) og en intens diagnostisk fragmention ved masse 223 (OC-CH=N-Nø2-ionfragment). Forsøg 10 foretages på et Finnigan GC/MS/DS Model 4023-instrument indstillet til 70 eV elektron-impact-ionisering og til en sendetemperatur på 220°C.Glucose gives a molecular ion at mass 556 (C28H32N2 ° lo) ° S a base ion at mass 168 (No2 ion fragment); D-glucosone provides a molecular ion at mass 512 (C2gH28N2Og) and an intense diagnostic fragment ion at mass 223 (OC-CH = N-No2 ion fragment). Experiment 10 is performed on a Finnigan GC / MS / DS Model 4023 instrument set for 70 eV electron impact ionization and to a transmit temperature of 220 ° C.
4. Kolometrisk testrørsprøve. Analysen af D-glucoson i nærvæ-15 relse af et overskud af glucose bestemmes let ved hjælp af to kolometriske metoder. Under anvendelse af triphenyltetrazoli-umchlorid (TTC) er bestemmelsesmetoden baseret på en differential reduktionshastighed af de to sukkerarter. Til et 20 ml testrør sættes 0,5 ml prøve plus 0,1 ml 1% vandig TTC plus 0,4 20 ml 6N NaOH. Efter nøjagtigt 5 min forløb tilsættes 15 ml eddi-kesyre/ethanol (1:9), og indholdet i testrøret omhvirvles. Med vand som blindprøve måles absorbansen ved 480 nm under anvendelse af en Varian 635 UV/VIS-spektrometer. Glucose reducerer TTC til et rødt pigment, en triphenylformazan, ca. 100 gange 25 langsommere end en ækvivalent mængde D-glucoson. Under anvendelse af diphenylamin/anilin/phosphorsyrereagens er bestemmelsesmetoden baseret på de forskellige farver, der dannes med sukkerarter af forskellige strukturer. Til et 20 ml testrør sættes 0,2 ml prøve plus 5 ml af den følgende reagensblanding: 30 diphenylamin 0,15 g anilin 0,80 ml isopropanol 100 ml phosphorsyre 11 ml 354. Colometric test tube sample. The analysis of D-glucosone in the presence of an excess of glucose is readily determined by two colometric methods. Using triphenyl tetrazolium chloride (TTC), the method of determination is based on a differential rate of reduction of the two sugars. To a 20 ml test tube is added 0.5 ml of sample plus 0.1 ml of 1% aqueous TTC plus 0.4 20 ml of 6N NaOH. After exactly 5 minutes, 15 ml of acetic acid / ethanol (1: 9) is added and the contents of the test tube are swirled. With water as the blank, absorbance is measured at 480 nm using a Varian 635 UV / VIS spectrometer. Glucose reduces TTC to a red pigment, a triphenylformazan, approx. 100 times 25 slower than an equivalent amount of D-glucosone. Using diphenylamine / aniline / phosphoric acid reagent, the assay method is based on the different colors formed with sugars of different structures. To a 20 ml test tube is added 0.2 ml of sample plus 5 ml of the following reagent mixture: 30 diphenylamine 0.15 g aniline 0.80 ml isopropanol 100 ml phosphoric acid 11 ml
Testrørene anbringes i 37°C vandbad i 60 min. Glucose giver en gulfarvet opløsning; D-glucoson giver en purpurfarvet opløsning.The test tubes are placed in a 37 ° C water bath for 60 minutes. Glucose gives a yellow-colored solution; D-glucosone provides a purple solution.
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Kilder for de rene sukkerarter, der anvendes i de forskellige analytiske aspekter, er angivet nedenfor: 1. D-glucose blev indkøbt fra Applied Science Laboratories, c 99% rent.Sources of the pure sugars used in the various analytical aspects are listed below: 1. D-glucose was purchased from Applied Science Laboratories, c 99% pure.
OISLAND
2. D-glucoson blev syntetiseret kemisk ved hjælp af følgende metode: 20 g glucose blandes med 1 liter destilleret vand indeholdende 27 ml iseddike. 44 g phenylhydrazin tilsættes. Reaktionen gennemføres i 3 timer ved 80°C under kraftig omrøring 10 ved hjælp af en mekanisk omrører og afkøles derpå til stuetemperatur natten over. Det faste stof filtreres fra og vaskes med 10% eddikesyre, vand og derpå ethylether. Det faste stof tørres omhyggeligt i en vakuumovn ved 50eC. Forsøgsudbyttet er 16,1 g glucosazon. Glucosazonet anbringes i en 3-halset, 3 15 liter kolbe, og 450 ml ethanol, 750 ml destilleret vand og 9 ml iseddike tilsættes. 27,8 g frisk benzaldehyd tilsættes og bringes til tilbagesvaling med kraftig omrøring ved hjælp af en mekanisk omrører. Reaktionen tilbagesvales i 5 timer. Svaleren vendes, og 450 ml destilleres over, idet der tilsættes 2 0 750 ml destilleret vand (via en tildrypningstragt) til kolbén. Reaktionsblandingen afkøles natten over for at tillade udfældning af benzaldehydphenylhydrazon. Opløsningen filtreres, og resten vaskes med destilleret vand (~1 liter), indtil vandet bliver klart. Filtratet plus vaskevæskerne koncentreres til 25 500 ml og ekstraheres derpå med 10 gange 300 ml portioner ethylether. For at slippe af med resterende ethylether i den vandige opløsning anbringes den på en rotationsfordamper i 30 min. Den vandige opløsning ledes gennem en 4 gange 100 cm søjle indeholdende omhyggeligt acetone-vasket Amberlite XAD-2.2. D-glucosone was chemically synthesized by the following method: Mix 20 g of glucose with 1 liter of distilled water containing 27 ml of glacial acetic acid. 44 g of phenylhydrazine are added. The reaction is carried out for 3 hours at 80 ° C with vigorous stirring 10 by means of a mechanical stirrer and then cooled to room temperature overnight. The solid is filtered off and washed with 10% acetic acid, water and then ethyl ether. The solid is carefully dried in a vacuum oven at 50 ° C. The experimental yield is 16.1 g of glucosazone. The glucose zone is placed in a 3-neck, 3-liter flask and 450 ml of ethanol, 750 ml of distilled water and 9 ml of glacial acetic acid are added. 27.8 g of fresh benzaldehyde are added and refluxed with vigorous stirring by means of a mechanical stirrer. The reaction is refluxed for 5 hours. The condenser is inverted and 450 ml is distilled over, adding 2 750 ml of distilled water (via a drip funnel) to the flask. The reaction mixture is cooled overnight to allow precipitation of benzaldehyde phenylhydrazone. The solution is filtered and the residue is washed with distilled water (~ 1 liter) until the water becomes clear. The filtrate plus the washings are concentrated to 25,500 ml and then extracted with 10 x 300 ml portions of ethyl ether. To get rid of residual ethyl ether in the aqueous solution, place it on a rotary evaporator for 30 minutes. The aqueous solution is passed through a 4 x 100 cm column containing carefully acetone-washed Amberlite XAD-2.
30 Søjlen vaskes med yderligere 200 ml vand for at fjerne resterende glucoson. De forenede vandige portioner lyofi 1iseres. Forsøgsudbytte af glucoson er 3,4 g (16% samlet udbytte).The column is washed with an additional 200 ml of water to remove residual glucosone. The combined aqueous portions are lyophilized. The experimental yield of glucosone is 3.4 g (16% overall yield).
De følgende eksempler belyser fremgangsmåden ifølge opfindel-35 sen. Med mindre andet er anført, er alle temperaturerne omgivelsernes temperatur (ca. 25°C) og alle tryk omgivelsernes tryk (ca. 1 atm).The following examples illustrate the method of the invention. Unless otherwise stated, all temperatures are ambient temperature (about 25 ° C) and all pressure ambient pressure (about 1 atm).
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Eksempel 1 I alt væsentligt fuldstændig omdannelse af · glucose til D-glu-coson under anvendelse af immobi1iseret carbohydratoxidase er 5 vist i dette eksempel.Example 1 Substantially complete conversion of glucose to D-glucosone using immobilized carbohydrate oxidase is shown in this example.
Glucose (2 g) sættes til 20 ml destilleret vand i en 100 ml Pyrex-kolbe, og sukkeropløsningen omrøres. Oxygengas bobles ind i kolben, og 3 mg katalase (Sigma Chemical Co., C-40, fra okselever) tilsættes. Agaroseimmobi1iseret carbohydratoxidase 10 fremstillet som angivet nedenfor ud fra 200 ml kultur sættes også til kolben.Glucose (2 g) is added to 20 ml of distilled water in a 100 ml Pyrex flask and the sugar solution is stirred. Oxygen gas is bubbled into the flask and 3 mg of catalase (Sigma Chemical Co., C-40, from bovine liver) is added. Agarose-immobilized carbohydrate oxidase 10 prepared as indicated below from 200 ml of culture is also added to the flask.
Til fremstilling af enzymet dyrkes mycelpuder af Polyporus ob-tusus ATCC nr. 26733 på skråstivnede gær/maltekstraktagarsub-15 strater som følger: gærekstrakt (3 g), maltekstrakt (3 g), agar (20 g), pepton (5 g) og glucose (10 g) sættes til destilleret vand (1 liter), og pH-værdien indstilles på 6,7. Mediet steriliseres ved 121eC i 15 min. pH-værdien indstilles så på 6,4. Organismen podes på de skråstivnede agarsubstrater og 20 . dyrkes i 7 dage ved 25°C. Den på det skråstivnede substrat dyrkede organisme anvendes derpå til podning af gær/maltek-straktmedium (20 ml medium i 125 ml Erlenmeyerkolbe), fremstillet som ovenfor (men uden tilsætning af agar). Organismen dyrkes i 9 dage på et roterende rysteapparat ved 25°C. Kultu-25 ren vakuumfiltreres gennem Whatman papir nr. 541 i en Buchner-tragt. De på filterpapiret tilbageholdte mycelier indeholder enzymet.For the preparation of the enzyme, mycelium pads of Polyporus obtusus ATCC No. 26733 are grown on oblique yeast / malt extract agar substrates as follows: yeast extract (3 g), malt extract (3 g), agar (20 g), peptone (5 g) and glucose (10 g) is added to distilled water (1 liter) and the pH is adjusted to 6.7. The medium is sterilized at 121 ° C for 15 min. The pH is then adjusted to 6.4. The organism is seeded on the oblique agar substrates and 20. grown for 7 days at 25 ° C. The organism grown on the inclined substrate is then used to inoculate yeast / malt extract medium (20 ml of medium into 125 ml of Erlenmeyer flask) prepared as above (but without the addition of agar). The organism is grown for 9 days on a rotary shaker at 25 ° C. The culture is vacuum filtered through Whatman paper # 541 in a Buchner funnel. The mycelia retained on the filter paper contain the enzyme.
Mycelierne, opnåede af 400 ml kultur, vaskes to gange med 0,05M 30 kal iumphosphatpuffer ved pH-værdi 7,0. Mycelierne anbringes så i et Waringblandeapparat, som indeholder 70 ml 0,05M kalium-phosphatpuffer ved pH-værdi 7,0 og homogeniseres derpå i 3 min. Blandingen centrifugeres så ved 6000 omdr. pr. min i 20 min, og supernatanten dekanteres fra det faste stof. Til su-35 pernatanten, anbragt i en 500 ml Erlenmeyerkolbe, sættes 19 g polyethylenglycol (vægt 4000), og opløsningen omrøres i 30 min. Suspensionen centrifugeres så ved 7000 omdr. pr. min i 20 min. Supernatanten dekanteres fra og kasseres. 15 ml 0,2M na-The mycelia, obtained from 400 ml of culture, are washed twice with 0.05 M 30 potassium phosphate buffer at pH 7.0. The mycelia are then placed in a Waring mixer containing 70 ml of 0.05M potassium phosphate buffer at pH 7.0 and then homogenized for 3 minutes. The mixture is then centrifuged at 6000 rpm. min for 20 min, and the supernatant is decanted from the solid. To the supernatant, placed in a 500 ml Erlenmeyer flask, add 19 g of polyethylene glycol (weight 4000) and the solution is stirred for 30 minutes. The suspension is then centrifuged at 7000 rpm. min for 20 min. The supernatant is decanted off and discarded. 15 ml 0.2M Na
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π triumchlorid plus 15 ml 0,05 M kaliumphosphatpuffer ved pH-værdi 7,0 sættes derpå til bundfaldet og omhvirvles. Opløsningen får lov til at henstå i 30 min, i hvilket tidsrum der dannes et bundfald. Blandingen centrifugeres ved 14000 omdr.π of tri-chloride plus 15 ml of 0.05 M potassium phosphate buffer at pH 7.0 is then added to the precipitate and swirled. The solution is allowed to stand for 30 minutes, during which time a precipitate is formed. The mixture is centrifuged at 14000 rpm.
5 pr. min i 20 min. Oer afdekanteres en uigennemsigtig, hvid supernatant indeholdende cellefrit, renset enzym.5 pr. min for 20 min. Oer is decanted into an opaque white supernatant containing cell-free, purified enzyme.
Immolibisering af enzymerne på agarose kan gennemføres som følger: Det cellefri rensede enzym dialyseres mod 500 ml de-10 stilleret vand natten over. Derpå tilsættes 5 ml 0,1 M natrium-bicarbonat ved pH-værdi 8,0. Til denne opløsning sættes 5 g aktiveret CH-sepharose 4B (vasket og genopkvældet på et sintret glasfilter under anvendelse af 500 ml ImM HC1). Under anvendelse af et ende-over-endeblandingsapparat blandes gelsus-pensionen i en time ved 25°C. Gelsuspensionen vaskes så først med 40 ml 0,1M natriumbicarbonat ved pH-værdi 8,0, derpå med 40 ml 0,05M Tris puffer ved pH-værdi 8,0 indeholdende Q,5M na-triumchlorid og derpå med 0,5M natriumformiatpuffer ved pH-værdi 4,0 også indeholdende 0,5M natriumchlorid.Immolization of the enzymes on agarose can be carried out as follows: The cell-free purified enzyme is dialyzed against 500 ml of distilled water overnight. Then 5 ml of 0.1 M sodium bicarbonate at pH 8.0 is added. To this solution is added 5 g of activated CH-sepharose 4B (washed and repurposed on a sintered glass filter using 500 ml of ImM HCl). Using an end-over-end mixing apparatus, the gel-gel pension is mixed for one hour at 25 ° C. The gel suspension is then washed first with 40 ml of 0.1M sodium bicarbonate at pH 8.0, then with 40 ml of 0.05M Tris buffer at pH 8.0 containing Q, 5M sodium chloride and then with 0.5M sodium formate buffer at pH 4.0 also containing 0.5M sodium chloride.
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Prøve af reaktionsblandingen udtages på forskellige tidspunkter og analyseres for glucose og D-glucoson. Under anvendelse af HPLC foretages kvantitativ bestemmelse af spidsarealerne af spidserne ved Rt på 11,5 min (glucose) og Rt på 14,0 min (D- glucoson), og koncentrationer af tilstedeværende sukker bereg- 25 nes. Der opnås følgende resultater:Samples of the reaction mixture are taken at different times and analyzed for glucose and D-glucosone. Using HPLC, quantitative determination of the peak areas of the peaks is done at Rt of 11.5 min (glucose) and Rt of 14.0 min (D-glucosone) and concentrations of sugar present are calculated. The following results are obtained:
Reaktionstid, Glucose, Q-glucoson, % glucose omdannet timer_g_g_til D-glucoson 0 2,0 0,0 0 30 1 1,2 0,8 40 2 0,5 1,5 75 3 0,2 1,8 90 4 0,1 1,9 99+ 35 Den væsentlige omdannelse af glucose til D-glucoson vises også ved forsvinden af pletten ved Rf på 0,58 (glucose) og tilsynekomsten af stregen ved Rf på 0,43-0,49 (D-glucoson) i løbet af reaktionen og ved forøgelsen af absorbansen ved 480 nm iReaction time, Glucose, Q-glucosone,% glucose converted hours_g_g_to D-glucosone 0 2.0 0.0 0 30 1 1.2 0.8 40 2 0.5 1.5 75 3 0.2 1.8 90 4 0 The significant conversion of glucose to D-glucosone is also shown by the disappearance of the spot at Rf of 0.58 (glucose) and the appearance of the dash at Rf of 0.43-0.49 (D-glucosone ) during the reaction and by increasing the absorbance at 480 nm i
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12 løbet af reaktionen under anvendelse af TTC-kolorimetritestrørsprøven .12 during the reaction using the TTC colorimetrit tube sample.
At produktet helt bestemt er D-glucoson, bekræftes ved deriva-^ tisering efterfulgt af massespektrometri som beskrevet tidligere, De diagnostiske D-glucosonmasseioner ved masse 512 (molekyl ion) og masse 223 (0C-CH=N-Nø2-ionfragment) opnås for produktet.That the product is definitely D-glucosone is confirmed by derivatization followed by mass spectrometry as described previously, The diagnostic D-glucosone mass ions at mass 512 (molecular ion) and mass 223 (0C-CH = N-No 2 ion fragment) are obtained for product.
Eksempel 2 10Example 2 10
Det følgende repræsenterer et eksempel på i alt væsentligt fuldstændig omdannelse af glucose til D-glucoson ved indvirkning af immobi1iseret carbohydratoxidase.The following represents an example of substantially complete conversion of glucose to D-glucosone by the action of immobilized carbohydrate oxidase.
Glucose (1 g) sættes til 50 ml destilleret vand i en 250 ml 1 oGlucose (1 g) is added to 50 ml of distilled water in a 250 ml 1 o
Pyrexkolbe, og sukkeropløsningen omrøres. Agarose-immobi1 i seret carbohydratoxidase, fremstillet som i eksempel 1 af 50 ml cellefrit, renset enzym, sættes så til kolben sammen med 1 mg katalase (som i eksempel 1).Pyrex flask and the sugar solution is stirred. Agarose immobilis in serous carbohydrate oxidase, prepared as in Example 1 of 50 ml of cell-free, purified enzyme, is then added to the flask with 1 mg of catalase (as in Example 1).
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Atten timer senere dekanteres den vandige opløsning fra det faste stof. Analyse af denne opløsning viser, at 99% af gi u-cosen er blevet omdannet til D-glucoson.Eighteen hours later, the aqueous solution is decanted from the solid. Analysis of this solution shows that 99% of the gi ucose has been converted to D-glucosone.
Eksempel 3 25Example 3 25
Dette eksempel viser i alt væsentligt fuldstændig enzymatisk omdannelse af glucose til D-glucoson under anvendelse af giucose-2-oxidase.This example shows essentially complete enzymatic conversion of glucose to D-glucosone using giucose-2 oxidase.
Reaktionen og betingelserne fra eksempel 1 (under anvendelse 30 af agarose som det immobi1iserende støttemateriale) gentages, idet giucose-2-oxidase anvendes i stedet for carbohydratoxidase. Efter 5 timers reaktion var mere end 99% af glucosen omdannet til D-glucoson.The reaction and conditions of Example 1 (using agarose as the immobilizing support) are repeated, using giucose-2 oxidase instead of carbohydrate oxidase. After 5 hours of reaction, more than 99% of the glucose was converted to D-glucosone.
35 Til fremstilling af enzymet giucose-2-oxidase dyrkes mycelkul-turer af Aspergillus oryzae ATCC no. 7252 i kød, gærekstrakt/-tryptonmedium som følger: kødekstrakt (5 g), gærekstrakt (5 g), trypton (3 g), dextrose (1 g) og Difco-stivelse (24 g) sættes DK 15357 13 til destilleret vand (1 liter), og pH-værdien indstilles p 7,3. Mediet steriliseres ved 121°C i 35 min. Under anvendeis af sporer opnået på almindelig kendt måde podes mediet til op nåelse βf ca. 3 x 10* sporer/ml og dyrkes i et roterende ry 5 steapparat (180 omdr. pr. min.) ved 30°C i 2 dage. Kulture vakuumfiltreres gennem Whatmanpapir nr. 541 i en Buchnertrag og vaskes adskillige gange med vand. Mycelierne, der er til bage på filterpapiret, indeholder enzymet.35 To prepare the enzyme giucose-2-oxidase, mycelial cultures are grown by Aspergillus oryzae ATCC no. 7252 in meat, yeast extract / tryptone medium as follows: meat extract (5 g), yeast extract (5 g), tryptone (3 g), dextrose (1 g) and Difco starch (24 g) are added to distilled water ( 1 liter) and the pH is adjusted to 7.3. The medium is sterilized at 121 ° C for 35 minutes. Using spores obtained in a generally known manner, the medium is germinated to reach βf ca. 3 x 10 4 spores / ml and grown in a rotary row 5 steamer (180 rpm) at 30 ° C for 2 days. Cultures are vacuum filtered through Whatman Paper No. 541 in a Buchner funnel and washed several times with water. The mycelia, which are baked on the filter paper, contain the enzyme.
Rensning og immobilisering af enzymet kan så forløbe under an vendelse af fremgangsmåden fra eksempel 1.Purification and immobilization of the enzyme can then proceed using the procedure of Example 1.
Eksempel 4 I eksempel 1 blev koncentrationen af frit hydrogenperoxid 15 dannet ved omsætningen af glucose med carbohydratoxidase forbindelse med D-glucosondannelsen, holdt så lav som muli ved anvendelse af katalase til dekomponer ing af hydrogenper oxidet. En alternativ metode til at holde koncentrationen a frit hydrogenperoxid så lav som mulig er at koble fremstillin 20 gen heraf til en hydrogenperoxidudnyttende reaktion, som give et ønskeligt (værdifuldt) co-produkt.Example 4 In Example 1, the concentration of free hydrogen peroxide 15 was formed by the reaction of glucose with carbohydrate oxidase compound with the D-glucose formation, kept as low as possible using catalase to decompose the hydrogen peroxide. An alternative method of keeping the concentration of free hydrogen peroxide as low as possible is to link its preparation to a hydrogen peroxide utilizing reaction which yields a desirable (valuable) co-product.
I dette eksempel kobles hydrogenperoxidproduktionen til pro duktionen af propylenbromhydrin, et mellemprodukt ved propy 25 lenoxidsynthesen ifølge begreber, som er angivet detaljeret US patentskrift nr. 4.284.723 og US patentskrift nr 4.247.641. Omsætningen af glucose og den immobi1iserede carbo hydratoxidase fra Polyporus obtusus ATCC no. 26733 til dan nelse af D-glucoson og hydrogenperoxid kobles til omsætninge 30 af immobi1iseret tang- eller algeperoxidase fra Coralina sp.In this example, hydrogen peroxide production is coupled to the production of propylene bromohydrin, an intermediate in the propylene oxide synthesis according to concepts set forth in detail U.S. Patent No. 4,284,723 and U.S. Patent No. 4,247,641. The reaction of glucose and the immobilized carbohydrate oxidase from Polyporus obtusus ATCC no. 26733 to form D-glucosone and hydrogen peroxide are coupled to turnover 30 of immobilized seaweed or algal peroxidase from Coralina sp.
nærværelse af bromid og propylen til dannelse af propylenbrom-hydrin. Slutresultatet af denne koblede reaktion er så co-produktionen af glucoson og af propy1enbromhydrin, som let omdannes til propylenoxid, som beskrevet i US patentskrift nr. 35 4.284.723 og US patentskrift nr. 4.247.641. Ethvert enzym, som er i stand til at oxidere hydroxy1 gruppen på 2-carbonatomet i glucose med ledsagende produktion af hydrogenperoxid, kan kobles til enhver halogenerende peroxidase og det sammensattepresence of bromide and propylene to form propylene bromine hydrine. The end result of this coupled reaction is then the co-production of glucosone and of propylene bromohydrin, which is readily converted to propylene oxide, as described in U.S. Patent No. 4,284,723 and U.S. Patent No. 4,247,641. Any enzyme capable of oxidizing the hydroxy1 group on the 2-carbon atom in glucose with concomitant production of hydrogen peroxide can be coupled to any halogenating peroxidase and the compound
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14 stof anvendes til alken-halohydrinproduktion i overensstemmelse med læren i de førnævnte US patentskrifter.14 substance is used for alkene halohydrin production in accordance with the teachings of the aforementioned US patents.
Cellefrit, renset tang- eller algeperoxidaseenzym fremstilles 5 som følger:Cell-free, purified seaweed or algal peroxidase enzyme is prepared as follows:
Coralina sp., opnået fra kysten af La Jolla, Californien, formales i en Virtis 45 homogen isator i 5 min i destilleret vand. Homogenatet roteres ved 20.000 omdr. pr. min i 20 min. Super-natanten dekanteres fra og gemmes. Pillen respuspenderes i de- 10 stilleret vand og centrifugeres igen. Supernatanten og den tidligere nævnte supernatant forenes. Opløsningen bringes først til 33¾ og derpå til 55¾ mætning i ammoniumsulfat. Centrifugering og separering af pillen gennemføres i hvert trin.Coralina sp., Obtained from the coast of La Jolla, California, is ground in a Virtis 45 homogeneous isator for 5 min in distilled water. The homogenate is rotated at 20,000 rpm. min for 20 min. The supernatant is decanted off and stored. The pellet is resuspended in distilled water and centrifuged again. The supernatant and the aforementioned supernatant are combined. The solution is first brought to 33¾ and then to 55¾ saturation in ammonium sulfate. Centrifugation and separation of the pill is performed at each step.
Den 33^55¾ pillefraktion sendes gennem en DEAE-søjle under 15 anvendelse af en 0,3M-1M phosphatpuffer-(pH-værdi 6,0)-gradi-ent. Fraktionen, som eluerer ved 1M, dialyseres mod 20 mM pho-sphatpuffer {pH-værdi 6) natten over.The 33 ^ 55¾ pill fraction is passed through a DEAE column using a 0.3M-1M phosphate buffer (pH 6.0) gradient. The fraction, eluting at 1M, is dialyzed against 20 mM pho-sphat buffer (pH 6) overnight.
Den immobi1 i serede tang- eller algeperoxidase fremstilles som 20 følger:The immobilis in serous seaweed or algal peroxidase is prepared as follows:
Glasperler (opnået fra Sigma Chemical Company, PG-700-200) aktiveres ved at suspendere 1 g plasperler i 18 ml deioniseret vand. 2 ml 10¾ (v/v)a-amino-propyltriethoxysilan tilsættes, og 25 blandingens pH-værdi indstilles på 3-5 med 6N HC1. Blandingen omrystes ved 75°C i 2 timer. Plasperlerne vakuumtørres så natten over ved 80°C. 3,2 ml renset Coralina sp.-enzym, fremstillet som ovenfor, og 50 mg vandopløseligt carbodiimid sættes til plasperlerne. pH-værdien indstilles på 4,5, og blandin-30 gen omrystes så ved 4eC natten over. Produktet (enzymbelagte perler) vaskes med vand. Aktiviteten måles som 2 monochlordi-medonenheder/g perler.Glass beads (obtained from Sigma Chemical Company, PG-700-200) are activated by suspending 1 g of plastic beads in 18 ml of deionized water. 2 ml of 10¾ (v / v) α-amino-propyl triethoxysilane is added and the pH of the mixture is adjusted to 3-5 with 6N HCl. The mixture is shaken at 75 ° C for 2 hours. The plasma beads are then vacuum dried overnight at 80 ° C. 3.2 ml of purified Coralina sp. enzyme prepared as above and 50 mg of water-soluble carbodiimide are added to the beads. The pH is adjusted to 4.5 and the mixture is then shaken at 4 ° C overnight. The product (enzyme-coated beads) is washed with water. Activity is measured as 2 monochlorodimon units / g beads.
Immobi1iseret carbohydratoxidase på agarose fremstilles som i eksempel 1 af 10 ml cellefrit, renset enzym.Immobilized carbohydrate oxidase on agarose is prepared as in Example 1 of 10 ml of cell-free, purified enzyme.
En reaktionsblanding indeholdende følgende bestanddele anbringes i en 100 ml Pyrexkolbe: 35 i. *A reaction mixture containing the following ingredients is placed in a 100 ml Pyrex flask: 35 i. *
DK 15357TBDK 15357TB
15 a) 1 g glasperler overtrukket med tang- eller algeperoxi-dase, b) den ovenfor fremstillede immobiiiserede carbohydrat-oxidase, 5 c) 800 mg kaliumbromid og d) 20 ml 0,01M kaliumphosphatpuffer, pH-værdi 7,0.15 a) 1 g glass beads coated with seaweed or algae peroxidase, b) the immobilized carbohydrate oxidase prepared above, 5 c) 800 mg of potassium bromide and d) 20 ml of 0.01M potassium phosphate buffer, pH 7.0.
Både propyl en og oxygen bobles til kolberne kontinuerligt. Reaktionen startes med 1 g glucose. Efter 20 timers forløb ud-tages en prøve af reaktionen, som analyseres for restglucose, D-glucoson og propylenbromhydrin. Det dannede propylenbromhy-drin analyseres som følger: 5 μΐ af reaktionsblandingen indsprøjtes i en Hewlett-Packard Model 402 gaskromatograf, forsynet med en 1,8 m gange 0,32 cm 15 glassøjle, pakket med Porapak R (80/100 mesh). Strømningshastigheden indstilles på 30 ml/minut for helium, øg søjletemperaturen indstilles på 200°C. Retentionstider for propylenbrom-hydrinerne er 9 min for l-brom-2-propanol og 10 min for 2-brom-l-propanol.Both propylene and oxygen are bubbled to the flasks continuously. The reaction is started with 1 g glucose. After 20 hours, a sample of the reaction is analyzed which is analyzed for residual glucose, D-glucosone and propylene bromohydrin. The resulting propylene bromine hydrine is analyzed as follows: 5 μΐ of the reaction mixture is injected into a Hewlett-Packard Model 402 gas chromatograph fitted with a 1.8 m by 0.32 cm 15 glass column packed with Porapak R (80/100 mesh). The flow rate is set to 30 ml / minute for helium, increase the column temperature to 200 ° C. Retention times for the propylene bromo hydrines are 9 min for 1-bromo-2-propanol and 10 min for 2-bromo-1-propanol.
2020
Produktidentitet blev bekræftet ved sammenligning med autentiske prøver af propylenbromhydrin: l-brom-2-prapanol leveres af Pfaltz og Bauer, Inc.; 2-brom-l-propanol synvtetiseres ved hjælp af 1ihtiumaluminumhydridreduktion af 1-brompropionyl-25 chlorid. Reaktionsprodukterne og de autentiske prøver viser de samme retentionstider og identiske massespektra: brom identificeres ved nærværelse af M- og (M+2)-isotopbi*ndter af ens intensitet. Molekyl ionen for begge isomerer bekræftes ved kemisk ionisering med isobutanreagensgas (M+; m/e 138+140); for 30 l-brom-2-propanol er hovedfragmenteringen det forventede tab af CH2=Br, medens hovedfragmenteringen for 2-brom-l-propanol er det forventede tab af CH3CHBr.Product identity was confirmed by comparison with authentic samples of propylene bromohydrin: 1-bromo-2-prapanol provided by Pfaltz and Bauer, Inc.; 2-Bromo-1-propanol is synthesized by means of lithium aluminum hydride reduction of 1-bromopropionyl chloride. The reaction products and authentic samples show the same retention times and identical mass spectra: bromine is identified by the presence of M and (M + 2) isotope bonds of similar intensity. The molecular ion of both isomers is confirmed by chemical ionization with isobutane reagent gas (M +; m / e 138 + 140); for 30 l-bromo-2-propanol, the main fragmentation is the expected loss of CH2 = Br, while the main fragmentation of 2-bromo-1-propanol is the expected loss of CH3CHBr.
Analysen af prøven viste en omdannelse på over 93% af glucose 35 til D-glucoson og propylenbromhydrinomdannelse på 20 g/1.The analysis of the sample showed a conversion of over 93% of glucose 35 to D-glucosone and propylene bromohydrin conversion of 20 g / l.
DK 153571BDK 153571B
1616
Eksempel 5Example 5
Dette eksempel tjener til yderligere belysning af begreberne, som er angivet og vist i eksempel 4. I dette tilfælde erstatt-5 es immobi1iseret carbohydratoxidase med immobi1iseret glucose-2-oxidase.This example serves to further elucidate the concepts set forth and shown in Example 4. In this case, immobilized carbohydrate oxidase is replaced with immobilized glucose-2-oxidase.
Det immobi1iserede tang- eller algeperoxidaseenzym fremstilles som beskrevet i eksempel 4. Den immobi1iserede glucose-2-oxidase fremstilles som beskrevet i eksempel 3.The immobilized seaweed or algal peroxidase enzyme is prepared as described in Example 4. The immobilized glucose-2-oxidase is prepared as described in Example 3.
1010
Reaktionsblandingen tilberedes som i eksempel 4, idet der i stedet for immobi1iseret carbohydratoxidase anvendes immobi-1iseret giucose-2-oxidase.The reaction mixture is prepared as in Example 4, using immobilized carbohydrate oxidase instead of immobilized giucose-2 oxidase.
Efter 20 timers forløb udtages der en prøve af reaktionsblandingen, som analyseres for restglucose, D-glucoson og propy-1enbromhydrin. Resultaterne viste en omdannelse på over 09% af glucose til D-glucoson og en propylenbromhydrinproduktion på 19,5 g/liter.After 20 hours, a sample of the reaction mixture is taken which is analyzed for residual glucose, D-glucosone and propylene bromohydrin. The results showed a conversion of over 09% of glucose to D-glucosone and a propylene bromohydrin production of 19.5 g / liter.
20 Eksempel 6Example 6
Dette eksempel belyser en høj omdannelse af glucose til D-glucososn i løbet af et udstrakt tidsrum under anvendelse af immobi1iseret carbohydratoxidase i en søjlereaktor.This example illustrates a high conversion of glucose to D-glucose over an extended period of time using immobilized carbohydrate oxidase in a column reactor.
2525
Carbohydratoxidase {cellefrit, renset enzym) (10 ml), fremstillet som i eksmepel 1, immobi1iseres på hydroxyapatit (kal-ciumphosphathydroxid) som følger:Carbohydrate oxidase (cell-free, purified enzyme) (10 ml), prepared as in Example 1, is immobilized on hydroxyapatite (calcium phosphate hydroxide) as follows:
Til 100 ml eellefrit, renset enzym sættes 20 g hydroxyapatit i 3 0 100 ml 1 mS*] kal iumphosphatpuffer ved pH-værdi 7,0. Blandingen omrøres i 3Θ min, hvorpå det faste stof skilles fra væsken ved dekantering, og det faste stof vaskes først med 200 ml 10 mM kaliumphospbatpuffer ved pH-værdi 7,0 og derpå med 200 ml destilleret vand.To 100 ml of cell-free, purified enzyme, 20 g of hydroxyapatite is added to 100 ml of 1 mS *] of calcium phosphate buffer at pH 7.0. The mixture is stirred for 3Θ minutes, after which the solid is separated from the liquid by decantation and the solid is first washed with 200 ml of 10 mM potassium phosphate buffer at pH 7.0 and then with 200 ml of distilled water.
3535
Dette stof pakkes så i en glassøjle {0,5 cm x 4,5 cm). En 1% giucoseopløsning ledes gennem søjlen ved en strømningshastighed på 1,5 ml pr. time. Eluanten analyseres periodisk for restglucose og dannet D-glucoson.This fabric is then packed in a glass column (0.5 cm x 4.5 cm). A 1% giucose solution is passed through the column at a flow rate of 1.5 ml per ml. hour. The eluent is periodically analyzed for residual glucose and formed D-glucosone.
Claims (5)
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Application Number | Priority Date | Filing Date | Title |
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US8810379 | 1979-10-24 | ||
US06/088,103 US4246347A (en) | 1979-05-29 | 1979-10-24 | Process for the production of fructose |
DK399880 | 1980-09-22 | ||
DK399880A DK153500C (en) | 1979-10-24 | 1980-09-22 | PROCEDURE FOR PREPARING FRUCTOSE |
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DK40083D0 DK40083D0 (en) | 1983-02-01 |
DK40083A DK40083A (en) | 1983-02-01 |
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DK153571C DK153571C (en) | 1988-12-05 |
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DK040083A DK153571C (en) | 1979-10-24 | 1983-02-01 | METHOD FOR PREPARING D-GLUCOSONE |
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US3114220A (en) * | 1959-07-20 | 1963-12-17 | Kaiser Aluminium Chem Corp | Furnace wall construction |
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