AU675205B2 - Method for the enzymatic production of alpha-glucosides and esters of alpha-glucosides, and utilizations of products thus obtained - Google Patents

Abstract

The present invention relates to a method for the enzymatic production of $g(a)-glucosides, characterized by contacting at least one alcohol whose only functional group or groups is/are a hydroxyl group or groups, with starch, maltodextrines or maltose, in the presence of a purified enzymatic preparation having an $g(a)-transglucosylation activity. The invention relates also to an enzymatic method for the esterification of the $g(a)-glucosides thus obtained.

Description

,0P1 DATE 16/03/93 AOJP DATE 27/05/93 APPLN. ID 24908/92 PCT NUMBER PCT/FR92/00782 1III11111111111111111111111111111 I 111111111 AU9224908 DFMANDE INTERNATIONALE PUBLIFE EN VERTU DU TRAITE DE COOPERATION EN MIATIERE DE BREVETS (PCT) (51) Classification internationale des brevets 5 C12P 19/44, C0714 3/02 B01IF 17/56 (11) Nurnro de publication internationale: Al (43) Date de publication internationale: WO 93/04185 4 mars 1993 (04.03.93) (21) Numno de ]a demnande interr'ationale: IPCT/FR92/00782 (22) Date de d~p6t international: 7 aoiit 1992 (07.08.92) 3.

9-l Donnies relatives i la priorit 91/10244 12 aot 1991 (12.08.9 1) FR UL/c SO9 (7 1) Dkposant (pour bousr ics Etats disigii~s saitf US: T-~f1ES [FR TFR]: B.P. 1. F 6;720 Ghtpe (F1 x,qe. dles flan/es 2e 6"r -c3Zc' ~ia, (72) Inventeurs; et X;Ame (75) Inventeurs/D~posants (US seuleinent) :PELENC. Vincent, Pascal [FR/FR]; 6 bis, rue Bernard-Mul&, F-3 1000 Toulouse PAUL, Franqois, Marie, Bernard [FR TR]; 69, chemin de Malep~re, F-3 1400 Toulouse MON- SAN, Pierre, Fr~d~ric [FR/FR]; Renoufail, F-31700 Mondonv'ille (FR).

(74) Mandataires: GUTMANN, Ernest etc. Ernest Gutmann- Yves Plasseraud 67, boulevard Haussmann, F- 75008 Paris (FR).

(81) Etats dtsign~s: AU, CA, Fl, JP, NO, US, brevet europ~en (AT, BE. CH, DE, DK, ES. FR. GB, OR, IEI, IT, LU, MC. NL. SE).

Publik A icc rapp~ort de rechierche inernationale.

(54)Title: METHOD FOR THE ENZYMATIC PRODUCTION OF c-GLUC0SIDFS AND IESTERS OF r-GLL'COSIDES, AND UTILIZATIONS OF PRODUCTS THUS OBTAINED (54)Titre: PROCEDE DE FABRICATION ENZYMATIQUE D'ri-GLUCOSIDES ET 1)'ESTERS f)L-GLUCOSIDES, ET UTILISATIONS DES PRODUlTS AINSI OBTENUS (57) Abstract The present invention relates to a method for the enzymatic production of r-glucosides. charact-,rized by Contacting at least one alcohol whose only functional group or groups is are a hydroNN I group or groups, %x ith star~h. maltodcxtrines or maltose, in the presence of a purified enzymatic preparition having an ri-transglucosylation activity. The invention relates also to an enzymatic method for the esterification of the u-glucosides thus obtained.

(57) Abr~lg6 La pr~sente invention concerne un proc~d de fabrication enzvrnatique dlri-glucos.ides, caracteris& par la mise en contact d'au moins un alcool dont Ic(s) seul(s) groupement(s) fonctionnel(s, est(sont) untdesJ groupementls) hydroxyl, avec de l'amidon, des maltodextrines ou du maltose, en presence d'une preparation enzymatique purifiec presentant une activit d'ri-transglucosvlation. L'invention concerne galement un proc~d enzymatique d'est6rification des r-glucosides ainsi obtenus, WO 93/04185 1 PCT/FR92/00782 PROCESS FOR THE ENZYMATIC MANUFACTURE OF a-GLUCOSIDES AND y-GLUCOSIDE ESTERS, AND USE OF THE PRODUCTS THUS OBTAINED The present invention relates to a process for the stereospecific enzymatic manufacture of a-glucosides from starting materials which are inexpensive and available in large quantities, especially starch and the maltodextrins, as well as directly from agricultural starting materials which may contain them, such as flours or semolinas. The invention also concerns a process for the esterification of the a-glucosides thus obtained.

Starch is very widely distributed in nature. In fact, it constitutes a major plant food source, and large quantities are placed in reserve by plant organisms order to maintain the life of the stem or the tuber during the winter dormancy, and to ensure that the embryo develops in the course of the germination.

Taking into account the abundance of this substance, numerous attempts at industrial exploitation have been made, most of them involving a hydrolysis reaction.

Starch is hydrolyzed by dilute acids, with complete acid hydrolysis giving D-glucose. Fragments of higher molecular weight, the dextrins, are obtained by controlled acid hydrolysis and by the action of temperature. Starch may also be hydrolyzed by enzymes (amylases).

The hydrolysis products of starch may be used in the manufacture of glucosides. These compounds, in particular the alkylglucosides, are used as biodegradable surfactants and nonionic detergents. They may be used as emulsifying agents in pharmaceutical, cosmetic and food WO 93/04185 2 -PCT/FH92 /00782 produects.

The chemical synthesis of thc alkyl glucosiden from glucose liar; already been described (for example, EP- A-0,301,298). This type of chemical process may, however, comprise a very high number of different steps and -always give an a~nomeric mixture of (/P-alkyltiono- and alkylpolyglucosides. These mixtures, which possess no precise physico-chemical characteristics, have all the disadvantages associated therewith and, especially, the absence of a precise melting point, the absence of regioselectivity and the possibility of secondary reactions.

The enzymatic manufacture of alkylglucosides from maltose has also been proposed Pan, Biochemistry, vol. 9, No. 8, 1970, pp 1833-1838 and Itano K. et al, Carbohydrate Research, 87, 1990, 27-34). Pan describes the transglucosylation of an alcohol such as butanol with, as glucose donor, maltose in the presence of an Aspercgillus nicier culture supernatent. The identity of the enzyme(s) contained in the supernatent and responsible for the transglucosylation has not been investigALed.

The anomeric configuration or 03) of the alkyiglucosides obtained has not been verified. Itano describes the enzymatic production of cyclohexanediol c-glucoaide using, as glucose donor, maltose and, as glucose acceptor, Lrans-l,2-cyclohexanediol. This reaction uses a crude mixture of various enzymes used in the agro-food -industry, most of which are hydrolases. These preparations additionally contain unidentified enzymes.

WO 93/04185 3 PCT/Fn92/00782 The yields obtained according to these enzymatic processes are low and do not permit uynthesls of the alkyiglucosides on tin industrial sc;Alc. Moreover, the anomeric configuration of the products obtained is sometimes unpredictable on account of the numerous enzymes present during the reactilon and, especially, on account of an unidentified P-glucosidase activity.

Finally, Fogarty et al. (Biotechnology Letters, vol. 4, 1, 61-64, 1982) describes the production of a~glyceroiglucoside by the enzymatic transglucosylation of glycerol with, as glucose donor, a-Inethylglucoside, The enzyme used in this reaction is purified tranuglucosidase from A. ,niger. Under the conditions described, the glycerol can only act as a glucose acceptor with a very limited number of substrates. These results show that transglucosidase, like many transferases, is capable of acting on a relatively high number of acceptors, but, on the other hand, the number of substrates or donors which it may use is very limited.

The subject of the present invention is to provide a method for the stereospecific manufacture of aglucosides giving rise to high yields which are suitable for an exploitation on an 'industrial scale and which make it possible to use, as starting materials, starch or ialtodextrins, as well as agricultural starting materials of high starch content such as flours or semolinas. Tho method sought must be simple, direct and inexpensive.

Another subject of the invention is to provide a process for the treatment of crude starchy material such as WA~~ Q1/OAI U U 2 starch or maltodextriris, which make~ it posisible to juduuu comupounds of high added value, comiprising, for example, a-glucoside euLers.

The inventors have now siinriginaly r,rrreh a-glucosides may be synthesized on an inceii'strial n~-l by enzymatic t-transglucosylation, using starch or nialtodextrins as substratef3 or "glucose donors" and alcohols as co-substrates or "glucose acceptors".

It is totally unexpected that polymeric molecules such as starch or ca-xaltodextrins can act as glucose donors in this reaction. As indicated above, a-transglucosiclase may carry out the transfer of a glucosyl residue onto a relatively high number of acceptors (alcohol in the present case), but can only use very few molecules as substrates.

The invention more particularly relates to a process for the enzymatic manufacture of ca-glucosides (also known under the name a -gl~icopyranos ides) characterized by placing at least one alcohol, whose only f unctional group is (are) hydroxyl group in contact with starch, maltodextrins or maltose, in the presence of a purified enzymatic preparation having an a-transglucosylation activity, The products obtained by this process are a~glucosides consisting of at lea~st one molecul.e of glucose and of: an alcohol, whose carbon chain is of variable length and structure according to the type of alcohol which has participated in the reaction. The product is devoid of P-glucoside. The biosynthesia is Acarried out WO 93/04105 5 PCT/F192/00782 under mild reaction conditions, at average temperature 0 C to 70 0 C, for example between 30°C and 60°C) and at pH between 3 and 7, preferably 4 and 6. The formation of glucose degradation products is thus avoided.

In the context of the invention, an enzymatic preparation "having an a-transglucosylation activity" is understood to refer to any enzymatic preparation capable of carrying out the transfer of a glucose molecule (from starting materials consisting of starch or containing starch, or consisting of starch hydrolysates or containing starch hydrolysates) onto the alcohol.

Purified should be understood to mean that the enzymatic preparation is deprived of any enzymatic activity capable, under the conditions used, of catalyzing the manufacture of substances other than aglucosides, that the enzymatic preparation is such that it avoids the formation of secondary products, that the enzymatic preparation is especially devoid of p-glucosidase activity.

The enzymatic preparation may consist of a single enzyme having an a-tranaglucosylation activity.

The enzymatic preparation may also consist of several enzymes such that either each one of them has an c-transglucosylation activity, or their combined enzymatic activities are such that they confer an a-transglucosylation activity on the abovementioned enzymatic preparation.

WO 93/04185 6 rPc'/-1192/o0782 It may be an "authentic" &-tranaglucosidaoc, also known "a-tranuxglucosylase" that is~ to say an enzyme of the Lrans1 feri113 Lypo. This typQ of etiyrne i.9 tio~ l 1i ly recognized by its capacity to synthesize oligosaccharides in dilute mtedium (f o2 example 100 gIL maltose) a-Transglucosidases which may be mentioned are fungal a-transglucosidases such as those from Aspergillufs niger, L. batatag. A oyzg or Tal-aromyces duponti. The a-traneglucosidases from Aspergillus nicier and Talaroinyces. duponti, are particularly preferred. It i~s advantageous to use these enzymes in purified form, the preparation thus being devoid of any P-glucooidase activity. The presence of P-glucosidase in the enzymatic preparation effectively prevents the stereospecific is synthesis of the ca-glucosides.

c-Transglucosidase from Aspergillus niger, available under the commercial name of TRANSGLUCOSIDASE-L (Amano Pharmaceutical Corporation Ltd, Japan) is normally devoid of A-glucosidase and may be used in the process of the invention without additional purification. on the other hand, that derived from Talaromyces duponti (also known under the nam!e of 1,4-a-D-glucan-6-ac-D-glycosyltransferase) is advantageously subjected to an additional purification step, by ion exchange, in order to remove the P-glucosidase, but has the advantage of a great therinotability. This enzyme is described In the European Patent Application EP-A-0,219,673.

It is also possible to use a mixture of purified enzymes, for example c&-transglucosidaseos from various WO 93/04185 7 PCT/FR92/00782 types of fungi, on condition that other enzymes are not present and that the various enzymer. funcLion uinder i t v may a yn. ,o n. a fl11Te -lCr~ wrc oo- cnc.

"marginal" property, the capacity to carry out a-transglucosylation reactions. This type of enzyme may gencrally be recognized by its capacity to form glucose in dilute medium (for example 100 g/L maltose). Examples of this type of enzyme which may be mentioned are the hydrolases such as a-glucosidase.

Although these enzymes are capable of carrying out types of reactionsi other than a-tranaglucosylation, the reaction medium, composed exclusively of starch, malV-odextrins or maltose, and alcohol, favor the formation of ca-glucosides.

it is possible to use, as substrates or "glucose donors", according to the invention, starch, xnaltodextrins or maltose or a mixture of these substances, as well as agricultural starting materials which may contain these substances, When maltose is used as substrate, it may be present in the form of a mixture with other substances originating from st~archy materilals, for example, when the substrate is a partial hydrolysate of starch, maltor-e may be present with malt-odextri-ns, Maltobe may also he used alone. The yields of 4-glucoside obtained from maltose are sometimes poorer than Lhose obLaIried f romn olher types of substrate, and are, for example, of the order of 10 to wo 93/04185 8 PCT/v92/00782 (calculated as in Example 1 below).

Maltodextrins constitute a particularly preferred substrate or "glucose donor" according to the process of the invention. They are partial hydrolysates of starch, consisting of a mixture of oligosaccharides of variable molar mass and displaying linkages between the glucose units. The maltodextrins may derive from a partial acid hydrolysis of starch or a thermal or enzymatic treatment of starch. This type of product is also available on the market and, on account of its purity, is particularly preferred when the a-glucosides obtained are intended to be used in the pharmaceutical industry.

According to another variant of the process of the invention, the substrate, or "glucose donor", may also be soluble starch. This product, which may be assimilated to maltodextrins, consists of starch which has been rendered soluble by an acid prehydrolysis, for example according to the Lintner process.

According to another particularly preferred embodiment of the invention, the substrate consists of native atarch. Sources of native starch which may be mentioned are cereals, tubers and leguminous plants, as well as any other plant. Among the preferred cereals there may be mentioned wheat, maize, barley, oats, rice, rye, triticale (a hybrid of rye and wheat), buckwheat and sorghum. The preferred tubers are potatoes and manioc.

Peas and beans are a source of leguminous starch. The cereals may be used in the form of whole grains, grain fractions (resulting, for example, from an enzymatic, WO 93/04185 9 PCT/PR92/00782 chemical, th-ermal or mechanical treatment of the grain) such asi flour or semolina.

of t-he enzyme which is capable of ua.LiyInkj out. Liiu atransglucosylation is associated with the action of a hydrolase, in order to render the starch accessible to the a-transglucosylation. The hydrolase liberates maltodextrins and maltose from the native starch into the medium. These substrates are then used as glucose donors by cc-transgluc.:Isidase which glucosylates the alcohols present in the medium. The hydrolaoc is preferably used simultaneously with ca-tranaglucosidase.

As hydrolases, it is preferable to use endoamylases, for example ca-amylase originating, for example, from fungi or from Bacillus ]icheni-n forrnis. Endo-amylases have the advantage of carrying out "endo" cleavages in the starch molecule, without liberating molecules of low molecular weight (that is to say glucose). In fact, c-transglucosidases are not capable of using glucose an a oubstrate. The production of glucose by amnylase would thus prevent the subsequent manufacture of ca-glucosides. As for maltose, although it may serve as a substrate for a-glucosidase, the alkylglucouldo yield thus obtained is lower -than with nialtodextrins or starch.

The ratio between the transferable glucose and the unused glucooe is highcr in the case of a maltodextrin than in the case of maltose.

Simultaneous use of alase and WO 93/04185 10 PCT/FR92/00782 a-transglucosidase is proecrred, competition for the soluble molecules beingj established between the two enzymes. The simultaneous progress of the two enzymatic processes leads in this way Lo an improvement in the yield.

The effectiveness of Einylasge on starch varies according to the botanical origin of the starch and according to the nature of the ainylacc, The relative concentrations of the substrate, the enzyme and the alcohol are for the substrate, approximately 10 q/l to approximately 800 g/1 and in particular approximdtely 100 g/l to approximately 400 g/1 for the alcohols, from approximately 10 g/1 to approximately 800 gil, and in particular from approximately g/l to approximately 400 g/l for the enzyme, from approximately 5 U/mI to approximately 1,000 U/ml, and in particular from approximately U/mi to approximately 200 U/mi.

The glucose acceptor according to the invention is an alcohol of simple functionality, that is to say an alc,.-hol which carries no functional groups other than the hydroxyl groupa. A mixture of various alcohols may be used. All the alcohols capable of acting as glucose acceptors in the veaction, and which do not inhibit the enzyme activity, may be used.

The preferred &~osare alkanolB, ethylenic alcohols, acetylenic aicohois, cyclic alcohols or phenols. Acyclic alcohols are particularly preferred, WO 93/04 185 11 l'CT/1l92/00782 especially uaturate.d alkanol s having, for example, between 2 and 24 carbon atoms, and saturated polyols.

According to the process of the invention, the alcohol inoy be a ruonohydriec alcohol, or may a] 8o be a polyol, for example a dial or a trial. Diols are parLicularly preferred, such as propanediols and butanediols. Primary, secondary and -tertiary alcohols may be used in the reaction of the invention, the primary and secondary alcohols being particularly advantageous.

It may be a water-maiscible or at least partially water-miscible alcohol. In this case, the alcohol adivantageously has a solubility of at least 2.7 v/v at In most casesg these alcohols contain between 1 and 6, and often between 1 and 5, carbon atoms.

Examples of preferred soluble alcohols which may be mentioned are isopropanoll n-butanol, isobutanol, isopentanol, propanol, pentanediol, hexanediol, 1-buten- 3-ol, 1-butyn-3-ol, cyclohexanol, etc., or a mixture of at least two of these alcohols. The reaction is conducted in aqueous medium, the alcohol often being used at the limit of its solubility.

When the alcohol is partially voluble In water, it is possible to carry out the process of the invention in a two-phase medium, that is to say an aqueous phase in which the alcohol(s) is (are) dissolved at a concentration lower than the iniL of their solubility, and an orqaiie phase in equilibrium with the aqueous phase and composed of the saturated buffer alcohol. The use of a two-phase medium exerts a favorable affect on the yield WO 93/04185 12 PCr/L1(92 /007 82 of a-glucosides. This system is particularly advantageous when the a-glucoside obtained by the ca-tranoglucosylation reaction may, above a certain concentration, constitute a substrate for the enzyme, For exainple, a-butylglucouide may act as a substrate for a-transglucosidase. In this case, the reaction proceeds in the aqueous phase, and the ca-gl"cosije is extracted from the aqueous phase into the organic phase, preventing destruction of the glucoside by the enzyme.

The use of an ethylenic alcohol such as 1-buten- 3-ol leads to the presence of an unsaturated bond in the glucoside. This permnits the subsequent use of the glucoside in polymarization reactions or the preparation of chiral synthons (intermediate molecules) for phaxinaceutical or chemical use.

According to a. particularly advantageous embodiment, the soluble alcohol is a monohydric alkanol having from 1 to 5 carbon atoms, These alcohols give rise to aalkyiglucosides in which the alkyl chain has from 1 to carbon atoms.

It is also possible to use water-insoluble alcohols, for example, alkanols having more than six carbon atoms, in particular between 12 and 18 carbon atoins, or else fatty alcohols (primary aliphatic alcohols) such as dodecyl alcohol, tetradecyl alcohol, hexadecyl alcohol, octadecyl alcohol, docyl alcohol, undecyl alcohol and tridecyl alcohol, anid the like.

Steroids may also be mentioned as water-insoluble alcohols. The uine of fatty alcohols is advantageously WO 93/04185 13 PCT/Vi(92/00782 accompanied by the use of short. alcohols (1 to 6 carbon atoms), which aze parLially water-soluble and which act as solvents for the fatty alcohocls arid as glucose acceptor.

Hydrtophobic alcohols, such as thore mentioned above, may participaLe in the enzymatic a-transglucosy1ation reaction by using a two-phaSe system (that is to say water dnd an organic solvent) The hydrophobic alcohol is thus dissolved in a sol-vent such as nitrobenzenef whereas the substrate and the enzyme are dissolved in an aqueous solution. The two solutions are subsequently mixed and stirred, The ca-transglucosylation takes place at the interface. It is also possible to use other organic solvents, for example acetone, on condition that the enzyme activity is not affected.

On account of the surfactant properties of the aalkyl glucosides, whose alkyl chain has between 12 and 18 carbon atoms, the use, as alcohol, of fatty alcohols is particularly preferred.

The a-glucoside yields obtained by the process of the invention vary according -to the glucose donor and according to the extraction method. With, for example, maltodextrin or starch as substrate and a two-phase organic solvent system for extraction of the product, the yields may reach 20 to 30 Maltose gives yields of to 15 The yields are calculated in the following way; c-qlucoside produced, moles X 100 c-glucoside produced glucose produced, moles The products obtained by the process of the Invention are stereospecific (ax) and are normally monoglucosides. It is possible, for example, by addingj an ccalkyiglucoside as "addiLiona1" subst-rate to the reaction medium, to bring about the forntation of d~tglu;ot~idcs,, that is to say inaltosides. These inaltos.ideq have very similar properties to those of the glucosides.

The steraospecificity of the product may be verified by carrying out the hydrolysis of a sample with an enzyme which can only act on the c-anomers, for example ca-glucosidase. Maltase is an example of an aglucosidase which is suitable for thIs verification.

On account of the absence of P1-anomier in the product of the invention, certain physical characteristics, such as the melting point and the solubility of the glucoside, are very narrowly defined. This precision is advantageous for a use in the pharmaceutical, cosmetic and chemical industry in general.

In addition, the enzyme used during the a-transglucosylation is in the purified state. The product of the invention is thus devoid of contaminants arising from secondary enzymatic reactions. The need to carry out numerous purification steps for the products of the reaction is thus removed.

The invenilon also relates to the a-glucosides, in particular the c-alkylglucos ides as obtained by the process of the invention. These products are thorn characterized by the absence of contamninants normally associated with glucosides produced by the chemnical route or by the enzymatic route using enzyme mixtures. a-flutylglucoside WO 93/04185 15 PCT/iiR2/ 007 82 is a particularly preferred produict of -the invention.

More particula~rly, the a-glucosidcs of the invention are ri R,1 f- An ri a A ri A e- _%7T nt e r nIr Ai n y- .4 secondary enzymnatic reac~tions.

t 'i on o~f t h 0 p-rodutc' f-r~m hr rn-i rcin medium is thus all the more 8iimple since, apart: from the prodruct, it only contains the excess alcohol, the remaining glucose donor and the glucose produced in the course of the reaction. The extraction may be done in a suitable organic solvent, chosen in accordance with the solubility of the product. According to a preferred embodiiment, extraction of the ca-glucoside, for example a-butylglucoside, is carried out in continuous fashion by means of a liquid column of extraction solvent, for example butanol.

According to this variant, the reaction medium is to begin with passed through a first column containing the Immobile transglucosidase, and then onto the liquid extraction column. The alcoholysis reaction and the extraction of the product then occur in continuous fashion. a-Butylglucoside is recovered in the form of a syrup after evaporation of the butanol, and the butanol is recycled for use as a substrate. By using this type of two-phase extraction, the yields of products reach 35 to The glucosidee produced according to the invention have a multitude of applications.

Alkylgiucosides, in particular those in which the alkyl chain has up to 6 carbon atoms, for example a-butylglucoside, may be used as liquid detergent additives, as WO 93/04185 16 PCT/FR92/00782 emulsion additives for modifying the viscosity without bringing about phase-separation, or as co-surfactant in microemulsions. In fact, the presence of hydrophobic and hydrophilic groups in'these molecules enables them to be used in emulsions, for example cosmetic and pharmaceutical creams, for modifying the properties of an emulsion, without destabilizing it. Since the glucosides of the invention are manufactured by a biosynthesis from natural materials, they are particularly suited to cosmetic and pharmaceutical applications of this type.

The alkylglucosides, and in particular a-butylglucoside, may also be used as additives in the plastics, rubber and PVC industries for example, as softeners in melamine resins. The use of a-butylglucoside prevents the resin from having an excessively brittle nature. They may also be used as additives in industrial and domestic cleaning compositions or in maintenance products.

Alkylglucosides in which the alkyl chain has at least 8 carbon atoms are used as biodegradable nonionic surfactants and detergents, displaying foaming, lubricating, emulsifying and moisturizing properties.

Since they are stereospecific and are obtained by an enzymatic synthesis process from plant materials, the products of the invention are particularly suitable for pharmaceutical, cosmetic or food use, for example as nontoxic and non-irritant emulsifying agents.

The glucosides of the invention also constitute an excellent starting material in the manufacture of polyether polyols, for example, of rigid foams based on WO 93/04185 17 PCT/FR92/00782 MIp-v Fb' ~rit h 4~rvr.

glucosides, in particular the alkyiglucosidos, may also be used as polyols in the manufacture of alkyd and polyes~ter res3ins or as a chemical intermediate for the sy l s J4 Of mJ"OnJILer WhILiSt 1Lncay bu- u- a .LIL JV LYUIBt-L kd.LL reactions.

The alkyiglucosides of the invention having short alkyl chains (Cl to may serve as starting materials in the manufacture of surface-active agents, for example by replacing the alkyl chain by an alkyl chain having at least 8, and preferably at least 12, carbon atoms. These alkylgiucosides of long alkyl chain have excellent emulsifying and foaming properties and are biodegradable.

Another type of surface-active agent may be produced by the eaterification, preferably in the positions C, and of lower alkylgiucosides of the invention. The einulsifiers thus obtained have moisturizing properties and are also biodegradable.

According to a particularly preferred variant of the invention, esterification of the ca-glucoside6 is~ carried out via the enzymatic route by placing the aglucoside in con-tact with a fatty acid or with a mixture of different fatty acids and an enzymatic preparation having lipase activity. Thi s type, of reaction has been described by Djmrkling et ,I Chemn. Soc., Chemn. Conu.m., 1989, p 934-935). Combining this en~ymnAtic estarification reaction with -the alcoholysis roaction of the invention makes it possible to manufacture pure ca-glucoside esters WO 93/04185 18 I9CT/FR92/00782 from staroh, maltodexLrosc or maltose. The esterification may be carried out after the alcoholysis reaction or simultaneously with the alcoholysi.

According to this variant of the invention, the a-glucoside is preferably an alkylgiucoside having between 1 and 5 carbon atoms or a mixture of these aal kyig luco sides. a-Butylglucoeide is particularly preferred, The fatty acid advantageously contains between 8 and carbon atoms. Caprylic capric (C 10 lauric and palinitic: (C, 6 acids are particularly preferred. It is also possible to use a mixture of 5everal fatty acids.

The esterification is catalyzed according to this embodiment by an enzymatic preparation having lipase activity, for example, a lipase originating from Mucor xniehei (for example Lipozynie(), Candida antartica, Humicola sp., Candida cylindracea and Pseudomonas ap..

Any lipase may be used which is capable of carrying out the reaction on the chosen fatty acid. It may be a purified Preparation containing a single enzyme or, alternatively, P, mixture of several enzymes showing lipase activity. The enzyme having lipase activity may be immobilized on a solid support.

The esterification reaction is carried out at a -temperature situated approximately between room temperattire and 80 6 C, on condition that it isi a temperature at which the fatty acid io in liquid form, and at which the enzyme is stable. The absence of ovcnt represents a considerable advantage from the economic and environmental points of view, WO 93/04185 19 PCT/IFl92/ 007 82 The products obtained by the enzymatic esterif ication, without solvent, of the a-glucosides of the invention are mono- 4nd dieo3Ler mixtures. The operating conditions may be optimized in ordeL Lo make iL possible to obtain approximately 80 of monoester3. The reaction io regiospecific, the esterification taking place to begin with in the pocition C4, and subsequently in the Position C 2 1 C3 or C 4 depending on the enzyme, The diester may be a mixture of 3,6- and 4,6-diesters. in thin case, the product comprises the monoester and a mixture of diesters. The diester is preferably a 2,6-diester. The production of a proportion of diesters is inevitable since, above a certain monoester concentration the enzyme recognizes the monoester as substrate rather than a-glucoside. when only a single fatty acid is used in the esterif ication, the diester carries two Identical groups.

on the other hand, if a mixture of two fatty acids are used, the diester is a mixed diester, f~or example butyl 2-0O-laury1-6-O--stearyl-cz-D-glucopyranoside. The choice of fatty acids rnakes it possible to vary the properties of the esters produced, in particular the H.L.B (hydrophilic/lipophilic balance).

The product of the esterification may be extracted from the reaction medium by a suitable organic solvent, such as diethyl ether or, on the industrial scale, hexane. Any solvent in which the cc-qlucoside and the f atty acid are insoluble, may be used. Recovery of 'the esters is also possible without addition of organic solvent. The produot may be used as it is without WO 93/04185 20 PCiILPR92/00782 purification, after removal of the iimtobilized enzyme.

The c-glucoside esters thus produced are free from t1-anomero and from secondary reaction producto normally associated with esters obtained from less pure glucosides or via a che-Acal route. These esters have a multitude of industrial applications arnd may be used for any standard application for esters of this type. Th'!3 aglucoside esters and, int particular, the ca-alkylglucoside esters are nonionic, non-irritant, non-toxic and biodegradable surfactants which have properties such as foaming, emulsifying, solubilizing, moisturizing, diopersing, wetting or lubricating proportien. They are advantageously used as additives in cosmetic and pharmaceutical and agro-food products, or alternatively In chemistry or in the detergent industry as raurfactants or whitening agent precursors. In agriculture, they may be used as inert adjuvants for augmenting the effectiveness of plant protection products, especially herbicides, fungicides and insecticides, and for the treatment of cultures and agricultural products in genezal.

The figures show various aspects of the invention, especially: Figure 1 shows the production of glucose arid of ccbutylglucoside from vialtodextrins with the aid of atrarisgiucosidase from TalaroMyLce duoni Figure 2 fillustrates 11PLC chromnatograims of the medium f or the synthesis of a-butylgluooside with transglucosidase from Aspergillus nicier -from insoluble starnh one-phase liquid inedium.

WO 93/04185 21 PCT/MR92/00782 a) reaction time 10 min b) reaction time 23 hours c) ca..f-butylglucoside rnix~ure, Figure 3 illustrates IIPLC chromatograins of the medium for the synthesis of a-butylglucoside with transgiucosidase from Aspergilius niger from insoluble starch two-phase liquid medium ax) reaction time 10 mmi b) reaction time 7 h1our&.

EXAMIPLES

Excample I Production of d.-butylglucoside from soluble Ptarchy substraters in the presence of but&nol with the aid of 4-tranaglucosidase from Talaromycesi duponti a) Assay of the e4-tranoglucosidase activity Assay of the a-transglucosidase activity is carried out at 601C in the following medium: m ialtose 100 gI sodium acetate buffer pH 4.5 50 mM -transglucosidaue I U/mi The medium is analyzed after reaction for 15 and 24 hours by an IIPLC technique (DIONEX Carbopack column, conductimetric detection) which allows the separation and the detection of -the panose formed in the course of -the reaction.

1 unit of a-transglucosidasc is the quantity of enzyme which catalyzes the production of 1. micromole of panose (6-O-a-D-glucopyranosylma-ttocse) per hour under the conditions presented above.

WU 93/04185 22 PCT/IV92/007G2 h) Prch!do ion of a-butv ilucoside The production of c-butyigluconide is carried out in the aqueons mediLuin of Lhc following copouilion -3utanoL 9 (v/vj c--transgiuoosidase from Ta.aLyumvyc duponti 100 u/rni Substrate 1 100 9/l This medium is incubated at the temperature of 50 0 C for 70 hours, Samples are withdrawn, diluted (1/10) in water, heated to 90 0 C to stop the reaction, and then analyzed by high performance liquid chromatography (IPLC) on a column of ion-exchange resin in calcium form. The eluent is ultrapure water.

Under these analysis conditions the a-butylglucoside is separated from the A-butylglucouide and the monoand oligosaccharidee, I-rom the area of the corresponding peak it is possible to calculate the ca-butylglycoaide concentration in the reaction medium and the glucose transfer yield defined in the following way; a-butylglucoside, mM (a-butyllucoside glucose), mM The results are presented in Table 1 WO 93/04185 23 PCT/F1192 /00782 Ta-ble 1 c-butylgJlucosidc produced front So~luble substrates and transfer yields after reaction for hours.

Substrate a-Dutylglucoside Yield in ial in Maltose 21 Maltopentaose 50 1s Naltodextrins 40 19 Soluble starch 40 The production of a-butylglucoside and glucose was followed in the course of the reaction. The change in the concentration of these two products is indiqated in Figure 1.

The production of a-butylglucoside takes place with the four starchy substrates tested in this example.

With inaltodextrine the a-butylglucoside concentration in~ greater than 10 cu/l.

Excample -2 z P'roduction of c-butylglucoside from insoluble starch in the presence of butanol with the aid of a-transqluoosidase from Talaromyces dui~onti- The reaction medium is constituted as in Example The substrate here is insoluble starch. ct-Amylasc from flnciliiun Iichiformis (5 U/ml) is added to the medium in order to make it possible to use the 4insolu~ble substrates.

Th(e samples withdrawn from the rcaction medium are treated aB in Example 1, but arc centrifuged and filtered before the BV'LC injection.

WO 93/04185 24 PCT/Ffl92/00782 The results are present.ed in Table 2: Table 2 a-bLylg1ucoside produced from insoluble atarch and transfer yields afteor reaction for hours.

Substrate a-lButyiglucoside YA s i nr in% Insoluble starch c-amylase 28 16 The insol'ible starch may also be used as a substrate of the reaction.

Example 3 z Production ol' a-buLylglucoside from insoluble starchy substiates in a liquid two-phase medium with the aid of a-transglucosidase from Talaromyces dupoqnti.

The reaction medium consists of two liquid phases of the following composition% Aqueous phase (volume 0.5 ml) -Butanol 9% (vfv) -Sodium acetate buffer pH1 4.5 5 50 mm -transglucosidase from T,tlqromvceas duponLi 100 U/nil 4 -amylase :5 to 500 U/nil -Substrate 100 9/L Oxjanic phaso (volume 0.5 or 2 tul) Butanol catura~od with 50 m14 sodium acetLt buffer, pil The reaction mecdia are vigorously stirred before each withdrawal and then trca ted as I n Examiple 2.

WO 93/04185 25 PCT/FR92/00782 Several media were investigated, with two recLiLon volumes and two a-amrylaue preparations. The influence of thc a-amylaa content of the medium~ was also studied.

The IIPLC analysis results for the reaction media arc presented in Tiablos 3 and 4 Taible 3 a-butylglucoside produced and transfer yields after reaction for 70 hours in a liquid twophase medium (ca-amylase from lacillus lichenif oris, 5 i/mI.) substrate '-Iutylglucoside Yield Reaction volume InM ml insoluble starch 146 23 Insoluble starch 25 23 WO 93/04185 26 PCT/FR92/00792 Table 4 a-butylglucosidc produced and tranafcr yields from cornflour with the aid of a-trainsgiucosidase from Talarermyces duponti in a liquid twophasc medium (reaction volume 1 ml) Source and activity a-Dutylglucoside Yield of the a-amylase mM B. licheniformis 14 24 U/ml '0 B. licheniformi6 15 19 U/mi B. licheniformis 9 14 500 U/mi 0 A, niger 10 14 U/mi 12 17 t/ml A. nigr 9 1.9 500 U/mi The use of crude cereal flour is entirely WO 93/04185 27 PCT/Fi(92/00782 possible for the synthesis of a-butylglucooide with the aid off a-transq] ucosidase ifrom Talaromyces duponti, The use of a liquid two-phase miediuminmakes it posoible to improve the production of a-butylglucoside.

In Table 3, it is seen that 4-6 lpmoles off c-butylgluc~oside were synthesized in the 1 mnl volume medium, whereas 62.5 innoles were synthesized in the 2.5 ml medium from the same amount of substrate and of enzymes.

Examplfe 4 :Production of a-alkylg lucos ides from soluble starchy substrates with the aid of a-transglucosidase from Talaromyces duponti in the presence of a mixture of alcohols.

Three media were constituted in the following way- Maltodextrins :100 g/l a-Transglucosidase from Talaromyces duponti ;100 U/mi Sodium acetate buffer pH 4.5 50 mm The three media contain a mixture of monoalcohols: Test 1 Test 2 Test 3 IButanol 9 45 Pentanol 25 Isopropanol -25 45 Water 91 30 30 The media were incubated at 50 0 C for 120 hours and then analyzed according to the method described in Example 1. The analysis results are indicated in Table WO 93/04185 20 PCIP/Pz9 2 00 702 Taible 5 Mkylglucosidcs produced in Lhe pro-oonce of alcohol inixtzuren (reaction voltime 1. ml) Tent 1 Test 2 Test 3 a-butylglycoside g/1, 10 Isopropylgluco3ide g1L 2 6 Pentylgiucoside g/L 2 Example 5 Production of c-butylglucoside from insoluble starch with the aid of ca-transglucosidase from Aspergillus njoeT Two media were prepared and then incubated according to the procedure indicated in Example 3.

One-phase aqueous medium (volume 20 ml) Butanol 9 (Vlv) Sodium acetate buffer pH 5.5 50 mm a-Transglucosidase from Aspergillus niier 100 U~/m1 ca-amylase from Bacillus licheniforii 5 U/mil Insoluble starch 100 g/l Two-phase liquid medium (volume 40 zal) Aqueous phase (volume 20 ml) Butanol 9% WO 93/04185 29 PCT/PR92/00782 Sodium acetate buffer p11 5.5 50 mm z-Transglucosi dase r Aspergillus nicler 100 U/il c-amylase from Bacillus lichenif orMiG 5 U/i -insoluble starch :100 g/l organic phase (volume 20 ml) Dutanol vatura~ed with 50 mM sodium acetate buffer, pH These media were analyzed as in Example 3. The chromatograms obtained are presented in Figures 2 and 3.

The ca-butylglucoside concentration is 12 gil (51 inN) in the one-phase liquid medium and 7 g/L (30 mM) in the twophase liquid medium.

is The ca-transglucosidase from Aspergillus niger is very effective for the synthesis of a-alkylglucouide.

Example 6 :Production of a-butylglucoside palmitic esters by enzymatic esterification of ca-butylglucoside A medium was prepared in the following way -palmitic acid (PROLA3O, France) 10.85 g -D-butylglucoside :10 9 The ax-D-butylglucoside was prepared according to the mothod of Example 1.

The mixture is brought to 75 0 C, the mealting temperature of palmitie acid. Af Ler homogenization, 1 g Of .Lipozymc-0, a lipase frova Mucor mighei, immobilized on a solid support (NOVO INDUSTRI, Denmark) le~ added.

After incubation for 3 days, the reaction medium.

WO 93/041.85 30 PCT/FR192/00782 ±13 diluted with 210 ml of diethyl ether (SDS, France) and oubsccjucntly filtered in order to remove the Lipozymnem. After filtration, 50 ml of sodium hydroxitde (NaOli 0. 02 N) are added in order to dianolve -the remaining palinitic acid in the aqueous phase, as well as -the remaining a-butylglucoside, in the form of a. sodium sait.

The organic phase (diethyl ether) containing the palmitic acid esters is evaporated under vacuum, in order to remove all trace of remaining solvent.

16.5 g of esters are finally obtained (mixture of monoesters and diesters of cc-D-butylglucopyranoside: butyl 6-O-palmityl-ca,D-giucopyranoside and butyl. 2,6-di- 0-palmi'tyl-cz,D-ylucopyranoside). Characterization of the reaction products was done by T.L.C. and by proton and "C~

N.M.R.

Hxmle 7 Production of *;-butylglucoside lauric eaters by enzymatic esterification of a-butylglucoaide: A medium was prepared in the following way: Lauric acid (ME~RCK, Germany) :8.5 g a-D-butylglucoside :10 g The a-D-butylglucoside was prepared according -to *the method of Example 1.

The mixture is brought to 451C, the melting temperature of lauric acid. After humognization, 1 g of LipozymeO, a lipase from Mu-cor mizuhi, immobilized on a solid support (tIOVO INDtJSTRI, Denmark) is added.

After incubation for 3 days, the reaction medium is diluted with 210 ml of diethyl ether (SDS, France) WYU '4 VLT/Ir 1i ZU U 110 ~nr n giih.~rsaw-nt- I~ -t rii f .c ,i n vrli h LipozymeS3. Af ter filtration, 50 ml of sodium hydroxide (Na~il 0.02 N) arc added in order to dissolve the remaining lauric acid in the aqueous phase, as well as the The organic phase (diethyl other) containing the lauric acid esters is evaporated under vacuum, in order to remove all trace of remaining solvent.

g of esters are finally obtained (mixture of monoesters and diesters of r-D-butylglucopyranosidn butyl 6-O-lauryl-a,D-glucopyranoside and butyl 2,G-di-Olauryl-ca,D-glucopyranoside). Characterization of the reaction products was done by T.L.C. and by proton and "~C

N.M.R.

Example 8 Production of ca-butylglucoeide etearic esters; The method of Example 7 was repeated using Stearic acid ;1.2 g ca--butylglucoside 1 g The mixture is broughL to 78*C and 0.15 g of LipozymeO is added.

The product was characLerized by 13C-N.M.R. It was a mixture mainly consisting of butyl 6-O-stearyl-a,Dglucopyranoside (approximately 00 and butyl 2,6-di-Ostearyl-caD-glucopyranoside (approximately 20

Claims (32)

1. Process for the enzymatic manufacture of a- glucosides, wherein at least one alcohol, whose only functional group(s) is(are) hydroxyl group(s), is a subs-trae selecfed om a nd placed in contact with starch, maltodextrins G- G' &66bsrdwe is 5 maltose, present in an initial concentration in the reaction medium of at least approximately 100 g/1, in the presence of a purified enzymatic preparation having an c-transglucosylation activity, said preparation .0 o sq actglucosidc.se being devoid of -ra eedse activity.

2. Process according to claim 1, wherein the enzymatic preparation consists of an enzyme having a- transglucosylation activity or of several enzymes such that each has an a-transglucosylation property or such that their combined enzymatic activities confer a- 15 transglucosylation activity on the enzymatic preparation.

3. Process according to either of claims 1 and 2, wherein the enzyme(s) is(are) a-transglucosidases, originating from a fungus.

4. Process according to claim 3, wherein the fungus is Talaromyces duponti, Aspergillus niger, Aspergillus orvzae or Asperqillus batatae. Process according to any one of claims 1 to 4, wherein the starch is in the form of native starch and in that the action of the enzymatic preparation, which is capable of carrying out the a-transglucosylation, is associated with the action of a hydrolase.

6. Process according to claim 5 wherein the hydrolase is an endo-amylase.

7. Process according to claim 5 wherein the hydrolase is a-amylase. IQ

8. Process according to claim 5, wherein +he two enzymatic reactions proceed simultaneously by simultaneous use of an endo-amylase and an a- transglucosidase.

9. Process according to any one of claims 5 to 8, wherein the native starch is derived from cereals, from tubers, from leguminous plants, or from any other plant. *0 0 .e 9** ~0 0t** a. a a a a .r *0 *e

10. Process according to claim 9 wherein 10 cereal is wheat, corn, barley, oats, rice, buckwheat, sorghum and triticale.

11. Process according to claim 9 wherein tuber is potato or manioc.

12. Process according to claim 9 wherein leguminous plant is peas or beans.

13. Process according to claim 9, wherein cereals are in the form of whole grains or fractior grains. the rye, the the the ns of

14. Process according to claim 13 wherein the grain fractions result from an enzymatic, chemical, thermal or mechanical treatment of the grain. Process according to claim 13 wherein the mechanical treatment is grinding.

16. Process according to any one of claims 1 to 4, wherein the starch is in the form of soluble starch.

17. Process according to claim 16 wherein the soluble starch results from an acidic or enzymatic prehydrolysis.

18. Process according to any one of the preceding claims, wherein the alcohol is an alkanol, an ethylenic alcohol, an acetylenic alcohol, a cyclic alcohol or a phenol.

19. Process according to claim 18, wherein the alcohol is a simple alcohol or a polyol.

20. Process according to claim 19 wherein the alcohol is a diol. O 34

21. Process according to any of claims 18 to wherein the alcohol contains from 1 to 6 carbon atoms, is at least partially water-soluble.

22. Process according to claim 21 wherein the alcohol has a solubility of at least 2.7 at 0 C.

23. Process according to any one of claims 18 to 21, wherein the alcohol is chosen from the following group isopropanol, n-butanol, isobutanol, tert- 10 butanol, isopentanol, hexanediol, 1-buten-3-ol, 1- butyn-3-ol, etc., or a mixture of at least two of these alcohols.

24. Process according to claim 18, wherein the alcohol is an alkanol containing from 1 to 5 carbon 15 atoms, the product of the process then being an a- S: alkylglucoside. 25, Process according to claim 24, wherein the alcohol is an alkanol containing 4 carbon atoms, the product of the process being a-butylglucoside. 20 26. Process according to claim 18, wherein the alcohol is a primary aliphatic alcohol.

27. Process according to claim 26, wherein the alcohol is a fatty alcohol having between 12 and 18 carbon atoms.

28. Process according to any one of claims 1 to 26, wherein it is carried out at a temperature of between 20 and 700C, and at a pH between 3 and 7.

29. Process according to claim 28, wherein it is carried out between 20 0 C and 500C, and at a pH between 4 and 6. Process for the enzymatic manufacture of a- glucoside esters from starch, maltodextrins or maltose, wherein a-glucosides are produced according to any one of claims 1 to 29, the a-glucoside(s) thus obtained 35 being subsequently placed in contact with one fatty acid at least and an enzymatic preparation having lipase activity, followed by recovery of the esters thus obtained.

31. Process according to claim 30, wherein the placing in contact of the a-glucoside with the fatty acid(s) and the lipase is carried out at a temperature at which the fatty acid(s) is(are) liquid, the reaction medium being devoid of solvent. 0006

32. Process according to claim 31, wherein the a- 10 glucoside in an a-alkylglucoside preferably having between 1 and 5 carbon atoms, and in that the fatty acid contains between 8 and 20 carbon atoms, preferably between 8 and 16. S. 33. a-glucosides as are produced according to the 15 process of any one of claims 1 to 29.

34. a-butylglucoside as is produced according to the process of any one of claims 1 to 29. Use of the a-glucosides according to claim 33 or 34 as surfactants, detergents or emulsifying agents 20 in the cosmetic, pharmaceutical or agro-food industry, or else as additives in the plastics, rubber and PVC industry.

36. Use of the a-glucosides according to claim as softeners in the plastics, rubber and PVC industry.

37. Use of the a-glucosides according to claim 33 or 34, for example the a-alkylglucosides, in which the alkyl chain contains up to 6 carbon atoms, as starting materials in the manufacture of surface-active agents, for example by esterfication or by transglucosidation, or as plyols in the manufacture of polyether polyols, polyesters or alkyd resins.

38. a-glucoside esters as are produced according to the process of any one of claims 30 to 32.

39. Use of the a-glucoside esters according to claim 38 as nonionic and biodegradable surfactants having foaming, emulsifying, moisturizing and solubilizing properties in cosmetic, pharmaceutical, agro-food or plant-protection compositions or as surface-active agent or whitening agent precursor in chemical compositions or in detergents. Process for the treatment of native starch, cereal flours or maltodextrins, characterized by the 10 placing in contact of these substrates, at an initial 5* Sb svrae In +ks rea4tion miediuAm A* concentration of at least approximately 100g/L,,,with an alcohol in which the only functional group(s) is(are) hydroxyl group(s), in the presence of a purified enzymatic preparation having a-transglucosylation 15 activity, said preparation being devoid of P- .r tefsglucosidase activity. DATED this 28th day of November 1996 a. ULICE S A By their Patent Attorneys By their Patent Attorneys CULLEN CO.