Abstract:
There is provided a regio-selective method for the resolution of carbohydrate monoester mixtures, by treating such mixtures with one or more selective enzymes. The resolution of such mixtures results in monoesters comprising significantly purer isolates of one desired isomer.

Description:
FILED OF THE INVENTION 
     This invention relates to the biosynthetic preparation of carbohydrate monoesters, which preparation is selective for the preparation of one or more positional isomers of such monoester. More particularly, this invention relates to the preparation of sucrose monoesters by selective hydrolysis of specific components of mixtures of sucrose monoesters by contacting such mixtures with specific enzymes. In addition, this invention relates to the use of monoesters made by the process of the present invention as excipients in pharmaceuticals, foods, cosmetics or as emulsifiers, coating agents, antiseptic agents and the like. 
     BACKGROUND OF THE INVENTION 
     Carbohydrate monoesters, and specifically sucrose monoesters, are used commercially as food emulsifying agents, as coating agents and as excipients in pharmaceutical, cosmetic and other products. Commercially available monoesters, typically made by chemical means, comprise mixtures of positional isomers or are often mixed with an amount of di- and tri-esters (usually at about 20% of the mixture). However, there is no commercially feasible synthesis that selectively yields pure monoesters, that is monoesters which substantially comprise one positional isomer of a monoester such as the 6-O, 6&#39;-O or 1&#39;-O monoester. If commercially available mixtures made by chemical means could be resolved such that specific isomers of the monoesters could be isolated, it would be advantageous since by selective isolation of the isomers the monoesters may have unique properties for food and cosmetic uses compared to the mixtures. 
     Therefore, there is a need for a process for the resolution of regio-selective isomers of carbohydrate monoesters, and particularly sucrose monoester. It is an object of this invention to provide a method to resolve a mixture of carbohydrate monoesters obtained by chemical reaction into monoesters of defined structure which comprise substantially the 6-O-ester, the 1&#39;-O-ester or the 6&#39;-O-ester. 
     SUMMARY OF THE INVENTION 
     This invention relates to a regio-selective method for the resolution of a mixture of carbohydrate monoesters, the method comprising incubating the ester mixture in an appropriate solvent system with one or more regio-selective enzymes and purifying the desired monoester. 
     In an embodiment of this invention, the carbohydrate monoester (or mixture thereof) is a sucrose or glucose monoester of a fatty acid of at least about 8 carbon atoms in length, and preferably about 8-22 carbon atoms. Most preferred are monoesters of a fatty acid of about 8-18 carbon atoms. Specifically preferred embodiments of the present invention comprise a carbohydrate which is glucose or sucrose and a fatty acid which is palmitate, laurate or caprate. The structure of sucrose monopalmitate and glucose-6-palmitate are provided below in order to show an illustration of what is meant by the 6-O, 1&#39;-O and 6&#39;-O esters, as well as to show varying fatty acid chain lengths. These structures should not be viewed as limiting the present invention. ##STR1## 
     Enzymes useful in the present invention are those enzymes which selectively hydrolyze certain components of the monoesters, and particularly those enzymes which hydrolyze primary alcohols on the carbohydrate. Useful enzymes include but are not limited to esterases, lipases, proteases or glycosidases or mixtures thereof. 
     DETAILED DESCRIPTION OF THE INVENTION 
     Presently, esters made by chemical, acid or base-catalyzed reactions of sucrose with fatty acid acyl donors are about 90% esters resulting from esterification of the three primary alcohol groups of sucrose. The remaining 10% is made up of esters of secondary alcohol groups on the sucrose. Such commercially available esters typically comprise a mixture of the 6-O-ester, the 1&#39;-O-ester and the 6&#39;-O-ester. Typically the 6-O and 6&#39;-O esters are present in approximately equal amounts and the 1&#39;-O-ester is about 20% of the combined amount of the 6-O and 6&#39;-O-esters. The remaining amount of monoesters consist of compounds with the five secondary alcohols in the sucrose molecule. The proportion of monoesters found in the eight alcohol functions on sucrose is in direct relationship to the chemical reactivity at those positions. 
     It has been found that by using regio-selective enzymes, it is possible to resolve a mixture of carbohydrate monoesters obtained by chemical reaction into monoesters of defined structure which defined structures are comprised substantially of one of the following esters: 6-O, 1&#39;-O or 6&#39;-O esters. The process of the present invention comprises incubating a mixture of carbohydrate monoesters in an appropriate solvent system with specific 
     
                                           TABLE 1__________________________________________________________________________Enzyme                                EsterIdentification         Source       Supplier   Specificity__________________________________________________________________________Amyloglucosidase         Rhizopus sp. Sigma      6-Amyloglucosidase         Aspergillus niger                      Sigma      6&#39;-, 1&#39;-AP-6          Aspergillus niger                      Amano      AAspergillus niger lipase         Aspergillus niger                      Biocatalysts Ltd.                                 6-AY-30         Candida sp.  Amano      6-CCL-VII       Candida cylindracea                      Sigma      6-CEH-I         Candida cylindracea                      E. Kodak   6-CEH-II        Candida cylindracea                      E. Kodak   ACEH grade 2   Candida cylindracea                      E. Kodak   Aα-Chymotrypsin type II         Bovine pancreas                      Sigma      BCVL           Chromobacterium viscosum                      Sigma      AFAP-15        Rhizopus sp. Amano      AGCI Lipase    Pseudomonas sp.                      Genencor Int&#39;l.                                 6-α-Glucosidase (maltase)         Baker&#39;s yeast                      Sigma      1&#39;-β-Glucosidase         Almonds      Sigma      6&#39;-, 1&#39;-Invertase.sup.1 grade V         Baker&#39;s yeast                      Sigma      6-L-700 (SAM-I) Pseudomonas fluoresens                      Mitsubishi AL-701 (SAM-II)         Pseudomonas fluoresens                      Mitsubishi AL-703 (L-10)  Candida lypolylica                      Amano      AL-706 (CE-10) Humicola lanuginosa                      Amano      6-L-707 (R-10)  Penicillium roqueforti                      Amano      ALP-160 (T-01) Chromobacterium viscostun                      Toyo Jozo  ALP-119        Mucor miehei Novo       ALP-302 (Lipase G)         Penicillim sp.                      knano      ALipozyme      Mucor miehei Novo       AM-AP10        Ymcor sp.    Amano      6&#39;-Mucor javanicus         Mucor javanicus                      Biocatalysts Ltd.                                 6&#39;-Mucor javanicus         Mucor javanicus                      Fluka      6&#39;-Mucor miehei  Micor miehei Biocatalysts Ltd.                                 AN conc        Rhizopus niveus                      Amano      AP (Super Pec P)         Aspergillus niger                      Amano      APenicillium cyclopium         Penicillim cyclopium                      Biocatalysts Ltd.                                 6&#39;-, 1&#39;-PL-105 (Super Pec PL)         Aspergillus niger                      Amano      6-PLE           Pig liver esterase                      Sigma      1&#39;-PPL type VI-S Porcine pancreas                      Sigma      BProleather (protease)         Bacillus subtilis                      Amano      BProtease Type XIV         Streptomyces griseus                      Sigma      1&#39;-Proteinase K  Aspergillus oryzae                      Int&#39;l Biotech.                                 6-Rhizopus javanicus         Rhizopus javanicus                      Biocatalysts Ltd.                                 ASAM-I         Pseudomonas fluoresens                      Amano      ASubtilopeptidase.sup.2          Bacillus subtilis                      Boehringer Mannheim                                 BWheat germ    Wheat germ   Sigma      A__________________________________________________________________________ A -- Activity with no selectivity B -- No activity .sup.1 Invertase incubation was performed at 45° C., pH 4.5, 50 mM citratephosphate buffer .sup.2 Subtilisin incubation was performed at 45° C., pH 8.4, 20 m potassium phosphate buffer 
    
     enzymes or combinations thereof, which enzymes selectively hydrolyze individual components while enriching the amount of a desired ester. The enriched, non-hydrolyzed ester is then purified by standard methods. 
     As used herein an &#34;appropriate solvent system&#34; means an aqueous solution, preferably water or an organic solvent system comprising solvents such as tetrahydrofuran, dimethylsulfoxide, dimethylformamide, pyridine, acetone and similar solvents mixed in varying amounts with water. 
     Enzymes useful in the present invention are those enzymes which selectively hydrolyze specific components of the carbohydrate monoesters. Preferably these enzymes are regio-selective for the primary alcohols of the monoester mixtures. Useful enzymes include but are not limited to esterases, lipases, proteases and glycosidases. Specifically useful enzymes are those listed in Table 1, below, which enzymes were screened using the methods described in Example 1. When glycosidases are used, the reaction is not a selective hydrolysis of a specific primary alcohol, rather, the desired reaction is the selective hydrolysis of a disaccharide glycosidic bond. 
     The carbohydrate monoesters of the present invention may be any carbohydrate monoester. Without intending to be limited, the applicants have demonstrated the present invention with sucrose and/or glucose esters of varying fatty acids. Sucrose monoesters are perhaps the most important from a commercial standpoint and, therefore, are used as the primary examples in this invention. The carbohydrates include but are not limited to glucose, sucrose, lactose, cellobiose, raffinose, maltose, mannose, galactose and ribose. Such carbohydrates are monoesters of a fatty acid which comprises at least about 8 carbon atoms. Such fatty acids may comprise about 8 to about 22 carbon atoms and preferably are palmitate, caprate, laurate, myristate, stearate and oleate. 
     Experimental: 
    
    
     EXAMPLE 1 
     Enzyme Screening Methods 
     Three types of hydrolytic enzymes were screened: lipases (esterases), proteases and glycosidases. The general method is as follows: 
     General Procedure: Sucrose monopalmitate (Ryoto P-1760), 100 mg, was dissolved in 10 ml of 50 mM potassium phosphate buffer, pH 7.0, with heating. After cooling to room temperature (23° C.) 100 mg (dried powder) enzyme was added. Mixture was stirred at room temperature for up to five days. Samples (200 μL) were taken at 3 hours 1 day and, in some cases, five day intervals. After drying on hot plate under air stream the samples were derivatized with 1 ml of 1/1 mixture of pyridine and BSTFA (Pierce) with heating at 100° C. for 1 hour. 
     Analytical Method: Preliminary assignment of structures was determined by gc chromatography. Gc chromatograms showed that the three major peaks belonged to the three primary esters. This determination is made on the assumption that primary alcohols in esterification reactions, among others, predominate when there is a choice between primary and secondary alcohols. As a rule the primary alcohols react at least 10 times faster than the secondaries. Acid hydrolysis followed by analysis of products via gc showed that only glucose6-palmitate, for which a standard was available, results from treatment with acid. From the other two primary esters, only free glucose was observed. 
     Gas chromatographic analysis was performed on a DB-5 column, 15 m (J &amp; W Scientific) with flame ionization detector, 1 μL injection. The starting temperature was 180° C. with a 10° per minute increase to 280° and a 6 minute holding time. Enzyme activity and specificity was estimated by comparing the areas under peaks corresponding to 6-O-, 1&#39;-O- and 6&#39;-O-esters. 
     Using the screening methods provided in this example, various classes of enzymes were tested. The screening summarized in Table 1 revealed five classes of enzymes: (1) those with no activity (&#34;B&#34;); (2) those with activity but no selectivity (&#34;A&#34;); (3) those with 6-O specificity; (4) those with 6&#39;-O specificity; and (5) those with 1&#39;-O specificity. In all cases, primary esters were attacked sit faster rates than secondary esters. 
     Based on the results of Example 1, three enzymes were selected for further study: Candida cylindracea lipase for 6-O position selectivity; Mucor javanicus lipase for 6&#39;-O position selectivity; and Penicillium cyclopium lipase for 1&#39;-O position selectivity. (In the case of P. cyclopium lipase, the 1&#39;-O-ester was hydrolyzed much faster than the 6&#39;-O-ester.) 
     EXAMPLE 2 
     CCL (Lipase) Hydrolysis of SMP 
     Sucrose monopalmitate (SMP) was used at a concentration of 10 mg/ml and Candida cylindracea lipase (CCL) (Sigma Type V) was added and dissolved at a concentration of 5 mg/ml. Incubation was performed with stirring at room temperature in water (no added buffers). Samples (200 μl ) were taken at 5, 10, 20, 25, 30 and 60 minutes. The reaction was stopped by immersion in a dry ice-propanol bath. Samples were freeze dried for 2 hours and derivatized for gc analysis as described in Example 1. Areas under peaks were calculated and plotted. It was found that CCL has a pronounced selectivity for the 6-O-ester of sucrose. 
     EXAMPLE 3 
     Map-10 Digestion of SMP 
     Following the procedure substantially as set forth in Example 2, Mucor javanicus lipase (M-AP10) (10 mg/ml) was incubated with SMP. It was found that Mucor javanicus lipase (M-AP10) has a selectivity for the 6&#39;-O-ester. 
     EXAMPLE 4 
     PC Digestion of SMP 
     Following the procedure substantially as set forth in Example 2, Penicillium cyclopium lipase (PC) (5 mg/ml) was incubated with SMP. It was found that Penicillium cyclopium lipase (PC) has a preference for the 1&#39;-O-ester, followed by preference for the 6&#39;-O-ester. 
     EXAMPLE 5 
     Assignment of GC Peaks 
     Candida cylindracea lipase (100 mg) was added to a sucrose monopalmitate mixture (100 mg) dissolved in 10 ml water. The mixture was stirred at room temperature for 1 hour and a 200 μl sample was taken for gc analysis. The mixture then was made up to 0.1N HCL and stirring was continued overnight at the same temperature. Samples were taken for gc analysis as described in Example 1. 
     A similar experiment was performed with Mucor javanicus lipase, except that the enzyme incubation time was 24 hours. 
     Review of the gc peaks revealed the following observations: Since only glucose-6-palmitate appeared in the hydrolysis sequence with Mucor lipase followed by acid hydrolysis, it was concluded that this enzyme had specificity for the 6&#39;-O position and, by similar deduction, the Candida lipase had 6-O specificity. Therefore, the two largest gc peaks must belong to the primary esters at 6- and 6&#39;- positions, while the next largest peak belongs to the 1&#39;-ester, since it is well known that chemical esterification favors primary alcohols strongly over secondary alcohols. 
     EXAMPLE 6 
     Digestion of Sucrose Di- and Tri-Esters to SMP 
     To a water solution/suspension of a mixture of 500 mg of sucrose dipalmitates and tripalmitates one of the three enzymes (200 rag) detailed in Examples 2-5 is added. Samples are taken at 30 minute intervals and analyzed by previous methods. 6- specific enzymes give mostly monoesters of 6&#39;-esters and 6&#39;-specific enzymes give mostly monoesters of 6-esters. 
     EXAMPLE 7 
     Specific Hydrolysis of Glucose-6-palmitate 
     To a mixture of glucose palmitate monoesters (50 mg) dissolved in 10 ml, pH 7 potassium phosphate, 50 mM, was added 25 mg of Candida cylindracea lipase. Incubation was at 23° for one hour. Gc analysis revealed that the major ester hydrolyzed was the primary ester in the 6-position. 
     EXAMPLE 8 
     Enzymatic Conversion 6f Sucrose Di- and Tri-Palmitate Esters 
     Enzymatic conversion of sucrose di- and tri-palmitate esters (Ryoto P-370, 100 mg) dissolved in 10 ml of a 50% solution of tetrahydrofuran (THF) in water is incubated with 100 mg of Candida cylindracea lipase for two hours at 45° C. After lyophilization and successive extractions with hexane and THF, purified 6-O-sucrose palmitate is obtained. By substituting Mucor javanicus lipase for Candida lipase and extending incubation to 20 hours, a purified sample of 6&#39;-O-sucrose palmitate is obtained. Further, if it is desired to remove the 1&#39;-ester from these preparations, a 20 minute incubation with Penicillium cyclopium lipase (10 mg/ml) is carried out.