CA1117047A - Preparation of high fructose syrups from sucrose - Google Patents

Abstract

ABSTRACT
This invention relates generally to (1) processes for the production and isolation of a novel fructosyl transferase enzyme from the fermentation broth of Pullularia pullulans, (2) enzymatic transfructosylation of sucrose to produce a novel fructose-polymer containing substrate, and (3) production of fructose syrups containing greater than 55% fructose from said novel substrate.

Description

FIELD OF THE INVENTION
This invention relates generally to enzymatic transEructosylation of sucrose. More particularly, thi~
invenltion relates to a unique proces~ for the production of fructose from sucro~e by way of a fructose polymer-containing substrate. This process provide~ a novel enzymatic approach for the prsduction of high fructose syrups having a fructose content significantly higher than presently obtainable by glucoæe isomerization of starch hydrolysates, without the necessity for physical separation of the resultant fructose end-product. This process i8 particularly adaptable to the production of fructose syrups containing greater than 55 fructose and higher. The invention al~o provides a novel transfructosylase enzyme from cultures of the yeast, Pullularia pullulans, found to be useful in such production.
This invention gives rise to a number of products. These include the ultimate fructose or a high fructose syrup, as well as various intermediate products such as the fructose polymer, initial fructose polymer (or polysaccharide) containing subætrate, substrate from which ~he polymer has been removed, and substrate (with or without the polymer) after isomerization. Each of these products is directly useful in its own right.
Each of these products has the properties of conventional sugars and syrups and may be employed in their customary applic-ationsO These include, ~or example, use as food sweetening agents and as raw materials for the preparation of pharmaceutical~.

1~7Q47 In addition, these produc~s may be employed in the common industrial ~ppLic~tiolls for su~ars and syrups. Thus they may be used in producing &dhesives, humectants, glassine paper, tanning agents, electric~l insulators, foundry core binders, S insecticides, dyes and ~he like or more generally as plasticizers, thickening agents, etc. In short, they are useful throughGut the broad spectrum of utilities in which analogo1ls products have already been employed.

1~7(~7 D~;IN_L~10NS
Because vf tlle many terms that are in common use in the art, the follow;ng definitions are provided to define the mcaning of thcse terrns as used herein:
Glucose and Dextrose The terms "g]ucose" and "dextrose" are em~loyed interchanyeably in this applica-tion to embrace this mono-saccharide in any form in solution or dry.
Sucrose The term "sucrose" refers to this disaccharide in lS refined or raw form, in solution or dry, from any sucrose raw material source, e.g. sugar cane or sugar beets. In the practice of this invention the sucrose starting material is typic~lly employed in aqueous medium.
Fructose and Levulose The terms "fructose" and "levulose" are generally employed interchangeably in the art to refer to the isomer of dextrose that is sweeter than dextrose. Fructose ;s found in honey and in invert sugar, along with dextr~se, and is va~uable because of its sweetness. The terms levulose and fructose will be used interchangeably in this specifica-tion to refer to this monosaccharide in any form, in solution ~r dry.

~il7~47 I n;~yrne L~re~.lrcltion qhe ~crm "en~me E~rcpclr.lt:ion" :i-; used hcrein to r~fe:r to clny composition of ma-tter that exhibits the ciesired enzylllatic a~ ity. The term is used to refer, for exaTnpl(-, to l:ive 7hole cells, dry cells, cell extracts, refined and concentrated p~eparations derived fr~m ~he cells and from culture liquors. The enzyme preparati~ns may be used ei.ther as a solution or in an immobilized form in the practice o this invention.
~somerase En2~nie Any enzyme preparation that isomerî%es deY~trose to levulose is referxed to herein as an "isomerase enzyme."
These enzymes are well known in the art and have been referred to as dextrose isomerase, xylose isomerase and glucose isomerase~ Such enzymes can be derived from a variety of suitable microorganisms. Examples oE such microorganisms include those of the genera Streptomyces, Bacillus, Arthrobacter, Actinoplanes, Curtobacterium and others.

A preferred isomerase en~yme useful :in the ~r~ctice of the presen-t invention is derived from Streetomyces olivo_hromoqenes ATCC No. 21, 713, ATCC No. 21, 714 or ATCC
No. 21,715, (the latter of which is a single colony iso].ate of ATCC ~o. 21,713) as disclosed in U. S. Patenl: 3,~13,318 1~17~ ~
and U.S. patent 3,957,5~7J particularly when prepared by the process described in U.S. Patent 3,770,589 or U.S. Patent 3,813,318.
Recently, the art has come to recognize processes where the isomerase enzyme is immobilized on a water-insoluble inert carrier. The immobilized enzyme i8 then suitable for use in continuously converting glucose to a high fructose syrup.
Examples of such processes are described in U.S. Patents 3,708,397: 3,788,945; 3,850,751; 3,868,304; Belgium Patent 819,859; and U.S. Patent 3,960,663 (Belgium Patent No. 810,480).

Isomerase Unit "Isomerase unit" i5 defined as the amount of enzyme activity that is required to produce one micromole of levulose per minute under the isomerization conditions described hereafter under the heading "Assay of Isomerase Activity".

AssaY of Isomerase ActivitY
As used herein this term refers to the assay procedure which involves making a spectrophotometric deter-mination of the ketose produced from a glucose solution under a standardized set of conditions.
A stock solution is made up in the following manner:

_`_C~_ Ol,tJTT~N FOR ~SS~Y
Corn~o-~ent ~mount .1 M MgS~,1-71~2O 1 ml 0.001 M CQC12 Gll2O 1 ml 1 M sodiurn phospate buffer, pH 7.5 0.5 ml Anhydrous D-ylucose 1,44 g .
Add distilled water to make up a total volume of 7.5 ml The enzyme preparation to be assayed is first diluted to contain frolQ 1 to 6 isomerase units per ml.
An enzymatic isomerization is conducted by adding 1 ml of the enzyme preparation to 3 ml of the stock solution, and incubating for 30 minutes at 60~C. At the end of the incubation period, a 1 ml aliquot is taken and quenched in a 9 ml volume of 0.5 N perchloric acid. The quenched aliquot is then diluted to a total volume of 250 ml. As a control, for comparative purposes, a glucose blank is also run by substituting 1 ml of water for the 1 ml of the enzyme preparation in-solution at the beginning of the incubation , 20 period.
The ketose is then determined hy a c~s~eine-sulfuxic acid method. For the purposes of this assay, one isomerase unit is defined as the amount of enzyme activity that is required to produce one micrornole of levu~ose per minute under the isomerization conditions described.

~117~4~

Tra s~rllctosy.lation This t~rm as used herein refers t~ the trans~er of a fructosyl moiety from donor, e.g., sucrose, to an acceptor, e.~ )olysaccharide.
ructosyl. Transferase Enæyme As used herein this term refers to any enzyme that catalyzes transfructosylation~and includes the enzyme prepara-tion derived from Puliularia pu].lulans ATCC 9348 (synonomous with Aureobasidium E~l~lulans _uct~sy~ ansferase Unit As used herein, one fructosyl transferase unit is defined as the amount of enzyme activity required to produce one micromole of reducing sugar, calculated as glucose, per minute under the fo.'.lowing conditions: (a) pH 5.5, (b) temperature 55C, and ~c). substrate concentration at 60 g food grade sucrose per 100 ml of an aqueous reaction mixture.
Reducing sugar determinations (calculated as - B glucose) are carried out using a "Technicon Autoanalyzer II"
(Technicon, Inc., Tarrytown, NQW York). Analysis is carried out by a conventional alkaline ferricyanide method, Analytical Bioch~ 45, No. 2, pp. 517-524 (1972~, adapted for use in ~he "Autoarlalyzer ~I". Unless othe~wise desi.gn~te~, enzyme acti~ity determinations are performed ~ continual monitoring of a reaction mixture consisting o the following composition: . - .
~r~de rn~r k ~117~47 7.5 ml of 80~ (w/v) aqueous food grade sucrose solution

2.3 ml 0.1 M citrate buffer p~I 5.5 0.2 ml enzyme s~mple conta~ning that amount of fructosyl transfcrase enzyme which will produce from 5 - 25 micrograms of reducing sugar (calcu-lated as glucose) per minute per ml of reaction mixture Primary Substrate The term "primary substrate" as used herein refers to those saccharides in a ~orm suitable for and having a fructosyl moiety available for participation in transfructo-sylation, as for example aqueous solutions of sucrose.
.Secondary Substrate The term "secondary substrate" as used herein is the reaction product resulting from subjecting the primary substrate to the action of a fructosyl transferase enzyme preparation, as defined herein.
Parts and Percentages In this application all parts are by weight and all percentages are weight by volume ~w/v) unless expressly stated to be otherwise.
Hi~h Pressure Liquid Chromatographic ~say This term as u.sed herein defines the procedure where~y the syrups of the inven-tion are analy;~ed using high pressure liquid chromatography in accordance w.ith the ~1~7~47 ~-llot~i2lg tcchni(l-!e- ~olnpolle~nt~ are chrom;lt:ocJraphed by elution wi~h wate l:rom a cation excllanc3e rcsill in the calcium form. ~lu~c:d (~o~pontnts are detected by means of a differential refractometer. Non-dextrose carbohydrates are quantitated usin~ an elcctronic inte~rator, and de~trose is obtained by difference. The general procedure is that given in "Analysis of Carbohydra~e Mixtures by Liquid Chromatogra-p~y", Am. Soc. Brew Chem. Proc., 1973, pp. 43-46. The resin used is "~minex Q" 15-5 in the calcium form, Bio- ~
Rad La~oratories, Richmond, California.
- SUMMARY O~ I~ENTIO~
In accordance with the invention there is provided a process for the production of syrups which comprises subjecting a primary substrate, e.g~, sucrose, to the action of a fructosyl transferase enzyme preparation capable of converting the sucrose to a produc~ comprising a monosaccharide fraction containing a major amount of glucose and a minor amount of fructose, and polysaccharides containing at least 66% (by weight) fructosyl moieties. Such product comprises a secondary substrate of this invention. These poly-saccharides of the secondary substrate include all fructose-cnntaining polymers (other than sucrose) having two or more ,nonosaccharide moieties. These polymers may be further characterized as including polysaccharides containing fructosyl moieties linked by (2 ~ beta linkages. As described hereinafter, the polysaccharidès resultant from given conditions of transfructosylation may lie pre-dominently within a predeterminable range. Thus, or example, secondary substrate may be produced in which most (e.g., at least 60%
by mole ratio) of the polysaccharide are oligomers having from 2 to 10 (i.e., DP 2-10, more normally 3 to 6 (i.e. DP 3-6) monosaccharide moieties. Thereafter, said glucose is isomerized (via the action of _g_ 1~7~7 is~merase ~nz~le) to fructose in th~ presence of said polysaccharides, fol]owed by hydrolysis of said polysaccharides in the absence of act:iv~ isom~rase cnzyme. The hydrolysis can be carried out enæymatically using invcrtase or by acid hyclrolysis under mild conditions.

~ n ~ furtller embodiment o~ the invention, the polysaccharides can be separated from said glucose and said fructose, and thereater, hydrol~zed, as previously described, to produce an ultra-high fructose syrup, e.~ having a fructose con~ent of greater han 66% ~y weight and preferably ~reater than 90Q by weight, directly from the secondary substrate of this invention without the necessity of isomeriza-tion of the dextrose in said substrate. Such-separation can conveniently be carried out by conventional physical separa-tion techniques based on molecular size, as for example, conventional membrane technology, ~e.g., ultrafiltration, I aialysis), solvent precipitation, carbon absorption and t~le li~e. Exemplary of such membrane technology are United States Patents 3,173,~67; Re 260~7; 3,541,006; and 3,691,06g A preferred embodiment is a process or the produc-tion of high fructose syrups which comprises subjecting ~; sucrose to the action of a fructosyl transferase enzyme /0 ~

1~17~4 ~

preparation, as for example that derived from Pullalaria pullulans such as ATCC 934~; ATCC 12535: NRRL 1673; NRRL
Y2311; NRRL YB3~92; NRRL YB 38617 NRRL 3937 and ATCC 15223.
The resulting product, or secondary substrate, is subjected to the action of isomerase enzyme. Thereafter the isomerized product is hydrolyzed in the absence of active isomerase enzyme.
The secondary substrate produced in accordance with the process of this invention is believed to be novel. This substrate is uniquely suitable for isomerization and subsequent hydrolysis to pxovide a syrup containing greater than 55~ fructose, and is produced by subjecting sucrose to 1~ 4~

the action of a fructosyl transferase enzyme preparation.
Therefore another cmbodiment oE this invention are substrates suitable for enzymatic isomerization ancl subsequent hydrolysis to syrtJps containing greater than 55~ fructose syrup, S comprising (1) from about 20% to about 60% by weight mono-sacchar:ides, containing a major amount of glucose and a minor amount o fructose, ana (2) from about 70~ to about 40% polysaccharides containing ~eatex than 66~ by weight - fructosyl moi`eties.

Especially pxeferred are secondary substrates derived from sucrose by transfructosylation in the presence of an effective amount of a fructosyl transferase enzyme pxeparation derived from a strain of Pullularia pullulans ATCC ~348, at a temperature ranging from about 25C to about 65C, and preferably frc?m about 50C to about 60C, and at a pH ranging from about 4.5 to about 6.5, and preferably about 5.4 to about 5.6. The starting sucrose concentrations employed can range as low as 10 g per 100 ml water. However, it i5 preferred to employ as high a dry substance concentration as possible, preferably ranging from about 30 g to about.
60 g pex 100 ml (for maximum reaction rate), up to the saturation point of sucrose (~nd highex, as clescxibcd more~
fully hexeinafter).
A minimum of 0.5 unit of fructosyl transferase per gram of sucrose can ~e employed to produce the novel substrate of this invention. ~enerally, the amount of enzyme used 1~L17~47 will no~ exceed liC uni-tc per ~ram oE sucrose because of economic considerat-i()ns. Especially pre~erred to obtain the desired secondary substrate in a commercially acceptable time, and within the above desc:ribed processing parameters, is a range of from .~bout 2 to about 30 units per gram of sucrose.
The foregoing and other embodiments OL this invention are descri~ed more fully hereinaftex.

DETAILED DESCRII'TION OF THE INVENTION
, The procedure for production of the novel ~ructosyl transferase of the invention can utilize conventiona' fermentation techniques, e.~. see United States Patent Nos. -

3,565,756; 3,806,419; 3,535,123; S. Ueda et al., Applied Microbio~ 215 (1963). Preferably certain novel separation or purification features, which shall be described in more detail hereinafter, are utilized. The following example is a typical fermentation procedure for production of the enzyme from Pullularia pullulans ATCC 9348.
Example 1 Production of Fructosyl Transferase Enzyme Preparation - Celite Carrier _ _ _ . The Fermentation Procedure Used to Produce the L:nzyme The medium used for inoculum development and fermentation to produce the enzyme is as follows:

lil7~47 0.5~ Di1~asi.c Potass.ium Phosphate O.l~ So~ium Chloride 0.02~ Magnesium Sulfate-~Ieptahydrate 0~ 06o Diammonium Sulfate 5 0.3~ Yeast Extract (Difco Lahoratories) 7.5% Sucrose (Food Grade) p~I of medium adjusted to 6r 8 The seed flasks, 500 ml Erlenmeyers containing lO0 ml of sterile meaium, are inoculated from a slant culture of the black yeast, Pul1uIar~a pullulans~ The paxticular strain o the yeast employed is designated in the catalogue of the American Type Culture Collection (Rockville, ~ryland) as ATCC 9348. The seed flasks, after development on a reciprocal shaker for 48 hours at 32C, are used to inoculate one liter Erlemneyer fermentation flasks, each containing 200 ml of tl1e previously defined medium. The inoculum concentration used is 0.5% w/v. The fermentation is conducted on a reciprocal shaker at 32C for 7 days.
B~ Recovery of the E~.zyme rom the Fermen tion Broth The fermentati.on broths from fort~ l-liter shaker flasl.s are pooled and the flasks are xinsed with ~7al~r ~rhich is a:~so added to the poo.Led broth. The final volurne o the .broth after dilution is l~ liters. The original. volume of broth is approximately a liters. The 12 liters of exmenta-tion ~roth are xun through a Sharples continuous centrifuge to remo~re the yeast cells and cellular debris~ The super-natant, which i.s a black viscous solution, is th~3- dosed 1~17047 with calcium chlc)rid~ ~o a 0.5~ w/v concentration and the p~l of the rcsultinc3 solu~ion is adjustcd to 7.0 with sodium hydroxidc. A second pass is then made throu~h the Sharples cen~ri~uge to procluce a viscous supernatant which is low in 5 eolor. The pH of the decolorized supernatant is adjusted to 5.5 with hydrochloric acid, followed by dosin~ with 1000 units (as defined in United States Pa-tent 3,806,~19) of pullulanase. The resultant broth is preserved with toluene (added to saturation) and the pullulanase is allowed to react at ambient temperature overnight. After digestion with pullulanase overnight, a 1% concentration of Grefco B #503 Celite (~ohns-Manville Products Corporation, Lompoc, California) is slurried in the broth followed by the addi-tion of 2 volumes (24 liters) of acetone. A precipitate forms and i5 collected by filtration, and the filter eake washed with acetone and dried at ambient temperature. The eollected filter ca]ce contains the insolubiliæed fructosyl transferase enzyme.
In Example 1 it should be noted that the addition of calcium chloride to the fermentation broth, with adjust-ment of the broth pH, results in the remova:i of the hlack pigment and the acidic polysacc~lari~es present. IFor discus-sion of these acidic polysaccharides see Acta. Chem. Scand.
16, 615-622 (1962).] The final enzyme pro~uct- is thereby rendered in the form of a relatively pure, colorless prepara-tion. This xefining procedure constitutes a preferred k -15- .

~1 7 C! 4 7 embodi.ment of thi5 invention and permits simple refining procedures, i.c. centriugation, filtration or precipitation, to obtai.n the final product. Thus, in accordance with this .
embodi.ment there is provided a process for separating acidic po].ysclccharides and black pigment by-products from final fermentation broths of the black yeast, Pullularia pullulans.
This method renders a final enzyme preparation free of undesirable pigment and acidic polysaccharide by-products, which are formed during the fermentation process. These by-products, unless remov~d, co-precipitate with the enzyme upon solvent addition to the fermentati.on broth.
As a further refinement for recovering purified enzyme preparations of this invention, it is desireable to remove the pullulan polysaccharide inherently present in the 15. fermentation broth becallse it too will co-precipitate with the fructosyl transferase enzyme upon solvent addition to the fermentation broth. Therefore, the supernatant obtained from the calcium chloride treatment and p~ adjustment can be *urther treated with the well-known hydrolyzing enzyme, pullulanase. Pullulanase enzyme randomly hydrolyzes the pullulan to produce a lower mo].ecular wei.ght polymer, thus avoidin~ coprecipitatiorl and consequent con-tamination of the frwctosyl transferase enzyme preparation during the sol~7ent (e.q. acetone, alcohol and t}3e li)ce) treatment. The purificat on of the desired fructosyl trans-ferase enzyme in this manner constitutes ailother novel embodiment of this invention.

1~17(~4';' The fructosyl transferase enzyme preparations. of this invention can be employed without the removal of the pullulan~ Thus, this invention can be practiced without hydrolysis of~ the~-pullu-la.~ by pullulanase, in which case pullulan serves as a carrier for the fructosyl transferase enzyme.

The followin~ Example demonstrates the use of pullulan as a carrier.
.
.

.

Example 2 .
Production of Secondary Substrate Using Fructosyl Transferase Enzyme On Pullulan Carrier 15 A 20% sucrose solution buffered with 0.0S ~
citrate buffer pH 5.5 is dosed with a 1% concentration of dry ~ullulan produced in accordance with the procedure of Example 1, except that the pullulan is not hydrolyzed with pullulanase and,.thereEore, serves as a carrier and no.
Celite is employed. Fructosyl transferase activity is 677 units/gram of pullulan. The ~eaction is carri.ed out at ambient temperature unt.il the mixture becomes ~azy. A
sample of this material is analyzed by high pressure liquid chromatography ~ith the following results Q~7 ~, .

S~CCEI~ D~: DISTRIBIITION B~ llr'LC AN~L~SIS
___ _ _ _ Fruc~ose Dextrose Dp2 DP3 DP4 6.9 ~0.6 6.2 11.1 35.2 Tlle following examples further characterize the 'enæyme produced in Example 1.
Example 3 Prod-cts of Enzymatic Action and Enzyme Thermal Stability To reaction bottles, equipped with scre-~-caps, are added 60 g of food grade sucrose and a fructosyl transferase enzyme preparation. The enzyme preparation is obtained from the enzyme product in ~xarnple 1 by dispersing suitable aliguots of the solid Celite-enzyme product into measured amounts of water to produce a suitable concentration of an enzyme solution. The Celite is then removed by filtration.
The filtrat~ is used for dosing the reaction mixture at 10, 20 and 30 units of enzyme per gram of sucrose substr~te.
These mixtures are then each diluted to a final volume of 100 ml with water. Conversions are carried out for 66 hours or 60C, at pH 5.5 and 55~C/respectively. Samples are taken at 24, 43 and 66 hours for reducing sugar determinations to ascertain the presence of enzymatic activity. After xeducing su~ar cle-~erminatiorls are run on the salllp]es, the remaininy r~action mixtures are frozen to stop the enzymatic action and samples submitted for determination of carbohydrate composition by high pressure liquid chromatography The follo~ing results were obtained:

.

~117~47 ¢

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a ~ o o u~
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~ o ~6 g ~ g 1~ 01 O O~ O ~
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~ P' C`l a 1~17(~ 7 ~ ample 3 demonstr-ates the thcrmal stability in the preC;ence of su~strate of the enæy}ne at 55C and 60C
throucJIout a 66 hour period at a pll of 5.5, ~hus demonstrating thc co~merci,~l potential of thc enzyTne. Moreover, the secondary S subs~rclte produced by enzymatic conversion of sucrose with the novel fructosyl transferase preparation of this inver-tion is demonstrated by the carbohydrate analysis to be a potentially valuable product suitable for the proauction of high fructose syrups from sucrose, because the predominant constituents are shown to be dextrose and polymers which upon subsequent hydrolysis yield fructose as the predominant monosaccharide. This example also demonstrates that the novel enzyme of the invention is effective at 60% (w/v) sucrose concentration. Also demonstrate~l is the functlonality of 5. the enzyme in the presence of high glucose concentrations.
Example 4 Effect of Temperature on Enzyme Activity .
'rhe effect of temperature on the reaction rate of fructosyl transferase enzyme is determined using the "Technicon Autoanalyzer II" as follows:
The reaction mixtu~e consists of 7.5 ml of 80%
(w/v) sucrose solution, 2.3 mL of a O.l ~ citrate buffer at a pH of 5.5, and 0.2 ml of a 2% w/v pullulaTl enzyTne solution (enzyme preparation of ~xamp e II). Final sucrose concentra-tion is 60% (w/v). The samples are held at the follo~ing Q~

temperatures and ~ssayed for 10 minutes OJ- the "Technicon Autoanalyzer II." Results demonstrate that at 40C, enzymatic rate of reaction is 1.89 times that at 30C; at 50C, 1,29 ,time.s ~rcater tllan at 40C, and at 60C, 1.48 times greater t},an at 50C. This demonstrated an incxeasing reaction rate with increasing temperature.
. Example 5 - ~emonstration Of The Michaelis-Menten Constant (Xm) Of The Fructosyl Transferase Enzyme The Km f an enzyme denotes the substrate con-rate o~ -centration at which the/product formation is at one-half of ,the Vma~. The following procedure was used to obtain the Km o~ the fructosyl transferase enzyme.
Using a 90% w/v sucrose stock solutior. adjusted to pH 5.5 with 0.1 M citrate buf~er, the proper concentrations of sucrose in 9.8 ml aliquots are prepared to give: 5, 10, 30, 40, 50, 60 and 70% concentrations of sucrose at a final volume of 10 ml. An enzyme solution at 0.2 ml containing 1.1 unit of fructosyl transferase enzyme is added to the at-io temperated samples. Then the samples are immediately.assayed at 55C on the "Technicon Autoanaly2er II" a~ prev.i.ous~
ly described (see fructosyl Lransferase unit). A glucose standard (calibrated in ~g/ml~ is included as a control.
Following is the rate of glucose formation ex-pressed as ~g/ml/minute at the various substrate concentra-tions us;.ng a constant dose (1.1 unit) of the fructosyl ~ransferase enzyme preparation of Example 1.

.

1~17~
.
96 SU}3STIU~TI~ llg/ml/min ~ ml/min ) 8.0 8.0 :10 11. 8 . 11. 6 ~0 15.7 15.2 18.0 . 17.6 18. 6 18. 2 19.2 17.2 17.8 18.2 7 0 15 . 6 a~ ~erun next day from a 60~ sucrose stock solution The K is 0.27 molar sucrose concentration. Maximum reaction m velocity occurred at a substrate concentration of 1. 374 molar sucrose, at pH 5.5 and 55C.
Example 6 The Preparation and Isomerization of Secondary Substrate From Sucrose A Production of Secondary Substrate .
Fooa Grade Sucrose, 600 g, is dissolved in water to a volume of 800 ml. The pH of this solution is adjusted to 5.5 with dilute hydrochloric acid. A dry Celite-enzyme preparation, 11 g, with an activity of 550 units~g, prepared as in Example 1, is slurried in 100 ml of water. The slurr~
is filtered under vacuum onto W~latman No. 1 filter paper in a Bùchner funnel. The filter cake is ~ashed ~ith an addi- -tional 100 ml of water. The 200 ml of filtrate is then added to the sucrose solution, which is in a 0.5 yallon bottle equipped with screw cap. The bott]e is then placed in a 5~C water ba~h and the reaction is .Illowed to continue for 20 hollrs a~ter which time a sample of the reaction product is assayed by high pressure liquid ~hromatography for cletermination o~ carbohydrate composition with the following results:
Carboh~drate Composition Fructose 2.4%
Dextrose 32.8%
2 10~6~
3 22.9%

4~ - 31.3%
Magnesium chloride is adaed to the remaining reaction product ti.e., secondary substrate~ to a concentra-tion of 5 millimolar, and the pH is.adjusted to 8.4 with dilute sodium hydroxide.
B. ontinuous Isomerization of Secondary Substrate Glucose isomerase derived from Streptomyces olivochromo~enes ATCC 21,715 (See United States Patent No.
Re: 29,152) is immob~lized on porous alumina (a controlled pore carrier produced by Corning Glass Co., Corning, New York, e.g., see United States Patent ~o. 3,992,329) as ollows:
1. Carrier is washed twice with water;
2. The carr;er is incubated with 0.1 M sodium citrate for 1 hour with agitation;
3. The sodium citrate is washed rom the carrier until conductivity of the wash solu~ion is 1000 micromhos;

-. 1i17~ 7 4. The ~r.ricr is .incubated wi.th 0~05 1~ rnagne~,ium chloride for one hour and the magnesium chloride solution .is decan-ted;

5. A volume of 0.05 M ma~nesium chloride i.s then added to provide for a final en~yme ooncentr~tion of 400 units/ml~

6. The isomerase enzyme concentr~te is adaed to the car.ri.er at a level o~ 14 mi.llion units per cub.ic foot;

7. The carrier and enzyme are con~acted or 22 2~ hours and then unbound enzyme .i.s washed xom the carrier wi.th distilled t.ratex;
. A jacketed glass column (3 cm x 18 cm~, equi.pped with a pump connected to a feed supply reservoi.r, is tllen ~5 loaded witli the immobilized enzyme thus prepared. The bed volume o~ th~ co].umn after loadi.ng is 45 ml. The column is opexated at 60C for all isomerizations. The loaded column i started on a dextrose syrup (50% w/w concentration) with S mil~imolar of magnesium chloride and adju~ted to p~l ~.4 to demonstrate that the co-umn .is aciive. The column flow rate is adjusted to 292 ml/hr. Th~ column :i.s l:hen ~rained to bed level. The introduction of the secondary sub~st:rat:e is conducted manually until ~0 ml o~ effJ.uent are c~:LIec1-ed anc~ then the f low-rate ~or the .secondaxy subs~rate :i.s 2r~ adjllsted to 292 ml/hrA The f.irst 10~ ml o~ syrup collected ~ is discarded to alloT.~ for ch~nge of suhstrate. q~he xemaini.ng :.

:. .

1117~
;econd;lxy ~ t:.rate (a~out ~50 ml) is then put t:hrough ~he ~olumn. ~ft.cr cne hour the flow rate incrcases to 570 ml~hr. The rlow rate .is ctdjusled and maintained at 300 ml/hr. to tht` end of the run. After comple-tion of the run wi.th secondlry substrate the colulNn is swi.tched back to d-xtrose to deMonstrate that isomerase act.ivity r~mains.
The follow.ing table compares the hiyh.prtssure liquid c:hromatography analy~es of the starting secondary su~)sl:rate and final pxoduct. from the i.somerization colu~n: -. _econdary.Substrate (A? Final Produc~ (B) Fructose~ 2.~. . 15.7 . . .
Dextrose 32.8 18.9 ~... ;

DP2 10.6 11.0 ~¦~P 3 2 2 ~ 9 ;2 2 r 9 . . ...
. 1 ~P4+ 31~3 . 31.5 These results indi~ate that 4~.38~ ~f the ~reedextrose in the secondary substrate is isomer-ized .in the : column to fructose. Also the fructose- polymexs present in the ~econdary substrate did nok appear to be ~fected or to af~ect the isomeriz~;ttion of dextrose to fructcse. X~ is noteworthy tlla.t the total monosacchari.de dis~ihuli.o~
consists o~ about 45~ fructose and 55~ dextx~se.
In the followlng exampl.e ~wo approaolles are emplc~etl ~o cleavt3 the pol~saccharides prtse~ in l~ e secon~a.r~ sllhstrate and also the isomerlzat.it~rl prod1lct - tie~x~ .Lo~rl. One approach :;s enzy~at.i.c a)l~ th~. o1:her ~Iti..~ es a ~ni.lcl a C:3 d hydxolysi.s .

'7~L'7 1 xamp le 7 Prcp~l:ra~ic)n oi~ lli.~h F`ructo.se ~yrup }~ ydro~is . ~n~,~n.~t.~ t.-c~
100 ml of procluct B rom ~xample 6 is dosed w;.th 10 mq of a puxified i.nvextase deri.ved from Ca~cl:~d~ util.~s.
'rhis mi~t:ure (preserved ~ith toluene) ;.s allowed to react ove~ni~llt at ~mbient temperaturc~ aft~r ~ ich a sample i~
wi~hdrawn and analyzed by high pre~ssuxe l:i.quid chromatct3raph~.
. 'rl~e remaining Prod~ct B is allowed ~o continue ~o react for an additional 6 days and ~hen a second sampl~ is assayed the same procedure with the following re~.ults: .

~A1:.er Startin~ Material Af~er Invertase ~dditional (Product B) Act on Six Fructose ~5~7% 41.9% 62.4 Dextrose 18.9g6 29.4~ 36~2 DP2 11. 0% 1. ~6 O
DP3 22.9% 12.9% . 0.5%
: ~P4~ 31.5% 13.9% . 0.9 . The invertase employed is an enzyme prepnrat.ion manufactuxed by Siekagakll Kogyo Co., Ltd., T~'~yor Jap~nt having an activit~ 123 units/~ng. ~here..l Ullit o in\rertase catal.ysen the cleav~ge of sucrose to form 1 m:i.cromo:l.e of glucose arld 1 micromole o fxuct:ose per mi.nute unde.r spaci.ied co~iti.o~s.
. : ~5 13. Ac~d Hydxolysis o Product B
Acid hy~rolysis of a sample ~rom P~oduc; ~ of E.xalllple 6 wa~; caxr;.ed out by addi.t~on of ~sul~wr:;.c ac:i.d ~o a concen~cat:i.on of o.or, l~l and heati.nc~ to 75-~)'C. Sas-nples ~rc~

. ~7-ta}~en, aLt~:r one ancl t~70 houl-s of h~drolysis, and analyzcd by hi~h pressurc ~iqu.i~ chromato~raphy. The results are as follows:

Starti.ng Material (Product B) 1 Hour2 Hours Fructose 15.7 60.3 59.1 ~extrose 18.9 38.7 37.9 ~P2 11.0 ~.9 2.6 ~P3 22.9 0.4 0.3 DP4~ 31.5 .. 0 0.2 The above re5ults in Step A show a predom.inant . increase in fructose and a les~ser.~ncrease in glllcose yield occurred with a corresponding drop in the DP~, DP3, and ..
~P4+ fractions, thus ~emonstrating the presence of a fructose ~olymer. ~ `
The results from acid hyarolysis in .Step B are in accord with those obtained in Step A, thus also demonstrating cleavage ~ fructose polymers. . . ~ -.
The foregoing discussion is directe~ to transfructo-;`;~20: s~lation using a primary substrate~wherein the.dry substance co~tent o~ starting sucrose does not exceed the saturat}on : point under given:reacti.on condi.tions. The ollow.in~ example ` . demonst~~a~es the use of a primar~ substrate havin~ an initial sucrose concentra~ion in excess of saturation arld which, when subjected to the action o~ the fr~ctQc;yl trans~erase : : .
: enzyme, results .in a product containing incr~ased levels o : DP3 mat.erl.a] (i..e~ ~ructosy~-sucrose~ and a ~ecreased concentra~
ti.on o~ D~4~ pro~ucts. Also ex.hibite~1 is an ;.ncxease in ~he ,: . ' , ;

dry subst~nc~ ~oi~on trati~n (w/w) of tlle secondary substrate over the dr~ su~st~nce concentration obtained in the absence of the ac~ion of the fructosyl transferase e~zyme. Moreover, it is ~emollstxated that, ~hen the dry substance concentration of the primary substrate increases, the degree of polymeriza--tion of the fxuctose polymer in the secondary substrate decreases and the DP4~ material is present in minor arnounts.
This is in marked contrast to the results ohtained in the previous examples usin~ sucrose substrates at less than saturation. ~n those examples the DP4~ ~aterial is the primary product.
~ Example 8 ; ¦Preparation of Secondary Substrate froM Sucrose ISl~lrries in Excess of Saturation -- __ __ __ ___ Food Graae Sucrose in 200 g al.iquots is placed in one pint jars w.i.th screw-cap lids. As a cont~ol, 50 ml of water is added to one jar and the other each xeceive 50 ml water containi.ng increasiny amounts of fructo~;yl transferase`

enzyme-Celite prepaxati.on (produced in accor~.nce with E~ample 1), as shown in tl~e following table. Each i.s capped and placed in a fihaking watex bath set at 54-55C. The 1ask.s are shalcen for 24 hou~s with occasi.orlal manual mi..x:;ng. A ~clmp:Le of ~uperna~ant is withdrawn from each bottle arld placed in a screw-ca~ test tube. in a ~oi.l.ing water bath ~o i.nacti.vate 25 the eAnY.~me. These samples are then analyzed hy high pres~iu:re liquid chl-omat:ography and supernata~ dry ~,ubstallce n.ethod de-t:er~l;.rlations a~.e ~acle (}~. ~isc~e~ 7i.l:h t.h~.~ fol3.owi.ny 3:C~ su !. t S:
- .

J~l D~r S~lbst~ncc (~ ition of Carboh~drate~;
L'~ttle ~o. S~lc~ose~ ;.ts in ~?ematant(~ ) in ~Solution~
~c~ DP2~E~4 200100 74. 5 0. G9 . 6 80. ~ 8. 9 N~
2 200200 75.6 . 0.7 lZ.772.2 13.7 ~I.7 3 200300 76. 3 1. 014. ~; 66. 8 16. 6 ~ 1 4 200~00 7~.1 0.~ 15.7~;~.6 19.0 .~.
20Q500 . 77. 7 1. .1. 17. 3 5~ S 21~ t~ r - 6 200 ~OC 78,1 1.3 .1~.0 56.0 2~,.9. ~,.g 7 . 200700 78.1 1.1 18.8 53.9 23.1 8r(}
- ~~ . 200 800 80 0 ~.. 0 .~.3 52~
9 200 90û 79 0 1.3 19.9 so.t?~5.0 3.7 : ~ .10 200 .. 1000 7g.6 1~3 .20.6 4~6 25.
. . ~ontrol . 200 None 72. 7 0. 3 ND2 99 . 7ND~ ~
.~ ~ ~ . - ' ' ' .
. ~Total un~ts c)f fructos~l transfer.ase en2yme in 5Q ml water = none del~:c~ed .
~- It is noteworthy that in the above e~ample, the control crystallized when cooled to- ambient temperature (about ~5C) whereas the enzymatically produced secondary substra es rem~in in solut:ion ana exhibit ex ellent shel~--life stability wi.thout cxystallizationa ,. .
Althou~h Example 8 employs 2U0 ~ suc:ros~ pel- !iO ml of water, i.. ~. 8096 wfw, the dry ~ul:~stance cc>n~entr~tion O:e - : ~ : the sucl-ose starting materi.al can be: increas;~d .

30~
.~-. . .
.. . . . .

~ - .

.

, 1~'7~
s~cc)n~ J
~ l'he n~vcljsu~str~lt~ o~ i..s embo~iment can be used as a l~ hly st<~ eA, non-crys~alliæ:i.rlcJ syruL~ in food applica-tions. It also provides a uni.quely high dry sub.stance composition, res:istant to microbial contamination and color S body ~orm~tion, whicll can be employed to ship and/or store t:he sugarat concerltrations heretofore not obta:inable in a form having the above described propexties. Moreover t this novel high dry substance secondary substrate can be subjected to hydrolysis as .shown in Example 7 to obtai.n an invert sugar mi.xture havi.n~ a desire~ble sweetness level. The novel secondary substrate of this embodim~nt ha.5 a dry substance content ~w/w) xanging from abc~ut 70% to about S2~
and contains a monosaccharide fraction consisti.ng essentially of dextrose and polysaccharide polymers, in excess of DP2, . consistlng predominately of DP3 product. A minor amount (4.1/~and helow) of DP4+ polymers is also present~
Although the transfructosylation step of th;.s invention has been demonstrated in terms of ba~ch unit operations, it will be apparent to those skilled in the art that continuous unit operations can likewise be employed.
In carryi.ng out such continuous transfruc:tosyIal:ion; the .~ tr~nsfructosylase enzyme is conveniently i~nob~ d usi.ntJ
- the techniques previously cli.scussed under the cle~inît.ion o~
i.~nob.iliæed enzyme ancl the continuous processi.ng methodo:Lo~,~y therein described.

,

Claims (15)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A method for producing a syrup comprising subjecting sucrose at an initial concentration of at least about 10% (w/v) to the action of an effective amount of a fructosyl transferase enzyme preparation to produce a secondary substrate comprising dextrose and a polysaccharide consisting predominantly of fructosyl moieties.

2. The method of claim 1 wherein the sucrose being acted upon by the enzyme is at a concentration of at least about 20 percent.

3. The method of claim 1, wherein said fructosyl transferase enzyme preparation is derived from Pullularia pullulans.

4. The method of claim 1, wherein the sucrose is subjected to the action of fructosyl transferase enzyme at a temperature of about 25°C to about 65°C, and a pH from about 4.5 to about 6.5.

5. The method according to claim 1, wherein the dextrose of the reaction product is isomerized to produce fructose.

6. The method of claim 5, wherein the isomerization is carried out using an immobilized glucose isomerase enzyme.

7. The method of claim 5, wherein the dextrose is isomerized to fructose in the presence of said polysaccharide,

8. The method of claim 1, wherein said polysaccharide is physically separated from the dextrose.

9. The method of claim 8, wherein said separation is accomplished by ultra-filtration.

10. The method according to claim 1, wherein the polysaccharide of the reaction product is hydrolyzed to produce fructose.

11. The method of claim 10, wherein the polysaccharide is hydrolyzed in the absence of active isomerase enzyme.

12. The method of claim 10, wherein the polysaccharide is hydrolyzed after physical separation from the dextrose.

13. The substrate produced in accordance with claim l and suitable for enzymatic isomerization and hydrolysis to syrups containing greater than 55% fructose, comprising (1) from about 20%
to about 60% monosaccharide, consisting predominantly of dextrose and fructose, and (2) from about 70% to about 40% polysaccharides consisting predominantly of fructosyl moieties, said percentages being by weight of said substrate.

14. The substrate of claim 13, wherein the polysaccharide contains at least 66% by weight fructosyl moieties.

15. The substrate produced in accordance with claim 1 and having a dry substance content (w/w) ranging from about 70% to about 82%
and containing a monosaccharide fraction consisting predominantly of dextrose and a polysaccharide fraction (other than sucrose) consisting predominantly of DP3 product.