CA1045569A - Process for producing dextrose using mixed immobilized enzymes - Google Patents

Process for producing dextrose using mixed immobilized enzymes

Info

Publication number
CA1045569A
CA1045569A CA234,067A CA234067A CA1045569A CA 1045569 A CA1045569 A CA 1045569A CA 234067 A CA234067 A CA 234067A CA 1045569 A CA1045569 A CA 1045569A
Authority
CA
Canada
Prior art keywords
amylase
alpha
dextrose
immobilized
starch
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Expired
Application number
CA234,067A
Other languages
French (fr)
Inventor
Kenneth N. Thompson
Richard A. Johnson
Norman E. Lloyd
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Standard Brands Inc
Original Assignee
Standard Brands Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Standard Brands Inc filed Critical Standard Brands Inc
Application granted granted Critical
Publication of CA1045569A publication Critical patent/CA1045569A/en
Expired legal-status Critical Current

Links

Classifications

    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12PFERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00Preparation of compounds containing saccharide radicals
    • C12P19/20Preparation of compounds containing saccharide radicals produced by the action of an exo-1,4 alpha-glucosidase, e.g. dextrose
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02Enzymes or microbial cells immobilised on or in an organic carrier
    • C12N11/10Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a carbohydrate
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/14Enzymes or microbial cells immobilised on or in an inorganic carrier
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/18Multi-enzyme systems
    • CCHEMISTRY; METALLURGY
    • C13SUGAR INDUSTRY
    • C13KSACCHARIDES OBTAINED FROM NATURAL SOURCES OR BY HYDROLYSIS OF NATURALLY OCCURRING DISACCHARIDES, OLIGOSACCHARIDES OR POLYSACCHARIDES
    • C13K1/00Glucose; Glucose-containing syrups
    • C13K1/06Glucose; Glucose-containing syrups obtained by saccharification of starch or raw materials containing starch
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10STECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00Chemistry: molecular biology and microbiology
    • Y10S435/813Continuous fermentation
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10STECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00Chemistry: molecular biology and microbiology
    • Y10S435/8215Microorganisms
    • Y10S435/822Microorganisms using bacteria or actinomycetales
    • Y10S435/832Bacillus
    • Y10S435/839Bacillus subtilis
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10STECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00Chemistry: molecular biology and microbiology
    • Y10S435/8215Microorganisms
    • Y10S435/911Microorganisms using fungi
    • Y10S435/913Aspergillus
    • Y10S435/918Aspergillus oryzae

Abstract

ABSTRACT OF THE DISCLOSURE
Process for converting starch to dextrose wherein a partially hydrolyzed starch solution containing at least 10 percent hydrolyzed starch is contacted with an enzyme system under conditions whereby substantially complete conversion of the starch to dextrose is achieved. The enzyme system comprises immobilized glucoamylase and alpha-amylase selected from the group consisting of soluble alpha-amylase, immobilized alpha-amylase and mixtures thereof.

Description

~4~5~9 This invention relates to a process for converting starch to dextrose. More particularly, this invention relates to a process for converting starch to dextrose by the use of an enzyme system comprising immobilized glucoamylase (E.C. #3.2.1.3.~
and alpha-amylase (E.C. #3.2.1.1.) selected from the group consisting of soluble alpha-amylase, im-mobilized alpha-amylase and mixtures thereof.
Processes for hydroly~ing starch to dex-trose are well known in the art. These methods can be grouped into two broad categories. These are the ; -acid-enzyme and the enzyme-enzyme conversion pro-cesses. In the acid-enzyme process, generally, starch is first partially hydrolyzed or liquefied, for in-stance, by forming an aqueous suspension containing from 35 to 40 percent starch and incorporating therein an acid such as hydrochloric acid. The suspension is then heated to relatively high temperatures to partially hydrolyze the starch and then cooled and treated with a glucoamylase preparation under suitable conditions to enzymatically convert the partially hydrolyzed starch to dextrose. The acid-enzyme process is dis-closed, for example in U.S. Patents 2,304,168,
2,531,999, 2,893,921 and 3,042,584.
: .
Glucoamylase has been referred to in the art as glucamylase ~

':
'-"~'.

- 1 - ,~ ,'.
. .

~ ~''' '.:
, .~, i .

1~45S6~
glucogenic enzyme, starch glucogenase and gama-amylase. Glucoamyl~se is an exo-amyloiytic enzyme which catalyzes the sequential hydrolysis of glucose moieties from the non-reducing ends of starch or amylo-dextrin molecules. Glucoamylase is elaborated by many types of micro organisms. Certain strains of fungi belonging to the Asper~,illus group such as strains belonging to the sper~illus ~ group and the~Aspergillus awamori group, certain strains of the Rhizopus species ; and certain strains of the Endomyces species elahorate glucoamylase.
- In the enzyme-enzyme conversion process, generally, a starch slurry is formed and a starch liquefying enzyme, for instance, bacterial alpha-amylase, is added thereto and the starch slurry heated ta a temperature in the range of 80 to 90C. to partially hydrolyze the starch. The partially hydrolyzed starch, which generally has a D.E. in the range of from about 10 to 20, is then treated with glucoamylase.
Alpha-amylase is an endo-amylolytic enzyme capable of pro-moting almost random cleavage of ~-1,4-glucosidic bonds within the starch molecule. Alpha-amylase is elaborated by many types of microorganisms such as members of the Bacillus subtilis species, AsPergillus niger and other species of the Aspergillus genus and malted cereal grains.
Alpha-amylase will not act upon the ~-1,6-glucosidic bonds .
in the starch molecule to any significant degree. Glucoamylase will act upon such bonds, but at a rate which is slower than is desired in commercial appllcatîons.
Recentlyg there has been a great deal of interest shown in - the use of starch debranching enzymes for dextrose production. The use of such enzymes increases the amount of dextrose formed since they can readily act upon bonds or linkages in the starch molecules which,are not acted upon by alpha-amylase or which are only slowly ` . . ' , ' 1¢~4556~

acted upon by glucoamylase. Debranching enzymes are generally referred to as ~-1,6-glucosidases. A number of enzymes having considerably different specificities have been iidentified in the art as being capable of hydrolyzing ~-1,6-gtucosidic linkages.
Of these, probab]~ the two ~ost ~mportant from the commercial ; standpoint are pullulanase (~.C,~t3.2 ~.4~) and i~oamylase (E.c.#3.2.l.68)~
The ma~or, dif~erence in regard to the ~pec~ficity o~ these enzymes is ~hat pullulanase will degrade the linear po~lysacchar-ids pulluIan whe~as isoa~ylase will not to any slgnificant degree.
There are a number of patents which disclose methods of~
producing isoamylase and pullulanase and ~he u~ilization thereof.
Canadian Patent 852,196 to Ueda et al. describes a process for producing isoamylase by cultivating a strain of ~ herLchl~l intermedLa in a fermentation medium compri~ing dextrins, poptone and inorganic salts. U. S. Patent 3,490,955 to Wallenfels et al.
; d~scloses a process for producing cellbound pullulanase from Aerobacter aero~enes in a culture mediu-n wherein the carbon sources ~-comprise maltose and pullulan or glycerin. U. S. Patent 3,560,34;
to Yokobayashi et al. describes a process for producing isoamylase by propagating Pseudomonas _myloderamosa in a culture medium con-taining as carbon sources, starch, starch derivatives or maltose.
Recently, there has been a great deal of interest shown in immobilized enzymes. Immobilized enzymes have a number of distinct advantages over soluble enzymes such as, for example, their use in continuous conversion systems.
Exemplary of publications which review the art directed to enzyme immobilization are the following:
Goldstein, in _ rmentation Ad~ances, A~ademic Press, New York, N.Y. (1969), pp. 391-4~4.
3~ Goldstein et al . ~ z. An~1. Gll~m., ~43, pp. 3~5-396 (1968).

.

i6~
Kay, ~rocess Biochem., 3 (8), pp. 36-39 (1968).
Tosa et al., a~aku_To Seibutsu, 7 (3), pp. 147-155 ~1967).
Silman et al., ~ D ochem., 35 (2), pp- 873-908 (1966).
Gryszkiewicz, Folia BioloRica, 19 (1), pp. 119-150 (1971)-Zaborsky, "Immobilized Enzymes", CRC Press, Cleveland, Ohio (1973)--10 In the art of enzyme immobilization, considerable intere~t has been directed to the immobilization of glucoamylase. T~is is probably due to the fact that in many commercial enzyme processes glucoamylase is used in large amounts. The art is repIeat with patents and publications directed to immobili~ation of glucoamylase.
Exemplary of such are the following:
U. S. Patents 2,717,852 to Stone; 3,619,371 to Crook et al.; 3,62i,638 to Barker et al.; 3,672,955 to Stanley; ;
3,715,277 to Dinelli et al.; Japanese Patents 1360/60 and 23560/68; British Patents 1,183,259 and 1,183,260; Gërman Patents 2,062,246, 2,146,390 and 2,206,360.
Also: Usami et al., Hakko_Kyokaishi, 25, pp. 513-516 ~1967); Barker et al., Carbo~!yd. ~es., 9, pp. 257-263 (1969);
Wilson et al., Biotechnol. Bioeng~, 11, pp. 349-362 (1969);
Usami et al., J. Ferment. Tech., 48, pp. 506-512 (1970);
~5 Gruesbeck, Ph.D. Thesis, Univ. Texas (1970); Bachler et al., Biotechnol Bioen~., 12, pp. 85-92 (1970); Maeda et al., NiPpon Nogei Ka~aku Kaishi, 44 (12), pp- 547~555 (1970); Maeda et al., Hakko Kyokaishi, 28 (10), pp. 391-- 397 (1970); Smiley, Biotec_nol. Bioen~., 13, pp. 309-317 .
,0 1 ~1971); Sorenson, MS Thesis, Purdue Univ. (1971); Miyamoto ~:- ' .~ '.

', . ~ . . ... . . .. .. . .. . . . . . ... . .. . . ... . . . . .. . . .

~9L5~69t et al., }lakko Kogaku Zasshi, 49 (6), pp. 565-573 tl971~;
Usami et al., Haklco_Kyokaishi, _9 (4), pp. 195-199 (1971);
O'Neill et al., Biotechnol. Rioen~., 13, pp. 337-352 (1971); Emery et al., Chem. En~. (London), No. 258, pp.
71-76 (1972); Gruesbeck et al., Ind. En~. Chem. Prod.
Res. Develop., 11 (1), pp. 74-83 (1972); Beck, Ph.D.
Thesis, Univ. Texas (1972); Gestrelius et al., Biochem.
Biophys. Acta, 276 (2), pp. 339-343 (1972); Maeda et al., A~r. Biol.-Chem., 36 (9), pp. 1581-1594 and pp. 1839-1842 (1972); Weetal et al., Biotechnol. Bioen~. Symp., No. 3, pp. 241-266 (1972); Christison, Chem. & Ind. (London), (5), pp. 215-216 (1972); Hough et al., Nature, 235, p. 389 (1972); Corno et al., Die Staerke, 24, pp. 420-424 (1972); Martensson et al., Bio ~ , 14 - lS (5), pp. 7i5-724 (1972); Park et al., J. Food Sci., 38, pp. 358-359 (1973).
There are also a number of patents and publira~ions whlch disclose the immobilization of alpha-amylase. Exemplary of such are ~~` .
the following:
U. S. Patents 3,627,638 to Barker et al. and 3,715,278 to Miller; German Patents 1,282,579, 1,943,490, 2,062,246 ; and 2,206,360.
,. Also: Grubhofer et al., Naturwissenschaften, 40, 508, `
.. . . . . .
~1953); Manecke, Pure Appl. Chem., 4, pp. 507-520 (1962);
Manecke et al., MakromoI. Chem., 51, pp. 199-216 (1962);
:~ ~ . . .' . :
- Bernfeld et al., Science, 142, pp. 678-679 (1963);
- Manecke et al., Makromol Chem., 91, pp. 136-154 ~1966,~;
i t Fukushi et al., J. Biochem., 64, pp. 283-292 (1968);
. .
Barker et al., Carbohxd. Res., 8, pp. 491-497 (1968~;
j Ledingham et al., Fed. Europ. Biocllem. Soc. I.ett., 5, _ 5_ .

.. . . .. .. . . .. . . . . .

4SS~
pp. 118-120 (1969);
Barker et al., Carbohyd. Res., 14, pp. 323-326 (1970);
Barker et al., Process Biochem., 5 (8), pp. 14-lS (1970);
Barker et al., Carbohyd. Res., 14, pp. 287-296 (1970);
Hough et al., Nature, 235, p. 389 (1972);
Epton et al., Carbohyd. Res , 22, pp. 301-306 (1972~.
~ Additionally, there have been several patents and publi-cations directed to processes for the immobilization of ~-1,6-glucosidases. Exemplary of such are the following~
British Patent 1,258,095, Martensson et al., Biotechnol.
Bioeng., 14 (5~, pp. 715-724 (1972).
From the above noted patents and publications, it is apparent that a number of enzyme immobilization techniques have been described. These techniques include covalently bonding an enzyme to a suitable insoluble carrier, encapsulation of an enzyme within a material which is impermeable to the enzyme but permeable to the substrate and the products of the catalyzed reaction, ad-sorption of an enzyme on an insoluble carrier and entrapment of an enzyme within a porous polymeric material wherein the pores are of such a size that will provide free access of the substrate and the catalyzed reaction products but which are sufficiently small to prevent the escape o f the enzyme.
At low starch substrate concentrations, e.g., about 1 percent, glucoamylase preparations will substantially quantitatively convert unhydrolyzed starch to dextrose. Marshall et al., Fed. ;~`
Europ. Biochem. Soc. ~ett., 9 (2), pp. 85-88 (1970) and Fukui et al., Agr. Biol. Chem., 33 (6), pp. 884-891 (1969) reported that gluco-amylase preparations inherently contain alpha-amylase. When the ; ~
alpha-amylase was removed from these preparat~ions and the alpha- ~ -amylase-free glucoamylase was used to saccharify a 1 percent starch ~
--6-- . .

.: ~
:'
4~iS69 .
~ . .
- solution, lesser amounts of dextrose were formed than when gluco-amylase preparations were used which inherently contained alpha-amylase.
When a glucoamylase preparation is immobilized, the resulting immobilized preparation is not capable of converting partially hydrolyzed starch to the same degrec as the glucoamylase pre~paration from which the immobilized enzyme was prepared. More-:::
over, reactions catalyzed by thc immobilized glucoamylase prepara-tion are not as rapid for a given number of glucoamylase units . , .
used, especially during the latter stages of the reaction period, as are reactions catalyzed by the glucoamylase preparation used for immobilization.
Therefore, it is the principal ob~ect of the present invention to provide a method whereby substantially complete con-version oi partially hydrolyzed starch to dextrose ls achieved using an immobilized enzyme system.
This object and other objects of the present invention which will be apparent from the following description are attained in accordance with the present invention by contacting a partially hydrolyzed sta~ch solution containing at least 10 percent hydrolyzed starch with an en~yme system comprising immobili~ed glucoamylase (E.c.#3.2.l.3) and alpha-amylase (E.C.#3,2.1,1.~ ~elected from the group cotlsis-ting of soluble nlpha-amylase, immobilized alpha-amylase and mixtures thereof un~er cond~tions whereby substantlally complete conversion of the hydrolyzed starch to dextrose is achieved.
As mentioned above, when a glucoamylase preparation is sub~ected to immobilization, the resulting lmmobilized glucoamylase does not convert partially hydroly~ed starch ~so rapidly nor so completely as the soluble glucoamylase PrePara~in from which the imrnobilized glucoamylase is prepared. We have found that d~trlng `
~:

. ', , 1~ :.

. ~J~

- 1~14SS69 the immobilization of a glucoamylase preparation, the alpl-a-amylase, which is inherently present therein, is rendered substantially inactive or inert regardless of the method of immobilization em-ployed. This is surprising in view of the many different methods that have been disclosed for the immobilization of alpha-amylases.
Although we do not wish to be bound to any theory, it is believed tha~t the methods which have been found suitable for the immobili-zation of glucoamylase are not suitable for the immobilization of the alpha-amylase inherently contained in glucoamylase preparations.
Apparently, the small amount of alpha-amylase which is inherently present in soluble glucoamylase preparations has a beneficial effect on the overall conversion of starch to dextrose with glucoamylase.
Thus, to obtain maximum utilization of immobilized glucoamylase in the corlversion of partially hydrolyzed starch to dextrose, there must also be present during the conversion soluble and/or immobilized `. alpha-amylase. Surprisingly, this finding is true even when the partially hydrolyzed starch has been prepared by treatment o~ un-modified starch with alpha-amylase and therefore would be assumed to be rendered readily susceptible to conversion with glucoamylase ~ ~`
, ~o by such treatment. Moreover, it has been discovered that alpha-amylase added to immobilized glucoamylase is effective for increasing the conversion of partially hydrolyzed starch to dextrose even during the latter stages of the conversion. Apparently, branched dextrins are formed during the initial stages of the hydrolysis reaction ~`~
wh1ch are not readily hydrolyzed by the immobilized glucoamylase but which are readily hydrolyzed by alpha-amylase and thus, the overall conversion of the starch hydrolysate is enhanced.
In the present process, the partially hydrolyzed starch r~ay be prepared either by an enzyme or acid treatment. In the case .
~ 30 of enzyme treatment, th~ partially hydrolyzed starch sh~uld have a -'' ~ ' ' ," ` "'",:
-8- ~

. `' ' . ~ ~, '; .:
.: : .
, . . . . .. . - -556~ ~:
D.~. in the range of irom about 10 to about 60. At substantially higher D.E. values, the amount of dextrose formed will be limited -due, presumably, to the presence of saccharides which are not readily acted upon by the immobilized glucoamylase, while at lower D.E.s, the hydrolyzed starch has a tendency to retrograde which includes the formation of a precipitate which may coat the immobilized ~nzymes to such an extent that their efficiency will be deleteri-ously affected. When a partial acid hydrolysate is used in the present process, the D.E. thereof should be in the range of from - 10 about 10 to about 30. At higher D.E.s substantial amounts of .
reversion products are present which are not acted upon by the present enzyme system.
The p}l of the partial hydrolysate being treated may be in the range of from about 3.5 to about 6.5 and preferably will lS be in the range of from about 4 to about 6.
The temperature of the partial hydrolyzate being treated in the present process may vary relatively widely, but the tempera-ture should not be sufficiently high to inactivate the enzymes within a relatively short period. Temperatures in the range of from about 30 to about 65C. are preferred and the most preferred temperatures are in the range of from about 50 to about 60~C. At these temperatures, the possibility of undesirable microbial growth in the hydrolyzed starch is reduced and optimum catalytic activity of the enzymes is generally obtained under normal operating condi-tions.
The present process may be periormed by a number of tech-niques. For instance, soluble or immobilized alpha-amylase and -immobilized glucoamylase may be used concurrently or sequentially.
It is preferred that they be used concurren.ly as, for example, when partially hydrolyzed starch is contacted with a mixture of _9_ : ~

: ' , 10~5569 immobilized glucoamylase and immobilized alpha-amylase. Of course, it will be realized that the alpha-amylase and gluco-amylase may be immobilized on or within the same carrier and results will be obtained which are substantially equivalent to those given by mixtures of alpha-amy]ase and glucoamylase immobilized on separate carriers. In the case where the enzymes are used sequentially, the conversion process will comprise at least three steps in the following sequence: (1) contacting the partial hydrolysate with immobilized glucoamylase, (2) contact-ing the resulting hydrolysate with a soluble or immobilized ~
alpha-amylase, and (3) contacting the resulting hydrolysate with -immobilized glucoamylase. The last two steps of the sequence may be repeated a nunlber of times depending on the conditions under which the reactions are conducted. The concurrent use of the enzyllles rcsults in greater amounts of the partially hydrolyzed starch being converted to dextrose than does sequential use except -~
when the steps employed in sequential use are repeated a large number of times. '~
The preferred method of preparing the immobilized alpha~
amylase for use in the present process is by covalently bonding r~ .
the alpha-amylase to carriers such as cellulose, porous ceramic, macroporous synthetic resins, crosslinked dextran and similar !~- ;
materials.

~he glucoamylase may be immobilized by any of the .^~
,,~
techniques known in the art, although, in the present process, it is preferred to use glucoamylase which has been immobilized ~ ~-on a cellulose derivative, such as DEAE-cellulose or immobilized , ~;~
covalantly to an inert carrier.
A number of different types of alpha-amylase may be usled, although it is preferred that saccharifying or pancreatic type ¦

,' , ' '' ~ ' ' ~' '' -10- ,~ ~"
' . ~:
;~ .

4551~9 alpha-amylase be used. Microorganisms such as Bacillus subtilis var. amylosacchariticus Fukumoto elaborate saccharifying type alpha-amylase. Generally, it is also preferred that alpha-amylase preparations which are to be used for immobilization have an S/L
value (hereinafter defined) of at least about 31 preferably at least about 50 a~d most preferably a value of at least about 100.
~ The ratio of the activities of the enzymes ~sed in the - present process should typically be above a certain minimum value to provide optimum catalytic action. In this regard, the amounts of immobilized glucoamylase and of alpha-amylase which may be us~d should be sufficient to provide a ratio of dextrinizing activity (hereinafter defined) to glucoamylase activity (hcreinafter defined) of at least 0.2 liquefons per glucoamylase unit. Preferably, the amounts of d~Trj~;zjng enzymes present will be sufficient to pro-vicle at least 1 liquefon per glucoamylase unit, and most preferably, ., I .: . .
the amounts will be sufficient to provide at least 3 liquefons per , ~ . ....
` glucoamylase unit.
- When the present enzyme system is used in a column or bed, or in other means whereby such can be used in a continuous manner, it is'important to remove any insoluble material which may be present in the partial starch hydrolysate so that such ma~erial does not plug the column or coat the immobilized enzymes to a degree which substantially reduces the efficiency of the enzyme system.
Removal of insoluble material may be accomplished in any convenient ;, ... .
manner such as filtration, centrifugation or the like.
Immobilized ~-1,6-glucosidases may also be used in the ` present process. ~xemplary of the preferred enzyme of this class is pullulanase. It is preferred to immobilize the pullulanase b~ covalently binding it to an inert carrier.
In order to more clearly describe the nature of ~he present -11- ;' ., ' .~ ~

S56~
invention, spccific examples will hereinafter be described. It should be understood, however, that this is done solely by way of example and is intended to neither delineate the scope of the invention nor limit the ambit of the appended clairns.
;5 Expressions and procedures referred ~o in the present specification and claims are defined below:
Dextrose Equivalent Dextrose equivalent (D.E.) was determined by Method E-26 described in "Standard Analytical Methods of the Member Companies of the Corn Industries Research Foundation", Corn Refiners Associa~
tion, Inc., 1001 Connecticut Avenue, N.W., Wasllington, D.C. 20036.
Dextrose Content '. ,:
Dextrose content was calculated rom the Mathews' Index~
For a discussion of Mathews' Index see Cayle and Viebrock, Cereal L5 Chem., 43, 237 (1966).
The Mathews' Index was determined from measurements of i optical rotation and reducing sugar content of the converted solu-tions. The converted solutions were diluted to about 3 percent dry solids and optical rotation (R) determined in degrees circular in ;
a 0.2-dm., jacketed cell maintained at 25C. using an automatic polarimeter (Bendix Scientific Instruments, Model NPL) equipped with a green light source (546.1 nm). A portion of the solution used for polarimetry was diluted four fold and titrated into 25 ml of Fehling's solution according to the method for determining D.E.
~`25 enumerated above. The titre (T) so obtained is the number of mls ~ of diluted solution ~hich contains reducing sugars equivalent to ; 0.12 g of dextrose. The Mathews' Index (M) was calculated from the ` rotation (R) and titre (T~ as follows: `
M = RT/4 . -:30 Percent dextrose (ash free, dry substance basis) was then calculated "
.

` ;` 1~;J4SS69 ;; :
from the Mathews' Index by the following equation:
Percent Dextrose = (170 - 2 M)/(0.2167 M -~ 1.0784) Preparation of Partially Hydrolyzed Starch Solution The partially hydrolyzed starch solutions used in the various analytical determinations and in the f~llowing examples were prepared using the following general procedure:
An 18 Be slurry of corn starch in water was adjusted to p~l 7.0 ~ith lime and alpha-amylase (B. subtilis origin, 33 liquefons per g dry starch) added. The mixture was instantaneously heated to 88C. to gelatinize the starch and initiate enzyme action-~ by blending with steam in a mixing jet and was then held at 88C.
; for about one hour. The mixture was then heated to 149C. by blending with steam under pressure in a mixing jet, held at 149C. for about one minute and then cooled to 88C. in a vacuum ~' 15 chamber. Additional alpha-amylase (11 liquefons per g of dry , . .
starch) was added to the mixture at 88C. and hydrolysis con- `
, tinued until the desired D.E. was obtained. After cooling to 60C., the solution was adjusted to p~l 3.5 to 4 using 4 M hydro-chloric acid and was then heated for 90 minutes at 100C. to inactivate any residual alpha-amylase activity. Three percent filter aid was added and the hot hydrolyzate was filtered to remove insoluble protein and fat. The above procedure provided partially hydrolyzed starch solution having a D.E. of 12 to 20 ; and 31 to 34 percent dry solids.
Glucoamylase Activit . ~
- A glucoamylase activity unit (GU) is defined as the amount of enzyme which catalyzes the production of one g of dextrose per hour at 60C. at pU 4.5 in the proced~lres described `~ below.
Drum-dried partially hydrolyzed starch was used for the ~, ' .

~ L5569 preparation of substrate solutions for glucoamylasc activity determinations. A partially hydrolyzed starch solution having a D.E. of 12, was treated with activated carbon (Nuchar CEE, West Virginia Pulp and Paper Co.) for 45 minutes at 60C. The carbon was removed by filtration and the filtrate was treated again with carbon and filtered in the same manner. The filtrate was c~ncentrated to about 50 percent dry solids and was then dried on a steam-heated drum drier and ground. Tlle drum-dried partially hydrolyzed starch contained 1.7 percent moisture and 0.5 percent ash. Substrate solutions for glucoamylase activity determinations were prèpared to contain 10 g of the dried hydrolyzed starch and 2 ml of p~l 4.5, 1 M sodium acetate buffer per 100 ml of solution.
Activi~y_~f Soluble GlucoamYlase Ten ml of substrate solution was pipetted into a capped reactor maintained at 60C. One ml of glucoamylase solution con-taining 0~03 to 0.15 GU was added and mixed therein and the mix- :
ture maintained for one hour at 60C. At the end of the l-hour incubation period, enzyme action was stopped by adding a predeter- "
mined volume of 1 ~ sodium hydroxide solution so as to obtain a pH of ~.5 to 10.5. The mixture was then cooled to room temperature.
2.5 ml of the assay hydrolysate so obtained was pipetted into 25 ml of Fehling's solution prepared as described in the above cited method for D.E. determination. The mixture was brought to a boil and titrated with standard dextrose solution containing 5 g of dextrose per liter according to the procedure cited above for D.E. determination. A control mixture was prepared and titrated in the exact same manner as for the assay hydrolysate above except that the 1 ml of glucoamylase solution was added to the substrate 0 solution after the one-hour incubation period and after the addi-` tion of sodium hydroxide solution. Glucoamylase activity was 556~
",~}
,~' calculated as follows:
GU~ml = 0.002 V (C-A) where V is the total volume (ml) of assay hydrolysate (usually 11.2 ml), C is the ml of standard dextrose solution used in the titratlon of the control mixture, and V is the ml of standard dextrose solu-tion used in titration of the assay hydrolysate.
Activi~ty_of Immobilized Glucoamylase The activity of immobilized glucoamylase was determined by a modification of the above procedure for determining the acti-vity of soluble glucoamylase. 10 ml of substrate solution prepared as described above was heated In a closed reactor to 60C. A
weighed amount (W) of immobilized glucoamylase containing frorn 3 to 10 GU was suspended in deminerallzed water and was diluted to 100 ml. The immobilized glucoamylase suspension was stirred and while stirring, a l-ml aliquot of the suspension was transferred to the 10 ml of the substrate solution held at ~0C. The mixture was stirred continually for exactly 1 hour at 60C. and was then filtered to remove the immobiliæed glucoamylase. 2.5 ml of the assay filtrate so obtained was added to 25 ml of Fehling's solu-tion and titrated with standard dextrose in the manner described , above for determining the activity of soluble glucnamylase. A
control filtrate was prepared and titrated by the exact same steps except that one ml of water was substituted for the one ml of immobiiized glucoamylase suspension. Immobilized glucoamylase . i .
activity was calculated as follows:
GU/g = 2.2 (Ci ~ Ai)~W
where Ci is the ml of standard dextrose solution used in the --titration of the control filtrat~, Ai is ~he ml of standard dex-trose solu~ion used in the titration of the assay filtrate and W
; 30 is the weight (g) of immobilized glucoamylase in the 100 ml of suspension.

-~5-. .

1~556g i Activity of Alpha-Amylase AlpIla-amylase preparations were assayed by two dif~erent methods. In one method, the ability of the alpha-amylase prepara-tion to hydrolyze soluble ~intner starch to dextrins too small to give a blue color with iodine was determinc!d as a measure of dex-trinizing activity. In the other method, the ability of the alpha-amylase preparation to produce reducing sugars by the hydrolysis of a reduced partially hydrolyzed starch was determi~ed as a measure of saccharifying activity.
Dextrinizin~ Activity o ~ lpha-Amylase ~ -The dextrinizing activity of soluble alpha-amylase pre-parations was determined by a modification of Standard Test Method, A~TCC 103, 1965, "Bacterial Alpha-Amylase Rnzymes Used in Desizing, Assay of~ published in the 1967 Edition of Technical Manual of the American Association of Textile Chemists and Colorists, Volume 43, pp. B-174 and B-175. The method was modified by substituting 10 ml of 1 M sodium acetate buffer, pH 5.0~ for the 1~ ml of pH 6.6 phosphate buffer solution used in the makeup of the buffered starch substrate. Also, 0.73 g of CaC12 2~120 was added per 500 ml of ; 20 buffered starch substrate. Results were calculated in terms of liquefons where one liguefon equals 0.35 Bacterial Amylase ~nit.
Dextrinizing Activity of Immobilized ~lpha-Amylase `~ The dextrinizing activity of immobilized alpha-amylase preparations was determined in the same manner as for soluble alpha-amylase preparations except that immobilized alpha-amylase was "diluted" for assay by suspension in 0.005 M calcium acetate solution at 30C. A 5-ml aliquot of the suspension was added to the lO ml of buffered starch substrate and the hydrolyzing mixture so formed was stirred continuously during the 30C.-hydrolysis step. At appropri-ate tlme intervals, 2-ml aliquots of ~he hydrolyzing mixture were ¦ taken tnd rapldly fittered and one ml oE the Eiltrate added to th~

. ,.
', .. :

. ~ . . . . , = . , , :

r ~ 4~5~
5 ml of dilute iodine solution. Time was counted starting at the instant the 5-ml aliquot of suspension wa~ added to the 10 ml of buffered starch substrate and finishing at the time that the 2-ml aliquot of hydroly~ing mixture was filtered.
S Saccharifyin~ Activity of Soluble ~lpha-Amylasç
Saccharifying activity of soluble alpha-amylase pre-parations was determined using a reduced partially hydroly~ed starch solution (RLS) as a suhstrate. One unit of saccharifying activity (S) was defined as the amount of enzyme which would produce an increase of 0.02 absorbance unit per minute in the procedure described below.
The RLS was prepared from a 1.8-liter sample of 12 D.E.
partially llydrolyzed starch solution containing 31 percent dry solids. ~he partially hydroly~ed starch solutlon was prepared as describec} previously except that the final steps in its pre-paration comprising adju~tment of the pH to 3.5 to 4.0 and heating to 90C. to inactivate residual alpha-amylase and fil-' tration were omitted. The hydrolyzed starch solution was ad- ~ -~usted to pH 6.5 to 7.0 and was heated to 70C. ~iquefying alpha-amylase preparation of B. subtilis origin containing 57,000 liquefons was added and the mixture held 3.5 hours at 70C. The pH was adjusted to 3.5 to 4.0 and the mixture heated one hour at 100C. 3 percent filter aid was added and the mix-ture was filtered. The filtrate was adjusted to pH 5.5 with 8 M
- ~5 NaOH solution, 10 g of DRAE-cellulose (Whatman D~ 23* Reeve Angel) added, and the mixture stirred for 30 minutes at ambient temperatllre. The mixture was then maintained for 18 hours at 5C. without stirring, heated to 60C. and filtered. The filtrate was refined twice by stirring for 60 minutes at 60C. with 16 g *
of ac~ivated carbon (Nuchar C~E) and ~boul: 50 g of filter aid followed by filtering. 975 ml of the twice-refined filtrate * Trade Mark -17-~'~ ;

~556~

,i was obtained containing 34.5 percent dry solids and having a - D.E. of 30.2. 400 nnl of the twice-refined filtrate was cooled to about 20C. and 0.5-g portions of sodi~lm l)orohydride dis-solved therein at 30-minute intervals until 6.0 g had been ~5 added. The resulting sollltion was stirred at ambient tempera-ture for about 16 hours, was recooled to about 2~C. and 0.5 g - of sodl~m borohydride dissolved therein. After stirring for 4 hours, a final 0.5-g portion of sodi~lm borohydride was added and the solution stirred for 26 hours at amhient temperature.
L0 435 ml of the resulting solution was refined by charging the same to an ion-exchange column containing an 89 x 2.5-cm bed *
of Borosorb (Calbiochem, CN 203667) and w~shing the charge thro~lgh the column with water. The efflucnt (charge plus wash-ings) was concentrated to ~00 ml and then was cllarged to three S columns placed in series as follows: an 89 x 2.5-cm bed of Borosorb, a 26 x 2.5-cm bed of strong acid resin ;n hydrogen form (Duolite C-3*, Diamond Shamrock Chemical Co.), and a 62 x 2.5-cm bed of weak base resin in free amine form (Duolite A-6~. The charge was washed through the columns ~ith water and the effluent collected until 4.9 liters had been recovered.
The effluent was concentra~ed to 450 ml, 0.09 g of sodium azide added nnd the mixt-lre filtered through a membrane filter (Nalge Corp.) having a maximum pore size of .2 microns. The RI.S solu- -tion so prepared had the following properties: D.E. less than 0.4, pH 6.4, 26.3 percent dry solids, 0.03 percent sulfated ash, 1.7 ppm boron.
An RLS substrate solution for measuring saccharifying activity was prepared to contain 2 g dry basis RLS, 2 ml of 1 M, pll 5.0 sodium acetate buffer, and 0.147 g of C1C12 2H20 in a total volume of 100 ml. For the saccharifying activity determination, * Trade Mark : .

._. . ,, , .. . . . , , : .:

1~ i6~
... :
;
5 ml of RLS substrate.solution equilibrated to 30C. was mixed with 5 ml of a solution of the alpha-amylase preparation diluted to contain 0.2 to 1.0 S/ml equilibrated to 30C. The hydrolyzing mixture was incubated at 30C. and l-ml aliquots removed at 1, 3 and 5 minutes after combination of the enzyme and substrate solutions. Each aliquot was immediately combined with 1 ml of dinitros~licylic acid reagent prepared according to P. Bernfeld in "Methods in Enzymology",.S. P. Colowick and N. 0. Kaplan, editors, Vol. I, p. 149, Academic Press, New York (1955). The mixture was heated for 5 minutes in a boiling water bath and O
was then cooled for at least 10 minutes in cold, runnin~ tap water (about 15C.). The mixture was diluted by adding 10 ml ,1 . .
j oE demineralized water and the absorbance of the resulting ¦ solution determined at 540 nm in a l-cm cell. A plot of ab-sorbance vs. incubation time was made and the slope (Y) of the -plot determined in absorbance units per minute. The activity of the diluted solution of alpha-amylase preparation was calcu-lated as follows: -Activity (S/ml) - 10 y S!~ Value The soluble alpha-amylase preparations derived from ; different sources and used for the preparation of immobili~ed ~ ;
l alpha-amylase were classified by their S/L Value which was de- - -', fined as one thousand times the saccharifying activity measured ~,; . , in saccharifying units (S) per g of alpha-amylase preparation I divided by the dextrinizing activity measured in liquefons per ; g of preparation.
Activity of Soluble Pullulanase Preparations ~ -PulIulanase activity was determined by its hydrolytic effect on pullulan using an alkaline ferricyanide reagent to , . ~ ':
.

~6~45~6~

J determine the maltotriose liberated. Activity was expressed in international ùnits (IU) where one IU is the amount of pullulanase which catalyzes the liberation oE 1 micromole of maltotriose per minute from a 0.5 percent soLution of pullulan S at pH 5.0 and 45C.
: The ferricyanide reagent was prepared by dissolving 0.85~g of potassium ferricyanide and 10 g of sodium carbonate in demineralized water and diluting to one liter. The reagent was calibrated against solutions of maltotriose (Pierce Chemical Co.). 2-ml aliquots of ferricyanide reagent were mixed in test tùbes with l-ml aliquots of maltotriose solutions containing 25, 100, 150, 200 or 250 micrograms of maltotriose per ml. The tubes were immersed in a boiling water bath for 10 minutes and then cooled for 10 minutes at ambient temperature and absorbance measured in a l-cm cell at 420 nm. Maltotriose concentration was plotted versus absorbance and a calibration factor (C) determined from the slope of the plot.
i To determine pullulanase activity, a test tube con-j taining 9.5 ml of substrate solution comprising 8.5 ml of .2 M, p~l S.0 sodium ac'etate buffer and 1.0 ml of a solution containing ' 50 mg of pullulan was incubated in a 45C. water bath for 5 minutes.
A O.S-ml aliquot of pullulanase solution was added to the test tube and mixed thercin. At 5, 10, 15, and 20 minutes after the addition , .:
of the pullulanase solution, l.0-ml aliquots of the reacting mix-ture were pipetted into test tubes containing 2 ml of the above calibrated ferricyanide reagent. The mixtures were heatedl cooled, and their absorbances determined as for the calibration of the ~erricyanide reagent above. Absorbance versus time was plotted and the slope (K) of the rate plot determined. Activity of the pullulanase solution was calculated from the following formula:

- -20- ;
.
~ , .

.~

~45~

Activity (IU/ml) = 0.0397 CK
: where C is the ferricyanide reagent calibration factor (micro-grams of maltotriose per ml per absorbance unit) and K is the slope of the rate plot (absorbance units per minute).
Activity of Immobilized Pullulanase Preparation ~ Activity of the immobilized pullulanase preparation was ; determ~ned by the method described above for soluble pullulanase with the following exceptions. A suspension was formed by stirring - 25 mg of immobilized pullulanase preparation in 5.0 ml of de mineralized water and a 0.5-ml aliquot of the suspension added to the 9.5 ml of substrate solution to form the reaction mixture. The reaction mixture was stirred constantly during the reaction period.
Aliquots were wlthdrawn at 5, 10, 15 and 20 minutes, were quickly ; filtered, and l.0-ml portions combined with 2-ml aliquots of the ferricyanide reagent.
Example I
This example illustrates the use of glucoamylase immo- ~ -;~ bilized on DEAE-cellulose and alpha-amylase derived from different sources immobilized on aminoethyl-cellulose for converting a partially hydrolyzed starch solution to dextrose.
~ Immobilization of Glucoamylase ; 53.0 g of a dry glucoamylase preparation (from Aspergillus awamori, free of transglucosylase activity) having a glucoamylase activity of 83.2 GU g 1 was incorporated into 3.8 liters of deionized water. The mixture was stirred for 30 minutes and filter aid added thereto. The mixture was filtered, the filter cake washed, the -- flltrate and washings combined, and the p~l of the combined solutions adjusted to 5.5 using 4 ~ 1. 13.3 g DEAE-cellulose (Whatman DE 23) was added, the mixture stirred for 60 minutes at ambient temperature and then fil~ered and the filter cake washed with deionized water.

-i6~
The recovered moist filter cake had a glucoamylase ~ctivity of 44 GU g . The moist filter calce is h~reinafter referred to in this example as "immobilized glucoamylase".
Immobilization of Alpha-Amylafie Alpha-amylase derived from various s~urce~ was immobilized by coupling the alpha-amylase with activated aminoethyl-cellulose (hereinafter referred to ~s "activated AE").
The activated AE was prepared by sl-lrrying 20 g of . *
aminoethyl-cellulose (Cellex-AE manufactured by Bio-Rad c Laboratories) in 500 ml of a 0.5 M phosphate buffer at pll 7, stirring for 20 mlnutes at ambient temperature and then main-taining the mixture for 7 hours without stirring. The mixture was filtered, the filter cake washed with deionized water and suspended for 12 hours in 500 ml of 0.5 M phosphate buffer at pH 7. 140 ml of a glutaraldehyde solution (50 percent) was added to the slurry, the slurry stirred for 90 minutes at ambient temperature, filtered and the filter cake washed wiLh deionized water. 74.9 g of filter cake (75.2 percent moisture) ;;
was recovered.
16.0-g portions of activated AE were added to 50 ml of each of the following four alpha-amylase solutions: ~ -a) Solution of Bacillus subtilis ~accharifying alpha-amylase (var. amylosacchariticus Fukumoto, twice recrystallized, Mile~ Laboratories, Inc., S/I. = 257) containing 0.33 mg protein per ml and having an activity of 134 liquefons per ml.
b) Solution of Bacillus subtilis liquefying alpha-amylase (Bacterial Type, II-A, 4x crystallized, Sigma Chemical Co., S/I, = 5) containing 0.45 mg protein per ml and having * Trade Mark -22-.

, ~; .

i~45S6~

an activity of 1166 liquefons per ml.
c) Solution of Aspergillus oryzae fungal alpha-amylase (3x crystallized, Calbiochem, S/L = 76) containing 0.45 mg protein per ml and having an activity of 322 liquefons per ml.
d) Solution of hog pancreatic alpha-amylase (2x crystallized, Wor~hington ~iochemical Corp., S/L = 227) containing 0.70 mg protein per ml and having an activity of 290 liquefons per ml.
The mixtures were stirred for 2 hours and filtered. The filter cakes were washed with 0.005 M calcium acetate at pH 7 with small portions of 0.5 M NaCl (total 100 ml) and then with about 50 ml of 0.005 ~ calcium acetate solution. The immobilized alpha-amylases exhibited the following potencies:
Potency A~ha-~mylase (liquefons Immobilized saccharifying 12.6 Immobilized liquefying125.0 Im~obilized fungal 7.3 Immobilized pancreatic41.5 :
To each of five stirred reactors maintained in a water bath at 50C. was added 138 g of partially hydrolyzed starch solution (pH
5.0, 16.9 D.E., 32.5 percent dry substance) prepared by the procedure described above which had been filtered through a cellulose ester membrane (~AWP 04700, 0.45 ~, Millipore Corp.) and then saturated with toluene. The purpose of the addition of the toluene was to pre-vent bacterial growth. ;;
0.51 g of immobilized glucoamylase was added to each of the reactors and sufficient immobilized alpha-amylase was added to ;~
-` four of the reactors to provide a total of 90 liquecons of alpha-', ~
.:`~ ' '. ~:

l~S~

~ir~ amylase activity per reactor. The reactors were continually f~' stirred and at various time intervals, samples were taken .~ from the reactors and filtered and the filtrates assayed for ..
. ,' percent dextrose. The results of this example are set forth ~ .
in Table I.

~'~

~'';''"~ .
':,"'.

~ ' .

' '' :

-24- ~
.~.

556~

1 ~ cu '` 1 `
/~ lu~ ~
,., l 1~ ~D O
E lo c~

~I N `

I j l_ ' Ci' ~ CO ' , ~'' ' O "
:
~
.
E-,., ~ . " .
h ~ U ~,~ O ~ "~

I td ~ . . ' '` O ~ ~
I ~ ::1 .'' , ,~ ¢P
. ~
I
,~: ....
I .

: ~ :
.~ ' . . .
-25~

, . .

~4556~ ~

From the above table, it is seen that a combination of immobilized glucoamylase and immobilized alpha-amylase resulted in a more complete conversion of starch to dextrose than when immobilized glucoamylase alone was used. Also, the combination of immobilized enzymes resulted in a more rapid-conversion of starch to dextrose. ~oreover, in general, the immobilized alpha-amylase preparations prepared from the soluble alpha-amylase ; preparation having a high S/L ratio are more beneficial in the conversion of partially hydrolyzed starch to dextrose.
Example II
.
; This example illustrates the effect of the ratio of immobilized alpha-amylase activity to immobilized glucoamylase activlty On the rate of production of dextrose.
60 g of activated AE prepared according to Example I
was added to 975 ml of 0.005 M sodium acetate solution containing , 5 5 x 10 liquefons of fungal alpha-amylase preparation derived from Aspergillus oryzae (Enzeco~ K768, Enzyme Development Corp.~. After slurrying for two hours at ambient temperature, the slurry was filtered, and the filter cake washed successively with deionized water, one liter of partially hydrolyzed starch solution (3.2 percent d.s., 16.4 D.E., p~l 5.0, 0.02 percent aaN3) and 2 ml of 0.02 percent NaN3 solution. The moist filter cake had an alpha-.' 1 ` amylase activity of 33.3 liquefons g Saccharification Using Immobilized Glucoamylase and Immobilized ` 25 Alpha-Amylase Derived from Asper~illus oryzae.
, Into 6 stirred reactors each containing 462 g of partially hydrolyzed starch solution (32.0 percent d.s., 16.7 D.E., pll 5.1, .
0.02 percent NaN3) at 50C. was added 1.13 g of immobiliæed gluco-` 30 amylase prepared according to the method described in Example I

~a~4556~3 :
/ and having a potency of 64.2 GU g . Then into the reactors were added, respectivèly, 21.8 g, 10.9 g, 5.45 g, 2.72 g~ 1.36 g and 0 g of immobilized fungal alpha-amylase prepared by the procedure described immediately above. The reactors were constantly stirred at 50C. and the percent dextrose determined at various periods.
Saccharification Using Immobilized Glucoamylase and Immobilized Alpha-Amylase of the Pancreatic Type.
Into 6 stirred reactors each containing 121 g of partially hydrolyzed starch solution (33.4 percent d.s., 16.9 D.E., pH 5.1, saturated with toluene) at 50C. was added 0.45 g of immobilized glucoamylase prepared according to Example I and having a potency of 44 GU g 1. Then into the reactors were added, respectively, 4.82 g, 2.41 g, 1.20 g, 0.60 g, 0.30 g and 0 g of immobilized pancreatic alpha-amylase prepared by the pro-cedure set forth in Example I. The reactors were constantly stirred at 50C. and the percent dextrose determined at various periods.
The results of these experiments are shown below in Tables II and III:

~ .
,' , ".
:: .
' ~ ~

''~', . ':

; 27 ~ ~
'' ~g~ss~

::
~( O ~t CJ` O ~ N N
~t co co o~

~ :
c~ N U~ N N ~
¢ `~:) ~ O ~I N
ct~
~ a~ .
O .C ~ o ~ ~, X CU ~~ o N ~::
N F ~ 00 CO I CO C~
~1 ~J . J_J
- ~:: ~c ~ a~ ~$ o N ~ `
O . ) O~ ~ ~ 00 C~ O Q~ :

~1 ~ N oo oo h H a) ~ ~ o ~ U~ ~ co rc .~ ~ ~ ~ r~ oo oo ~o o~ oO ~,~ , O ¦ ~D O O N U~
l ~ r~
r JJ r~ '.
N U~
., .,~
., ' ~r~ ~r~
~ a ~, 0~ -~
. ~ ¢ ~ ~
.
,: ~ ~ ~ .~
~ ~ .~ ~ ~
~ ~ .~ O
O ¢ ~
U ¢ ~ N u~ O
t~ r-l ul --~ ~D N U~ o o . ~ ~r~ r~l O O ~I N U~ 0 t~ ; ~
~r~ 41 ~ ~ .. ' ' .
. ` ~ O ~ ~ ,'.,:
: .C O O C~l U :
C~ U ~

'Z ~d . ~ ~ ~
~ ~ :L
' ~' ~ .

~` . ' ;"','.
' ' :~' ' , .~

- - - . . . . . . . _ . . . .
.. . . . .
6~

a) ),-r N ~ ~ I~ ~O
~1 O . C~
~ ~ oo ~
:. t'd ." ',.
~ ~ ~D CO1- ~ C~l ~ ', ~1 ~D ~ O ~ ~ u~ In . , .~ ~
~d O r ¦ ,~ ~ ~ ~ . N
C X N O O ~ ~ u) u~
P~ . ~ ~ ~
C ~ ` O ~ D N
~rl S~ 00 1~ 1~N ~ ~ u :~ ,( ~1 ~o ~ o~

1 I . r~~ N a~ o N
"J ~ C~ Nco ~I N ~ o H C~ ~ l~ ooco ~ a~ cl~,o 1-~a~ ~ .
_ N N 00 ~~ O N
i~ O C~l ~r a) ,- 'oo ~'' . N V O
rl ~rl D ~ O u~
El ~rl ~ . O
E3 rl t11 ~ C '' ': ':
a u~ ~ ~r~
~rl 111 V ~
r rl O O
g ¢ V C C~ ~ :
. ~ ~ ¢ ~ J U) O O .,~;,"~
rl ~ O O~1 C~ O t~
~rl ~1 ' 1~1 O E~ ~ :'' .
.; ~ O O V U

V ~ l ~ ;,, ~d ~

" ~ .
' , '' ' ;':,' ~, ~
. ~ ~' ', .
' ': ,: --2~ -~, ~ "' . .
~. : :'.
, .
-, . - - ~. . : ~ . ' : - . . . .

/ :
~ 55~,~

From the abovc tables it is seen that as the ratio of alpha-amylase activity to glucoamylase activity is increased, the faster and the more complete is the conversion. ~lowever, at the higher ratios the difference in the conversion is small which indicates that there is a maximum ratio where no substantial increase in conversion is obtained.
Example III
This example illustrates the utilization of covalently immobilized glucoamylase and various other immobilized enzymes.
Immobilization of Glucoamylase 20 g of DEAE;cellulose (Whatman ~E 23) was slurried in 500 ml of lN ~aOII, stirred for 30 minutes at ambient temperature and the slurry filtered. The filter cake was slurried in 30 ml of acetone containing 4.0 g cyanuric chloride for one minute and then 600 ml of 20 percent acetic acid solution was added. After about one minute, the slurry was filtered, the filter cake washed ~-:, :
with deionized water and suspended in 800 ml of a 50 percent (v/ v) ` mixture of 0.2 M tris(hydroxymethyl)aminomethane and SN HCl. After stirring for 7 minutes, 600 ml of 2O percent acetic acid was added ;~
to the slurry and stirring continued for another minute. The slurry was filtered, the filter cake washed extensively with deionized - water and then with 500 ml of acetone. The filter cake was dried by applying partial vacuum thereto. 19 g of filter cake was recovered.
The filter cake was added to 2000 ml of a glucoamylase . .
~ 25 solution (3.8 GU ml 1) free of transglucosylase prepared by thoroughly .
dialyzing a glucoamylase concentrate against tap water and then against pH 8.1 borate buffer (0.05 M). After stirring for 20 hours at ambient temperature, the slurry was filtered and the filter cake washed extensively with deionized water. ThP moist filter cake was then suspended in S00 ml of lM NaCl, stirred 30 minutes and filtered.
' . .

5i569 / The filter cake was washed with 500 ml of a lM NaCl solution and then with deioni~ed water. The filter cake weighed 74.3 g and had an activity of 15 GU g 1, Immobilization of Pullulanase Aerobacter aero~enes ATCC 15050 waa propagated and the pH of the fermentation broth was adjusted to 7 by the addition of a t,2 M solution of Nall2P04. 80 g of Triton X-100 (Rohm & Haas) was also added to the broth. The broth was stirred for 16 hours at 35C., centrifuged at 18,000 ~ g for 10 minutes and the sediment discarded. The supernate had a pullulanase activity of 1.07 IU
ml ~ 1 The p-~ of a 4000-ml portion of the supernate was ad-Justed to 7.6 by the addition of a solution of 0.2 M Na211PO~ and 6 g of DEAE-Scphadex A-50 ~Pharmacia) was added. The slurry was stirred for 30 minutes at ambient temperature, fil~ered and the filter cake washed with 1000 ml of deionized water. The filter cake was suspended in 100 ml of pH 7.0, 0.01 M phosphate buffer containing 5.4 g NaCl and stirred for 30 minutes. The slurry was filtered and the filtrate concentrated to 290 ml by ultrafiltration ; 20 in an Amicon model 401 ultrafiltration cell equipped with an XM-50 membrane. This filtrate was then dialyzed against deionized water ;~
to obtain a solution having a pullulanase activity of 10.5 IU ml 1, 80 g of ~hatman standard grade powdered cellulose was i suspended in 500 ml of 5 M Na~l solution and allowecl to stand for 16 hours. The supernate was removed by decantation and the cellulose washed several times with deionized water. The supernate was again removed by decantation and the cellulose filtered and suspended in 500 ml of deionized water. A 200 ml aliquot of the suspension con-taining about 20 g dry basis cellulose was adjusted to pll 10.5 by the addition of 1 M NaO~ solution. 50 ml of a solution containing * Trade Mark -31- ~-, . . . . . .

~4~569 5 g of cyanogen bromide was added and during a 45-minute reaction period, the pH of the mixture was maintained in the range of from ~' 10.0 to 10.5 by the periodic addition of 1 M NaOH solution. The cyanoge~n bromide-activated cellulose was collected by filtration, the filter cake washed with 10~0 ml of deionized water and then with 200 ml of 0.01 M sodium phosphate buffer at pH 7.9.
~ 200 ml of the dialyzed filtrate having a pullulanase activity of 10.5 IU ml was adjusted to pH 7.9 by the addition of 0.2 M Na2HPO~ solution. 10 g of the cyanogen bromide-activated cellulose was added, the suspension stirred for 16 hours at a temperature of about 3C. and filtered, and the filter cake washed with 50 ml of I M NaCl solution. The washed filter cake had a pullulanase activity of 80.2 IU g 1.
X~nobilization of Saecharifyin~ Alpha-AmYlase
7 g of cyanogen bromide-activated cellulose (prepared by the procedure described above) was added to 99 ml of cold 0.1 M phosphate buffer at pH 8 having dissolved therein sufficient saceharifying alpha-amylase (B. subtilis var. amylosacchariticus) to obtain an activity of 260 liquefons ml~l. The suspension was stirred for 2 hours while being maintained at 5C. and was filtered, the filter cake washed, successively, with 0.1 M phosphate buffer, with deionized water, with 2 percent Lintner starch solution at pH 5 and finally again with deionized water.
The washed filter cake had an alpha-amylase activity of 74 liquefons g 1.
Utilization of the Immobilized EnzYmes Four stirred reactors each containing 400 ml of partially hydrolyzed stareh solution (25.6 percent dry substance, 12.1 D.~
pll 5.2) were set up. The immobilized enzymes prepared as described above were introduced into the reactors and stirring commenced.

~, ' ' '.

1~455~9 / .:
Periodically, the D.E.s of the converted solutions were determined.
After 70 hours, the contents of each of tlle reactors was filtered, the filter cakes washed extensively with deionized water and then ..
added to 400 ml of partially hydrolyzed starch solution (30.8 per-S cent dry substance, 12.1 D.E., pH 5.2, 0.02 M.in acetate buffer).
:
Periodically, the D.E.s of the converted solutions were determined.
Afte~ 94 hours the contents of each of the reactors were filtered, the filter cakes washed extensively with deionized water and then added to 400 ml of partially hydrolyzed starch solution (30.8 per-cent dry substance, 12.1 D.R., pH 5.2, 0.02 M in acetate buffer). .
The D.E.s of the converted solutions were determined periodically.
The results of these experiments are set forth in Tables IV to VII below~
,. .
.:
','', ' . ~ ,', ' ~ i :: , :
: . .-.. , :~

~!
'~ ' - ' :1 :
' i ~ ' ' : : .
.... ..
: ! ~ . . .
,~: : ' ' ' ,. '' .

`:!: ` . :
:1 . . ~

, ~ :
_ ~: ' .
:.
, : :`
:'''' '':'~' ;'''' '"' ;`"''; ' ~'.'' `.' ' ,` .' . " `' ' , .' ,"" " ' 45569 ~ :
:~ ,,' , ' ' ':
. ~

.~ ~ :
,, a) :
? h ~
. ~ O ~ O ~ '.'~., ., l ': -.' l ~1 ~s~
~ ~ ',' ¦ ~sl ~, ~I . :, . '''` .
~ ~ ~1) .
~: _l a~ c~ ~ ~ ai , I ~ I r'--v _~ ~Oo ~1 . ~ U~ ~~ ~1~ O O ~ ~ O

3~,~ 3, 3 ~ o o h ~ ~, 04-'~o ~ ~

:. `

::

:
. : :

~ ~4556~

.

¦ h~

C

, . ~ o .. ~ .
.' ~ ~ ~ . . .
., ~ ~ . ..
, . ,~ U~ .......
., ~ V U~ ,~

. . , ¦ _ ~ CO 00 O
.^ . I ~ ~ .. .
.-~
.
.~ ~ u O O a~

, . l :' ' ~ U~

~,-"~ : ~ o,~o~ o,.~Co ~ ~

:: :
;.. : .. :: ," . .: . ... . . , ~ . ; . . . ,: . : . : . . :: . . .

- / :
~¢~45569 ::
:-.
:.
. :
s .~ ~ a~
a~
: V
, ~
s . G O O~
a) U~ . -h . - . .
X .
C) U) O ~
. . . . ....
1 . ~ :' "'':' ., tl~ . .~ ~ :'. ' ~ :' W . .
.
:' ~ ~
~ O~ ~U O
.. ; ~ .~: . . .
., ' .. C~l ''' a) C~l . ,.
W ~ ,: :
~, W ~ ~ ~ ~ ' ' ' :.
C : N ~rl O
., ~ I .,~
,,,, -1 W ~ W
C~ S ~ V , ~i ~ ~
a~ ¢ E~ o N . E
.r/ OC .~
w :~ a ,~ ~ E cr~ o ~::
O W ~ ~ O
~ 4~ . oo w w ~
~ o n C
~ ~ u~ o w ,, S . ~ oO ~,q O
hO t~ C~ ~. ~ ~1 a) ~ C4 C U ~ C ~

;': W
. ~ ~ ~ ~
O W , ~1 ~ W ~ C ~ ?
.j .,1 u~ N ~ ~ O ~I C
,~ c u ~ u ~ o w ~ ~ ~ ~ E ~ u ~ o ~ e ~ I o ~ ~ o j: ~ . ~ ~ ~ W W ~ . .
I W ~ U) ~1 rC W ~
u w a o ~ s~ . E cJ
,, u o w w w ~ w s~
~': w '~ ~ u O ~~ '`
., . --I w 1~ ~ u ~ o ., u~ ~ ~ a) ~ u . ~ O ~0 ~ ~ bO Cl ~ ,,, O ~ JJ
, : N ~ tJ
.~
:~ : ~1 0 ~ :~ O
.D C U~ ,~ ~ to ,~
N bO O O ~ O
c ~ E u O a r O
~ o ~ U S
: O ,~ :~ ~ 0 ~1 ~3 ~ o a) ~ o ~ a~
: E ~ ,~
?~ : ~I U~ ~ ~I) W ~ ~I) W ~
:~ ~ o ~ u :::) u ~?~ W 6 '~1 ,-~ ~ ~ ~ D ,~ C~ ,~ ' :
~ 00U~ , W W ~L~ W ~d " .
: ~ : , . . .
36- :
:: . .: .:
- ~ -. ~ .
: ~ :` ~' :-:

~ 45S69 .

- cn ,~ .S
'~ ~ u~ ' .
,_ ., .
J~
. ~: '.::
, ' ~ t I
O t~ ~ t~ .
tn .; ~ :., tu tn~
cn~ ~ol t~ a~ oO
E .
. o~ a ~.
3 ~n~n . ...
,' ~ t~ r trl ~J
N I t U t~ 00 CO
t~
~1 ~ .C
t~ t~
bO ~ a~ ~ ~ ' ' ' a ~ ~ o ~n h :
. j ~ cJ o a) ~ a o c~ . E 0 ~ ~ tq o tO t-E t~ ttl t~ ~ t~ o ~ ~n E ~ tu W ~ ~ E O n~ ~ E 3 ~I tll ~1o W ~1 o s~ 4~
.~' . u~oo t~ ~ t o ~ 4~ 0 . ' '1 ` ~d t~tr~ ~ V t~~ tU tO ~ ~ 0 C~ ~ ~ ~~ rlt`l~ h 04 tU ~1 .:1 U N '~ O~ tU O
., t~ W tU O ,C tU
i tU tU O~ U ~t N ~N bOtUO tU tU O ~ (U
1 ~ ~ ~ .0 ~ e tU~V
~3 ~ V h 0 0 ~ O ~h ~U~
~u 5 tu tU.~
to ~ ,~ t~ ~ V ~d ~ t~h 0 ~,0~ ~ tU
~: : . : O r-l tr(U t~ U ~d ~ ''`~
O ~1 0 ~ ~_1 E O ,~ E O
: : ,:: .
i: ' :
:
i, : .

; ! ; ' . . :

~: , . . ... , , ' , ' ' ~ , , , .. , ' ' , ' : : ., ,, : ' . :: ' ' . ' '

Claims (34)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE PROPERTY OR
PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A process for converting starch to dextrose comprising contacting a partially hydrolyzed starch solution containing at least 10 percent hydrolyzed starch with an enzyme system comprising immo-bilized glucoamylase (E.C.#3.2.1.3) and alpha-amylase selected from the group consisting of soluble alpha-amylase (E.C.#3.2.1.1.), immobilized alpha-amylase and mixtures thereof under conditions whereby substantially complete con-version of the hydrolyzed starch to dextrose is achieved.
2. A process for converting starch to dextrose as defined in Claim 1, wherein the alpha-amylase is immobilized alpha-amylase.
3. A process for converting starch to dextrose as defined in Claim 1, wherein the amount of immobilized glucoamylase and the amount of alpha-amylase are such as to provide a ratio of dextrinizing activity to glucoamylase activity of at least 0-2 liquefons per glucoamylase unit.
4. A process for converting starch to dextrose as defined in Claim 1, wherein the amount of immobilized glucoamylase and the amount of alpha-amylase are such as to provide a ratio of dextrinizing activity to glucoamylase activity of at least 1 liquefon per gluco-amylase unit.
5. A process for converting starch to dextrose as defined in Claim 1, wherein the amount of immobilized glucoamylase and the amount of alpha-amylase are such as to provide a ratio of dextrinizing activity to glucoamylase activity of at least 3 liquefons per gluco-amylase unit.
6. A process for converting starch to dextrose as defined in Claim 2, wherein the immobilized alpha-amylase is prepared from a soluble alpha-amylase preparation having a S/L value of at least about 3.
7. A process for converting starch to dextrose as defined in Claim 2, wherein the immobilized alpha-amylase is prepared from a soluble alpha-amylase preparation having a S/L value of at least about 50.
8. A process for converting starch to dextrose as defined in Claim 2, wherein the immobilized alpha-amylase is prepared from a soluble alpha-amylase preparation having a S/L value of at least about 100.
9. A process for converting starch to dextrose as defined in Claim 2. wherein the partially hydrolyzed starch is contacted with a mixture of immobilized glucoamylase and immobilized alpha-amylase.
10. A process for converting starch to dextrose as defined in Claim 2, wherein the alpha-amylase and the glucoamylase are immo-bilized on or within the same carrier.
11. A process for converting starch to dextrose as defined in Claim 9, wherein the partially hydrolyzed starch solution is con-tacted, sequentially, with immobilized glucoamylase, with immobilized alpha-amylase and with immobilized glucoamylase.
12. A process for converting starch to dextrose as defined in Claim 1, wherein the partially hydrolyzed starch solution is pre-pared by an enzyme treatment and has a D.E. from about 10 to about 60.
13. A process for converting starch to dextrose as defined in Claim 1, wherein the partially hydrolyzed starch solution is prepared by an acid treatment and has a D.E. of from about 10 to about 30.
14. A process for converting starch to dextrose as defined in Claim 2, wherein the temperature of the partially hydrolyzed starch solution being contacted with the enzyme system is from about 30° to about 65°C.
15. A process for converting starch to dextrose as defined in Claim 14, wherein the pH of the partially hydrolyzed starch solution being contacted with the enzyme system is from about 3.5 to about 6.
16. A process for converting starch to dextrose as defined in Claim 1, wherein the enzyme system includes an immobilized alpha-1,6-glucosidase.
17. A process for convering starch to dextrose as defined in Claim 16, wherein the immobilized alpha-1,6-glucosidase is immobilized pullulanase (E.C. #3.2.1.41).
18. A process for converting starch to dextrose com-prising treating starch with alpha-amylase to obtain a partially hydrolyzed starch solution containing at least 10 percent hy-drolyzed starch and then treating the partially hydrolyzed starch solution with an enzyme system comprising immobilized gluco-amylase selected from the group consisting of glucoamylase covalently bonded to an insoluble carrier and glucoamylase ad-sorbed on an insoluble carrier and immobilized alpha amylase selected from the group consisting of alpha-amylase covalently bonded to an insoluble carrier and alpha-amylase adsorbed on an insoluble carrier under conditions whereby a hydrolysate con-taining at least about 92 percent dextrose on an ash free, dry basis is produced.
19. A process for converting starch to dextrose as defined in Claim 18, wherein the amount of immobilized gluco-amylase and the amount of immobilized alpha-amylase are such as to provide a ratio of dextrinizing activity to glucoamylase activity of at least 0.2 liquefons per glucoamylase unit.
20. A process for converting starch to dextrose as defined in Claim 18, wherein the amount of immobilized gluco-amylase and the amount of immobilized alpha-amylase are such as to provide a ratio of dextrinizing activity to glucoamylase activity of at least 1 liquefon per glucoamylase unit.
21. A process for converting starch to dextrose as defined in Claim 18, wherein the amount of immobilized gluco-amylase and the amount of immobilized alpha-amylase are such as to provide a ratio of dextrinizing activity to glucoamylase activity of at least 3 liquefons per glucoamylase unit.
22. A process for converting starch to dextrose as defined in Claim 18, wherein the immobilized alpha-amylase is prepared from a soluble alpha-amylase preparation having a S/L
value of at least about 3.
23. A process for converting starch to dextrose as defined in Claim 18, wherein the immobilized alpha-amylase is prepared from a soluble alpha-amylase preparation having a S/L
value of at least about 50.
24. A process for converting starch to dextrose as defined in Claim 18, wherein the immobilized alpha-amylase is prepared from a soluble alpha-amylase preparation having a S/L
value of at least about 100.
25. A process for converting starch to dextrose as defined in Claim 18, wherein the partially hydrolyzed starch is contacted with a mixture of immobilized glucoamylase and im-mobilized alpha-amylase.
26. A process for converting starch to dextrose as defined in Claim 18, wherein the alpha-amylase and the gluco-amylase are immobilized on or within the same carrier.
27. A process for converting starch to dextrose as defined in Claim 25, wherein the partially hydrolyzed starch solution is contacted, sequentially, with immobilized gluco-amylase, with immobilized alpha-amylase and with immobilized glucoamylase.
28. A process for converting starch to dextrose as defined in Claim 18, wherein the partially hydrolyzed starch solution is prepared by an enzyme treatment and has a D.E. from about 10 to about 60.
29. A process for converting starch to dextrose as defined in Claim 18, wherein the temperature of the partially hydrolyzed starch solution being contacted with the enzyme system is from about 30° to about 65° C.
30. A process for converting starch to dextrose as defined in Claim 29, wherein the pH of the partially hydrolyzed starch solution being contacted with the enzyme system is from about 3.5 to about 6.
31. A process for converting starch to dextrose as defined in Claim 18, wherein the enzyme system includes an im-mobilized alpha-1,6-glucosidase.
32. A process for converting starch to dextrose as defined in Claim 31, wherein the immobilized alpha-1,6-gluco-sidase is immobilized pullulanase.
33. A process for converting starch to dextrose as defined in Claim 32, wherein the pullulanase is covalently bonded to an inert carrier.
34. A process for converting starch to dextrose as defined in Claim 18, wherein the glucoamylase is adsorbed on a cellulose derivative.
CA234,067A 1974-08-26 1975-08-25 Process for producing dextrose using mixed immobilized enzymes Expired CA1045569A (en)

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
US05/500,975 US4011137A (en) 1974-08-26 1974-08-26 Process for producing dextrose using mixed immobilized enzymes

Publications (1)

Publication Number Publication Date
CA1045569A true CA1045569A (en) 1979-01-02

Family

ID=23991639

Family Applications (1)

Application Number Title Priority Date Filing Date
CA234,067A Expired CA1045569A (en) 1974-08-26 1975-08-25 Process for producing dextrose using mixed immobilized enzymes

Country Status (7)

Country Link
US (2) US4011137A (en)
BE (1) BE832776A (en)
CA (1) CA1045569A (en)
DE (1) DE2538322A1 (en)
FR (1) FR2283225A1 (en)
GB (1) GB1521112A (en)
NL (1) NL188103C (en)

Families Citing this family (29)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4011137A (en) * 1974-08-26 1977-03-08 Standard Brands Incorporated Process for producing dextrose using mixed immobilized enzymes
US4116771A (en) * 1976-07-02 1978-09-26 Novo Industri A/S Immobilized saccharifying enzyme product and process for preparation thereof
GB1571987A (en) * 1976-07-02 1980-07-23 Novo Industri As Enzyme products
US4132595A (en) * 1976-12-13 1979-01-02 Cpc International Inc. Dextrose production with immobilized glucoamylase
US4121974A (en) * 1977-06-29 1978-10-24 The United States Of America As Represented By The Secretary Of Agriculture Preparation of retrogradation-resistant starches with immobilized amylases
US4202939A (en) * 1977-09-01 1980-05-13 Cpc International Inc. Glucoamylase immobilized on cationic colloidal silica
US4206284A (en) * 1977-11-15 1980-06-03 Novo Industri A/S Saccharification of glucose raffinate or mother liquors
US4206285A (en) * 1977-12-27 1980-06-03 Novo Industri A/S Saccharification of enriched fructose content syrups
DE2911192A1 (en) * 1979-03-22 1980-10-02 Boehringer Sohn Ingelheim INNOVATIVE IMMOBILIZED GLUCOSE OXIDASE CATALASE PREPARATION AND ITS USE FOR ENZYMATIC GLUCOSE OXIDATION
US4226937A (en) * 1979-04-27 1980-10-07 Cpc International Inc. Method using glucoamylase immobilized on porous alumina
US4259445A (en) * 1979-06-01 1981-03-31 Cpc International Immobilized enzyme catalyst
US4477569A (en) * 1982-02-18 1984-10-16 Canadian Patents & Development Limited Pentose fermentation with selected yeast
DD238305A3 (en) * 1984-04-23 1986-08-20 Maisan Werke Barby Veb PROCESS FOR THE PREPARATION OF D-GLUCOSE AND STAERKEHYDROLYSATES
US4654216A (en) * 1985-07-31 1987-03-31 Novo Laboratories, Inc. Bread antistaling method
US4681845A (en) * 1985-08-16 1987-07-21 Uop Inc. Increased glucose levels in starch saccharification using immobilized amyloglucosidase
EP0257535B1 (en) * 1986-08-28 1994-01-26 Nihon Shokuhin Kako Co., Ltd. Process for production of starch sugar
JPH04218394A (en) * 1990-04-10 1992-08-07 Kanzaki Paper Mfg Co Ltd Analysis of starch or related saccharide and analytical equipment therefor
US20050213187A1 (en) * 1994-08-25 2005-09-29 University Of Iowa Research Foundation Methods for forming magnetically modified electrodes and articles produced thereby
US20040115779A1 (en) * 2002-03-19 2004-06-17 Olsen Hans Sejr Fermentation process
EP1720897B1 (en) * 2004-02-27 2010-03-31 Dow Global Technologies Inc. Method for the extraction of intracellular proteins from a fermentation broth
US7815741B2 (en) * 2006-11-03 2010-10-19 Olson David A Reactor pump for catalyzed hydrolytic splitting of cellulose
US7815876B2 (en) 2006-11-03 2010-10-19 Olson David A Reactor pump for catalyzed hydrolytic splitting of cellulose
WO2009003111A2 (en) 2007-06-27 2008-12-31 H R D Corporation High shear process for dextrose production
US20130231281A1 (en) 2011-11-02 2013-09-05 Adocia Rapid acting insulin formulation comprising an oligosaccharide
CN107952065A (en) 2012-11-13 2018-04-24 阿道恰公司 Include the Insulin Aspart for being substituted anionic compound
AU2013346623A1 (en) * 2012-11-13 2015-05-14 Adocia Substituted anionic compounds consisting of a backbone consisting of a discrete number of saccharide units
FR3020947B1 (en) 2014-05-14 2018-08-31 Adocia AQUEOUS COMPOSITION COMPRISING AT LEAST ONE PROTEIN AND A SOLUBILIZING AGENT, ITS PREPARATION AND ITS USES
US9795678B2 (en) 2014-05-14 2017-10-24 Adocia Fast-acting insulin composition comprising a substituted anionic compound and a polyanionic compound
FR3043557B1 (en) 2015-11-16 2019-05-31 Adocia RAPID ACID COMPOSITION OF INSULIN COMPRISING A SUBSTITUTED CITRATE

Family Cites Families (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US2304168A (en) * 1939-11-27 1942-12-08 Standard Oil Co Making hydrocarbon conversion catalyst
US2531999A (en) * 1947-10-13 1950-11-28 Wallerstein Co Inc Manufacture of crystalline dextrose
US2891869A (en) * 1953-06-03 1959-06-23 Staley Mfg Co A E Process for preparing starch syrups
US2893921A (en) * 1955-01-12 1959-07-07 Staley Mfg Co A E Production of amyloglucosidase
US3042584A (en) * 1960-12-22 1962-07-03 Corn Products Co Treatment and use of enzymes for the hydrolysis of starch
US3560345A (en) * 1967-06-02 1971-02-02 Hayashibara Co Process for producing bacterial isoamylase
IL32406A (en) * 1968-06-26 1973-01-30 Snam Progetti Enzyme preparations comprising a solution or dispersion of enzyme occluded in filaments of cellulose esters or synthetic polymers
GB1232619A (en) * 1968-10-23 1971-05-19
US3720583A (en) * 1968-12-20 1973-03-13 Staley Mfg Co A E Enzyme hydrolysis
US3810821A (en) * 1969-12-18 1974-05-14 Ranks Hovis Mcdougall Ltd Enzymes attached to cellulose carbonate
US3809613A (en) * 1971-05-28 1974-05-07 Research Corp Biocatalytic module
BE789195A (en) * 1971-09-24 1973-03-22 Gist Brocades Nv ENZYME COMPOSITIONS
US3783101A (en) * 1972-02-17 1974-01-01 Corning Glass Works Enzymes bound to carriers having a metal oxide surface layer
US3849253A (en) * 1972-10-10 1974-11-19 Penick & Ford Ltd Process of immobilizing enzymes
NL7400363A (en) * 1973-01-30 1974-08-01
US4011137A (en) * 1974-08-26 1977-03-08 Standard Brands Incorporated Process for producing dextrose using mixed immobilized enzymes

Also Published As

Publication number Publication date
US4102745A (en) 1978-07-25
DE2538322A1 (en) 1976-03-11
GB1521112A (en) 1978-08-09
NL7510099A (en) 1976-03-01
BE832776A (en) 1976-02-26
FR2283225B1 (en) 1980-09-19
US4011137A (en) 1977-03-08
NL188103C (en) 1992-04-01
NL188103B (en) 1991-11-01
FR2283225A1 (en) 1976-03-26

Similar Documents

Publication Publication Date Title
CA1045569A (en) Process for producing dextrose using mixed immobilized enzymes
CA1128885A (en) Thermostable glucoamylase from talaromyces duponti
CA1199292A (en) Debranching enzyme product, preparation and use thereof
US4916064A (en) Carbohydrate refining process and novel enzyme compositions suitable for use therein
US4591560A (en) Process for saccharification of starch using enzyme produced by fungus belonging to genus Chalara
CA1173766A (en) Inulinase
US4970158A (en) Beta amylase enzyme product, preparation and use thereof
US4132595A (en) Dextrose production with immobilized glucoamylase
US4211842A (en) Starch-degrading benzymes derived from Clacosporium resinae
WO1986001832A1 (en) THERMOSTABLE beta-AMYLASE
EP0405283A2 (en) Novel thermoduric and aciduric pullulanase enzyme and method for its production
US3862005A (en) Process for producing aldonic acids and starch sugars containing aldonic acids
EP0138428A2 (en) Acid-stable alpha-amylase composition, preparation and use thereof
GB2123001A (en) Preparation of fructose syrups
CA1178550A (en) Process for producing glucose/fructose syrups from unrefined starch hydrolysates
US4111750A (en) Process for converting liquefied starch to a mixture of glucose and fructose utilizing a multi-component immobilized enzyme system
US3108928A (en) Treatment and use of enzymes for the hydrolysis of starch
EP0860500B1 (en) Purified acid-stable alpha-amylase from fungal origin
EP0157638A2 (en) Method for production of high conversion syrups and immobilized alpha-amylase employed in the process
US3660236A (en) Production of glucoamylase
Lee et al. Continuous ethanol production from sago starch using immobilized amyloglucosidase and Zymomonas mobilis
CA1213235A (en) Process for preparing high dextrose starch hydrolysates from immobilized glucoamylase
US4254225A (en) Novel neutral glucoamylase and method for its production
US3332851A (en) Process of purifying glucoamylase
Freire et al. Characterization of a glucoamylase immobilized on chitin