Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
1iD7Z~:~9
This invention rela-tes to insolubilized enzymes and to a process
for preparing insolubilized enzyme products. ~.
More particularly, this invention relates to particulate physi-
cally stabilized forms of insolubilized saccharifying enzymes se-
lected from the ~roup consisting of amyloglucosidase and maltoge-
nic alpha-amylase, both usually of fungal origin, and to a process ~ :~
for the preparation of such insolubilized enzyme products.
,
Insolubilization ~or immobilization) of extracellular and of in-
tracellular water soluble enzymes, that is, the fixation of cata- . :
lytically active (native) enzymes in an insolubilized, solid or
quasisolid, shaped or shapable structure has become of increasin~ ;~
'cechnological as well as economical importance, as a means of ren- :.
dering a particular enzyme product reusable in batch-type proces-
ses or suitable for a continuous mode of operation in a so-called
enzyme reactor. ~ :
.
During recent years efforts have been devoted particula.rly to the
developmenc of immobilization technology in the starch processing
industry in connection with the manufacture of such products as `
high dextrose and high fructose syrups. Lately, the relative im-
portance of the latter has been increasing in food and related
industries, high ~ructose syrup being an advantageous substitute
~; for sucrose and ~or (the less-sweet) high dextrose syru~. Two ad-
ditional types of sugar syrup ~ased on starch hydrolysis and both
.having a substantial conten-t of maltose, namely high maltose and
high conversion syrup are being used to an increasing extent,
particularly in the confectionary (hard candy? and canning indu-
stries, respectively.
The over-all industrial process of converting starch via high
dextrose syrup into hi~h fructose syrup colnprises three consecu- :
~tive steps, namely the starch thinnin~ or liqueEaction process.
to dextrins, catalyzed by acid and/or by a bacterial alpha-amylase,
followed by s~ccharification to a high dextrose syrup and then
the conversion of ~he latter into high fructose syrup, each process
. catalyzed by its specific enzyme, viz. amyloglucosidase and glu-
cose isomerase, respectively. I~hereas the most recent technc,logi-
cal development has made cont.inuous processes available for ~oth
the first and the thir~ step of the above production sequence, in- .
~ ' ~
.
2~
dustrial saccharification is still predomin~ntly a batch-t~pe
process. The starting material of a hiyh maltose type of syrup
is also liquefied starch produced ~y either one of the methods
just described. However, in this case the subsequent saccharifica- ;
tion step resulting in a syrup o~ high maltose content (and usual-
ly containing relatively li-ttle glucose) is effected by means of
a maltogenic amylase, preferably a fungal alpha-amylase. Like
glucogenic saccharification, the corresponding maltooenic process
is usually conducted batch-wise using the soluble enzvme,
Although the convenience of having at ones disposal a totally
continuous production sequence is evident, the development of in-
dustrial enzyme reactor techniques involving the use o insolubi- ~ ;
lized amylo~lucosidase in the saccharification step is only at a
prelimanary stage.
~ , .
The prior art contains a number of references to the insolubili-
zation of amyloglucosidase, for example by bonding the enzyme to
insoluble inorganic or organic carriers. However, only few of ~ -
these methods appear to have been developed be~ond the laboratory
- hench scale. Among the methods that apparently have Dassed that
stage specific mention should be made of the immobil-zation of
amyloglucosidase by covalent bonding to porous partïcles of glass
or ceramic material. In this connection reference is made to a
recent article published by D.D. Lee et al. in "Die Starke", Vol.
27 (lg75~ pp 384-387.
. . . .
In a continuous process in which a pilot plant size column was
packed with this particular enzyme product and fed with solutions
of commercially available dextrins, degrees of conversioll to dex-
trose were attained which approached the minimal conversion of
about 92 percent generally required in a batch saccharification -~
-process with soluble amyloglucosidase. However, product analyses `
_ indisated that reversion reactions, i.e. amyloglucosidase cata-
lyzed repolymerization of glucose into maltcse, isomaltose and -
higher oligomers, occurs to a greater extent in the column process
than in the batch-type, free enzyme ~rocess.
The phenomenon of increased reversion encountered wi-th an im~obi-
lized enzyme product of this type avpears at least partly to be
~an inherent disadvanta~e of using porous enzyme carrier materials. ;~
~'~
"` ' ~L~7~2g
Clearly, the porous structure contributes to a very substantial
degree to the total surface area lending itself to enzyme bondin~
and, consequently, to the maximal enzyme activity obtainable of
the immobilized enzyme product. ~;
.:
However, the high concentration of amyloglucosidase attached to
the carrier pore surface combined with a reduced di~fusion rate
within the pores inevitably results in locally high ~lucose con-
centrations, thus constituting favourable conditions for the pro-
motion of enzyme processes having high Km-values. This is exactly
the case with the undesirable reversion reactionsf and particular-
ly those in which isomaltose and isomaltriose are formed.
An insolubilized maltogenic alpha-amylase ~roduct, prepared by
bonding the soluble enzyme to aminoethylcellulose by means of
glutaraldehyde, is disclosed in U.S. Patent Mo. 4.oll.137 tThompson
et al.). However, according to this reference the intended use oE
the immobilized enzyme is limited to increasing the degree of con~
version of dextrinized starch to glucose by means of a similarly
immobilized amyloglucosidase preparation, the saccharification
process being conducted by means of a mixture of the two immobili-
zed enzymes. There is no demonstration whatsoever in thç patent
of the use of the i~nobilized maltogenic alpha-amylase for the pro-
duction of a high maltose syrup.
: ! '
An additional disadvantage of using a particulate immobilized ~
saccharifying enzyme, in which the enzyme is bonded to a porous
or reticular material which is essentially homogeneous throughout
the particulate structure can be foreseen when takin~ into account
the heterogeneity in molecular size of the dextrinized starch
serving as substrate. All dextrins, whether produced by acid or
by enzymatic hydrolysis of starc~, contain substantial fr~ctions
~of glucose oligomers. Nor~ally, the starch thinning process is
stopped ~hen the average chain ~ ~ of the hydrolysate is in
the range of from 6 to `Lo glucose residues, and it is conceivable
that mere steric hindrance may impede the diffusion of the larger
dextrin molecules to enzyme sites embedded beyond a certain depth
within the pGrous or reticular particle structure.
~7263;~:~
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,,~:
It is one object of the present invention to overcome or at least
to mitigate the principal disadvantages encountered with the prior
art immobilized saccharifying enzyme products described above,
by providing an insolubilized product having process characteri-
stics essentially similar to those of the soluble enzyme, irres-
pective of whether the immobilized product is used in a batch
process or in a continuous mode of operation.
In the case of immobilized amyloglucosidase this requirement en-
tails that a continuous saccharafication process can be conducted
under such practically acceptable process conditions (e.g. in terms
of composition ancl flow rate of the dextrin feed) as to yield a
glucose concentration of at least 92 percent, the total concentra-
tion of disaccharides ~maltose and isomaltose) and trisaccharides
(mainly panose and isomaltotriose) not exceeding about 4 and 1
percent, respectively.
In the case o~ the immobilized maltogenic alpha-amylase the corres-
ponding conversion parameters required would be: approximately 40-60
percent mal-tose, 25-35 percent maltotriose and, preferably, less
than lo percent glucose.
.. ~ . ,
Since the commercially available soluble saccharifying enzyrnes
are relatively cheap and highly active, another preferable but
not essential object of the pr-esent invention is the provision-af
economically favourable, insolubilized products. The ~ursuance,of
this goal necessitates the utilization of comparatively inexpensi-
ve, cor~mercially readily available carrier and other auxiliary ma-
terials and, furthermore, th~ achievements of substantial recoveri-
es of enzyme activities in the immobilization process. In addition,
from a toxicological point of view all materials used should be
acceptable for food processing purposes.
-Briefly, these objects are attained according to the present in- ~ ;
vention which provides an immobilized saccharifying enzyme pro-
duct in particulate form in which enzymatically inert, water-in-
soluble carrier particles are essentially coated with a liauid-
per~eable, proteinaceous layer in ~hich the saccharifying enzyme
is immobilized by cross-linking with glutaraldehyde.
,
,
72029
Attempts to prepare such insolubiliz~d products in which the pro-
teinaceous layer is composed of the glutaraldehyde cross-linked
saccharifying enzyme per se proved unsuccessful in the sense that,
irrespective of the choice of core material, they resulted in in-
completely immobilized products from which the enzyme would
rapidly léak away. Similar problems were encountered with a
variety of water-insoluble particulate carrier materials of
inorganic (for example mineral or ceramic) origin or with certain
types of proteinaceous core material (such as granular soy pro-
tein), presumably owing to an insufficient number of cross-link
binding sites at the surface of such particles.
However, it has now been found that these and other
obstacles can be overcome and the physical and enzymatic pro-
perties of the immobilized enzyme coated products greatly
improved, provided that granular casein is used as core material ;;
and, furthermore, that glutaraldehyde cross-linking of the enzyme
in the coating layer is effected in the presence of a water-
soluble, non-enzymatic binder protein, such as egg albumen, the
choice of such materials evidently constituting a favourable com~
bination for creating a sufficient number of inter-protein
enzyme stabilizing cross-links.
Thus, more specifically, according to one aspect of the
present invention there is provided an immobilized saccharifying
enzyme product in particulate form/ the saccharifying enzyme being
selected from the group consisting o amyloglucosidase and malto-
genic alpha-amylase, which prcduct comprises carrier particles
of granular casein having a coating of a liquid-permeable pro-
teinaceous layer comprising the saccharifying enzyme and egg
albumen cross-linked by reaction with glutaraldehyde.
The rPlative amounts of the constituent saccharifying
enzyme, casein and albumen may vary within rather wide limits,
depending inter alia on the unit activity (as hereinafter defined)
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~ .
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of a soluble saccharifying enzyme starting material. However, in
a preferred embodiment of this invention the ratio of saccharify-
ing enzyme:casein is in the range of from 1:200 to 1:3, preferably
1:20 to 1:5, the amount o~ saccharifying enzyme being calculated
on the basis of a product having a purity of at least 5~/~. Typi-
cally the amount of amyloglucosidase and maltogenic ~-amylase is
calculated on the basis of products having activities of 10,000
and 3,000 units per gram respectively. ~ikewise a preferred range
of the ratio of saccharifying enzyme:egg albumen is from 1:5 to
1:0.05 and s~ill more preferred is the range from 1:2 to 1:0.2.
According to a further aspect of this invention there i5
provided a process for the preparation of an immobilized sac- ;
charifying enæyme product in particulate form, the saccharifying
enzyme being selected from the group consisting of amyloglucosi- ~-
dase and maltogenic alpha-amylase, which process comprises coat-
; ing carrier particles of granular casein with a reaction mixture ~ ~-
of the saccharifying enzyme, egg albumen and glutaraldehyde,
whereby the granular casein becomes coated by a li~uid-pexmeable :`
proteinaceous layer, in which the saccharifying enzyme and egg
'~ 20 albumen are cross-linked by reaction with glutaraldehyd~. ;
In ~urther detail, this process comprises the step of ~;
wetting a dry mixture of granular ca~ein and egg albumen powder
with an aqueous mixture consisting of the saccharifying enzyme
and glutaraldehyde dissolved at pH 4-7, with vigorous stirring,
followed by the step of maintaining the resulting wetted mixture
` in a quiescent state, usually at ambient temperature, to complete-
the combined carrier coating and protein cross-linking process.
Wetting may be conducted either manually, for example by
thorough mixing and kneading in a mortar, or mechanically, or
example in a plough type horizontal mixer obtained from Gebr.
Lodige Maschinenbau G.M.B.H., Pader~orn, West Germany, or a
similar type of industrial mixing apparatus. Optionally the
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, ......
~i'''~''~;
... ... . ... :.: - : .. .. .
~.~7~az9
albumen may be dissolved together with the enzyme and the
glutaraldehyde instead of being mixed in the dry state with the
casein.
The weight ratio of glutaraldehyde ~usually added as a
50 percent (w/w) aqueous solution) to coating layer proteins
(enzyme plus aIbumen) may vary considerably, the preferred range
being 0.1 to 1. The water content of the aqueous solution is
usually adjusted to be in the range of from 30 to 60 percent
of the total mixture.
; The properties of the granular casein pr3duct selected ~;
as carrier material are significant for attaining the objects of
this invention. Thus, the casein granules should possess .
sufficient physical stability to resist substantial deformation
upon soaking under packed bed column conditions. In addition,
the degree of swelling in water should be reasonably low, pre-
ferably not exceeding 200 percent. Exemplary of a product meet-
ing such requirements is acid precipitated granular casein.
Food grade products of this type, preferably having particle dia-
., .
meters in the range of from 100 to 500 microns are obtainable
from various sources, for example from the French company Scerma
S.A~ Scanning electron mlcroscope examination reveals that the
surface of such particles has a very uneven and irregularly
folded structure, bearing a certain resemblance to the macro-
scopic appearance of pumice. It is conceivable that a surface
micro-structure of this type would expose a large number of
glutaraldehyde bonding sites~
It is essential for practising the present invention
that a substantial fraction of the albumen used as binder protein
be almost instantly soluble in water. Hence, the preferred grade
of albumen is a generally available spray-dried product~
The mixing process may entail a certain degree of
agglomeration of the coated particles, resulting upon completion
of the cross-linking reaction in the formation of a moist, coarse
- 8-
granular and lumpy product~ This pro~uct may be subjected to
further disintegration, for example by granulation, to form a
particulate product with particle sizes within a preferred range.
Granulation may be effected on an oscillatory granulator of the
type supplied by several companies ~Diaf, Copenhagen or Manosty
Liverpool). The granulate product passing through the granulator
screen, for example one with holes of 1-2 mm, may be freed of
fine material by conventional means. The product so obtained
may optionally be washed, followed by drying to a desired water
content.
Prior to being used or a saccharification process, the
dried enzyme product is usually conditioned by pre-soaking in an
aqueous solution. At this stage and also during the initial
phase of the subsequent use of the product a certain leakage oE
enzyme activity may be observed. It has been found that the
'i inclusion of an additional cross-linking step, optionally in
connection with the soaking process, may serve to substantially
- reduce this loss of enzyme activity. ~ence, according to one
embodiment of this invention pre-treatment of the enzyme product
i~ performed in an aqueous solution containing a suitable amount,
preferably 0.5 to 5 percent of glutaraldehyde, usually followed
by removal of excess reagent by washing and, optionally, recovery
of the dried product.
` It has been found that pre-treatment of the enzyme
product with a salt of sulphurous acid may effect a significant
increase in the degree of conversion (as expressed in percent
of dextrose equivalent or DE-value) of the saccharified product~
A theory has been advanced that the sulphite action is an "opening
up" of certain reticular structures involving the proteinaceous
enzymatically active layer but apparently not affecting the bond-
ing of the enzyme, thus facilitating the access of higher oligo-
saccharides to enzyme active sites. Accordingly, a further embodi-
_ g _
~72alZ~
ment of this invention implies treating the enzyrne product witha dilute aqueous solution of sodium sulphite, preferably 0.1~2
percent of solution at pH 4-5. The sulphite treatment is
preferably conducted at ambient temperature and terminated after ~-
120 minutes by washing with water, optionally followed by
recovery of the dried product. After the sulphite treatment
enzyme leakage does not usually reappear.
The particulate enzyme products prepared according to
~i the above procedure may be used in batch-type saccharification
process with separation and reuse of ~he enzyme product or in a
continuous type of process in an enzyme reactor.
A preferred commercial amylo~lucosidase product for
the practice of this invention is obtained by cultivating strains -
~belonging to the genera of Aspergillus or Rhizopus, e.g. Asper-
qillus niqer or Rhizopus delemar. Likewise, preferred maltogenic
alpha-amylases may be o~tained from strains of Asperq llus oryzea.
Determination of amylo~lucosidase activitY ;-
The unit activity of soluble amyloglucosidase as well as that of
the immobilized enzyme, defined as the amount of enzyme or
enzyme product which hydrolyses an aqueous solution containing
30 percent (w/v) of m~ltose, at an initial rate of 1 micromole
of maltose per minute, is assayed under standard conditions,
which are pH 4.5 and a temperature of 55,0C. The activity
of the immobilized enzyme is assayed batch-wise on a sample
which is kept suspended by means of a shaking device.
Determination of funqal alpha-am~lase activity
- The unit activity of soluble fungal alpha-amylase as well as
; that of the im~obilized enzyme, defined as the amount of enzyme
or enzyme product which hydrolyses an aqueous solution containing
soluble starch (Merck Amylum solubile, DAB, Erg. B VI), (6,95 g
per 1000 ml) at an initial rate of 5.26 mg of starch per hour,
is assayed under standard conditions, which are pH 5.6-5.7, a
-- 10 --
t~mperature of 37~0c + o.05c and in the presence of 4 . 3 mmolar
Ca+~ion. The formation of a blue colour with iodine is used to
follow the reaction. During breakd~wn of the starch this colour
becomes weaker and changes gradually to red-brown~ The colour
change is checked visually by comparison with coloured glass
standards. The activity of the immobilized enzyme is assayed
under conditions analogous to those indicated for amyloglucosidase.
Soluble saccharifying enzymes are commercially avail-
able. Examplary of such products are AMYLOG~UCOSIDASE NOVO 150*
(an aqueous solution) and FU~GAMYL 1600* (a powder). Aqueous
solution enzyme sources are usually converted into a spray-dried
powder prior to being used for the purpose of this invention.
Optionally, the drying process may be preceded by ultrafiltration,
usually combined with a washing process to remove low-molecular
contaminants. By using such concentration and purification pro-
cesses, all well known to the worker skilled in the art, a solid
amyloglucosidase product with an activity of 5000 units per
gram or higher may easily be obtained.
FU~GAMYL 1600* has an activity of 1600 units per gram.
~0 The commercial product as such may serve as a satisfactory start-
ing material. If desired, purification and concentration maybe
used to increase the enzyme unit activity.
The overall recovery of activity in the insolubilized
saccharifying enzyme product will vary with the detailed para- - '
meters of the immobilization process, but is usually not less
than 40 percent, and frequently higher
The immobilized enzyme products of this invention may
~` be employed on any oligosaccharide undergoing saccharification
in the presence of the corresponding soluble enzyme. Exemplary
of preferred substrates are dextrins obtained by acid and/or
enzyme liquefaction of starch and having DE-values in the range
of from 5 to 40, high maltose syrups (DE-values from 35 to 60),
*trademark
'1- - 11 -
. ~. . ,... - :.. . - - :
~7;~Z~
and residual oligosaccharides occurring in mother liquors from
dextrose crystallisation or in raffinates obtained from
fructose-glucose fractionation. The immobilized saccharifying
enzyme~products are particularly applicable to saccharification
: of dextrin solution having a dry solids content in the range
of from 20 to 55 percent (w/v).
The following examples further illustra~e the present
~`. invention.
.
i: :
`~ ' '"
'.
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( ~ `
._- . . . ..
~72~
` EX~IPI.F. 1.
'A relatively coarse ~rade granular hydrochloric-acid-precipitated
casein (9 g of "caseine alimentair~' (Scerma S.A., ~rance), consis~
ting of 100-500 micron particles, the 300-500 micron fraction being
about 60 percent) was mi~ed with commercial spray-dried egg albumen
(1.2 g). To this mixture ~as added a pre-mi~ed solution o~ amyloglu-
cosidase pot~der (6.5 ml of 18.5 percent (w/v) aqueous solution, con-
taining a total of 12.500 amyloglucosidase units) and glutaraldehyde
(1.2 ml of 50 percent (w/~) aqueous solution). The amyloglucosidase
po~;dex was an ultrafiltrated, spray-dried product prepared from
A~IYLOGL~COSIDASE ~'OVO (NOVO INDus~rRI A/S, Denmark). The mixture was .
kneaded carefully in a mortar and then allowed to stand at room tem- '
perature for one hour. The resulting aggregate was disintegrated by
mortaring to form a granular product which was allowed to dry at
room temperature for one day.
.` ..
The dried granular product (11.4 g) had an activity of 425 units
per g, thus ~epresenting a recovery after immobilization of 38.8.
percent.
~ E ~ lPLE 2.
Granular acid-precipitated casein (1500 g of type M60 (Scerma S.A.),' `~
consisting of loo 500 micron particles, the 150-350 micron fraction ~'
being about 70 percent~ was mixed in the dry state with commercial
spr'ay-dried albumen (120 ~) in a 20 litre plough-type horizontal
mixer (Gebr. Lodige ~lachinenbau, G.m.b.H., Wes~ Germany?. ~
An ultrafiltrated concentrate of amyioglucosidase, prepared from ~; '
AMYLOGLUCOSIDASE NOVO (650 g of a solution havin~ a dry matter con~
tent of about 25 percent and containing ca. 3200 units of soluble
am~loglucosidase per g of concentrate) was mixed thoroughly with
glutaraldehyde (180 ml of a 50 percent (W/~7) aqueous solution) at '
pH 4.9 and a temperature of 18C, whereafter the resulting solution
was poured into the mixing apparatus. Vigorous mixing was continued for ~'
an additional o.5 to'l minute, followed by removal of the wetted '
particulate product from the mi~er. The moist produc~ was left
"~,
~ - 13 ~ 2~
. ~ .
quiescent for about 45 minutes ~ comp]ete the cross-linkin~ pro-
cess, whereby lumps of a~gregated particles ~lere ~ormed. Granula-
tion of the product was conducted on an oscillatory gran~lator
~for example of the type supplied by Diaf ~/S, Copenhagen) provided ;-`
with a 1.5 mm dialneter screen.
The granulate was ~Jashed ~ith de-~onized ~ater (lo 1) for lo minu-
tes, recovered by filtration and tlIen subjected to ~luid-bed dr~-
ing. The dried produc~ (1600 g, 450 amyloa1ucosidase units p~r g)
was freed of fines by sieving.
EX~IPLE 3.
.
solution of spray-dried amyloglucosidase powder (105 g, activity:
11.400 units per g, prepared as described in Example 1) and spray-
dried albumen (105 g) was prepared in tap water-(400 ml) and left
overnight in the refrlgerator at b-7C. The pH of the solution was
5.4.
Glutaraldehyde !loo ml of 50 percent (w/w) aqueous solution) was
then added and the resulting solution ~as transferred in the course
of 3 minutes to a viqorously stirred batch of granular hydrochloric-
acid-precipitated casein (725 g of the same qrade as that used
in Exa~ple 1). Mixing was conducted in an industrial mixer of the -
same ~yp~ as that of E~ample 2. Vigorous stirring of the reaction ~ ;
mixture was continued for an additional few minutes, ~lhereafter
i~ was left quiescent until the cross-linking reaction was comple-
ted after about 1 hour. The resulting aggreqated and lumpy product
was granulated on an oscillatory granulator through a 2 mm diameter
screen.
The granulate was washed ~Ith an aqueous solution of sodium ace-
_tate (pH 4.2), followed by vacuum drying at ~5C~
The dried granular product (925 g) had a.~ activit~y of 800 units
per g, representing a recoveFy alter immobilization of 67.8 percent.
~7~
~ P ~
The i~ohilized amyioglucosidase product prepared according to ' ,~
Example 3 (20 g) ~as soaked for one hour at ambient temperature in '~
an aqueous solution of glutaraldehyde (500 ml of 1 ~ercent solu- ,;
tion adjusted to pH 7), followed by rec~very,of the dried product
(~o g),
, ` ` `
~, , ~he activity of this product was 625 units per g, thus represen-
"i ting a recovery of 78 percent from the second immohilization step
and an overall recovery from soluble amyloylucosidase of 53 percent..
.,~, , , . , :
EX~IPI~E_ 5 -
~, An aqueous solution of maltogenic àlpha-amylase ~2 ml of 5 percent
(w/v) solution of FUNG~IYL 1600 (NOVO INDUSTRI A/S, Denmark) in , ,
, . ,~
de-ionized water at pH 6.2) was mixed with an aqueous solution of
glutaraldehyde (o.3 ml of 50 percent (w/w) solution) and then ,i,
quickly added to a dry mixture of granular acid-precipitated casein
(4.o g of type M60) and commercial spray--dried egg albumen (o.S g).
After thorough kneading in a morter in the resultin~ product was ~'
left for 1 hour at room temperature. Lumps were disintegrated by ;,
mortaring wherea~ter the product was,allowed to dry at room tempe- -;
rature for 1 day. '
~ .
EX~lPLE 6.
.~
A 15 mm inner diam. jacketed column, maintained at 55C., was loa- `'
ded with the immobilized amyloglucosidase product (5.7 g) prepared
according to Example 1.
- , . .
down-flow feed consisting of a 30 percen-~ (W/V) commercial acid/
enzyme-hydrolyzed dextrin (CPC Snow Flake Maltodextrin ol915 of
.E. 20) having the following approximate composition: ' ~
percent , ,
glucose (DPl): 4-5
disaccharides (DP2): 8-9
trisaccharides (DP3): 6-7
tetra-and oligosaccharides (DP4+):79-~2 ~'~
- 15 ~
to which was added o.2 percent (w/v) sodium sulphite followed by
adjustment of pi~ to 4.5, was apr~lied to tlIe column at a constant
flow rate of 15 ml per hour.
The effluent was analyzed by high pressure liquid chromatography
(IIPLC) to give the following results:
' _I
: Dsys DPl percent¦DP2 percent DP3 ~ cent DP~+ ~cent Calc~lat,ed D E
1 92.o ~ 5.6 o.7 1.7 95.3
~ _ _ _ ~ _ .
4' 93.2 4.2 o.6 2.o 95.8 ,
92.9' 3.9 o.7 2.5 95.4
_ . ~ .
7 , 93.6 3.2 o.6~ 2.5 95.8
_ __ . i ~
9 93.3 3.1 o.7. '- 2.9 95.5
. _ _ . _
.11 92.6 3.2 o.8 3.3 94.9 , ~:
13 92.6 2.9 o.9 3.6 9~.9 ~ ~
,~
. EX~IPLE 7.
A 25 mm inner ~iam. ~ac~eted column, maintained at 55C., was
loaded with the immobilized amyloglucosidase roduct ~20 g) pre-
pared according to Example 2.
. .
An up-flow substrate feed having the same concentration, composi- ~ :
tion and pH as that used in Example 6 was applied to the column
at a constant flow rate of So ml per hour. The glucose content
(percent DPl) was analyzed by the hexokinase method to ~ive the
following results: .
.
_
. Days percent DPl
~ _ ,
1 B9.o
4 ' 93.~ _
__ . 93.6 ,'
' 93.1
.
1 6 ~ ~ 7~113;~
EXA~PLF 8
: .
A 25 mm inner diam..jacketed column, maintained at 55C, was
loaded with the i~mobilized amyloglucosidase product (25 g)
prepared according to Example 2. ~n up-flow substrate feed of the
same composition as that used in Example 5 was applied to the column.
The feed (pH q.5) had a dry solids content of 30 percent (T,~/w1 and
potassium sorbate ~2 g per litre) was aidded as a preservative.
The flow-rate was adjusted so as to maintain an approximately
constànt degree of conversion.
The effluent was analyzed by means of ~PLC to give the following .
conversion to glucose (DPl~ with time:
~, . ..
Days DPl percen~ Flow rate ml/hour
. __ -- : ~ : .,.
1 - 92.6 115 `~
2 _ 112
_ :
3 92.4 lol ~
. _ _
~ 4 72.0 82 ~
. _ , , .
92.2 75 ~
_
6 92.3 70 1 ~,
7 92.1 - _ 66
8 92.6 6
60.
, .
lo 92.o 57
i.
After completion of the run no changes were observed in the hard-
-ness or other physical properties of the column material.
'~ -
EX~PLE 9.
:. . .
By using the procedure of Example 7, but substituting the immobi-
lized amyloglucosidase product of Example 2 with that prepared ac- I
cording to Example 4, the following results ~ere obtained: ~;
,.
- 17 - , ~ ~ ~2
~.
.` ~ ,
Day~ percent
l 90.3
2 92.9
4 ~3.7
13 93.3 _
. _ . "
EXAMPLE lo.
,
The immobilized maltoqenic aIpha-amylase prepared according to
the procedure of Example 5 1 4 g~ was packed into a ~acketed column
maintained at 45C, and a down-flow feed of commercial dextrin
(CPC Snow Flake ~1altode~trin ol91,3'~ containing 30 percent (w/v)
dry solids and sodium sulphite (o,2 percent) as a preservative
~as applied. The pH of the feed was adjusted to 4.5 and the flow-
rate to 15 ml per hour. The following table shows the composition
o~ the column effluent; DP2 mainly representing the maltose content:,
,
, . _
Days DPl percent¦DP2 percent 1~P3 percent DP4~ percellt ¦ Calculated D.E. -
_ .
l 5.3 ~5.4 21.o 2~3.3 42.5
_ .:
2 5.o ~4.9 - 21.6 28.5 42.1 ~
. . _ . _
3 4.7 44.1 22.o 29.2 41.7
...... _. _
4 4.4 ~3.5 22.7 29.~ 41.2 `
'
EXA*IPLE ll.
A~ '
A 15 mm inner diam. jacketed column, maintained at 55C was loaded
with immobilized a'~yloglucosidase (12 g) prepared according to
the method of Example 2.
' ~
A down-flow feed of dextrin (31 percent (w/v) based on dry mat-
ter content), prepared by liquefaction of starch with TE~1~YL ~
.. . .. . . . .. . . .. . . . .. .. . .
..... - . ~-.
.. ~ . .
.~ , ',
,
- 18 ~ 7~02~
~.
L6Q (NOVO INDUST~I A/S, Denmark) and havinq the followincr compo-
sition:
percent
glucose (DP~
disaccharides (DP2): 7-8
trisacchdxides (DP3): 10-12
tetra- and oligosaccharides (DP4~): 7~-82
D.E. 21
was applied to the column.
The feed contained sodium sulphite (o.2 percent (~/v)) as a preser-
vative and had a pH of 4.5. The flow was regulated to maintain a
constant degree of conversion (in terms of percent DPl). The efflu-
ent was analyzed by HPLC to give the following results:
. .
_ _
Days DPl percent DP2 percent DP3 percent DP4+ percent Flow rate ml/hour
_ _ _ . . .
3 92.1 4.2 o.7 3.1 35 -
. .
7 9~.3 3.6 o.8 3.3 33
l _ :--
14 g2.2 3.6 o.7 3.5 27
. _ . _ , I
28 92.4 3.1 o.9 3.6 18
..
56 92.1 3.3 o.8 3.7 15 ~`
A dextrin substrate prepared according to the method described
under ~A) and having the following composition:
percent
glucose (DPl): 1.1
disaccharides (DP2): 6.8
_ T trisaccharides ~DP3): ll.o
tetra- and oligosaccharides (DP4~3: 81.o
was treated with o.ol percent (based on total dry solids3 of malto-
genic alpha-amylase (FUNG~IYL 1600, NOVO I~DUST~I A/S) at pEI 5.o
and 50C for two hours. After treatment the substrate had the
following compositio :
~.
.. . ..
.. . ... ... ~ ..
~a~7~:~z9
-- 19 - ~
percent
glucose (DPl )~ 1. 3
disaccharides (DP2): 13.9
trisaccharides (DP3): 22.3
tetra- and oligosaccharides (DP4): 62.5
Each of two 15 mm inner diam. jacketed columns, maintained at
55C, were loaded with immobilized amyloglucosidase (lo g, pre-
pared according to Example 2). Down flow fe~ds consistin~ of 30
percent lw/v) of the two types of dextrin, prepar~d as described
above, to which was added o.2 percent (w/v) of potassium sorbate,
followed by adjustment o~ pl-l to 4.5, were applied to the columns.
The followin~ results were obtained:
' ; . :
Peed ; Dex1 rin
before FUNGAMYL treatment after FUNG~IYL treatment
_ i~ ~'
Maximum DX-
value of 92.7 93.3
effluent,
percent
, , _
.
EX~IPEI. 12.
.
Portions (lo g) of immobilized amyloglucosidase, prepared according
to the method of Example 2, were soaked in loo ml solutions con-
taining different concentrations of sodium sulphite. The pH of the
solution was adjusted to 4.5 by the addition of acetic acid. ~fter
soaking for 2 hours at ambient temperature the enzyme samples were
washed with de-ionized water and packed into 15 mm inner diam.
jaketed columns maintained at 55C.
Down-flow feeds consisting of dextrin (30 percent (~/v)), prepared
according to the method of Example 11 A to which was added potas-
sium sorbate (o.2 percent (w/v)) followed by p~ adjustment to 4.5 `;
were applied to the columns.
By adjusting the flow rate of each column to give the maximum D~-
value of the effluent the follo~/ing rcsults we~e obtained:
.
:. .
~ 2~z9
- 20 - ' :
. _ _ _ ,' .:
Sodiunl sulphite -o o.ol o.l o.2 o.5 l.o .
g/loo ml . :
~Iaximum DX 90.3 90.2 91.2 ~1.7 91.9 92.4 : `-
: __ . _ ,i~ ~ ,
,
.'~
E~AMPLE 13. ;
The collected high maltose (D.E. about 42) effluent from the
immobilized FUNG~IYI, column of Example lo ~as used as a feed for
the immobilized amyloglucosi.dase column of Example 6. The flow ~:
rate was adjusted (to 73 ml per hour) to yield an effluent having
the following composition of a high conversion (D.E. about 65)
syrup: ~ ~
percent : :.
glucose (DPl~: 38.1
disaccharides (DP2): 39.6 :
trisaccharides (DP3): . 2.8
tetra- and oligosaccharides (DP4~): 19.6 . : ;:
'' `. '',
EXAMPLE 14. . ::
A 25 mm inner diam. jacketed column, maintained at 55C, was
loaded with immobilized amyloglucosidase product (20 g) prepared
according to Example 3. ~
An up-flow substrate feed consisting of the mother liquor (or) ::
raffinate) obtained from fructose-glucose fractionation and having . :
the following composition~
- - percent
fructose 3.25 .:~ :
- - glucose . 85.71 ~.
disaccharides 9.17
. trisaccharides 1.42
higher saccharides o.54
'
: and p~l adjusted to 4.5 was applied to the column at a flow rate
of 250 ml per hour at a concentration of 25 percent (~I/w). The
.
` ~7~2~
- 21 - ~
~.
composition of the effluent, de~ermined hy ll~LC ~as shown to be:
percent
fructose 3.25
glucose 91.16
disaccharides 4.14
trisaccharides 1.15
higher saccharides o.30
'~;
'~
- ':
'~
