Note: Descriptions are shown in the official language in which they were submitted.
~1 ~)572~
~ he invention of this application relates to the con-
version of starch to levulose, and in particular, to such con-
version which is effected wholly by enzymes. More ~pecially
the invention provides such a process which provides an improved
yield of levulose.
Starch is a polymeric carbohydrate material of very
high molecular weight. Its monomeric units are termed anhydro-
glucose units, and the complete hydrolysis of starch yields
dextrose, Dextrose in turn is susceptible of isomerization
to levulose, either by alkaline or enzyme catalysis. The
latter is of increasing importance at the present time because
of recent improvements in the conversion of dextrose to
levulo9e by means of enzyme catalysi~.
Of all the "sugar" consumed throughout the world,
sucrose is by far the most commonly u~ed, It is what is
i commonly known as table ~ugar. It is a remarkably stable
product and has very good sweetenins~ properties. It is not
.
entirely without shortcomings, however, because at high
concentrations it does tend to crystallize and thu~
~a~versely affects the texture and appearance of food~ in
!
.. . ....
'' ~ ~ , ..
,'
:,: . : .
~2 ~
. ~ .
: A~ 1
.: , - .
... . . ., . ;............ . -
which it is containé~. ~urthermore, it~ sweetne~6 is said by some
to lack depth and fullnews. Dextrose i8 an alternative, but dextrose
lack~ the high degree of sweetness which characterizes uucrose.
Dextrose i~ generally rated as being about 60 to 80% as sweet as
sucrose and the price a~ which dextrose is sold 1~ correspondingly
lower ~han that of sucrose. Like sucrose, dextro~e tends to crystallize
easily.
Levulose, on ~he other hand, iB even sweeter than sucrose, and
it doeA not have the undesirable tendency to crystalli~e readily.
Unfortunately, levulose does not occur naturally in
large quantities andit5 preparation ha~ heretofore been difficult. Its
~ preparatlon from sucrose by hydrolysis with hydrochloric acid or with the ~'-
'1 enzyme invertase has long been known and this hydrolysis produces
so-called inver~ sugar, half of which i~l levulose and the oth~r
half of which i8 dextrose.
.. i .
1 The overall conversion of starch to levulose ordinarily
~ 1 , .
involves three principal, separate step3: a thinning of the starch,
followed by saccharification, followed in turn by isomerization. -~
In the f~rs~ step, an aqueous slurry of starch ie heated to
20~ gelatinize the starrh, and simultaneously, treated with an alpha-
amylase or acid,~to convert it to an intermedia~e hydrolysi~ product
i~ ~havin8 a considerably reduced viscosi~y with re~pect to that of
the original pa~ted aqùeous starch mixture. Then, in the second
step, this intermediate hydrolysis product is saccharified, i.e. 9
~ 25 converted to dextrose by treatment with a ~accharifying enzyme, i.e.,
`, ~ a glucoamylase. In the third step, thi~ dextrose produc~ i8
;'~ ' ' ". '
' o ' .
" , '
... , .
_ ~ _
. .
~L~357Z~3
treated with an isomerase wi~h the re~ul~ing for~ation of a
product containing about half de~trose and half levulose, or ~lth
a base such as sodium hydro~ide to produca ~ product contalnlng
a maximum of about 30~ levulose.
~ach of the above steps are carried out under diferent
condition~ of pH and temperature, 30 as to optlmi~e the efficlency
of each s~ep. Thus, it is necessary to make significant ad~ustments
in these conditions at the conclu~ion of each step, with the
re~ults that the overall efficiency of the procesB iB ConBiderably
; 10 dimlnished.
It is accordingly a prl~cipal ob~ect of the present inventlon
to prov~de an improved proce~s for the conver3ion of ~tarch ~o
levulo~e.
It is another obJect of the present invention to provide such
a process which results in high yields of levulose.
It is ano~her ob~ect of ~he present lnvention to provide
such a proce~s which is charac~eriæed al60 by relatively low
~: :
, temperatures.
- j , "
It is yet another obJect of the pre~ent lnvention to provite
such a process which can be carried out conveniently and economically ~; -
; in one step.
~ 3
~; '` ' ~' '''
.' .
': l, ' :
~5~ 8
These and other objects are accomplished by the
process of converting starch to levulose comprising forming
an aqueous slurry of granular starch, bacterial alpha-amylase,
glucoamylase, and glucose isomerase derived from Streptomyces
albus, at a temperature of from about 40C to about 70C, and
at a pH of from about 5.0 to about 7.0; and maintaining , ---
the starch in essentially granular form until a soluble
hydrolysate is produced, wherein any residual insoluble
starch is in es~entially granular, ungelatinized form.
. ~ .. .
It is appropriate to maintain the temperature -
below the gelatinization temperature of the starch. -
The process of the invention accomplishes the
above objectives both because of the combined synergistic
action of the bacterial alpha-amylase, glucoamylase and ~;
glucose isomerase which results in efficiènt production of
,~ levulose at a ~ingle temperature ancl pH; and by virtue of
`1 the glucose isomerase derived from ';treptomyces albus which
i :
~ result~ in a marked increase in the yield of levulose.
', ~ ' , ',` ' . .'
~ I j .
, : '
' / ,~``: ,. .
.. , . .
. ' . .
` ~:
`
`: ,' ~':
'" ` ~ ''
'', ' , .,
,
' ~ , ", '
'. ...
",, ~'' ' ' '' "' ' ' ',, ', ', "''' ' ' '' ,,," ' " "' ' "''' ,' .," " ' '' ; ~ ",' '"', ' ' " ''',: ,- . ' ~
2~
The starch may be any of those commonly available,
including corn starch, waxy maize, tapioca, potato starch,
white sweet potato starch, wheat starch, sago, sorghum and the
like. Wi~xy and the non-waxy starches are suitable. As
indicated, the starch is granular. Corn grits and other
raw materials high in starch content may be used satisfactorily.
Corn starch is a preferred raw material because of its ready
availability.
An important advantage of the process is that it
10 may be carried out in an aqueous slurry at relatively high `
concentrations. The solids content of the starch slurry
generally is within the range of from about l~/o to about 7C%
ordinarily, the solids content will be 20-5C%. Lesser
concentrations can of course be used, and in general
as the concentration i9 decreased, so is the extent
,
'`~ ' '
`' :
~ .
:-'~ : ' ` :
: i ,, .
'`,` .'' :''.':
:; - .': :
.
J'i'~
,. _ 5 _ ~
~S~Z~L~ .`;
of 6tarch ffolubilization, and thus the ~ield of levulo~e is
increased. As a prac~ical mat~er, however9 it i9 highly desirable
in mo~t instances ~o use ~mall volusefl, i.e., high ooncentrations
of starch. Thie avoids or a~ least dimlnishes the co~slderable
expen~e of concentrating the conver~ion mi~ure prior to ultimate
separation of lew lose. In some cases, however, ~he advantage of
~ a hlgher yield may be sufficient ~o outwe~gh this disadvantage,
- and a concentratio~ of about 10~ solids would be preferre.d.
The process herein.p~rmit~ the solubillzatlon of 90%
or more of ~he starch in a 30-40% aqueou~ ~lurry. Furthermore,
the undls301ved s~arch can be recycled eo as to lmprove the
overall efficiency; i.e., to solubilize the prevlou31y undlssolved
~tarch and thsreafter to convert it to levulose. An incldent&l
15 advantage of such recycling step 18 the fact that a significant c~
proportion of en~yme activity 1~ thu~ al~o recovered. The
~olublllzed starch thu~ obtained has a dextrose equivalent (D.E.
~, of 90-95. The term "D.E." 18 used to indicate the reducing
sugar content of the i~omerized hydroly~ate, calculated as dextrose,
l ~ ,
and expressed as;percent by welght of the dry substance present.
; The bacteriai alpha-amyla~e preferably is one which i8
: .
active at a relatively low pH, i.e., within the range of fro~
about 5.0 to about 7.0, a~d al~o at relatively low temperature~,
; l.e., bel~w the temperature at which a par~lcular ~tarch gelatinizes,
Pre~erred source-~of ~uch alpha-~myla~es include certaln ~pecie~
.'1'.~ , ~ . . .
''1~ ~ ' - , ' , :'
.1 . .
: .
` ~ - 6 - ~:
:: ~ ' ' , , :.
`, ' ': ':, ., '
~S7Z~
of the Bacillus microorganism, viz., B. subtillis, B. licheni-
formis, B. coaqulans and B. amyloliquefaciens. Suitable alpha-
amylases are described in West German Offenlegungsschrift
2,025,748 and in U.S. 3,697,378. Especially suitable amylases
are those derived from B. licheniformis as described in the
above German Offenlegungsschrift. Particularly preferred is
that alpha-amylase derived from B. licheniformis strain
~CIB 8061 other specific microorganism includi~ B. licheni-
formis strains NCIB 8059, ATCC 6598, ATCC 6634, ATCC 8480,
.
ATCC 9945A and ATCC 11945. One such alpha-amylase preparation ;~
is identified by the trademark "THERMAMYL", available from
~ovo Terapeutisk Laboratorium, Copenhagen, Denmark. THERMAMYL ~^
is characterized by the following properties:
(a) it i5 thermally stable,
(b) it has a broad range of pH activity, and
(c) its activity and heat stability are independent
of the presence of added calcium ion.
Analysis of a suitable preparation is as follows:
Dry Substance, % 94.6
.
~lpha-amylase activity, U/g (as is) 9,124
Protein, % d.b. - 21 2
Ash, % d~b. 64.4
Calcium, % d~b. 4.9
''''
':'
.~ :.. .-'.'
' ,'' '' .
~'.
: ' ,.
,
~A'; - -
~57''~
Other suitable alpha-amylases include THERM~MYL 60*
(a liquid) and THERMAMYL 120* (a solid) having the following
analyses:
THERM~MYL THERMAMYL
60* 120*
Dry Substance, % 35.4 98.8
Alpha-amylase activity, U/g (as is)1,156 2,105
Protein, % d.b, 26.5 21.2
Ash, % d.b. 60.1 91.2
Calcium, % d.b. 0.04 0.72 -
Sodium, % d.b. 12.3 12.2
Still other suitable alpha-amylases which are avail-
able include the following:
TABLE I
Enæyme
Preparation ComE~any FormActivity -
Rhozyme H-39* Rohm & HaasPowder4,874 ~/g
Takamine HT-1000* MilesPowder 3,760 ~/g
~enase* Miles Liquid2,043 ~/ml
Dex-Lo MM* WallersteinLiquid1,213 ~/ml
ovo SP-96* Novo Powder, 7,310 ~/g
'~ ~ovo B. substillis* ~ovo Liquid1,599 ~/ml
Klelstase GM-16* Daiwa KasaiPowder 26,593 ~/g
Kleistase L-l* Daiwa KasaiLiquid1,918 ~/ml
Rapidase SP-250* SocietePowder 11,~S5 ~g
,~ "Rapidase"
France
~,'i , , :.
j *tradem~rk
, ,;. :. : .. ,
. .:
' ,i` . .,,. ~'
'`, " , ' ~'.':
~ 8 - i ~
7;2~
The ii-~iOUllt of bac~rial alpha~ iJ~ e Lo i~P u~ed
ranges from about 1.0 to about 25 unies per gr~ , of starch tdry
basis). The use of larger amount# provlde~i no prac~ical
advantage; the increased ~itarch solubilization ~hich res~ult,3
5 fro~ the use of more than 25 units peE gram does not Justify ~-
I the additional cor~it of en2~me.
.
The alpha-a~ lase activltlJ of an enzyme i8 detenmined
as follows:
The en~y~ie i3 allowed to react with a standard starch
solution under controlled conditions. En~yme acti~ity i~
determined by the exten~ of s~arch hydrolyais, a,3 reflected by a
decrease ln iodine-stainlng capaci~y, ~hich ii~i measured i3pectro-
phoitome~rlcally. The unit of bacterial alpha-amylase activity
', i8 the amount o enzyme required to hydrolyze 10 mg. of starch per
minute under the condi~oni3 of ~he procedure. The method is
applicable to bacterial alpha-amyla~e,3, includlng lndu~itrial
preparatlons, except materlal~ which posse3i~ ~3igniflcant
~accbarlfying ac~i~ity.
From 0.3 to 0.5 gram~ of solid sample or from 0.3 to 1.0
: ~: : , : .-
- 20 ml. of a llquid sample i,~, dicisolved in a ~iufflcent quanti~y of
~1 0.0025~M aqueous calcium chloride to give ~n en~iyme ~301utlon
co~taining approximately 0.25 unit of actlvity per ml.
A mlx~ure of 10 ml. of lX Llntner starch solution,
equilibrated ~o 60C, and 1 ml. of the eni~yme sample to be ;
! ~ : 25~ te9ted i3 mixed and held m a 60~ c0~3ti~t temperature bath
'l ' '
. ~
g~
:~, : . .
. : ' '
.,, . ~ ' - .
. . - - -: . . . . . .: .
, , : . , ,:, ; ~, . ,l.j . .
~)57~
for exactly lO minutes. A l-ml. sample is remo~ed and added
~o a mixture of l ml. of 1 M aqueous hydrochloric acid and about
50 ml. of distilled water. The lodine-staining capacity of such
acidified sample then is determined by adding 3.0 ml. of 0.05%
aqueous iodine solution, diluting to 100 ~l. wi~h dlstilled water~
and mixing well. The ab~orbance of the solution, relative to that
of distilled water, i9 measured at 620 nm, in a 2-cm. cell.
similar measurement is made of the standard starch ~olution (to
which water i9 added instead of the en~yme solution) to pro~ide
a blank absorbance.
The en~yme activity, in units/gram or /ml. i8 equal
to
(Blank Absorbance - Sample Ab90rbance) x Dilution Factor x 50
Blank Absorbance x 10 x 10
The glucoamylase may be any of the well-known amylase
preparations, particularly those derived from members of the
Asper~illus genus, the Endomyces genus, and the Rhizopus genus.
.. . .
A particularly preferred glucoamylase is that available fro~ the
proces3 described ln U. S. 3,042,584 (~oi et al) whereby a fungal
amylase preparation is freed of unde~ired transglucosidase activity
by treatment in an aquPous medium with a clay mineral. The am~unt
of glucoamylase to be used ranges from about 0.1 unit to about 5.0
,. . . . .
;, ~ units per gram of starch (dry basis). Preferably, on an enzyme
cost/performance ba~is, about 0.25 unit of glucoamylase per gram
f starch (dr~ baal~ i8 used.
''' ~:
-- 10 --
' - ~
~57'~
Glucoamylase activity units are determined as follows:
The substrate is a 15-18 D.E acid hydrolysate of
corn starch dissolved in water and diluted to 4.0 grams of dry
substance per 100 ml. of solution. Exactly 50 mlO of the
solution is pipetted into a 100 ml. volumetric flask. To the
flask is added 5.0 ml. of 1.0 molar sodium acetate-acetic
acid buffer (pH: 4~3). The flask is placed in a water bath
at 60C and a~tar 10 minutes the proper amount of the enzyme
preparation is added. At exactly 120 minutes after addition
of the enzyme preparation the ~olution is adjusted to a phenol- ~ -
phthalein end-point with one normal ~odium hydroxide~ The
solution is then cooled to room temperature, and diluted to
volume. A reducing sugar value, calculated as dextrose, is -
; determined on the diluted sample and on a control with no
enzyme preparation added. Glucoamylase activity is
calculated as follows:
A = 2 X B
,
~ where
; A = glucoamyla~e ac~ivity unit~ per ml. tor per gram) of enzyme
preparation.
- .
S = reducing sugar~ in enzyme converted sample, grams per 100 ml.
B = reducing sugars in control, grams per 100 ml.
~ . . . .
E~= amounts of enzyme preparation used, ml. (or grams).
"S" should not exceed 1.0 grams per 100 ml.
The glucose isomerase is one derived from
Streptomyces albus capable of converting dextrose (gluco~e) to
levulose (fructose3~ Subsequent references to "isomerase"
are to be understood as a reference to glucose isom~rase~
:
' ' ~-
,:
-- 11 --
d
~57;~
An especially preferred species of the micro-
organism is S. albus YT-~o 5 ~ATCC No. 21,132 ).
Glucose isomerase derived from other microbial
sources are also useful in converting starch to levulose as
described in Canadian Patent Application S.~. 220,947, Ronald E.
Hebeda et al, filed February 26, 1975; however, in accordance
with the present invention the glucose isomerase derived from
Streptomyce-~ albus results in a significantly i~proved levulose
yield~ Other microorganisms from which the glucose isomerase
10 may be derived and which are outside the scope of this invention
incluae S. bobiliae, S. fradiae, SO roseochromoqenes, S. -
olivacens, S. californlcus, S. vinacens, S. virqiniae, S. olivo- ;
chromoaenes, and S. phaeochromoaenes: glucose isomerases ;
elaborated by microorganism of the ~rthrobacter genus, for
;~ example, A. nov. sp. ~RRL B-3724, A. nov. sp. ~;[RRL B-3725, A.
nov. p. ~RRL B-3726, A. nov. sp. ~IRRL B-3727 and A. nov. sp.
~RL B-3728; and gluco~e isomerases lelaborated by microorganism ~;
of the Lactobacillus genus, e.g., k~ brevi3, k~ mannitopens and
, . . .
L. buchneri; and also Aerobacter cloacae and A. aeroqenesO
The amount of glucose isomerase of the invention to
be u~ed ranges from about 0.1 unit to about 20 units per gram ;~
of ~tarch (dry basis), In the usual, preferred instance, an ~-
amount within the range of from about 0.2 to about 2.() will be
used, ~
~ , ,
Isomerase activ1ty units are detarmined as follows:
he procedure involves making a spectrophotometric
;~ determ~nation of the keto~e produced from a glucose solution
under a standardized set oE condition~
,~: : . \
The enzyme preparation to be assayed i9 first
30 diluted to contain from 1 to 6 isomerase units per ml.
;'~ ' '
~:'' ' .
` A` 1~
~57Z18
A RtoCk ~olu~ion i8 prep~red ~ ~o310~
Co~ponent Amou~t
; 0-1 M MgS04 7H20 1 ml,
0.01 M CoC12 6H20 1 m~.
5 1 M Phosphate Buffer, pH 7.5 0.5 ml.
Anhydrous D-glucose 1.44 g.
Distilled Water To n~ake up a total volume
o~ 7-5 ~1.
'
An enzymatic isomerization is conducted by adding 1 ml.
of the enzyme preparation to 3 ml. of the stock solu~ion, then
incubating it for 30 minutes at 60C. At the end of this
incubation period, a 1 ml. aliquot i8 take~l and quenched in
9 ml. of O.S N perchloric acid. The quenched aliquo~ then is
diluted to a total volume of 250 ml. As ~I control9 for
comparatlve purposes, the procedure i8 repeated substituting 1 ml.
of water for the 1 ~11. of the enzyme preparatlon in solution
form, at the beginning of the incubati~n period. ~ -
The ketose then is determinad by a cy6te~sulfuric -
acid method. See Dische et al, J. Biol. Chem. 192, pg. 583 (1951). `-
20 ~ For the purposes of this assay, one isomerase unit is deflned
~ a6 the amount of enzyme activity requir d to produce one mlcromole
.. : :
~ o levulo e per minute under the iso~erization conditions described.
' ' . ' ::'
` 13 -
~ .
.~' ~ - , . .
,
7~
The te~perature of the reaction mixture of the
proces~ herein should as indicated be from about 40C to about
70C. Ordinarily, the temperature will be at the upper end of
~his range, consistent with the requirement that it be below
the tempera~ure at which the starch i9 gelatinized. A particular
advantage of the process i9 the fact that high temperatures are
avoided. This permits a considerable sa~ings in the cost of
supplying heat to the process and minlmizes the formation of
color bodies with a subsequent saving~ in refining costs.
,
The selection of ~H depends upon the par~icular
enzymes used in the process. Ideally, the thinning, saccharlfylng
and i~omerase enæymes would exhibit ~heir optimum activitiea at
: : :.
about the same pH, but a8 a practical matter thls is unlikely.
Glucoamylase i8 of course the saccharifying enzyme and its optimum
15 activity is in the range of ~.5 - 5.0 pH. Alpha-amylase's optimum ~ -
i activity is at a pH within the range of 5.5 - 7 and i8 not
sufficiently active at a pH below 5 to promote the deslred ~tarch
solubilization. The isomerases generally are mo~t active at 8till
. ! :
higher pH's, e.g., in the order of 7.0 - 9Ø It is thus unexpected
to find that all three of these enzymes will act cooperatlvely at one
p~, as in fact they do. A su~table pH for the purposes of the
, .
inventlon herein i8 one falling within the range of from a~out 5.0
`'~:
1 to about 7Ø
. I .
The hydroIysis mixture should contain magnesium and
: ` ` :
j' ~ cobal~ ions. These may be supplied in the Porm of magnesium
sulfate hexahydrate (MgS04 6H20~ and cobalt chloride heptahydrate
(CoC12~7H20).~ The amounts of the~e ~alt~ or of other water aoluble
,~:
:.
" .
, ' '
. . .
~ - 14 -
::
. ., ~ .
`,
.::
. .
;, , ; . , ~ . , : . .
~(~5i72~
magne~lum and cobal~ sali~ ~hclnLJI b~ ~UC~I a~ pLo~l;le ~ro~
abou~ 0.005 ~o abou~ O.:L0 mol~c~ per li~er o~ iLIlgnes:L~m and frcm
abou~ 0.0001 to abou~ 0.005 mole~ peL' l~ er or cobalt Lond.
These ions in these concen~ra~ions enhan2e the ac~ivit~ oE the
isomerase and appear not to have an ad~erse af~ect on Ihe
acti~ity of the othe-r en~yme4.
Al~hough the calcL~Im :Lon i~ known to h~e a beneficlal af~ecc
on ~he activity of alpha-amylasas, it ls unneces~ary to add lt to
the conversion mixtures of this inven~ion and, in cPrtaln preferred
ln3tances, it is advisable tlOt to add any because it appear3 to have
an adverse effec~ on the ac~lvi~y of ~he isornerase and, correspondingly,
on the ultimate yield of levulose.
As shown in E~camp:le 1, 73Z o~ ~he ~tarch ls ~ol.ubillzed
in 18 hours, with a yield of 29.3% (of the solubillzed starch) of
levulose. At 42 hour~, the corre~ponding figures are 81X
solublll~ed starch and 36.3% levulose; and at 67 hours, the
correspondlng figures are 91X and 3~.9X. In Example 2, over 98%
of the gtarch is solubili~ed at 48 hours and 40.7% of
solubilized starch has been converted to levulose.
` 20 The inventlon i8 illustrated in some detail by the
followlng e~amples which, hGwever, are no~ ~o be taken as llmiting
in any respect.
. . . .'.':
~ EXAMPLE 1
::
To a 32.7X (18.4 Baumé) aqueou3 ~u3pen~ion of granular
~5 corn atarch there is added 0401 mole o magnesium iOTI (as ~agne~lurn
' ":
. ~ 15- '
: ` .
::
.:
.. , ,, . . ~ ,.. . . . . . . . : ....... .. .
,, , ... . . : :.. , .; ,:,:, ,. ,: . . ... - . :: . :. :~
1~572~ ~
sulEate he~ahydrate) and 0.001 mole of cobalt ion ~a~ cobal~ou~
chlorlde heptahydraLe) and the pH adJusted at 5.-7. Sodlum
carbonate (a one s~ormal aqueous solutlon) is added as nece~sary to
; maintain the pH at this level. The temperature is maintained at
60C and 0.075% (6.8 activity units per gram of starch), 0.1%
(0.2 activity unit per gram of starch) of glucoamylase and 0.8%
(0.33 activity unit~ per gram of 8 tarch) of isomerase (Streptomyces
albus YT-5) are added. The following results were obtained: -
Time (Hours) i 18 42 67
D. S. in Filtrate 24Z ?6.4% 29.8%
D. E. of Filtrate 93.6~94.4% 94.1%
Levulo~e in Filtrate 29.3%* 36.5~* 3~.9%*
Dextrose in Flltrate 62%* 57.4X* 53.8%*
*based on solids
The soluble fraction ~g of co~r~e the filtrate obtained
upon filtration of the conversion mixture. The filtration proceeds
easily because there i8 no gelatinized 3tarch in the sonversion
mixture. The amount of granular starch obtained after the
20~ completion o~ the reaction as above, amounted ~o 4% of the whole.
EXAMPLE 2
To a 40.9% (23 Baumé) aqueous suspension of granular
corn starch there i~ added cobalt ion and magnesium ion a~ ~n Example
., .
~, 1. The pH 18 ad~usted at 5.7 and maintained at thi~ level throughou~
-
- 16 ~
' ' ; ' '
~q)572~
the conversion by the addition of a one nonnal Rodium carbonate
solution as needed. The tempera~ure i8 maintained at 60C and
0.15% (13.7 activity unies per gram of starch) of THEgMAMYL alpha-
amylase9 0.1~ (0~2 activity unit per gram of s~arch) of glucoamylase
and 0.8% (0.33 activity unlt per gram of s~arch) of i~omerase
(Streptomyces albus YT-5) are addcd. The slurry is kept at 60C for - -
48 hours, then filtered. The filtrate iB characterized by the
following analytical data:
.
D.S. 40.2%
D.E. 91.5%
Levulose 40.7%* ;
Dextrose 512*
Ash (% Sulfate d.e.) O.35~
lS Starch Test ~egative
I *based on solids
EXAMPLE 3
A 25% by weigh~ aqueous slurry of gra~ular corn s~arch
i~ prepared containing the following lngredients~
125 g. of corn starch ~:
... .
'~ ~ 250 ml. of 1.0 N aqueous potassium phosphate buffer, pH: 7.5
1 5 ml. of l.0 N magnesium suifate hexahydrate ~ ::-:
5 mlO of 0.1 N cobalt chloride heptahydra~e ~:. :
~` Sufficien~ aqueous calciu~ chloride to provide lO0 ppm of
~: 25 c~lcium lon
. .
.. :
,
~ ' ' ' .
- 17 ~
, '.' ~:'
''''
'l ,: ,
lV57~
Alpha-amylase (Bacillus lich iformis, 5 activity
units/g of starch (dry basis))
Glucoamylase (1.0 activity units/g of starch (dry
Isomerase (Streptomyces olivochromoqenes, 10
activity units/g of starch (dry basis))
The above aqueous slurry is maintained at 60C for -
24 hours, the pH being adjusted to 6.0 as necessary by additions
of aqueous potassium hydroxide. The conversion mixture is
filtered and the filtrate ad~usted to a pH of 4.5 by the
addition of hydrochloric acid, then boiled to inactivate the
enzymes. The ~Colids material comprises 44.9% of the original
granular starch, which means that 55.1% of the granular starch
i9 solubilized. Thi~ solubilized starch product is found to
have a dextrose content of 51.2% and a ketose (levulose)
content of 20.6%, both based on solids content.
EXAMPLE 4
The procedure of Example 3 is repeated except that
~'~ the ~pH is maintained at 6.5 throughout. The proportion of
starch ~olubilized is 48.~/o and the dextrose content of that
solubilized portion is 36O1, the ketose ~levulose) content is
23~ o~ `
All parts and percentages herein unless otherwise
expressly stated are by weight.
By reference to the foregoing examples it will be
:, l
~ een ~hat the use o~ a glucose isomerase derived from
, ~ ~ ! ; . .` .
treptomYces aIbus~as in Examples 1 and 2 results in a significant
increase in the yield of levuIose as compared with the use of
glucose isomerase in other origin as in Examples 3 and 4.
~: 30 The following eDzyme numbers may be of as~istance in
:: reading the disclosuré and claims~ - -
1 ~ .
alpha-amylase E.C. 3.2.1,1, ~ -
~; glucoseamylase E.C~ 3.2,1.3.
- glucose isomerase E.C. 5.3.1.18,
:
~ 18 _
;7~
It must be recognized, however, that enzyme
classifications are based on the primary function of the en~yme;
on the other hand an enzyme may have more than one function
For example, xylose isomerase (E.C. 5.3.1. 5) is commonly
employed as a glucose isomerase in the isomerization of glucose
to levulose, although the primary function for which it is
named is its a~ility to promote the isomerization of D-xylose
to D-xylylose.
In the present invention an enzyme derived from
Streptomyces albus and having the ability to isomerize glucose
to levulose is employed and so is designated glucose isomerase.
While the invention has been described in connection
with specific embodiments thereof, it will be understood that
it is capable of further modification, and this application
iCi intended to cover any variations, uses or adaptations of
the invention following, in general, the principles of the
invention and including such departures from the present ,;~
disclosure as come within known or customary practice in the
art to which the invention pertains and as may be applied to
the e~sential features hereinbefore set forth, and as fall
;..: :.: .,
~ within the scope of the invention.
., , . ~ .
.:~- :.
.: . -' :
,.: , ::.
! ~
.. ' :',
: ~, ~ ~ ' \ ' ' . -'
, :.
: i -
,
'.: ' ~
':'.
. ~ ,. . .
~. j .
,
