Note: Descriptions are shown in the official language in which they were submitted.
ACKGROUND OF THE INVENTION
1. Field of the Invention: The present invention
is concerned with a PrOCeSS for producing maltopentaose
frQm amylose, and in particular, to a process involving
p:roduction of soluble amylose and the reaction of amylose
w:ith the enzyme amylase.
2. Discussion of the Prior Art: It has recently
been discovered that maltopentaose is one of a limited
number o~ polysaccharides which can be used as a sub-
strate in a preerred diagnostic procedure to measureserum amylase. See, for example, U.S. Patent 3,879,263
which issued to Thomas Adams on April 22, 1975.
~ lthough maltopentaose is one of the hydrolysis
products of starch reacted with various amylases, the
other products of this r~action (:i.e., glucose and the
- other polysaccharides formed by the linked glucose
molecules) predominate. Since on:ly a very small percen-
tage of the reaction product is maltopentaose, the
hydrolysis of starch by amylase does not lend itself
well to a commercial process for producing maltopentaose.
- ~t i.s known that amylase obtained from a
particular source, Bacillus licheniformis, whPn reacted
with amylose ox amylopectin produces a higher percentage
of m~ltopentaose than produced when other sources of
amylase are used. See the article by Saito in the
Arslhives of Biochemistry and Biophysics, 155, 290 (1953jo
However, the yield of maltopentaose from such reactions
is still small because of the limited solubility of
' amylose in t.he aqueous medium required for the blological
hyclrolysis.
' ' ~ ` ` ~ .
SUM~RY_OF THE INVENTION
A multi-step process for obtaining a high yield of
soluble amylose and maltopentaose has now been found. According
to this invention, the process comprises the steps of-
(a) dissol~ing amylose in a first organic solvent
capable o dissociating and dissolving the amylose to
form a first solution;
(b) mixing the first solution with an aqueous acid
solution capable of partially hydrolyzing the amylose,
thereby forming a second solution;
(c) heating the second solution to partially hydro-
lyze the dissociated amylose into lower molecular weight
components;
(d) mixing the second solution containing the par~
tially hydrolyzed amylose with a second organic solvent
capable of stripping the first orc~anic solvent from the
hydrolyzed amyl.ose to form a thircl solution, and allowing
a precipitate containing partially hydrolyzed amylose to
fo~m;
(e) collecti.ng, washing and drying the precipitate
to form soluble amylose;
(f) mixing amylase obtained rom Bacillus licheni-
formis andsoluble amylose in a buffered aqueous solu-
tion to form a fourth solution;
?5 (g) incubating the fourth solution for a time sufi-
c1~ent to produce a fifth solution containing a high percen-
talge of maltopentaose;
(h) deactivating the amylase in the Eifth solution;
and
~i) fractionating the fifth solution to obtain a
sixth solution predominant in maltopentaose.
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The sixth solution can be dried to obtain a solid
maltopentaose containing product. In the Dreferred embodi-
ment, the first organic solvent is dimethyl sulfoxide, the
aqueous acidic solution is an aqueous sulfuric acid solution,
and incubation of the fourth solution is accomplished at an
elevated temperature.
BRIEF DESCRIPTION OF THE DRAWINGS
The present invention will be described by reference
to the single figure which represents a flow diagram for a
specific embodiment of the process.
DETAILED DESCRIPTION OF THE I~VENTION
The procedure begins by mixing amylose, a long
chain linear or lightly branched glucose polysaccharide,
with an organic solution capable of dissolving the amylose.
The linear molecules of amylose are normally entwined with
one another and, in this form, are insoluble in water at
concentrations greater than 2 gm. per 100 ml. solution.
The organic solvent used in this process dissociates the
a~ylose by solvatlng the linear molecules so that they are
dissolved to form a solution (hereinafter referred to as
the first solution). Any organic solvent, well known to
those skilled in the art, which will dissolve amylose, can
be used in the process of this invention. Dimethyl sul-
foxide (DMSO) is an example of a suitahle organic solvent.
The first amylose solution is then mixed with an
aqueous acid solution capable of partially hydroly~ing the
individual amylose molecule into lower molecular weight
- segments. Many acids well known to those skilled in the
art can be used in this second solution. Sulfuric acid
is one suitable acid. An aqueous solution is necessary~
s:ince water is needed as a reactant in the hydrolysis
process. The mixture of the first (dissociated amylose
containing) solution with the aqueous acidic solution
produces a solution (hereinafter referred to as the second
solution~, and partial hydrolysis is accomplished by rais-
ing the second solution to an elevated temperature for a
time sufficient to accomplish the partial hydrolysis.
Some hydrolysis will occur at any temperature if the
solution is maintained at that temperature for a sufficient
time, however, to produce sufficient hydrolysis in a reason-
able length of time, the second solution should be hea~ed
to above about 100C.
The second (hydrolyzed amylose containing) solu-
~ion is then mixed with a second organic solvent to form a
third solution. The second organic solvent is chosen for
its ability to strip the first organic solvent molecules
from the partially hydrolyzed amyLose, rendering the par-
tially hydrolyzed amylose insoluble in the third solution.
The amylose segments which are no longer soluble in the
thixd solution form a precipitate which is a mixture of
linear glucose polysaccharide~ ranging in length from 2 to
approximately 75 glucose unitsO The particular second
orcJani~ solvent used depends upon what first organic
solvent is used, but a number of organic solvents capable
~5 of stripping the first organic solvent from the partially
hydrolyzed amylose can easily be chosen by one skilled in
the art. Suitable second organic solvents which can be
u~ed when DMSO is used as the first organic solvent are
methanol, acetone, and mixtures thereof.
3Q The precipitate~ which will be referred to
as soluble amylose, is removed from the third solution,
washed with a suitable organic solvent well known to those
skilled in the art, and dried to form a solid. The liquid
r~sidue is discarded.
In the preferred process of the invention, the solu~le
amylose prepared as described is used in the preparation of
maltopentaose described below. However, water-soluble
amylose from any source may be used.
The soluble amylose can be mixed with amylase obtained
from the bacterium Bacillus licheniformis in an aqueous solu-
tion buffered to optimize amylase activity. Any pH between
about 4 and 10 will suf~ice. The mixing of these two com-
ponents, to form a fourth solution, is preferably accomplished
at a temperature close to but not exceeding 100C. The amyl-
ase and soluble amylose are mixed at an elevated temperature
because the higher molecular weight amylose segments will
remain dissolved in the fourth solution longer at elevated
temperatures, giving the amylase an opportunity to break down
these high molecular weight segments into lower molecular
weicJht fragments. After the amylase has been added, the fourth
~amylose~amylase containing) solution is incubated at an
ele~ated temperature for a time sufficient to produce a
fifth solution containing a high maltopentaose concentra~ion.
This step in the process is a time/temperature~amylase
dependent step. At a given temperature and a given amylase
concentration, the concentration of maltopentaose in the
fifth solution will increase to a maximum in a given time,
after which the maltopentaose concentration will begin to
decrease in favor o~ lower chain-length polysaccharides
(l.e., maltotetraose, maltotriose, maltose and glucose).
At a given temperature and amylase concentration, the time
requir~d to produce the maximum concentration of malto-
pentaose in the fifth solution can be easily determined
by sampling and testing the fifth solutionO ~ temperature
of between about 50 and 60C. and a reaction time of about
24 hours is routinely used to produce a maximum maltopentoase
concentration.
Once the maximum maltopentaose concentration is
obtained, the amylase in the fifth solution must be deacti-
vatled. This can be done in a number of ways well known tothose skilled in the art. One way is to heat the ifth
solution to a temperature above 100C., preferably 120C.,
for a time sufficient to destroy the amylase activity. The
(amylase deactivated) fifth solution is then filtered to
remove particulate contaminants, and fractionated to collect
the maltopentaose. One way to obtain the maltopentaose
components of the fifth solution is to use a separation
column. The technique of separating various organic frac-
tions from aqueous solutions is well known and will not be
di~cussed here. With little or no experimentation, a
maltopentaose-rich solution, hereinafter referred to as
the sixth solution, can be ohtained from the separation
colu.mn.
This sixth solution is then dried to produce a
~5 maltopentaose-rich solid product. Drying can be accomplished
in a number of ways, but one satisfactory way is to lyo-
philize the product. The sixth solution can be made to
contain as much as 98% of maltopentaose with small percen-
tages of maltotetraose and maltohexaose as contaminants.
XAMPLE
Preparation of soluble amylose
A ten liter graduated cylinder with a contained
stirring bar is placed on a Cole-Parmer magnetic stirrerO
4.,i3 liters of ~ISO are added to the graduated cylinder,
ancl the stirring bar is rotated at moderate speed. One
kilogram of amylose is added to the graduated container so
that the amylose mixes evenly in the DMSO and no clumps are
formed. The stirring bar is adjusted to maximum speed and
this first solution is allowed to stir overnight or until
the amylose goes completely into solution. Any concentra-
tion of amylose in the D~ISO will work~ but, in the prefèrred
embodiment, this first solution contains between about 15
and about 20 w/v of amylose in the DMSO.
Sixty milliliters of 10~ sulfuric acid tv/v) are
then added to the graduated cylinder and mixed thoroughly
with the contents of the graduated cylinder to form a second
solution. The graduated cylinder with its contents is then
placed in an autoclave and heated for 55 minutes at 121C.
to partially hydrolyze the amylose in the second solution.
Again~ any concentration of acid in the solution will produce
some hydrolyzation, but~ in the preEerred embodiment, the
second solution contains between about 0.05 and about 0.20
percent of H~SO4.
Fifteen liters of methanol and 7.5 liters of
acet:one are ~rought to a temperature of 4C. and then mixed
in 21 38 liter container. After the graduated cylinder with
its contents has cooled to 100C., the contents of the
graduated cylinder are poured into the cold methanol
acet:one solution and stirred. The resulting solution,
--8--
reerred to as the -third solution, is ~hen left for a time
sufficient to allow a precipitate to form. By allowing the
mixture to stand overnight, most of the precipitated soluble
amylose settles from the third solution. The liquid residue
is decanted off and discarded, and the precipitate is fil-
tered using a Buchner funnel and coarse filter paper. The
precipitate is washed in 3.79 liters of methanol and then
removed from the methanol by filtration, again using a
Buchner funnel and filter flask. In both collection stages,
care must be taken not to allow moisture in the air to
dissolve the amylose.
The precipitate is placed in a lyophilization
tray and dried overnight in a Hull lyophilizer to provide
dry soluble amylose.
P~paration of maltopentaose
.
An aqueous buffer solution is ~repared using
5 liters of Trisma buffer to which is added 12.1 grams of
Trisma base and sufficient acetic acid to bring the pH of
the buffer solution to 8.0 ~ 0.2. This buffer solutlon is
placed in a 20 litex stainless steel container and auto-
claved for 15 minutes at 121C.
When the buffer is removed from the autoclave,
and while the temperature is greater than 90C., 0.5 milli-
liters of amylase obtained from Bacillus licheniformis is
added to the buf~er solution and stirred wi~h a magnetic
stirring bar. Such amylase is sold under the tradenameThermamyl~ 60 by NQVO Chemical Company.
After the amylase is dispersed in the ~uffer
solu~tion, 1000 grams of soluble amylose prepared according
to the procedure set forth above is added to the buffer
so that no large agqregates of soluble a~ylose are formed.
This fourth sol~ltion is autoclaved for 15 minutes at 121C.
The solution is then removed from the autoclave while it
is still hotter than g0C~, and 0~5 milliliters of the same
~mylase is again added to the buffer solution. When the
temperature of the buffer solution drops to 85C., an
additional 1.0 milliliters of amylase is added, and 1.0
additional milliliters of amylase are added at every 5
decrease in temperature, down to and including 55C., the
total amount of amylase added being ~.0 milliliters.
This fourth solution is incubated overnight at
50 to 55C. using a heated stirring plate. A fifth solu-
tion containing a high maltopentaose concentration is
f ormed .
~fter about 24 hours, a 1 milliliter sample of
this fifth solution is removed and the carbohydrate compo-
sition OL the sample is determi.ned by standard chromato-
graphic techniques. If, on the basis of this analysis, it
is determined that the maltopentaose fraction of the total
carbohydrate is greater than about 25~, then, the entire
solution is placed in an autoclave for 15 minutes at 121C.
to deactivate the amylase enzyme. If the maltopentaose
fraclion of the carbohydrate in the fi th solution is below
about 25~, then additional amylase is added to the solution
and t:he incubation continues until the proper maltopentaose
content is achieved.
Once the amylase activity in the solution has
been destroyed by autoclaving, the solution is cooled to
about 50C. and all particulate matter is removed from the
solution by filtration or centrifu~ation. The supernatant
is decanted, and the solid impurities are discarded.
-10-
The supernatant is then separated using d separa-
tion column filled with -400 mesh P2 gel (obtainPd from
Bic) Rad). Using conventional techniques, fractions eluting
from the column containing primarily maltopentaose are
collected. These fractions, referred to as a sixth
(ma,ltopentaose-rich) solution when pooled, contain about
95 to 98% of maltopentaose, with small concentrations of
maltotetraose and maltohexaose. The pooled fractions are
then lyophilized by conventional techniques to produce a
solid maltopentaose-rich product.
The above disclosure has been made to describe
the invention to those skilled in the art and is not intended
to limit the scope of that invention as defined in the
appended claims. Many modifications to the above well
within the skill of the art are intended to be incorporated
by these claims.
