U.S. patent application number 14/242060 was filed with the patent office on 2014-07-31 for xylanase composition with increased stability.
This patent application is currently assigned to YFY Biopulp Technology Limited. The applicant listed for this patent is YFY Biopulp Technology Limited. Invention is credited to Chunhui HSIEH, Yahui HUANG, Hsuanyu LAI.
Application Number | 20140212948 14/242060 |
Document ID | / |
Family ID | 44065812 |
Filed Date | 2014-07-31 |
United States Patent
Application |
20140212948 |
Kind Code |
A1 |
HUANG; Yahui ; et
al. |
July 31, 2014 |
XYLANASE COMPOSITION WITH INCREASED STABILITY
Abstract
A xylanase composition and a method for manufacturing the
xylanase composition are provided, wherein the xylanase composition
comprises a xylanase and a stabilizer, and the xylanase is from an
anaerobic fungus, the stabilizer comprises a polyol, and the
content of the polyol is at least 40 wt %, based on the total
weight of the xylanase composition.
Inventors: |
HUANG; Yahui; (Hsinchu City,
TW) ; HSIEH; Chunhui; (Ershui Township,Changhua
County, TW) ; LAI; Hsuanyu; (New Taipei City,
TW) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
YFY Biopulp Technology Limited |
Road Town |
|
VG |
|
|
Assignee: |
YFY Biopulp Technology
Limited
Road Town
VG
|
Family ID: |
44065812 |
Appl. No.: |
14/242060 |
Filed: |
April 1, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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13505310 |
May 1, 2012 |
8735118 |
|
|
PCT/CN2009/075103 |
Nov 24, 2009 |
|
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14242060 |
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Current U.S.
Class: |
435/200 |
Current CPC
Class: |
C12Y 302/01008 20130101;
C12N 9/96 20130101; C12N 9/2482 20130101 |
Class at
Publication: |
435/200 |
International
Class: |
C12N 9/24 20060101
C12N009/24 |
Claims
1. A method for manufacturing a xylanase composition with increased
stability comprising: providing a xylanase; providing a stabilizer,
comprising a polyol; and mixing the xylanase and the stabilizer to
form a xylanase composition; wherein the xylanase is from
Neocallimastix genus, and the content of the polyol is at least 60
wt %, based on the total weight of the xylanase composition,
wherein the polyol is selected from the group consisting of
glycerol, sorbitol, sucrose, and combinations thereof.
2. The method according to claim 1, wherein the xylanase is from
Neocallimastix frontalis.
3. The method according to claim 1, wherein the polyol is
glycerol.
4. The method according to claim 1, wherein the content of the
polyol is at least 80 wt % based on the total weight of the
xylanase composition.
Description
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] This application is a Divisional Application of U.S. patent
application Ser. No. 13/505,310, entitled "Xylanase Composition
With Increased Stability", filed May 1, 2012, now U.S. Pat. No.
______. U.S. patent application Ser. No. 13/505,310 claims priority
to PCT Patent Application No. PCT/CN2009/075103 filed on Nov. 24,
2009. The priority of U.S. patent application Ser. No. 13/505,310
and PCT Patent Application No. PCT/CN2009/075103 are hereby claimed
and their disclosures are incorporated herein by reference in their
entirety.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates to a xylanase composition with
increased stability, and the method for manufacturing the same.
[0004] 2. Descriptions of the Related Art
[0005] Enzymes are proteins which can function as bio-catalysts and
can be widely applied in various chemical reactions to increase the
rate of reaction.
[0006] Inactive enzymes are costly for researchers and factory
owners because once an enzyme is inactivated and loses its
function, it cannot exert its intrinsic catalytic action. As a
result, researchers or factory owners have to pay additional costs
to increase the amount of the enzyme used or purchase new enzymes
for replacement to achieve the desired efficiency. Therefore, a
method or a formulation that prevents enzymes from being
inactivated due to environment factors, such as high temperature,
or increases the stability of enzymes during storage or transport
is urgently desired in the industry.
[0007] Xylanase is the main enzyme that degrades hemicellulose
among sugar hydrolysis enzymes, and is widely applied in various
fields such as foods, animal feeds, textiles or papermaking. For
example, xylanase may be used to treat chicken feed to degrade
anti-nutrient factors in the feed, thereby, promoting the uptake of
nutrient and the growth of chickens. In addition, if xylanase is
added in the dough, the mechanical strength of the dough may be
improved, and its appearance and storability can also be improved
accordingly.
[0008] A method for increasing the stability of xylanase from
aerobic fungi has been disclosed in the literature, which improves
the storability of xylanase by using a formulation comprising a
polyol, salts and antibiotics (Fisk et al. 1992. Development of A
Method for the Stabilization and Formulation of Xylanase from
Trichoderma Using Experimental Design. Studies in Organic
Chemistry, 47: 323-328, which is incorporated hereinto by
reference).
[0009] However, according to the literature, the stabilizing effect
(e.g., the stabilizing duration and temperature) provided by the
formulation is quite limited. The activity of xylanase from an
aerobic fungus, Trichoderma, was at about 80% after 42 hours under
48.degree. C.
[0010] As known by persons with ordinary knowledge in the art of
enzymes, different enzymes have different characteristics, such as
the structure, heat resistance, or optimum storage condition, etc.
The interaction between different enzymes and the same compound or
formulation is also different. Therefore, it has been indicated in
literatures that for different enzymes, the same component or
formulation cannot provide the same stabilizing effect, and polyols
do not necessarily provide a stabilizing effect (Asther et al.
1990. Increased thermal stability of B. licheniformis alpha-amylase
in the presence of various additives. Enzyme and Microbial
Technology. 12: 902-905, which is incorporated hereinto by
reference).
[0011] Among the known xylanses from different sources, xylanases
from anaerobic fungi (also called rumen fungi) are the most
interesting because anaerobic fungi generally grow in the rumen
(such as the digestive tract of ruminants or herbivores with a
single stomach) in which competition stress for survival is high.
Thus, these fungi have evolved to produce xylanases with high
activity (Anthony et al. 1994. Anaerobic fungi in herbivorous
animals. Mycol. Res. 98: 129-152, which is incorporated hereinto by
reference). Compared to general xylanases, xylanases from anaerobic
fungi are more widely applicable due to their advantages, such as
high enzyme activity, high specificity, heat resistance, etc.
Hence, a method for preserving xylanases from anaerobic fungi
effectively is required for persons in this field to prevent the
enzyme activity from decreasing during the storage.
[0012] The present invention provides a composition for increasing
the stability of xylanase from anaerobic fungi. The inventors of
the present application found that a composition comprising polyols
with specific components and ratio can achieve the effect of
significantly increasing enzyme storability.
SUMMARY OF THE INVENTION
[0013] The primary objective of the present invention is to provide
a xylanase composition with increased stability, comprising a
xylanase and a stabilizer comprising a polyol.
[0014] Another objective of the present invention is to provide a
method for manufacturing a xylanase composition with increased
stability, comprising providing a xylanase and a stabilizer
comprising a polyol, and mixing the xylanase and the stabilizer to
form a xylanase composition.
[0015] The first objective of the present invention is to provide a
method for manufacturing a xylanase composition with increased
stability, comprising the following: [0016] providing a xylanase;
[0017] providing a stabilizer, comprising a polyol; and [0018]
mixing the xylanase and the stabilizer to form a xylanase
composition; [0019] wherein the xylanase is from an anaerobic
fungus, and the content of the polyol is at least 40 wt %, based on
the total weight of the xylanase composition.
[0020] The second objective of the present invention is to provide
a xylanase composition, comprising a xylanase and a stabilizer,
wherein the xylanase is from an anaerobic fungus, the stabilizer
comprises a polyol, and, based on the total weight of the xylanase
composition, the content of the polyol is at least 40 wt %.
[0021] The detailed technology and preferred embodiments
implemented for the subject invention are described in the
following paragraphs accompanying the appended drawings for people
skilled in this field to well appreciate the features of the
claimed invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0022] FIG. 1 is a bar chart showing the enzyme stabilizing effect
of xylanase compositions comprising different polyols at different
concentrations; and
[0023] FIG. 2 is a curve chart showing enzyme stabilizing effect of
xylanase compositions comprising glycerol at different
concentrations under 50.degree. C.
DETAILED DESCRIPTION OF THE INVENTION
[0024] Unless otherwise stated herein, the terms "a (an)", "the" or
the like used in this specification (especially in the Claims
hereinafter) shall be understood to encompass both the singular and
plural form.
[0025] In the context, the term "stability of an enzyme" is defined
as the resistance or tolerance of the enzyme to environmental
factors, especially temperature. Generally, the stability of an
enzyme can be determined by the decay rate of enzyme activity under
a specific environment. The slower the decay rate, the higher the
enzyme stability.
[0026] As described above, xylanase is widely applied in fields
such as food, animal feed, textile, papermaking, etc. For
papermaking application, it is known that when xylanase is added
into the pulp during the process of pulp making, the power required
for pulping can be significantly reduced, and the energy
consumption can be also reduced accordingly. Furthermore, a large
amount of chlorine-containing bleach is usually required in the
process of pulp bleaching; however, chlorine leads to the
generation of highly toxic organic chloride compounds, resulting in
waste disposition problems. If the pretreatment by xylanase is
performed before the bleaching process, the amount of
chlorine-containing substances used can be significantly reduced,
and the waste disposition problems can be effectively resolved.
Therefore, if xylanase can perform its function under high
temperature and does not degrade cellulose, the reaction rate of
pulp treatment can be improved without damaging cellulosic fibers.
In addition, if xylanase can specifically hydrolyze hemicellulose
(i.e., without hydrolyzing cellulose) and is heat-resistant, the
paper making process can be improved more effectively thereby.
[0027] Because xylanases from anaerobic fungi has the desired high
specificity and heat-resistance properties, it is important to also
elevate the stability of these enzymes. Thus, the present invention
provides a xylanase composition, comprising a xylanase and a
stabilizer, wherein the xylanase is from, for example,
Neocallimastix genus, Caecomyces genus, or Piromyces genus,
Orpinomyces genus, Cyllamyces genus, Anaeromyces genus, etc., and
is preferably from Neocallimastix genus. In the preferred
embodiment of the present invention, a xylanase from Neocallimastix
frontalis is used to prepare the xylanase composition.
[0028] In addition, the xylanase in the present invention may be
obtained by isolating natural xylanase or by an artificial
synthesizing method (such as using genetic engineering or a peptide
synthesizer). Furthermore, for an isolated or synthesized xylanase,
molecular biology technique may be applied to further modify its
gene to increase its activity, specificity and/or stability. For
example, when xylanase from the anaerobic fungus Neocallimastix
frontalis is used to prepare the composition of the present
invention, the removal of the dockerin domain in the xylanase gene
may be performed before expression of the xylanase gene to further
increase the stability and activity of xylanase. Specifically, the
removal of the dockerin domain in the xylanase gene may be
performed using the following method. First, a polymerase chain
reaction (PCR) is used to amplify the xylanase gene of
Neocallimastix frontalis (about 1,011 bp), and then restriction
enzymes are used to remove the dockerin domain in the xylanase
gene. The generated gap is eliminated with a ligase. After that,
another polymerase chain reaction is conducted to amplify the
xylanase gene that does not contain a dockerin domain (about 729
bp), thereby, obtaining the gene of xylanase that has high activity
and stability. The above method can be seen in Taiwan Patent
Application Publication No. 200720435, which is incorporated
hereinto by reference.
[0029] The stabilizer in the composition of the present invention
comprises a polyol. The term "polyol" indicates any compound with
two or more hydroxyl groups. For example, but not limited thereto,
the polyol in the composition of the present invention may consist
of one or more C.sub.3-C.sub.12 polyols, such as propylene glycol,
glycerol, erythrose, sorbitol, glucose, mannose, fructose,
galactose, sucrose, maltose, lactose, and the like. Preferably, the
polyol in the composition of the present invention is selected from
the group consisting of glycerol, sorbitol, sucrose, and
combinations thereof.
[0030] In the xylanase composition of the present invention, based
on the total weight of the xylanase composition, the content of the
polyol is at least 40 wt %, preferably at least 50 wt %, more
preferably at least 60 wt %, and most preferably at least 80 wt
%.
[0031] In an example of the present invention, when the xylanase
compositions comprising a polyol at different contents were used to
stabilize xylanase, it was found that the stabilizing effect of the
polyol on xylanase from the anaerobic fungus is approximately
proportional to the content of the polyol in the composition. In
other words, the higher the content of the polyol in the
composition, the better the stabilizing effect of the composition
on xylanase.
[0032] The discovery above is quite different from the findings in
the existing literatures. For example, Fisk et al. uses a polyol to
stabilize xylanase from an aerobic Trichoderma fungus, and
discloses that a composition comprising glycerol at a content of
40% has the best stabilizing effect, but when the content of
glycerol is higher above 40%, the stabilizing effect provided is
not increased but rather decreased with the increase of the content
of glycerol (Fisk et al. 1992. Development of A Method for the
Stabilization and Formulation of Xylanase from Trichoderma Using
Experimental Design. Studies in Organic Chemistry, 47: 323-328,
which is incorporated hereinto by reference). In other words,
according to the literature, the stabilizing effect of a polyol is
not proportional to its content, and the content of the polyol is
preferably not more than 40%.
[0033] In addition, the other literature also indicates that the
enzyme stabilizing effect of a polyol depends on the number of
hydroxyl groups on the polyol, and the stabilizing effect is better
with the increase of the number of hydroxyl groups (Viswanathan et
al. 1995. Effect of polyols on heat inactivation of Aspergillus
niger van Teighem inulinase. Letters in Applied Microbiology, 21:
282-284, which is incorporated hereinto by reference). However, as
shown in the following examples, compared to sorbitol (with six
hydroxyl groups) and sucrose (with eight hydroxyl groups), glycerol
(with three hydroxyl groups) can provide a better stabilizing
effect on xylanase from Neocallimastix frontalis.
[0034] The xylanase composition of the present invention may
further comprise other additives as long as the additives do not
deteriorate the activity of xylanase and have no substantially
adverse influence on the stabilizing effect of the composition. In
an embodiment of the present invention, in addition to a xylanase
and a polyol, the xylanase composition further comprises water, so
that the composition is in the form of an enzyme solution (i.e.,
xylanase dissolved in water).
[0035] The xylanase composition of the present invention can be
applied to a variety of fields, such as food, animal feed, textile,
papermaking, etc.
[0036] The present invention also provides a method for
manufacturing a xylanase composition with increased stability,
comprising providing a xylanase; providing a stabilizer comprising
a polyol; and mixing the xylanase and the stabilizer to form a
xylanase composition. The xylanase, stabilizer, polyol and xylanase
composition involved in the method of the present invention are
those as described above.
[0037] The method of the present invention may further comprise
adding water into at least one of the following substances:
xylanase, the stabilizer, and the xylanase composition.
Specifically, water may be used to adjust the properties of
components in the composition of the present invention to achieve
the following purposes: 1) by mixing xylanase and water, xylanase
may be in the form of an enzyme solution, and the activity unit of
xylanase can be controlled; 2) the concentration of a polyol in the
stabilizer may be adjusted by mixing the stabilizer and water; and
3) the final adjustment may be performed by adding water into the
xylanase composition. In an embodiment of the present invention,
xylanase from an anaerobic fungus Neocallimastix frontalis was
mixed with glycerol to prepare a xylanase composition, and water
was then used to adjust the activity unit of xylanase and make the
final content of glycerol in the composition be 90 wt %. The
composition was stored for two weeks and eight weeks respectively
under 50.degree. C., and xylanase contained therein still had the
residual activity of 97.+-.4.8% and 59.6.+-.4.7%, respectively.
[0038] Therefore, compared to the conventional art, the xylanase
composition of the present invention may provide xylanase with a
better stabilizing effect under common storage conditions (e.g.,
room temperature) or even more stringent conditions (e.g., high
temperature), thereby extending the storage period of xylanase.
[0039] The detailed technology and preferred embodiments
implemented for the present invention are described in the
following paragraphs; however, the scope of the present invention
is not limited thereby.
EXAMPLE 1
Preparation of the Xylanase Compositions
[0040] First, xylanase compositions comprising glycerol with
different contents were prepared by adding glycerol into an enzyme
solution containing xylanase from an anaerobic fungus
Neocallimastix frontalis (i.e., an solution in which xylanase was
dissolved in water, and the activity of xylanase was about 100,000
unit (U)/ml), and followed by stirring the mixture, so that the
final content of glycerol, based on the total weight of the
xylanase composition, was about 20 wt %, 40 wt % or 50 wt %,
respectively.
[0041] Second, xylanase compositions comprising 20 wt %, 40 wt % or
50 wt % of sorbitol or sucrose were prepared with the same method
and ratio. The control group (i.e., a xylanase composition
comprising 0 wt % of a polyol) was prepared by substituting
glycerol, sorbitol or sucrose with water.
Measurement of the Activity of Xylanase in the Compositions
[0042] The measurement of the activity of xylanase in the
compositions was conducted based on the following mechanism. After
a dinitrosalicyclic acid (DNS) solution and the reducing sugars
generated from hydrolysis by xylanase are heated together, a brown
red amino compound is formed, and a colorimetric method is used to
measure the content of the reducing sugars in the sample to
determine the activity of xylanase.
[0043] The detailed method for measuring activity was based on
Georis' method (Georis et al. 1999. Sequence, overproduction and
purification of the family 11 endo-beta-1,4-xylanase encoded by the
xyll gene of Streptomyces sp. S38. Gene 2437:123-33, which is
incorporated hereinto by reference). In this method, 90 .mu.l of 3
wt % of xylan (oat spelts xylan dissolved into a
tris(hydroxymethyl) aminomethane or (Tris) buffer solution of 25
mM, pH 8.0) was used as a substrate for the enzyme reaction, and
was uniformly mixed with 10 .mu.l of the above xylanase
compositions, of which the content was properly adjusted. After the
mixture was reacted at 60.degree. C. for 5 minutes, 125 .mu.l of a
dinitrosalicyclic acid reagent was added to terminate the reaction,
and the resulting mixture was then reacted at 98.degree. C. for 5
minutes to undergo the color reaction. Finally, the absorbance was
measured at wavelength of 540 nm to calculate the content of the
reducing sugars, and the activity of xylanase in the compositions
was thus determined, wherein one (1) activity unit (U) is defined
as the amount of the enzyme required for releasing/hydrolyzing 1
.mu.mole of reducing sugars per minute per ml.
Determination of the Stability of Xylanase
[0044] First, before this test was conducted, the original activity
of xylanase in the xylanase compositions prepared above was
determined, and the measured original activity was defined as
residual activity of 100%. The xylanase compositions were then set
in the environment at 50.degree. C. for four weeks (i.e.,
continuously stored for four weeks).
[0045] After four weeks, the activity of xylanase in the xylanase
compositions was determined respectively, and the residual activity
of xylanase was calculated using the following formula:
Residual Activity (%)=(the activity of xylanase after storage/the
original activity of xylanase).times.100%.
The higher the residual activity of xylanase measured, the better
the enzyme stabilizing effect of a xylanase composition.
[0046] The measurement results of the enzyme stability are shown in
Table 1 and FIG. 1 below.
TABLE-US-00001 TABLE 1 Polyol Glycerol Sorbitol Sucrose Content (wt
%) Residual activity of xylanase (%) 0 0 0 0 20 3.9 .+-. 0.3 3.2
.+-. 0.3 2.8 .+-. 0.1 40 43 .+-. 1.2 43 .+-. 3.2 10.1 .+-. 0.3 50
63.7 .+-. 0.9 38.2 .+-. 3 21.5 .+-. 1.4
[0047] It can be seen from FIG. 1 and Table 1 that for the
composition without a stabilizer, after being stored at 50.degree.
C. for four weeks, the residual activity of xylanase was 0%,
indicating that the xylanase had completely lost its activity.
However, if a stabilizer, such as a polyol like glycerol, sorbitol,
sucrose, etc., was added into the composition, the residual
activity of xylanase was significantly increased, and the increase
effect was approximately proportional to the content of the
polyol.
[0048] In addition, for the polyols, glycerol showed the best
improvement for the stability of the enzyme, and after continuously
being stored at 50.degree. C. for four weeks, the composition
containing 50 wt % glycerol retained a residual activity of
63.7.+-.0.9%.
EXAMPLE 2
[0049] First, xylanase compositions comprising glycerol at
different contents were prepared by adding glycerol into a solution
containing xylanase from an anaerobic fungus Neocallimastix
frontalis (i.e., a solution in which xylanase, with an activity of
100,000 unit (U)/ml, was dissolved in water), and then followed by
stirring the mixture, so that the final content of glycerol, based
on the total weight of the xylanase composition, was 20 wt %, 40 wt
%, 50 wt %, 70 wt %, or 90 wt %, respectively. The control group
(i.e., a xylanase composition comprising 0 wt % of a polyol) was
prepared by substituting glycerol with water.
[0050] Second, the activity and stability of xylanase in the
compositions prepared above were measured by the same methods as
described in Example 1. The xylanase compositions were stored in
the environment at 50.degree. C. for eight weeks (i.e.,
continuously stored for eight weeks), and the samples were
collected every two weeks to measure the activity of xylanase in
the compositions. The residual activity was thus calculated.
[0051] The measurement results of the enzyme stability are shown in
Table 2 and FIG. 2 below.
TABLE-US-00002 TABLE 2 Polyol Glycerol Residual activity of
xylanase (%) Content (wt %) 1 week 2 weeks 4 weeks 6 weeks 8 weeks
0 1 0 0 0 0 20 21.9 .+-. 1.6 3.9 .+-. 0.3 0.7 .+-. 0 0.2 .+-. 0 40
72.8 .+-. 4.2 .sup. 43 .+-. 1.2 27.3 .+-. 0.2 20.7 .+-. 0.6 50 84.2
.+-. 3.sup. 63.7 .+-. 0.9 46.7 .+-. 2.sup. 34.3 .+-. 1.0 70 101
.+-. 5.4 81.1 .+-. 6.3 73.7 .+-. 5.2 58.4 .+-. 2.6 90 .sup. 97 .+-.
4.8 76.8 .+-. 3.sup. 73.8 .+-. 5.3 59.6 .+-. 4.7
[0052] It can be seen from FIG. 2 and Table 2 that if no glycerol
was added into the xylanase composition, the residual activity of
xylanase, after being continuously stored at 50.degree. C. for one
week, remained about 1%. However, if glycerol was added into the
xylanase composition, the activity of xylanase can be maintained
longer. After continuously being stored at 50.degree. C. for eight
weeks, in the xylanase compositions comprising different glycerol
contents, the residual activity of the enzyme, from the lowest to
the highest, was 20 wt %: 0.2.+-.0%; 40 wt %: 20.7.+-.0.6%; 50 wt
%: 34.3.+-.1.0%; 70 wt %: 58.4.+-.2.6%; and 90 wt %: 59.6.+-.4.7%,
respectively.
[0053] In addition, it can be seen from FIGS. 1 and 2 and Tables 1
and 2 that as the polyol content increased, the residual activity
of xylanase also increased, which illustrates that the stabilizing
effect of the xylanase composition on xylanase was approximately
proportional to the content of a polyol. When the content of
glycerol was higher than 70 wt %, this proportional relation became
less obvious.
[0054] Examples 1 and 2 proved that the composition of the present
invention can provide an excellent stabilizing effect on xylanase
from the anaerobic fungus, and thus, may extend the storage period
of xylanase.
[0055] The above disclosure is related to the detailed technical
contents and inventive features thereof People skilled in this
field may proceed with a variety of modifications and replacements
based on the disclosures and suggestions of the invention as
described without departing from the characteristics thereof
Nevertheless, although such modifications and replacements are not
fully disclosed in the above descriptions, they have substantially
been covered in the following claims as appended.
* * * * *