U.S. patent application number 17/134296 was filed with the patent office on 2021-07-01 for enterobacter and its application.
This patent application is currently assigned to JIANGNAN UNIVERSITY. The applicant listed for this patent is JIANGNAN UNIVERSITY, Shandong Focusfreda Biotech Co., Ltd. Invention is credited to Jinsong GONG, Chuanli KANG, Hui LI, Qing LI, Lei LIU, Wei LIU, Jianying Qian, Jinsong SHI, Zhenghong XU, Mengyi ZHANG.
Application Number | 20210198620 17/134296 |
Document ID | / |
Family ID | 1000005330347 |
Filed Date | 2021-07-01 |
United States Patent
Application |
20210198620 |
Kind Code |
A1 |
SHI; Jinsong ; et
al. |
July 1, 2021 |
ENTEROBACTER AND ITS APPLICATION
Abstract
The invention belongs to the field of cosmetic biotechnology,
and specifically relates to an Enterobacter that degrades
hyaluronic acid and a cultivation method and application thereof.
The Enterobacter sp. CGJ001 of the present invention was deposited
in the China General Microbiological Culture Collection Center on
Oct. 10, 2019, and the preservation number is CGMCC NO. 18661. The
Enterobacter strain can efficiently produce hyaluronidase, and can
be used in the process of preparing low molecular hyaluronic acid
and oligomeric hyaluronic acid from high molecular hyaluronic acid.
The enzyme has high specificity towards hyaluronic acid, excellent
thermal stability and pH stability, and is suitable for large-scale
industrial application. Thus it can replace the traditional
hyaluronidase extracted from expensive animal tissues. There should
be broad application prospects in the fields of medicine and
cosmetics.
Inventors: |
SHI; Jinsong; (Wuxi,
Jiangsu, CN) ; XU; Zhenghong; (Wuxi, Jiangsu, CN)
; GONG; Jinsong; (Wuxi, Jiangsu, CN) ; LIU;
Wei; (Wuxi, Jiangsu, CN) ; LI; Hui; (Wuxi,
Jiangsu, CN) ; Qian; Jianying; (Wuxi, Jiangsu,
CN) ; LIU; Lei; (Qufu, Jining,Shandong, CN) ;
LI; Qing; (Qufu, Jining,Shandong, CN) ; ZHANG;
Mengyi; (Qufu, Jining,Shandong, CN) ; KANG;
Chuanli; (Qufu, Jining,Shandong, CN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
JIANGNAN UNIVERSITY
Shandong Focusfreda Biotech Co., Ltd |
Wuxi
Qufu |
|
CN
CN |
|
|
Assignee: |
JIANGNAN UNIVERSITY
Shandong Focusfreda Biotech Co., Ltd
|
Family ID: |
1000005330347 |
Appl. No.: |
17/134296 |
Filed: |
December 26, 2020 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12R 2001/01 20210501;
C12N 1/205 20210501; C12Y 302/01036 20130101; C12N 1/20
20130101 |
International
Class: |
C12N 1/20 20060101
C12N001/20; C12R 1/01 20060101 C12R001/01 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 26, 2019 |
CN |
201911365498.0 |
Claims
1. Enterobacter sp. CGJ001 deposited at the China General
Microbiological Culture Collection Center on Oct. 10, 2019 at No.
3, Yard. 1, Beichen West Road, Chaoyang District, Beijing,
Institute of Microbiology, Chinese Academy of Sciences under
Accession Number CGMCC NO. 18661.
2. The Enterobacter of claim 1 wherein the Enterobacter CGJ001 has
the effect of degrading hyaluronic acid.
3. A method of producing hyaluronidase comprising subjecting the
Enterobacter CGJ001 of claim 1 to plate culture, seed culture, and
fermentation culture.
4. The method of claim 3 comprising: (1) plate culturing the
Enterobacter CGJ001 to obtain plate strains; (2) inoculating the
plate strains into sterilized seed culture medium and incubating at
30-40.degree. C. and 150-300 rpm for 12-24 h to obtain seed liquid;
(3) inoculating the seed liquid into a sterilized fermentation
medium and incubating at 30-40.degree. C. and 150-300 rpm for 12-24
h to obtain a hyaluronidase-containing bacterial liquid.
5. The method of claim 4 wherein the seed medium and fermentation
medium components comprise 1.about.10 g/L hyaluronic acid,
1.about.5 g/L K3PO4, 0.1.about.1 g/L MgSO4, 1.about.10 g/L L
peptone, 1.about.10 g/L yeast powder.
6. The method of claim 3 wherein the degradation product of
hyaluronidase by the enzyme is 2-6 sugars.
Description
RELATED APPLICATIONS
[0001] This application claims the priority from China Patent
Application Serial Number CN 201911365498.0, filed on Dec. 26,
2019, the content of which is incorporated here by reference.
BACKGROUND OF THE INVENTION
1.Technical Field
[0002] The present invention relates to a strain of Enterobacter
degrading hyaluronic acid and its culture method and application,
which belongs to the field of cosmetic biotechnology.
[0003] 2.BACKGROUND ART
[0004] Hyaluronic acid (HA) is an acidic mucopolysaccharide.
Hyaluronic acid is the main component of mammalian extracellular
matrix, which is widely present in the intercellular substance of
animal tissues and the capsule of some bacteria. Hyaluronic acid
(HA) is a straight-chain polysaccharide composed of repeated
-D-glucuronic acid and N-acetyl-D-glucosamine disaccharide units,
linked by alternating-1,3 glycosidic bonds and-1,4 glycosidic
bonds. The molecular weight of hyaluronic acid from different
sources is very different and the molecular weight range is very
wide.
[0005] Hyaluronic acid plays a prominent role in many important
physiological and pathological processes in mammals. Hyaluronic
acid with different molecular weight has different important
meanings for its biological function. High molecular weight HA
(MW-HA) exists in healthy tissues and acts as a space filler and
lubricant with immunosuppressive properties, while low molecular
weight HA (MW-HA) plays an opposite role in immune activation as an
endogenous danger signal. HA is often used in the pharmaceutical
and cosmetic industries, including drug delivery systems, daily
cosmetics, etc., and is an important biocompatible material.
However, since the immune properties of HA depend on its molecular
weight, the molecular weight of HA must be strictly controlled.
Especially for the pharmaceutical industry, it is particularly
important to strictly control the molecular weight of hyaluronic
acid.
[0006] Hyaluronidase degrades hyaluronic acid by breaking the-1,4
glycosidic bond or-1,3 glycosidic bond. According to the substrate
specificity of hyaluronidase, the catalytic mechanism and the types
of degradation products, hyaluronidase was classified into three
types. The first type of hyaluronidase (EC 3.2.1.35) is of bovine
testis type. It degrades hyaluronic acid by acting on-1,4-glucoside
bonds. The main product is tetrasaccharide, which can also act on
chondroitin sulfate and chondroitin sulfate, and the hydrolytic
product is corresponding tetrasaccharide. Typical examples include
bovine testis hyaluronidase, bee venom hyaluronidase and lysosomal
hyaluronidase. The second type of hyaluronidase (EC 3.2.1.36) is
hirudo type. It degrades hyaluronic acid by acting on-1,3-glucoside
bonds. Its degradation products contain tetrasaccharides and
hexoses and cannot act on chondroitin sulfate and chondroitin
sulfate. Typical examples are hirudo hyaluronidase and hookworm
salivary gland hyaluronidase. The third type of hyaluronidase (EC
4.2.2.1) is from microbial sources and widely distributed in
bacteria, pathogenic fungi and phages. It degrades hyaluronic acid
to produce unsaturated disaccharides by acting on-1,4-glucoside
bonds and elimination reactions, and also acts on chondroitin and
chondroitin sulfate. Examples include clostridium, micrococcus,
streptococcus and Streptomyces.
[0007] Hyaluronidase is widely used in cardiology, ophthalmology,
plastic surgery, oncology, dermatology and gynecology, etc.
Hyaluronidase can effectively slow down myocardial infarction by
reducing myocardial hyaluronic acid content, reducing arterial
resistance caused by myocardial ischemia, and increasing blood
flow. The combined action of hyaluronidase and anticancer drugs can
enhance the efficacy of anticancer drugs and play an important role
in enhancing the anti-breast cancer effect of adriamycin and
reducing the recurrence rate of bladder cancer. Hyaluronidase can
"destroy" the hyaluronic acid around the tumor tissue, and has a
certain inhibitory effect on the tumor growth. Using hyaluronidase
as a dispersant can hydrolyze mucopolysaccharide, promote the
absorption of therapeutic drugs, and accelerate the diffusion and
absorption of local accumulation fluid and exudate. In addition, as
an anesthetic assistant, hyaluronidase can effectively promote the
dispersion and absorption of anesthetic drugs, shorten the duration
of anesthesia, and increase the depth of anesthesia. However, many
problems have been found in the application of hyaluronidase.
Therefore, the new hyaluronidase with new properties will become
the research hotspot in the future. So far, hyaluronidase has been
reported mainly from streptococcus, while the enzymes from
Enterobacter sp. has never been reported.
SUMMARY OF THE INVENTION
[0008] In view of the present hyaluronidase used widely, but the
shortage of enzyme resources, the present invention provides a kind
of Enterobacter CGJ001 (Enterobacter sp.), the species preservation
in China General Microbiological Culture Collection Center (CGMCC)
on Oct. 10, 2019, microbial preservation management committee
address: Beijing Chaoyang District Beichen West Road No. 1 Hospital
No. 3, the Institute of Microbiology, Chinese Academy of Sciences,
preservation number CGMCC NO. 18661.
[0009] The above-mentioned Enterobacter sp. CGJ001 is gram-negative
with short rod-shaped bacteria, as shown in FIG. 1.about.3.
[0010] The Enterobacter sp. CGJ001 strain has been molecularly
identified and sequenced, and its 16S rRNA gene sequence length is
1381 bp, as shown in SEQ NO. 1. By using the BLAST program of the
National Center for Biotechnology Information (NCBI), the results
show that the 16S rDNA sequence of this strain is comparable to
that of Enterobacter (JQ795804.1, KJ184972.1, MH883957.1, etc.) The
related sequences have more than 99.8% homology, and they are
finally classified as Enterobacter strains.
[0011] In one embodiment, the Enterobacter CGJ001 has the effect of
degrading hyaluronic acid.
[0012] Another object of the present invention is to provide an
application of Enterobacter CGJ001 in the production of
hyaluronidase.
[0013] In one embodiment, hyaluronidase was prepared from the
Enterobacter CGJ001 through plate culture, seed culture and
fermentation culture.
[0014] In one embodiment, the application specifically includes the
following steps:
[0015] (1) Plate culture of Enterobacter CGJ001 to obtain plate
strains;
[0016] (2) Inoculate the plate strains into the sterilized seed
culture medium and incubate at 30-40.degree. C. and 150-300 rpm for
12-24 h to obtain seed liquid;
[0017] (3) Inoculate the seed liquid into a sterilized fermentation
medium and incubate at 30-40.degree. C. and 150-300 rpm for 12-24 h
to obtain a hyaluronidase-containing bacterial liquid.
[0018] In one embodiment, the seed medium and fermentation medium
components include 1-10 g/L hyaluronic acid, 1.about.5 g/L
K.sub.3PO.sub.4, 0.1.about.1 g/L MgSO.sub.4, 1.about.10 g/L
peptone, 1.about.10 g/L yeast powder.
[0019] In one embodiment, the pH value of the seed medium and the
fermentation medium is 6-8.
[0020] In one embodiment, the composition per liter of the plate
culture medium is as follows:
[0021] Peptone 1.about.10 g, yeast powder 1.about.10 g, sodium
hyaluronate 1.about.10 g, K3PO4.3H2O 1.about.5 g, MgSO4.7H20
0.1.about.1 g, water 1000 mL, agar powder 15.about.25 g, The pH
value is 6-8.
[0022] The above-mentioned Enterobacter CGJ001 bacterial liquid
prepares the crude hyaluronidase liquid, the steps are as
follows:
[0023] (i) Centrifuge the bacterial liquid of Enterobacter CGJ001
obtained in step (3) to obtain the bacterial cells, and the
conditions of the centrifugation are: 6000 rpm for 10-15 min;
[0024] (ii) Discard the supernatant of the above centrifugal
liquid, add an equal volume of PBS solution to resuspend the
bacteria;
[0025] (iii) The above-mentioned resuspended bacterial cells were
subjected to ultrasonic cell disruption for 20 minutes to prepare a
crude hyaluronidase solution.
[0026] The components per liter of the ABOVE PBS solution are as
follows:
[0027] 2.about.3 g of sodium dihydrogen phosphate, 0.5.about.2 g of
anhydrous disodium hydrogen phosphate and 5.about.10 g of sodium
chloride, 1000 mL of water, pH5-7.
[0028] Hydrochloric acid and sodium hydroxide were used to adjust
the pH of the above PBS solution.
[0029] The parameters of the ultrasonic crushing instrument were
set as follows: ultrasonic time 20 min, breaking for4 S and
stopping for 6 S, power 300 W.
[0030] In one embodiment, the hyaluronidase prepared by any of the
above-mentioned hyaluronidase preparation methods is analyzed by
mass spectrometry, and the degradation product of hyaluronidase by
the enzyme is 2-6 sugars.
[0031] The invention has the advantages of:
[0032] The Enterobacter CGJ001 of the present invention has
excellent enzyme activity, strong specificity for hyaluronic acid,
good thermal stability and pH stability, and is suitable for
scale-up production, thereby replacing hyaluronidase extracted from
expensive animal tissues. It has a broad application prospect in
the fields of medicine and cosmetics.
BRIEF DESCRIPTION OF THE DRAWINGS
[0033] FIG. 1 shows the colony morphology of Enterobacter CGJ001
strain on solid medium.
[0034] FIG. 2 shows the optical microscope photos of Enterobacter
CGJ001 strain.
[0035] FIG. 3 shows the electron microscope images of Enterobacter
CGJ001 strain.
[0036] FIG. 4 shows the effect of degradation of crude
hyaluronidase enzyme solution on the viscosity of sodium
hyaluronate.
[0037] FIG. 5 shows the optimal reaction temperature of the crude
hyaluronidase solution.
[0038] FIG. 6 shows the optimal pH of the crude hyaluronidase
enzyme solution in Example 5.
[0039] FIG. 7 shows the mass spectrometric analysis of unsaturated
oligosaccharides prepared by crude enzyme solution in example
5.
DESCRIPTION OF PREFERRED EMBODIMENTS
[0040] The detailed implementation of the invention is further
described as follows. The following embodiments are used to
illustrate the invention, but not to limit the scope of the
invention. Technicians in this field can clearly understand the
characteristics and efficacy of the invention from the contents
explained in this specification, and the invention can also be
implemented or applied in other specific ways.
EXAMPLE 1
[0041] A polluted river water sample from Wuxi, Jiangsu province
was taken and 1 mL supernatant was added to 9 mL normal saline,
which was diluted to 5 concentration gradients of 10.sup.-4,
10.sup.-5, 10.sup.-6, 10.sup.-7, 10.sup.-8, respectively. The
diluted bacterial suspension was coated on the screening
medium.
[0042] Two parallel cultures under each concentration were carried
out at a concentration of 30.degree. C. for 5 days. The single
colonies with good growth were picked out, then seeded in liquid
culture and coated in solid medium. Then the single colony was
selected in liquid culture medium for cultivation at 30.degree. C.
and 220 rpm for 24 h. 0.9 mL of the culture substance was added to
0.9 mL40% glycerol, which was mixed and stored in the refrigerator
at -80.degree. C. for a long time.
[0043] The components of the above screening medium per liter are
as follows:
[0044] Sodium hyaluronate (5 g), K.sub.3PO.sub.4.3H.sub.2O (2 g),
MgSO.sub.4.7H.sub.2O (0.5 g), water (1000 mL), and agar (20 g) were
added as the screening medium.
[0045] The components per liter of the seed medium are as
follows:
[0046] Peptone 5 g, yeast 5 g, sodium hyaluronate 5 g,
K.sub.3PO.sub.4.3H.sub.2O 2 g, MgSO.sub.4.7H.sub.2O 0.5 g, water
1000 mL, pH 6.
[0047] The components of the solid medium per liter are as
follows:
[0048] Peptone 5 g, yeast 5 g, sodium hyaluronate 5 g,
K.sub.3PO.sub.4.3H.sub.2O 2 g, MgSO.sub.4.7H.sub.2O 0.5 g, water
1000 mL, pH 6, and agar 20 g were added.
[0049] The application of Enterobacter CGJ001 in the production of
hyaluronidase is as follows:
[0050] (1) Take Enterobacter CGJ001 for plate culture to obtain
plate strains;
[0051] (2) Inoculate the plate strains into the sterilized seed
culture medium, and incubate at 30.degree. C. and 150 rpm for 12
hours to obtain seed liquid;
[0052] (3) Inoculate the seed liquid into a sterilized fermentation
medium, and cultivate for 12 hours at 30.degree. C. and 150 rpm to
obtain a hyaluronidase-containing bacterial liquid.
[0053] The seed medium and fermentation medium components include 1
g/L hyaluronic acid, 1 g/L K.sub.3PO.sub.4, 0.1g/L MgSO.sub.4, 1
g/L peptone, and 1 g/L yeast powder. The pH value of the seed
medium and the fermentation medium is 6.
[0054] The components per liter of the flat culture medium are as
follows:
[0055] Peptone 5 g, yeast 5 g, sodium hyaluronate 5 g,
K.sub.3PO.sub.4.3H.sub.2O 2 g, MgSO.sub.4.7H.sub.2O 0.5 g, water
1000 mL, agar 20 g, pH 6.
[0056] The above-mentioned bacterial solution of Enterobacter
CGJ001 was used to prepare crude hyaluronidase solution. The steps
are as follows:
[0057] (i) Take step (3) The hyaluronidase containing bacteria
solution prepared by centrifugation to obtain the bacteria body,
the centrifugation conditions are as follows: 6000 rpm for 10
min;
[0058] (ii) Discard the supernatant of the centrifugal liquid and
add the bacteria to PBS solution for resuspended suspension at the
same volume;
[0059] (iii) The resuspended bacteria were broken by ultrasonic
disruption for 20 min to prepare the crude hyaluronidase
solution.
[0060] The components per liter of the above PBS solution are as
follows:
[0061] 2.5 g sodium dihydrogen phosphate, 1.0 g disodium hydrogen
phosphate and 8.2 g sodium chloride anhydrous, 1000 mL water, pH
6.2.
[0062] Hydrochloric acid and sodium hydroxide are used to adjust
the pH of the above PBS solution.
[0063] The parameters of the ultrasonic disruption instrument are
set as follows: ultrasonic time 20 min, breaking 4 S and stopping 6
S, power 300 W.
EXAMPLE 2
[0064] The specific method for measuring the enzyme activity of
crude hyaluronidase solution is as follows:
[0065] Preparation of DNS solution:
[0066] Weigh (10.+-.0.1) g of 3,5-dinitrosalicylic acid, place it
in about 600 mL of water, gradually add 10 g of sodium hydroxide,
stir to dissolve in a 50.degree. C. water bath (magnetic force),
and then add 200 g of potassium tartrate Sodium, phenol 2 g, and
anhydrous sodium sulfite 5 g sequentially. After all above were
dissolved and clarified, cool it to room temperature, dilute to
1000 mL with water, and filter. Store it in a brown reagent bottle,
and place it in a dark place for 7 days. (Preparation of standard
DNS reagent according to the standard procedure of Ministry of
Light Industry)
[0067] The total reaction system was 3 mL. Add 0, 50, 100, 150, 200
.mu.L glucose standard solution (2 mg/mL) to 2 mL DNS solution
respectively, add water to make up to 3 mL, boil it in a boiling
water bath for 10 minutes, and then cool it to room temperature,
and add water to make up to 10 mL. The absorbance was measured at
540 nm, the absorbance was taken as the abscissa, and the glucose
concentration was taken as the ordinate to make a standard
curve.
[0068] The determination steps of the crude hyaluronidase solution
sample are as follows:
[0069] The following reagents are required: 2 mg/mL hyaluronic
acid, 50 mmol/L, pH 6.0 PBS buffer. The reaction system was 1 mL,
including 800 .mu.L hyaluronic acid, 100 .mu.L of crude
hyaluronidase solution, adding PBS buffer to make up 1 mL, reacting
in a 39.degree. C. water bath for 15 min. The absorbance value was
measured and substituted into the standard curve to obtain the
reduced equivalent glucose mass concentration, replacing the
glucose standard solution with 1 mL of the reaction sample.
[0070] A unit of enzyme activity is defined as: under the above
experimental conditions, the amount of enzyme required to produce 1
.mu.g of glucose per 1 h is defined as a unit of enzyme
activity.
[0071] The Enterobacter CGJ001 of the present invention has a
fermentation enzyme activity of 8139 U/mL.
EXAMPLE 3
[0072] As shown in FIG. 1, the solid medium plate, optical
microscope and electron microscope morphology of Enterobacter
CGJ001 were observed and identified. The results showed that the
colony of the strain on the hyaluronic acid solid medium plate was
a convex colony, the surface was smooth, moist, crystal clear, and
the color was milky white, which was easy to pick. When observed
under a light microscope, the cells are short rod-shaped. This
strain is a short gram-negative rod-shaped bacterium, without
capsule, spores and flagella, and can grow with hyaluronic acid as
the only carbon source.
[0073] The above general primers for gene amplification of
bacterial strains are:
TABLE-US-00001 The forward primer is 27f: 5'-AGTTTGATCCTG GCT
CAG-3'; The reverse primer is 1492r:
5'-GCTTACCTTGTTACGACTT-3'..
[0074] The above reaction system for strain gene amplification is
as follows, the total volume is 20 .mu.L:
TABLE-US-00002 Forward primer 27f with a concentration of 1 .mu.L
10 mmol/L Reverse primer 1492r with a concentration of 1 .mu.L 10
mmol/L 2xES-taq enzyme 10 .mu.L Ultra-pure water 8 .mu.L
[0075] And pick a single colony on the solid medium and mix it in
the system.
[0076] gene amplification reagents used were purchased from Sangon
Biotech (Shanghai) Co., Ltd.
[0077] The above procedures for strain gene amplification were
pre-denaturation at 95.degree. C. for 10 min, denaturation at
95.degree. C. for 30 S, annealing at 55.degree. C. for 30 S,
extension at 72.degree. C. for 90 S, a total of 34 cycles,
extension at 72.degree. C. for 15 min, and insulation at 4.degree.
C. .
[0078] The 16S rDNA sequence length of this strain is 1381bp. Blast
comparison of the sequence in NCBI database shows that 16S rDNA
sequence of this strain is over 99.8% homologous with relevant
sequences of Enterobacter (JQ795804.1, KJ184972.1, MH883957.1,
etc.), and finally it is classified as Enterobacter strains.
Enterobacter sp. CGJ001 was identified by combining its
morphological and physiological and biochemical characteristics.
This strain has been stored in the China General Microbiological
Culture Collection Center, with the storage address being No. 3,
Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the
preservation number is CGMCC No. 18661.
EXAMPLE 4
[0079] The crude hyaluronidase enzyme solution prepared by Example
1 was added to 50 mL 10 g/mL sodium hyaluronate solution according
to 5% enzyme dosage, and was put into a thermostatic water bath at
39.degree. C. for reaction. The initial viscosity of the system was
1405 cP. The viscosity of the system was measured every 10 min, 1
mL was sampled, and the absorbance was inactivated by boiling for 2
min. Then, DNS method was used to measure the absorbance at
different time periods.
[0080] As shown in FIG. 4, with the increase of enzymatic
hydrolysis time, the viscosity of sodium hyaluronate solution
decreases, and the absorbance of DNS reaction of the whole system
increases. This result indicates that as the time of enzymatic
hydrolysis increases, the viscosity of sodium hyaluronate
decreases, and the number of oligosaccharides with reducing ends
produced increases.
[0081] Using the crude enzyme preparation from Enterobacter CGJ001
to analyze the degradation ability of different
polysaccharides:
[0082] After mixing the polysaccharide substrate with a
concentration of 2 mg/mL, PBS buffer and the crude hyaluronidase
enzyme solution prepared in Example 1 in a ratio of 8:1:1 (volume
ratio), the mixed solution was reacted at 39.degree. C. for 4
hours, incubated in a boiling water bath for 5 minutes to
inactivate the enzyme, and centrifuged at 8000 rpm at 4.degree. C.
for 10 minutes. Then the supernatant was taken as the enzymolysis
product of the crude enzyme liquid preparation prepared by
Enterobacter CGJ001. The reducing sugar produced was detected by
the DNS method.
[0083] The results are shown in Table 1. The crude hyaluronidase
enzyme solution prepared by Enterobacter CGJ001 has high
specificity for hyaluronic acid, and no degradation ability for
sodium alginate and chitosan, so it has potential application
value.
TABLE-US-00003 TABLE 1 Degradation ability on different
polysaccharides of crude enzyme solution prepared by Enterobacter
CGJ001 Types of Sodium Sodium polysaccharides hyaluronate
Chitooligosaccharides Alginate Ability of + - - degradation Note:
"+" means biodegradable, "-" means non-biodegradable.
[0084] The results of mass spectrometry in anion mode showed that
the oligosaccharide products obtained by degrading hyaluronic acid
with the crude hyaluronic acid solution prepared in Example 1 were:
hyaluronic acid disaccharide with a molecular weight of 379, the
transparent hyaluronic acid with a molecular weight of 758, and the
tetrasaccharide of tetrasaccharide, hyaluronic hexasaccharide with
a molecular weight of 1137, as shown in FIG. 7.
EXAMPLE 5
[0085] After mixing hyaluronic acid with a concentration of 2
mg/mL, PBS buffer and the crude hyaluronidase enzyme solution
prepared in Example 1 at a ratio of 8:1:1 (volume ratio), three
parallel experiments were performed. Put them in a water bath at
35.degree. C., 37.degree. C., 39.degree. C., 41.degree. C., and
43.degree. C. separately for 1 hour, and then remove them into
another water bath at 100.degree. C. for 5 minutes to inactivate.
Then add 2 ml DNS reagent separately and put them in a 100.degree.
C. water bath to react for 10 minutes, then take them out and cool
to room temperature. Measure their absorbance with UV
spectrophotometer at 540 nm.
[0086] Relative enzyme activity is defined as: the percentage of
the average absorption value of each group and the maximum
absorption value. The maximum absorption value corresponds to the
optimal reaction temperature of the crude enzyme solution.
[0087] The results of the optimal reaction temperature of the crude
hyaluronidase enzyme solution are shown in FIG. 5, and the optimal
reaction temperature of the crude hyaluronidase enzyme solution is
39.degree. C.
[0088] Research on the optimum Reaction pH of crude hyaluronidase
enzyme solution
[0089] Prepare hyaluronic acid solutions with a concentration of 2
mg/mL using phosphate buffers with pH 4, 5, 6, 7, and 8,
respectively. After the hyaluronic acid is completely dissolved, it
is incubated at the optimum temperature of 39.degree. C., and then
100 .mu.L of the crude hyaluronic acid solution prepared in Example
1 is added to every 900 .mu.L of the hyaluronic acid solution of
different pH. After mixing them uniformly, they were placed in a
39.degree. C. water bath to react for lh, and 3 parallel samples
were set up under each condition.
[0090] Relative enzyme activity is defined as: the percentage of
the average absorption value of each group and the maximum
absorption value. The maximum absorption value corresponds to the
optimum pH of the crude enzyme solution.
[0091] The results of the optimal reaction pH of the crude
hyaluronidase solution are shown in FIG. 6, and the optimal
reaction pH of the crude hyaluronidase solution is 6-7.
[0092] The above-mentioned examples are only preferred examples for
fully explaining the present invention, and the protection scope of
the present invention is not limited thereto. The equivalent
substitution or transformation made by technicians in the technical
field on the basis of the present invention shall be within the
protection scope of the present invention. The scope of protection
of the invention shall be governed by the claim.
Sequence CWU 1
1
111381DNAEnterobacter sp. 1gtagcgccct cccgaaggtt aagctaccta
cttcttttgc aacccactcc catggtgtga 60cgggcggtgt gtacaaggcc cgggaacgta
ttcaccgtag cattctgatc tacgattact 120agcgattccg acttcatgga
gtcgagttgc agactccaat ccggactacg acgcacttta 180tgaggtccgc
ttgctctcgc gaggtcgctt ctctttgtat gcgccattgt agcacgtgtg
240tagccctact cgtaagggcc atgatgactt gacgtcatcc ccaccttcct
ccagtttatc 300actggcagtc tcctttgagt tcccggcctg accgctggca
acaaaggata agggttgcgc 360tcgttgcggg acttaaccca acatttcaca
acacgagctg acgacagcca tgcagcacct 420gtctcagagt tcccgaaggc
accaatccat ctctggaaag ttctctggat gtcaagagta 480ggtaaggttc
ttcgcgttgc atcgaattaa accacatgct ccaccgcttg tgcgggcccc
540cgtcaattca tttgagtttt aaccttgcgg ccgtactccc caggcggtcg
acttaacgcg 600ttagctccgg aagccacgcc tcaagggcac aacctccaag
tcgacatcgt ttacggcgtg 660gactaccagg gtatctaatc ctgtttgctc
cccacgcttt cgcacctgag cgtcagtctt 720tgtccagggg gccgccttcg
ccaccggtat tcctccagat ctctacgcat ttcaccgcta 780cacctggaat
tctacccccc tctacaagac tctagcctgc cagtttcgaa tgcagttccc
840aggttgagcc cggggatttc acatccgact tgacagaccg cctgcgtgcg
ctttacgccc 900agtaattccg attaacgctt gcaccctccg tattaccgcg
gctgctggca cggagttagc 960cggtgcttct tctgcgggta acgtcaatcg
acaaggttat taaccttatc gccttcctcc 1020ccgctgaaag tactttacaa
cccgaaggcc ttcttcatac acgcggcatg gctgcatcag 1080gcttgcgccc
attgtgcaat attccccact gctgcctccc gtaggagtct ggaccgtgtc
1140tcagttccag tgtggctggt catcctctca gaccagctag ggatcgtcgc
ctaggtgagc 1200cgttacccca cctactagct aatcccatct gggcacatct
gatggcaaga ggcccgaagg 1260tccccctctt tggtcttgcg acgttatgcg
gtattagcta ccgtttccag tagttatccc 1320cctccatcag gcagtttccc
agacattact cacccgtccg ccgctcgtca cccaagagca 1380a 1381
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