CN108588157A - The method that combination enzymic degradation degreasing euphausia superba powder recycles polypeptide intermediate product and prepares N-Acetyl-D-glucosamine - Google Patents
The method that combination enzymic degradation degreasing euphausia superba powder recycles polypeptide intermediate product and prepares N-Acetyl-D-glucosamine Download PDFInfo
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
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Abstract
Method enzymic degradation degreasing euphausia superba powder recycling polypeptide intermediate product the invention discloses combination and prepare N acetylglucosamines；The pretreatment for first relating to degreasing euphausia superba powder recycles the polypeptide in degreasing euphausia superba powder by basic protein enzyme pretreatment, so that raw material is fully used, and improve chitin relative amount in degreasing euphausia superba powder.Next is related to the united hydrolysis of three kinds of chitinases, so that hydrolysis efficiency is greatly improved by united hydrolysis, and the yield of reduced sugar is made to be improved.Finally, N acetylglucosamines are obtained by mixing addition OfHex1 enzymes in enzyme system, and hydrolysis efficiency is very high, makes it possible that combination enzyme process utilizes degreasing euphausia superba powder to produce N acetylglucosamines.
Description
Technical field
The invention belongs to biotechnologies, and in particular among combination enzymic degradation degreasing euphausia superba powder recycling polypeptide
Product and the method for preparing N-Acetyl-D-glucosamine.
Background technology
N-Acetyl-D-glucosamine (GlcNAc) is a kind of glucosan derivative, often with β-Isosorbide-5-Nitrae glucosides key connection at high polymer
It is present in the constituent of a variety of biologies.Because it is with multiple biological activities, such as repair joint injury, treat enterogastritis and
Skin-whitening etc. is widely used in edible cosmetic product additive.
The method of industrial production N-Acetyl-D-glucosamine is presently mainly to be synthesized using chemical method, and low output is of high cost,
Environmental pollution is serious.Another method is to produce N-Acetyl-D-glucosamine, chitin conduct by chitin (chitin) of degrading
Second largest biomass of reserves in nature is a kind of raw material cheap and easy to get, and the method for traditional chitin degrading is using strong
The shortcomings of sour water solution, it is more serious that there are environmental pollutions, and product is difficult to detach there are by-product.
In recent years, enzymic degradation chitin production N-Acetyl-D-glucosamine is because of its product quality height, and bioactivity is good, environment
It pollutes small as a kind of emerging technique full of potentiality.However, there are still challenges at present for Production by Enzymes.One be raw material selection
With processing, the other is the production efficiency of enzyme.It, should be in the selection of raw material and with upper alternative costs for two above problem
Low raw material improves the production efficiency of enzyme again.Known krill rich content, every year catching in the case where not influencing the ecological balance
The amount of obtaining is 50,000,000 tons, and this quantity is far not achieved in the quantity of the catch in the world.And krill is used only for the extraction of shrimp sauce at present,
And remaining residue is effectively utilized not yet.
Invention content
The present invention in order to solve the problems, such as described in background technology and, disclose a kind of combination enzymic degradation degreasing South Pole phosphorus
The method of shrimp med polypeptide intermediate product recycling, and the method for preparing N-Acetyl-D-glucosamine.
First, the method for the degradation degreasing euphausia superba powder recycling polypeptide intermediate product comprising following step：
Degreasing euphausia superba powder is dissolved in mixing in the buffer solution of pH7.5-8.5, adds 10000-15000U basic proteins
Enzyme, water-bath 20-24h under the conditions of 40-45 DEG C；Suction filtration obtains pretreatment degreasing euphausia superba powder and supernatant containing albumen；With spraying
Processing is dried to pretreated degreasing euphausia superba powder in seasoning, obtains drying sample.For using obtained by the method
Drying sample in content of peptides be detected, respectively to untreated samples and processing after degreasing euphausia superba powder carry out kelvin
Determine nitrogen, it is about 40% that pretreatment recycling content of peptides, which is calculated,；Wherein, it includes at least 17 kinds of ammonia based on aspartic acid to contain
Base sour component.
In the case of preferred, in method as discussed above, the degreasing euphausia superba powder is dissolved in the ratio of buffer solution
For (1~2) g:(3~8) mL.Further preferably, the ratio that degreasing euphausia superba powder is dissolved in buffer solution is 1g：4mL.Preferred feelings
Under condition, in method as discussed above, the buffer solution be selected from phosphate buffer, PBS buffer solution, Tris buffer solutions and
Bis-Tris buffer solutions.
A kind of method that combination enzymic degradation degreasing euphausia superba powder prepares N-Acetyl-D-glucosamine of the present invention,
Include the following steps：
The drying sample obtained in the method for the recycling polypeptide intermediate product is added into the buffer solution of pH5.5-6.5,
Make its final concentration of 15-25mg/mL, adds final concentration of 6~24 μM of group synthase, 40 DEG C of water-baths 4~for 24 hours；The group
Synthase is SmChiA mutant, SmChiB, SmChiC and OfHex1；The additive amount of four kinds of enzymes is respectively 43 in said combination enzyme
~45%, 35~37%, 17~19%, 0~1.5%.
In the case of preferred, in method as described above, group synthase SmChiA mutant, SmChiB,
The additive amount of SmChiC and OfHex1 is respectively 43.90%, 35.90%, 18.90%, 1.30%.
In the case of preferred, in method as discussed above, group synthase SmChiA mutant, SmChiB,
Final concentration of 6 μM of SmChiC, OfHex1.
In the case of preferred, in method as discussed above, the obtained final concentration of 20mg/mL of drying sample.Institute
It is pH6.0 to state buffer solution.
Advantageous effect
Method as described above through the invention first degrades to degreasing euphausia superba powder and recycles polypeptide intermediate product,
It is about 40% to obtain recycling content of peptides；Wherein, it includes at least 17 kinds of aminoacid ingredients based on aspartic acid to contain；As it can be seen that
Combination enzymic degradation degreasing euphausia superba powder of the present invention can harvest the higher polypeptide intermediate product of content, raw material is made to obtain
To making full use of, and improve chitin relative amount in degreasing euphausia superba powder.
Then the synergetic hydrolysis for carrying out SmChiA mutant, SmChiB, SmChiC to recycling polypeptide intermediate product again, takes
Hydrolysate measures the light absorption value A405 at 405nm with ELISA Plate.Synergetic hydrolysis is measured to obtain (GlcNAc)2It is a concentration of
1.38mM.This hydrolysis effect is that single enzyme hydrolysis is maximum (GlcNAc)22 times of concentration (0.69mM).
In order to further increase hydrolysis efficiency, on the basis of former reaction system, increases OfHex1 enzymes in group synthase, that is, use
SmChiA mutant, SmChiB, SmChiC and OfHex1 synergetic hydrolysis are after testing using the method products therefrom
GlcNAc.It measures synergetic hydrolysis and obtains a concentration of 5.63mM of GlcNAc.The reaction time is advanced optimized, will be improved in the reaction time
To 24 hours, a concentration of 11.57mM of GlcNAc are measured, hydrolysis efficiency reaches 29.7% (such as Fig. 3).Further to improve hydrolysis
Efficiency increases the concentration of enzyme to 24 μM under conditions of concentration of substrate and constant final volume.Obtain a concentration of of GlcNAc
12.23mM, hydrolysis efficiency are promoted to 34.5% (such as Fig. 4).Products therefrom is handled through high performance liquid chromatography (HPLC), detection production
The purity of object, it can be found that products therefrom is GlcNAc (such as Fig. 5 A) compared with standard items.
Description of the drawings
Fig. 1 is SmChiA mutant, SmChiB and the single hydrolysis degreasing euphausia superba powder of tri- enzymes of SmChiC and the combination of three enzymes
Hydrolysis degreasing euphausia superba powder obtains (GlcNAc)2Concentration.The result shown from figure clearly can be seen that SmChiA
Hydrolysis efficiency after the combination of tri- enzyme of mutant, SmChiB, SmChiC is significantly larger than the efficiency of single enzyme hydrolysis, is single enzyme hydrolysis
2-4 times of efficiency.Fig. 2 is the mould using Minitab software predictions SmChiA mutant, SmChiB and SmChiC optimum proportionings
Quasi- figure.Hydrolysis efficiency is higher when SmChiA mutant and SmChiB concentration is high in degreasing krill hydrolytic process, and right
SmChiC concentration necessaries want lower.Fig. 3 is to increase the GlcNAc and (GlcNAc) obtained after the reaction time2Concentration and anti-
Relationship change figure between seasonable.SmChiA mutant, the hydrolysis efficiency of tri- enzyme of SmChiB, SmChiC and SmChiA mutant,
The hydrolysis efficiency of tetra- enzyme of SmChiB, SmChiC, OfHex1 all increases as time increases, it was demonstrated that the experiment can pass through
Increase the reaction time to improve hydrolysis efficiency.
Fig. 4 is GlcNAc and (GlcNAc) after the concentration for increasing enzyme2Concentration and enzyme concentration relationship change figure.From diagram
SmChiA mutant, the hydrolysis efficiency of tri- enzyme of SmChiB, SmChiC and SmChiA mutant known to interpretation of result, SmChiB,
The hydrolysis efficiency of tetra- enzyme of SmChiC, OfHex1 all increases with the increase of enzyme concentration, it was demonstrated that the experiment can pass through increase
Enzyme concentration in reaction system improves hydrolysis efficiency.
Fig. 5 is the purity that hydrolyzate detects product through high performance liquid chromatography.Fig. 5 A be SmChiA mutant, SmChiB,
Product after SmChiC and OfHex1 synergetic hydrolysis.After Fig. 5 B are SmChiA mutant, SmChiB and SmChiC synergetic hydrolysis
Product.All it is monosaccharide from can be seen that system hydrolysate from Fig. 5 A known to graphical results analysis, and can be seen in Fig. 5 B
Go out, system hydrolysate is all disaccharides, it was demonstrated that the product of this method hydrolysis is all single product and purity is higher.
Specific implementation mode
Following non-limiting embodiments can make those skilled in the art be more fully understood the present invention, but not with
Any mode limits the present invention.Any one skilled in the art in the technical scope of present disclosure, according to
Technical scheme of the present invention and its inventive concept carry out equivalent substitution or change and belong to protection category of the present invention.
In order to facilitate statement, in the examples below, " mM " indicates mmol/L；" μM " expression " μm ol/L "；The item of sterilizing
Part is high pressure sterilization 20min at 121 DEG C.
Degreasing euphausia superba powder：It is provided by Liao Yu groups, by the krill residue for taking out shrimp sauce processing.
Embodiment 1
1) degreasing euphausia superba powder pre-processes
It weighs 100g degreasing euphausia superba powders and adds 400mL phosphate buffers (pH 8.0) mixing, then be charged with
1000U alkali proteases (be purchased from Novozymes), water-bath is for 24 hours under the conditions of 40 DEG C.Sample is filtered with Buchner funnel,
Obtain pretreatment degreasing euphausia superba powder and supernatant containing albumen.With spray drying process to pretreated degreasing euphausia superba powder into
Row is dried, and obtains drying sample.Kjeldahl determination is carried out to degreasing euphausia superba powder after untreated samples and processing, is calculated
It is about 40% to pretreatment recycling content of peptides.
The aminoacid ingredient of gained supernatant is analyzed with amino-acid analyzer, the ingredient for obtaining each amino acid in product is：
It is aspartic acid, threonine, serine, glutamic acid, glycine, alanine, cystine, valine, methionine, isoleucine, bright
Propylhomoserin, tyrosine, phenylalanine, lysine, histidine, arginine, proline, content are respectively：9.68%, 1.59%,
1.36%, 4.16%, 1.48%, 1.83%, 0.59%, 1.56%, 1.74%, 1.71%, 2.66%, 1.75%, 1.67%,
2.61%, 0.94%, 2.13%, 1.08%.(note：Other unlisted amino acid expressions do not detect amino acid, do not indicate that be free of and be somebody's turn to do
Amino acid)
1) preparation of SmChiA mutant, SmChiB and SmChiC
The preparation of SmChiA mutant：The coli strain for taking the expressing genes of mutant containing SmChiA of freezing,
37 DEG C activate to OD600For 0.6-0.8 when, add 0.5mM IPTG.Then, cell is in 37 DEG C of induced expression 5h.In 4 DEG C of conditions
Under, 8000rpm centrifuges 5min and collects thalline, and is resuspended in Buffer A (20mM sodium dihydrogen phosphates, 500mM sodium chloride, pH
7.4) in.Using high pressure homogenization crusher machine thalline, pressure 800bar.Under the conditions of 4 DEG C, 10000rpm centrifuges 10min, removal
Unbroken thalline and inclusion body, 0.22 μm of filter filter supernatant.Albumen is isolated and purified using metal chelate chromatography, albumen exists
Impurity protein is eluted under Buffer C (20mM sodium dihydrogen phosphates, 500mM sodium chloride, 50mM imidazoles, pH 7.4), albumen exists
It elutes and collects under Buffer E (20mM sodium dihydrogen phosphates, 500mM sodium chloride, 150mM imidazoles, pH 7.4).It is bright with coomassie
Blue laws detects albumen concentration, uses the purity of SDS-PAGE test-target albumen.
The preparation of SmChiB：The coli strain for taking the expressing gene containing SmChiB of freezing is activated at 37 DEG C to OD600
For 0.6-0.8 when, add 0.05mM IPTG.Then, cell is in 16 DEG C of induced expression 12-14h.Under the conditions of 4 DEG C, 8000rpm
It centrifuges 5min and collects thalline, and be resuspended in Buffer A (20mM sodium dihydrogen phosphates, 500mM sodium chloride, pH 7.4).Using height
Press homogeneous crusher machine thalline, pressure 800bar.Under the conditions of 4 DEG C, 10000rpm centrifuges 10min, removes unbroken thalline
And inclusion body, 0.22 μm of filter filter supernatant.Albumen is isolated and purified using metal chelate chromatography, albumen is in Buffer E
Impurity protein is eluted under (20mM sodium dihydrogen phosphates, 500mM sodium chloride, 150mM imidazoles, pH 7.4), albumen is in Buffer F
It elutes and collects under (20mM sodium dihydrogen phosphates, 500mM sodium chloride, 300mM imidazoles, pH 7.4).It is detected with Coomassie Brilliant Blue
Albumen concentration uses the purity of SDS-PAGE test-target albumen.
The preparation of SmChiC：The coli strain for taking the expressing gene containing SmChiC of freezing is activated at 37 DEG C to OD600
For 0.6-0.8 when, add 0.1mM IPTG.Then, cell is in 16 DEG C of induced expression 12-14h.Under the conditions of 4 DEG C, 8000rpm
It centrifuges 5min and collects thalline, and be resuspended in Buffer A (20mM sodium dihydrogen phosphates, 500mM sodium chloride, pH 7.4).Using height
Press homogeneous crusher machine thalline, pressure 800bar.Under the conditions of 4 DEG C, 10000rpm centrifuges 10min, removes unbroken thalline
And inclusion body, 0.22 μm of filter filter supernatant.Albumen is isolated and purified using metal chelate chromatography, albumen is in Buffer D
Impurity protein is eluted under (20mM sodium dihydrogen phosphates, 500mM sodium chloride, 75mM imidazoles, pH 7.4), albumen is in Buffer E
It elutes and collects under (20mM sodium dihydrogen phosphates, 150mM sodium chloride, 300mM imidazoles, pH 7.4).It is detected with Coomassie Brilliant Blue
Albumen concentration uses the purity of SDS-PAGE test-target albumen.
Embodiment 2
1) making of standard curve
Respectively using chitobiose and chitin monosaccharide as standard items, by standard items be configured to various concentration (be respectively 0,0.5,
1,1.5,2mM) sample, take 60 μ L samples that 180 μ L potassium ferricyanide solutions (2g/L), boiling water bath 15min is added to take 200 μ L to survey and inhale
Shading value A405 makes reduced sugar standard curve.
2) SmChiA mutant, SmChiB, SmChiC group synthase hydrolyze degreasing euphausia superba powder respectively
SmChiA mutant hydrolyze degreasing euphausia superba powder：Reaction system is 200 μ L, and the end of degreasing euphausia superba powder is dense
Degree is 20mg/mL, and the final concentration of SmChiA mutant is 6 μM, insufficient volume 20mM phosphate buffer polishings.It is right
It is same as above according to a group each component, then boiling water bath 5min makes enzyme lose activity.It is put into 40 DEG C of water-baths and reacts 8h；60 μ L are taken out to be added to
Rear that 180 μ L 2g/L potassium ferricyanide solutions are added in another 1.5mL centrifuge tube, then boiling water bath boils 15min, and taking-up is put into
12000rpm centrifuges 3min in centrifuge；It finally draws 200 μ L supernatants and is added to ELISA Plate, measure the light absorption value at 405nm
A405。
SmChiB and SmChiC hydrolysis degreasing euphausia superba powder step is same as above.
Combine enzyme hydrolysis degreasing euphausia superba powder：By software (minitab17) simulate SmChiA mutant, SmChiB,
When the optimal proportion of tri- enzyme hydrolysis degreasing euphausia superba powders of SmChiC is 44.50%, 36.30%, 13.30%, hydrolysis can be made to imitate
Rate reaches maximum, using the practical hydrolysis efficiency of experiment detection combination enzyme and theoretical value, determines optimal proportion (such as Fig. 2).Degreasing
The final concentration of euphausia superba powder is about 20mg/mL, SmChiA mutant, final concentration of 6 μM of SmChiB, SmChiC, three enzymes
Adding proportion is 44.50%, 36.30%, 13.30%, insufficient volume phosphate buffer (pH 6.0) polishing.Control
The reaction system of group is 200 μ L, final concentration of 6 μM of three kinds of enzymes, the same experimental group of ratio of three enzymes, insufficient volume phosphate
Buffer solution (pH 6.0) polishing.Boiling water bath 5-10min makes enzyme lose activity, then with phosphate buffer (pH 6.0) by body
System supplies to 200 μ L.Above-mentioned experimental group and control sample are put into respectively after reacting 4h in 40 DEG C of water-baths；60 are taken out respectively
μ L measuring samples are for detecting, specially：Two groups of measuring samples are added separately in another 1.5mL centrifuge tube, are separately added into
180 μ L 2g/L potassium ferricyanide solutions, then boiling water bath boil 15min, taking-up is put into 12000rpm in centrifuge and centrifuges 3min；
200 μ L supernatants are finally drawn respectively and are added to ELISA Plate, measure the light absorption value A405 at 405nm.
The results are shown in Figure 1, is SmChiA mutant from highest group of single enzyme hydrolysis efficiency known to graphical results analysis
Hydrolyze degreasing euphausia superba powder group, maximum (GlcNAc)2A concentration of 0.69mM, SmChiB and SmChiC hydrolyze degreasing south respectively
(GlcNAc) that pole krill meal generates2A concentration of 0.48mM and 0.43mM, and SmChiA mutant, SmChiB, SmChiC combination
(GlcNAc) that enzyme hydrolysis degreasing euphausia superba powder generates2A concentration of 1.53mM, the significantly larger than hydrolysis efficiency of single enzyme are single
2-4 times of one enzyme hydrolysis efficiency..
3) SmChiA mutant, the experiment of SmChiB, SmChiC and OfHex1 synergetic hydrolysis
The final concentration of degreasing euphausia superba powder is about 20mg/mL, SmChiA mutant, SmChiB, SmChiC, OfHex1
Final concentration of 6 μM, the additive amounts of four enzymes is 43.9%, 35.9%, 18.9%, 1.3%, insufficient volume phosphate-buffered
Liquid (pH 6.0) polishing.The reaction system of control group is 200 μ L, and final concentration of 6 μM of four kinds of enzymes, the ratio of four enzymes is the same as experiment
Group, insufficient volume phosphate buffer (pH 6.0) polishing.Boiling water bath 5-10min makes enzyme lose activity, and then uses phosphoric acid
Salt buffer (pH 6.0) supplies system to 200 μ L.Above-mentioned experimental group and control sample are put into 40 DEG C of water-baths respectively
After middle reaction 4h；60 μ L measuring samples are taken out respectively for detecting, specially：Two groups of measuring samples are added separately to another
In 1.5mL centrifuge tubes, 180 μ L 2g/L potassium ferricyanide solutions are separately added into, then boiling water bath boils 15min, and taking-up is put into centrifugation
12000rpm centrifuges 3min in machine；200 μ L supernatants are finally drawn respectively and are added to ELISA Plate, measure the light absorption value at 405nm
A405。
When OfHex1 additive amounts are 0%, measure synergetic hydrolysis and obtain (GlcNAc)2A concentration of 1.38mM.To improve water
Efficiency is solved, increases the reaction time on the basis of former reaction system.It will improve in reaction time to 24 hours, measure (GlcNAc)2It is dense
Degree is 4.37mM, and hydrolysis efficiency reaches 21.5% (such as Fig. 3).To further increase hydrolysis efficiency, in concentration of substrate and final volume
Under conditions of constant, increase the concentration of enzyme to 24 μM, meanwhile, SmChiA mutant, tri- enzyme of SmChiB, SmChiC ratio
It is constant.It obtains (GlcNAc)2A concentration of 4.8mM, hydrolysis efficiency is promoted to 28.4% (such as Fig. 4).By products therefrom through efficient
Liquid chromatography (HPLC) processing, detects the purity of product, it can be found that products therefrom is compared with standard items
(GlcNAc)2(such as Fig. 5 B).
When OfHex1 additive amounts are 1.3%, the product that synergetic hydrolysis obtains is by (GlcNAc)2Become GlcNAc, measures association
A concentration of 5.63mM of GlcNAc is obtained with hydrolysis.To improve hydrolysis efficiency, increase the reaction time on the basis of former reaction system.
It will improve in reaction time to 24 hours, and measure a concentration of 11.57mM of GlcNAc, hydrolysis efficiency reaches 29.7% (such as Fig. 3).For into
One step improves hydrolysis efficiency and increases the concentration of enzyme to 24 μM under conditions of concentration of substrate and constant final volume.Obtain GlcNAc
A concentration of 12.23mM, hydrolysis efficiency is promoted to 34.5% (such as Fig. 4).Products therefrom through high performance liquid chromatography (HPLC) at
Reason, detects the purity of product, it can be found that products therefrom is GlcNAc (such as Fig. 5 A) compared with standard items.
Sequence table
<110>Dalian University of Technology
<120>Combination enzymic degradation degreasing euphausia superba powder recycling polypeptide intermediate product and the side for preparing N-Acetyl-D-glucosamine
Method
<130> 2015
<160> 6
<170> PatentIn version 3.3
<210> 1
<211> 1626
<212> DNA
<213>SmChiA mutant nucleotide sequences
<400> 1
atggatgccg cgccgggcaa gccgaccatc gcctggggca acaccaagtt cgccattgtt 60
gaagttgacc aggcggctac cgcttataat aatttggtga aggtaaaaaa tgccgccgat 120
gtttccgtct cctggaattt atggaatggc gacaccggca cgacggcaaa agttttatta 180
aatggcaaag aggcgtggag tggtccttca accggatctt ccggtacggc gaattttaaa 240
gtgaataaag gcggccgtta tcaaatgcag gtggcattgt gcaatgccga cggctgcacc 300
gccagtgacg ccaccgaaat tgtggtggcc gacaccgacg gcagccattt ggcgccgttg 360
aaagagccgc tgctggaaaa gaataaaccg tataaacaga actccggcaa agtggtcggt 420
tcttatttcg tcgagtgggg cgtttacggg cgcaatttca ccgtcgacaa gatcccggcg 480
caaaacctga cccacctgct gtacggcttt atcccgatct gcggcggcaa tggcatcaac 540
gacagcctga aagagattga aggcagcttc caggcgttgc agcgctcctg ccagggccgc 600
gaggacttca aagtctcgat ccacgatccg tgggccgcgc tgcaaaaagc gcagaagggc 660
gtgaccgcct gggatgaccc ctacaagggc aacttcggcc agctgatggc gctgaagcag 720
gcgcatcctg acctgaaaat cctgccgtcg atcggcggct ggacgctgtc cgacccgttc 780
ttcttcatgg gcgacaaggt gaagcgcgat cgcttcgtcg gttcggtgaa agagttcctg 840
cagacctgga agttcttcga cggcgtggat atcgactggg agttcccggg cggcaaaggc 900
gccaacccta acctgggcag cccgcaagac ggggaaacct atgtgctgct gatgaaggag 960
ctgcggacga tgctggatca gctgtcggcg gaaaccggcc gcaagtatga gctgacctcc 1020
gccatcagcg ccggtaagga caagatcgac aaggtggctt acaacgttgc gcagaactcg 1080
atggatcaca tcttcctgat gagctacgac ttctatggcg cctgggatct gaagaacctg 1140
gggcatcaga ccgcgctgaa tgcgccggcc tggaaaccgg acaccgccta caccacggtg 1200
aacggcgtca atgcgctgct ggcgcagggc gtcaagccgg gcaaaatcgt ggtcggcacc 1260
gccatgtatg gccgcggctg gaccggggtg aacggctacc agaacaatat tccgttcacc 1320
ggcaccgcca ccgggccggt taaaggcacc tgggagaacg gtatcgtgga ctaccgccaa 1380
atcgccggcc agttcatgag cggcgagtgg cagtatacct acgacgccac ggcggaagcg 1440
ccttacgtgt tcaaaccttc caccggcgat ctgatcacct tcgacgatgc ccgctcggtg 1500
caggccaaag gcaagtacgt gttggataag cagctgggcg gcctgttctc ctgggagatc 1560
gacgcggata acggcgatat tctcaacagc atgaacgcca gcctgggcaa cagcgccggc 1620
gttcaa 1626
<210> 2
<211> 563
<212> PRT
<213>SmChiA mutant amino acid sequences
<400> 2
MRKFNKPLLA LLIGSTLCSA AQAAAPGKPT IAWGNTKFAI VEVDQAATAY NNLVKVKNAA 60
DVSVSWNLWN GDTGTTAKVL LNGKEAWSGP STGSSGTANF KVNKGGRYQM QVALCNADGC 120
TASDATEIVV ADTDGSHLAP LKEPLLEKNK PYKQNSGKVV GSYFVEWGVY GRNFTVDKIP 180
AQNLTHLLYG FIPICGGNGI NDSLKEIEGS FQALQRSCQG REDFKVSIHD PWAALQKAQK 240
GVTAWDDPYK GNFGQLMALK QAHPDLKILP SIGGWTLSDP FFFMGDKVKR DRFVGSVKEF 300
LQTWKFFDGV DIDWEFPGGK GANPNLGSPQ DGETYVLLMK ELRTMLDQLS AETGRKYELT 360
SAISAGKDKI DKVAYNVAQN SMDHIFLMSY DFYGAWDLKN LGHQTALNAP AWKPDTAYTT 420
VNGVNALLAQ GVKPGKIVVG TAMYGRGWTG VNGYQNNIPF TGTATGPVKG TWENGIVDYR 480
QIAGQFMSGE WQYTYDATAE APYVFKPSTG DLITFDDARS VQAKGKYVLD KQLGGLFSWE 540
IDADNGDILN SMNASLGNSA GVQ 563
<210> 3
<211> 1836
<212> DNA
<213>SmChiB nucleotide sequences
<400> 3
gaattcattc acgctgaacg ttggcacaac acataaacgc caagacaggc ggcagtaaat 60
aaaaaattca ttcttatggt gatttatttc gacttttgtt tttacgaaaa ataaacatta 120
atggcggtgg ggaatacttc cccatcataa aaacatccac tctggagaaa taccatgtcc 180
acacgcaaag ccgttattgg gtattatttt attccaacca accaaatcaa taactacacc 240
gagaccgata cgtccgtcgt gccattcccg gtttccaaca ttacgccggc caaagccaaa 300
cagctgacgc acatcaactt ctcgttcctg gatatcaaca gcaatctgga atgcgcctgg 360
gatccggcca ccaacgacgc caaggcgcgc gatgtggtca accgtctgac cgcgctcaaa 420
gcgcacaacc ccagcctgcg catcatgttc tccatcggcg gctggtacta ctccaacgat 480
ctgggcgtgt cgcacgccaa ctatgtcaac gcggtgaaaa ccccggcgtc gcgcgccaag 540
ttcgcccaat cctgcgtgcg catcatgaag gattacggct tcgacggtgt ggacatcgac 600
tgggagtacc cgcaagcggc ggaagtggac ggcttcatcg ccgcgctgca ggagatccgc 660
accttgctga accagcaaac catcacagac ggccgccagg cgttgccgta ccagttgacc 720
atcgccggcg ccggcggcgc cttcttcctg tcgcgctatt acagcaagct ggcgcagatc 780
gtcgcgccgc tcgattacat caacctgatg acctacgatc tggccggccc ctgggagaag 840
gtaaccaacc accaggcggc gctgttcggc gacgcggccg ggccgacctt ctacaacgcg 900
ctgcgcgaag ccaatctggg ctggagctgg gaagagctga cccgcgcctt ccccagcccg 960
ttcagcctga cggtcgacgc cgccgtgcag caacacctga tgatggaagg cgtgccgagc 1020
gccaaaatcg tcatgggcgt gcccttctat ggccgcgcct tcaagggcgt cagcggcggc 1080
aacggtgggc aatacagcag ccacagcacg ccgggcgaag atccgtatcc gagcaccgac 1140
tactggctgg tgggctgcga agagtgcgtg cgcgacaagg atccgcgcat cgcctcctat 1200
cgccagttgg agcagatgct gcagggcaac tacggctatc agcggttgtg gaacgacaag 1260
accaaaaccc cttatctgta tcatgcgcag aacgggctgt tcgtcaccta tgacgatgcc 1320
gagagcttca aatacaaagc gaagtacatc aagcagcagc agctgggcgg cgtgatgttc 1380
tggcatctgg ggcaagacaa ccgcaacggc gatctgctgg ccgcgctgga tcgctatttc 1440
aacgccgcgg actacgacga cagccagctg gatatgggca ccgggctgcg ctacaccggc 1500
gtcggccccg gcaacctgcc tatcatgacc gcgccggcct atgtgccggg caccacttac 1560
gcgcagggcg cgctggtgtc ctaccagggc tacgtctggc agaccaagtg gggttacatc 1620
acctctgcac cgggttcaga cagcgcctgg ctgaaggtgg gccgcgtagc gtaaaccata 1680
aaaaaacccc gtagccgaat gctgcggggt tttcattgag ttaaccgttt gattttcgcg 1740
tcccttcgtc tctattcctt cagttgtggc accatggata gccgccatcc cgcaccactt 1800
cgcggcccat caggctgtag acatcgcttt tacgcg 1836
<210> 4
<211> 499
<212> PRT
<213>SmChiB amino acid sequences
<400> 4
MSTRKAVIGY YFIPTNQINN YTETDTSVVP FPVSNITPAK AKQLTHINFS FLDINSNLEC 60
AWDPATNDAK ARDVVNRLTA LKAHNPSLRI MFSIGGWYYS NDLGVSHANY VNAVKTPASR 120
AKFAQSCVRI MKDYGFDGVD IDWEYPQAAE VDGFIAALQE IRTLLNQQTI TDGRQALPYQ 180
LTIAGAGGAF FLSRYYSKLA QIVAPLDYIN LMTYDLAGPW EKVTNHQAAL FGDAAGPTFY 240
NALREANLGW SWEELTRAFP SPFSLTVDAA VQQHLMMEGV PSAKIVMGVP FYGRAFKGVS 300
GGNGGQYSSH STPGEDPYPS TDYWLVGCEE CVRDKDPRIA SYRQLEQMLQ GNYGYQRLWN 360
DKTKTPYLYH AQNGLFVTYD DAESFKYKAK YIKQQQLGGV MFWHLGQDNR NGDLLAALDR 420
YFNAADYDDS QLDMGTGLRY TGVGPGNLPI MTAPAYVPGT TYAQGALVSY QGYVWQTKWG 480
YITSAPGSDS AWLKVGRVA 499
<210> 5
<211> 1862
<212> DNA
<213>SmChiC nucleotide sequences
<400> 5
cccttccgtc gccgatatca ccatcttctg atgcgaccat ggcggccgcc cggccgccgt 60
tatttccttc tcccccagcg tcagtttgaa tttaattcgt tcatggccgt aaaaggtttc 120
agcctgcctg cgttaaaaat cctcattata acgttacgcc ccgccaatag ctgatattgc 180
cggcgagcgg aaaactctta cccctaatta atgaggccac catgagcaca aataacacta 240
ttaatgccgt cgccgccgat gacgcggcca ttatgccgtc tatcgccaat aaaaagatcc 300
tgatgggttt ctggcacaac tgggccgccg gcgccagtga cggctaccag caagggcagt 360
tcgccaatat gaacctgacc gacattccca ccgagtacaa cgtggtggcc gtcgccttta 420
tgaaaggcca gggcatcccg accttcaagc cttacaacct gtccgacacc gagttccgcc 480
gccaggtggg cgtgctgaac agccagggcc gcgcggtgct gatctccctc ggcggcgcag 540
acgcgcatat cgagctaaag accggcgatg aagacaagct gaaagacgag attattcgcc 600
tggtggaagt ctatggcttc gacggcctgg atatcgatct ggaacaggcg gcgatcggcg 660
ccgccaataa taaaaccgtc ttgcctgcgg cattgaaaaa agtaaaagac cattacgccg 720
cgcagggaaa aaactttatt atcagcatgg cgccggaatt cccgtattta cgcaccaacg 780
gcacctatct ggattatatc aacgccctcg aaggctatta cgactttatc gcgccgcaat 840
attacaatca gggcggcgac ggtatttggg tggatgaact caatgcctgg atcacgcaga 900
ataacgacgc catgaaagag gacttcctct actacctgac ggaaagcctg gttaccggca 960
cccgcggcta tgcgaagatc ccggcggcga aattcgtcat cggcctgccg agcaacaacg 1020
atgccgccgc caccggctac gtggtcaaca aacaggcggt gtataacgct ttctcgcgtc 1080
tcgacgccaa aaacctgtcg atcaagggcc tgatgacctg gtcaatcaac tgggataacg 1140
gcaagagcaa agccggcgtc gcctacaatt gggagttcaa aacccgctat gcgccgctga 1200
ttcagggcgg cgtcaccccg ccgccgggaa agcctaatgc gccgacggcg ctgacggtcg 1260
ccgaactggg cgccacctcg ctgaaactga gctgggccgc cgccaccggc gctttcccga 1320
tcgccagtta caccgtctac cgcaacggca acccgatcgg ccagaccgcc ggtctgtcgc 1380
tggctgacgg cggtctgacg ccggcgaccc agtacagcta cttcgttacc gcgaccgata 1440
gccagggcaa tacctcgctg ccgagcagcg cgctggcggt caaaaccgcc aacgacggca 1500
cgccgcccga tccgggggcg cccgagtggc agaacaacca cagttacaag gctggcgacg 1560
tggtgagcta taaaggcaag aaatatacct gtatccaggc gcacacctcc aacgccggct 1620
ggacgccgga cgccgccttc accctgtggc agctcatcgc ctaatcgcta atcgattgcc 1680
ggccaaactg gccggcaatc ccgccatcac gctaaaaatt gcataatcga taatttttca 1740
ggtcgataac tgaacatccg ttaaaaacca cacctaagca aacaactatt tctcaacgca 1800
tggctaaaac gcttgcatct cccgcagctt ttgacgcatt ttcataacca cagcgcagca 1860
aa 1862
<210> 6
<211> 480
<212> PRT
<213>SmChiC amino acid sequences
<400> 6
MSTNNTINAV AADDAAIMPS IANKKILMGF WHNWAAGASD GYQQGQFANM NLTDIPTEYN 60
VVAVAFMKGQ GIPTFKPYNL SDTEFRRQVG VLNSQGRAVL ISLGGADAHI ELKTGDEDKL 120
KDEIIRLVEV YGFDGLDIDL EQAAIGAANN KTVLPAALKK VKDHYAAQGK NFIISMAPEF 180
PYLRTNGTYL DYINALEGYY DFIAPQYYNQ GGDGIWVDEL NAWITQNNDA MKEDFLYYLT 240
ESLVTGTRGY AKIPAAKFVI GLPSNNDAAA TGYVVNKQAV YNAFSRLDAK NLSIKGLMTW 300
SINWDNGKSK AGVAYNWEFK TRYAPLIQGG VTPPPGKPNA PTALTVAELG ATSLKLSWAA 360
ATGAFPIASY TVYRNGNPIG QTAGLSLADG GLTPATQYSY FVTATDSQGN TSLPSSALAV 420
KTANDGTPPD PGAPEWQNNH SYKAGDVVSY KGKKYTCIQA HTSNAGWTPD AAFTLWQLIA 480
Claims (9)
1. the method for degreasing euphausia superba powder of degrading recycling polypeptide intermediate product, it is characterised in that：The method includes following steps
Suddenly：
Degreasing euphausia superba powder is dissolved in mixing in the buffer solution of pH7.5-8.5, adds 10000-15000U alkali proteases,
Water-bath 20-24h under the conditions of 40-45 DEG C；Suction filtration obtains pretreatment degreasing euphausia superba powder and supernatant containing albumen；Use spray drying process
Processing is dried to pretreated degreasing euphausia superba powder, obtains drying sample.
2. according to the method described in claim 1, it is characterized in that：The ratio that the degreasing euphausia superba powder is dissolved in buffer solution is
(1~2) g:(3~8) mL.
3. according to the method described in claim 2, it is characterized in that：The ratio that the degreasing euphausia superba powder is dissolved in buffer solution is
1g：4mL.
4. according to the method described in claim 1, it is characterized in that：The buffer solution is selected from phosphate buffer, PBS is buffered
Liquid, Tris buffer solutions and Bis-Tris buffer solutions.
5. a kind of method that combination enzymic degradation degreasing euphausia superba powder prepares N-Acetyl-D-glucosamine, it is characterised in that：It is described
Method includes the following steps：
The drying sample that claim 1 obtains is added into the buffer solution of pH5.5-6.5, makes its final concentration of 15-25mg/mL,
Add final concentration of 6~24 μM of group synthase, 40 DEG C of water-baths 4~for 24 hours；The group synthase be SmChiA mutant,
SmChiB, SmChiC and OfHex1；In described group of synthase the additive amount of four kinds of enzymes be respectively 43~45%, 35~37%, 17~
19%, 0~1.5%.
6. according to the method described in claim 5, it is characterized in that：Group synthase SmChiA mutant, SmChiB,
The additive amount of SmChiC and OfHex1 is respectively 43.90%, 35.90%, 18.90%, 1.30%.
7. according to the method described in claim 6, it is characterized in that：Group synthase SmChiA mutant, SmChiB,
Final concentration of 6 μM of SmChiC, OfHex1.
8. according to the method described in claim 5, it is characterized in that：The drying sample final concentration that the claim 1 obtains
For 20mg/mL.
9. according to the method described in claim 5, it is characterized in that：The buffer solution is pH6.0.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109762863A (en) * | 2019-03-11 | 2019-05-17 | 青岛琛蓝医药科技发展有限公司 | A kind of preparation method of krill active oligopeptide, the krill active oligopeptide of this method preparation and application |
| CN110628854A (en) * | 2019-09-20 | 2019-12-31 | 华南理工大学 | Enzyme method green process for producing chitosan oligosaccharide, astaxanthin, protein and calcium powder by using shrimp shells |
| CN113322293A (en) * | 2021-05-27 | 2021-08-31 | 浙江工业大学 | Method for catalyzing chitin in low hydration mode through ball milling auxiliary combined enzyme method |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109762863A (en) * | 2019-03-11 | 2019-05-17 | 青岛琛蓝医药科技发展有限公司 | A kind of preparation method of krill active oligopeptide, the krill active oligopeptide of this method preparation and application |
| CN110628854A (en) * | 2019-09-20 | 2019-12-31 | 华南理工大学 | Enzyme method green process for producing chitosan oligosaccharide, astaxanthin, protein and calcium powder by using shrimp shells |
| CN113322293A (en) * | 2021-05-27 | 2021-08-31 | 浙江工业大学 | Method for catalyzing chitin in low hydration mode through ball milling auxiliary combined enzyme method |
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