CN116333157B - Modified D-psicose3-epimerase and application thereof - Google Patents
Modified D-psicose3-epimerase and application thereof Download PDFInfo
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- CN116333157B CN116333157B CN202210856853.XA CN202210856853A CN116333157B CN 116333157 B CN116333157 B CN 116333157B CN 202210856853 A CN202210856853 A CN 202210856853A CN 116333157 B CN116333157 B CN 116333157B
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- 108030002106 D-psicose 3-epimerases Proteins 0.000 title claims abstract description 21
- BJHIKXHVCXFQLS-PUFIMZNGSA-N D-psicose Chemical compound OC[C@@H](O)[C@@H](O)[C@@H](O)C(=O)CO BJHIKXHVCXFQLS-PUFIMZNGSA-N 0.000 claims abstract description 20
- 108010011110 polyarginine Proteins 0.000 claims abstract description 9
- 210000004899 c-terminal region Anatomy 0.000 claims abstract description 4
- 238000000034 method Methods 0.000 claims description 10
- 102000040430 polynucleotide Human genes 0.000 claims description 8
- 108091033319 polynucleotide Proteins 0.000 claims description 8
- 239000002157 polynucleotide Substances 0.000 claims description 8
- 150000001413 amino acids Chemical group 0.000 claims description 7
- 238000004519 manufacturing process Methods 0.000 claims description 6
- 238000000746 purification Methods 0.000 claims description 6
- 241000894006 Bacteria Species 0.000 claims description 5
- RFSUNEUAIZKAJO-VRPWFDPXSA-N D-Fructose Natural products OC[C@H]1OC(O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-VRPWFDPXSA-N 0.000 claims description 5
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 claims description 5
- 239000013612 plasmid Substances 0.000 claims description 5
- 238000006243 chemical reaction Methods 0.000 claims description 3
- 229920001184 polypeptide Polymers 0.000 claims description 3
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 3
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 3
- 229920000724 poly(L-arginine) polymer Polymers 0.000 claims 2
- 230000004927 fusion Effects 0.000 claims 1
- 108090000790 Enzymes Proteins 0.000 abstract description 31
- 102000004190 Enzymes Human genes 0.000 abstract description 31
- 102000004169 proteins and genes Human genes 0.000 abstract description 16
- 108090000623 proteins and genes Proteins 0.000 abstract description 16
- 241000193403 Clostridium Species 0.000 abstract description 2
- 230000001461 cytolytic effect Effects 0.000 abstract description 2
- 235000018102 proteins Nutrition 0.000 description 13
- 210000004027 cell Anatomy 0.000 description 8
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 6
- 230000001580 bacterial effect Effects 0.000 description 5
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 241000588724 Escherichia coli Species 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 235000001014 amino acid Nutrition 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 239000013604 expression vector Substances 0.000 description 3
- 239000001963 growth medium Substances 0.000 description 3
- 238000009776 industrial production Methods 0.000 description 3
- 238000009630 liquid culture Methods 0.000 description 3
- 239000012139 lysis buffer Substances 0.000 description 3
- 239000008188 pellet Substances 0.000 description 3
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 2
- 150000000837 D-psicose derivatives Chemical class 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000000703 high-speed centrifugation Methods 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 150000002772 monosaccharides Chemical class 0.000 description 2
- 238000011296 nano differential scanning fluorimetry Methods 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 239000013598 vector Substances 0.000 description 2
- AEMOLEFTQBMNLQ-UHFFFAOYSA-N 3,4,5,6-tetrahydroxyoxane-2-carboxylic acid Chemical compound OC1OC(C(O)=O)C(O)C(O)C1O AEMOLEFTQBMNLQ-UHFFFAOYSA-N 0.000 description 1
- WQZGKKKJIJFFOK-IVMDWMLBSA-N D-allopyranose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@H](O)[C@@H]1O WQZGKKKJIJFFOK-IVMDWMLBSA-N 0.000 description 1
- 241001198387 Escherichia coli BL21(DE3) Species 0.000 description 1
- 208000008589 Obesity Diseases 0.000 description 1
- 241000193453 [Clostridium] cellulolyticum Species 0.000 description 1
- 241000883281 [Clostridium] cellulolyticum H10 Species 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 238000001261 affinity purification Methods 0.000 description 1
- 235000009697 arginine Nutrition 0.000 description 1
- 150000001484 arginines Chemical class 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 235000015872 dietary supplement Nutrition 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 235000003599 food sweetener Nutrition 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 235000020824 obesity Nutrition 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 238000001742 protein purification Methods 0.000 description 1
- 238000011897 real-time detection Methods 0.000 description 1
- 238000002407 reforming Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 239000003765 sweetening agent Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 208000001072 type 2 diabetes mellitus Diseases 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/90—Isomerases (5.)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/02—Monosaccharides
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y501/00—Racemaces and epimerases (5.1)
- C12Y501/03—Racemaces and epimerases (5.1) acting on carbohydrates and derivatives (5.1.3)
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
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- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Molecular Biology (AREA)
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Abstract
The application discloses a modified D-psicose3-epimerase and application thereof, and relates to the technical field of protein engineering, wherein the modified DTease enzyme consists of a DTease sequence and a C-terminal fused poly-arginine tag, and is named as DTease-5R. By adding RRRRRRR labels to the C end of a clostridium cellulolytic DTease protein sequence, the yield is improved by 5.5 times compared with the prior art, the Tm value is increased by 7 ℃, and the DTease provided by the application has better application scene in the application of industrially producing D-psicose.
Description
Technical Field
The application relates to the technical field of protein engineering, in particular to an improved D-psicose3-epimerase and application thereof.
Background
D-psicose, which belongs to the carbon-3 isomer of D-fructose, is a rare sugar. It is a very low energy monosaccharide compared to conventional monosaccharides and is therefore commonly used in the food industry as a palatable sweetener or dietary supplement. The D-psicose also has the activities of resisting oxidation, reducing blood sugar, reducing blood fat and the like, and has wide application prospect in the industrial field. At present, the difficulty of producing D-psicose by adopting a chemical synthesis method is high. Because D-psicose is only present in a few fruits in nature, it is difficult to extract, and a common production method is to produce D-psicose by a bioconversion process, and the conversion of D-fructose into D-psicose is catalyzed by a D-psicose3-epimerase (DTease) [1.Hossain,Akram et al ] "Rare surer D-allose: potential role and therapeu tic monitoring in maintaining obesity and type 2diabetes mellitus ]" Pharmacology & therapeutics vol.155 (2015): 49-59 ]. However, since some of the D-psicose 3-epimerases are relatively limited in stability, most of which are unsuitable for industrial use, finding a thermostable DTEase is very important for industrial production of D-psicose.
The DTease enzyme derived from clostridium cellulolyticum is a novel DTease discovered in 2011, has the function of converting D-fructose into D-psicose, has better thermal stability compared with the DTease of the prior species source, can play an active role at 55 ℃ and has long half-life, thus having wide application prospect in the industrial production of D-psicose [2.Chan, hsiu-Chien et al, "Crystal structures of D-psicose3-epimerase from Clostridium cellulolyticum H10 and its complex with ketohexose subscribers," Protein & cell vol.3,2 (2012): 123-31 ].
Disclosure of Invention
The application aims to provide an improved D-psicose3-epimerase and application thereof, wherein the improved D-psicose3-epimerase has higher heat stability and purification yield compared with a wild type DTease enzyme.
The application realizes the above purpose through the following technical scheme:
the application provides an engineered D-psicose3-epimerase, which consists of a DTease sequence and a C-terminal fused poly-arginine tag.
A further improvement is that the polypeptide sequence of the poly-arginine tag is RRRRR, and the modified DTease enzyme is named as DTease-5R.
A further improvement is that the amino acid sequence of the modified D-psicose3-epimerase is shown as SEQ ID NO. 1.
The application also provides a polynucleotide encoding the modified D-psicose 3-epimerase.
A further improvement is that the sequence of the polynucleotide is shown as SEQ ID NO. 2.
The application also provides a recombinant plasmid which is an expression vector containing the polynucleotide and capable of translating and expressing the modified D-psicose 3-epimerase.
A further improvement is that the expression vector is a pET-28a vector.
The application also provides a recombinant engineering bacterium which comprises the recombinant plasmid.
The further improvement is that the engineering bacteria are escherichia coli BL21 (DE 3).
The application also provides an application of the modified D-psicose3-epimerase in the production of D-psicose.
A further improvement consists in the production of D-psicose by means of a bioconversion process, the conversion of D-fructose into D-psicose being catalyzed by said modified 3-epimerase of D-psicose.
The application also provides a method for improving the purification yield and the thermal stability of the DTease, and the C-terminal of the wild type DTease is fused with the poly-arginine label.
A further improvement is that the wild type DTease is derived from Clostridium cellulolytic bacteria.
The application has the following beneficial effects:
the application provides a method for optimizing and reforming the original DTease sequence by adding 5 extra arginines at the C end of the DTease, which obviously improves the yield of the DTease by up to 5.5 times after the sequence is reformed, thereby greatly reducing the cost of producing the DTease; and secondly, after the sequence is modified, the Tm value of the DTease is also improved, the temperature is increased by 7 ℃, and the thermal stability of the DTease is greatly improved. Previous research reports indicate that DTease has activity and long half-life at 55 ℃, and the Tm value of the DTease is improved to 63.5 ℃ after the sequence of the DTease is optimized, so that the modified DTease is more heat-resistant in the industrial production process of D-psicose, the half-life of the modified DTease is prolonged, and the production efficiency of the D-psicose is improved.
Drawings
FIG. 1 is a graph showing the expression test of a wild-type and modified DTease protein in a small amount;
FIG. 2 is a graph showing the result of purification of a wild-type and engineered DTease-5R enzyme;
FIG. 3 is a graph showing the results of the purification of modified DTease-7R and modified DTease-9R enzymes
FIG. 4 is a graph showing the Tm value of a wild-type and modified DTease enzyme.
Detailed Description
The present application will be described in further detail with reference to the accompanying drawings, wherein it is to be understood that the following detailed description is for the purpose of further illustrating the application only and is not to be construed as limiting the scope of the application, as various insubstantial modifications and adaptations of the application to those skilled in the art can be made in light of the foregoing disclosure.
1. Material
All reagents used in the experiment are conventional reagents unless specified, all reagents are prepared by deionized water, and all used instruments are laboratory conventional instruments.
2. Method of
2.1 wild-type and engineered DTease protein expression
The recombinant plasmids (the expression vector is pET-28a vector) of wild DTease (the amino acid protein sequence is shown as SEQ ID NO. 3) and modified DTease (the amino acid protein sequence is shown as SEQ ID NO. 4) and DTease-7R (the amino acid protein sequence is shown as SEQ ID NO. 5) are respectively transformed into BL21 (DE 3) escherichia coli competent cells according to the conventional molecular biological means, the amino acid sequence of the DTease-5R is shown as SEQ ID NO.1, and the sequence of the polynucleotide for encoding the DTease-5R is shown as SEQ ID NO. 2.
The monoclonal bacterial plaque is selected to be cultured in 5mLLB liquid culture medium at 37 ℃ overnight, 0.5mM IPTG15 ℃ is added to be cultured overnight when the bacterial liquid OD600 is 0.6-0.8, and the bacterial cells are collected by centrifugation at 5000 rpm. After the cells were homogenized with 50mM Tris-HCl (pH 7.5) as a lysis buffer and 250mM NaCl, the cells were lysed using an ultrasonic breaker, and the lysed cells were collected by high-speed centrifugation at 16000rpm, and the supernatant and the pellet samples were taken and subjected to SDS-PAGE detection, respectively, with the results shown in FIG. 1. According to the results, it was found that both DTease-5R, DTEase-7R, DTEase-9R and DTease were expressed in E.coli, but the expression level of DTease-5R was higher than that of DTEase, DTEase-7R, DTEase-9R.
2.2 wild type and modified DTease protein purification and yield comparison
To compare the yields of DTease-5R, DTEase-7R, DTEase-9R and DTease in E.coli, four enzymes were expressed in 1L of BL21 strain: the obviously expressed strain is inoculated to 50mLLB liquid culture medium for culturing overnight at 37 ℃, the bacteria cultured overnight are inoculated to 1LLB liquid culture medium according to the proportion of 1:100, 0.5mM IPTG15 ℃ is added for culturing overnight when the bacterial liquid OD600 is 0.6-0.8, and the bacterial cells are collected by centrifugation at 5000 rpm. The collected pellet was weighed, a corresponding volume of lysis buffer (50 mM Tris-HCl (pH 7.5), 500mM NaCl,5% glycerol) was added in a ratio of 1:10, and the pellet was crushed by using a high-pressure homogenizer, and the supernatant was collected by high-speed centrifugation at 16000 rpm. The method comprises the steps of enriching and purifying proteins by using affinity chromatography HisFF, balancing the HisFF column by using a lysis buffer before purification, eluting the supernatant of all cells by using imidazole solutions with different gradients, collecting proteins eluted by imidazole with different gradients for SDS-PAGE detection, collecting proteins with better purity, measuring the protein concentration by using Nanodrop, and calculating the protein yield.
As shown by SDS-PAGE results, the DTease enzyme, the DTease-5R enzyme, the DTease-7R enzyme and the DTease-9R enzyme with higher purity are obtained through affinity purification. The yield of DTease-5R enzyme calculated from the protein concentration measured by Nanodrop was 6.47mg/L as shown in the left graph of FIG. 2, the yield of DTease-7R enzyme was 35.5mg/L as shown in the right graph of FIG. 2, the yield of DTease-7R enzyme was 20.85mg/L as shown in the left graph of FIG. 3, and the yield of DTease-9R enzyme was 24.35mg/L as shown in the right graph of FIG. 3. This example shows that the yield of DTease-5R is improved by a factor of 5.5 compared with the previous one, and that the purity of DTease-5R is significantly improved compared with the DTease-7R enzyme and the DTease-9R enzyme.
2.3 comparison of Heat stability of wild type and modified DTease enzymes
Tm values of proteins were detected using micro differential scanning fluorescence (nanoDSF) techniques, the specific experimental procedure was as follows:
20 mu L of DTease enzyme, DTease-5R enzyme, DTease-7R enzyme and DTease-9R enzyme with the concentration of 0.5mg/ml are respectively added into 384-hole experimental plates, after shaking and centrifugation, the experimental plates are placed on a sampling frame, and a NanoDSF capillary is used for sucking samples, so that the samples are ensured to be full of the whole capillary. The capillary tube was placed in a nanoDSF instrument, set to an initial temperature of 20 ℃, and terminated by a final rise to 90 ℃ at a rate of 2.0 ℃ per minute. The instrument will perform temperature rise and real-time detection according to the set parameters.
The results of the Tm value test are shown in FIG. 4, and it is found from the results of the test that the Tm value of the DTease enzyme is 56.5℃which is close to 55℃reported in the literature in the background art. The Tm value of the DTease-5R enzyme was 63.4℃and the Tm value of the DTease-7R enzyme was 58.8℃and the Tm value of the DTease-9R enzyme was 59.1℃wherein the Tm value of the DTease-5R enzyme was the highest and 7℃higher than that of the DTease enzyme. The DTease-5R enzyme provided by the application has higher thermal stability than the original DTease, so that the DTease-5R enzyme has better application scene.
The foregoing examples illustrate only a few embodiments of the application and are described in detail herein without thereby limiting the scope of the application. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the application, which are all within the scope of the application.
Claims (8)
1. The modified D-psicose3-epimerase is characterized by comprising a DTease sequence and a C-terminal fused polyarginine tag, wherein the polypeptide sequence of the polyarginine tag is RRRRR, and the amino acid sequence of the modified D-psicose3-epimerase is shown as SEQ ID NO. 1.
2. A polynucleotide encoding the engineered D-psicose3-epimerase of claim 1.
3. A polynucleotide according to claim 2, wherein the sequence of said polynucleotide is as shown in SEQ ID No. 2.
4. A recombinant plasmid comprising the polynucleotide of any one of claims 2-3 and capable of translationally expressing the engineered D-psicose3-epimerase of claim 1.
5. A recombinant engineering bacterium comprising the recombinant plasmid of claim 4.
6. Use of the engineered D-psicose3-epimerase of claim 1 for the production of D-psicose.
7. Use of an engineered D-psicose3-epimerase according to claim 6 for the production of D-psicose, wherein D-psicose is produced by a bioconversion process and wherein the engineered D-psicose3-epimerase is used to catalyze the conversion of D-fructose into D-psicose.
8. A method for improving the purification yield and thermal stability of DTease is characterized in that a poly-arginine tag is fused at the C end of a wild type DTease, the polypeptide sequence of the poly-arginine tag is RRRRR, and the amino acid sequence of the DTease after the fusion of the poly-arginine tag is shown as SEQ ID NO. 1.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4532207A (en) * | 1982-03-19 | 1985-07-30 | G. D. Searle & Co. | Process for the preparation of polypeptides utilizing a charged amino acid polymer and exopeptidase |
| CN112695006A (en) * | 2021-02-05 | 2021-04-23 | 江南大学 | Recombinant bacillus subtilis for expressing D-psicose-3-epimerase |
| CN113801866A (en) * | 2021-09-02 | 2021-12-17 | 无锡佰翱得生物科学有限公司 | High-efficiency expression recombinant TEV enzyme with high activity and stability, and preparation method, determination method and application thereof |
-
2022
- 2022-07-20 CN CN202210856853.XA patent/CN116333157B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4532207A (en) * | 1982-03-19 | 1985-07-30 | G. D. Searle & Co. | Process for the preparation of polypeptides utilizing a charged amino acid polymer and exopeptidase |
| CN112695006A (en) * | 2021-02-05 | 2021-04-23 | 江南大学 | Recombinant bacillus subtilis for expressing D-psicose-3-epimerase |
| CN113801866A (en) * | 2021-09-02 | 2021-12-17 | 无锡佰翱得生物科学有限公司 | High-efficiency expression recombinant TEV enzyme with high activity and stability, and preparation method, determination method and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| 南极嗜冷杆菌脂肪酶的原核可溶性表达优化及酶学性能表征;刘弘忍;王亮亮;何琦阳;王飞;李迅;;林业工程学报(05);第75页右栏 * |
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