CN104762280A - Technology for biotransformation preparation of panoxadiol saponins by recombinant glucoside hydrolase - Google Patents
Technology for biotransformation preparation of panoxadiol saponins by recombinant glucoside hydrolase Download PDFInfo
- Publication number
- CN104762280A CN104762280A CN201510036363.5A CN201510036363A CN104762280A CN 104762280 A CN104762280 A CN 104762280A CN 201510036363 A CN201510036363 A CN 201510036363A CN 104762280 A CN104762280 A CN 104762280A
- Authority
- CN
- China
- Prior art keywords
- ginsenoside
- compound
- protein
- ccbgl1a
- gyp xvii
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- 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
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
-
- 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
- C12P33/00—Preparation of steroids
- C12P33/20—Preparation of steroids containing heterocyclic rings
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Enzymes And Modification Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses a method for preparing ginsenosides of Gyp XVII, compound O, Mb and F2. The method comprises the following steps that panoxadiol saponins as substrates and a protein undergo a catalytic reaction to produce reactants containing ginsenosides of Gyp XVII, compound O, Mb and F2, wherein the protein is a protein (a) or (b), the protein (a) has an amino acid sequence shown in the formula of SEQ ID No.2 or shown in the formula of SEQ ID No.4, and the protein (b) is derived from the amino acid sequence of the protein shown in the formula of SEQ ID No.2 or shown in the formula of SEQ ID No.4 by replacement and/or deletion and/or addition of one or more amino acid residues and has ginsenoside hydrolase activity. An experiment proves that the method for preparing ginsenosides of Gyp XVII, compound O, Mb and F2 has good practicality.
Description
Technical field
The present invention relates to a kind of method preparing ginsenoside in biological technical field.
Background technology
Panoxadiol type saponin(e Gyp XVII, compound O, Mb and F2, be in the news and have good pharmacological activity, such as anti-tumor activity, anti-inflammatory activity etc., have certain application potential.But these ginsenosides content in natural ginseng is very micro-even not to be existed, from natural ginseng, carry out extraction and isolation very difficult, this greatly limits their application.
In Radix Ginseng total saponins, the content of diol type ginsenoside Rb1, Rb2, Rc and Rd, exceedes more than 60% of total Ginsenosides Content.Contrast ginsenoside Rb1 and Gyp XVII, Rb2 and compound O, Rc and Mb, Rd and F2 structure known, their sapogenin structure is identical, and the glycosyl number be just connected on C-3 position is different.Therefore, by the outside glucosyl group of the C-3 position of hydrolysis panoxadiol type saponin(e Rb1, Rb2, Rc and Rd, ginsenoside Gyp XVII, compound O, Mb and F2 can be obtained respectively.
There is investigator to prepare the less ginsenoside of some glycosyl numbers by Partial acid hydrolysis Radix Ginseng total saponins in the past.But acid-hydrolyzed poor stability, between different batches, quality is unstable.In addition, acid-hydrolyzed specificity is poor, easily produces by product, and the productive rate causing target product is on the low side, and follow-up separation and purification of products is more difficult.Also investigator is had to utilize the glycoside hydrolase of the microorganism secretion such as bacterium, fungi to come biologic converted ginseng total saponins, panoxadiol type saponin mixture or certain panoxadiol type saponin monomer, preparation Gyp XVII, compound O, Mb and F2, or wherein one or more.Bio-transformation, compared with acid hydrolysis, has the superiority such as mild condition, specificity be good.But due to the glycoside hydrolase comparatively small amt of bacterium or fungus secretion, and microorganism easily makes a variation, degenerates, the efficiency causing this biotransformation is very low, and this production process is not suitable for amplifying, add unstable to production process, be not suitable for plant-scale application.Present urgent need is by engineered means, and the gene of Direct Cloning and the enzyme required for expression, realizes the stable preparation of ginsenoside Gyp XVII, compound O, Mb and F2.
Summary of the invention
The object of this invention is to provide a kind of method preparing ginsenoside Gyp XVII, compound O, Mb and F2.
The method preparing ginsenoside Gyp XVII, compound O, Mb and F2 provided by the invention, comprise with panoxadiol type saponin(e for substrate protein carries out the step that catalyzed reaction obtains containing ginsenoside Gyp XVII, compound O, Mb and F2 reactant, the name of described protein is called CcBgl1A, deriving from fiber bacterium (Cellulosimicrobium sp.) 21, is following protein a) or b) or c) or d):
A) aminoacid sequence is the protein of SEQ ID No.2;
B) aminoacid sequence is the protein of SEQ ID No.4;
C) by the protein with ginsenoside hydrolytic enzyme activities that the aminoacid sequence shown in SEQ ID No.2 obtains through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation;
D) by the protein with ginsenoside hydrolytic enzyme activities that the aminoacid sequence shown in SEQ ID No.4 obtains through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation.
Wherein, SEQ ID No.2 is made up of 390 amino-acid residues, is the aminoacid sequence of natural ginseng saponin(e lytic enzyme CcBgl1A (name is called N CcBgl1A); SEQ ID No.4 is made up of 410 amino-acid residues, is the aminoacid sequence of restructuring ginsenoside lytic enzyme CcBgl1A (name is called R CcBgl1A); SEQ ID No.4 is the aminoacid sequence obtained at N-terminal interpolation aminoacid sequence: the MGSSHHHHHHSSGLVPRGSH of SEQ ID No.2.
Above-mentioned c) in protein can synthetic, also can first synthesize its encoding gene, then carry out biological expression and obtain.
Above-mentioned c) in the encoding gene of protein by the codon by lacking one or several amino-acid residue in the DNA sequence dna shown in SEQ ID No.1, and/or the missense mutation carrying out one or several base pair obtains.Above-mentioned d) in protein can synthetic, also can first synthesize its encoding gene, then carry out biological expression and obtain.Above-mentioned d) in the encoding gene of protein by the codon by lacking one or several amino-acid residue in the DNA sequence dna shown in SEQ ID No.3, and/or carry out the missense mutation of one or several base pair, and/or the encoding sequence connecting the His-Tag shown in SEQ ID No.3 is held to obtain at its 5 ' end and/or 3 '.
In aforesaid method, described method comprises purifying from described reactant and obtains the step of ginsenoside Gyp XVII, compound O, Mb and F2.
In aforesaid method, describedly to carry out in a liquid for substrate CcBgl1A carries out catalyzed reaction with panoxadiol type saponin(e, in described liquid, the proportioning of CcBgl1A and described panoxadiol type saponin(e is panoxadiol type saponin(e described in 1IU CcBgl1A:0.5-2mg (as described in 0.8IU CcBgl1A:1mg panoxadiol type saponin(e), and described IU is that the beta-glucosidase enzyme of CcBgl1A is lived.
In aforesaid method, described catalyzed reaction carries out 1-3 hour, and described 1-3 hour can be 1-2 hour or 1.5-2 hour or 2-3 hour or 2-2.5 hour or 2 hours.
In aforesaid method, described catalyzed reaction is at 25-35 DEG C, and described 25-35 DEG C can be 25-30 DEG C or 30-35 DEG C.
In aforesaid method, described purifying comprises the following steps: undertaken centrifugal by described reactant, ginsenoside is made to enter in supernatant liquor, collect supernatant liquor, obtain the supernatant liquor containing ginsenoside Gyp XVII, compound O, Mb and F2, described supernatant liquor is dry, obtain the xeraphium containing ginsenoside Gyp XVII, compound O, Mb and F2, remove the impurity in described xeraphium, obtain ginsenoside Gyp XVII, compound O, Mb and F2.
In aforesaid method, the impurity removed in described xeraphium comprises the following steps: be dissolved in solution A by described xeraphium, obtain the liquid mixture containing ginsenoside Gyp XVII, compound O, Mb and F2, described liquid mixture is carried out silica gel column chromatography, with described solution A wash-out, collect the fraction containing ginsenoside Gyp XVII, compound O, Mb and F2 respectively, described fraction is dry, obtain ginsenoside Gyp XVII, compound O, Mb and F2.
In aforesaid method, describedly centrifugally under normal temperature, 6000rpm condition, carry out 10min; Described solution A is made up of chloroform, methyl alcohol, water, and in described solution A, the volume ratio of chloroform, methyl alcohol, water is 65:35:10 (lower floor's solution).
CcBgl1A is preparing the application in ginsenoside Gyp XVII, compound O, Mb and F2 also in protection scope of the present invention; as CcBgl1A biologic converted ginseng diol type saponin(e and/or Rb1 and/or Rb2 and/or Rc and/or Rd prepare ginsenoside Gyp XVII, compound O, Mb and F2, or wherein one or more.
The biomaterial relevant to CcBgl1A is preparing the application in ginsenoside Gyp XVII, compound O, Mb and F2 also in protection scope of the present invention; as as described in biomaterial biologic converted ginseng diol type saponin(e and/or Rb1 and/or Rb2 and/or Rc and/or Rd prepare ginsenoside Gyp XVII, compound O, Mb and F2, or wherein one or more.Described biomaterial is following B1) to B16) in any one:
B1) nucleic acid molecule of coding CcBgl1A;
B2) containing B1) expression cassette of described nucleic acid molecule;
B3) containing B1) recombinant vectors of described nucleic acid molecule;
B4) containing B2) recombinant vectors of described expression cassette;
B5) containing B1) recombinant microorganism of described nucleic acid molecule;
B6) containing B2) recombinant microorganism of described expression cassette;
B7) containing B3) recombinant microorganism of described recombinant vectors;
B8) containing B4) recombinant microorganism of described recombinant vectors;
B9) containing B1) the transgenetic animal cell system of described nucleic acid molecule;
B10) containing B2) the transgenetic animal cell system of described expression cassette;
B11) containing B3) the transgenetic animal cell system of described recombinant vectors;
B12) containing B4) the transgenetic animal cell system of described recombinant vectors;
B13) containing B1) the transgenic plant cells system of described nucleic acid molecule;
B14) containing B2) the transgenic plant cells system of described expression cassette;
B15) containing B3) the transgenic plant cells system of described recombinant vectors;
B16) containing B4) the transgenic plant cells system of described recombinant vectors.
Wherein, described nucleic acid molecule can be DNA, as cDNA, genomic dna or recombinant DNA; Described nucleic acid molecule can be also RNA, as mRNA or hnRNA etc.
In above-mentioned biomaterial, B1) described nucleic acid molecule specifically can be following 1) or 2) or 3) or 4) or 5) shown in nucleic acid molecule:
1) encoding sequence is DNA molecular or the cDNA molecule of the 1-1173 position Nucleotide of SEQ ID No.1 in sequence table;
2) encoding sequence is DNA molecular or the cDNA molecule of the 1-1233 position Nucleotide of SEQ ID No.3 in sequence table;
3) with 1) or 2) nucleotide sequence that limits has more than 75% or 75% identity, and the cDNA molecule of coding CcBgl1A or genomic DNA molecule;
4) under strict conditions with 1) or 2) or 3) nucleotide sequence hybridization that limits, and the cDNA molecule of coding CcBgl1A or genomic DNA molecule.
Wherein, SEQ ID No.1 is made up of 1173 Nucleotide, the aminoacid sequence shown in the nucleotide coding SEQ ID No.2 of 1-1173 position of SEQ ID No.1.The protein of the genomic gene coding SEQ ID No.2 of the CcBgl1A gene in fiber bacterium (Cellulosimicrobium sp.21).SEQ ID No.3 is made up of 1233 Nucleotide, aminoacid sequence shown in coding SEQ ID No.4, the 1-60 position Nucleotide of SEQ ID No.3 is the sequence on pET-28a (+) carrier, the aminoacid sequence shown in the nucleotide coding SEQ ID No.2 of 61-1233 position of the 13-30 position nucleotide coding His-Tag of SEQ ID No.3, SEQ ID No.3.
Those of ordinary skill in the art can adopt known method easily, the method for such as orthogenesis and point mutation, suddenly change to the nucleotide sequence of coding CcBgl1A of the present invention.Those are through manually modified, have and be separated the nucleotide sequence 70% of the CcBgl1A obtained or the Nucleotide of higher identity with the present invention, as long as coding CcBgl1A and have ginsenoside hydrolytic enzyme activities is all be derived from nucleotide sequence of the present invention and be equal to sequence of the present invention.
Term used herein " identity " refers to the sequence similarity with native sequence nucleic acid." identity " comprises and has 75% or higher with promotor nucleotide sequence of the present invention, or 85% or higher, or 90% or higher, or the nucleotide sequence of 95% or higher identity.Identity can with the naked eye or computer software evaluate.Use computer software, the identity between two or more sequence can represent with per-cent (%), and it can be used for evaluating the identity between correlated series.
In above-mentioned biomaterial, described stringent condition is in the solution of 2 × SSC, 0.1%SDS, hybridizes and wash film 2 times, each 5min at 68 DEG C, again in the solution of 0.5 × SSC, 0.1%SDS, hybridizes and wash film 2 times, each 15min at 68 DEG C.
More than above-mentioned 75% or 75% identity, can be the identity of 80%, 85%, 90%, more than 95%.
In above-mentioned biomaterial, the expression cassette (CcBgl1A expression casette) of the nucleic acid molecule containing coding CcBgl1A B2), refer to the DNA that can express CcBgl1A in host cell, this DNA not only can comprise the promotor starting CcBgl1A genetic transcription, also can comprise the terminator stopping CcBgl1A and transcribe.Further, described expression cassette also can comprise enhancer sequence.
Available existing expression vector establishment contains the recombinant vectors of described CcBgl1A expression casette.
In above-mentioned biomaterial, described carrier can be plasmid, glutinous grain, phage or virus vector.
In above-mentioned biomaterial, B5)-B8) described in microorganism can be yeast, bacterium, algae and fungi, as intestinal bacteria.
In above-mentioned biomaterial, B9)-B12) described in transgenic plant cells system and transgenetic animal cell system do not comprise reproductive material.Described transgenetic animal cell system can be insect cell.
In an embodiment of the invention, the encoding gene of CcBgl1A is imported in E. coli BL21 (DE3) by the recombinant vectors of the expression cassette of the encoding gene containing CcBgl1A.The recombinant vectors pET-28a/CcBgl1A that fragment between the Nde I that described recombinant vectors is DNA molecular replacement pET-28a (+) shown in the 1-1173 position with SEQ ID No.1 and BamH I recognition site obtains.CcBgl1A prepares application in ginsenoside Gyp XVII, compound O, Mb and F2 also in protection scope of the present invention at catalysis panoxadiol type saponin(e.
Above-mentioned B1) to B16) any one biomaterial relevant to CcBgl1A prepares application in ginsenoside Gyp XVII, compound O, Mb and F2 also in protection scope of the present invention at catalysis panoxadiol type saponin(e.
Experiment proves, CcBgl1A can be utilized as ginsenoside lytic enzyme, carry out catalyzed reaction obtain ginsenoside Gyp XVII, compound O, Mb and F2 using panoxadiol type saponin(e as substrate.CcBgl1A of the present invention is utilized to prepare ginsenoside Gyp XVII, compound O, Mb and F2, reaction conditions is gentle, low for equipment requirements, production process is without the need to high temperature or cooling, and energy consumption is low, because enzyme catalysis has efficient, single-minded selectivity, and transformation efficiency is high, bioconversion rate is fast, can complete in 2h, this method produces ginsenoside Gyp XVII, compound O, Mb and F2 no coupling product produces, and purifying is convenient.Reacting most solvent is water, three waste discharge is low, environmental protection, and achieve the stable preparation of ginsenoside Gyp XVII, compound O, Mb and F2, solve the problems such as ginsenoside Gyp XVII, compound O, the yielding poorly of Mb and F2 are prepared in traditional bio-transformation and acid hydrolysis, wastage of material, cost are high, quality is unstable.Provided by the inventionly prepare ginsenoside Gyp XVII, compound O, the method for Mb and F2 has stronger practicality.
Biomaterial preservation explanation
The Classification And Nomenclature of biomaterial: fiber bacterium (Cellulosimicrobium sp.)
The strain number of biomaterial: 21
Depositary institution's title of biomaterial: China Committee for Culture Collection of Microorganisms's common micro-organisms center
The depositary institution of biomaterial is called for short: CGMCC
The depositary institution address of biomaterial: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode: 100101
The preservation date of biomaterial: on 05 14th, 2013
Register on the books numbering in the preservation center of biomaterial: CGMCC No.7587
Accompanying drawing explanation
Fig. 1 is the PCR agarose gel electrophoresis figure of CcBgl1A gene.Wherein, swimming lane 1 is DNA marker, and swimming lane 2 is the pcr amplification product of CcBgl1A gene.
Fig. 2 is the Nde I of recombinant vectors pET-28a/CcBgl1A and the agarose gel electrophoresis figure of BamH I double digestion qualification.Wherein, swimming lane 1 is DNA marker, and swimming lane 2 is pET-28a (+), and swimming lane 3 is pET-28a/CcBgl1A, and swimming lane 4 is NdeI and the BamH I double digestion product of pET-28a/CcBgl1A.
Fig. 3 is the poly-propionic acid amide gel electrophoresis figure of protein induced expression.Wherein, swimming lane 1 is protein molecular weight marker, swimming lane 2 is total protein solution before recombinant bacterium pET-28a/E.coli BL21 (DE3) induction, swimming lane 3 is total protein solution after recombinant bacterium pET-28a/E.coli BL21 (DE3) induction, swimming lane 4 is total protein solution before recombinant bacterium pET-28a/CcBgl1A/E.coli BL21 (DE3) induction, swimming lane 5 is recombinant bacterium pET-28a/CcBgl1A/E.coli BL21 (DE3) total protein solution after IPTG induction, and swimming lane 6 is the CcBgl1A albumen after Ni column purification.
Fig. 4 is the HPLC-C18 reversed-phase column analysis collection of illustrative plates that restructuring ginsenoside lytic enzyme CcBgl1A is hydrolyzed the reaction product of panoxadiol type saponin(e.Wherein, curve a is conversion of substrate-panoxadiol type saponin(e collection of illustrative plates, and curve b is the collection of illustrative plates after converted product-panoxadiol type saponin(e transforms 2h, and curve c is ginsenoside standard substance collection of illustrative plates.
Fig. 5 is the structure schematic diagram of recombinant expression vector pET-28a/CcBgl1A.
Fig. 6 is the biological characteristics of restructuring ginsenoside lytic enzyme CcBgl1A.Wherein, a is optimal pH curve, and b is pH beta stability line, and c is optimum temperuture curve, and d is temperature-stable linearity curve.
Embodiment
Below in conjunction with embodiment, the present invention is further described in detail, the embodiment provided only in order to illustrate the present invention, instead of in order to limit the scope of the invention.
Experimental technique in following embodiment, if no special instructions, is ordinary method.
Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
Fiber bacterium (Cellulosimicrobium sp.) 21 is screened by Northeast Normal University and obtains from soil, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on May 14th, 2013, register on the books numbering in this preservation center: CGMCC No.7587.
The structure of embodiment 1, ginsenoside lytic enzyme recombinant vectors
The polynucleotide (SEQ ID No.1) of the ginsenoside lytic enzyme from fiber bacterium (Cellulosimicrobium sp.) 21 (preserving number is CGMCC No.7587) are obtained by the mode of PCR, be cloned in expression vector pET-28a, obtain the recombinant plasmid can expressing ginsenoside lytic enzyme.Concrete steps comprise:
1) extraction of fiber bacterium (Cellulosimicrobium sp.) 21 genomic dnas:
By fiber bacterium (Cellulosimicrobium sp.) 21 on LB nutrient agar, 37 DEG C of activation 12h.The bacterial classification of activation connects a ring in LB liquid nutrient medium, 37 DEG C, 200rpm shaking culture 12h.5000rpm, 4 DEG C of centrifugal 10min collect thalline, with bacterial genomes DNA extraction kit (Tian Gen biochemical technology company limited), extract fiber bacterium (Cellulosimicrobium sp.) 21 genomic dnas to specifications.
2) amplification of CcBgl1A gene:
Be that template carries out PCR with the genomic dna of fiber bacterium (Cellulosimicrobium sp.) 21, amplification CcBgl1A gene.Amplimer is: Primer-F 5 '-GGAATTCCATATGGTGTCGATCACCTTCCCCGAGTC-3 '; Primer-R 5 '-CGGGATCCTTATCAGTCGTCGAGCGAGACGGTC-3 '.Amplification condition is: 98 DEG C of denaturation 30s; 98 DEG C of 10s, 65 DEG C of 45s, 72 DEG C of 1min, 30 circulations; 72 DEG C extend 10min.1% agarose gel electrophoresis detects PCR result, as shown in Figure 1.The fragment obtained is checked order, sequencing result shows that the fragment obtained contains the DNA molecular shown in SEQ ID No.1, DNA molecular shown in SEQ ID No.1 is the encoding sequence of natural ginseng saponin(e lytic enzyme CcBgl1A, ginsenoside lytic enzyme CcBgl1A shown in coding SEQ ID No.2, by this natural ginseng saponin(e lytic enzyme CcBgl1A called after N CcBgl1A.
3) structure of recombinant vectors pET-28a/CcBgl1A
As shown in Figure 5, PCR primer is reclaimed with PCR primer purification kit.The PCR primer cut back to close with restriction enzyme Nde I and BamH I (New England Biolabs) enzyme and pET-28a (+) carrier (Novegan) (Fig. 2), reclaim test kit (Tian Gen biochemical technology company limited, DP209) with sepharose after enzyme cuts end and reclaim digestion products.What glue reclaimed that test kit reclaims cuts pET-28a (+) carrier of process and PCR primer connection under the effect of T4DNA ligase enzyme (TAKARA, 2011A) is spent the night through same enzyme.Connect product conversion in E. coli BL21 (DE3), screening positive clone also serves the order-checking of Hai Sheng work Biological Co., Ltd., the exactness of authentication sequence.By the recombinant vectors called after pET-28a/CcBgl1A that the fragment (other sequence of maintenance pET-28a (+) is constant) of replacing with CcBgl1A gene shown in SEQ ID No.1 between the Nde I of pET-28a (+) and BamH I site obtains.By positive colony called after recombinant bacterium pET-28a/CcBgl1A/E.coli BL21 (DE3) containing pET-28a/CcBgl1A recombinant expression vector.Recombinant bacterium pET-28a/CcBgl1A/E.coli BL21 (DE3) expresses the restructuring ginsenoside lytic enzyme CcBgl1A shown in SEQ ID No.4, by this restructuring ginsenoside lytic enzyme CcBgl1A called after R CcBgl1A.The 21-410 amino acids sequence of SEQ ID No.4 is the aminoacid sequence shown in SEQ ID No.2.
Empty carrier pET-28a (+) vector introduction E.coli BL21 (DE3) is obtained the recombinant bacterium pET-28a/E.coli BL21 (DE3) containing pET-28a (+), as empty vector control bacterium.
The preparation of embodiment 2, restructuring ginsenoside lytic enzyme CcBgl1A
1, the recombinant bacterium pET-28a/CcBgl1A/E.coli BL21 (DE3) of embodiment 1 is inoculated on the LB solid medium containing 50 μ g/ml kantlex, 37 DEG C of overnight incubation.Choose a ring to be inoculated into 5ml and to contain in the LB liquid nutrient medium of 50 μ g/ml kantlex, 37 DEG C, 200rpm cultivates 12h (with the LB liquid nutrient medium containing 50 μ g/ml kantlex for blank), is seed liquor.Seed liquor is inoculated into 200ml according to the ratio of 1:50 to be contained in the LB liquid nutrient medium of 50 μ g/ml kantlex, in 1L shaking flask, and 37 DEG C, 200rpm cultivation.When OD600 arrives 0.5 (taking fermention medium as blank), be cooled to 25 DEG C, and add 0.4mM IPTG induction 20h.After induction terminates, the centrifugal 10min of 5000rpm collects thalline, after washing thalline 1 time with PBS, Eddy diffusion is in the PBS damping fluid of 50ml, sonicated cells, 15000g, 4 DEG C of centrifugal 30min, collect supernatant liquor, supernatant liquor is total protein solution after the induction of recombinant bacterium pET-28a/CcBgl1A/E.coli BL21 (DE3).Get recombinant bacterium pET-28a/CcBgl1A/E.coli BL21 (DE3) thalline before induction simultaneously, after washing thalline 1 time with PBS, Eddy diffusion is in the PBS damping fluid of 50ml, sonicated cells, 15000g, 4 DEG C of centrifugal 30min, collect supernatant liquor, obtain total protein solution before the induction of recombinant bacterium pET-28a/CcBgl1A/E.coli BL21 (DE3).
2, according to the method for step 1 in the present embodiment, the recombinant bacterium pET-28a/CcBgl1A/E.coli BL21 (DE3) of embodiment 1 is replaced with recombinant bacterium pET-28a/E.coli BL21 (DE3), other step is constant, obtains total protein solution before the induction of total protein solution and recombinant bacterium pET-28a/E.coli BL21 (DE3) after the induction of recombinant bacterium pET-28a/E.coli BL21 (DE3) respectively.
3, the purifying of restructuring ginsenoside lytic enzyme CcBgl1A
After the induction of recombinant bacterium pET-28a/CcBgl1A/E.coli BL21 (DE3), add imidazoles in total protein solution makes its final concentration be 10mM, obtain the imidazole solution of total protein, slowly vibrate the Ni Sepharose fastflow gel (GE healthcare) of the imidazole solution of total protein and 5ml under 4 DEG C of conditions 30min, obtain albumen-gel mixture, it is in the glass chromatography column of 1.6cm, high 10cm that albumen-gel mixture is loaded diameter.Then use the 10mM imidazoles/PBS solution wash-out of 2 times of chromatography column volumes, then use the imidazoles/PBS solution wash-out of 300mM of 5 times of chromatography column volumes.Collect the fraction of 300mM imidazoles wash-out, obtain the enzyme liquid containing restructuring ginsenoside lytic enzyme CcBgl1A, remove the imidazoles in enzyme liquid with the ultra-filtration membrane (Millipore company) that molecular weight cut-off is 10kDa, obtain the restructuring ginsenoside lytic enzyme CcBgl1A of purifying.
4, by total protein solution after the induction of recombinant bacterium pET-28a/CcBgl1A/E.coli BL21 (DE3), total protein solution before the induction of recombinant bacterium pET-28a/CcBgl1A/E.coli BL21 (DE3), total protein solution after the induction of recombinant bacterium pET-28a/E.coli BL21 (DE3), before the induction of recombinant bacterium pET-28a/E.coli BL21 (DE3), the restructuring ginsenoside lytic enzyme CcBgl1A of total protein solution and purifying carries out polyacrylamide gel electrophoresis respectively, result as shown in Figure 3, before showing to only have the induction of recombinant bacterium pET-28a/CcBgl1A/E.coli BL21 (DE3) total protein solution and recombinant bacterium pET-28a/CcBgl1A/E.coli BL21 (DE3) induction after total protein solution and purifying restructuring ginsenoside lytic enzyme CcBgl1A in have albumen (i.e. CcBgl1A) band of 46kDa, after the induction of recombinant bacterium pET-28a/E.coli BL21 (DE3) total protein solution and recombinant bacterium pET-28a/E.coli BL21 (DE3) induction before albumen (i.e. CcBgl1A) band of equal not this 46kDa of total protein solution.
The protein content measuring the rear total protein solution of recombinant bacterium pET-28a/CcBgl1A/E.coli BL21 (DE3) induction by Coomassie brilliant G-250 method is 10mg/ml.
The mensuration of ginsenoside lytic enzyme CcBgl1A activity of 5, recombinating
1) enzyme activity of restructuring ginsenoside lytic enzyme CcBgl1A and wild type strain ginsenoside lytic enzyme is detected with artificial substrates p-nitrophenyl-β-D-glucopyranoside (pNPG).Ginsenoside lytic enzyme enzyme activity determination system is: in 100 μ l reaction systems, add the total protein solution of total protein solution or wild type strain fiber bacterium (Cellulosimicrobium sp.) 21 after the induction of total protein solution or recombinant bacterium pET-28a/E.coli BL21 (DE3) after the induction of the recombinant bacterium pET-28a/CcBgl1A/E.coli BL21 (DE3) that 10 μ l suitably dilute successively, 10 μ l pNPG (5mM) and 80 μ l NaAc-HAc damping fluid (50mM, pH 5.5), after 37 DEG C of reaction 10min, add 100 μ l 1M NaOH termination reactions.The enzyme calculating glycoside hydrolase with reference to pNP typical curve is lived.
It is 1IU that the Mei Huo unit (IU) of beta-glucosidase is defined as the per minute catalytic substrate enzyme amount generated required for 1 μm of ol pNP.
Live as 17IU/mg total protein according to the enzyme of beta-glucosidase in total protein after the induction of recombinant bacterium pET-28a/CcBgl1A/E.coli BL21 (DE3) that the result detected calculates, after the induction of recombinant bacterium pET-28a/E.coli BL21 (DE3), total protein there is no beta-glucosidase enzyme and lives.Experimental result shows that CcBgl1A albumen has beta-glucosidase enzyme and lives, and proves that CcBgl1A albumen is beta-glucosidase.
After the induction of recombinant bacterium pET-28a/CcBgl1A/E.coli BL21 (DE3), in total protein, the enzyme of beta-glucosidase is lived as 17IU/mg total protein, in wild type strain fiber bacterium (Cellulosimicrobium sp.) 21 total proteins, beta-glucosidase enzyme is lived as 0.5IU/mg total protein, after showing the induction of recombinant bacterium pET-28a/CcBgl1A/E.coli BL21 (DE3), in total protein, the enzyme work of beta-glucosidase is 34 times that in wild type strain total protein, beta-glucosidase enzyme is lived, illustrate that the total protein after the induction of recombinant bacterium pET-28a/CcBgl1A/E.coli BL21 (DE3) has higher beta-glucosidase enzyme and lives.Wherein, total protein content all measures by Coomassie brilliant G-250 method.
2) by the restructuring beta-glucosidase CcBgl1A of purifying according in step 5 in upper the present embodiment 1) the mensuration enzyme method of living measure beta-glucosidase enzyme and live, result shows that the beta-glucoside specific activity of enzyme of the restructuring beta-glucosidase CcBgl1A of purifying is 32IU/mg albumen.
The biological characteristics of embodiment 3, restructuring beta-glucosidase CcBgl1A
1, pH is on the impact of restructuring beta-glucosidase CcBgl1A vigor
By the restructuring beta-glucosidase CcBgl1A of purifying that suitably dilutes from substrate pNPG in the damping fluid of different pH value, react 10min under 37 DEG C of conditions, measure the enzyme activity of restructuring beta-glucosidase CcBgl1A under condition of different pH.The damping fluid adopted is respectively: pH 2.0-6.0,50mM NaAc-HAc damping fluid; PH 6.0-8.0,50mM Na
2hPO
4-NaH
2pO
4damping fluid; PH 8.0-11.0,50mM Glycine-NaOH damping fluid.Experimental result shows, the optimum pH of restructuring beta-glucosidase CcBgl1A is 5.5 (in Fig. 6 a).
2, pH is on the impact of restructuring beta-glucosidase CcBgl1A stability
Restructuring beta-glucosidase CcBgl1A enzyme liquid and the 50 μ l concentration of the purifying suitably diluted by 10 μ l are that the damping fluid of the various different pH value (4.0-10.0) of 100mM mixes that (damping fluid of employing is respectively: pH 4.0-6.0,50mM NaAc-HAc damping fluid; PH 6.0-8.0,50mM Na
2hPO
4-NaH
2pO
4damping fluid; PH 8.0-10.0,50mM Glycine-NaOH damping fluid), after being incubated 24h respectively in 4 DEG C of refrigerators, the enzyme liquid got after 10 μ l insulations measures remnant enzyme activity.Control group is that the enzyme liquid that 10 μ l suitably dilute mixes with 50 μ l deionized waters (enzyme is lived and is defined as 100%), result shows that restructuring beta-glucosidase CcBgl1A is after pH 6.0-10.0 scope inside holding 24h, still retains the enzyme activity of more than 50% (in Fig. 6 b).
3, temperature is on the impact of restructuring beta-glucosidase vigor
In 20-80 DEG C of water-bath, take pNPG as substrate, according to 1 in step 5 in embodiment 2) method measure the enzyme activity of restructuring beta-glucosidase CcBgl1A of purifying.Experimental result shows, the optimal reactive temperature of restructuring beta-glucosidase CcBgl1A be 35 DEG C (in Fig. 6 c).
4, temperature is on the impact of restructuring beta-glucosidase CcBgl1A stability
The restructuring beta-glucosidase CcBgl1A of purifying is incubated respectively in the water-bath of 30,35,40,45,50 DEG C 1h and measures enzyme activity.With the restructuring beta-glucosidase CcBgl1A of untreated purifying in contrast (enzyme is lived as 100%).Experimental result finds, the restructuring beta-glucosidase CcBgl1A of purifying is respectively after 30,35,40,45,50 DEG C of insulation 1h, and its residual enzyme activity is respectively 95%, 21%, 6%, 1.2%, 0.2% (in Fig. 6 d).
Embodiment 4, prepare panoxadiol type saponin(e Gyp XVII, compound O, Mb and F2 with restructuring beta-glucosidase CcBgl1A
1, the detection of each component of panoxadiol type saponin(e PPDGM and content thereof
The restructuring beta-glucosidase CcBgl1A that the present embodiment utilizes embodiment 2 step 3 purifying to obtain with panoxadiol type saponin(e PPDGM for substrate prepares panoxadiol type saponin(e Gyp XVII, compound O, Mb and F2.Wherein, panoxadiol type saponin(e PPDGM, according to the method for document (" the former bacterium of leaf muld of tomato transforms the research of ginsenoside ", Wang Juan, Northeast Normal University's master thesis), extracts and obtains from dry ginseng.Each component and the percentage of this panoxadiol type saponin(e PPDGM are respectively: Rb1:37.8%; Rb2:17.1%; Rc:24.3%; Rd:9.5%.The method detecting the panoxadiol type each component of saponin(e PPDGM and content thereof is HPLC-C18 reversed-phase column method, and method is as described below:
Chromatographic column: Shim-pack PREP-ODS (H) reversed-phase column (250 × 4.6mm, 5 μm)
Applied sample amount: 20 μ l
Moving phase: A: water; B: acetonitrile
Program: 0-10min, 32%B, 68%A; The volumn concentration of 10-40min (not comprising 10min), B is at the uniform velocity reduced to 40% by the volumn concentration that 32% is at the uniform velocity increased to 60%, A by 68%; 40-50min (not comprising 40min), 60%B, 40%A
Column temperature: 25 DEG C
Detector: UV-detector, 203nm
Flow velocity: 1.0mL/min
Wherein, standard substance are respectively ginsenoside Rb1, and Rb2, Rc, Rd, Gyp XVII, compound O, Mb and F2 (national standard physical resources net, cat. no is respectively Rb1SS0769; Rb2SS0770; Rc SS0767; Rd SS0768; Gyp XVII A0638; F2SG-G021; Compound O and Mb is according to document (Efficient biotransformation for preparation of pharmaceutically active ginsenoside Compound K by Penicillium oxalicum sp.68.Annals of Microbiology, 63,139-149,2013) prepare and identify).
2, ginsenoside Gyp XVII is prepared, compound O, Mb and F2 with recombinant beta-glycoside hydrolase CcBgl1A
Prepare ginsenoside Gyp XVII with recombinant beta-glycoside hydrolase CcBgl1A, in triplicate, the concrete steps of each experiment are as follows in compound O, Mb and F2 experiment:
The recombinant beta obtained by embodiment 2 step 3 purifying-glycoside hydrolase CcBgl1A 50mM NaAc damping fluid (pH 5.5) adjustment protein concentration is the enzyme liquid of 1mg/ml (i.e. 32IU/ml), 2.5g panoxadiol type saponin(e PPDGM is added in the above-mentioned enzyme liquid of 100ml, its final concentration is made to be 25mg/ml, 30 DEG C, react 2h under 100rpm agitation condition.After reaction terminates, 80 DEG C of heating 10min make enzyme deactivation, and centrifugal, supernatant liquor rotary evaporation is to 20ml, and after-70 DEG C of pre-freezes, lyophilize obtains converted product xeraphium.According to the present embodiment above-mentioned HPLC-C18 reversed-phase column method with ginsenoside Gyp XVII, and compound O, Mb and F2 (national standard physical resources net, cat. no is respectively Gyp XVII A0638, F2SG-G021, compound O and Mb is according to document (Efficient biotransformation for preparation of pharmaceutically active ginsenoside Compound K by Penicillium oxalicum sp.68.Annals of Microbiology, 63, 139-149, 2013) prepare and identify) be standard substance detection converted product, quantitative analysis (Fig. 4) is carried out with employing calibration curve method (external standard method) according to the retention time of standard substance is qualitative, result shows the ginsenoside Gyp XVII in converted product, compound O, Mb and F2 content is respectively 35%, 16.5%, 23.9%, 8.9% (mass percentage).
Converted product xeraphium is dissolved on a small quantity by volume ratio be the chloroform of 65:35:10 (v/v/v, lower floor), in the liquid that forms of first alcohol and water, loading is to 100mL silica gel column chromatography.With being 65:35:10 (v/v/v by volume ratio, lower floor) chloroform, first alcohol and water composition liquid carry out wash-out, collect respectively containing ginsenoside Gyp XVII, the fraction of compound O, Mb and F2, rotary evaporation flows out to organic solvent-free, lyophilize respectively after-70 DEG C of pre-freezes, obtain ginsenoside Gyp XVII, compound O, Mb and F2 sterling.Described ginsenoside Gyp XVII, compound O, Mb and F2 sterling shows content all more than 95% through above-mentioned HPLC analytical results.Result shows that this 2.5g panoxadiol type saponin(e PPDGM obtains ginsenoside Gyp XVII respectively, compound O, Mb and F2 sterling 0.75 ± 0.1g, 0.35 ± 0.05g, 0.46 ± 0.05g, 0.20 ± 0.04g, ginsenoside Gyp XVII, the productive rate of compound O, Mb and F2 is respectively 30%, 14%, 18.4%, 8% (each ginsenoside output/PPDGM that feeds intake measures * 100%).
Claims (10)
1. prepare the method for ginsenoside Gyp XVII, compound O, Mb and F2, comprise with panoxadiol type saponin(e for substrate protein carries out the step that catalyzed reaction obtains containing ginsenoside Gyp XVII, compound O, Mb and F2 reactant, described protein is following protein a) or b) or c) or d):
A) aminoacid sequence is the protein of SEQ ID No.2;
B) aminoacid sequence is the protein of SEQ ID No.4;
C) by the protein with ginsenoside hydrolytic enzyme activities that the aminoacid sequence shown in SEQ ID No.2 obtains through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation;
D) by the protein with ginsenoside hydrolytic enzyme activities that the aminoacid sequence shown in SEQ ID No.4 obtains through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation.
2. method according to claim 1, is characterized in that: described method comprises purifying from described reactant and obtains the step of ginsenoside Gyp XVII, compound O, Mb and F2.
3. method according to claim 1 and 2, it is characterized in that: describedly to carry out in a liquid for substrate protein carries out catalyzed reaction with panoxadiol type saponin(e, the proportioning of protein described in described liquid and described panoxadiol type saponin(e is protein described in 1IU: panoxadiol type saponin(e described in 0.5-2mg, and described IU is that the β-glycoside hydrolase enzyme of described protein is lived.
4., according to the arbitrary described method of claim 1-3, it is characterized in that: described catalyzed reaction carries out 1-3 hour, described catalyzed reaction is at 25-35 DEG C.
5. according to the arbitrary described method of claim 1-4, it is characterized in that: described purifying comprises the following steps: described reactant is carried out centrifugal, ginsenoside Gyp XVII, compound O, Mb and F2 is made to enter in supernatant liquor, collect supernatant liquor, obtain the supernatant liquor containing ginsenoside Gyp XVII, compound O, Mb and F2, described supernatant liquor is dry, obtain the xeraphium containing ginsenoside Gyp XVII, compound O, Mb and F2, remove the impurity in described xeraphium, obtain ginsenoside Gyp XVII, compound O, Mb and F2 respectively.
6. according to the arbitrary described method of claim 1-5, it is characterized in that: the impurity removed in described xeraphium comprises the following steps: be dissolved in solution A by described xeraphium, obtain the liquid mixture containing ginsenoside Gyp XVII, compound O, Mb and F2, described liquid mixture is carried out silica gel column chromatography, with described solution A wash-out, collect the fraction containing ginsenoside Gyp XVII, compound O, Mb and F2 respectively, by dry for each described fraction, obtain ginsenoside Gyp XVII, compound O, Mb and F2;
Described solution A is made up of chloroform, first alcohol and water, and in described solution A, the volume ratio of chloroform, first alcohol and water is 65:35:10 (lower floor's solution).
7. the described protein in method described in claim 1 is preparing the application in ginsenoside Gyp XVII, compound O, Mb and F2.
8. the application in ginsenoside Gyp XVII, compound O, Mb and F2 prepared by the biomaterial relevant to the described protein in method described in claim 1.
9. the described protein in method described in claim 1 prepares the application in ginsenoside Gyp XVII, compound O, Mb and F2 at catalysis ginsenoside.
10. the application in ginsenoside Gyp XVII, compound O, Mb and F2 prepared by the biomaterial relevant to the described protein in method described in claim 1 at catalysis ginsenoside.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510036363.5A CN104762280B (en) | 2015-01-25 | 2015-01-25 | A kind of glycoside hydrolase that recombinates is used for the technique that bioconversion prepares panoxadiol type saponin(e |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510036363.5A CN104762280B (en) | 2015-01-25 | 2015-01-25 | A kind of glycoside hydrolase that recombinates is used for the technique that bioconversion prepares panoxadiol type saponin(e |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104762280A true CN104762280A (en) | 2015-07-08 |
| CN104762280B CN104762280B (en) | 2017-11-10 |
Family
ID=53644372
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510036363.5A Active CN104762280B (en) | 2015-01-25 | 2015-01-25 | A kind of glycoside hydrolase that recombinates is used for the technique that bioconversion prepares panoxadiol type saponin(e |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104762280B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108138211A (en) * | 2015-10-22 | 2018-06-08 | 株式会社爱茉莉太平洋 | The method for selectively preparing Ginsenoside F2, Mc compounds and O compounds from the saponin of ginseng using enzyme process |
-
2015
- 2015-01-25 CN CN201510036363.5A patent/CN104762280B/en active Active
Non-Patent Citations (10)
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108138211A (en) * | 2015-10-22 | 2018-06-08 | 株式会社爱茉莉太平洋 | The method for selectively preparing Ginsenoside F2, Mc compounds and O compounds from the saponin of ginseng using enzyme process |
| CN108138211B (en) * | 2015-10-22 | 2021-08-10 | 株式会社爱茉莉太平洋 | Method for selectively preparing ginsenoside F2, Mc compound and O compound from ginsenoside of ginseng by enzyme method |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104762280B (en) | 2017-11-10 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106978360B (en) | High-yield cellulase trichoderma reesei recombinant strain and application thereof | |
| Hooker et al. | Leveraging anaerobic fungi for biotechnology | |
| CN105821020B (en) | A kind of 'beta '-mannase mRmMan5A and its encoding gene and application | |
| Yan et al. | Transcriptomic profiling and genetic analyses reveal novel key regulators of cellulase and xylanase gene expression in Penicillium oxalicum | |
| Li et al. | Diversity of cellulase-producing filamentous fungi from tibet and transcriptomic analysis of a superior cellulase producer Trichoderma harzianum LZ117 | |
| Han et al. | Regulation of expression of cellulosomes and noncellulosomal (hemi) cellulolytic enzymes in Clostridium cellulovorans during growth on different carbon sources | |
| Long et al. | Enhancing cellulase and hemicellulase production in Trichoderma orientalis EU7-22 via knockout of the creA | |
| CN111808831A (en) | Preparation method of recombinant manganese peroxidase and application of recombinant manganese peroxidase in degradation of Chinese herbal medicine lignin | |
| CN103981112A (en) | Double-promoter multi-copy recombinant pichia pastoris strain for highly producing endo-inulinase | |
| CN102898512B (en) | Recombinant plectasin as well as preparation method and application of recombinant plectasin | |
| CN101469325A (en) | Secretory expression method for exoinulinase from Kluyveromyces marxianus | |
| CN108753741A (en) | A kind of extracellular AA9 families polysaccharide monooxygenase AnLPMO15g and its application | |
| CN104762280A (en) | Technology for biotransformation preparation of panoxadiol saponins by recombinant glucoside hydrolase | |
| Ding et al. | Molecular cloning and transcriptional expression analysis of an intracellular β-glucosidase, a family 3 glycosyl hydrolase, from the edible straw mushroom, Volvariella volvacea | |
| CN106929425B (en) | High-temperature-resistant acidic thermoascus thermophilus cellulase, and preparation method and application thereof | |
| CN116790542A (en) | Glycosyltransferase PpUGT3 for biosynthesis of rhizoma paridis saponin | |
| CN110655554B (en) | Protein active peptide for improving saccharomyces cerevisiae proliferation and ethanol tolerance as well as preparation method and application thereof | |
| CN109402090B (en) | Beta-1,3 endoglucanase with immune enhancing activity and derived from scapharca broughtonii and encoding polynucleotide thereof | |
| CN109182361B (en) | Cellulose endoenzyme gene, protein and application thereof | |
| CN109234293B (en) | β -glucosidase gene and expression vector and protein thereof | |
| CN101824401B (en) | Glucanase and coding nucleic acid and expression thereof | |
| CN113980939B (en) | Glucose-resistant beta-glucosidase, and expression gene and application thereof | |
| Reeta Rani et al. | Beta-Glucosidase from Aspergillus niger NII 08121-Molecular characterization and applications in bioethanol production | |
| JP2014168424A (en) | Filamentous fungus mutant with cre1 gene not functioning | |
| CN104004727B (en) | A kind of preparation method with the protein of sialic acid hydrolytic enzyme activities |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| EXSB | Decision made by sipo to initiate substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| CB03 | Change of inventor or designer information |
Inventor after: Tai Guihua Inventor after: Zhou Yifa Inventor after: Gao Juan Inventor after: Yuan Ye Inventor after: Hu Yanbo Inventor after: Sun Chengxin Inventor after: Chen Honglei Inventor after: Hu Chenxing Inventor before: Zhou Yifa Inventor before: Gao Juan Inventor before: Yuan Ye Inventor before: Hu Yanbo Inventor before: Sun Chengxin Inventor before: Chen Honglei Inventor before: Hu Chenxing |
|
| CB03 | Change of inventor or designer information | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |