CN105039371A - Trehalose synthase-trehalose hydrolase fusion enzyme, expression gene thereof and application - Google Patents
Trehalose synthase-trehalose hydrolase fusion enzyme, expression gene thereof and application Download PDFInfo
- Publication number
- CN105039371A CN105039371A CN201510431597.XA CN201510431597A CN105039371A CN 105039371 A CN105039371 A CN 105039371A CN 201510431597 A CN201510431597 A CN 201510431597A CN 105039371 A CN105039371 A CN 105039371A
- Authority
- CN
- China
- Prior art keywords
- enzyme
- trehalose
- oligosaccharide based
- malt oligosaccharide
- based mycose
- 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.)
- Pending
Links
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/52—Improvements relating to the production of bulk chemicals using catalysts, e.g. selective catalysts
Landscapes
- Enzymes And Modification Thereof (AREA)
Abstract
The invention relates to a trehalose synthase-trehalose hydrolase fusion enzyme, an expression gene thereof and application. Amino acid sequences of the maltooligosyl trehalose synthase-maltooligosyl trehalose tetrahydrolase fusion enzyme are shown as SEQ ID NO.2, and nucleotide sequences of the expression gene are shown as SEQ ID NO.1. The trehalose synthase-trehalose hydrolase fusion enzyme, the expression gene and the application have the advantages that the novel maltooligosyl trehalose synthase-maltooligosyl trehalose tetrahydrolase fusion enzyme is constructed by the aid of genetic engineering technologies and has the activity of maltooligosyl trehalose synthase and the activity of maltooligosyl trehalose tetrahydrolase, and accordingly production processes can be effectively simplified as compared with the traditional two-enzyme methods.
Description
Technical field
The present invention relates to TreP-hydrolysis of trehalose enzyme and merge enzyme and expressing gene thereof and application, merge enzyme and expressing gene thereof and application in particular to malt oligosaccharide based mycose synthetase-malt oligosaccharide based mycose lytic enzyme, belong to genetically engineered and technical field of enzyme engineering.
Background technology
Trehalose is a kind of ubiquitous irreducibility disaccharide, is connected by α-1,1 glycosidic link by glucose, and wherein α, α-1,1-trehalose is separated at present, and is found extensively to be present in plant, animal and microorganism.Natural trehalose has good and nonspecific provide protection to organism or biomacromolecule, and the organism possessing trehalose can be made can to spend the severe environment such as hunger, drying, low temperature, high temperature, radiation.Along with the increase to the trehalose degree of awareness, trehalose application in every respect is constantly expanded, and is extensively promoted in fields such as medicine, food, agriculturals.
The production technique of current trehalose mainly contains three kinds:
(1) extraction method
Extraction method obtains mainly through cultivating the higher natural biological of content of trehalose, extraction object main is at present yeast (content of trehalose accounts for 15% of yeast dry weight), but because trehalose belongs to yeast content, need during extraction to carry out the broken wall that copies and separation-extraction technology, therefore prepared trehalose method by enzyme process gradually at present and replace.
(2) single enzyme process
Single enzyme process was proposed by people such as Japanese Nishimoto first in nineteen ninety-five, namely adopted trehalose synthase to transform the technique of maltose production trehalose.Trehalose synthase can by α, α-1, the maltose that 4-glycosidic link connects is converted into α, the trehalose that α-1,1-glycosidic link connects, this conversion reaction does not need phosphatic existence, do not need to consume anakinetomer, but the enzyme heat stability that the method adopts is general lower, and transformation efficiency mostly is about 60%, in addition because this enzyme has slight hydrolytic activity simultaneously, the glucose of a small amount of by product therefore in single enzymatic conversion method process, also can be produced.
(3) double-enzyme method
Double-enzyme method by people's reported first such as Lama, namely adopted malt oligosaccharide based mycose synthetase (MTSase) and malt oligosaccharide based mycose lytic enzyme (MTHase) two kinds of enzymes to utilize starch for the method for substrate production trehalose in 1991.In the method, the first enzyme is used for the maltodextrin that catalyzed polymerization degree (DP) is greater than 3, and the α-Isosorbide-5-Nitrae connecting key transforming its reducing end generates α-1,1 connecting key.α-1, the 1 key trehalose that the second enzyme specificity catalytically hydrolyzing alpha-Isosorbide-5-Nitrae key generates and low-molecular-weight malto-oligosaccharide.Current double-enzyme method most with reductibility starch for raw material, transformation efficiency mostly is about 70-80%, and therefore more single enzyme process is compared more economical, except producing except trehalose, can also produce the trisaccharide maltose had compared with high added value, therefore this operational path is used to suitability for industrialized production simultaneously.
Although double-enzyme method has above plurality of advantages, still Shortcomings part: double-enzyme method transforms two kinds of enzyme requires used and carries out respectively fermenting and purifying could obtain, and therefore the production cost of enzyme is apparently higher than single enzyme process.
Summary of the invention
The present invention is directed to the deficiencies in the prior art, provide malt oligosaccharide based mycose synthetase-malt oligosaccharide based mycose lytic enzyme to merge enzyme and expressing gene thereof and application.
Technical solution of the present invention is as follows:
Malt oligosaccharide based mycose synthetase-malt oligosaccharide based mycose lytic enzyme merges the expressing gene of enzyme, and nucleotide sequence is as shown in SEQIDNO:1.
Malt oligosaccharide based mycose synthetase-malt oligosaccharide based mycose lytic enzyme merges enzyme, and aminoacid sequence is as shown in SEQIDNO:2.
A kind of recombinant vectors, inserts nucleotide sequence shown in above-mentioned SEQIDNo.1 in plasmid.
Preferred according to the present invention, described plasmid is pPIC3.5K vector plasmid or pPIC9K vector plasmid.
A kind of transgenic cell line, transforms in host cell and has above-mentioned recombinant vectors.
Preferred according to the present invention, described host cell is Pichia pastoris GS115.
Above-mentioned malt oligosaccharide based mycose synthetase-malt oligosaccharide based mycose lytic enzyme merges the expressing gene of enzyme, recombinant vectors or transgenic cell and ties up to and prepare malt oligosaccharide based mycose synthetase-malt oligosaccharide based mycose lytic enzyme and merge application in enzyme.
The expressing gene that above-mentioned malt oligosaccharide based mycose synthetase-malt oligosaccharide based mycose lytic enzyme merges enzyme is preparing the application in trehalose and trisaccharide maltose.
Beneficial effect
Technical scheme of the present invention compared with prior art has the following advantages:
1, malt oligosaccharide based mycose synthetase-malt oligosaccharide based mycose lytic enzyme that the present invention adopts genetic engineering technique to construct a kind of novelty merges enzyme, described fusion enzyme has the activity of malt oligosaccharide based mycose synthetase and malt oligosaccharide based mycose lytic enzyme two kinds of enzymes simultaneously, effectively simplifies production technique compared with traditional double enzyme process.
2, the ratio enzyme after the fusion enzyme that the present invention obtains merges is lived and is respectively 147.4U/mg and 160.1U/mg, active reasonable ratio, without the need to considering that the proportioning of two kinds of enzymes can realize Efficient Conversion again compared with traditional double enzyme process, therefore the fusion enzyme produced of the present invention is more easy in use.
3, because the substrate specificity of malt oligosaccharide based mycose lytic enzyme fusion enzyme is the product of malt oligosaccharide based mycose synthetase, therefore the fusion of two kinds of enzymes malt oligosaccharide based mycose lytic enzyme is melted time that synthase contacts substrate plays a role shortens greatly, the enzyme reaction of use fusion under the same conditions only 20 hours trehalose transformation efficiencys can reach 85.3%, comparatively traditional double enzyme process is compared and has been saved nearly 4 hours, effectively can save the production time, reduce costs, reduce microbiological contamination probability, be therefore more suitable for suitability for industrialized production and application.
4, the present invention adopts pichia spp as the production bacterial strain merging enzyme, and can effectively avoid endotoxic pollution compared with escherichia expression system, have higher security, the trehalose that therefore the present invention produces is more suitable for being applied to food service industry.
Accompanying drawing explanation
Fig. 1 malt oligosaccharide based mycose synthetase-malt oligosaccharide based mycose lytic enzyme merges enzyme gene PCR and builds result;
In figure: M, marker, 1 and 2, PCR primer.
Embodiment
The following examples will illustrate working method of the present invention, but can not as limitation of the invention.
The extraction of embodiment 1:Arthrobactersp. genome DNA.
Contriver is through a large amount of screening, strain oxidation Arthrobacter (Arthrobactersp.) QYW623 is found in the contaminated agricultural land soil of Jinan, Shandong Province suburban area one, its fermented liquid has higher malt oligosaccharide based mycose synthetase simultaneously and malt oligosaccharide based mycose lytic enzyme merges enzymic activity, and the ratio enzymic activity of purifying latter two enzyme relatively, optimum temperuture is all at about 55 DEG C, all there is higher catalytic activity (remnant enzyme activity > 85%) between 5.0-5.8, through cloning the malt oligosaccharide based mycose synthetase existed in this bacterium and maltooligosyltrehalohydrolase hydrolase gene sequence, find that the gene order of two kinds of enzymes is comparatively close to the gene order of corresponding enzyme in Arthrobactersp.L77 genome (NZ_JWSU00000000.1), sequence identity is all greater than 99%, but not yet there is people to study its zymologic property.
By oxidation Arthrobacter (Arthrobactersp.) the QYW623 inoculation of preservation at-80 DEG C to LB liquid nutrient medium (peptone 10g/L, yeast powder 5g/L, NaCl10g/L) activation culture 48 hours in, thereafter be seeded in fresh LB according to 1% inoculum size and cultivate 24 hours, the method for going 10mL bacterium liquid to provide according to Shanghai raw work bacterial genomes extraction test kit extracts this bacterium genome DNA.
Embodiment 2: malt oligosaccharide based mycose synthetase-malt oligosaccharide based mycose lytic enzyme melts the structure of synthase gene
Design primers F 1 and R1 (amplification malt oligosaccharide based mycose synthetase), F2 and R2 (amplification malt oligosaccharide based mycose lytic enzyme) respectively according to the Arthrobactersp. genome sequence that NCBI logs in, primer sequence is as follows:
F1:GAATTCGTGTTGACACCGAAATCGACCTACC
R1CCTCGGGGGTGAACGTGC
F2:ATGAGTTCGCCATTCGAGGT
R2:GCGGCCGCGTCGAGCAGGTGGATGGAGG
The STb gene obtained with embodiment 1 is for template, F1 and R1 is primer, utilizes TaKaRaExTaq
tMthe system that specification sheets provides carries out PCR;
PCR condition is: 95 DEG C of sex change 5min; 95 DEG C of sex change 30sec, 54 DEG C of annealing 30sec, 72 DEG C extend 2.5min, totally 30 circulations; 72 DEG C extend 10min, 4 DEG C of preservations;
About 1700bp band is reclaimed in rubber tapping, for subsequent use as product 1;
The STb gene obtained with embodiment 1 is for template, F2 and R2 is primer, utilizes TaKaRaExTaq
tMthe system that specification sheets provides carries out PCR;
PCR condition is: 95 DEG C of sex change 5min; 95 DEG C of sex change 30sec, 56 DEG C of annealing 30sec, 72 DEG C extend 2min, totally 30 circulations; 72 DEG C extend 10min, 4 DEG C of preservations;
About 2300bp band is reclaimed in rubber tapping, for subsequent use as product 2;
Subsequently the product 1 of acquisition and product 2 are realized two intergenic splicings by overlapping PCR method;
PCR condition is: after product 1 and product 2 mix with enzyme liquid, 95 DEG C of sex change 5min; 95 DEG C of sex change 30sec, 56 DEG C of annealing 30sec, 72 DEG C extend 2.5min, 5 circulations; Then primers F 1 and R2 is added; 95 DEG C of sex change 30sec, 56 DEG C of annealing 30sec, 72 DEG C extend 4.5min, totally 30 circulations; 72 DEG C extend 10min, 4 DEG C of preservations;
1% agarose gel electrophoresis is adopted to analyze (Fig. 1) PCR primer, result shows that 4000bp right position has specificity object band to occur in swimming lane 1 and 2, close with theoretical value 4059bp, show that the goal gene sequence merging enzyme is successfully built.
Rubber tapping is reclaimed about about 4000bp malt oligosaccharide based mycose synthetase-malt oligosaccharide based mycose lytic enzyme and is merged enzyme goal gene sequence, and be connected with pMD18-T carrier and obtain pMD18-MM, screen positive recombinant after transformation of E. coli JM109 and check order and preserve; After testing, its nucleotide sequence is as shown in SEQIDNO.1, and the aminoacid sequence of expression is as shown in SEQIDNO.2.
Embodiment 3: melt the expression of synthase gene in pichia spp
Described Pichia anomala expression used carrier is to realize the carrier that in pichia spp born of the same parents, (for pPIC3.5K) or born of the same parents outer (for pPIC9K) express.
Extract pMD18-MM plasmid and use EcoRI and NotI double digestion, pPIC3.5K with pPIC9K afterwards after cutting through identical two kinds of enzyme enzymes is connected, obtain heterologous expression vector pPIC3.5K-MM and pPIC9K-MM, by obtained carrier after BglII linearizing, method (the 2500V transformed by electricity, 5ms) transform in Pichia pastoris GS115, and adopt G418 to screen positive recombinant.
Embodiment 4: expression, the Purification and properties mensuration of enzyme are merged in restructuring
Express handbook according to Pichia pastoris GS115 and heterogenous expression is carried out to obtained pichia spp recon.
This project bacterium is with after 1.0wt% methanol induction 72h, collected by centrifugation fermented supernatant fluid (first need carry out cytoclasis to intracellular expression recombinant yeast pichia pastoris), adopt respectively saltout, dialyse, ion-exchange and gel-filtration carry out purifying, zymologic property restructuring being merged to enzyme measures.
Result show obtain merge enzyme can show simultaneously malt oligosaccharide based mycose synthetase (than enzyme live: 147.4U/mg) and malt oligosaccharide based mycose lytic enzyme (than enzyme work: the 160.1U/mg) activity of two kinds of enzymes, TreP and hydrolysis of trehalose enzyme enzyme are lived than being about 1:1.1, the reaction ratio (1:1) of this ratio and theory the best is close, and the zymologic property merging enzyme is similar to two kinds of enzymes before merging, and (optimum pH is all about 5.5, optimum temperuture is 50 DEG C), the trehalose transformation efficiency transforming starch emulsion 20 hours gained of 20wt% with the addition of 500U/L at 50 DEG C can reach 85.3%, trisaccharide maltose content is 1.2%, arrival same conversion time more existing double-enzyme method is compared and has been saved nearly 4 hours.
Claims (8)
1. malt oligosaccharide based mycose synthetase-malt oligosaccharide based mycose lytic enzyme merges the expressing gene of enzyme, and nucleotide sequence is as shown in SEQIDNO:1.
2. malt oligosaccharide based mycose synthetase-malt oligosaccharide based mycose lytic enzyme merges enzyme, and aminoacid sequence is as shown in SEQIDNO:2.
3. a recombinant vectors, inserts nucleotide sequence shown in SEQIDNo.1 described in claim 1 in plasmid.
4. recombinant vectors as claimed in claim 3, it is characterized in that, described plasmid is pPIC3.5K vector plasmid or pPIC9K vector plasmid.
5. a transgenic cell line, transforms in host cell and has above-mentioned recombinant vectors.
6. recombinant vectors as claimed in claim 5, it is characterized in that, described host cell is Pichia pastoris GS115.
7. malt oligosaccharide based mycose synthetase described in claim 1-malt oligosaccharide based mycose lytic enzyme merges transgenic cell described in recombinant vectors described in the expressing gene of enzyme, claim 3 or claim 5 and ties up to and prepare malt oligosaccharide based mycose synthetase-malt oligosaccharide based mycose lytic enzyme and merge application in enzyme.
8. the expressing gene that malt oligosaccharide based mycose synthetase described in claim 1-malt oligosaccharide based mycose lytic enzyme merges enzyme is preparing the application in trehalose and trisaccharide maltose.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510431597.XA CN105039371A (en) | 2015-07-21 | 2015-07-21 | Trehalose synthase-trehalose hydrolase fusion enzyme, expression gene thereof and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510431597.XA CN105039371A (en) | 2015-07-21 | 2015-07-21 | Trehalose synthase-trehalose hydrolase fusion enzyme, expression gene thereof and application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN105039371A true CN105039371A (en) | 2015-11-11 |
Family
ID=54446343
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510431597.XA Pending CN105039371A (en) | 2015-07-21 | 2015-07-21 | Trehalose synthase-trehalose hydrolase fusion enzyme, expression gene thereof and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105039371A (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105296524A (en) * | 2015-11-23 | 2016-02-03 | 齐鲁工业大学 | Construction method and application of aspergillus niger engineering bacteria for preparation of food grade trehalose |
| CN105886573A (en) * | 2016-05-16 | 2016-08-24 | 齐鲁工业大学 | Method for preparing trehalose by continuous exoenzyme biological process |
| CN105925642A (en) * | 2016-06-14 | 2016-09-07 | 湖南汇升生物科技有限公司 | Method of industrial production for trehalose by way of microbial fermentation |
| CN106190880A (en) * | 2016-07-20 | 2016-12-07 | 江南大学 | The genetic engineering bacterium of high yield malt oligosaccharide based mycose synthetase and application thereof |
| CN108118047A (en) * | 2016-11-28 | 2018-06-05 | 中国科学院微生物研究所 | A kind of preparation method of bifunctional enzyme and its application in trehalose production |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030097673A1 (en) * | 1996-01-12 | 2003-05-22 | Oscar Johannes Maria Goddijn | Enhanced accumulation of trehalose in plants |
| CN101100658A (en) * | 2007-06-08 | 2008-01-09 | 中国农业科学院饲料研究所 | Trehalose synthetase and application thereof |
| CN101880681A (en) * | 2010-04-30 | 2010-11-10 | 成都大学 | Preparation method of maltooligosyltrehalose hydrolase gene sequence and recombinant protein thereof |
-
2015
- 2015-07-21 CN CN201510431597.XA patent/CN105039371A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030097673A1 (en) * | 1996-01-12 | 2003-05-22 | Oscar Johannes Maria Goddijn | Enhanced accumulation of trehalose in plants |
| CN101100658A (en) * | 2007-06-08 | 2008-01-09 | 中国农业科学院饲料研究所 | Trehalose synthetase and application thereof |
| CN101880681A (en) * | 2010-04-30 | 2010-11-10 | 成都大学 | Preparation method of maltooligosyltrehalose hydrolase gene sequence and recombinant protein thereof |
Non-Patent Citations (2)
| Title |
|---|
| SAXENA,A.K.等: "Arthrobacter sp. L77 scaffold1.1, whole genome shotgun sequence NCBI Reference Sequence: NZ_JWSU01000001.1", 《GENBANK》 * |
| YONG HWAN KIM 等: "Trehalose Synthesis by Sequential Reactions of Recombinant Maltooligosyltrehalose Synthase and Maltooligosyltrehalose Trehalohydrolase from Brevibacterium helvolum", 《APPLIED AND ENVIRONMENTAL MICROBIOLOGY》 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105296524A (en) * | 2015-11-23 | 2016-02-03 | 齐鲁工业大学 | Construction method and application of aspergillus niger engineering bacteria for preparation of food grade trehalose |
| CN105886573A (en) * | 2016-05-16 | 2016-08-24 | 齐鲁工业大学 | Method for preparing trehalose by continuous exoenzyme biological process |
| CN105925642A (en) * | 2016-06-14 | 2016-09-07 | 湖南汇升生物科技有限公司 | Method of industrial production for trehalose by way of microbial fermentation |
| CN105925642B (en) * | 2016-06-14 | 2019-08-13 | 湖南汇升生物科技有限公司 | With the method for microbe fermentation method industrialized production trehalose |
| CN106190880A (en) * | 2016-07-20 | 2016-12-07 | 江南大学 | The genetic engineering bacterium of high yield malt oligosaccharide based mycose synthetase and application thereof |
| CN106190880B (en) * | 2016-07-20 | 2020-03-24 | 江南大学 | Genetic engineering bacterium for high-yield maltooligosyl trehalose synthase and application thereof |
| CN108118047A (en) * | 2016-11-28 | 2018-06-05 | 中国科学院微生物研究所 | A kind of preparation method of bifunctional enzyme and its application in trehalose production |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102199581B (en) | Zearalenone toxin degradation enzyme and coding gene and application thereof | |
| CN105039371A (en) | Trehalose synthase-trehalose hydrolase fusion enzyme, expression gene thereof and application | |
| CN109385413B (en) | Glucoamylase TlGA1931 and gene and application thereof | |
| CN103205475A (en) | Novel application of malt oligosaccharide based mycose synthetase and malt oligosaccharide based mycose hydrolase in mycose production | |
| CN102787130A (en) | Acid and high temperature resistant alpha-amylase, and its gene, engineering bacterium and preparation method | |
| CN105062997A (en) | L-asparaginase mutant with improved enzyme activity and construction method thereof | |
| CN103687947B (en) | Saltant type β glucosidase, biomass decomposition enzymatic compositions and the manufacture method of liquid glucose | |
| CN105039374A (en) | Starch induction type recombinant bacillus subtilis as well as preparation method and application thereof | |
| CN103834629A (en) | Recombinant high-temperature pullulanase and preparation method thereof | |
| CN105950640A (en) | Preparation method of marine bacteria-derived kappa-carrageenase gene and recombinase | |
| CN101993863B (en) | Glucamylase as well as encoding gene and application thereof | |
| CN105039191B (en) | A kind of surface display trehalose synthetase, the method for hydrolysis of trehalose enzyme and application | |
| CN103146726B (en) | Aspergillus niger alpha-glucosidase gene and high-efficiency expression method thereof | |
| CN102994476B (en) | Saccharifying enzyme | |
| CN102367448A (en) | Construction method of genetic engineering strain for high expression and easy purification of beta-mannanase | |
| CN102517307B (en) | Beta-glucosidase Mut1b as well as expressed gene and application thereof | |
| JP2023534879A (en) | N-Acetylglucosamine-producing strain and method for construction and use thereof | |
| CN103013956A (en) | Saccharifying enzyme and recombinant expression strain thereof | |
| CN110106154A (en) | A kind of limonene synzyme SynLS2 and its application | |
| CN108410903A (en) | The endo-xylanase and its encoding gene of a kind of resistance to low ph value and application | |
| CN105316274A (en) | Recombined bacillus subtilis with improved asparaginase secretion capacity and application thereof | |
| CN102634558B (en) | A kind of genetically engineered co-immobilization xylanolytic enzyme prepares the method for xylo-oligosaccharide and wood sugar | |
| CN102952790B (en) | Multifunctional cellulose as well as expression gene and application thereof | |
| CN102676476B (en) | Dextranase with improved enzyme activity and thermal stability | |
| CN105112348A (en) | Recombinant brevibacillus brevis highly yielding pullulanase and application of recombinant brevibacillus brevis |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20151111 |