CN102876705A - Method for breeding high-yield leucine aminopeptidase strain by protoplast transformation - Google Patents
Method for breeding high-yield leucine aminopeptidase strain by protoplast transformation Download PDFInfo
- Publication number
- CN102876705A CN102876705A CN2012104186053A CN201210418605A CN102876705A CN 102876705 A CN102876705 A CN 102876705A CN 2012104186053 A CN2012104186053 A CN 2012104186053A CN 201210418605 A CN201210418605 A CN 201210418605A CN 102876705 A CN102876705 A CN 102876705A
- Authority
- CN
- China
- Prior art keywords
- strain
- leucine aminopeptidase
- lap
- smm
- recombinant
- 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
Images
Abstract
The invention relates to a method for breeding a high-yield leucine aminopeptidase strain by protoplast transformation, belonging to the technical fields of enzyme preparations and food additives. In the invention, a recombinant plasmid PMA-BSAP and a protoplast prepared from Bacillus subtilis Zj016 are mixed in a hypertonic buffer by a protoplast transformation technique; under the mediation of PEG, the protoplast and PMA-BSAP are mutually coagulated to carry out genetic transformation; and cell walls are regenerated under proper conditions to successfully obtain a recombinant transformed strain ZH-Zi016. The recombinant transformed strain is collected in China General Microbiological Culture Collection center, and the collection number is CGMCC No.6559. The repeated subculture experimentation proves that the recombinant transformed strain has stable heredity. After primarily optimizing the shake culture medium components and fermenting conditions of the recombinant transformed strain, the enzymatic activity of the leucine aminopeptidase produced by fermenting the recombinant transformed strain ZH-Zj016 is 161 U/mL, which is 23 times of the initial strain.
Description
Technical field
The method of protoplast transformation breeding high-yield leucine aminopeptidase(LAP) bacterial strain, specifically utilize the protoplast transformation breeding technique to change recombinant plasmid PMA-BSAP over to subtilis Zj016 protoplastis, regenerative cell's wall under the suitable condition successfully obtains a strain recombinant conversion bacterium ZH-Zj016; The many experiments that goes down to posterity proves that the heritability of this recombinant conversion bacterium is stable.Behind the shake-flask culture based component and fermentation condition of this transformed bacteria of initial optimization, the leucine aminopeptidase(LAP) enzyme 161U/mL of being alive is produced in recombinant conversion bacterium ZH-Zj016 fermentation, reaches 23 times of original strain Zj016.Belong to zymin, technical field of food additives.
Background technology
The protoplast transformation technology is an important Protocols in Molecular Biology that grows up the sixties in 20th century, use the protoplast transformation technology and carry out microbes screening and culturing, because enzymolysis has been removed cell walls, having removed cell is in contact with one another and the natural cover for defense that exchanges with foreign DNA, can break the kind boundary of microorganism, realize the gene recombination between the bacterial strain of source far away; And the genetic information amount is large, and the transmission of genetic material is more complete, is conducive to improve breeding speed, thereby so that people at short notice seed selection to desirable strain.
Microbial strains is the core of fermentation industry, the enzyme that is produced by its metabolism has been applied to the every aspect that people live, proteolytic enzyme just has important application at field of industrial productions such as food, leather, washing composition, zymin is the core product of fermentation industry, the zymin industry development of fermentation industry and relevant industries, and produced huge economic and social benefit.Aminopeptidase is the general name that a class is cut the exopeptidase of amino-acid residue from the nitrogen end enzyme of protein or peptide chain, and it all has at aspects such as food service industry, medical science, chemical industry widely uses.The domestic stage that is in fast development in the research aspect the aminopeptidase production, for realizing industrialization, fill up the blank of home products, import substitutes strengthen China's zymin industrial competition, break this product and are had great significance by external monopolization.
Summary of the invention
The purpose of this invention is to provide a kind of method of utilizing protoplast transformation technology screening high yield leucine aminopeptidase(LAP) bacterial strain, the relative starting strain of superior strain enzymatic productivity that screens is significantly increased.
Technical scheme of the present invention: a kind of method of protoplast transformation breeding high-yield leucine aminopeptidase(LAP) bacterial strain, protoplastis preparation and regeneration condition to the subtilis Zj016 that can produce leucine aminopeptidase(LAP) are optimized, on this basis, the protoplastis of recombinant plasmid PMA-BSAP and prepared subtilis Zj016 is mixed in the high osmotic buffer, under the PEG mediation, make protoplastis and PMA-BSAP that the phase mutual coagulation occur, carry out genetic transformation, regenerative cell's wall under the suitable condition, success obtains a strain recombinant conversion bacterium ZH-Zj016, be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, deposit number: CGMCC No.6559, shake-flask culture based component and the fermentation condition of this recombinant conversion bacterium of initial optimization obtain high yield leucine aminopeptidase(LAP) bacterial strain, and technique is:
A. high yield leucine aminopeptidase(LAP) bacterial strain screening:
(1) starting strain: subtilis (
Bacillus subtilis) the Zj016([food and fermentation industries] the 3rd phase of 32 volumes in 2006 is open, the preservation of laboratory, this school, the applying date rises in 20 years and provides to the public);
(2) plasmid: recombinant plasmid PMA-BSAP, see Chinese patent CN102492645A for details;
(3) high osmotic buffer (SMM liquid) (mol/L): sucrose 0.5, maleic acid 0.02, MgCl
20.02 with the deionized water preparation, transferring pH is 7.0;
(4) fusogen: with SMM liquid preparation mass concentration 40% PEG6000, transferring pH is 7.0;
(5) lysozyme soln: be mixed with 20 mg/mL with the high sepage of SMM, filtration sterilization ,-20 ℃ of preservations;
(6) liquid nutrient medium (g/L): peptone 10, sodium-chlor 5, extractum carnis 5, with the deionized water preparation, 7.2,121 ℃ of sterilizations of pH, 20 min;
(7) solid medium (g/L): peptone 10, sodium-chlor 5, extractum carnis 5, agar 20, with the deionized water preparation, 7.2,121 ℃ of sterilizations of pH, 20 min;
(8) regeneration culture medium (g/L): peptone 10, extractum carnis 5, NaCl 5, agar powder 40, with the preparation of SMM liquid, pH 7.2;
(9) transformant regeneration culture medium (g/L): peptone 10, extractum carnis 5, casamino acids hydrolyzate 5, glucose 6, sodium-chlor 1.2, agar 20, kantlex 0.05 is used the SMM solution preparation, and pH 7.2;
(10) Screening of strain with high productivity substratum (g/L): Zulkovsky starch 6, dregs of beans 18, yeast extract paste 24, glycerine 2.5, K
2HPO
43H
2O 6, CoCl
20.015, L-Leu-4-N-methyl-p-nitroaniline 0.5, kantlex 0.05, agar 20, with the deionized water preparation, pH 8.0;
(11) seed culture medium (g/L): peptone 10, sodium-chlor 5, extractum carnis 5, with the deionized water preparation, pH 7.2;
(12) fermention medium (g/L): Zulkovsky starch 6, dregs of beans 18, yeast extract paste 24, glycerine 2.5, K
2HPO
43H
2O 6, CoCl
20.015, kantlex 0.05, with the deionized water preparation, 8.0,121 ℃ of sterilizations of pH, 20 min;
(13) subtilis Zj016 protoplastis preparation: centrifugal 10 min of Zj016 bacterium liquid 5000 r/min that will be cultured to logarithmic phase, remove supernatant, wash thalline 2 times with stroke-physiological saline solution, SMM liquid is resuspended after washing 2 times, then adding N,O-Diacetylmuramidase liquid to enzyme final concentration is 2.0 mg/mL, 37 ℃ of lower vibration enzymolysis 30 min, the centrifugal collection protoplastis of 2000r/min, with resuspended after the washing of SMM liquid, the Zj016 protoplasma scale of construction is 8 * 10 in the resuspended liquid
9Individual/mL;
(14) protoplast transformation: get 8 * 10
9The subtilis Zj016 protoplastis suspension 500 μ L of individual/mL add the plasmid PMA-BSAP of 20 μ L, 0.1 μ g/ μ L and the high osmotic buffer SMM of 20 μ L successively, use gently pressure-vaccum mixing of rifle head; Add again the 1.5mL fusogen, put upside down mixing gently after, 37 ℃, 80 r/min are processed 5 min, centrifugal 10 min of 2000 r/min; With resuspended after the SMM washing 2 times;
(15) regeneration: the resuspended liquid dilution spread of gained in the transformant regeneration culture medium, is cultivated 24 h, got transformant for 37 ℃; Recombinant plasmid PMA-BSAP self is with kalamycin resistance, and the bacterium colony that grows at the transformant regeneration culture medium is transformant;
(16) Screening of strain with high productivity: primary dcreening operation: the transformant point that obtains is connected on the Screening of strain with high productivity substratum, cultivate 24h for 37 ℃, time and shade thereof that the variation of record periphery of bacterial colonies color produces, colony characteristics and variable color loop diameter size thereof, according to being changed significantly and the greater of variable color loop diameter of single periphery of bacterial colonies color on the flat board, preliminary screening is produced the leucine aminopeptidase(LAP) bacterial strain;
Multiple sieve: the bacterial strain behind the primary dcreening operation is carried out the shake flask fermentation test, and rotary type shaking speed 225 r/min cultivate 30 h for 37 ℃, measure enzyme and live.Obtain the good conversion bacterial strain of a strain, produce the enzyme enzyme activity and reach 126 U/mL, produce enzyme level and be 18 times of bacterium Zj016 of setting out.
B. transformed bacteria shake-flask culture base and fermentation condition optimization:
Investigated carbon source, nitrogenous source, initial pH, culture temperature, liquid amount by experiment of single factor, inoculum size is on the impact of enzymatic production.The top condition that draws is: Zulkovsky starch 6 g/L, dregs of beans 18 g/L, yeast extract paste 24 g/L, glycerine 2 mL/L, K
2HPO
43H
2O 6g/L, CoCl
20.6mmol/L kantlex 0.05g/L prepares with deionized water; Initial pH 8.0,37 ℃ of leavening temperatures, inoculum size 4%, liquid amount 50mL/250mL.Reciprocating type shaking table 225 r/min, under the 30 h conditions of fermenting, it is 161 U/mL that the work of leucine aminopeptidase(LAP) enzyme is produced in CGMCC No.6559 fermentation, reaches 23 times of starting strain Zj016.
Beneficial effect of the present invention: the present invention successfully obtains a strain recombinant conversion bacterium ZH-Zj016, and the many experiments that goes down to posterity proves that the heritability of this recombinant conversion bacterium is stable.Behind the shake-flask culture based component and fermentation condition of this recombinant conversion bacterium of initial optimization, it is 161 U/mL that the work of leucine aminopeptidase(LAP) enzyme is produced in recombinant conversion bacterium ZH-Zj016 fermentation, reaches 23 times of original strain.
The biological material specimens preservation: a plant height produces the recombinant conversion bacterium of leucine aminopeptidase(LAP), this recombinant conversion bacterium called after subtilis (
Bacillus subtilis) ZH-Zj016, be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, be called for short CGMCC, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, deposit number: CGMCC No.6559, preservation date: on September 7th, 2012.
Description of drawings
Fig. 1 protoplast transformation breeding high-yield leucine aminopeptidase(LAP) bacterial strain process schematic diagram
The microscopic examination of Fig. 2 protoplastis
Fig. 3 high yield leucine aminopeptidase(LAP) bacterial strain primary dcreening operation is dull and stereotyped
The different carbon sources of Fig. 4 are on producing the impact of enzyme
Fig. 5 different nitrogen sources is on producing the impact of enzyme
The initial pH of Fig. 6 is on producing the impact of enzyme
Fig. 7 enzymatic production curve.
Embodiment
Embodiment 1
With 500 μ L protoplastis suspensions, add respectively plasmid PMA-BSAP and the isopyknic SMM damping fluid of 20 μ L, 0.1 μ g/ μ L, use gently pressure-vaccum mixing of rifle head; Add again 1.5 mL fusogens, put upside down mixing gently after, 37 ℃, 80 r/min process 5 min, centrifugal 10 min of 2000 r/min; With resuspended after the SMM washing 2 times, dilution spread transformant regeneration culture medium is cultivated 24 h for 37 ℃, and recombinant plasmid PMA-BSAP self is with kalamycin resistance, grows at above-mentioned substratum and is transformant.The transformant point that obtains is connected on the Screening of strain with high productivity substratum, cultivate 24h through 37 ℃ of primary dcreening operations, time and shade thereof that the variation of record periphery of bacterial colonies color produces, colony characteristics and variable color loop diameter size thereof, according to being changed significantly and the greater of variable color loop diameter of single periphery of bacterial colonies color on the flat board, the leucine aminopeptidase(LAP) bacterial strain is produced in screening.
Bacterial strain behind the primary dcreening operation is carried out the shake flask fermentation test, and rotary type shaking speed 225 r/min cultivate 30 h for 37 ℃, measure enzyme and live.Obtain the good conversion bacterial strain of a strain, produce the enzyme enzyme activity and reach 126 U/mL, produce enzyme level and be 18 times of bacterium Zj016 of setting out.
Shake-flask culture base and fermentation condition optimization: at Zulkovsky starch 6 g/L, dregs of beans 18 g/L, yeast extract paste 24 g/L, glycerine 2mL/L, K
2HPO
43H
2O 6 g/L, CoCl
20.6 mmol/L, kantlex 0.05g/L prepares with deionized water; Fermentation condition is: initial pH 8.0,37 ℃ of leavening temperatures, inoculum size 4%, liquid amount 50mL/250mL, reciprocating type shaking table 225 r/min are under the 30 h conditions of fermenting, it is 161 U/mL that the work of leucine aminopeptidase(LAP) enzyme is produced in recombinant conversion bacterium ZH-Zj016 fermentation, reaches 23 times of original strain.
Claims (3)
1. the method for a protoplast transformation breeding high-yield leucine aminopeptidase(LAP) bacterial strain is characterized in that:
(1) transforms: utilize the protoplast transformation technology, with 8 * 10
9The subtilis Zj016 protoplastis suspension 500 μ L of individual/mL add the plasmid PMA-BSAP of 20 μ L, 0.1 μ g/ μ L and the high osmotic buffer SMM of 20 μ L successively, use gently pressure-vaccum mixing of rifle head; Add again 1.5 mL fusogens, put upside down mixing gently after, 37 ℃, 80r/min are processed 5min, centrifugal 10 min of 2000 r/min; With resuspended after the SMM washing 2 times;
Described high osmotic buffer SMM counts with mol/L: sucrose 0.5, maleic acid 0.02, MgCl
20.02 with the deionized water preparation, transferring pH is 7.0;
Described fusogen is: 40% PEG6000, and with the preparation of SMM liquid, transferring pH is 7.0;
(2) regeneration: the resuspended liquid dilution spread of step (1) gained in the transformant regeneration culture medium, is cultivated 24 h, got transformant for 37 ℃;
Described transformant regeneration culture medium is counted with g/L: peptone 10, and extractum carnis 5, casamino acids hydrolyzate 5, glucose 6, sodium-chlor 1.2, agar 20, kantlex 0.05 is used the SMM solution preparation, and pH 7.2;
(3) screening: select the line of step (2) gained transformant and coat the Screening of strain with high productivity substratum, successful seed selection obtains high yield leucine aminopeptidase(LAP) bacterial strain;
Described Screening of strain with high productivity substratum is counted with g/L: Zulkovsky starch 6, dregs of beans 18, yeast extract paste 24, glycerine 2.5, K
2HPO
43H
2O 6, CoCl
20.015, L-Leu-4-N-methyl-p-nitroaniline 0.5, kantlex 0.05, agar 20, with the deionized water preparation, pH 8.0.
2. obtain the recombinant conversion bacterium that a plant height produces leucine aminopeptidase(LAP) with the method for the described protoplast transformation breeding high-yield of claim 1 leucine aminopeptidase(LAP) bacterial strain, this recombinant conversion bacterium Classification And Nomenclature be subtilis (
Bacillus subtilis) ZH-Zj016, be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, deposit number: CGMCC No.6559, it produces the enzyme enzyme activity and reaches 126 U/mL, produce enzyme level and be 18 times of bacterium Zj016 of setting out, the many experiments that goes down to posterity proves that the heritability of this recombinant conversion bacterium is stable.
3. the method for producing leucine aminopeptidase(LAP) with the described CGMCC No.6559 of claim 2 strain fermentation, it is characterized in that by behind the shake-flask culture based component and fermentation condition initial optimization of experiment of single factor to this transformed bacteria, at the shake-flask culture based component be: Zulkovsky starch 6 g/L, dregs of beans 18 g/L, yeast extract paste 24 g/L, glycerine 2 mL/L, K
2HPO
43H
2O 6g/L, CoCl
20.6mmol/L kantlex 0.05g/L prepares with deionized water; Fermentation condition is: initial pH 8.0,37 ℃ of leavening temperatures, inoculum size 4%, liquid amount 50mL/250mL, reciprocating type shaking table 225 r/min are under the 30 h conditions of fermenting, the leucine aminopeptidase(LAP) enzyme 161U/mL of being alive is produced in CGMCC No.6559 fermentation, reaches 23 times of starting strain Zj016.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201210418605 CN102876705B (en) | 2012-10-29 | 2012-10-29 | Method for breeding high-yield leucine aminopeptidase strain by protoplast transformation |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201210418605 CN102876705B (en) | 2012-10-29 | 2012-10-29 | Method for breeding high-yield leucine aminopeptidase strain by protoplast transformation |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102876705A true CN102876705A (en) | 2013-01-16 |
| CN102876705B CN102876705B (en) | 2013-10-30 |
Family
ID=47478220
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 201210418605 Expired - Fee Related CN102876705B (en) | 2012-10-29 | 2012-10-29 | Method for breeding high-yield leucine aminopeptidase strain by protoplast transformation |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102876705B (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104293749A (en) * | 2014-10-11 | 2015-01-21 | 江南大学 | Method for preparing high-yield leucine aminopeptidase through fermentation of recombinant bacillus subtilis |
| CN106119173A (en) * | 2016-08-22 | 2016-11-16 | 广东轻工职业技术学院 | A kind of shrimps low molecular peptide without bitterness and preparation method and application |
| CN107400666A (en) * | 2017-09-11 | 2017-11-28 | 广东轻工职业技术学院 | A kind of aminopeptidase and its encoding gene and application |
| CN109504624A (en) * | 2018-11-20 | 2019-03-22 | 广东肇庆星湖生物科技股份有限公司 | A kind of screening technique of leucine producing strain |
| CN111925953A (en) * | 2019-12-30 | 2020-11-13 | 河南工业大学 | Glutamic acid producing strain and preparation method and application thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1998051163A2 (en) * | 1997-05-16 | 1998-11-19 | Novo Nordisk Biotech, Inc. | Methods of producing protein hydrolysates |
| CN1282369A (en) * | 1997-12-16 | 2001-01-31 | 诺沃挪第克公司 | Polypeptides having aminopepyidase activity and nucleic acids encoding same |
| CN101139580A (en) * | 2007-08-07 | 2008-03-12 | 江南大学 | Method for preparing debittering aminopeptidase by fermentation of bacillus subtilis |
| CN101717763A (en) * | 2009-11-23 | 2010-06-02 | 江南大学 | Method for improving stability of liquid aminopeptidase and application of aminopeptidase |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1282369C (en) * | 2003-04-15 | 2006-10-25 | 明基电通股份有限公司 | Method of correcting display device colour image and its device |
-
2012
- 2012-10-29 CN CN 201210418605 patent/CN102876705B/en not_active Expired - Fee Related
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1998051163A2 (en) * | 1997-05-16 | 1998-11-19 | Novo Nordisk Biotech, Inc. | Methods of producing protein hydrolysates |
| CN1282369A (en) * | 1997-12-16 | 2001-01-31 | 诺沃挪第克公司 | Polypeptides having aminopepyidase activity and nucleic acids encoding same |
| CN101139580A (en) * | 2007-08-07 | 2008-03-12 | 江南大学 | Method for preparing debittering aminopeptidase by fermentation of bacillus subtilis |
| CN101717763A (en) * | 2009-11-23 | 2010-06-02 | 江南大学 | Method for improving stability of liquid aminopeptidase and application of aminopeptidase |
Non-Patent Citations (1)
| Title |
|---|
| 吴庆勋: "氨肽酶高产菌株的选育及发酵条件优化", 《中国优秀硕士学位论文全文数据库》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104293749A (en) * | 2014-10-11 | 2015-01-21 | 江南大学 | Method for preparing high-yield leucine aminopeptidase through fermentation of recombinant bacillus subtilis |
| CN106119173A (en) * | 2016-08-22 | 2016-11-16 | 广东轻工职业技术学院 | A kind of shrimps low molecular peptide without bitterness and preparation method and application |
| CN106119173B (en) * | 2016-08-22 | 2019-09-17 | 广东轻工职业技术学院 | A kind of shrimps low molecular peptide of no bitter taste and the preparation method and application thereof |
| CN107400666A (en) * | 2017-09-11 | 2017-11-28 | 广东轻工职业技术学院 | A kind of aminopeptidase and its encoding gene and application |
| CN109504624A (en) * | 2018-11-20 | 2019-03-22 | 广东肇庆星湖生物科技股份有限公司 | A kind of screening technique of leucine producing strain |
| CN111925953A (en) * | 2019-12-30 | 2020-11-13 | 河南工业大学 | Glutamic acid producing strain and preparation method and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102876705B (en) | 2013-10-30 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102876705B (en) | Method for breeding high-yield leucine aminopeptidase strain by protoplast transformation | |
| CN101555461B (en) | Bacterial strain LT3 producing alkalescence cellulase and breeding method and initial optimization of cellulase production conditions thereof | |
| CN113215031B (en) | Bacillus belgii 19573-3 and application thereof | |
| CN106190921A (en) | A kind of corynebacterium glutamicum and application | |
| CN103667150B (en) | Bacillus subtilis capable of producing neutral protease with strong heat stability and application of bacillus subtilis | |
| CN105176883A (en) | Bacillus subtilis of high-yield mesophilic alpha-amylase and liquid fermentation method for bacillus subtilis | |
| CN110484471A (en) | The method that one plant height produces the acidproof bacterial strain of bacteria cellulose and its produces bacteria cellulose | |
| CN102634464A (en) | High-temperature-resistant saccharomyces cerevisiae and separating and culturing method thereof | |
| CN101619299B (en) | Rhodococcus ruber and method for preparing 5-cyanovaleramide by utilizing same | |
| CN102864113A (en) | Strain capable of producing succinic acid, method for producing succinic acid and application thereof | |
| CN105368729B (en) | One plant of acidproof flocculability an industrial strain of S.cerevisiae strain and construction method | |
| CN102329747B (en) | Culture medium and culture method for high-density culture of Staphylococcus xylosus A2 | |
| CN104450571A (en) | Bacillus thuringiensis strain for efficient degradation of fly larvae protein | |
| CN108841743B (en) | Cold region straw rotten bacterial strain and preparation method and application thereof | |
| CN114015612B (en) | Xanthomonas leucovorin and application thereof in fermentation production of non-pigmented xanthan gum | |
| CN103146776A (en) | Method for producing indigo pigment with bacillus subtilis | |
| CN107641602B (en) | Candida utilis and application thereof in protein production through fermentation | |
| CN107557311B (en) | Lactobacillus acidophilus and application thereof in fermentation production of antibacterial polypeptide | |
| CN108913629A (en) | A kind of bacterium of cellulase-producing and the preparation method and application thereof | |
| CN109136100A (en) | One plant of Tabin aspergillus bacterial strain and the application on fermentation green brick tea | |
| CN114075520B (en) | Bacillus belicus for producing gamma-polyglutamic acid and method for producing gamma-polyglutamic acid by solid fermentation of bacillus belicus | |
| CN110903994B (en) | Bacillus licheniformis for producing high-temperature protease and application thereof | |
| CN103074283A (en) | Bacillussp., microbial agent and applications of Bacillussp. and microbial agent | |
| CN113502282A (en) | Method for producing pectinase preparation by solid-state fermentation of penicillium | |
| CN112779207A (en) | Rejuvenation method of ganoderma lucidum strain special for liquid fermentation |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20131030 Termination date: 20141029 |
|
| EXPY | Termination of patent right or utility model |