CN105200076A - Bacillus subtilis recombined to express gamma-lactamase and immobilization and application - Google Patents
Bacillus subtilis recombined to express gamma-lactamase and immobilization and application Download PDFInfo
- Publication number
- CN105200076A CN105200076A CN201510612331.5A CN201510612331A CN105200076A CN 105200076 A CN105200076 A CN 105200076A CN 201510612331 A CN201510612331 A CN 201510612331A CN 105200076 A CN105200076 A CN 105200076A
- Authority
- CN
- China
- Prior art keywords
- gamma
- lactam
- bacillus subtilis
- cell
- enzyme
- 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
Landscapes
- Enzymes And Modification Thereof (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses a method for catalytically producing optically pure (-)-gamma-lactam by applying recombined bacillus subtilis whole cells, and belongs to the field of gene engineering and bioengineering. According to the method, recombined bacillus subtilis (B.subtilis 168/pMA5-delm) containing (+)-gamma-lactamases genes which are derived from Delftia sp.CGMCC 5755 is established, a produced enzyme is expressed to prepare a whole-cell catalyst, immobilization is conducted on recombined whole cells, the recombined bacillus subtilis is applied to enantioselective hydrolysis of racemic gamma-lactam, and the (-)-gamma-lactam with the optical purity larger than 99% is obtained finally. The method for microbial catalysis preparation of the (-)-gamma-lactam has the advantages of being high in stereoselectivity, high in reaction yield and high in product concentration.
Description
Technical field
The invention belongs to genetically engineered and bioengineering field, be specifically related to the recombined bacillus subtilis of a strain heterogenous expression (+)-gamma-lactam enzyme, the preparation of its reconstitution cell and process for fixation, and the application of applying that this catalyzer enantioselective hydrolysis racemize gamma-lactam produces optical purity (-)-gamma-lactam.
Background technology
(–)-2-azabicyclo-[2,2,1]-heptan-5-alkene-3-ketone (being called for short this lactone of literary composition, (–)-gamma-lactam), be the important chiral precurser of a kind of carbocyclic nucleoside compounds synthesis.The cyclopentenes structure of gamma-lactam, gamma-lactam can be made can to participate in a lot of chemical reaction, such as epoxidation, nitrogen is monocyclopropanated, fluoridizes, hydroxylation, selenizing, ciprofloxacin eye drops reactions etc., thus can synthesize multiple chiral drug, such as anti-AIDS drug A Baka big (Abacavir), anti-influenza A and bird flu resistant medicine peramivir (Peramivir) and newly-developed hypoglycemic agent melogliptin (Melogliptin) etc.At present, numerously prepare (Physical, chemical method and biological process) in the method for optical purity gamma-lactam, biological process because have that reaction efficiency is high, stereoselectivity is high, reaction conditions is gentle, the advantage such as reusable, less energy-consumption, pollution are few, have and apply widely.Microbial enzyme method resolution of racemates gamma-lactam has a good application prospect.
Gamma-lactam enzyme refers to can the class of enzymes of Hydrolysis Resolution raceme gamma-lactam.Excavating the gamma-lactam enzyme with High level of stereoselectivity selectivity and catalysis activity, is the key that biological process produces optical purity gamma-lactam.Have report to utilize the complete pure gamma-lactam of cell asymmetric hydrolysis gamma-lactam synthesizing optical such as Pseudomonassp., Rhodococcusequi and Aurobacteriumsp., but the stereoselectivity of whole-cell catalyst is not high.From the innovation of gene sequencing technology, disclosing of magnanimity genomic data, and the maturation etc. of gene engineering method, utilize genetic engineering bacterium heterogenous expression object gamma-lactam enzyme, the difficult problem that wild mushroom genetic background is complicated and regulation and control are difficult can be avoided, thus obtain efficient catalyzer.After this research of gamma-lactam enzyme mostly is and adopts intestinal bacteria Escherichiacoli heterogenous expression gamma-lactam enzyme.(+)-gamma-lactam enzyme investigation and application deriving from sulfolobus solfataricus (Sulfolobussolfataricus) is comparatively deep, it has the enantioselectivity of >99%, but its optimal reactive temperature too high (80 DEG C), utilizes this enzyme to carry out the energy consumption of production process very large.By the right method of protein ratio find soybean slowly raw root nodule bacterium (BradyrhizobiumjaponicumUSDA6) also contain gamma-lactam enzyme, but the optical purity of its product is not high, and ee value is 96%.Wang Jianjuns etc. utilize the method for gene excavating, have found the RutB albumen from intestinal bacteria (E.coli) Isochorismatase family, have (+)-gamma-lactam enzymic activity equally, but its soluble-expression are poor.(+)-gamma-lactam enzyme of current report mainly contains the shortcoming existed in racemize gamma-lactam split process: the selectivity that (1) (+)-gamma-lactam enzyme splits racemize gamma-lactam is poor; (2) poor stability of (+)-gamma-lactam enzyme; (3) solubility heterogenous expression is poor, thus limits its industrial applications.
The Delftiasp.CGMCC5755 bacterial strain with (+)-gamma-lactam enzymic activity has been screened in earlier stage in this laboratory, and (this bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on February 14th, 2012, address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City), by potential (+)-gamma-lactam enzyme heterogenous expression in intestinal bacteria of this bacterium source, find that major part exists with the inclusion bodies of non-activity, solubility expression is lower.The present invention is by construction recombination plasmid pMA5-delm, achieve the solubility expression of (+)-gamma-lactam enzyme gene in subtilis 168 in Delftiasp.CGMCC5755 source, embedded immobilization is carried out to improve operational stability to restructuring transformant, utilizing the asymmetric fractionation gamma-lactam of this recombined bacillus subtilis further, providing Research foundation for preparing optical purity (-)-gamma-lactam.
Summary of the invention
The technical problem to be solved in the present invention is to provide the method for (+)-gamma-lactam enzyme that a kind of solubility heterogenous expression Delftiasp.CGMCC5755 originates, and improve the operational stability of reconstitution cell further, for resolution of racemic gamma-lactam, prepare optically pure (-)-gamma-lactam.
The present invention by following technical proposals to solve the problems of the technologies described above:
The aminoacid sequence of (+) of the present invention-gamma-lactam enzyme is as shown in the SEQIDNo.2 in sequence table; Described Delftiasp. is preserved in China General Microbiological DSMZ, and preserving number is CGMCC5755, and this bacterial strain screens and obtains from soil, is described in detail in patent of invention (CN102719378A).
The invention still further relates to one (+)-gamma-lactam enzyme gene, its base sequence is as shown in sequence table SEQ IDNo.1, or the protein of the aminoacid sequence composition shown in its coding sequence SEQIDNo.2.
(+) of the present invention-gamma-lactam enzyme gene can derive from Delftiasp.CGMCC5755, concrete preparation method can be: according to the gene order design and synthesis primer of the Ntn hydrolase in the Delftiasp. source of including in GenBank, then with the genomic dna of Delftiasp.CGMCC5755 for template, polymerase chain reaction is utilized to carry out gene amplification, obtain complete (+)-gamma-lactam enzyme gene, base sequence is as shown in SEQIDNo.1, called after delm, total length 1230bp, intronless in this sequence, the aminoacid sequence of its coded protein is as shown in sequence table SEQ IDNo.2.
The present invention relates to the recombinant expression vector of the nucleotide sequence comprising (+)-gamma-lactam enzyme gene.The nucleotide sequence of (+) of the present invention-gamma-lactam enzyme gene is connected to structure on various carrier by this area ordinary method and forms by it.Described carrier can be the cloning vector of this area routine, as commercially available plasmid or virus vector etc., and preferred pMA5.Preferably, recombinant expression vector of the present invention is obtained: by by (+)-gamma-lactam enzyme gene product of pcr amplification gained and pMA5 restriction enzyme NdeI and BamHI double digestion by following method, form complementary sticky end, connect through T4DNA ligase enzyme again, form the recombinant expression plasmid pMA5-delm containing the present invention (+)-gamma-lactam enzyme gene.
The invention further relates to the recombinant expressed transformant comprising (+) of the present invention-gamma-lactam enzyme gene or its recombinant expression vector.It is by being converted in host microorganism can obtaining recombinant expression vector of the present invention.The preferred subtilis of the present invention, more preferably subtilis (Bacillussubtilis) 168, to improve the solubility expression of (+)-gamma-lactam enzyme.Aforementioned recombinant expression plasmid pMA5-delm is converted in subtilis 168, the preferred engineering strain of the present invention, i.e. subtilis (Bacillussubtilis) 168/pMA5-delm.
The invention still further relates to a kind of preparation method of restructuring (+)-gamma-lactam enzyme, it comprises the following steps: cultivate recombinant expressed transformant of the present invention, obtains restructuring (+)-gamma-lactam enzyme.The recombinant expressed transformant of wherein said cultivation substratum used can be that this area is any to be made transformants grew and produce the substratum of (+) of the present invention-gamma-lactam enzyme, for bacterial strain, be preferably as follows substratum: Tryptones 20g/L, yeast extract 16g/L, glucose 4g/L, NaCl5g/L, Na
2hPO
44g/L, KH
2pO
40.5g/L, (NH4)
2sO
43g/L.Cultural method and culture condition do not have special restriction, can carry out appropriate selection according to the difference of the factor such as host type and cultural method by this area general knowledge, as long as enable transformant grow and produce (+)-gamma-lactam enzyme.Preferred following method: in 37 DEG C of incubated overnight in picking list bacterium colony to substratum, by the inoculum size of 2 ~ 5%, be inoculated into fermentor tank, 37 DEG C of cultivations, control dissolved oxygen and be not less than 15%, control pH is about 7.0, fermentation 12h, collecting cell, and carry out vacuum lyophilization, can high expression (+) of the present invention-gamma-lactam enzyme.
The invention still further relates to the process for fixation of recombinant expressed transformant, it comprises the steps: above-mentioned expression (+)-gamma-lactam enzyme recombined bacillus subtilis intact cells to mix with fixation support, obtains the recombinant expressed transformant of immobilization.Specific as follows: 1g freeze drying cell to be fully dissolved in 10mL deionized water, with 3% sodium alginate soln 40mL mix and blend, by syringe pillow, the calcium chloride solution that above-mentioned mixed solution highly injects 1% with 5cm is formed smooth microsphere immediately, and this immobilized spherule is remained in the above-mentioned calcium chloride solution of 4 DEG C the 30min that hardens, and then harden further in the glutaraldehyde solution of 0.3%, for subsequent use with immobilized cell being stored in 4 DEG C of refrigerators after brine.
The invention further relates to utilization (+)-gamma-lactam enzyme as catalyzer at enantioselective resolution gamma-lactam to prepare the application in optical activity gamma-lactam.Preferably, described application is carried out as follows: utilize recombined bacillus subtilis freeze drying cell 10 ~ 15g/L or immobilization recombined bacillus subtilis intact cells 10 ~ 15g/L, catalytic hydrolysis 100g/L gamma-lactam substrate, reaction conditions is temperature of reaction 30 DEG C, pH8 ~ 10, preferred pH9.0, mixing speed 200 ~ 400rpm, preferred 400rpm.After reaction terminates, through dichloromethane extraction, underpressure distillation and drying treatment, final (-)-gamma-lactam obtaining purity and be greater than 99%.
Agents useful for same of the present invention and raw material are all commercially.
Positive progressive effect of the present invention is: (+)-gamma-lactam enzyme gene heterogenous expression in subtilis successfully achieving Delftiasp.CGMCC5755 source, immobilization is carried out to the full cell of restructuring transformant and has further increased operational stability, and utilized this recombinant conversion body whole-cell catalytic High-efficient Production optical purity (-)-gamma-lactam.This is for utilizing the first report of recombined bacillus subtilis production optical purity (-)-gamma-lactam.In this invention, fermentation method prepares recombined bacillus subtilis is that whole-cell catalytic reaction provides stable catalyzer; Recombined bacillus subtilis embodies higher transformation efficiency and stereoselectivity, and the recombined bacillus subtilis transformation system of optimization has good industrial application DEVELOPMENT PROSPECT.
Embodiment
Mode below by embodiment further illustrates the present invention, but does not therefore limit the present invention among described scope of embodiments.The experimental technique of unreceipted actual conditions in the following example, conventionally and condition, or selects according to catalogue.
Material source in the following example is:
Expression plasmid pMA5 is that laboratory builds.
EscherichiacoliJM109, Bacillussubtilis168 competent cell, High fidelity PCR enzyme and sepharose DNA reclaim test kit purchased from Beijing Tian Gen biochemical technology company limited.
Primer:
Forward：5’-CGC
CATATGGCCGAAACCCTGATCAAGGTCG-3
Reverse：5’-CGC
GGATCCTTACTTGTCCTGGGCGATG-3’
Wherein, upstream primer underscore part is NdeI restriction enzyme site, and downstream primer underscore part is BamHI restriction enzyme site.
Substratum is (g/L): Tryptones 20, yeast extract 16, glucose 4, NaCl5, Na
2hPO
44, KH
2pO
40.5, (NH4)
2sO
43.3L fermentor tank, 1.5L fermented liquid, 115 DEG C of sterilizings, 20min.
Enzyme activity determination method: containing 200 μ L fermented liquid cells in 1mL reaction system, 800 μ LpH7.0 phosphate buffer soln 1g/L substrates, under 30 DEG C of conditions, reaction 30min, termination reaction.Enzyme activity is defined as:
HPLC analyzes: normal-phase HPLC analysis gamma-lactam substrate selective and transformation efficiency, chromatographic column is DaicelChiralpackAS-H, and moving phase is acetonitrile and Virahol (ratio is 8:2), and flow velocity is 0.3mL/min, and wavelength is 230nm.
HPLC chemical purity is analyzed: product purity detects: chromatographic column is DiamonsilC18 (2), 250 × 4.6mm, and moving phase is methyl alcohol and water (ratio is 9:1), and flow velocity is 1mL/min, and ultraviolet detection wavelength is 225nm.
Embodiment 1: restructuring Bacillussubtilis168/pMA5-delm strain construction
With Delftiasp.CGMCC5755 genome for template, with Forward and Reverse for upstream and downstream primer amplification obtains goal gene fragment (about 1230bp), called after delm.After PCR primer purifying, through NdeI and BamHI double digestion, reclaim, connect, proceed to E. coli JM109 after connection with the carrier pMA5 through identical restriction enzyme linearization process, coating amicillin resistance is dull and stereotyped, checking positive colony.Again transform Bacillussubtilis168 competent cell to the recombinant plasmid pMA5-delm of successful connection, coating kalamycin resistance is dull and stereotyped.Checking transforms successful recombinant bacterial strain Bacillussubtilis168/pMA5-delm.
Embodiment 2: the enzymatic production of recombined bacillus subtilis
With the restructuring Bacillussubtilis168/pMA5-delm of incubated overnight for seed liquor, be forwarded to fermention medium according to the inoculum size of 2 ~ 5%, 37 DEG C of cultivations, mixing speed 300 ~ 700rpm, controlling dissolved oxygen minimum is that 15%, pH maintains about 7.0.Fermentation 12h, with 6000 × g normal temperature collected by centrifugation thalline, with the phosphoric acid salt of 0.02mol/L (pH7.0) buffered soln washed cell, freeze-drying cryopreservation.
Embodiment 3: the immobilization of recombined bacillus subtilis
1g freeze drying cell is fully dissolved in 10mL deionized water, with 3% sodium alginate soln 40mL mix and blend, by syringe pillow, the calcium chloride solution that above-mentioned mixed solution highly injects 1% with 5cm is formed smooth microsphere immediately, and this immobilized spherule is remained in the above-mentioned calcium chloride solution of 4 DEG C the 30min that hardens, and then harden further in the glutaraldehyde solution of 0.3%, for subsequent use with immobilized cell being stored in 4 DEG C of refrigerators after brine.After measured, the activity recovery of gained immobilized cell is 48%.
Embodiment 4: recombined bacillus subtilis asymmetric hydrolysis gamma-lactam condition optimizing
(1) optimum temperuture of resting cell: in 1mL reaction system, containing the phosphate buffer soln (pH7.0 that final concentration is 1g/L recombined bacillus subtilis freeze drying cell, 0.1mol/L), respectively at preheating 10min at 10,20,30,40,50,60,70,80 DEG C of temperature, adding substrate gamma-lactam to final concentration is that under 5g/L continues at said temperature, 30min is reacted in concussion, heating termination reacts, the transformation efficiency of HPLC detection substrate.Result is as shown in table 1, integrated temperature stability, determines the suitableeest invert point 30 DEG C.
Table 1 temperature of reaction is on the impact of recombined bacillus subtilis intact cells Hydrolysis Resolution gamma-lactam
(2) optimal pH of resting cell: in 1mL reaction system, it is buffered soln (pH6.0 – 8.0 phosphate buffered saline buffer of the different pH value of 0.1mol/L of the recombined bacillus subtilis freeze drying cell of 1g/L containing final concentration, pH9.0-11.0Tris-HCl damping fluid), 30 DEG C of temperature bath 10min, add the substrate gamma-lactam that final concentration is 5g/L, under optimum temperuture, conversion reaction 5h, termination reaction, the change of HPLC detection substrate.The suitableeest conversion pH is 9.0.
Table 2 pH of buffer is on the impact of recombined bacillus subtilis intact cells Hydrolysis Resolution gamma-lactam
(3) the suitableeest mixing speed: containing 10g/L freeze drying cell in 50mL reaction system, concentration of substrate is 50g/L, under the optimum temperuture and pH condition of above-mentioned optimization, investigates mixing speed to the impact of hydrolysis reaction, speed setting is 200,300 and 400r/min.Sample at set intervals, carry out HPLC analysis.Through determining, the suitableeest mixing speed is 300r/min.
Table 3 mixing speed is on the impact of recombined bacillus subtilis intact cells Hydrolysis Resolution gamma-lactam
Embodiment 5: utilize the recombined bacillus subtilis intact cells that dissociates to prepare optical purity (-)-gamma-lactam
Apply above-mentioned optimum condition (pH9.0,30 DEG C, 300r/min), in 500mL transformation system, utilize 10g/L and 15g/L recombined bacillus subtilis freeze drying cell respectively, fractionation hydrolysis is carried out to prepare optical purity (-)-gamma-lactam to 100g/L gamma-lactam.Finally, under 10g/L catalyzer adding conditional, reaction 6h, transformation efficiency be 55.2% and ee be 98.6%; Under 15g/L catalyzer adding conditional, reaction 4h, transformation efficiency be 56.3% be 99.2% with ee.
Table 4 utilize recombined bacillus subtilis dissociate intact cells amplify prepare optical purity (-)-gamma-lactam
Embodiment 6: utilize recombined bacillus subtilis immobilized cell to prepare optical purity (-)-gamma-lactam
Apply above-mentioned optimum condition (pH9.0,30 DEG C, 300r/min), in 500mL transformation system, utilize 10g/L and 15g/L recombined bacillus subtilis immobilized cell respectively, fractionation hydrolysis is carried out to prepare optical purity (-)-gamma-lactam to 100g/L gamma-lactam.Finally, under 10g/L catalyzer adding conditional, reaction 12h, transformation efficiency be 54.5% and ee be 98.7%; Under 15g/L catalyzer adding conditional, reaction 10h, transformation efficiency be 55.6% and ee be 99.0%.
Table 4 utilize recombined bacillus subtilis dissociate intact cells amplify prepare optical purity (-)-gamma-lactam
Embodiment 7:(-) extraction of-gamma-lactam product separation
To above-mentioned conversion reaction, utilize isopyknic dichloromethane extraction reaction solution three times, merge organic phase, after anhydrous sodium sulfate drying, underpressure distillation, drying, obtain product (-)-gamma-lactam.After testing, the optical purity of (-)-gamma-lactam is greater than 99%, and chemical purity is greater than 98%.
Claims (10)
1. a recombinant expression vector, it is characterized in that: the skeleton of described recombinant expression vector is pMA5, it includes (+)-gamma-lactam enzyme gene deriving from Delftiasp.CGMCC5755 source, nucleotide sequence is as shown in SEQ ID No .1, and its encoding amino acid sequence is as shown in SEQ ID No .2.
2. a recombinant expressed transformant, is characterized in that: it comprises recombinant expression vector as claimed in claim 1 or lactamase gene.
3. recombinant expressed transformant as claimed in claim 2, is characterized in that: it is recombinant expression vector according to claim 1 is converted into genetic engineering bacterium obtained in host microorganism; Described expressive host is subtilis, is preferably subtilis (Bacillussubtilis) 168.
4. a preparation method for restructuring (+)-gamma-lactam enzyme, is characterized in that comprising recombinant expressed transformant as claimed in claim 3, obtains restructuring (+)-gamma-lactam enzyme.
5. recombinate the preparation method of (+)-gamma-lactam enzyme as claimed in claim 4, it is characterized in that it comprises the steps: recombinant expressed transformant as claimed in claim 3 to be seeded to incubated overnight in substratum and obtains seed liquor, by seed liquor by 2 ~ 5% inoculum size seed liquor be forwarded in 3L fermentor tank cultivate, control dissolved oxygen in process and be not less than 15%, pH is about 7.0, collecting cell after 37 DEG C of cultivation 12h, and lyophilize obtains restructuring (+)-gamma-lactam enzyme; Described substratum comprises: Tryptones 20g/L, yeast extract 16g/L, glucose 4g/L, NaCl5g/L, Na
2hPO
44g/L, KH
2pO
40.5g/L, (NH4)
2sO
43g/L, pH are 7.0.
6. produce a process for fixation for (+)-gamma-lactam enzyme recombined bacillus subtilis, it is characterized in that recombinant conversion body according to claim 5 to carry out embedded immobilization, obtain immobilized catalyst.
7. preparation method as claimed in claim 6, it is characterized in that it comprises following steps: 1g freeze drying cell be fully dissolved in 10mL deionized water, with the sodium alginate soln mix and blend of 40mL3%, by syringe needle, the calcium chloride solution that above-mentioned mixed solution highly injects 1% with 5cm is formed smooth microsphere immediately, and this immobilized spherule is remained in the above-mentioned calcium chloride solution of 4 DEG C the 30min that hardens, and then harden further in the glutaraldehyde solution of 0.3%, for subsequent use with immobilized cell being stored in 4 DEG C of refrigerators after brine.
8. the immobilized cell as described in claim 5 or 7 as catalyzer at enantioselective resolution gamma-lactam to prepare the application in optical activity gamma-lactam.
9. apply as claimed in claim 8, it is characterized in that: utilize recombined bacillus subtilis freeze drying cell 10 ~ 15g/L as claimed in claim 5, catalytic hydrolysis 100g/L gamma-lactam substrate, reaction conditions is temperature 30 DEG C; PH8 ~ 10, preferred pH9.0; Mixing speed 200 ~ 400rpm, preferred 400rpm.After reaction terminates, through dichloromethane extraction, underpressure distillation and drying treatment, final (-)-gamma-lactam obtaining purity and be greater than 99%.
10. apply as claimed in claim 8, it is characterized in that: utilize recombined bacillus subtilis immobilized cell as claimed in claim 7 10 ~ 15g/L, hydrolysis 100g/L gamma-lactam substrate, reaction conditions is temperature 30 DEG C, pH9.0, mixing speed 300rpm, reaction terminates rear isolated cell, rejoin 100g/L gamma-lactam substrate and continue reaction, reaction solution is merged after operate continuously 5 batches, through dichloromethane extraction, underpressure distillation and drying treatment, final (-)-gamma-lactam obtaining purity and be greater than 99%.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510612331.5A CN105200076A (en) | 2015-09-23 | 2015-09-23 | Bacillus subtilis recombined to express gamma-lactamase and immobilization and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510612331.5A CN105200076A (en) | 2015-09-23 | 2015-09-23 | Bacillus subtilis recombined to express gamma-lactamase and immobilization and application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN105200076A true CN105200076A (en) | 2015-12-30 |
Family
ID=54948037
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510612331.5A Pending CN105200076A (en) | 2015-09-23 | 2015-09-23 | Bacillus subtilis recombined to express gamma-lactamase and immobilization and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105200076A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106011119A (en) * | 2016-05-23 | 2016-10-12 | 电子科技大学 | Preparation method of cross-linked beta-lactamase aggregate for treating pharmaceutical wastewater |
| CN108192888A (en) * | 2018-01-29 | 2018-06-22 | 大连大学 | A kind of method of marine low temperature gamma-lactam enzyme production (-) gamma-lactam |
| CN112442474A (en) * | 2020-12-09 | 2021-03-05 | 江南大学 | Preparation method of (-) gamma-lactam |
| CN112481320A (en) * | 2020-12-09 | 2021-03-12 | 江南大学 | Method for preparing (-) gamma-lactam with high catalytic efficiency |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050158843A1 (en) * | 2001-11-06 | 2005-07-21 | Pertti Koski | Non-sporulating bacillus subtilis having parts of the gene encoding sigma g deleted |
| CN101063156A (en) * | 2007-04-30 | 2007-10-31 | 江南大学 | Method for preparing (R)-6-hydroxy-8-chlorine octanoic acid ethyl by enzyme resolution |
| CN101544972A (en) * | 2009-05-15 | 2009-09-30 | 北京化工大学 | Method for purifying and immobilizing gamma-lactamase and splitting (+/-) gamma-lactam by one step |
| CN101875959A (en) * | 2010-06-04 | 2010-11-03 | 江西省驰邦药业有限公司 | Method for preparing abacavir chiral intermediate by biological split |
| CN101979646A (en) * | 2010-11-05 | 2011-02-23 | 河南省南街村（集团）有限公司 | Method for preparing guanosine by using immobilized cell |
| CN102002509A (en) * | 2010-05-25 | 2011-04-06 | 江南大学 | Escherichia coli-bacillus subtilis shuttle expression vector and application thereof |
| CN102618601A (en) * | 2012-04-17 | 2012-08-01 | 广西大学 | Method for preparing sucrose-6-ethyl ester by using biological fermentation and immobilized enzyme methods |
| CN104928278A (en) * | 2015-07-13 | 2015-09-23 | 通威股份有限公司 | Preparation method for composite bacillus microcapsule preparations |
-
2015
- 2015-09-23 CN CN201510612331.5A patent/CN105200076A/en active Pending
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050158843A1 (en) * | 2001-11-06 | 2005-07-21 | Pertti Koski | Non-sporulating bacillus subtilis having parts of the gene encoding sigma g deleted |
| CN101063156A (en) * | 2007-04-30 | 2007-10-31 | 江南大学 | Method for preparing (R)-6-hydroxy-8-chlorine octanoic acid ethyl by enzyme resolution |
| CN101544972A (en) * | 2009-05-15 | 2009-09-30 | 北京化工大学 | Method for purifying and immobilizing gamma-lactamase and splitting (+/-) gamma-lactam by one step |
| CN102002509A (en) * | 2010-05-25 | 2011-04-06 | 江南大学 | Escherichia coli-bacillus subtilis shuttle expression vector and application thereof |
| CN101875959A (en) * | 2010-06-04 | 2010-11-03 | 江西省驰邦药业有限公司 | Method for preparing abacavir chiral intermediate by biological split |
| CN101979646A (en) * | 2010-11-05 | 2011-02-23 | 河南省南街村（集团）有限公司 | Method for preparing guanosine by using immobilized cell |
| CN102618601A (en) * | 2012-04-17 | 2012-08-01 | 广西大学 | Method for preparing sucrose-6-ethyl ester by using biological fermentation and immobilized enzyme methods |
| CN104928278A (en) * | 2015-07-13 | 2015-09-23 | 通威股份有限公司 | Preparation method for composite bacillus microcapsule preparations |
Non-Patent Citations (4)
| Title |
|---|
| SCHLEHECK, D.等: "Delftia acidovorans SPH-1, complete genome", 《GENBANK》 * |
| TIAN-YUN XUE等: "Soluble Expression of (+)-γ-Lactamase in Bacillus subtilis for the Enantioselective Preparation of Abacavir Precursor", 《APPL BIOCHEM BIOTECHNOL》 * |
| 秦小溪: "产γ-内酰胺水解酶菌株的固定化及在手性药物合成中的应用", 《中国优秀硕士学位论文全文数据库 基础科学辑》 * |
| 黄艳红灯: "巨大芽孢杆菌青霉素酰化酶在枯草杆菌中的表达及其分离纯化", 《中国生物化学与分子生物学学报》 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106011119A (en) * | 2016-05-23 | 2016-10-12 | 电子科技大学 | Preparation method of cross-linked beta-lactamase aggregate for treating pharmaceutical wastewater |
| CN106011119B (en) * | 2016-05-23 | 2020-03-24 | 电子科技大学 | Preparation method of crosslinked β -lactamase aggregate for pharmaceutical wastewater treatment |
| CN108192888A (en) * | 2018-01-29 | 2018-06-22 | 大连大学 | A kind of method of marine low temperature gamma-lactam enzyme production (-) gamma-lactam |
| CN112442474A (en) * | 2020-12-09 | 2021-03-05 | 江南大学 | Preparation method of (-) gamma-lactam |
| CN112481320A (en) * | 2020-12-09 | 2021-03-12 | 江南大学 | Method for preparing (-) gamma-lactam with high catalytic efficiency |
| CN112481320B (en) * | 2020-12-09 | 2022-07-05 | 江南大学 | Method for preparing (-) gamma-lactam with high catalytic efficiency |
| CN112442474B (en) * | 2020-12-09 | 2022-08-23 | 江南大学 | Preparation method of (-) gamma-lactam |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108424900B (en) | Nitrilase mutant and construction method and application thereof | |
| CN106191025B (en) | A method of utilizing graphene oxide-metallic ion coordination immobilised enzymes | |
| CN105200076A (en) | Bacillus subtilis recombined to express gamma-lactamase and immobilization and application | |
| CN106636020A (en) | Mutant short-chain dehydrogenase, recombinant expression vector, genetic engineering bacterium and application | |
| CN109055327A (en) | Aldehyde Ketoreductase mutant and its application | |
| CN110396505A (en) | Ketone group pantoic acid lactone reductase and its application | |
| CN106995808B (en) | A kind of recombination transaminase and its application | |
| CN104152506A (en) | Method catalytically synthesizing (S)-N, N-dimethyl-3-hydroxy-(2-thiofuran)-1-propylamine((S)-DHTP) by aldehyde ketone reductase recombinant strain crude enzyme system | |
| CN107858340A (en) | The phosphate aldolase A mutant of D fructose 6, recombinant expression carrier, genetic engineering bacterium and its application of high catalytic activity | |
| Ates et al. | Enhancement of citric acid production by immobilized and freely suspended Aspergillus niger using silicone oil | |
| CN105420154A (en) | Double knockout recombinant rhodococcus as well as construction method and application thereof | |
| CN101463358B (en) | Nitrile hydratase gene cluster and use thereof | |
| CN102796720A (en) | (+)gamma-lactamase with racemate gamma-lactam resolution activity and its application | |
| CN102827853B (en) | Halogenohydrin dehalogenation enzyme gene mutant and application thereof | |
| CN104561076A (en) | Methods for constructing and fermenting L-serine high-yielding recombinant corynebacterium glutamicum | |
| CN104673814B (en) | A kind of L threonine aldolases for coming from enterobacter cloacae and its application | |
| CN103555646B (en) | A kind of coexpression L-arabinose isomerase gene and the genetic engineering bacterium of mannose-6-phosphate isomerase | |
| CN104726355A (en) | Method for converting and preparing (S)-phenylglycol asymmetrically through (S)-carbonyl reductase II expressed by Saccharomyces cerevisiae | |
| CN104212850A (en) | Method for preparing 1-cyancyclohexylacetic acid by using nitrilase engineering bacterium | |
| CN101285085B (en) | Process for synthesizing adenosine methilanin by intact cell catalysis | |
| CN114350630B (en) | L-pantolactone dehydrogenase, mutant and application thereof | |
| CN102417900B (en) | ATC racemase and coding gene thereof, and application of recombinant expression protein thereof | |
| CN107299074B (en) | Construction method and application of formate dehydrogenase engineering strain | |
| CN108929883A (en) | II E of sporulation related gene spo is influencing the application in strain growth and producing enzyme | |
| CN106244566B (en) | A kind of chondroitin synthase mutant and its application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20151230 |