CN107475170A - A kind of colibacillus engineering and its application for being used to express Pseudomonas putidas Creatininase - Google Patents

A kind of colibacillus engineering and its application for being used to express Pseudomonas putidas Creatininase Download PDF

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CN107475170A
CN107475170A CN201710873266.0A CN201710873266A CN107475170A CN 107475170 A CN107475170 A CN 107475170A CN 201710873266 A CN201710873266 A CN 201710873266A CN 107475170 A CN107475170 A CN 107475170A
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creatininase
cre
colibacillus engineering
pseudomonas putidas
putidas
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林影
梁书利
侯赣生
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South China University of Technology SCUT
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y305/00—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
    • C12Y305/02—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in cyclic amides (3.5.2)
    • C12Y305/0201—Creatininase (3.5.2.10)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/978—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • G01N2333/986—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in cyclic amides (3.5.2), e.g. beta-lactamase (penicillinase, 3.5.2.6), creatinine amidohydrolase (creatininase, EC 3.5.2.10), N-methylhydantoinase (3.5.2.6)

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Abstract

A kind of colibacillus engineering for being used to express Pseudomonas putidas Creatininase, the colibacillus engineering is by the way that the expression vector containing following exogenous DNA is transformed into Escherichia coli to obtain：The exogenous DNA is the DNA encoding sequence of Pseudomonas putidas Creatininase, and the amino acid sequence of the Pseudomonas putidas Creatininase is as shown in Seq No.2.Application of the restructuring Pseudomonas putidas Creatininase obtained present invention also offers the construction method of the colibacillus engineering and using colibacillus engineering fermentation of the invention in creatinine detection.The colibacillus engineering culture of the present invention is quick and easy, and inductive condition is easily controlled, and production cost is low, has the potentiality of industrialized production；The restructuring Pseudomonas putidas Creatininase prepared simultaneously by its fermentation has good creatinine enzymatic activity, and shows excellent temperature stability, temperature capacity and pH stability.

Description

It is a kind of be used for express Pseudomonas putidas Creatininase colibacillus engineering and its Using
Technical field
The present invention relates to technical field of bioengineering, more particularly to a kind of it is used to express Pseudomonas putidas Creatininase Colibacillus engineering and its application.
Background technology
Creatininase (creatininase；EC 3.5.2.10) it is a kind of hydrolase, belong to the related hydroamidase of urase Family, it is distributed in a few bacterium.Contain zinc ion, the catalyzing hydrolysis synthetic reaction of Creatininase in the activated centre of Creatininase What i.e. catalysis cyclic amides creatinine was reversible is hydrolyzed to creatine.
Objective indicator can be provided for clinical evaluation detection of glomeruli filtration function with creatinine assay in urine in serum, thus be to face The biochemical investigation project often done on bed, traditional creatinine assay chemical method is because atopic is poor, easily by sample The interference of non-specific material, is progressively substituted by Enzymology method.The substrate of creatinine enzyme effect has specificity, and substrate is only Can be creatinine either creatine, enzyme process detection creatinine has the advantages of atopic is strong, simple to operate, and is used as therein one Individual key enzyme, Creatininase have important application value in medical science detection.
In natural environment, have in the metabolin of microorganism such as pseudomonas putida, urea arthrobacterium and nicotianae It was found that Creatininase.The more Creatininase of research is the Creatininase in wild mushroom mostly at present, but wild mushroom culture is difficult, creatinine Production of enzyme is low, and specific enzyme activity is low, and purification has difficulties.With the exploitation of enzyme process detection creatinine reagent box, for the demand of Creatininase It is increasing, using wild mushroom carry out Creatininase production be difficult to industrialization amplification and also production often cost it is too high.
Therefore, the engineering bacteria of high Creatininase of the expression with good nature is constructed, realizes the life of Creatininase in the industry Production, can be greatly lowered production cost.
The content of the invention
In view of this, technical problem solved by the invention is to overcome insufficient in the prior art, there is provided one kind is used for Express the colibacillus engineering and its construction method of Pseudomonas putidas Creatininase.
Technical problem solved by the invention, which also resides in, provides a kind of restructuring Pseudomonas putidas Creatininase, utilizes this The colibacillus engineering of invention is fermented and obtained.
Technical problem solved by the invention, which also resides in, provides a kind of restructuring Pseudomonas putidas Creatininase in creatinine Application in detection.
In order to solve the above-mentioned technical problem, it is used to express the big of Pseudomonas putidas Creatininase the invention provides a kind of Enterobacteria engineering bacteria, the colibacillus engineering are by the way that the expression vector containing following exogenous DNA is transformed into large intestine bar Bacterium (Escherichia coli) and obtain：
The exogenous DNA be Pseudomonas putidas Creatininase DNA encoding sequence, the Pseudomonas putidas creatinine The amino acid sequence of enzyme is as shown in Seq No.2.
Preferably, the exogenous DNA array is as shown in Seq No.1.
Preferably, the expression vector is that the exogenous DNA is cloned into coli expression carrier pET-28a (+), is obtained The expression vector pET28a-cre arrived.
Preferably, the colibacillus engineering be transformed into Escherichia coli after the expression vector pET28a-cre and , the expression cassette that the DNA encoding sequence and its regulatory gene are formed is incorporated into the genome of Escherichia coli.
Correspondingly, the present invention also provides a kind of colibacillus engineering for being used to express Pseudomonas putidas Creatininase Construction method, comprise the following steps：
Step 1: the acquisition of encoding gene：Based on the amino acid sequence of Pseudomonas putidas Creatininase, to sequence Optimized according to the codon preference of Escherichia coli, obtain the corresponding coded sequence of codon optimization；Pseudomonas putida bar Bacterium creatinine enzyme amino acid sequence is as shown in SEQ ID NO.2, and its coded sequence is as shown in SEQ ID NO.1；
Step 2: the structure of expression vector：The encoding gene that step 1 obtains is cloned into coli expression carrier PET-28a (+), obtain expression vector pET28a-cre；
Step 3: expression vector conversion and colibacillus engineering obtain：Expression vector pET28a-cre will be expressed and carried Body pET28a-cre converts colibacillus engineering BL21 (DE3) competent cell, and positive transformant is screened after culture, and to sieve The positive transformant of choosing is identified that it is colibacillus engineering BL21/pET28a-cre to identify correct transformant.
Preferably, the screening of positive transformant is carried out in kalamycin resistance flat board.
Preferably, identifying for being carried out to positive transformant enters performing PCR identification, wherein cre- by primer of cre-S and cre-A S sequence is as shown in Seq No.3, and cre-A sequence is as shown in Seq No.4.
In addition, present invention also offers one kind to recombinate Pseudomonas putidas Creatininase, it is characterised in that：The restructuring stench Pseudomonas alba Creatininase is to be fermented by the colibacillus engineering disclosed in such scheme and obtained.
Preferably, its fermentation process is as follows：Picking recombination bacillus coli engineering bacteria is inoculated into LB culture mediums, 37 DEG C, 250rpm cultivates 12h, then the bacterium solution of culture is forwarded to 100mL TB fluid nutrient mediums (50 μ g/mL cards by 1% inoculum concentration That mycin) in, 30 DEG C, it is about 0.7-0.8 that 250rpm, which is cultivated to OD600,.1M IPTG are added into bacterium solution, control IPTG end Concentration is 0.8mM, and 30 DEG C, 12-14h is cultivated under the conditions of 250rpm；8000rpm centrifugation 15min processing zymotic fluids, remove supernatant, 50mM PBS (pH8.0) suspension cell is added after thalline is washed twice with distilled water, it is thin using ultrasonic wave under condition of ice bath Born of the same parents' pulverizer crushes to recombination bacillus coli；Carry out refrigerated centrifuge processing to the suspension after broken, 10000rpm, 4 DEG C, 30min, centrifuged supernatant is taken, purified with restructuring Creatininase of the affinity chromatography method to fermented supernatant fluid.
Moreover present invention also offers a kind of restructuring Pseudomonas putidas Creatininase of the invention in creatinine detection Using.
Compared with prior art, the invention has the advantages that：
First, restructuring Pseudomonas putidas Creatininase colibacillus engineering culture of the invention is quick and easy, lures Sliver part is easily controlled, and production cost is low, has the potentiality of industrialized production.What is more important is by restructuring stench of the invention The restructuring Pseudomonas putidas Creatininase that Pseudomonas alba Creatininase colibacillus engineering ferments and obtained has Creatininase Activity.Most suitable action pH, pH stability, most suitable action temperature also are carried out to the restructuring Pseudomonas putidas Creatininase of acquisition simultaneously Degree and temperature stability analysis.As a result show that temperature stability and the pH stability of the restructuring Pseudomonas putidas Creatininase are non- Chang Hao, show that the restructuring Pseudomonas putidas Creatininase has good temperature capacity.
The present invention is described in further detail with reference to the accompanying drawings and detailed description, but the present invention is not limited to these realities Mode is applied, any improvement or replacement on essence spirit of the present invention, is still fallen within claimed in claims of the present invention Scope.
Brief description of the drawings
Fig. 1 is (a) SDS-PAGE analyses and the figure of purifying Creatininase；SDS-PAGE collection of illustrative plates, wherein M：Standard protein point Son amount；1：The purification of Recombinant Creatininase of heat denatured.
Fig. 2 is (a) optimal pH curve and (b) pH stability curves of purification of Recombinant Creatininase.
Fig. 3 is the optimum temperature curve and temperature stabilization linearity curve of purification of Recombinant Creatininase.
Embodiment
Embodiment and Application Example are prepared with reference to specific, the present invention is described in further detail, but the present invention Embodiment not limited to this.
Embodiment 1
The synthesis of new creatinine enzyme gene
With US National Biotechnology Information center (NCBI, http://www.ncbi.nlm.nih.gov/) on announce genbank GI:Based on 34810367 gene orders, the sequence is optimized according to the codon preference of Escherichia coli, New creatinine enzyme gene is obtained, its sequence passes through biotech company's (Shanghai JaRa bioengineering as shown in SEQ ID NO.1 Co., Ltd) full genome synthesis, the gene cloning of synthesis obtains on pGH plasmids (being purchased from JaRa bioengineering Co., Ltd) Plasmid pGH-cre.
Embodiment 2
Recombinant plasmid pET28a-cre containing the new creatinine enzyme gene and bacterial strain E.coliTop10/ for carrying the plasmid PET28a-cre structure.
The codon that 6 encoding histidines have been carried behind plasmid pET-28a (+) multiple cloning sites region is marked as His Sign, come with terminator codon TGA behind label simultaneously, terminator codon is eliminated when designing anti-sense primer.According to new flesh The nucleotide sequence design PCR primer of acid anhydride enzyme gene：Forward primer cre-S：5'-CGGCCATGGATGTCTAAGTCTGTT-3' (underscore is NcoI restriction enzyme sites) and reverse primer cre-A：5'-ATACTCGAGAGTTGGTGGGAACTC-3'(underscores are XhoI restriction enzyme sites, do not contain terminator codon).
Using the plasmid pGH-cre in embodiment 1 as template, cre-S and cre-A are that primer enters performing PCR amplification.By glue reclaim PCR primer after purification carries out double digestion with restriction enzyme NcoI and XhoI, with also passing through NcoI and XhoI double digestions Plasmid pET-28a (+) with the connection of 16 DEG C of T4 ligases overnight, connection product chemical conversion E.coliTop10 (is purchased from the U.S. Invitrogen hero's life technology Co., Ltd), through kalamycin resistance plate screening positive transformant, positive transformant carries Plasmid is taken to obtain recombinant plasmid pET28a-cre after the identification of NcoI and XhoI double digestions is correct.Contain correct recombinant plasmid PET28a-cre clone strain E.coliTop10/pET28a-cre adds 15% glycerine in -80 DEG C of preservations.
Embodiment 3
New Creatininase production bacterial strain Escherichia coliBL21/pET28a-cre structure and restructuring Creatininase table Reach
Recombinant plasmid pET28a-cre Transformed E scherichia coliBL21 (DE3) (are purchased from U.S. Invitrogen Hero's life technology Co., Ltd) competent cell, and carry out positive transformant screening in kalamycin resistance flat board.With card that The transformant that chloramphenicol resistance plate screening obtains is template, enters performing PCR identification by primer of cre-S and cre-A, PCR is identified just True transformant is initially identified as new Creatininase production bacterial strain Escherichia coliBL21/pET28a-cre.
3 PCR of picking identify that correct transformant is inoculated into BMGY culture mediums, 37 DEG C, 250rpm culture 12h, then will The bacterium solution of culture is forwarded to by 1% inoculum concentration in 100mL TB fluid nutrient mediums (50 μ g/mL kanamycins), 30 DEG C, 250rpm It is about 0.7-0.8 to cultivate to OD600.1M IPTG are added into bacterium solution, control IPTG final concentration of 0.8mM, 30 DEG C, 12-14h is cultivated under the conditions of 250rpm；8000rpm centrifugation 15min processing zymotic fluids, remove supernatant, are washed thalline with distilled water 50mM PBS (pH8.0) suspension cell is added afterwards twice, using ultrasonic cell disruptor to recombinating large intestine under condition of ice bath Bacillus is crushed；Refrigerated centrifuge processing is carried out to the suspension after broken, 10000rpm, 4 DEG C, 30min, takes centrifugation supernatant Liquid.The restructuring Creatininase in bacterial cell disruption supernatant is purified with affinity chromatography method.Purification process reference HisTrapTMFF crude 5mL (GE Healthcare, Sweden) operating guidance.Purified restructuring Creatininase is in 4 DEG C of guarantors Deposit.
Embodiment 4
The restructuring Creatininase cre of purifying zymologic property
The assay method of creatinine enzyme activity：
Using creatine as substrate, 0.9ml 0.1mol/L creatine solutions (0.05M PBS dissolve, PH8.0) are taken into test tube, 37 DEG C place 5 minutes；0.l m1 enzyme liquids are added, are mixed, are reacted 10 minutes at 37 DEG C；Pipette 0.1ml reaction solutions extremely rapidly L.9mlNaOH in；L.0ml picric acid is added, is placed 20 minutes at 25 DEG C；Absorbance is determined at 520nm.
Creatininase enzyme activity defines：Under these conditions, it is 1 enzyme activity to produce 1 μm of ol/min picric acid creatinines compound Unit (U).
Creatinine enzyme activity calculation formula：Enzyme activity (U/L)=A × Vt × 10 × df/ (520 × 1.0 × t of ε × Vs)；
n:Enzyme liquid extension rate；A：The changing value of absorbance；Vt：The cumulative volume of enzyme reaction solution；Vs:Enzyme liquid in enzyme reaction solution Volume；ε520：Picric acid creatinine compound mM absorptivity is 4.65.
Optimal pH and pH stability：Using 0.1M creatines as substrate, cre is determined at 37 DEG C in pH3.0-10.0 The change of enzyme activity between (Britton-Robinson buffer solutions).Cre is placed in pH3.0-11.0 Britton-Robinson In buffer solution, taken out after handling 24h at 25 DEG C, the enzyme activity of the cre after being processed is determined under the conditions of pH8.0,37 DEG C, simultaneously 100% is set to using the undressed enzyme liquid of equivalent as positive control, vigor.Using creatine as substrate, cre is in pH3.0-11.0 for measure Optimal pH be 7.0 (Fig. 2 a), moreover, cre is highly stable under pH7.0-8.0 environment, enzyme activity is still after being handled by 24h More than 94% (Fig. 2 b) can be so kept, shows that cre has good tolerance under the conditions of neutral meta-alkali.
Optimum temperature and temperature stability：Cre under the conditions of pH8.0, using 0.1M creatines as substrate, is being determined it 30 Enzyme activity change in the range of DEG C -80 DEG C.By cre respectively in 50 DEG C of -70 DEG C of processing, sample, take out in 0.5h, 1h, 2h and 3h Ice bath afterwards, and determine under the conditions of the pH8.0,37 DEG C it is processed after cre enzyme activity, while with the undressed enzyme liquid of equivalent As positive control, vigor is set to 100%.As a result showing, cre optimum temperature is up to 60 DEG C (Fig. 3 a) during using creatine as substrate, And more than 77% enzyme activity (Fig. 3 b) can be kept in 50 DEG C of processing 3h of high temperature range.And cre has higher thermostabilization Property, 3h is handled even in 70 DEG C, still can keep more than 35% enzyme activity.Experimental result fully shows to recombinate Creatininase tool There is resistant to elevated temperatures advantage.
Above-described embodiment is the preferable embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment Limitation, other any Spirit Essences without departing from the present invention with made under principle change, modification, replacement, combine, simplification, Equivalent substitute mode is should be, is included within protection scope of the present invention.
Sequence table
<110>South China Science ＆ Engineering University
<120>A kind of colibacillus engineering and its application for being used to express Pseudomonas putidas Creatininase
<130> 2017
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ggtgctttgg tcttgccagg tttgcaatac ggttacaagt ctcagcaaaa gtctggtggt 240
ggtaaccatt ttccaggtac tacttctttg gatggagcaa ctttgactgg tactgtccag 300
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tatgagaact ctatgtttat tgttgaggga attgacttgg cattgagaga gttgagatat 420
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gctgttattc agagattgta tcctgaaggt ttcttgggtt gggatattga acacggaggt 540
gttttcgaga catctttgat gttggctttg tacccagatt tggttgattt ggaaagagtc 600
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Met Ser Lys Ser Val Phe Val Gly Glu Leu Thr Trp Lys Glu Tyr Glu
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Pro Thr Ala Val Cys Gln Arg Val Ala Glu Arg Ile Gly Ala Leu Val
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Leu Pro Gly Leu Gln Tyr Gly Tyr Lys Ser Gln Gln Lys Ser Gly Gly
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Gly Asn His Phe Pro Gly Thr Thr Ser Leu Asp Gly Ala Thr Leu Thr
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Gly Thr Val Gln Asp Leu Leu Arg Glu Leu Ala Arg His Gly Val Arg
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Arg Leu Val Leu Met Asn Gly His Tyr Glu Asn Ser Met Phe Ile Val
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Glu Gly Ile Asp Leu Ala Leu Arg Glu Leu Arg Tyr Ala Gly Ile His
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Claims (10)

  1. A kind of 1. colibacillus engineering for being used to express Pseudomonas putidas Creatininase, it is characterised in that the large intestine bar Bacterium engineering bacteria is by the way that the expression vector containing following exogenous DNA is transformed into Escherichia coli to obtain：
    The exogenous DNA is the DNA encoding sequence of Pseudomonas putidas Creatininase, the Pseudomonas putidas Creatininase Amino acid sequence is as shown in Seq No.2.
  2. 2. the colibacillus engineering as claimed in claim 1 for being used to express Pseudomonas putidas Creatininase, its feature exist In：The exogenous DNA array is as shown in Seq No.1.
  3. 3. the colibacillus engineering as claimed in claim 2 for being used to express Pseudomonas putidas Creatininase, its feature exist In：The expression vector is that the exogenous DNA is cloned into coli expression carrier pET-28a (+), obtained expression vector pET28a-cre。
  4. 4. the colibacillus engineering as claimed in claim 3 for being used to express Pseudomonas putidas Creatininase, its feature exist In：The colibacillus engineering is transformed into Escherichia coli and obtained after the expression vector pET28a-cre, the DNA encoding The expression cassette that sequence and its regulatory gene are formed is incorporated into the genome of Escherichia coli.
  5. A kind of 5. construction method for being used to express the colibacillus engineering of Pseudomonas putidas Creatininase, it is characterised in that Comprise the following steps：
    Step 1: the acquisition of encoding gene：Based on the amino acid sequence of Pseudomonas putidas Creatininase, to sequence according to The codon preference of Escherichia coli optimizes, and obtains the corresponding coded sequence of codon optimization；Pseudomonas putidas flesh Acid anhydride enzyme amino acid sequence is as shown in SEQ ID NO.2, and its coded sequence is as shown in SEQ ID NO.1；
    Step 2: the structure of expression vector：The encoding gene that step 1 obtains is cloned into coli expression carrier pET-28a (+), obtain expression vector pET28a-cre；
    Step 3: expression vector conversion and colibacillus engineering obtain：By expression vector pET28a-cre by expression vector PET28a-cre converts colibacillus engineering BL21 (DE3) competent cell, and positive transformant is screened after culture, and to screening Positive transformant identified that it is colibacillus engineering BL21/pET28a-cre to identify correct transformant.
  6. 6. the structure side as claimed in claim 5 for being used to express the colibacillus engineering of Pseudomonas putidas Creatininase Method, it is characterised in that：The screening of positive transformant is carried out in kalamycin resistance flat board.
  7. 7. the structure side as claimed in claim 5 for being used to express the colibacillus engineering of Pseudomonas putidas Creatininase Method, it is characterised in that：Identifying for being carried out to positive transformant enters performing PCR identification, wherein cre-S by primer of cre-S and cre-A Sequence as shown in Seq No.3, cre-A sequence is as shown in Seq No.4.
  8. 8. one kind restructuring Pseudomonas putidas Creatininase, it is characterised in that：The restructuring Pseudomonas putidas Creatininase be by Colibacillus engineering in claim 1-4 described in any one is fermented and obtained.
  9. 9. restructuring Pseudomonas putidas Creatininase as claimed in claim 8, it is characterised in that its fermentation process is as follows： Picking recombination bacillus coli engineering bacteria is inoculated into LB culture mediums, 37 DEG C, 250rpm culture 12h, the bacterium solution of culture then is pressed into 1% Inoculum concentration is forwarded in 100mL TB fluid nutrient mediums (50 μ g/mL kanamycins), and 30 DEG C, 250rpm, which is cultivated to OD600, is about 0.7-0.8.1M IPTG are added into bacterium solution, IPTG final concentration of 0.8mM is controlled, 30 DEG C, 12- is cultivated under the conditions of 250rpm 14h；8000rpm centrifugation 15min processing zymotic fluids, remove supernatant, 50mM PBS are added after thalline is washed twice with distilled water (pH8.0) suspension cell, recombination bacillus coli is crushed using ultrasonic cell disruptor under condition of ice bath；To broken Suspension afterwards carries out refrigerated centrifuge processing, 10000rpm, 4 DEG C, 30min, takes centrifuged supernatant, with affinity chromatography method to hair The restructuring Creatininase of ferment supernatant is purified.
  10. A kind of 10. application of restructuring Pseudomonas putidas Creatininase as claimed in claim 8 or 9 in creatinine detection.
CN201710873266.0A 2017-09-25 2017-09-25 A kind of colibacillus engineering and its application for being used to express Pseudomonas putidas Creatininase Pending CN107475170A (en)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108085291A (en) * 2018-01-04 2018-05-29 哈尔滨瀚邦医疗科技有限公司 A kind of detection method of genophore genetic stability of encoding muramidase release albumen and application
CN109957538A (en) * 2019-04-04 2019-07-02 大连大学 A kind of genetic engineering bacterium and its preparation method and application preparing sarcosine oxidase
CN111676180A (en) * 2020-03-12 2020-09-18 北京达成生物科技有限公司 Escherichia coli engineering bacterium for expressing pseudomonas stutzeri creatininase

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108085291A (en) * 2018-01-04 2018-05-29 哈尔滨瀚邦医疗科技有限公司 A kind of detection method of genophore genetic stability of encoding muramidase release albumen and application
CN109957538A (en) * 2019-04-04 2019-07-02 大连大学 A kind of genetic engineering bacterium and its preparation method and application preparing sarcosine oxidase
CN111676180A (en) * 2020-03-12 2020-09-18 北京达成生物科技有限公司 Escherichia coli engineering bacterium for expressing pseudomonas stutzeri creatininase

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