CN105695441A - Oxalate decarboxylase and recombinant expression method of oxalate decarboxylase - Google Patents
Oxalate decarboxylase and recombinant expression method of oxalate decarboxylase Download PDFInfo
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- CN105695441A CN105695441A CN201610217032.6A CN201610217032A CN105695441A CN 105695441 A CN105695441 A CN 105695441A CN 201610217032 A CN201610217032 A CN 201610217032A CN 105695441 A CN105695441 A CN 105695441A
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- oxalate decarboxylase
- expression
- recombinant expression
- oxalate
- recombinant
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- 229930003231 vitamin Natural products 0.000 description 1
- 150000003722 vitamin derivatives Chemical class 0.000 description 1
- 235000004416 zinc carbonate Nutrition 0.000 description 1
- 239000011667 zinc carbonate Substances 0.000 description 1
- 229910000010 zinc carbonate Inorganic materials 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/88—Lyases (4.)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y401/00—Carbon-carbon lyases (4.1)
- C12Y401/01—Carboxy-lyases (4.1.1)
- C12Y401/01002—Oxalate decarboxylase (4.1.1.2)
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Abstract
The invention discloses an oxalate decarboxylase and a recombinant expression method of the oxalate decarboxylase. The oxalate decarboxylase is subjected to recombinant expression through an Escherichia coli expression system, and is stable and active when the pH value is less than 3.0; and compared with the primary zymoprotein sequence, a signal peptide segment is deleted in the protein sequence of the oxalate decarboxylase in the expression process. The recombinant expression method is suitable for oxalate decarboxylase which is still active when the pH value is lower than 3.0: the DNA segment of the optimized oxalate decarboxylase coding gene and an Escherichia coli expression vector are mixed according to the mole ratio of (3-9):1 to perform connection so as to establish the recombinant expression vector, and the recombinant expression vector is used for transforming DH5a competent cells. PCR (polymerase chain reaction) and sequencing verification are utilized to screen the successfully established recombinant expression vector transformed bacteria, the recombinant expression vector is extracted after culture and is used for transforming the molecular-chaperone-containing Escherichia coli expression strain, and 15-30-DEG C culture and protein expression induction are performed. The method enhances the expression efficiency, simplifies the production steps and lowers the production cost.
Description
Technical field
The present invention relates to and stablize under a kind of new low ph conditions and efficient oxalate decarboxylase, and the recombinant expression method of oxalate decarboxylase。
Background technology
Oxalic acid (oxalicacid) is a kind of molecular formula is C2O4H2, molecular weight be the binary organic acid of 90.04, be present in common most foods, in specific food (such as Herba Spinaciae or other green vegetables, green tea, cacao bean, bean milk, coffee etc.), content is significantly high。Oxalic acid is end product in vivo, it is impossible to be further divided, and Major excretion mode is to discharge with urine。Oxalic acid in human body includes exogenous oxalic acid and endogenous oxalic acid, exogenous oxalic acid refers to by the oxalic acid that diet is taken in, endogenous oxalic acid refers to the oxalic acid that human liver's own metabolism produces, and exogenous oxalic acid and the content of endogenous oxalic acid in normal human are essentially identical。Some renal calculus group of people at high risk then exogenous oxalic acid is far longer than endogenous oxalic acid。
When oxalic acid content in blood in human body in liquid and urine is too high, it is easy to form insoluble calcium oxalate crystals with calcium ion, calcium oxalate crystals is easy in bladder, kidney and other organs formation of deposits calculus。It is abnormal that II type hyperoxaluria refers to that food-borne oxalic acid absorbs, and causes that urine oxalic acid calibration ordinary person is higher, is lead lithogenic key factor outside primary hyperoxaluria, therefore, control the oxalic acid of picked-up from diet, largely just can reducing urine oxalic acid amount, suffering from calculus risk thus reducing。The strategy preventing and treating calcium oxalate stone of low oxalic acid diet or degraded food mesoxalic acid has become common recognition in medical circle。Diet mesoxalic acid is mainly released under one's belt, concentration is also of a relatively high, it is very beneficial for enzymic degradation to remove, but the vigor of oxalate-degrading enzyme is had very big challenge by the low ph conditions in stomach and pepsin, therefore find and the enzyme of efficient degradation oxalic acid can become the key of this treatment technology at low ph conditions。
The enzyme with decomposing oxalic acid function having now been found that, has oxalate decarboxylase (hereinafter referred to as " OXDC "), Aero-oxalo dehydrogenase and oxalyl CoA decarboxylase。The effect of OXDC is that catalysis oxalic acid is decomposed into formic acid and carbon dioxide。Hitherto known, the OXDC having been reported belongs to essentially from bacillus cereus (Bacillus), aspergillosis (Aspergillus) belongs to, Flammulina velutiper (Fr.) Sing (Flammulinavelutipes), Coriolous Dersicolor (Fr.) Quel (Coriolusversicolor), whiterot fungi (Trametesversicolor) etc.。Oxalate decarboxylase expression in above-mentioned species is extremely low, and majority growth is slow, complicated component, separation and Extraction purification is very difficult, do not possess commercial applications to be likely to, so oxalate decarboxylase being carried out recombinant expressed production become the inevitable choice of its commercial applications。That realize RT-PCR expression in current oxalate decarboxylase is the YvrK gene source OXDC of bacillus subtilis Pseudomonas, and it is carried out the parsing of zymologic property, but its enzyme activity ranges for pH3.0～6.0, lower than 3.0 debilities。The OXDC in Flammulina velutiper (Fr.) Sing source has by tobacco expressed one-tenth merits and demerits report, but expression is extremely low, it has been also carried out protokaryon (E.coli) to express but without enzyme activity (referring to document: MeenuKesarwani simultaneously, et.alOxalateDecarboxylasefromCollybiavelutipes, THEJOURNALOFBIOLOGICALCHEMISTRY, 2000), other kinds or gene source OXDC, especially Eukaryotic OXDC, still find no and successfully carry out recombinant expressed report, carry out recombinant expressed particularly with E. coli system。
Summary of the invention
This technical problem of Recombinant protein expression is cannot be carried out in order to solve the stable and great-hearted OXDC of low pH in prior art (pH < 3.0), the present invention passes through high flux screening, obtain multiple under gastrointestinal tract environment (pH1.5～7.5) all have relatively top grass acid degradation enzyme activity, and have the oxalate decarboxylase of high vigor low pH gastric environment (pH1.5～3.0), and clone and obtain its encoding gene to achieve it by transformation recombinant expressed。It is a further object to provide the Recombinant protein expression method of the stable and great-hearted OXDC of low pH。
A kind of oxalate decarboxylase provided by the invention, it is for be undertaken recombinant expressed by escherichia expression system, and stablizes when pH < 3.0 and have vigor；The protein sequence of this oxalate decarboxylase more primary pheron sequence deletion signal peptide fragment in expression process。Time recombinant expressed, the DNA sequence of these oxalate decarboxylase signal peptide parts is removed, make improved gene without signal coding sequence, achieve Recombinant protein expression, the problem that when overcoming in prior art for pH < 3.0, stable and great-hearted oxalate decarboxylase cannot be produced by escherichia expression system, reduces production cost。
During described pH < 3.0 stable and have vigor, refer under the pH environment lower than 3.0, still have vigor, and enzyme function-stable。This oxalate decarboxylase, it is preferable that at pH < 2.8, pH < 2.6, pH < 2.4, stablize under pH < 2.2, pH < 2.0, pH < 1.8 or pH < 1.5 environment and have vigor。
The present invention also provides for a kind of edible composition, and it is containing above-described oxalate decarboxylase, for preventing and/or treat primary hyperoxaluria, II type hyperoxaluria, intestinal source property hyperoxaluria and containing one or more in calcium oxalate urinary calculi disease。Live and activity stabilized owing to the oxalate decarboxylase of the present invention still has higher enzyme under low pH (pH < 3.0) environment, therefore may be used for preparation prevention and/or treat the edible composition of above-mentioned multiple disease。
Preferably, above-mentioned edible composition is pharmaceutical composition, health food or special medicine purposes formula food。Described special medicine purposes formula food is primarily to and meets limited, the Disorder of Digestion and Absorption of feed, metabolism disorder or the particular disease states crowd special requirement to nutrient or meals, the formula food that special processing is formulated。This series products under doctor or clinical nutrition teacher instruct, individually must eat or matched with other food。
Present invention also offers a kind of food additive, it is containing above-described oxalate decarboxylase, for preparing low oxalic acid/without the Foods or drinks of oxalic acid。
Present invention also offers the recombinant expression method of a kind of oxalate decarboxylase, the Expression product of still great-hearted oxalate decarboxylase during suitable in pH < 3.0, it is to be attached according to the mixed in molar ratio of 3～9:1 with coli expression carrier by the DNA fragmentation of oxalate decarboxylase encoding gene after optimization, building recombinant expression carrier, recombinant expression carrier converts DH5a competent cell；Being screened the recombinant expression carrier transformed bacteria successfully constructed by PCR and sequence verification, extract recombinant expression carrier, convert the E. coli expression strains containing molecular chaperones after cultivation, the Low-temperature culture the induced protein that carry out 15～30 DEG C are expressed。
This recombinant expression method, it is preferably applied at pH < 2.8, pH < 2.6, pH < 2.4, pH < 2.2, the Expression product of stable and great-hearted oxalate decarboxylase under pH < 2.0, pH < 1.8, pH < 1.5 environment。
Competent cell (competentcells) be a kind of have take in foreign DNA ability by hold bacterium, it can absorb the plasmid DNA etc. of external source。
During pH < 3.0, still great-hearted oxalate decarboxylase is generally beyond expression in escherichia coli, the present invention is by being optimized process to the DNA fragmentation containing its encoding gene, and increase molecular chaperones realizes its expression in escherichia coli in expression strain, it is provided that another easy production ways。
Preferably, in the recombinant expression method of above-mentioned oxalate decarboxylase, described optimization includes the signal peptide fragment deleting oxalate decarboxylase encoding gene DNA fragmentation；Or delete the signal peptide fragment of oxalate decarboxylase encoding gene DNA fragmentation and merge hydrotropy label at C end；Or delete the signal peptide fragment of oxalate decarboxylase encoding gene DNA fragmentation and merge hydrotropy label at C end and carry out codon optimized；Or oxalate decarboxylase encoding gene DNA fragmentation is carried out codon optimized and erasure signal fragments of peptides。
Described codon optimized be that its codon is replaced by the codon being more suitable for escherichia coli expression, signal peptide is to affect the key factor that the stable and great-hearted oxalate decarboxylase of low pH is expressed, therefore the gene encoding signal peptide part must go to remove, and could realize its normal expression at escherichia expression system；Merge hydrotropy label and can be greatly improved the dissolubility of expressing protein, reduce the formation of inclusion body, improve production efficiency。
Preferably, in the recombinant expression method of above-mentioned oxalate decarboxylase, described hydrotropy label is polyglutamic acid fragment, many poly-aspartates fragment, poly-D-lysine fragment, any one in poly glumine fragment and many poly-asparagines fragment, or the fragment by glutamic acid, aspartic acid, agedoite, lysine and glutamine five seed amino acid random combine。Random combine refers to the arrangement of five seed amino acid residues and the random collocation of quantity。
Preferably, in the recombinant expression method of above-mentioned oxalate decarboxylase, described expression vector is lactose-induced expression vector, galactose inducible expression carrier, IPTG inducible expression carrier, thermal induction expression vector or cold inducible expression carrier。Pass through abduction delivering, it is possible to accurately control expression process。
Preferably, in the recombinant expression method of above-mentioned oxalate decarboxylase, described E. coli expression strains is BL21, BL21 (DE3), K12, K12 (DE3), Origami, Origami (DE3), OrigamiB, OrigamiB (DE3), Rosetta or Rosetta (DE3)。
Wherein, OrigamiB (DE3) is derived from the BL21 bacterial strain of lacZY sudden change, and this sudden change can accurately regulate expression product according to the concentration of IPTG so that expression product amount presents IPTG concentration dependent。
Rosetta (DE3) (RosettaCompetentCell (DE3)) adopts escherichia coli Rosetta bacterial strain to process the escherichia coli Competent cell obtained through special process, there is chlorampenicol resistant, supplement 6 kinds of rare codon (AUA that escherichia coli lack, AGG, AGA, CUA, CCC, GGA) corresponding tRNA, improves exogenous gene, the especially decarboxylase gene of eukaryotic source expression in escherichia expression system。
Preferably, in the recombinant expression method of above-mentioned oxalate decarboxylase, described molecular chaperones be in pG-KJE8, pGro7, pKJE7, pGTf2 and pTf16 any one or several。These molecular chaperoneses are Takara company commercialization escherichia coli molecular chaperones, it is possible to help the Exogenous Oxalic Acid decarboxylase protein of escherichia coli expression correctly to fold, improve expression efficiency。
Preferably, oxalate decarboxylase of the present invention, shown in its aminoacid sequence such as SEQIDNO:1～11 are arbitrary。
Compared with prior art, the method have the advantages that
The oxalate decarboxylase of the present invention is the enzyme that low pH is stable, it is possible to adapt to the low ph conditions of gastric completely, has stronger antipepsin degradation function, enzyme is lived high, food-borne oxalic acid can be decomposed rapidly, reduce the gastrointestinal absorption of oxalic acid, thus prevention and treatment hyperoxaluria。
The recombinant expression method of the present invention, successfully passes prokaryotic expression system by OXDC gene stable for low pH and expresses, and improves expression efficiency, simplifies production stage, reduces production cost。
Accompanying drawing explanation
Fig. 1 is OXDC gene diffusion carrier pET-22b linearisation electrophoretogram；
Fig. 2 is the recombinant expressed electrophoresis result of Flammulina velutiper (Fr.) Sing oxalate decarboxylase, wherein, upper: bacterial cell disruption supernatant, complete: the full liquid of bacterial cell disruption, not: do not induce the full liquid of bacterial cell disruption；A figure is 18 DEG C of inductions, and B figure is 28 DEG C of inductions, and C figure is 37 DEG C of inductions；
Fig. 3 is the pET-22b-12E-OXDC and the pET-22b-OXDC plasmid expression result without hydrotropy label that have hydrotropy label, and wherein A figure is the oxalate decarboxylase containing hydrotropy label；B figure is the oxalate decarboxylase without hydrotropy label；
Fig. 4 is the OXDC degraded food mesoxalic acid effect of cb01；
Fig. 5 is the OXDC degraded food mesoxalic acid treatment hyperoxaluria effect of recombinant expressed separate sources。
Detailed description of the invention
Below in conjunction with specific embodiment, the invention will be further described, so that those skilled in the art can be more fully understood that the present invention and can be practiced, but illustrated embodiment is not as a limitation of the invention。
Embodiment 1: the screening of oxalate decarboxylase and vigor range test
The present inventor has successively screened over one hundred kind of species, wherein comprise agrocyb eaegerita (Agrocybeaegirit), Tricholoma lobayense Heim (TricholomaLobayenscHeim), Agrocybe aegerita (Brig) Sing (AgrocybeCylindracea), bacillus subtilis (Bacillussubtilis), Coriolous Dersicolor (Fr.) Quel (Coriolusversicolor), brown rot fungus (Postiaplacenta), the species such as cyanophyceae (Cyanobacteria), screen a series of oxalate decarboxylase having efficient vigor in gastrointestinal tract pH environment (pH1.5～7.5)。
Screening technique is as follows:
Inoculation shake-flask culture thalline, abduction delivering OXDC is carried out with phosphorus acid for adjusting pH to 2.5～3.0, collect thalline, weigh each 2g of wet thallus, 18s is smashed with high-speed homogenization machine 9500rpm after adding the PBS mixing of pH3.0, centrifugal thalline or homogenate supernatant add the oxalic acid reactant liquor (pH1.5～7.5) of different pH, and 37 DEG C, 1000rpm reacts 30min；100 μ L2.5mol/LH2SO4Terminate reaction。12000rpm is centrifuged, and takes and lives with high performance liquid chromatograph (HPLC) enzyme analysis after supernatant filters。
High-efficient liquid phase chromatogram condition: mobile phase is 2.5mMH2SO4；Analytical column is CarbomixH-NP10 (5%) organic acid post；Column temperature 55 DEG C；Sample size 20 μ L；Flow velocity 0.6mL/min。
1 enzyme activity unit (IU) refers under specified conditions (37 DEG C, pH3.0), the enzyme amount of 1 micromole's oxalic acid of degrading in 1 minute, or generates the enzyme amount of 1 micromole's formic acid。
The oxalate decarboxylase activity pH scope in table 1. different genera source and optimum pH
Note: in table, optimum pH refers to that the crude enzyme liquid of zymogenic bacteria to be screened vigor under this pH value is maximum。
Embodiment 2: decarboxylase gene is cloned and recombinant expressed
By the product oxalate decarboxylase species of the above-mentioned function admirable screened, carry out shake-flask culture, and carry out producing enzyme induction, collect thalline mid-term extract its total serum IgE and to carry out reverse transcription be cDNA producing enzyme, design degenerate primer, carry out cDNA amplification with RACE (RapidAmplificationofcDNAEnds) PCR, operate the SMARTer in strict accordance with Clontech companyTMRACEcDNAAmplificationKit workbook performs, and wherein use 5 ' and 3 ' universal primers and gene mapping primer (OXDCGSPprimer) are as shown in table 2。The gene that amplification obtains obtains gene order through order-checking screening, and the protein sequence after translation is as shown in table 3。
Table 2. degenerate primer sequence
The oxalate decarboxylase stable for low pH that table 3 filters out
| Oxalate decarboxylase source strain code name | Aminoacid sequence |
| A0 | SEQ ID NO:1 |
| D3 | SEQ ID NO:2 |
| Cb01 | SEQ ID NO:3 |
| Cb03 | SEQ ID NO:4 |
| Vr | SEQ ID NO:5 |
| Vl | SEQ ID NO:6 |
| Pl | SEQ ID NO:7 |
| B5 | SEQ ID NO:8 |
| B15 | SEQ ID NO:9 |
| Asp | SEQ ID NO:10 |
| A5 | SEQ ID NO:11 |
The Recombinant protein expression of embodiment 3:OXDC
The present inventor to screening and cloning to OXDC stable for low pH (include it have been reported that cross Flammulina velutiper (Fr.) Sing OXDC) carry out the recombinant expressed of E. coli system。Flammulina velutiper (Fr.) Sing OXDC gene has more than 10 years of relevant report, referring to document (MeenuKesarwani, et.alOxalateDecarboxylasefromCollybiavelutipes, THEJOURNALOFBIOLOGICALCHEMISTRY, 2000), not yet success is recombinant expressed in escherichia expression system success so far for Flammulina velutiper (Fr.) Sing OXDC gene。In consideration of it, the present embodiment carries out the elaboration of OXDC Recombinant protein expression method for Flammulina velutiper (Fr.) Sing OXDC。
(1) gene clone
Cloning primer (table 4) is designed according to expression vector and Flammulina velutiper (Fr.) Sing OXDC gene order, need during design of primers to remove the coded sequence of signal peptide part, directly expand the DNA sequence of OXDC albumen, the Flammulina velutiper (Fr.) Sing gene (Vl) being cloned in embodiment 2 carries out pcr amplification for template, PCR system (table 5) and amplification condition is as mentioned below, carries out glue recovery by PCR primer。Expression vector pET-22b PCR method is carried out linearisation (amplification pET-22b-12E containing hydrotropy label and two kinds of sequences of the pET-22b without hydrotropy label), primer (table 6, table 7), PCR system (table 8) and reaction condition is as shown below。Vector linearization product is carried out DpnI collagenase treatment, finally carries out glue recovery。It is that 3:1 to 9:1 carries out seamless link reaction, construction of expression vector by gene outcome and linearized vector product according to mol ratio。Seamless link product is converted DH5a strain, and by the bacterial strain that PCR and sequence verification screening vector successfully construct, shake-flask culture extracts expression plasmid, finally converts the expression strain BL21 (DE3) containing molecular chaperones pGro7。
Table 4.OXDC gene clone primer
Table 5.PCR system (50 μ L)
| Reagent | Volume μ L |
| 2×PCRmix | 25 |
| Forward primer 22b-VL-F (10 μMs) | 2 |
| Downstream primer 22b-VL-R (10 μMs) | 2 |
| OXDC gene template | 1 |
| ddH2O | 20 |
PCR condition:
94 DEG C of denaturation 2min；98 DEG C of degeneration 10sec；60 DEG C of annealing 30sec, 68 DEG C extend 45sec, circulate 30 times, and 68 DEG C extend 5min, 12 DEG C of stopped reaction。
(2) vector linearization
The table 6. vector linearization primer without hydrotropy label
| Primer | Primer sequence (5 '-3 ') |
| pET-22b-F | TAATGGATCCGAATTCGAGCTCC(SEQIDNO:17) |
| pET-22b-R | CATATGTATATCTCCTTCTTAAAG(SEQIDNO:18) |
Table 7. merges the vector linearization primer of hydrotropy label (12E)
Table 8.PCR system (50 μ L)
| Reagent | Volume μ L |
| 2×PCRmix | 25 |
| Forward primer pET-22b-F (10 μMs) | 2 |
| Downstream primer pET-22b-R (10 μMs) | 2 |
| OXDC gene template | 1 |
| ddH2O | 20 |
PCR condition:
94 DEG C of denaturation 2min；98 DEG C of degeneration 10sec；55 DEG C of annealing 30sec, 68 DEG C extend 3min, circulate 30 times, and 68 DEG C extend 5min, 12 DEG C of stopped reaction。
Product after PCR vector linearization is carried out glue recovery, and the enzyme action then carrying out DpnI processes, and reaction system is as shown in table 9。
Table 9.DpnI reaction system (50 μ L)
DpnI processes reaction condition: hatch 2h for 37 DEG C。
After the PCR primer of OXDC and DpnI being processed, linearized vector (containing hydrotropy label and without two kinds of hydrotropy label) sample reclaims through glue recovery test kit respectively, operates and operates in strict accordance with test kit description。
(3) seamless link (10 μ L) of genes of interest and linearized vector
Table 10. seamless link reaction system
Note: the mol ratio between genes of interest and carrier is 3:1 to 9:1。
After composition each in pipe is fully mixed, it is placed in PCR instrument and reacts by following response procedures: 25 DEG C of enzyme action 30min；60 DEG C connect 15min；4 DEG C of stopped reaction。Sample is immediately placed on ice after terminating by reaction。Obtain recombiant plasmid (pET-22b-12E-OXDC containing hydrotropy label and two kinds without hydrotropy label pET-22b-OXDC)。
(4) recombinant plasmid transformed escherichia coli DH5a
Recombiant plasmid pET-22b-OXDC and pET-22b-12E-OXDC complete for seamless link is converted escherichia coli DH5a Efficiency Competent, 37 DEG C of incubated overnight are to growing monoclonal, picking monoclonal carries out PCR checking and sequence verification, and the correct strain that checks order carries out conservation, and extracts plasmid after cultivation。
(5) expression plasmid converts E. coli expression strains BL21 (DE3)
PET-22b-OXDC plasmid (without hydrotropy label) is converted BL21 (DE3) strain containing pGro7 molecular chaperones, respectively different temperatures (18 DEG C, 28 DEG C and 37 DEG C) under carry out inducing culture, abduction delivering result is as shown in Figure 2。Result shows, low temperature contributes to the solvable activity expression of Flammulina velutiper (Fr.) Sing oxalate decarboxylase。
To merge the PET-22b-12E-OXDC and the pET-22b-OXDC plasmid without hydrotropy label that there are hydrotropy label, and convert BL21 (DE3) strain containing pGro7 molecular chaperones respectively, carry out inducing culture at 37 DEG C, abduction delivering result is as shown in Figure 3。Result shows, merges hydrotropy label and can significantly improve the solubility expression of Flammulina velutiper (Fr.) Sing OXDC。
(6) enzyme property
After recombinant expressed OXDC is carried out chromatography, measure the Rate activity of enzyme and vigor pH scope and optimum pH scope。Table 11 show the restructuring OXDC enzymatic property result of wherein several separate sources:
The recombinant expressed OXDC enzyme property of table 11.
| Strain code name | PH field of activity | Optimum pH | Optimum pH scope (more than 50% vigor) | Rate activity |
| A0 | 1.5～6.5 | 3.0～3.5 | 2.5～5.0 | 15U/mg |
| D3 | 1.5～7.5 | 3.5 | 3.0～4.0 | 12U/mg |
| Cb01 | 1.5～5.0 | 2.5 | 1.5～4.0 | 50U/mg |
| Cb03 | 1.5～6.0 | 2.5 | 1.5～4.0 | 50U/mg |
| Vl | 2.0～6.0 | 2.5 | 2.0～4.0 | 10U/mg |
Note: in table, optimum pH refers to that the crude enzyme liquid of zymogenic bacteria to be screened vigor under this pH value is maximum。
Embodiment 4: the oxalate-degrading enzyme oxalic acid (salt) for degrading in food
This embodiment is only for the OXDC of Cb01, and research oxalate decarboxylase, for the oxalic acid degraded in conventional food, prepares low oxalic acid food。
(1) oxalic acid (salt) in degraded tea
Weighing tippy tea, green tea, black tea, each 5g in Pu'er respectively, add 50ml deionized water, boiled water simmers about 15min, and filter paper filtering removes tea leaf residual, obtains tea sample。Detect immediately after being cooled to room temperature or put 4 DEG C to be measured。
Adding citric acid in tea sample is regulated to pH2.8, adds this product to 10mg/1000ml tea, after stirring evenly, react 30min in room temperature (25 DEG C)。
Take the forward and backward tea sample of process, respectively by the oxalic acid content in HPLC method detection sample, assess this product degradation effect to tea mesoxalic acid。
(2) oxalic acid (salt) in degraded coffee
Weighing commercially available Blue Mountain Coffee and each 5g of espresso coffee bean respectively, add 250ml deionized water, boiled water simmers about 60min, and filter paper filtering removes tea leaf residual, obtains coffee concentrating sample。Detect immediately after being cooled to room temperature or put 4 DEG C to be measured。
Regulate coffee samples adds hydrochloric acid to pH2.4, add this product to 10mg/1000ml coffee liquid, after stirring evenly, react 30min in room temperature (25 DEG C)。
Before taking process, rear coffee samples, respectively by the oxalic acid content in HPLC method detection sample, assess this product degradation effect to coffee mesoxalic acid。
(3) oxalic acid (salt) in fruit juice is removed
Weighing commercially available Citrus, red cloth woods, each 100g sarcocarp such as Herba Apii graveolentis and Amaranthus mangostanus L. respectively, juice extractor squeezing juice, filter paper filtering removes residue, obtains concentration of juices sample。Detect immediately after being cooled to room temperature or put 4 DEG C to be measured。
Regulate samples of juice adds hydrochloric acid to pH2.0, add this product to 10mg/1000ml fruit juice, after stirring evenly, react 30min in room temperature (25 DEG C)。
Before taking process, rear samples of juice, respectively by the oxalic acid content in HPLC method detection sample, assess this product degradation effect to fruit juice mesoxalic acid。
(4) oxalic acid (salt) in sowens is removed
Another name take commercially available oatmeal 30g, add water 200ml, and heated and boiled is about 5min, in atherosclerotic, detect immediately after being cooled to room temperature or put 4 DEG C to be measured。
Regulate sowens sample adds hydrochloric acid to pH1.8, add this product to 10mg/1000g sowens, after stirring evenly, react 30min in room temperature (25 DEG C)。
Before taking process, rear sowens sample, through 10000g high speed centrifugation, take supernatant filter paper filtering, for testing sample, the oxalic acid content in use HPLC method detection sample, assesses this product degradation effect to Herba bromi japonici mesoxalic acid respectively。
Result as shown in Figure 4, before in bar diagram, post height expression on the left of same food processes, after the post height expression on right side processes。
Embodiment 5: oxalate-degrading enzyme is for the treatment of food-borne hyperoxaluria
6 beasle dogs, male, body weight 7～8kg, buy from Rui Kesen laboratory animal company limited of Anlu Hubei, raise in standard independence dog cage, after adapting to 3～5 days, feed (the table 12 of food without oxalic acid of preparation, it is not added with oxalic acid), collect 24h urine, measure urine total oxalate and urine creatine total amount, to urinate oxalic acid/urine creatine measurement urine oxalic acid excretion, treat that than value stabilization postscript be low oxalic acid excretion (Basal), then feeding height oxalic acid food (table 12), collect urine, measure urine oxalic acid excretion, after stable, take average and be designated as high oxalic acid excretion (HOD), then while feeding height oxalic acid food, OXDC enzyme recombinant expressed for feeding 10mg, collect urine, measure urine oxalic acid excretion, the ability of the OXDC enzymatic degradation food mesoxalic acid of test separate sources, result is as shown in Figure 5。It is shown that the oxalic acid that OXDC can significantly degrade in diet, reduce oxalic acid and absorb, thus the oxalic acid excretion reduced in urine, treat food-borne hyperoxaluria。
Table 12. dog grain formula
| Nutrient | G kg |
| Casein | 242.0 |
| Sucrose | 392.6096 |
| Corn starch | 130.0 |
| Maltodextrin | 100.0 |
| Adeps Sus domestica | 50.0 |
| Soybean oil | 15.0 |
| Cellulose | 25.0 |
| Vitamin mixtures | 10.0 |
| TBHQ | 0.005 |
| Calcium phosphate dihydrate | 8.47 |
| Citric acid monohydrate potassium | 13.26 |
| One water potassium phosphate | 6.14 |
| Magnesium oxide | 1.7 |
| Sodium chloride | 5.0 |
| Ferric citrate | 0.6 |
| Zinc carbonate | 0.12 |
| Manganese sulfate monohydrate | 0.04 |
| Copper sulfate | 0.03 |
| Ten sulfate dihydrate chromium potassium | 0.02 |
| Potassium iodate | 0.004 |
| Four water ammonium molybdates | 0.001 |
| Sodium selenite pentahydrate | 0.0004 |
| Disodium oxalate. | 6.1 |
Embodiment described above is only the preferred embodiment lifted for absolutely proving the present invention, and protection scope of the present invention is not limited to this。Equivalent replacement that those skilled in the art make on basis of the present invention or conversion, all within protection scope of the present invention。Protection scope of the present invention is as the criterion with claims。
SEQUENCELISTING
<110>Wuhan Kangfude Biotechnology Co., Ltd.
<120>recombinant expression method of a kind of oxalate decarboxylase and oxalate decarboxylase
<130>
<160>20
<170>PatentInversion3.3
<210>1
<211>470
<212>PRT
<213>artificial sequence
<400>1
MetIleSerValAlaSerCysThrIleAlaLeuLeuLeuSerSerVal
151015
AlaPheAlaAlaProAlaProSerSerAlaAlaSerSerIleValVal
202530
SerAlaThrSerSerSerThrValSerSerAlaProValSerValSer
354045
SerPheLeuProThrThrSerIleAlaAlaAlaThrProSerSerIle
505560
AlaValAlaLeuSerSerThrAlaThrValProPheIleAspLeuAsn
65707580
ProAsnGlyProLeuTrpAspProSerValSerGlyValProGlnAla
859095
GluArgGlySerLeuGlyAlaThrIleMetGlyProThrAspValAsp
100105110
ThrThrLysAlaAsnProAspLeuLeuAlaProProThrThrAspHis
115120125
GlySerValAspAsnAlaLysTrpAlaPheSerLeuSerHisAsnArg
130135140
LeuGlnThrGlyGlyTrpAlaArgGluGlnAsnIleGlyAlaMetPro
145150155160
IleAlaThrGluMetAlaSerValAsnMetArgLeuGluProGlyAla
165170175
IleArgGluLeuHisTrpHisLysThrAlaGluTrpAlaTyrValLeu
180185190
LysGlyAsnThrGlnValThrAlaValAspGlnAsnGlyLysAsnPhe
195200205
IleGlyThrValGlyProGlyAspLeuTrpTyrPheProProGlyIle
210215220
ProHisSerLeuGlnAlaThrGlyAspAspProGluGlySerGluPhe
225230235240
IleLeuValPheAspSerGlyAlaPheSerGluAspSerThrPheLeu
245250255
LeuThrAspTrpMetSerHisValProValGluValLeuAlaLysAsn
260265270
PheGlnThrAspIleSerAlaPheAlaArgIleProAlaGluGluLeu
275280285
TyrIlePheProAlaAlaValProProAspSerGlnGlnAspProThr
290295300
SerProGluGlyThrValProAsnProPheThrPheAlaLeuSerLys
305310315320
ValProProMetGlnLeuSerGlyGlyThrAlaLysIleValAspSer
325330335
ThrThrPheThrValSerLysAlaIleAlaAlaAlaGluValThrIle
340345350
GluProGlyAlaIleArgGluLeuHisTrpHisProThrGlnAspGlu
355360365
TrpSerPhePheIleGluGlyArgAlaArgMetThrIlePheAlaAla
370375380
GlnSerAsnAlaArgThrPheAspTyrGlnAlaGlyAspIleGlyTyr
385390395400
ValProAlaThrMetGlyHisTyrValGluAsnIleGlyAsnThrThr
405410415
ValArgTyrLeuGluIlePheAsnThrAlaValPheGluAspIleSer
420425430
LeuSerAsnTrpLeuAlaLeuThrProProGluLeuValLysAlaHis
435440445
LeuGlyPheAspAspAlaThrMetAlaHisLeuAlaLysValLysPro
450455460
IleValValGlyProAla
465470
<210>2
<211>470
<212>PRT
<213>artificial sequence
<400>2
MetIleSerPheAlaSerCysValCysAlaLeuLeuPheAlaArgLeu
151015
AlaLeuSerAlaProAlaProAlaAlaSerSerSerAlaProThrVal
202530
SerThrValSerSerValIleAlaProIleSerProThrAlaGluSer
354045
SerValAlaAlaSerSerAlaSerAsnLysProArgProThrSerThr
505560
AlaThrAlaThrGluProThrAlaThrValProPheIleAspLeuAsp
65707580
ProAsnGluProLeuTrpAsnGluAspThrProGlyIleHisGlnPro
859095
IleHisGlySerLeuGlyAlaLysLeuLeuGlyProThrAsnAsnAla
100105110
IleValLysGlnAsnProAspLeuLeuAlaProProThrThrAspHis
115120125
GlySerValProAsnAlaLysTrpProPheSerLeuSerHisAsnArg
130135140
LeuGlnThrGlyGlyTrpAlaArgGluGluAsnIleAlaValMetPro
145150155160
ValAlaGlnAlaMetAlaSerValAsnMetArgLeuGluAlaGlyAla
165170175
ValArgGluLeuHisTrpHisLysThrAlaGluTrpAlaTyrValLeu
180185190
LysGlySerThrGlnValThrAlaValAspAlaAspGlyArgAsnPhe
195200205
ValSerThrValGlyProGlyAspLeuTrpTyrPheProProGlyIle
210215220
ProHisSerLeuGlnAlaThrAsnAspAspProAspGlySerGluPhe
225230235240
ValLeuValPheAspSerGlySerPheSerGluAspSerThrPheLeu
245250255
LeuThrAspTrpLeuAspHisValProAlaGluValLeuAlaLysAsn
260265270
PheGlnValAsnIleSerAlaPheAlaHisIleProAlaGluGluLeu
275280285
TyrIlePheProAlaAlaLeuProGluProAspSerAlaAlaProLys
290295300
SerProGlnGlyThrValProAspProPheSerPheSerMetSerLys
305310315320
ValLysProThrGlnLeuThrGlyGlyThrValLysValValAspSer
325330335
ThrThrPheLysIleSerLysThrIleAlaAlaAlaGluValThrVal
340345350
GluProGlyAlaIleArgGluLeuHisTrpHisProThrGlnAspGlu
355360365
TrpSerPhePheIleGluGlyGluGlyArgMetThrIlePheAlaSer
370375380
GlnSerAsnAlaArgThrPheAsnTyrGlnAlaGlyAspIleGlyTyr
385390395400
ValProAlaThrMetGlyHisTyrLeuGluAsnThrGlyAsnThrThr
405410415
LeuArgPheLeuGluIlePheLysSerGluLysPheGlnAspIleSer
420425430
LeuAlaGlnTrpLeuAlaLeuThrProProLysLeuValLysGluHis
435440445
LeuGlyPheSerAspAspValIleAlaArgLeuSerLysThrLysLeu
450455460
ThrValValGlyProLys
465470
<210>3
<211>389
<212>PRT
<213>artificial sequence
<400>3
MetGlnLysLysSerLysPhePheLeuGlyLeuLeuGlyValIleThr
151015
CysPheValLeuIleGlySerPheCysLeuProSerLeuAlaGlnThr
202530
GlnThrTrpArgSerLeuSerAsnValValTrpGlyLysAspLeuPro
354045
AlaPheSerTyrProPheSerLysThrProLeuValAspTyrAspGly
505560
GlyValThrLysGlnValGlyThrTyrAsnPheProValSerLysGly
65707580
MetAlaGlyValTyrMetThrLeuLysProGlyAlaIleArgGluLeu
859095
HisTrpHisAlaAsnAlaAlaGluTrpAlaTyrValIleGluGlyArg
100105110
ThrArgValThrLeuThrAsnProAspGlyGlnValGlnIleAlaAsp
115120125
ValAspGlnGlyGlyLeuTrpTyrPheProArgGlyTrpGlyHisSer
130135140
IleGluGlyIleGlyProGlyThrAlaLysPheLeuLeuValPheAsn
145150155160
AspGlyThrPheSerGluGlyAlaThrPheSerIleThrAspTrpLeu
165170175
SerHisThrProIleSerTrpValGlnGlnAsnPheGlyTrpSerGln
180185190
AspGluValGluLysLeuProLysLysGlnValTyrIleSerArgTyr
195200205
AsnProGluValLysProLeuAspLysThrGlnSerArgAsnProLys
210215220
ValSerArgIleValLeuProTyrThrHisAsnLeuLeuAlaGluLys
225230235240
ProArgThrSerGlnAlaGlyAsnThrLeuLysLeuAlaSerAlaLys
245250255
GluPheProAlaSerPheAsnMetAlaGlyAlaLeuLeuArgLeuGlu
260265270
ProGlyAlaMetArgGlnLeuHisTrpHisProAsnAlaAspGluTrp
275280285
GlnTyrValLeuAsnGlySerMetAspLeuAlaValPheAlaSerGlu
290295300
GlyLysAlaSerMetSerArgLeuGlnLysGlyAspValGlyTyrVal
305310315320
ProLysGlyTyrGlyHisAlaLeuArgAsnSerSerAspGlnProLeu
325330335
AspValLeuIleValPheAsnAspGlyAspTyrGlnSerIleAspLeu
340345350
AsnAspTrpIleMetSerAsnProAsnThrValLeuAspAspValPhe
355360365
GlnLeuSerProGlnLeuLeuAspLysLeuProLysGluSerGluIle
370375380
LeuIleProArgSer
385
<210>4
<211>394
<212>PRT
<213>artificial sequence
<400>4
MetValAsnSerValIleGlyTrpLeuArgArgArgPheLeuLeuVal
151015
GlyLeuSerValLeuLeuIleThrPheLeuGlyIlePheThrProThr
202530
IleAlaGlnSerGluGlnTrpArgSerLeuSerAsnValValTrpGly
354045
LysAspLeuProAlaPheThrTyrAlaPheSerLysThrProLeuVal
505560
LeuTyrAspGlyGlyThrThrLysGlnValGlyThrTyrAsnPhePro
65707580
ValSerLysGlyMetAlaGlyValTyrMetSerLeuGluProGlyAla
859095
IleArgGluLeuHisTrpHisAlaAsnAlaAlaGluTrpAlaTyrVal
100105110
MetGluGlyArgThrArgIleThrLeuThrSerProGluGlyLysVal
115120125
GluIleAlaAspValAspLysGlyGlyLeuTrpTyrPheProArgGly
130135140
TrpGlyHisSerIleGluGlyIleGlyProAspThrAlaLysPheLeu
145150155160
LeuValPheAsnAspGlyThrPheSerGluGlyAlaThrPheSerVal
165170175
ThrAspTrpLeuSerHisThrProIleAlaTrpValGluGluAsnLeu
180185190
GlyTrpThrAlaAlaGlnValAlaGlnLeuProLysLysGlnValTyr
195200205
IleSerSerTyrGlyProAlaSerGlyProLeuAlaSerAlaThrPro
210215220
GlnGlyGlnThrAlaLysIleGluValProHisThrHisAsnLeuLeu
225230235240
GlyGlnGlnProLeuValSerLeuGlyGlyAsnGluLeuArgLeuAla
245250255
SerAlaLysGluPheProGlySerPheAsnMetThrGlyAlaLeuIle
260265270
HisLeuGluProGlyAlaMetArgGlnLeuHisTrpHisProAsnAla
275280285
AspGluTrpGlnTyrValLeuAspGlyGluMetAspLeuThrValPhe
290295300
AlaSerGluGlyLysAlaSerValSerArgLeuGlnGlnGlyAspVal
305310315320
GlyTyrValProLysGlyTyrGlyHisAlaIleArgAsnSerSerGln
325330335
LysProLeuAspIleValValValPheAsnAspGlyAspTyrGlnSer
340345350
IleAspLeuSerThrTrpLeuAlaSerAsnProSerSerValLeuGly
355360365
AsnThrPheGlnIleSerProGluLeuThrLysLysLeuProValGln
370375380
AspThrIlePheSerLeuProThrGlnPro
385390
<210>5
<211>455
<212>PRT
<213>artificial sequence
<400>5
MetGlyLysPheLeuAlaThrValLeuCysAlaValLeuTyrGlySer
151015
LeuAlaAlaAlaIleProValGlyAspValSerAlaSerSerSerAla
202530
SerSerSerIleAlaGluAlaAlaThrSerThrSerGlyGlyAlaSer
354045
ProSerProThrValProLeuAlaSerAspAspProAsnTyrTrpLeu
505560
TrpAsnGluThrThrThrThrAspProGlnProGluArgGlySerLeu
65707580
GlyAlaAsnIleLeuGlyProGlnAsnValAlaIleAspLysGlnAsn
859095
ProAspIleLeuAlaProProThrThrAspGlnGlyThrValGlyAsn
100105110
AlaLysTrpProPheSerLeuSerLysGlnGlnLeuAsnThrGlyGly
115120125
TrpValArgGlnGlnAsnValGlnGlnMetProIleAlaThrAlaMet
130135140
AlaGlyValAsnMetArgLeuGluSerGlyAlaIleArgGluLeuHis
145150155160
TrpHisGlnThrAlaGluTrpAlaTyrValLeuSerGlySerThrGln
165170175
IleSerSerValAspGlnLeuGlyArgAsnTyrValAlaThrValArg
180185190
GlnGlyAspLeuTrpTyrPheProProGlyIleProHisSerLeuGln
195200205
AlaThrAsnAspSerSerGluGlyThrGluPheLeuLeuIlePhePro
210215220
AspGlyAsnPheAsnAspAspAspThrLeuLeuLeuThrAspTrpLeu
225230235240
AlaHisThrProLysGluValIleAlaLysAsnPheGlnAspAsnIle
245250255
AlaAspTrpAspAspIleProGlySerGlnLeuTyrIlePheProGly
260265270
ValProProProAspAsnGlnGlnProProThrSerProAlaGlyGlu
275280285
IleProGlnProPheSerTyrAlaPheSerGluIleThrProThrGln
290295300
TyrThrGlyGlyThrAlaLysIleAlaAspSerThrThrPheLysVal
305310315320
AlaThrLysIleAlaValAlaGluValThrValGluProGlyAlaMet
325330335
ArgGluMetHisTrpHisProThrGlnSerGluTrpGlyPhePheLeu
340345350
GluGlyThrAlaArgValThrLeuPheAlaGlyThrAlaIleAlaGln
355360365
ThrPheAspTyrGlnProGlyAspIleSerTyrIleProThrAlaTyr
370375380
GlyHisTyrValGluAsnThrGlyAsnThrThrLeuLysPheLeuGlu
385390395400
IlePheAsnSerAspValPheGlnAspValSerLeuAlaGlnTrpLeu
405410415
AlaLeuThrProProAlaLeuValLysGlnHisLeuGlnLeuSerAsp
420425430
AlaThrIleSerArgPheAsnArgThrLysGlyValValValGlyGly
435440445
ProGlyAlaAsnValSerSer
450455
<210>6
<211>447
<212>PRT
<213>artificial sequence
<400>6
MetPheAsnAsnPheGlnArgLeuLeuThrValIleLeuLeuSerGly
151015
PheThrAlaGlyValProLeuAlaSerThrThrThrGlyThrGlyThr
202530
AlaThrGlyThrSerThrAlaAlaGluProSerAlaThrValProPhe
354045
AlaSerThrAspProAsnProValLeuTrpAsnGluThrSerAspPro
505560
AlaLeuValLysProGluArgAsnGlnLeuGlyAlaThrIleGlnGly
65707580
ProAspAsnLeuProIleAspLeuGlnAsnProAspLeuLeuAlaPro
859095
ProThrThrAspHisGlyPheValGlyAsnAlaLysTrpProPheSer
100105110
PheSerLysGlnArgLeuGlnThrGlyGlyTrpAlaArgGlnGlnAsn
115120125
GluValValLeuProLeuAlaThrAsnLeuAlaCysThrAsnMetArg
130135140
LeuGluAlaGlyAlaIleArgGluLeuHisTrpHisLysAsnAlaGlu
145150155160
TrpAlaTyrValLeuLysGlySerThrGlnIleSerAlaValAspAsn
165170175
GluGlyArgAsnTyrIleSerThrValGlyProGlyAspLeuTrpTyr
180185190
PheProProGlyIleProHisSerLeuGlnAlaThrAlaAspAspPro
195200205
GluGlySerGluPheIleLeuValPheAspSerGlyAlaPheAsnAsp
210215220
AspGlyThrPheLeuLeuThrAspTrpLeuSerHisValProMetGlu
225230235240
ValIleLeuLysAsnPheArgAlaLysAsnProAlaAlaTrpSerHis
245250255
IleProAlaGlnGlnLeuTyrIlePheProSerGluProProAlaAsp
260265270
AsnGlnProAspProValSerProGlnGlyThrValProLeuProTyr
275280285
SerPheAsnPheSerSerValGluProThrGlnTyrSerGlyGlyThr
290295300
AlaLysIleAlaAspSerThrThrPheAsnIleSerValAlaIleAla
305310315320
ValAlaGluValThrValGluProGlyAlaLeuArgGluLeuHisTrp
325330335
HisProThrGluAspGluTrpThrPhePheIleSerGlyAsnAlaArg
340345350
ValThrIlePheAlaAlaGlnSerValAlaSerThrPheAspTyrGln
355360365
GlyGlyAspIleAlaTyrValProAlaSerMetGlyHisTyrValGlu
370375380
AsnIleGlyAsnThrThrLeuThrTyrLeuGluValPheAsnThrAsp
385390395400
ArgPheAlaAspValSerLeuSerGlnTrpLeuAlaLeuThrProPro
405410415
SerValValGlnAlaHisLeuAsnLeuAspAspGluThrLeuAlaGlu
420425430
LeuLysGlnPheAlaThrLysAlaThrValValGlyProValAsn
435440445
<210>7
<211>440
<212>PRT
<213>artificial sequence
<400>7
MetArgArgGlnLeuValThrArgLeuGlnSerLeuValValAlaAla
151015
ValCysAlaAlaSerValThrAlaIleProLeuAlaProSerLeuThr
202530
GluSerAlaProAlaTyrProSerProThrValProTyrAlaThrAsp
354045
AspProAsnArgGluLeuTrpAsnProLeuSerAsnValAspProGln
505560
ProIleArgGlyThrLeuGlyAlaAspIleIleAlaGlnGlnAsnVal
65707580
ProLeuGlnLeuGlnAsnSerAspLeuLeuAlaProProThrThrAsp
859095
HisGlySerValProAsnIleLysTrpProPheThrLeuSerHisAsn
100105110
ArgLeuHisThrGlyGlyTrpAlaArgGlnGlnAsnIleHisAspLeu
115120125
ProIleSerThrGluMetAlaGlyValAspMetArgLeuGluAlaGly
130135140
AlaIleArgGluLeuHisTrpHisThrAlaAlaGluTrpAlaTyrVal
145150155160
LeuLysGlySerThrGlnValSerThrValThrProAspGlyGlnAsn
165170175
TyrValAlaThrAlaAsnGlnGlyAspLeuTrpTyrPheProProGly
180185190
GlnProHisSerLeuGlnAlaThrAlaGlnAspProAspGlyThrGlu
195200205
PheLeuLeuValPheAspAsnGlyGluPheSerGluAspSerThrPhe
210215220
LeuLeuThrAspTrpLeuAlaHisValProLysGluValLeuValArg
225230235240
AsnPheGlnAlaThrLysSerAlaPheAspHisIleProAspArgGlu
245250255
LeuTyrIlePheProGlyValProProAspProAsnAlaGlnProPro
260265270
SerSerProGlnGlyGlnThrProLeuProTyrThrPheProLeuSer
275280285
GlnValGluAlaThrLysPheProGlyGlyThrThrLysIleValAsp
290295300
SerThrThrPheLysValSerLysThrMetAlaValAlaGluValThr
305310315320
LeuGluProGlyAlaMetArgGluLeuHisTrpHisProThrGlnThr
325330335
GluTrpAspTyrPheMetSerGlyTyrAlaArgValThrValPheAla
340345350
AlaAsnAlaAspAlaArgThrPheAspPheGlnAlaGlyAspIleGly
355360365
TyrIleProGlnSerTyrGlyHisTyrIleGluAsnThrGlyAsnThr
370375380
ThrLeuHisPheLeuGluIleLeuLysThrAspIleAspLysPheGln
385390395400
AspValSerLeuAlaGlnTrpLeuAlaLeuThrProProAlaValVal
405410415
LysAlaHisLeuAspValSerAspAspThrIleAlaAlaPheSerLys
420425430
ThrLysGlnArgIleValGlyLys
435440
<210>8
<211>398
<212>PRT
<213>artificial sequence
<400>8
MetLysLysArgThrValAsnGluAlaGlyArgAsnValProGlnPro
151015
IleArgSerAspGlyAlaGlyAlaIleAspSerGlyProArgAsnVal
202530
MetArgAspIleGlnAsnProAsnMetLeuValProProIleThrAsp
354045
AlaGlyLeuValProAsnLeuLysPheSerPheSerAspThrSerMet
505560
IleLeuLysGlnGlyGlyTrpSerArgGluIleThrAlaArgGluLeu
65707580
ProValSerThrThrIleAlaGlyValAsnMetSerLeuThrAlaGly
859095
GlyValArgGluLeuHisTrpHisLysGluAlaGluTrpAlaTyrMet
100105110
LeuLeuGlyArgAlaArgIleThrAlaValAspGlnAsnGlyArgAsn
115120125
PheIleAlaAspValGlyProGlyAspLeuTrpTyrPheProProGly
130135140
IleProHisSerIleGlnGlyLeuGluHisCysGluPheLeuLeuVal
145150155160
PheAspAspGlyHisPheSerAspLeuSerThrLeuAlaIleSerAsp
165170175
TrpPheAlaHisThrProLysGluValLeuSerAlaAsnPheGlyVal
180185190
ProGluSerValPheArgSerLeuProSerAspGlnValTyrIleTyr
195200205
GlnGlyGluValProGlySerLeuGluSerGlnGluValGlnSerPro
210215220
LysGlyGluValProLeuThrPheLysHisGluLeuLeuLysGlnLys
225230235240
ProValLysThrProGlyGlySerValArgIleValAspSerThrAsn
245250255
PheProIleSerLysThrIleAlaAlaAlaLeuValGluValGluPro
260265270
GlyGlyMetArgGluLeuHisTrpHisProAsnAsnAspGluTrpGln
275280285
TyrTyrLeuThrGlyGluAlaArgMetThrValPheLeuGlyAsnGly
290295300
ThrAlaArgThrPheAspTyrArgAlaGlyAspValGlyTyrValPro
305310315320
PheAlaThrGlyHisTyrIleGlnAsnThrGlyThrGluThrLeuTrp
325330335
PheLeuGluMetPheArgSerAsnArgPheGluAspValSerLeuAsn
340345350
GlnTrpMetAlaLeuThrProLysGluIleValGluSerAsnIleHis
355360365
ValGlyProGlnValMetAspSerLeuArgLysGluLysTrpProVal
370375380
ValLysTyrProGlyPheSerTyrSerProLysSerAspGlu
385390395
<210>9
<211>394
<212>PRT
<213>artificial sequence
<400>9
MetLysArgGlyAspAsnValLysProLeuLysGlyAsnProAsnIle
151015
ProGlnProIleArgAlaAspGlyAlaGlyGlyValAspArgGlyPro
202530
ArgAsnLeuMetArgAspLeuGlnAsnProAsnIleLeuValProPro
354045
GluThrAspArgGlyLeuIleProAsnLeuArgPheSerPheSerAsp
505560
AlaHisMetGlnLeuAsnHisGlyGlyTrpSerArgGluIleThrGln
65707580
ArgAspLeuProIleAlaThrThrLeuAlaGlyValAsnMetSerLeu
859095
ThrProGlyGlyValArgGluLeuHisTrpHisLysGlnAlaGluTrp
100105110
SerTyrMetLeuLeuGlyHisAlaArgIleThrAlaValAspGlnAsn
115120125
GlyArgAsnPheIleAlaAspValGlyProGlyAspLeuTrpTyrPhe
130135140
ProProGlyIleProHisSerIleGlnGlyLeuAspAspGlyCysGlu
145150155160
PheLeuLeuValPheAspAspGlyMetPheSerAspLeuSerThrLeu
165170175
SerLeuSerAspTrpMetAlaHisThrProLysAspValLeuSerAla
180185190
AsnPheGlyValProGluSerValPheAlaThrIleProThrGluGln
195200205
ValTyrIleTyrGlnAspGluValProGlyProLeuGlnSerGlnGln
210215220
IleAsnSerProTyrGlyAlaValProGlnThrPheLysHisGluLeu
225230235240
LeuLysGlnProProLeuValThrProGlyGlySerValArgIleVal
245250255
AspSerArgAsnPheProValSerLysThrIleAlaAlaAlaLeuVal
260265270
GluValGluProGlyAlaMetArgGluMetHisTrpHisProAsnAsn
275280285
AspGluTrpGlnTyrTyrLeuThrGlyGlnAlaArgMetThrValPhe
290295300
ThrGlyAsnGlyValAlaArgThrPheAspTyrArgAlaGlyAspVal
305310315320
GlyTyrValProPheAlaThrGlyHisTyrIleGlnAsnThrGlyAsn
325330335
GluSerValTrpPheLeuGluMetPheLysSerAspArgPheGluAsp
340345350
ValSerLeuAsnGlnTrpLeuAlaLeuThrProThrGluLeuValGln
355360365
HisAsnIleHisValAspSerLysPheThrAsnLysLeuArgLysGlu
370375380
LysTrpProValValLysTyrProThrIle
385390
<210>10
<211>472
<212>PRT
<213>artificial sequence
<400>10
MetLysProSerThrLeuTyrSerSerLeuProTrpValIleThrSer
151015
LeuLeuThrValAlaValHisGlyAlaProThrGlyThrLysSerAsn
202530
ProProLeuArgGlySerGluAsnLeuLeuGlyTyrSerAlaSerAsn
354045
ThrValThrAspGlnSerThrAspGluIleProTyrValProValPro
505560
GlyGlnThrAspAlaAlaAspLeuGlyValTyrLeuAspPheGluAsp
65707580
IleGluAsnProGlnProValArgGlySerThrGlyGlyThrAspPro
859095
GlyProArgAsnAspTyrTyrAspArgIleAsnSerAspLysLeuAla
100105110
ProProGlyThrAspAsnGlyGlnThrIleAsnAlaGlnTrpProMet
115120125
GlyLeuSerHisAsnArgLeuGlyLeuAsnGluSerGlyTrpAlaArg
130135140
GlnGluAsnGluValValMetProGlyAlaThrGluMetAlaGlyVal
145150155160
AspMetArgLeuGluAlaGlyAlaTyrArgGluLeuHisTrpHisVal
165170175
AlaSerGluTrpSerLeuValLeuAsnGlySerCysArgIleGluAla
180185190
ValAsnGluAsnGlyGlnThrPheValAspAspValSerAlaGlyAsp
195200205
ValTrpPhePheProProGlyValProHisSerIleGlnAlaLeuAsp
210215220
SerGlyValGluPheLeuLeuIlePheAspAspGlySerPheSerGlu
225230235240
AspAsnThrPheLeuAlaThrGluValPheAlaHisGlnProArgGlu
245250255
ValLeuAlaLysAsnPheAspLeuProValAlaAlaPheAspAspIle
260265270
ProGluAspGluLeuTyrIlePheProGlyThrProAlaProGlnAsn
275280285
IleGluGluGlnAsnValThrGlySerAlaGlyValLeuProLysSer
290295300
GlnSerTyrSerTyrHisPheSerGluGlnProAlaHisGluValGln
305310315320
GlyGlySerValLysIleValAspSerLeuThrPheProIleSerThr
325330335
AsnThrAlaAlaAlaLeuValThrValHisProGlyGlyMetArgGlu
340345350
IleHisTrpHisProSerSerAspGluTrpThrPhePheIleSerGly
355360365
LysAlaArgAlaThrLeuPheThrAlaProSerThrAlaThrThrPhe
370375380
AspTyrArgProGlyAspValGlyTyrPheProGlnSerAsnSerHis
385390395400
TyrIleGluAsnThrGlyAspGluAspLeuValPheLeuGluValLeu
405410415
GlnThrGluGlnPheSerAspIleSerLeuGlyGlnTrpIleGlySer
420425430
ThrProLysGlnIleValSerAspThrLeuAsnLeuProGlnSerAla
435440445
LeuAspArgLeuLysThrGluLysMetTyrValValAlaGlySerAsn
450455460
GluThrAspValAlaAlaThrAla
465470
<210>11
<211>455
<212>PRT
<213>artificial sequence
<400>11
MetIleArgLeuSerSerCysLeuCysAlaLeuLeuLeuAlaThrPhe
151015
AlaAlaAlaAlaProAlaAlaSerAspSerAlaSerSerValProAla
202530
SerIleSerSerGluHisProSerSerThrValLysProThrGlyThr
354045
ThrThrGlySerThrValProAlaSerGluThrValProLeuIlePro
505560
LeuAspProAsnPheProLeuTrpAsnGluSerThrThrValLysPro
65707580
AspAlaIleArgGlySerLeuGlyAlaHisValLeuGlyProThrAsn
859095
GluProIleAspLysGlnAsnProAspPheLeuAlaProProThrThr
100105110
AspHisGlySerLeuProAsnAlaLysTrpProPheSerLeuSerHis
115120125
AsnArgLeuGlnThrGlyGlyTrpAlaArgGlnGluAsnThrGlyVal
130135140
MetProIleAlaGluGlnMetSerSerValAsnMetArgLeuGluPro
145150155160
GlyAlaValArgGluLeuHisTrpHisLysThrAlaGluTrpAlaTyr
165170175
ValLeuLysGlyThrThrGlnIleAlaAlaThrAspProAsnGlyArg
180185190
AsnTyrValAlaAsnValGluProGlyAspLeuTrpTyrPheProAla
195200205
GlyThrProHisSerLeuGlnAlaThrGlyAspAsnProGluGlySer
210215220
GluPheIleLeuValPheAspValGlyAspPheSerGluAspSerThr
225230235240
PheLeuLeuThrAspTrpLeuAlaHisValProValGluValLeuAla
245250255
LysAsnPheGlnValAspProGluAlaPheLysThrValProAlaGlu
260265270
GluLeuTyrIlePheProAlaAsnProProGlnThrGluAspAlaPro
275280285
LysSerProGlnGlyThrValGluAsnProPheSerPheProPheSer
290295300
LysValLysGluThrGlnLeuGluGlyGlySerValLysValValAsp
305310315320
SerThrThrPheLysIleSerLysThrIleAlaAlaAlaGluValThr
325330335
ValGluProGlyAlaMetArgGluLeuHisTrpHisProThrGlnAsp
340345350
GluTrpSerPhePheLeuSerGlyAsnAlaArgValThrIlePheAla
355360365
AlaGlnSerAsnAlaArgThrPheAspTyrGlnAlaGlyAspIleGly
370375380
TyrValProGlyAlaMetGlyHisTyrValGluAsnThrGlyAsnThr
385390395400
ThrLeuArgPheLeuGluIlePheArgAspAspValPheGlnAspVal
405410415
SerLeuAsnGlnTrpLeuAlaLeuThrProProGluLeuValLysAla
420425430
HisLeuGlyPheSerAspGluValIleSerLysLeuThrLysLysLys
435440445
LysThrValValGlyProAla
450455
<210>12
<211>45
<212>DNA
<213>artificial sequence
<400>12
ctaatacgactcactatagggcaagcagtggtatcaacgcagagt45
<210>13
<211>22
<212>DNA
<213>artificial sequence
<400>13
acgaggaagctgtcacttggca22
<210>14
<211>23
<212>DNA
<213>artificial sequence
<400>14
agtgccaagtgacaagcttcctc23
<210>15
<211>35
<212>DNA
<213>artificial sequence
<400>15
ggagatatacatatggtcccattggcatccactac35
<210>16
<211>35
<212>DNA
<213>artificial sequence
<400>16
aattcggatccattagttaactggaccaacaacag35
<210>17
<211>23
<212>DNA
<213>artificial sequence
<400>17
taatggatccgaattcgagctcc23
<210>18
<211>24
<212>DNA
<213>artificial sequence
<400>18
catatgtatatctccttcttaaag24
<210>19
<211>59
<212>DNA
<213>artificial sequence
<400>19
gaggaagaagaggaggaagaggaggaggaagaagaataatggatccgaattcgagctcc59
<210>20
<211>24
<212>DNA
<213>artificial sequence
<400>20
catatgtatatctccttcttaaag24
Claims (11)
1. an oxalate decarboxylase, it is characterised in that: for being undertaken recombinant expressed by escherichia expression system, and stablize when pH < 3.0 and have vigor；The protein sequence of this oxalate decarboxylase more primary pheron sequence deletion signal peptide fragment in expression process。
2. an edible composition, it is characterized in that, containing the oxalate decarboxylase described in claim 1, for preventing and/or treat primary hyperoxaluria, II type hyperoxaluria, intestinal source property hyperoxaluria and containing one or more in calcium oxalate urinary calculi disease。
3. edible composition according to claim 2, it is characterised in that for pharmaceutical composition, health food or special medicine purposes formula food。
4. a food additive, it is characterised in that containing the oxalate decarboxylase described in claim 1, for preparing low oxalic acid/without the Foods or drinks of oxalic acid。
5. the recombinant expression method of an oxalate decarboxylase, the Expression product of still great-hearted oxalate decarboxylase during suitable in pH < 3.0, it is characterized in that, the DNA fragmentation of oxalate decarboxylase encoding gene after optimization is attached according to the mixed in molar ratio of 3～9:1 with coli expression carrier, building recombinant expression carrier, recombinant expression carrier converts DH5a competent cell；Being screened the recombinant expression carrier transformed bacteria successfully constructed by PCR and sequence verification, extract recombinant expression carrier, convert the E. coli expression strains containing molecular chaperones after cultivation, the Low-temperature culture the induced protein that carry out 15～30 DEG C are expressed。
6. the recombinant expression method of oxalate decarboxylase according to claim 5, it is characterised in that described optimization includes the signal peptide fragment deleting oxalate decarboxylase encoding gene DNA fragmentation；Or delete the signal peptide fragment of oxalate decarboxylase encoding gene DNA fragmentation and merge hydrotropy label at C end；Or delete the signal peptide fragment of oxalate decarboxylase encoding gene DNA fragmentation and merge hydrotropy label at C end and carry out codon optimized；Or oxalate decarboxylase encoding gene DNA fragmentation is carried out codon optimized and erasure signal fragments of peptides。
7. the recombinant expression method of oxalate decarboxylase according to claim 6, it is characterized in that, described hydrotropy label is polyglutamic acid fragment, many poly-aspartates fragment, poly-D-lysine fragment, any one in poly glumine fragment and many poly-asparagines fragment, or the fragment by glutamic acid, aspartic acid, agedoite, lysine and glutamine five seed amino acid random combine。
8. the recombinant expression method of oxalate decarboxylase according to claim 5, it is characterised in that described expression vector is lactose-induced expression vector, galactose inducible expression carrier, IPTG inducible expression carrier, thermal induction expression vector or cold inducible expression carrier。
9. the recombinant expression method of oxalate decarboxylase according to claim 5, it is characterized in that, described E. coli expression strains is BL21, BL21 (DE3), K12, K12 (DE3), Origami, Origami (DE3), OrigamiB, OrigamiB (DE3), Rosetta or Rosetta (DE3)。
10. the recombinant expression method of oxalate decarboxylase according to claim 5, it is characterised in that described molecular chaperones be in pG-KJE8, pGro7, pKJE7, pGTf2 and pTf16 any one or several。
11. oxalate decarboxylase according to claim 1, it is characterised in that shown in aminoacid sequence such as SEQIDNO:1～11 are arbitrary。
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106418120A (en) * | 2016-07-13 | 2017-02-22 | 武汉康复得生物科技股份有限公司 | Low-oxalic-acid food or beverage and preparation method thereof |
| CN107868776A (en) * | 2016-09-23 | 2018-04-03 | 武汉康复得生物科技股份有限公司 | Glycosylate oxalate decarboxylase and its preparation and application |
| CN108977455A (en) * | 2018-08-01 | 2018-12-11 | 武汉康复得生物科技股份有限公司 | For producing the recombinant plasmid, escherichia expression system and methods and applications of oxalate decarboxylase |
| CN108998462A (en) * | 2018-08-01 | 2018-12-14 | 武汉康复得生物科技股份有限公司 | The escherichia expression system and its application method of the recombinant protein containing manganese ion |
| CN110656077A (en) * | 2019-11-07 | 2020-01-07 | 江南大学 | Method for producing sucrose phosphorylase and application thereof |
| JP2021514679A (en) * | 2017-03-07 | 2021-06-17 | 武漢康復得生物科技股▲ふん▼有限公司 | Recombinant oxalate decarboxylase expressed by filamentous fungal host cells |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101918551A (en) * | 2008-01-04 | 2010-12-15 | 天野酶株式会社 | Recombinant expression plasmid vector and recombinant strain to be used in producing oxalate decarboxylase, and method of producing recombinant oxalate decarboxylase |
| CN102725403A (en) * | 2009-11-25 | 2012-10-10 | 凯普托杰姆有限责任公司 | Methods and compositions for treating oxalate-related conditions |
| CN102732542A (en) * | 2012-05-22 | 2012-10-17 | 江苏省农业科学院 | Bacillus subtilis Bs-916 oxalate decarboxylase gene sequence and applications thereof |
-
2016
- 2016-04-08 CN CN201610217032.6A patent/CN105695441A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101918551A (en) * | 2008-01-04 | 2010-12-15 | 天野酶株式会社 | Recombinant expression plasmid vector and recombinant strain to be used in producing oxalate decarboxylase, and method of producing recombinant oxalate decarboxylase |
| CN102725403A (en) * | 2009-11-25 | 2012-10-10 | 凯普托杰姆有限责任公司 | Methods and compositions for treating oxalate-related conditions |
| CN102732542A (en) * | 2012-05-22 | 2012-10-17 | 江苏省农业科学院 | Bacillus subtilis Bs-916 oxalate decarboxylase gene sequence and applications thereof |
Non-Patent Citations (6)
| Title |
|---|
| COPELAND, A., ET AL.: "Genbank accession number:ANN58418.1", 《GENBANK》 * |
| KESARWANI,M., ET AL.: "Genbank accession number:AAF13275.1", 《GENBANK》 * |
| SUN-KI KIM, ET AL.: "Simple Amino Acid Tags Improve Both Expression and Secretion of Candida antarctica Lipase B in Recombinant Escherichia coli", 《BIOTECHNOLOGY AND BIOENGINEERING》 * |
| 于俊杰 等: "枯草芽孢杆菌Bs-916草酸脱羧酶基因Bacisubin 的克隆、原核表达及其表达产物的酶活性分析", 《江苏农业学报》 * |
| 李江姣 等: "辛德毕斯病毒E2包膜蛋白可溶性表达条件的优化", 《中国生物工程杂志》 * |
| 沈裕虎: "农杆菌草酸脱羧酶的原核表达与分泌特性验证", 《中国博士学位论文全文数据库基础科学辑》 * |
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106418120A (en) * | 2016-07-13 | 2017-02-22 | 武汉康复得生物科技股份有限公司 | Low-oxalic-acid food or beverage and preparation method thereof |
| CN107868776A (en) * | 2016-09-23 | 2018-04-03 | 武汉康复得生物科技股份有限公司 | Glycosylate oxalate decarboxylase and its preparation and application |
| JP2021514679A (en) * | 2017-03-07 | 2021-06-17 | 武漢康復得生物科技股▲ふん▼有限公司 | Recombinant oxalate decarboxylase expressed by filamentous fungal host cells |
| JP2022110110A (en) * | 2017-03-07 | 2022-07-28 | 武漢康復得生物科技股▲ふん▼有限公司 | Recombinant oxalate decarboxylase expressed in filamentous fungal host cell |
| CN108977455A (en) * | 2018-08-01 | 2018-12-11 | 武汉康复得生物科技股份有限公司 | For producing the recombinant plasmid, escherichia expression system and methods and applications of oxalate decarboxylase |
| CN108998462A (en) * | 2018-08-01 | 2018-12-14 | 武汉康复得生物科技股份有限公司 | The escherichia expression system and its application method of the recombinant protein containing manganese ion |
| CN108998462B (en) * | 2018-08-01 | 2021-06-18 | 武汉康复得生物科技股份有限公司 | Escherichia coli expression system of manganese ion-containing recombinant protein and application method thereof |
| CN108977455B (en) * | 2018-08-01 | 2021-07-20 | 武汉康复得生物科技股份有限公司 | Recombinant plasmid for producing oxalate decarboxylase, escherichia coli expression system, method and application |
| CN110656077A (en) * | 2019-11-07 | 2020-01-07 | 江南大学 | Method for producing sucrose phosphorylase and application thereof |
| CN110656077B (en) * | 2019-11-07 | 2021-10-08 | 江南大学 | Method for producing sucrose phosphorylase and application thereof |
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