CN105543187A - Demanding bacillus uricase mutant V145 A - Google Patents
Demanding bacillus uricase mutant V145 A Download PDFInfo
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- CN105543187A CN105543187A CN201610093491.8A CN201610093491A CN105543187A CN 105543187 A CN105543187 A CN 105543187A CN 201610093491 A CN201610093491 A CN 201610093491A CN 105543187 A CN105543187 A CN 105543187A
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- C12Y107/03—Oxidoreductases acting on other nitrogenous compounds as donors (1.7) with oxygen as acceptor (1.7.3)
- C12Y107/03003—Factor-independent urate hydroxylase (1.7.3.3), i.e. uricase
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Abstract
The invention provides a demanding bacillus uricase mutant V145 A and collaborative mutants A2R/V145A, V145A/Y320R, V145A/Y320H, A2R/V145A/Y320R and A2R/V145A/Y320H thereof. The mutant and parents of the collaborative mutants thereof correspond to wild type uricase from the amino acid sequence of the invention CN101875922 in China. Compared with wild type uricase, activity of the mutant V145A, activity of the collaborative mutant V145A/Y320R and activity of the collaborative mutant V145A/Y320H are improved by about 30%, while activity of the collaborative mutants A2R/V145A, A2R/V145A/Y320R and A2R/V145A/Y320H is improved by about 50%. Stability of the mutant V145A and the collaborative mutant A2R/V145A is equivalent to that of wild type uricase under the physiological condition, and the heat inactivation process conforms to an index attenuation model basically. Under the physiological condition, the heat inactivation process of the collaborative mutants V145A/Y320R, V145A/Y320H, A2R/V145A/Y320R and A2R/V145A/Y320H does not conform to the index attenuation model, activity is kept over 90% basically within 6 days during the initial period of heat inactivation, and thereafter, enzyme activity is of index attenuation basically.
Description
Technical field
The invention belongs to biotechnology of enzymes field, disclose bacillus fastidiosus uriKoxidase mutant V145A and multidigit point works in coordination with mutant; Relative to the uriKoxidase of wild-type bacillus fastidiosus disclosed in Chinese invention patent ZL200910103741.1, this mutant and collaborative mutant thereof the catalytic activity when pH9.2 is or/and the thermostability under physiological condition is improved.
Background technology
UriKoxidase can specific oxidation uric acid be wallantoin and hydrogen peroxide, being the important tool enzyme that the fields such as Clinical Laboratory measure serum uric acid, is also the medicinal enzyme that clinical treatment tumour dissolves syndromes (TLS), gout and other hyperuricemia relative disease.
High reactivity and stable wild-type uriKoxidase or its mutant have prior using value.
Wild-type bacillus fastidiosus uriKoxidase has satisfactory stability and higher activity, is suitable for serum uric acid, but also needs to significantly improve activity and thermostability as treatment enzyme, the thermostability especially under physiological condition.
The present invention designs and screens and obtains bacillus fastidiosus uriKoxidase mutant V145A and multidigit point thereof and work in coordination with mutant, and its specific activity wild-type bacillus fastidiosus uriKoxidase is improved, and collaborative mutant has more high thermal stability simultaneously.
Summary of the invention
Design of the present invention is compared with uriKoxidase in the born of the same parents of wild-type bacillus fastidiosus disclosed in Chinese invention patent ZL200910103741.1, active or/and the better mutant of thermostability.
Described uriKoxidase mutant with bacillus fastidiosus wild-type uriKoxidase for parent.
This wild-type uriKoxidase sequence is shown in Chinese invention patent ZL200910103741.1, publication number CN101875922.
Described uriKoxidase mutant specifically comprises following six kinds:
1, mutant V145A, wild-type uriKoxidase the 144th valine mutation is L-Ala.
Mutant V145A aminoacid sequence is as shown in SEQIDNO.1.
The alanine mutation of the 2nd is arginine according to mutant V145A by 2, collaborative mutant A2R/V145A.
Collaborative mutant A2R/V145A aminoacid sequence is as shown in SEQIDNO.2.
3, collaborative mutant V145A/Y320R, sports arginine according to mutant V145A by the 320th tyrosine.
Collaborative mutant V145A/Y320R aminoacid sequence is as shown in SEQIDNO.3.
4, collaborative mutant V145A/Y320H, sports Histidine according to mutant V145A by the 320th tyrosine.
Mutant V145A/Y320H aminoacid sequence is as shown in SEQIDNO.4.
5, collaborative mutant A2R/V145A/Y320R, sports arginine according to collaborative mutant A2R/V145A by the 320th tyrosine.
Collaborative mutant A2R/V145A/Y320R aminoacid sequence is as shown in SEQIDNO.5.
6, collaborative mutant A2R/V145A/Y320H, sports Histidine according to collaborative mutant A2R/V145A by the tyrosine of the 320th.
Collaborative mutant A2R/V145A/Y320H aminoacid sequence is as shown in SEQIDNO.6.
Beneficial effect of the present invention:
Table 1 compares wild-type uriKoxidase and mutant thereof at the activity of pH9.2 and physiological condition stability inferior
As table 1, mutant V145A improves more than 30% than wild-type uricase activity and physiological condition stability inferior is suitable; Collaborative mutant A2R/V145A activity comparatively wild-type uriKoxidase improves nearly 1 times and stability and slightly improves; Collaborative mutant V145A/Y320R and V145A/Y320H is active suitable with mutant V145A, but it has active substantially unattenuated plateau at the heat inactivation initial stage, is thereafter exponential attenuation; Collaborative about 6 days mutant V145A/Y320R decay of activity plateaus, now wild-type uriKoxidase remaining activity about 75% (accompanying drawing 1), collaborative about 6 days mutant V145A/Y320H decay of activity plateaus; Collaborative mutant A2R/V145A/Y320R and A2R/V145A/Y320H is active suitable with mutant A2R/V145A, but about 8 days and 6 days respectively A2R/V145A/Y320R and A2R/V145A/Y320H decay of activity plateau.
Accompanying drawing explanation
Fig. 1: collaborative mutant V145A/Y320R and wild-type uriKoxidase, under physiological condition, heat inactivation process compares
Embodiment
Term used in the present invention, unless otherwise specified, is generally the general implication of those skilled in the art.
Embodiment 1: the recombinant expression vector of bacillus fastidiosus uriKoxidase series mutants builds.
Choose and intend mutational site, entrust Beijing Sino-U.S. calm and peaceful Bioisystech Co., Ltd composite coding gene, and construction of expression vector, be transformed into carry out in e. coli bl21 (DE3) recombinant expressed.
Embodiment 2: the expression and purification of bacillus fastidiosus uriKoxidase mutant.
By the Bacillus coli cells of conversion after LB substratum (containing 0.1g/L kantlex) enlarged culturing 4-6h, the IPTG adding final concentration 0.24g/mL cultivates 18h in 16 DEG C, and cell collects thalline at 4 DEG C with the centrifugal 10min of 8000rpm.Thalline 0.1MTris-HCl (pH8.0) damping fluid is resuspended, ultrasonic bacterial cell disruption is discharged uriKoxidase in born of the same parents, by cell pyrolysis liquid in 4 DEG C, the centrifugal 10min of 12000rpm collects supernatant.The supernatant collected is used for the purifying of uriKoxidase mutant.
Carry out the separation of target protein with DEAE-cellulose anion displacement chromatography post, purification step is specially: (1) balances: by 0.1MTris-HCl (pH8.0) the damping fluid equilibrate overnight of 10 times of column volumes; (2) loading: the sample of anticipating is with the flow velocity loading of 1mL/min; (3) wash-out: carry out gradient elution by high density NaCl solution, Fraction collection detects enzyme and lives.Primary ions displacement chromatography is all repeated when measuring thermostability and specific activity.
Embodiment 3: bacillus fastidiosus uriKoxidase series mutants Activity and stabill is analyzed.
(1) determination of activity
Unit of enzyme activity defines: the per minute enzyme amount be oxidized needed for 1 μm of ol substrate uric acid is a unit of activity.
Enzyme assay step: with 0.2M borate buffer solution (pH9.2), measure system uric acid concentration to 75 μm ol, time delay 30s, timed interval 10s, measure Change of absorption initial velocity in 1 minute at 293nm.Corresponding damping fluid and uric acid are all preheated to (25 ± 0.5) DEG C, and uric acid optical extinction coefficient is fixed as 11.5 (mmolL
-1cm)
-1.
(2) stability analysis
Concentrated dialysis uriKoxidase enzyme liquid after purifying, bacteriological filtration, add (pH7.4 in 0.2M phosphate buffered saline buffer, containing 0.1g/ml ammonia benzyl mycin and kantlex, the EDTA of 0.1mM, 2mM p-Aminobenzamidine dihydrochloride, uses after bacteriological filtration), end-body is 4.0mL, wherein adds enzyme liquid and amasss and be no more than 10% of total system.37 degree of constant temperature cell culture incubators put into by sample, and after 20 minutes, super clean bench sampling (1st) measures active, and record decay of activity process, representative is as accompanying drawing 1.
Claims (4)
1. bacillus fastidiosus uriKoxidase mutant V145A, its aminoacid sequence as shown in SEQIDNo.1, and describes the position of its amino-acid residue by the mode containing initial amino acid methionine(Met); Compared with the uriKoxidase of wild-type bacillus fastidiosus disclosed in Chinese invention patent ZL200910103741.1 sequence, its 145th amino acids becomes L-Ala A from α-amino-isovaleric acid V, therefore by this bacillus fastidiosus uriKoxidase mutant referred to as mutant V145A; This mutant V145A is recombinant expressed in intestinal bacteria is the same tetramer of active, soluble, and in its active tetramer, first methionine(Met) of each subunit is divested, therefore after expressing, active mutant only contains 322 amino-acid residues.
2., according to bacillus fastidiosus uriKoxidase mutant V145A described in claim 1, there is following collaborative mutant:
A, collaborative mutant A2R/V145A: compared with the aminoacid sequence of mutant V145A, the 2nd amino acids becomes arginine R from L-Ala A, and its complete amino acid sequence position is as shown in SEQIDNo.2;
B, collaborative mutant V145A/Y320R: compared with the aminoacid sequence of mutant V145A, the 320th amino acids becomes arginine R from tyrosine Y, and its complete amino acid sequence is as shown in SEQIDNo.3;
C, collaborative mutant V145A/Y320H: compared with the aminoacid sequence of mutant V145A, the 320th amino acids becomes Histidine H from tyrosine Y, and its complete amino acid sequence is as shown in SEQIDNo.4;
D, collaborative mutant A2R/V145A/Y320R: compared with the aminoacid sequence of mutant V145A, 2nd amino acids becomes arginine R from L-Ala A, 320th amino acids becomes arginine R from tyrosine Y simultaneously, and its complete amino acid sequence is as shown in SEQIDNo.5;
E, collaborative mutant A2R/V145A/Y320H: compared with the aminoacid sequence of mutant V145A, 2nd amino acids becomes arginine R from L-Ala A, 320th amino acids becomes Histidine H from tyrosine Y simultaneously, and its complete amino acid sequence is as shown in SEQIDNo.6.
3., according to bacillus fastidiosus uriKoxidase mutant V145A described in claim 1, compared with wild-type uriKoxidase disclosed in Chinese invention patent ZL200910103741.1, active improve about 30% and stability is suitable.
4., according to the collaborative mutant of bacillus fastidiosus uriKoxidase mutant V145A described in claim 2, feature is as follows:
A, compared with wild-type uriKoxidase, collaborative mutant A2R/V145A activity improves about 50% and stability is suitable;
B, compared with wild-type uriKoxidase, collaborative mutant V145A/Y320R and V145A/Y320H activity all improve about 30%; Activity not exponentially decay in heat inactivation process under these two kinds collaborative mutant physiological conditions, its activity maintains time of initial value more than 90% and plateau all close to 6 days;
C, compared with wild-type uriKoxidase, collaborative mutant A2R/V145A/Y320R and A2R/V145A/Y320H activity all improve about 50%, and activity not exponentially decay under the two physiological condition, all about 6 days its plateaus.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109022282A (en) * | 2018-08-08 | 2018-12-18 | 大连大学 | A kind of screening technique producing marine low temperature urate oxidase bacterial strain |
| WO2021068925A1 (en) * | 2019-10-11 | 2021-04-15 | 上海君实生物医药科技股份有限公司 | Improved uricase and method for treating hyperuricemia using same |
| CN114480320A (en) * | 2022-02-28 | 2022-05-13 | 复旦大学 | Recombinant nocturnal monkey uricase and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101875922A (en) * | 2009-04-30 | 2010-11-03 | 重庆医科大学 | Two uricases from baccilus fastidious and mutants thereof as well as polyethylene glycol modification and application thereof |
-
2016
- 2016-02-18 CN CN201610093491.8A patent/CN105543187B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101875922A (en) * | 2009-04-30 | 2010-11-03 | 重庆医科大学 | Two uricases from baccilus fastidious and mutants thereof as well as polyethylene glycol modification and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| LIAO F.等: "uricase[Bacillus fastidiosus]", 《GENBANK：ACR09749.1》 * |
| 廖娟等: "尿酸酶突变体的高通量筛选方法", 《西北大学学报(自然科学版)》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109022282A (en) * | 2018-08-08 | 2018-12-18 | 大连大学 | A kind of screening technique producing marine low temperature urate oxidase bacterial strain |
| WO2021068925A1 (en) * | 2019-10-11 | 2021-04-15 | 上海君实生物医药科技股份有限公司 | Improved uricase and method for treating hyperuricemia using same |
| CN114502728A (en) * | 2019-10-11 | 2022-05-13 | 上海君实生物医药科技股份有限公司 | Improved uricase and methods for its use in treating hyperuricemia |
| CN114480320A (en) * | 2022-02-28 | 2022-05-13 | 复旦大学 | Recombinant nocturnal monkey uricase and application thereof |
| CN114480320B (en) * | 2022-02-28 | 2024-01-16 | 复旦大学 | Recombinant night monkey uricase and application thereof |
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| CN105543187B (en) | 2020-07-10 |
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