CN105543187B

CN105543187B - Fasted bacillus uricase mutant V145A

Info

Publication number: CN105543187B
Application number: CN201610093491.8A
Authority: CN
Inventors: 廖飞; 冯涛; 杨晓兰; 胡小蕾; 景一娴; 饶菁菁; 廖娟
Original assignee: Chongqing Medical University
Current assignee: Chongqing Medical University
Priority date: 2016-02-18
Filing date: 2016-02-18
Publication date: 2020-07-10
Anticipated expiration: 2036-02-18
Also published as: CN105543187A

Abstract

The fastidious bacillus intracellular uricase synergistic mutant A2R/V145A, V145A/Y320R, V145A/Y320H, A2R/V145A/Y320R, A2R/V145A/Y320H; the amino acid sequence of the wild type uricase corresponding to the synergistic mutant is obtained from the Chinese patent CN 101875922; compared with wild type uricase, the heat inactivation processes of the synergistic mutants V145A/Y320R, V145A/Y320H, A2R/V145A/Y320R and A2R/V145A/Y320H do not conform to an exponential decay model under physiological conditions, the activity is basically kept above 90 percent in the initial 6 days of heat inactivation, and the activity is basically exponentially decayed thereafter.

Description

Fasted bacillus uricase mutant V145A

Technical Field

The invention belongs to the technical field of enzyme biology, and discloses a fasted bacillus uricase mutant V145A and a multi-site synergistic mutant thereof, wherein the mutant and the synergistic mutant thereof have improved catalytic activity at pH9.2 or/and thermal stability under physiological conditions compared with wild type fasted bacillus uricase disclosed in Chinese patent Z L200910103741.1.

Background

Uricase can specifically oxidize uric acid into allantoin and hydrogen peroxide, is an important tool enzyme for measuring serum uric acid in the fields of clinical examination and the like, and is also a medicinal enzyme for clinically treating tumor lysis syndrome (T L S), gout and other diseases related to hyperuricemia.

The wild uricase or the mutant thereof with high activity and stability has more important application value.

The wild-type demanding bacillus uricase has good stability and higher activity, and is suitable for serum uric acid determination, but the activity and the thermal stability, especially the thermal stability under physiological conditions, are required to be obviously improved as a therapeutic enzyme.

The invention designs and screens and obtains the demanding bacillus uricase mutant V145A and the multi-site synergistic mutant thereof, the activity of the demanding bacillus uricase mutant is improved compared with that of wild-type demanding bacillus uricase, and the synergistic mutant has higher thermal stability.

Disclosure of Invention

Compared with the wild type fastidious bacillus intracellular uricase disclosed in the Chinese invention patent Z L200910103741.1, the mutant of the invention has better activity or/and thermal stability.

The uricase mutant takes the wild type uricase of bacillus fastidiosa as a parent.

The sequence of the wild type uricase is shown in Chinese patent Z L200910103741.1 with publication number CN 101875922.

The uricase mutant specifically comprises the following six types:

1. mutant V145A, valine 144 of wild-type uricase mutated to alanine.

The amino acid sequence of the mutant V145A is shown in SEQ ID NO. 1.

2. The mutant V145A mutated alanine to arginine at position 2, in synergy mutant A2R/V145A.

The amino acid sequence of the synergistic mutant A2R/V145A is shown as SEQ ID NO. 2.

3. The mutant V145A/Y320R was coordinated, and the 320 th tyrosine was mutated to arginine by the mutant V145A.

The amino acid sequence of the synergistic mutant V145A/Y320R is shown as SEQ ID NO. 3.

4. The mutant V145A/Y320H is synergistic in that the 320 th tyrosine is mutated to histidine by the mutant V145A.

The amino acid sequence of the mutant V145A/Y320H is shown as SEQ ID NO. 4.

5. Synergistic mutant A2R/V145A/Y320R mutated tyrosine 320 to arginine according to synergistic mutant A2R/V145A.

The amino acid sequence of the synergistic mutant A2R/V145A/Y320R is shown as SEQ ID NO. 5.

6. The synergistic mutant A2R/V145A/Y320H mutated tyrosine at position 320 to histidine according to the synergistic mutant A2R/V145A.

The amino acid sequence of the synergistic mutant A2R/V145A/Y320H is shown as SEQ ID NO. 6.

The invention has the beneficial effects that:

table 1 compares the activity and stability under physiological conditions of pH9.2 of wild-type uricase and its mutants

As shown in table 1, the mutant V145A has an activity improved by more than 30% compared with wild-type uricase and has comparable stability under physiological conditions; compared with wild uricase, the activity of the synergistic mutant A2R/V145A is improved by nearly 1 time, and the stability is slightly improved; the activity of the synergistic mutants V145A/Y320R and V145A/Y320H is equivalent to that of the mutant V145A, but the synergistic mutants have a plateau period with basically non-attenuated activity in the initial period of heat inactivation and then are exponentially attenuated; the activity-attenuating plateau period of synergistic mutant V145A/Y320R is about 6 days, at which time the remaining activity of wild-type uricase is about 75% (FIG. 1), and the activity-attenuating plateau period of synergistic mutant V145A/Y320H is about 6 days; the activity of the synergistic mutants A2R/V145A/Y320R and A2R/V145A/Y320H is equivalent to that of the mutant A2R/V145A, but the activity of A2R/V145A/Y320R and A2R/V145A/Y320H is attenuated by about 8 days and 6 days respectively.

Drawings

FIG. 1: synergistic mutant V145A/Y320R and wild type uricase are compared in heat inactivation process under physiological conditions

Detailed Description

The terms used in the present invention have the general meanings of those skilled in the art unless otherwise specified.

Example 1: constructing a recombinant expression vector of the bacillus fastidious uricase series mutant.

Selecting a pseudo-mutation site, entrusting Beijing Zhongmeitai and biotechnology limited company to synthesize a coding gene, constructing an expression vector, and transforming the expression vector into escherichia coli B L21 (DE3) for recombinant expression.

Example 2: expression and purification of Bacillus fastidious uricase mutant.

After the transformed Escherichia coli cells are subjected to amplification culture in L B medium (containing 0.1 g/L kanamycin) for 4-6h, IPTG with the final concentration of 0.24g/M L is added to be cultured at 16 ℃ for 18h, the cells are centrifuged at 8000rpm for 10min at 4 ℃ to collect thalli, the thalli are resuspended in 0.1M Tris-HCl (pH 8.0) buffer solution, the thalli are broken by ultrasound to release intracellular uricase, cell lysate is centrifuged at 4 ℃ and 12000rpm for 10min to collect supernatant, and the collected supernatant is used for purifying uricase mutants.

Separating target protein with DEAE-cellulose anion exchange chromatographic column, wherein the purification steps comprise (1) balancing with 10 times column volume of 0.1M Tris-HCl (pH 8.0) buffer solution overnight, (2) loading the sample at flow rate of 1M L/min, and (3) eluting with high concentration NaCl solution, collecting and detecting enzyme activity step by step, and repeating once ion exchange chromatography when measuring thermal stability and specific activity.

Example 3: and (3) analyzing the activity and stability of the bacillus fastidiosa uricase series mutant.

(1) Activity assay

Definition of enzyme activity units: the amount of enzyme required to oxidize 1. mu. mol of substrate uric acid per minute is one unit of activity.

The enzyme activity determination step comprises using 0.2M borax buffer solution (pH 9.2), determining uric acid concentration of system to 75 μmol, delaying for 30s, time interval of 10s, determining absorption change initial speed within 1 min at 293nm, preheating corresponding buffer solution and uric acid to (25 + -0.5) deg.C, and fixing uric acid extinction coefficient to 11.5(mmol L)^-1·cm)^-1。

(2) Stability analysis

After purification, the uricase solution was concentrated and dialyzed, filtered, added to 0.2M phosphate buffer (pH 7.4 containing 0.1g/ml ampicillin and kanamycin, 0.1mM EDTA, 2mM p-aminobenzamidine dihydrochloride, used after filtration), and the final system was 4.0M L, wherein the volume of the added enzyme solution was not more than 10% of the total system, the sample was placed in a 37 ℃ incubator, after 20 minutes, a superclean bench sample (1st) was taken to measure the activity, and the activity decay process was recorded, as represented in FIG. 1.

Claims

1. Fastidious bacillus uricase mutants, whose amino acid residue positions are described in a manner that they contain the initial amino acid methionine, are recombinantly expressed in escherichia coli as actively soluble homotetramers and the first methionine of each subunit is removed; the amino acid sequence characteristics of the uricase mutant of the fastidious bacillus are as follows:

A. synergistic mutant A2R/V145A: the amino acid sequence is shown as SEQ ID No. 2;

B. synergistic mutant V145A/Y320R: the amino acid sequence is shown as SEQ ID No. 3;

C. synergistic mutant V145A/Y320H: the amino acid sequence is shown as SEQ ID No. 4;

D. synergistic mutant A2R/V145A/Y320R: the amino acid sequence is shown as SEQ ID No. 5;

E. synergistic mutant A2R/V145A/Y320H: the amino acid sequence is shown in SEQ ID No. 6.

2. The bacillus fastidious uricase mutant according to claim 1, characterized as follows: compared with wild type uricase, the activity of the synergistic mutant V145A/Y320R, V145A/Y320H, A2R/V145A/Y320R and A2R/V145A/Y320H in the heat inactivation process under physiological conditions is not exponentially attenuated, and the activity is maintained at the time of more than 90% of the initial value, namely the plateau period is close to 6 days.