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Pichia guilliermondii uricase mutant

A mutant, uricase technology, applied in the field of enzyme biology, can solve the problem of low solubility

Pending Publication Date: 2021-10-01
CHONGQING UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] MGU is currently the only known uricase with an optimum pH close to neutral, with good stability but low solubility

Method used

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  • Pichia guilliermondii uricase mutant
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  • Pichia guilliermondii uricase mutant

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1: Construction of recombinant expression vectors of Pichia mongoliana uricase series mutants

[0037] Select the pseudo-mutation site, entrust Beijing Zhongmei Taihe Biotechnology Co., Ltd. to synthesize the coding gene, construct an expression vector, transform Escherichia coli BL21 (DE3), and confirm the inserted coding sequence by picking a single clone on a conventional kanamycin plate.

Embodiment 2

[0038] Example 2: Expression and purification of uricase mutants from Pichia pastoris

[0039] Transformed E. coli cells were cultured in LB medium (containing 0.1g / L kanamycin) for 4-6h, added 0.24g / mL IPTG and induced at 16°C for 18h, centrifuged at 8000rpm at 4°C for 10min to collect cells, 0.1M Tris The cells were resuspended in -HCl (pH 8.0) buffer solution, ultrasonically disrupted, and centrifuged at 12,000 rpm for 10 min at 4°C to collect the supernatant as a crude sample of soluble uricase.

[0040] DEAE-cellulose chromatography purification: (1) equilibration: equilibrate with 10 times the column volume of 0.1M Tris-HCl (pH 8.0) buffer; (2) loading: the crude sample is loaded at a flow rate of 1.0mL / min; (3 ) elution: directly collect the enzyme activity fraction in the unadsorbed effluent; (4) repeat: repeat the above-mentioned ion exchange column equilibration, sample loading, and elution process to remove as much impurity protein as possible.

Embodiment 3

[0041] Example 3: Comparative analysis of activity, optimum pH and solubility of Bacillus fastidiosa uricase series mutants

[0042] (1) Definition of activity measurement: the amount of enzyme required to oxidize 1 μmol of substrate uric acid per minute is an activity unit.

[0043] Enzyme activity measurement steps: use 0.2M borax buffer solution (pH 9.2), measure the uric acid concentration in the system to 75 μmol, delay for 30 s, time interval 10 s, and measure the initial speed of absorption change within 1 minute at 293 nm. The corresponding buffer and uric acid were preheated to (25±0.5)°C, and the extinction coefficient of uric acid was fixed at 11.5 (mmol L -1 cm) -1 .

[0044] (2) Determination of optimum pH Select two buffer systems: 0.10M Tris-HCl, pH from 7.4 to 9.2; mix 0.10M borax and boric acid in different proportions to measure pH. The same amount of enzyme was used to measure the change of enzyme activity with pH; the results are shown in Figure 1.

[0...

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Abstract

The pichia guilliermondii uricase mutants are I260K, I260R, S96D, T241D, L246D, S96D / T241D, T241D / I260R, S96D / T241D / L246D and S96D / T241D / L246D / I260K, and the optimal pH value of the pichia guilliermondii uricase mutants is close to the neutral; under the optimal pH, compared with the uricase activity of wild type pichia guilliermondii, the activity of mutants I260K and I260R is improved; in a neutral aqueous solution, compared with the wild type pichia guilliermondii uricase, the solubility of the mutants S96D, T241D, L246D, S96D / T241D, T241D / I260R, S96D / T241D / L246D and S96D / T241D / L246D / I260K is increased.

Description

technical field [0001] The invention belongs to the field of enzyme biotechnology, and discloses nine mutants of Pichia uricase (Meyerozymaguilliermondii Uricase, abbreviated as MGU, referring to its wild type); compared with the MGU recorded in NCBI database record KY706244.1, The pH optima of the published mutants and MGU are both close to neutral, and the maximum catalytic activity or solubility of the published mutants is increased at neutral pH. Background technique [0002] Uricase can specifically oxidize uric acid to allantoin and hydrogen peroxide. It is an important tool enzyme for the determination of serum uric acid and an important medicinal enzyme for the treatment of tumor lysis syndrome (TLS), gout and other hyperuricemia-related diseases. [0003] Highly active uricase at neutral pH is more valuable as a biotechnology product for the determination of serum uric acid and the treatment of hyperuricemia; high solubility of uricase can reduce the production cost...

Claims

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Application Information

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IPC IPC(8): C12N9/06
CPCC12N9/0048C12Y107/03003
Inventor 廖飞汪佳琪张露瑶
Owner CHONGQING UNIV OF TECH
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