WO2017031839A1

WO2017031839A1 - L-asparaginase mutant with improved enzyme activity and construction method thereof

Info

Publication number: WO2017031839A1
Application number: PCT/CN2015/094554
Authority: WO
Inventors: 饶志明; 龙水清; 张显; 杨套伟; 徐美娟
Original assignee: 江南大学
Priority date: 2015-08-25
Filing date: 2015-11-13
Publication date: 2017-03-02
Also published as: CN105062997A

Abstract

Provided are an L-asparaginase mutant with improved enzyme activity and a construction method thereof, wherein the mutant is constructed on the basis of the amino acid sequence as shown in the SEQ ID NO.2, in which glycine at position 107 is mutated into aspartic acid. Also provided is a genetically-engineered bacteria expressing the L-asparaginase mutant.

Description

[Correct according to Rule 26 22.03.2016] An L-asparaginase mutant with improved enzyme activity and its construction method

Technical field

The invention relates to an L-asparaginase mutant with improved enzyme activity and a construction method thereof, and belongs to the technical field of genetic engineering.

Background technique

L-asparaginase amidohydrolase (E.C. 3.5.1.1) is capable of hydrolyzing deamination of L-asparagine to form L-aspartic acid and ammonia. L-asparaginase has anti-tumor activity and has been used in the treatment of acute lymphoblastic leukemia and Hodgkin's disease. In recent years, it has been found that L-asparaginase can also reduce the formation of acrylamide in fried foods. The size, structure and nature of L-asparaginase vary from source to source. L-asparaginase is widely available, and L-asparaginase is found in guinea pig serum, plants and microorganisms.

The problem of heterologous expression of L-asparaginase is that the protein expression is low and the L-asparaginase enzyme activity is low. Therefore, site-directed mutagenesis of L-asparaginase and enhancement of extracellular enzyme activity are of great significance for improving the industrial application prospect of L-asparaginase.

Summary of the invention

The present invention first provides an L-asparaginase mutant having improved enzymatic activity, the amino acid sequence of which is the sequence shown in SEQ ID NO.

The nucleotide sequence encoding the mutant is the sequence shown in SEQ ID NO.

The mutant is based on an amino acid such as the amino acid shown in SEQ ID NO. 2, and amino acid 107 is mutated from glycine to aspartic acid.

The present invention also provides a genetically engineered bacteria expressing the L-asparaginase mutant.

The method for preparing the genetically engineered bacteria is to mutate the codon encoding the 107th glycine into a codon encoding aspartic acid based on the nucleotide sequence shown in SEQ ID NO. The recombinant gene is ligated to the expression vector to obtain a recombinant plasmid, and the recombinant plasmid is transformed into a Bacillus subtilis host strain to obtain a Bacillus subtilis genetically engineered strain.

In one embodiment of the invention, the expression vector is pMA5.

In an embodiment of the present invention, the preparation method is specifically:

(1) using the nucleotide sequence shown in SEQ ID NO. 4 as a template, Flprimer (the sequence is shown in SEQ ID NO. 5), and Rlprimer (the sequence is shown in SEQ ID NO. 6) as primers, and PCR is obtained. Recombinant gene shown in SEQ ID NO. G107D.

(2) The recombinant gene sequence obtained in the previous step was ligated into the pMA5 expression vector to obtain a recombinant plasmid pMA5-G107D, and the recombinant plasmid was transformed into B. subtilis 168 to obtain a recombinant Bacillus subtilis engineering strain, which was named pMA5-G107D/B. .subtilis 168.

Based on the natural L-asparaginase, the present invention modifies the molecular structure of L-asparaginase by site-directed mutagenesis, and the pure enzyme solution of the mutant enzyme is 83% higher than that before the mutation. The substrate affinity K _{m of} the mutant enzyme G107D was reduced by 50% compared to before the mutation, and the catalytic efficiency was increased (the ratio of k _cat to K _m ) was 84%. The invention shows that the 107 amino acid residue has a great influence on the catalytic action of the enzyme, provides a certain basis for the research on the catalytic mechanism of the enzyme, and improves the industrial application potential of the enzyme. The invention can be used for preparing medicines for treating acute lymphocytic leukemia and Hawkinson's disease, and can also be used for reducing the formation of acrylamide in fried foods.

detailed description

Example 1 Construction of Recombinant Vector Containing L-Asparaginase Mutant

(1) Obtaining the G107D mutant: using the nucleotide sequence shown in SEQ ID NO. 4 as a template, Fprimer (the sequence is shown in SEQ ID NO. 5), and Rprimer (the sequence is shown in SEQ ID NO. 6) as The primer was subjected to PCR to obtain the recombinant gene shown in SEQ ID NO.

(2) The recombinant gene and pMA5 were digested with BamHI and MluI, respectively, and purified, and then ligated with T4 DNA ligase at 16 ° C overnight. The ligation product chemically transforms JM109 competent cells. The transformant was coated with kanamycin (50 mg/L) LB plate, the plasmid was extracted, and the constructed recombinant plasmid was verified by double digestion, and named pMA5-G107D. The sequencing work was completed by Shanghai Biotech.

Example 2 Construction of L-asparaginase-producing Bacillus subtilis engineering bacteria

The recombinant plasmid pMA5-G107D obtained in Example 1 was chemically transformed into B. subtilis 168 competent cells by the following methods:

(1) The solution required for the transformation experiment is as follows (g/L):

Sp-A: (NH ₄ ) ₂ SO ₄ 4, K ₂ HPO ₄ 28, sodium citrate 12Sp-B: MgSO ₄ ·7H ₂ O 0.4

100×CAYE:Casamino acid 20, yeast powder 100Sp I medium: Sp-A 49%, Sp-B 49%, 50% glucose 2%, 100×CAYE 2% Sp II medium: Sp I medium 98%, 50 mmol/LCaCl ₂ 1%, 250 mmol/L MgCl ₂ 1%. Damp heat sterilization at 115 °C.

(2) Single colonies of B. Subtilis 168 were inoculated into 2 mL Sp I medium (50 mL centrifuge tube), and cultured overnight at 37 ° C, 200 r / min;

(3) Take 100 μL of the culture solution to 5 mL of Sp I medium, and incubate at 37 ° C, 200 r / min to log phase (OD600 value) It is about 1), about 4 to 5 hours;

(4) Take 200 μL of the culture solution into 2 mL of Sp II medium, incubate at 37 ° C, 200 r / min for 90 min, remove 20 μL of 10 mmol / L EGTA, continue to culture at 37 ° C, 200 r / min for 10 min, and then dispense into 500 μL per Tube, add 5 μL of recombinant plasmid pMA5-G107D, mix, incubate at 37 ° C, 200r / min for 90min, and take the bacterial solution to coat the resistant plate. Incubate at 37 ° C for 12 h, and pick positive transformants to verify. The recombinant strain pMA5-G107D/B.subtilis 168 was obtained.

Example 3 High-efficiency expression and enzyme activity assay of recombinant strain pMA5-G107D/B.subtilis 168L-aspartate.

(1) The recombinant strain pMA5-G107D/B.subtilis 168 constructed in Example 2 and the control strain pMA5-ansz/B.subtilis 168 expressing the unmutated enzyme were inoculated into 10 mL of LB medium containing kanamycin, respectively. The culture was shaken overnight at 37 ° C, and transferred to the fermentation medium of Bacillus subtilis by 4% inoculation on the next day, and cultured at 37 ° C for 24 h. The fermentation broth was centrifuged at 4 ° C, 10000 r / min for 10 min, and the supernatant was extracellular. The crude enzyme solution and the cell disrupted supernatant are intracellular crude enzyme solutions for the determination of enzyme activity.

(2) Fermentation medium of Bacillus subtilis: soybean protein 胨 10g / L, K ₂ HPO ₄ 2.3g / L, KH ₂ PO ₄ 1.7g / L, corn syrup 15g / L, urea 3g / L, glucose 40g / L , MgSO ₄ 0.75 g / L, NaCl 5 g / L. Adjust pH 6.8-7.0.

(3) Enzyme activity definition: The amount of enzyme required to catalyze the conversion of L-aspartamide to 1 μmol of NH ₃ per minute under a reaction condition of 40 ° C is an enzyme unit.

(4) L-asparaginase enzyme activity assay method: The enzyme activity was determined by measuring the amount of NH ₃ released in the catalytic reaction using L-asparagine as a substrate. The reaction mixture (1 mL) was composed of: 400 μL of 25 mM L-asparagine (dissolved in 50 mM pH 7.5 Tris-HCl); 400 μL of 50 mM pH 7.5 Tris-HCl; 100 μL of an appropriate concentration of the enzyme solution. The reaction mixture was reacted at 40 ° C, pH 7.5 for 15 min, and then the reaction was terminated by adding 100 μL of a 15% (W/V%) trichloroacetic acid solution. The reaction solution in which the reaction was terminated by adding trichloroacetic acid before the enzyme reaction was used as a blank control. The reaction mixture was centrifuged at 20,000 g for 10 min, and 200 uL of the supernatant was added to 4.8 mL of deionized water. 200 μL of the Nessler reagent was added to the above system, and the absorbance was measured at a wavelength of 450 nm, and the amount of NH ₃ released by the enzyme reaction was measured by a color reaction.

(3) The results showed that the total enzyme activity (sum of intracellular and extracellular enzyme activities) of L-asparaginase expressed by recombinant strain pMA5-G107D/B.subtilis 168 was 961 U/mL, which was higher than the control strain pMA5-ansz/ .subtilis 168 (534.2 U/mL) L-asparaginase enzyme activity increased by 80%.

(4) The extracellular crude enzyme solution obtained in the step (1) is purified to obtain L-asparaginase G107D _ansz , and the purified L-asparaginase G107D _ansz enzymatic properties are analyzed, as shown in Table 1, substrate The affinity K _{m was} reduced by 50% before the mutation, the catalytic efficiency k _cat /K _{m was} increased by 84%, and the enzyme activity was increased by 83%. Due to the increase in catalytic efficiency, the specific enzyme activity of G107D _ansz is increased.

Table 1 G107D _ansz reaction kinetic parameters

Although the present invention has been disclosed in the above preferred embodiments, the present invention is not limited thereto, and various modifications and changes can be made thereto without departing from the spirit and scope of the invention. The scope of the invention should be determined by the scope of the claims.

Claims

An L-asparaginase mutant characterized in that the amino acid sequence of the mutant is as shown in SEQ ID NO.
A gene encoding the mutant of claim 1.
A recombinant expression vector comprising the gene of claim 2.
A genetically engineered bacterium expressing the L-asparaginase mutant of claim 1.
A method for producing the genetically engineered bacteria according to claim 4, characterized in that, based on the sequence shown in SEQ ID NO. 4, the 107th glycine is mutated to aspartic acid to obtain a recombinant gene, and the recombinant gene is obtained. The recombinant plasmid is obtained by linking to the expression vector, and the recombinant plasmid is transformed into the B. subtilis host strain to obtain the Bacillus subtilis genetically engineered bacteria.
The preparation method according to claim 5, wherein the method is specifically: (1) using the nucleic acid sequence shown in SEQ ID NO. 4 as a template, and the sequence is SEQ ID NO. 5, SEQ ID NO. The primers shown are subjected to PCR to obtain the G107D mutant gene sequence in which the 107 amino acid is mutated from glycine to aspartic acid; (2) the recombinant gene sequence obtained in the previous step is ligated into the pMA5 expression vector to obtain a recombinant The plasmid pMA5-G107D was transformed into B. Subtilis by recombinant plasmid to obtain recombinant Bacillus subtilis genetically engineered bacteria.
Use of the L-asparaginase mutant of claim 1 for the preparation of a medicament for the treatment of acute lymphocytic leukemia and Hodgkin's disease.
Use of the L-asparaginase mutant of claim 1 for reducing acrylamide production in fried foods.