CN106636048A - Phenylalanine deaminase mutant with improved enzyme activity - Google Patents

Abstract

The invention discloses a mutant of phenylalanine deaminase with improved enzyme activity, which belongs to protein engineering. The specific enzyme activity of the phenylalanine deaminase mutant S73N provided by the present invention is 2.22U/mg, which is 25% higher than the 1.78U/mg of the original enzyme; k _cat (s ^-1 ) is 1.650s compared with the original enzyme ^‑1 improved by 26.7%.

Description

A Mutant of Phenylalanine Deaminase with Enhanced Enzyme Activity

technical field

The invention relates to a mutant of phenylalanine deaminase with improved enzyme activity, which belongs to protein engineering.

Background technique

Phenylalanine deaminase (PAL) can be used for the effective and long-term treatment of phenylketonuria (PKU). PAL derived from Anabaena variabilis (Av-PAL) has shown good potential in the treatment of PKU because of its stability and low immunogenicity. However, the enzymatic activity of Av-PAL from prokaryotic sources is quite different from that of PAL from eukaryotic sources, and it has also been found that the loop of Tyr78 (Ser73 to Ala97) has extraordinary significance for the MIO family of enzymes, including enzymatic activity and substrate material selectivity.

Contents of the invention

The present invention aims to provide a mutant of phenylalanine deaminase with improved enzyme activity, encoded by the nucleotide sequence shown in SEQ ID NO.1.

The present invention also provides a method for recombinantly expressing the phenylalanine deaminase mutant, which uses Escherichia coli BL21 as the host and pET28a as the expression vector, and inoculates the obtained recombinant bacterial seed solution in the kanamycin-containing In the 2YT expression medium, shake culture at 37°C and 200r/min until the OD ₆₀₀ reaches 0.8-1.0, add the inducer IPTG to 0.1mM, and induce the expression of the phenylalanine deaminase mutant for 16-18h at 20°C.

2YT expression medium contains peptone 16g/L, yeast extract 10g/L, NaCl 5g/L.

In one embodiment of the present invention, it also includes crushing the recombinant bacteria, collecting the supernatant, and filtering the supernatant through a membrane, and separating the phenylalanine deaminase mutant with a His Trap HF column.

The specific enzyme activity of the phenylalanine deaminase mutant S73N provided by the present invention is 2.22 U/mg, which is 25% higher than the 1.78 U/mg of the original enzyme; k _cat (s ^-1 ) is 1.650s compared with the original enzyme ^-1 improved by 26.7%.

detailed description

Definition of enzyme activity: the amount of trans-cinnamic acid converted from L-phenylalanine per milligram of PAL per minute (μmols).

Enzyme activity assay method: in 100mM Tris-Hcl buffer solution with pH 8.5, with 10mM L-phenylalanine as substrate, react at 37°C for 20min, measure the concentration of cinnamic acid generated according to the absorption value at 290nm, and determine the enzyme activity live.

Determination of kinetic parameters: at 37°C, with 0.01 to 0.3mM L-phenylalanine as substrate, add 0.5μg of pure enzyme, react in 100mM Tris-Hcl buffer at pH 8.5 for 12min, during which 96 wells The plate continuously detects the product.

Example 1

(1) Construction of mutant S73N:

Using pET28a-PAL as a template, using the primers shown in Table 1 and the conditions shown in Table 2, the recombinant vector pET28a-PAL/S73N carrying the gene encoding mutant S73N was obtained by PCR. For the construction method of pET28a-PAL, see Moffitt, M.C., Louie, G.V., Bowman, M.E., Pence, J., Noel, J.P. and Moore, B.S. (2007) Discovery of twocyanobacterial phenylalanine ammonia lyases: Kinetic and structural characterization. Biochemistry-Us, 46 ,1004-1012.

Table 1 Primers

Table 2 Whole plasmid PCR amplification reaction system

The PCR amplification reaction conditions are:

PCR products were identified by agarose gel electrophoresis. Then the PCR product was purified and digested and transferred to BL21 host.

(2) Inoculate recombinant Escherichia coli BL21/pET28a-PAL/S73N in 4 mL of LB medium (peptone 10 g/L, yeast extract 5 g/L, NaCl 10 g/L) with a kanamycin concentration of 100 μg/L, 37 Cultivate overnight with shaking at 200r/min.

Inoculate the above overnight culture into 100 mL of 2YT expression medium (peptone 16 g/L, yeast extract 10 g/L, NaCl 5 g/L) containing 100 μg/mL of kanamycin at an inoculum size of 1%, at 37 °C , 200r/min shaking culture to OD ₆₀₀ to 0.8-1.0, add inducer IPTG to 0.1mM, induce 16-18h at 20°C to obtain bacteria, and centrifuge at 5000g to collect bacteria.

(3) Dissolve the recombinant cells in 20mL binding buffer solution (50mmol/L Na ₂ HPO ₄ , 50mmol/L NaH ₂ PO ₄ , 500mmol/L NaCl, 20mmol/L imidazole), sonicate, centrifuge at 13000g for 25min, and the supernatant Filter with a 0.22 μm membrane filter. Equilibrate a 1mL His Trap HF column with 10 times the column volume of the binding buffer solution, wash off the non-specifically adsorbed protein with 15 times the column volume of the binding buffer solution, and use 8 times the column volume of 150, 300 and 500mmol/L imidazole respectively Proteins were eluted with buffer, and samples were collected and identified by SDS-PAGE analysis.

(4) Determination of enzyme activity: 3 μg of purified mutant enzyme S73N and wild enzyme (SEQ ID NO.2) were added to 400 μl of Tris-Hcl buffered reaction system with pH 8.5, and substrate L-phenylalanine was added to 10mM, react at 37°C for 20min, and determine the corresponding specific enzyme activity. The specific enzyme activity of S73N is 2.22U/mg, which is 1.78U/mg of wild enzyme.

(5) Determination of kinetic parameters: using different concentrations of L-phenylalanine as the substrate (0.01 to 0.3 mM), the kinetic parameters were determined.

Table 1 Kinetic parameters of wild enzyme (WT) and S73N

Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore The scope of protection of the present invention should be defined by the claims.

SEQUENCE LISTING

<110> Jiangnan University

<120> A mutant of phenylalanine deaminase with enhanced enzyme activity

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Claims (9)

1. a kind of PD mutant, it is characterised in that nucleotide sequence coded by shown in SEQ ID NO.1.

2. the gene of the PD mutant described in claim 1 is encoded.

3. the carrier or cell of gene described in claim 2 are carried.

4. the recombinant bacterium of PD mutant described in a kind of recombinant expressed claim 1, it is characterised in that with large intestine Bacillus BL21 is host, with pET28a as expression vector.

5. the method for PD mutant described in a kind of recombinant expressed claim 1, it is characterised in that be with large intestine Bacillus BL21 is host, with pET28a as expression vector, the seed liquor of gained recombinant bacterium is inoculated in the 2YT tables containing kanamycins Up in culture medium, 37 DEG C, 200r/min shaken cultivations to OD₆₀₀During to 0.8-1.0, derivant IPTG to 0.1mM is added, in 20 DEG C induction 16-18h PD mutant expression；The 2YT expression culture medium 16g/L containing peptone, yeast are carried Take thing 10g/L, NaCl 5g/L.

6. method according to claim 5, it is characterised in that also include that supernatant, supernatant will be collected after restructuring bacterial cell disruption After membrane filtration, with His Trap HF post separations PD mutant is obtained.

7. the PD mutant described in claim 1 is in answering that preparation is used in the medicine for treating PKU With.

8. application of the carrier or cell described in claim 3 in the medicine for treating PKU is prepared.

9. application of the recombinant bacterium described in claim 4 in the medicine for treating PKU is prepared.