Mutant of Acid Phosphatase and Application Thereof

Abstract

Disclosed is a mutant of acid phosphatase and an application thereof, belonging to the technical field of biological engineering. The disclosure provides a mutant of acid phosphatase PaAPase.sub.Mu3. By expressing the mutant of acid phosphatase PaAPase.sub.Mu3 in Escherichia coli and using a whole-cell conversion method, L-ascorbic acid is transformed into L-ascorbate-2-phosphate. Moreover, a catalytic system of the mutant of acid phosphatase PaAPase.sub.Mu3 is optimized from the aspects of reaction pH and temperature, so that a yield of L-ascorbate-2-phosphate can reach 90.1 g/L and a molar yield can reach 75.1%. Therefore, the problems of a high substrate cost, environmental pollution and the like before are greatly reduced, laying a foundation for the industrial green production of L-ascorbate-2-phosphate.

Claims

1. A mutant of an acid phosphatase, wherein the acid phosphatase is from Pseudomonas aeruginosa and is a parent enzyme having an amino acid sequence set forth in SEQ ID NO: 1, and the mutant comprises the amino acid sequence of SEQ ID NO: 1 except for mutations of at least one of sites 57, 58, 94 and 135 of the parent enzyme.

2. The mutant according to claim 1, wherein the mutant has any one of the mutations in (a)-(f): (a) the site 58 is mutated into phenylalanine; (b) the site 94 is mutated into phenylalanine; (c) the site 135 is mutated into arginine; (d) the site 94 is mutated into phenylalanine, and the site 135 is mutated into arginine; (e) the site 58 is mutated into proline, and the site 135 is mutated into arginine; and (f) the site 57 is mutated into histidine, the site 58 is mutated into proline, and the site 135 is mutated into arginine.

3. A microbial cell expressing the mutant according to claim 1.

4. The microbial cell according to claim 3, taking Escherichia coli as an original strain.

5. A method of using the microbial cell according to claim 4 for synthesizing L-ascorbate-2-phosphate, comprising transforming L-ascorbic acid as a substrate to produce L-ascorbate-2-phosphate.

6. The method according to claim 5, wherein the microbial cell is added to a reaction system containing L-ascorbic acid and sodium pyrophosphate, the reaction system having a pH of 3.8-4.3.

7. The method according to claim 6, wherein the reaction at 25-37° C. lasts for not less than 8 h.

8. The method according to claim 7, wherein a concentration of the microbial cells in the reaction system is 20-40 g/L, a concentration of the L-ascorbic acid is 70-100 g/L, and a concentration of the sodium pyrophosphate is 180-240 g/L.

9. The method according to claim 8, wherein the concentration of the microbial cells in the reaction system is 30 g/L, the concentration of the L-ascorbic acid is 88 g/L, and the concentration of the sodium pyrophosphate is 223 g/L.

Description

BRIEF DESCRIPTION OF FIGURES

[0029] FIG. 1 is an SDS-PAGE diagram of the mutant of acid phosphatase PaAPase.sub.Mu3.

[0030] FIG. 2 is a diagram illustrating the enzyme activities of the mutant of acid phosphatase at different pHs.

[0031] FIG. 3 is a diagram illustrating the enzyme activities of the mutant of acid phosphatase at different temperatures.

[0032] FIG. 4 is a diagram illustrating a relationship between the pH of a buffer solution of the conversion system and the yield of L-ascorbate-2-phosphate.

[0033] FIG. 5 is a diagram illustrating a relationship between the conversion temperature and the yield of L-ascorbate-2-phosphate.

DETAILED DESCRIPTION

[0034] (1) Liquid Chromatographic Analysis:

[0035] A liquid chromatograph Waters ACQUITY UPLCTM is adopted for sample analysis.

[0036] Composition of a mobile phase: 1 L of 0.1 mol.Math.L.sup.−1 KH.sub.2PO.sub.4 solution is prepared with ultrapure water, then 500 mL of acetonitrile is added, and the two were mixed together. The pH of the mixed solution is adjusted with phosphoric acid to 3.0. The mobile phase is subjected to vacuum filtration by a 0.22 μm organic filter membrane, and then 20 min of ultrasonic degassing is performed.

[0037] Sample introduction procedure: One sample is introduced every 8 minutes, where the sampling amount of each sample is 12 μL.

[0038] Elution procedure: The flow rate of the mobile phase is constant at 1 mL.Math.min.sup.−1.

[0039] Chromatographic column: APS-2 HYPERSIL column (150 mm×4.6 mm, 5 μm) at 35° C.

[0040] Detector: The ultraviolet absorption wavelength is 254 nm.

[0041] (2) Protein purification:

[0042] An ultrasonic cell disruptor is employed to perform an ultrasonic treatment on cells containing a target plasmid, at a power of 36% and at an interval of 3 s for every 2 s of work, till complete disruption, to obtain a whole-cell disruption liquid. The cell disruption liquid is centrifuged in a refrigerated centrifuge at a temperature of 4° C. and at a speed of 12,000 rpm for 30 min. After the centrifugation, the supernatant is filtered with a 0.22 μm water-based filter membrane, and then the filtrate is collected. The filtrate is purified with a purifier of an AKTAxpress system, using a 5 mL HisTrap HP purifying column as a purifying column. In elution, a buffer solution (20 mM Tris-HCl, 150 mM NaCl) containing 1 M imidazole is used for elution at a flow rate of 2-3 mL.Math.min.sup.−1. Then, the purified components are verified through 10% (w/v) sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The purest component is desalted using a PD-10 desalting column and a low-salt buffer solution (10 mM Tris-HCl, 0.1 M NaCl; pH 6.0). The purified and desalted proteins are collected.

[0043] (3) Sequences of primers used in examples:

TABLE-US-00001 TABLE 1 Primers used during mutation Name Primer (5′-3′) D56-F GCGCATNNKCAGTGGGAAGAT SEQ ID NO: 3 D56-R CCACTGMNNATGCGCGCGCAC SEQ ID NO: 4 W58-F GATCAGNNKGAAGATAACGTG SEQ ID NO: 5 W58-R ATCTTCMNNCTGATCATGCGC SEQ ID NO: 6 V94-F CAAGAAAACCTGNNKGAAGTG SEQ ID NO: 7 V94-R CATCAGCACTTCMNNCAGGTT SEQ ID NO: 8 D135-F GGCCATACCATGNNKAGCTAT SEQ ID NO: 9 D135-R TTTAAAATAGCTMNNCATGGT SEQ ID NO: 10 W58P/D135R/Q57L-F CGCGCGCATGATAAACCAGAA SEQ ID NO: 11 W58P/D135R/Q57L-R GTTATCTTCTGGTTTATCATG SEQ ID NO: 12 W58P/D135R/Q57R-F CGCGCGCATGATAGGCCAGAA SEQ ID NO: 13 W58P/D135R/Q57R-R GTTATCTTCTGGCCTATCATG SEQ ID NO: 14 W58P/D135R/Q57H-F CGCGCGCATGATCACCCAGAA SEQ ID NO: 15 W58P/D135R/Q57H-R GTTATCTTCTGGGTGATCATG SEQ ID NO: 16

Example 1: Construction and Screening of Mutants of a PaAPase Enzyme

[0044] Taking a PaAPase enzyme having an amino acid sequence shown by SEQ ID NO: 1 as a parent (the nucleotide sequence of a coding gene thereof was shown by SEQ ID NO: 2), saturation mutation (the primers used were shown in Table 1) was performed at sites 56, 58, 135 and 94 thereof, and beneficial mutants were screened out.

[0045] A fragment obtained by PCR was connected to pET-28a (+) carriers (Bam H I and Hind III enzymes), and whole-plasmid PCR was performed using corresponding primers in Table 1, to obtain recombinant plasmids containing mutants. The recombinant plasmids were transferred into Escherichia coli, to construct recombinant bacteria. The recombinant bacteria were spread on a LB plate having kanamycin resistance, and cultured at 37° C. to grow monoclones.

[0046] A sterile 96-well deep-well plate (first-level plate) was opened in a super clean bench. 400 μL of a LB medium (containing 0.1 g.Math.L.sup.−1 kanamycin) was added into each well with a multichannel pipette. Escherichia coli transformants for saturation mutation on the LB plate were picked with a toothpick and inoculated into the 96-well deep-well plate, where original strains were added into the plate wells in column 12 as controls. Overnight culture was performed in a high-throughput shaker at 37° C. and 600 rpm. 50 μL of a seed solution in the first-level plate was fetched with a multichannel pipette and inoculated into a 96-well deep-well plate (second-level plate) containing 800 μL of a lactose auto-induction medium (containing 0.1 g.Math.L.sup.−1 kanamycin) in each well, and cultured at 37° C. and 600 rpm for 3-4 h (with OD.sub.600 around 1). The temperature of the high-throughput shaker was adjusted to 25° C., and induction was performed for 12 h. Then, the resulting product was centrifuged at 3,000×g for 5 min. The supernatant in the 96-well deep-well plate (second-level plate) was discarded while the cells were collected and stored at −80° C. for later use. The 96-well deep-well plate containing mutant cells frozen at −80° C. was taken out, and placed at room temperature for 30 min for defrosting. Then, 200 μL of a substrate solution (4 g.Math.L.sup.−1 L-ascorbic acid, 30 g.Math.L.sup.−1 pyrophosphoric acid, pH 4.0) was added into each well with a multichannel pipette. Through repeated blowing and suction with the multichannel pipette, the cells were suspended in the substrate solution. Then the cell suspension was maintained at a temperature of 25° C. for 16 h. The cell suspension was centrifuged at 3,000×g for 20 min for separation and precipitation. The supernatant in the deep-well plate was taken out, diluted by 5 times, and measured with HPLC. A molar conversion rate was calculated.

[0047] According to the HPLC results and by sequencing, 3 beneficial mutants, W58F, V94F and D135R, were screened out, with molar conversion rates of 37.6%, 41.3% and 45.3% respectively.

[0048] Based on the 3 beneficial mutants, W58F, V94F and D135R were subjected to combinatorial mutations according to the foregoing steps (mutations using corresponding primers based on single mutants, to construct double mutants), and conversion efficiency was determined according to step 2.

[0049] The results showed that the conversion rates of the combinatorial mutants, W58F/D135R, W58F/V94F and V94F/D135R, were 19.7%, 17.9% and 40.9% respectively, all lower than that of D135R. Therefore, PaAPase.sub.Mu1 (D135R) was selected as a template to carry out site-saturation mutation on Trp58 and Val94.

[0050] The results showed that in the double mutants, the conversion rate of PaAPase.sub.Mu2 (W58P/D135R) was increased by 1.56 times. Taking PaAPase.sub.Mu2 (W58P/D135R) as a template, Gln57 was mutated into positive amino acid (Lys, Arg and His), where the conversion rate of PaAPase.sub.Mu3 (Q57H/W58P/D135R) was increased by 1.9 times from that of a wild type.

TABLE-US-00002 TABLE 2 Conversion effects of mutants Conversion rate Mutant (%) Yield WT 25.9 31.08 W58F 37.6 45.12 V94F 41.3 51.96 D135R 45.3 54.36 W58F/D135R 19.7 23.64 V94F/D135R 40.9 49.08 W58F/V94F 17.9 21.48 W58P/D135R 62.1 79.32 Q57H/W58P/D135R 75.1 90.10

Example 2: Enzymatic Properties of Mutants

[0051] The obtained recombinant bacteria containing PaAPase.sub.Mu3 (Q57H/W58P/D135R) were introduced into a LB medium, and cultured for 12 h to obtain an activated liquid. The activated liquid was inoculated into a fresh TB medium, and cultured for 2 h. Then, IPTG with a final concentration of 0.2 mM was added. The mixture was cultured at 25° C. for 14 h, to induce expression of a recombinant target protein. The enzyme liquid was collected and subjected to ultrasonic disruption, and then albumin glue verification was performed (FIG. 1). In FIG. 1, M is marker, 1 is a band of a crude enzyme liquid, and 2 is a band of the purified target protein (29 KDa). The other mutants were expressed and purified with the same method.

[0052] (1) Determination of Specific Enzyme Activity

[0053] The activities of PaAPase and mutants thereof were determined with high-performance liquid chromatography. 500 μL of a substrate solution (500 mM L-ascorbic acid and 500 mM sodium pyrophosphate) (pH 5.0) and 5 μL of the enzyme liquid react at 30° C. for 30 min, and 95 μL of 42% phosphoric acid was added to terminate the reaction, where an activity unit was defined as the amount of enzyme required for producing 1 μmol of L-ascorbate-2-phosphate within 1 min (Table 3).

[0054] (2) Determination of Kinetic Parameters

[0055] The kinetic parameters, including V.sub.max, K.sub.m and k.sub.cat, were calculated by measuring the generation rates of initial products of L-ascorbic acid and L-ascorbate-2-phosphate (1-1,000 mM) at different concentrations at 30° C. and under other conditions consistent with those of the determination of specific enzyme activity. The determination of all activities was repeated three times, with the results shown in Table 3.

TABLE-US-00003 TABLE 3 Specific enzyme activities and kinetic parameters of mutants Specific Enzyme Activity K.sub.m k.sub.cat k.sub.cat/K.sub.m Enzyme (U mg.sup.−1) (mM) (s.sup.−1) (min.sup.−1 .Math. mM.sup.−1) WT 14.8 289 16.3 3.38 Mu1/(D135R) 28.1 245 18.7 4.58 Mu2 (W58P/D135R) 32.3 223 19.2 5.17 Mu3 (Q57H/W58P/D135R) 39.7 211 20.1 5.72

[0056] (3) Impact of pH on PaAPase.sub.Mu3 Activity

[0057] The pH stability of PaAPase was detected within a pH range from 2.5 to 8.0. From FIG. 2, the enzyme activity of PaAPase was lost a little after 12 h of storage at 4° C. and at a pH of 3.0-8.0, and the remaining enzyme activity was 90% or above. However, when the pH was below 2.5, the remaining activity of PaAPase was only 60% of the initial enzyme activity. The above-mentioned results indicate that PaAPase has excellent pH stability at a pH of 3.0-8.0, and the enzyme activity undergoes a relatively large loss in a strong acid condition.

[0058] (4) Impact of Temperature on PaAPase.sub.Mu3 Activity

[0059] The temperature stability of PaAPase was detected at 0-55° C., with the results shown in Table 3. PaAPase was relatively stable in an environment below 20° C., and 90% or above of the initial activity of PaAPase could be maintained. When the temperature was between 20° C. and 55° C., the loss of enzyme activity was increased gradually. After standing for 1 h at 50° C., the loss of enzyme activity was close to 50%. The above-mentioned results indicate that an increasing temperature promotes catalysis of protease molecules. But as the temperature rises, a trend of protein denaturation and inactivation becomes more obvious. When the temperature exceeds 50° C., the enzyme activity starts to decrease sharply mainly because a large number of proteins are denaturalized and inactivated at the time. At 25-50° C., an increasing temperature increases the enzyme catalytic activity in a reaction system by an amount larger than the loss of activity caused by protein denaturation, thereby leading to an overall increasing trend of the enzyme activity.

Example 3: Whole-Cell Production of L-Ascorbate-2-Phosphate Using Recombinant Bacteria PaAPase.SUB.Mu3

[0060] (1) Whole-Cell Optimum Reaction pH

[0061] The strains stored in a glycerin tube were spread on a LB solid medium, and cultured at a constant temperature of 37° C. to grow monoclones. The monolclones were picked and placed into a fresh LB liquid medium, and cultured at a constant temperature of 37° C. and at 200 rpm for 12 h, to obtain an activated liquid. The activated liquid was inoculated into a fresh TB medium, and cultured for 2 h. Then, IPTG with a final concentration of 0.2 mM was added, and induction culture was performed at 25° C. for 14 h. After the culture, the cells were collected.

[0062] 3 g of the whole cells expressing PaAPase.sub.Mu3 protein after the induction culture were added into each of 100 mL conical flasks respectively, and were reacted at 25° C. for 12 h in reaction systems with a pH of 3.0, 3.5, 4.0, 4.5 and 5.0 respectively, the reaction systems containing 88 g/L L-ascorbic acid, 223 g/L sodium pyrophosphate and hydrochloric acid for adjusting pH. Then, the resulting products passed through a 0.22 μm water membrane, and liquid chromatographic analysis was carried out.

[0063] The yield of L-ascorbate-2-phosphate was determined with the foregoing detection method. The results are shown in Table 4 and FIG. 4 that the conversion rate of the PaAPase.sub.Mu3 enzyme at a pH of 3.0-4.0 is increased as the pH rises. The yield of L-ascorbate-2-phosphate can reach 85 g/L or more at a pH of 3.8-4.3 and 90.1 g/L at a pH of 4.0 while the molar yield is 75.1%. The yield of L-ascorbate-2-phosphate is reduced with further increase of the pH.

TABLE-US-00004 TABLE 4 Yield of L-ascorbate-2-phosphate at different pHs pH Yield of L-ascorbate-2-phosphate (g/L) 3 10.4 3.5 56.7 3.6 66.2 3.7 72.3 3.8 85.6 3.9 88.5 4.0 90.1 4.1 91.1 4.2 89.1 4.3 85.5 4.4 76.5 4.5 55 5 22.5

[0064] (2) Whole-Cell Optimum Reaction Temperature

[0065] According to the steps in (1), the yield of L-ascorbate-2-phosphate after 14 h of conversion of PaAPase.sub.Mu3 at different temperatures (20° C., 25° C., 30° C. and 37° C.) was determined every 2 hours at a pH of 4 of the reaction system, and a molar yield was calculated. The results are shown in FIG. 5 that the PaAPase.sub.Mu3 enzyme can lead to a relatively high yield of L-ascorbate-2-phosphate at 25-30° C. The conversion temperature of 25-30° C. is more favorable for the catalysis of a phosphorylation reaction by the PaAPase.sub.Mu3 enzyme. At 25° C., the conversion rate is the highest, the yield of L-ascorbate-2-phosphate is 90.1 g/L, and the molar yield is 75.1%. At 30° C., the yield of L-ascorbate-2-phosphate is 84 g/L, and the molar yield is 70%.

[0066] Although the disclosure is described above using preferred examples, the disclosure is not limited thereto. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the disclosure. Therefore, the scope of the disclosure shall be subject to the definition of the claims.