Method for Preparing Ascorbic Acid-2-phosphate Using a Recombinant Strain

Abstract

The present invention provides a recombinant strain, construction method thereof and a method for producing acid phosphatase using the recombinant strain. In the invention, the phosphatase gene is obtained from Pseudomonas aeruginosa by a molecular biology method, the constructed expression plasmid is transformed into E. coli BL21 (DE3). The purified enzyme and whole cells were used for the conversion of ascorbic acid to ascorbic acid-2-phosphate. Ascorbic acid-2-phosphate can be efficiently produced by controlling the ratio of substrates. When the conversion reaction is performed at pH4.5 under 40 C. for 8 h, the output of ascorbic acid-2-phosphate reaches 54.8 /L, the conversion is 42.9% and the space time yield is 6.9 g/L/h.

Claims

1. A recombinant strain for producing acid phosphatase, wherein the recombinant strain is obtained by overexpressing an encoding gene of acid phosphatase shown in SEQ ID NO. 1 in Escherichia coli via a vector.

2. The recombinant strain as claimed in claim 1, wherein the encoding gene of acid phosphatase is amplified from Pseudomonas aeruginosa.

3. The recombinant strain as claimed in claim 1, wherein the vector is selected from the group consisting of pET28a, pET22b, pET20a.

4. The recombinant strain as claimed in claim 1, wherein the Escherichia coli is Escherichia coli BL21 (DE3).

5. A construction method of a recombinant strain for producing acid phosphatase, comprising combining an encoding gene of acid phosphatase shown in SEQ ID NO. 1 with a vector to obtain a constructed vector, and overexpressing the constructed vector in Escherichia coli.

6. The construction method as claimed in claim 5, wherein the encoding gene of acid phosphatase is amplified from Pseudomonas aeruginosa.

7. The construction method as claimed in claim 5, wherein the vector is selected from the group consisting of pET28a, pET22b, pET20a.

8. The construction method as claimed in claim 5, wherein the Escherichia coli is Escherichia coli BL21 (DE3).

9. A method for preparing ascorbic acid-2-phosphate using the recombinant strain of claim 1, comprising reacting ascorbic acid with pyrophosphate under the catalytic conversion of the recombinant strain, to obtain the ascorbic acid-2-phosphate.

10. The method as claimed in claim 9, wherein the conversion is performed at pH 3-7 under a temperature of 30-50 C.

11. The method as claimed in claim 9, wherein the molar ratio of ascorbic acid and pyrophosphate is 2.5-6:6.

12. The method as claimed in claim 9, wherein wet cells of the recombinant strain are used for conversion, and the amount of the wet cells is 60-100 mg/mmol ascorbic acid.

13. The method as claimed in claim 10, wherein wet cells of the recombinant strain are used for conversion, and the amount of the wet cells is 60-100 mg/mmol ascorbic acid.

14. The method as claimed in claim 9, wherein the acid phosphatase produced by the recombinant strain is used for the conversion, and the mount of the acid phosphatase is 0.5-1 U/mmol ascorbic acid.

15. The method as claimed in claim 10, wherein the acid phosphatase produced by the recombinant strain is used for the conversion, and the mount of the acid phosphatase is 0.5-1 U/mmol ascorbic acid.

16. The method as claimed in claim 11, wherein the acid phosphatase produced by the recombinant strain is used for the conversion, and the mount of the acid phosphatase is 0.5-1 U/mmol ascorbic acid.

17. The method as claimed in claim 9, wherein when the recombinant strain is cultured to an OD.sub.600 of 0.4-0.6, IPTG is added to induce the expression of the acid phosphatase.

18. The method as claimed in claim 14, wherein the acid phosphatase is purified by Ni-NTA agarose by the steps of: eluting impurities from the acid phosphatase using 0, 10, 20, 30, 40 and 50 mM imidazole respectively; specifically eluting the target protein using a 500 mM buffer solution of imidazole; and then preforming gradient dialysis to obtain the acid phosphatase.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0039] FIG. 1 shows the SDS-PAGE electrophoretogram of expressed proteins, wherein M: marker; 1: non-induced cells; 2: cells induced for 12 h; 3: control cells.

[0040] FIG. 2 shows the SDS-PAGE electrophoretogram of proteins, wherein M: marker; 1: crude enzyme; 2: the eluent of protein impurities; 3: the purified enzyme.

[0041] FIG. 3 shows the HPLC chromatogram of converted products, wherein (A): Standard samples of AsA and AsA-2-P, (B): supernatant of the reaction solution (0 h), (C): supernatant of the reaction solution (8 h)

[0042] FIG. 4 shows the effect of mole ratio of AsA to PPi on AsA-2-P production.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0043] The invention will be further illustrated in more detail with reference to accompanying drawings. It is noted that, the following embodiments only are intended for purposes of illustration and are not intended to limit the scope of the invention.

[0044] Enzyme Activity Assay:

[0045] ACP activity is quantified by HPLC. One unit of ACP is defined as the amount of enzyme that catalyzes the formation of 1 mol AsA-2-P per min at 40 C. and pH4.5.

[0046] Sample preparation: the culture broth was centrifuged at 1000 rpm for 2 min and the supernatant was collect for HPLC analysis.

[0047] The concentration of AsA-2-P was determined by high performance liquid chromatography (HPLC) equipped with a C18 ODS HYPERSIL column (Agilent 1200; Agilent Technologies, Palo Alto, Calif., USA), an UV detector (model LC-9A; Shimadzu, Kyoto, Japan) at 254 nm, with mobile phase combined buffer A (0.1 M KH2PO4, pH 3.0) and buffer B (acetonitrile) on 2:1 by volume, elution at 35 C. and a flow rate of 1 mL/min. Space-time yield(g/L/h)=AsA-2-P yield (g/L)/convertion time (h).

Embodiment 1

[0048] Construction of Recombinant Strain Containing Phosphatase Gene

[0049] (1)The Pseudomonas aeruginosa ACP gene (SEQ ID NO.1, GenBanK: CP016214.1) was amplified by PCR with primers PaACP-F (SEQ ID NO. 3) and PaACP-R (SEQ ID NO. 4). The PCR cycle comprised: the first step at 94 C. for 3 minutes; 30 cycles of the second step at 94 C. for 30 seconds (denaturation), at 55 C. for 30 seconds (annealing), and at 72 C. for 1 min (elongation), and then at 72 C. for 10 min (elongation). PCR was carried out adding with LAtaq.

[0050] (2) The PCR products were cleaved at the restriction enzyme cleavage sites at both of its ends with BamH I and Hind III and then ligated to pET28a using T4 ligase at 16 C. for 10 h.

[0051] (3) The resulting pET28a-PaACP was transformed into E. coli BL21 (DE3) competent cells, and then incubated for 12 h. The pET28a-PaACP plasmid was extracted and sequence analysed.

[0052] (4) The plasmid containing the target gene was sequenced to select the strain with desired gene, i.e., the recombinant strain containing phosphatase gene E. coli BL21-PaACP.

[0053] By the similar method, genes from Staphylococcus aureus and Pseudomonas azotocolligans (with the Genbank ID CP010998.1 and E03360.1) were respectively expressed in E. coli BL21 (DE3) according to the appropriate method, and resulting recombinant strain E. coli BL21-SaACP and E. coli BL21-PazACP were harvest.

Embodiment 2

[0054] Induction Expression of Recombinant Strain

[0055] (1) The positive recombinant strain E. coli BL21-PaACP was cultivated in LB inclined medium for 12 h.

[0056] (2) A ring of seed culture was incubated in LB culture for 6 h.

[0057] (3) The seed solution of E. coli BL21-PaACP was inoculated in TB fermentation medium, when the optical density at 600 nm (OD.sub.600) reached 0.6, 0.4 mM IPTG (final concentration) was added, and induction was performed at 25 C. for 12 h, then the strains were collected and washed with sterile saline solution. The culture of recombinant strain without IPTG induction was used as the control. The SDS-PAGE electrophoretogram of protein expression is illustrated in FIG. 1, it can be seen that the phosphatase can be correctly expressed in BL21 strains, and the molecular weight is 29 kDa, and the enzyme activity is 0.3 U/mL.

[0058] (4) The E. coli BL21-SaACP and E. coli BL21-PazACP was incubated under same condition, and the resulting phosphatase activity is only 0.18 U/mL and 0.21 U/mL, separately.

Embodiment 3

Purification of Acid Phosphatase

[0059] The recombinant strain constructed in the embodiment 1 was cultured and induced according to the method of the embodiment 2, the culture broth was centrifuged to harvest the cells; the cells were washed by stroke-physiological saline solution for twice, and then suspended in 50 mM phosphate buffer (pH 7.2), and sonicated by Ultrasonic Cell Disruptor (power 285 W, ultraphonic 4 s, pause 4 s, total 10 min). The resulting solution was centrifuged at 8000 g for 30 min, supernatant filtered by a 0.45 m filter membrane was the crude enzyme. The crude enzyme was purified by Ni-NTA agarose, and washed with 0, 10, 20, 30, 40 and 50 mM imidazole respectively to remove the impurities, and washed with a 500 mM buffer solution of imidazole to specifically elute the target protein, finally gradient dialysis was performed to obtain the purified acid phosphatase (as shown in FIG. 2). The results of purification was shown in table 1, it can be seen that the specific activity of the purified enzyme produced from E. coli BL21-PaACP strains can reach 14.8 U/mg protein, and the specific activities of the purified enzymes produced from Pseudomonas azotocolligans and Flavobacterium devorans are 6.9 U/mg and 9.4 U/mg protein. Thus, the acid phosphatase of the invention has competitive advantages.

TABLE-US-00001 TABLE 1 Purification of Phosphatase Specific Total enzyme Total activity Purifi- Yield activity (U) protein(mg) (U/mg protein) cation (%) Crude 434.7 74.1 5.9 1 100 enzyme Purified 137.6 9.3 14.8 8 31.7 enzyme

Embodiment 4

[0060] Production of AsA-2-P by AsA

[0061] The substrates and purified acid phosphatase were mixed to form a 2 mL reaction system. The reaction mixture included 20 mM phosphate (pH 4.5), 100 mM AsA, 200 mM PPi and 10 L purified enzyme (60 U/mL) prepared in embodiment 3. After reacting at 40 C. for 8 h, the mixture was filtered through a 0.22 m filter and analyzed by HPLC. Two prominent peaks corresponding to AsA and AsA-2-P is shown in the HPLC chromatogram (as shown in FIG. 3). It is noted that the output of AsA-2-P reaches 11.8 g/L, the conversion rate was 46.4% and the space time yield was 1.5 g/L/h.

Embodiment 5

[0062] Effect of Mole Ratio of AsA to PPi on Production of AsA-2-P

[0063] Utilizing the similar method as the embodiment 4, the concentration of PPi in the reaction mixture was 600 mM, AsA-2-P was produced by different mole ratios of AsA to PPi (1:6, 2:6, 3:6, 4:6, 5:6.). The concentration of AsA-2-P in the supernatant of the reaction solution was determined by HPLC. When the mole ratio of AsA to PPi was 5:6, the concentration of AsA-2-P reached 54.8 g/L, the conversion was 42.9% and the space time yield was 6.9 g/L/h. When the mole ratio of AsA to PPi was 3:6, the highest conversion of AsA-2-P reached 50.8%, the concentration of AsA-2-P was 38.9 g/L and the space time yield was 4.9 g/L/h. While the mole ratio of AsA and PPi was 1:6 and 2:6, the output of AsA-2-P only reached 11.5 g/L and 24.4 g/L respectively. The results is shown in FIG. 4.

Embodiment 6

Production of AsA-2-P by Whole Cell Conversion of AsA

[0064] The strains obtained by the embodiment 2 were not purified, and the wet strains were directly used for the whole cell conversion. The concentration of PPi in the reaction mixture was 600 mM, the mole ratio of AsA: PPi was 5:6, and the conversion cycle was 8 h. When the additive amount of strains was 40-50 mg/mmol AsA, the concentration of AsA-2-P was 8.5-23.5 g/L, the conversion was 6.7-18.4% and the space time yield was 1.1-2.9 g/L/h. When the additive amount of strains was 60-100 mg/mmol AsA, the concentration of AsA-2-P reached 32.9-52.7 g/L, the conversion was 25.8-41.3% and the space time yield was 4.1-6.6 g/L/h.

[0065] The above preferred embodiments are described for illustration only, and are not intended to limit the scope of the invention. It should be understood, for a person skilled in the art, that various improvements or variations can be made therein without departing from the spirit and scope of the invention, and these improvements or variations should be covered within the protecting scope of the invention.