# US 10,308,919 B2 · Method for preparing ascorbic acid-2-phosphate using a recombinant strain

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- Number: US 10,308,919 B2
- Document ID: US10308919B2
- Status: Granted
- Kind: B2
- Application number: 15/386,588
- Filed: 2016-12-21
- Published: 2018-03-01
- Granted: 2019-06-04
- Earliest claimed: 2016-08-31
- Assignees: Jiangnan University
- Applicants: Jiangnan University
- Inventors: Liming Liu; Wei Song; Jing Wu; Kai Zheng; Xiulai Chen
- Web page: https://patentgrep.com/patent/US10308919B2

## CPC

- C12N 9/16: acting on ester bonds (3.1)
- C12N 15/70: Vectors or expression systems specially adapted for E. coli
- C12P 17/04: containing a five-membered hetero ring, e.g. griseofulvin {, vitamin C}
- C12Y 301/03002: Acid phosphatase (3.1.3.2)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}
- C12Y: ENZYMES

## 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 pH 4.5 under 40° C. for 8 h, the output of ascorbic acid-2-phosphate reaches 54.8 g/L, the conversion is 42.9% and the space time yield is 6.9 g/L/h.

## Claims

### Claim 1

A method for preparing ascorbic acid-2-phosphate, comprising:
providing a recombinant strain for producing an acid phosphatase by overexpressing the acid phosphatase gene shown in SEQ ID NO. 1 in Escherichia coli via a vector, wherein the acid phosphatase gene is amplified from Pseudomonas aeruginosa and the Escherichia coli is Escherichia coli BL21 (DE3) and the acid phosphatase has a specific activity of about 14.8 U/mg protein; and
reacting ascorbic acid with pyrophosphate under the catalytic conversion of the recombinant strain, to obtain the ascorbic acid-2-phosphate,
wherein the molar ratio of ascorbic acid and pyrophosphate is 4:6.

### Claim 2 (depends on 1)

The method as claimed in claim 1, wherein the conversion is performed at pH 3-7 under a temperature of 30-50° C.

### Claim 3 (depends on 1)

The method as claimed in claim 1, 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.

### Claim 4 (depends on 2)

The method as claimed in claim 2, 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.

### Claim 5 (depends on 1)

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

### Claim 6 (depends on 2)

The method as claimed in claim 2, wherein the acid phosphatase produced by the recombinant strain is used for the conversion, and the amount of the acid phosphatase is 0.5-1 U/mmol ascorbic acid.

### Claim 7 (depends on 1)

The method as claimed in claim 1, wherein when the recombinant strain is cultured to an OD600 of 0.4-0.6, and IPTG is added to induce the expression of the acid phosphatase.

### Claim 8 (depends on 5)

The method as claimed in claim 5, wherein the acid phosphatase is purified by Ni-NTA agarose by the steps of: removing impurities from the acid phosphatase by using 0, 10, 20, 30, 40 and 50 mM imidazole; eluting the acid phosphatase by using a 500 mM buffer solution of imidazole; and then performing gradient dialysis to obtain the acid phosphatase.

### Claim 9 (depends on 1)

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