# US 12,180,528 B2 · Strain for producing lipase and application thereof

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- Number: US 12,180,528 B2
- Document ID: US12180528B2
- Status: Granted
- Kind: B2
- Application number: 17/754,062
- Filed: 2019-10-31
- Published: 2022-10-20
- Granted: 2024-12-31
- Earliest claimed: 2019-09-27
- Assignees: Changshu Institute of Technology
- Applicants: Changshu Institute of Technology
- Inventors: Bin Qi; Limei Wang; Manting Qi; Tiantian Xu; Yamei Ji; Zhenghong Li
- Web page: https://patentgrep.com/patent/US12180528B2

## CPC

- C12P 23/00: Preparation of compounds containing a cyclohexene ring having an unsaturated side chain containing at least ten carbon atoms bound by conjugated double bonds, e.g. carotenes (containing heterorings C12P17/00)
- C12N 1/20: Bacteria; Culture media therefor
- C12N 1/205: Bacterial isolates
- C12N 9/20: Triglyceride splitting, e.g. by means of lipase
- C12P 7/6436: Fatty acid esters
- C12R 2001/125: Bacillus subtilis {; Hay bacillus; Grass bacillus}
- C12Y 301/01003: Triacylglycerol lipase (3.1.1.3)
- 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)}
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12R: INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12Y: ENZYMES

## Abstract

The present invention relates to a lipase-producing strain and application thereof. The strain is classified and named Bacillus subtilis CS1802, with a preservation number of CCTCC NO: M2018262. The strain can be used to produce vitamin A palmitate through whole-cell transformation of vitamin A and palmitic acid. The Bacillus subtilis CS1802 of the present invention is derived from traditional natural fermented food and is a microorganism generally recognized as safe. The strain can be easily cultured and preserved. The highest content of vitamin A palmitate obtained through whole-cell transformation of vitamin A and palmitic acid is 15.35 mg/L. The highest transformation efficiency is 76.75%. The strain provides a new path for enzymatic synthesis of vitamin A palmitate and has important application prospects.

## Claims

### Claim 1

A method for production of vitamin A palmitate by a lipase-producing strain classified and named Bacillus subtilis CS 1802, with a deposit number of CCTCC NO: M2018262, the method comprising:
(1) inoculating the strain CS1802 into a beef extract peptone liquid medium and culturing in a shaking apparatus;
(2) inoculating the culture from step (1) into a fermentation medium, culturing in a shaking apparatus, centrifuging the fermentation broth, and collecting the supernatant as the fermented bacterial solution;
(3) dissolving vitamin A and palmitic acid at a 1:1 mass ratio in n-hexane to produce an organic phase system; and
(4) centrifuging the fermented bacterial solution from step (2) to collect the strain CS1802 and inoculating the strain CS1802 into the organic phase system from step (3) for fermentation to produce vitamin A palmitate.

### Claim 2 (depends on 1)

The method according to claim 1, wherein the fermentation medium in step (2) consists of: tryptone 10 g/L, yeast powder 5 g/L, NaCl 10 g/L, MgSO4·7H2O 1 g/L, KH2PO4 0.5 g/L, K2HPO4 0.5 g/L, and olive oil emulsion 12 ml/L, wherein the olive oil emulsion is prepared by mixing polyvinyl alcohol (PVA) and olive oil at a volume ratio of 3:1.

### Claim 3 (depends on 1)

The method according to claim 1, wherein each concentration of vitamin A and palmitic acid is 10-25 g/L, respectively.

### Claim 4 (depends on 1)

The method according to claim 1, wherein the strain CS1802 are inoculated into the organic phase system and fermented for 0.5-2 hours in step (4).

### Claim 5 (depends on 1)

The method according to claim 1, wherein each concentration of vitamin A and palmitic acid is 15 g/L, respectively.

### Claim 6 (depends on 4)

The method according to claim 4, wherein the strain CS1802 are inoculated into the organic phase system and fermented for 1 hour in step (4).