# US 8,785,169 B2 · Cutinase-producing genetically engineered microorganism and use thereof

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- Number: US 8,785,169 B2
- Document ID: US8785169B2
- Status: Granted
- Kind: B2
- Application number: 13/264,560
- Filed: 2011-02-28
- Published: 2012-06-14
- Granted: 2014-07-22
- Earliest claimed: 2010-12-08
- Assignees: Jiangnan University
- Applicants: Jian Chen; Jing Wu; Dan Wu; Lei Wang
- Inventors: Jian Chen; Jing Wu; Dan Wu; Lei Wang
- Web page: https://patentgrep.com/patent/US8785169B2

## CPC

- C12N 9/18: Carboxylic ester hydrolases {(3.1.1)}
- C12N 1/20: Bacteria; Culture media therefor
- C12N 1/32: Processes using, or culture media containing, lower alkanols, i.e. C1 to C6
- C12Y 301/01074: Cutinase (3.1.1.74)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12Y: ENZYMES

## Abstract

The present invention relates to the field of bioengineering and discloses a cutinase-producing genetically engineered microorganism and use thereof. Recombinant plasmid Tfu_0883-hlyAs/pET20b(+) was constructed and transformed into E. coli BL21(DE3) to obtain recombinant E. coli strain Tfu_0883-hlyAs/pET20b(+)/E. coli BL21(DE3). Specific growth rate was maintained at a certain value using fed-batch fermentation mode. After fermenting 30-34 hours, the enzyme activity in the supernatant reached 700-750 U/mL. The present invention uses glycerol as the main raw material and employs semi-synthetic medium, has the advantages of good stability and ease of control, and is suitable for large-scale production.

## Claims

### Claim 1

A cutinase-producing genetically engineered microorganism, comprising cutinase Tfu—0883 gene and hlyAs gene introduced into E. coli.

### Claim 2

A cutinase-producing genetically engineered microorganism, wherein the genetically engineered microorganism is E. coli BL21 (DE3) carrying recombinant plasmid Tfu—0883-hlyAs/pET20b(+).

### Claim 3 (depends on 1)

The genetically engineered microorganism according to claim 1, wherein the genetically engineered microorganism is constructed as follows:
1) cutinase Tfu—0883 gene is amplified using plasmid Tfu—0883/pET20b(+) as template;
2) hlyAs gene is amplified using E. coli CFT073 total DNA as template;
3) PCR amplification is performed using the PCR fragments of Tfu—0883 gene and hlyAs gene recovered by gel cutting as templates to obtain Tfu—0883-hlyAs gene;
4) pET20b(+) and Tfu—0883-hlyAs are subjected to double enzyme digestion, and the products recovered by gel cutting are transformed into E. coli JM109 competent cells to obtain plasmid Tfu—0883-hlyAs/pET20b(+);
5) the recombinant plasmid Tfu—0883-hlyAs/pET20b(+) is transformed into E. coli BL21(DE3) to obtain E. coli strain Tfu—0883-hlyAs/pET20b(+)/E. coli BL21(DE3).

### Claim 4 (depends on 1)

A method for producing cutinase from the genetically engineered microorganism according to claim 1, said method comprising the following steps:
1) during fermentation, the temperature of the fermentation medium or seed culture medium is maintained at 36-38° C., the dissolved oxygen is maintained at 20-40% by increasing or decreasing stirring rotation speed or supplying oxygen-rich air, and the pH is maintained at 7.0-7.2 in the growth stage and at 6.4-6.6 in the stage of induction of production of the enzyme by supplementing ammonia water;
2) after fermenting for 5-6 hours or when the concentration of dissolved oxygen rises to more than 70%, 500 g/L of glycerol is supplemented at an initial flow rate of 3.5-5.0 mL·L−1·h−1, and subsequently, glycerol is fed in exponential feeding mode to control the specific growth rate of the microorganism in the range of 0.15-0.3 h−1;
3) after fermenting for 12-13 hours or when OD600 reaches 25-35, IPTG is added at a final concentration of 0.02-0.04 mM/L for induction, and at the same time 50 g/L of lactose is supplemented at a rate of 8-10 mL·L−1·h−1 and the supplementation of ammonia water is stopped until the pH drops to 6.4-6.6, at which time the supplementation of ammonia water is resumed to control the pH at 6.4-6.6;
4) after fermenting for 15-17 hours or inducing for 3-4 hours, glycerol is fed at a constant flow rate of 25-35 mL·L−1·h−1;
5) after fermenting for 19-21 hours or when OD600 no longer increases, the flow rate of lactose is decreased to 2-4 mL·L−1·h−1, and the flow rate of glycerol is gradually decreased to half of that in step 4) over 6-8 hours.

### Claim 5 (depends on 4)

The method according to claim 4, wherein the seed culture medium or fermentation medium consists of 10 g/L of peptone, 5 g/L of yeast powder, 10 g/L of NaCl and 100 mg/L of ampicillin.

### Claim 6 (depends on 5)

The method according to claim 5, wherein the fermentation medium consists of 1 g/L of peptone, 2 g/L of yeast powder, 4 g/L of (NH4)2HPO4, 13.5 g/L of KH2PO4, 4.1 g/L of MgSO4.7H2O, 0.85 g/L of citric acid, 8 g/L of glycerol, 5 mL/L of a solution of trace elements and 100 mg/L of ampicillin.

### Claim 7 (depends on 6)

The method according to claim 6, wherein the solution of trace elements consists of 5 M/L of HCl, 10 g/L of FeSO4.7H2O, 2.25 g/L of ZnSO4.7H2O, 1.0 g/L of CuSO4.5H2O, 0.5 g/L of MnSO4.4H2O, 0.23 g/L of Na2B4O7.10H2O, 2.0 g/L of CaCl2.2H2O and 0.1 g/L of (NH4)6Mo7O24.