# US 2019/0376153 A1 · Pseudomonas sp. and a preparation method and application of bifunctional enzyme preparation of pseudomonas sp.

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- Number: US 2019/0376153 A1
- Document ID: US20190376153A1
- Status: Published application
- Kind: A1
- Application number: 16/312,309
- Filed: 2018-06-12
- Published: 2019-12-12
- Earliest claimed: 2017-06-28
- Assignees: Ecology Institute, Shandong Academy of Sciences
- Applicants: Ecology Institute, Shandong Academy of Sciences
- Inventors: Lei JI; Qiang ZHANG; Guanhong CHEN; Jianing WANG; Xiaowen FU; Fanyong SONG; Tianyuan LI
- Web page: https://patentgrep.com/patent/US20190376153A1

## CPC

- C12N 1/205: Bacterial isolates
- A62D 3/02: by biological methods, i.e. processes using enzymes or microorganisms
- C12N 1/20: Bacteria; Culture media therefor
- A62D 2101/22: containing halogen
- C12N 2500/74: from fungi, e.g. yeasts
- C12R 2001/38: Pseudomonas
- C12R 2001/40: Pseudomonas putida
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A62D: CHEMICAL MEANS FOR EXTINGUISHING FIRES OR FOR COMBATING OR PROTECTING AGAINST HARMFUL CHEMICAL AGENTS; CHEMICAL MATERIALS FOR USE IN BREATHING APPARATUS
- C12R: INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS

## Abstract

A Pseudomonas sp. ECO-1 strain was preserved at the China General Microbiological Culture Collection Center (CGMCC) on Mar. 31, 2017, with the preservation number of CGMCC No. 13960. The Pseudomonas sp. ECO-1 strain was separated from POPs (Persistent Organic Pollutants) polluted soil for the first time. The bifunctional enzyme preparation capable of efficiently degrading polychlorinated biphenyl and atrazine was prepared by utilizing the strain for the first time; especially, the bifunctional enzyme preparation has remarkable degradation activity on the polychlorinated biphenyl which is difficult to degrade under an aerobic condition, which is completely different from functions of existing known Pseudomonas sp. and enzyme preparations thereof.

## Claims

### Claim 1

A Pseudomonas sp. ECO-1 strain deposited at the China General Microbiological Culture Collection Center (CGMCC) under CGMCC accession number CGMCC No. 13960.

### Claim 2 (depends on 1)

A method for producing a bifunctional enzyme preparation by using the Pseudomonas sp. ECO-1 strain according to claim 1, comprising the following steps:
(1) scribing the Pseudomonas sp. ECO-1 strain on an LB solid medium, and carrying out inverted activation culture at 28-37° C. for 1-2 days to prepare an activated strain;
(2) inoculating the activated strain prepared in Step (1) into the LB liquid medium, and carrying out a shake culture for 1-2 days under the conditions of a temperature of 28-37° C. and a rotation speed of 100-200 rpm to prepare a seed solution;
(3) inoculating the seed solution prepared in Step (2) into an inorganic salt medium at a volume percentage of 1-10%, and carrying out enlargement culture under the conditions of a temperature of 28-37° C. and a rotation speed of 100-200 rpm for 3-5 days to prepare a Pseudomonas sp. ECO-1 solution; and
(4) centrifuging the Pseudomonas sp. ECO-1 solution prepared in Step (3) at a rotation speed of 3,000-10,000 rpm for 2-10 minutes, collecting thalli, and suspending them in a 10-30-fold by volume phosphate buffer solution with the pH of 6.0-8.0, carrying out ultrasonic cell disruption, centrifuging under the conditions of a temperature of 4-25° C. and a rotation speed of 3,000-8,000 rpm for 2-5 minutes, and collecting the supernatant to obtain a bifunctional enzyme preparation.

### Claim 3 (depends on 2)

The method of claim 2, wherein the LB solid medium in Step (1) comprises the following components per liter:
10 g of peptone, 5 g of a yeast extract, 10 g of sodium chloride, 20 g of agar, and the balance of water, with a natural pH.

### Claim 4 (depends on 2)

The method of claim 2, wherein the LB liquid medium in Step (2) comprises the following components per liter:
10 g of peptone, 5 g of yeast extract, 10 g of sodium chloride, and the balance of water, with a natural pH.

### Claim 5 (depends on 2)

The method of claim 2, wherein the inorganic salt medium in Step (3) comprises the following components per liter:
0.5 g of monopotassium phosphate, 0.5 g of disodium hydrogen phosphate, 0.2 g of magnesium sulfate, 0.1 g of calcium chloride, 0.2 g of sodium chloride, 1.0 g of ammonium sulfate, 2.0 g of peptone, 0.5 g of biphenyl, 0.025 g of atrazine and the balance of water, with a pH of 7.0.

### Claim 6 (depends on 2)

The method of claim 2, wherein the ultrasonic cell disruption in Step (4) is carried outer under the following conditions:
the disruption time/gap time is 2 s/2 s, the total time is 17 min, and the power is 165 W.

### Claim 7 (depends on 2)

A process for degrading polychlorinated biphenyls and atrazine by a bifunctional enzyme that is prepared by a method of claim 2.

### Claim 8 (depends on 7)

The process of claim 7, comprising the following steps:
taking the prepared bifunctional enzyme preparation, adjusting the concentrations of polychlorinated biphenyls and/or atrazine in the system to 0.5-5 mg/L and 50-500 mg/L, respectively, adding the bifunctional enzyme preparation until the bifunctional enzyme preparation concentration in the system reaches 0.05-0.25 g/L, and reacting for 3-24 h at a temperature of 20-37° C.