CN108823116B - Rhodococcus equi mutant strain for producing chitin deacetylase and application thereof - Google Patents

Abstract

The invention belongs to the technical field of biology, and particularly relates to a rhodococcus equi mutant strain F6-C7 for producing chitin deacetylase and application thereof. The chitin deacetylase high-yield strain is Rhodococcus equi F6-C7 with the preservation number of CGMCC No. 15723. The bacterial strain is obtained by ARTP and ultraviolet composite mutagenesis on the basis of an original bacterial strain Rhodococcus equi F6, the content of chitin deacetylase in fermentation liquor reaches 3400 plus 3850U/mL after fermentation for 18-24h, the speed of producing the chitin deacetylase by the bacterial strain is extremely high, the bacterial strain is a bacterial strain which is reported to produce chitin deacetylase and has the highest enzyme activity, and the bacterial strain is easy to culture in a large scale.

Description

Rhodococcus equi mutant strain for producing chitin deacetylase and application thereof

The technical field is as follows:

the invention belongs to the technical field of biology, and particularly relates to a rhodococcus equi mutant strain F6-C7 for producing chitin deacetylase and application thereof.

Background art:

chitin, also known as chitin, (1, 4) -2-acetamido-2-deoxy- β -D-glucan, is an aminopolysaccharide that is second to cellulose in nature and is found primarily in invertebrates such as shrimps, insects, algae, fungi and yeasts. However, chitin is not soluble in water, acid, alkali and organic solvents, so that it has no great commercial value, and chitosan, which is a product obtained after deacetylation, is widely applied to industries such as medicine, food, chemical industry and cosmetics. The chemical method mainly used for producing chitosan at present has a plurality of problems, such as long reaction time, high energy consumption, unstable product quality, and particularly, huge environmental pollution caused by emissions.

The chitosan product with stable deacetylation degree and narrow molecular mass distribution range can be produced by removing acetyl on the chitin by the chitin deacetylase, and a new way is provided for solving the problems of chitosan production by a chemical method. At present, only a few documents report about the research on chitin deacetylase producing bacteria at home and abroad, and the chitin deacetylase derived from microorganisms mainly takes fungi as main materials and has few bacteria. At present, the production of chitin deacetylase by microbial fermentation has a series of problems of long fermentation time, low enzyme activity and the like, and the enzyme has not been industrialized. Therefore, the breeding of the enzyme-producing strain with excellent performance is still one of the important problems for solving the industrial application of the CDA, and has important practical value and academic research value.

The invention content is as follows:

aiming at the defects of the prior art, the invention aims to provide a rhodococcus equi mutant strain F6-C7 capable of highly producing chitin deacetylase, and a fermentation method and application thereof, the rhodococcus equi mutant strain for highly producing the chitin deacetylase is a high-yield mutant strain which is obtained by screening and increasing the fermentation enzyme yield by more than 20 times on the basis of an original strain rhodococcus equi F6(CGMCC No.14861) through ARTP and ultraviolet composite mutagenesis, is a strain with the highest enzyme activity of all chitin deacetylation unit produced by fermentation in the prior art, and is easy for large-scale culture and industrial application.

The first purpose of the invention is to provide a rhodococcus equi mutant strain F6-C7 capable of highly producing chitin deacetylase, which is preserved in China general microbiological culture Collection center in 2018, 5 and 3 months, and the address is as follows: the microbial research institute of Chinese academy of sciences, No. 3 Xilu-Beijing, Chaozhou, Chao code 100101, with the collection number of CGMCC No. 15723.

The physical and chemical properties of Rhodococcus equi F6-C7 are as follows: gram-positive bacteria, wherein colonies on an LB culture medium are spherical and moist, the color of the colonies is soft pink, the optimal growth pH is 6-8, and the optimal growth temperature is 32-38 ℃. The colony picture is shown in FIG. 1.

The second purpose of the invention is to provide the application of the rhodococcus equi.

The third purpose of the invention is to provide a production method for producing chitin deacetylase by fermenting rhodococcus equi F6-C7, which comprises the following steps:

(1) seed culture

The culture conditions are as follows: the stirring speed is 160-200rpm, the temperature is 30-37 ℃, and the fermentation is carried out for 12-24 h;

seed culture medium: 5-10g/L of peptone, 3-8g/L of yeast extract powder, 5-10g/L of sodium chloride and 6.0-7.0 pHs;

(2) fermentation culture

Fermentation conditions are as follows: the inoculation amount is 2-10%, the stirring speed is 160-200rpm, the temperature is 30-40 ℃, and the fermentation is carried out for 18-24 h;

fermentation medium: 5-10g/L of yeast extract powder, 0.5-2.0g/L of glucose, 1.0g/L of magnesium sulfate, 0.3g/L of monopotassium phosphate, 1.0g/L of dipotassium phosphate, 0.5-2.0g/L of sodium chloride and pH 6.0-7.0;

after fermentation for 18-24h, the content of chitin deacetylase in the fermentation liquor reaches 3400-3850U/mL.

Has the advantages that:

the Rhodococcus equi CGMCC No.15723 provided by the invention can utilize common carbon sources and nitrogen sources to quickly carry out cell culture and accumulate a large amount of chitin deacetylase. Through condition optimization, after fermentation for 18-24h, the content of chitin deacetylase in the fermentation liquid reaches 3400-3850U/mL. Compared with the original strain, the fermentation enzyme production is improved by 22.1 times, compared with the prior art, the Rhodococcus equi CGMCC No.15723 provided by the invention can realize the rapid accumulation of chitin deacetylase, achieves the effect of high yield, and has wide industrial application prospect.

Description of the drawings:

FIG. 1F6-C7 colony picture and atomic force microscope photograph

Wherein A is a colony picture; b is atomic force microscope photograph;

FIG. 2F6-C7 high throughput screening method;

FIG. 3 rescreening results of mutagenized strains;

FIG. 4 fermentation enzyme production curves of the mutagenic highly productive strain F6-C7.

The specific implementation mode is as follows:

in order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the present patent and are not intended to limit the present invention.

Example 1 construction of high throughput screening method

Culturing original strain F6 (with the strain preservation number of CGMCC No.14861) in a triangular flask seed to prepare a bacterial suspension, then carrying out ARTP and ultraviolet composite mutagenesis, diluting the mutagenized bacterial liquid, coating the diluted bacterial liquid on an LB solid culture medium containing 200mg/L p-nitroacetanilide for culture at 37 ℃, primarily screening positively mutated strains through the size of a color development ring after 24 hours of culture, then carrying out 37 ℃ and 200r/Min fermentation culture on the strains in a 24-deep-hole plate filled with an LB liquid culture medium, after 4 hours of culture, sampling, carrying out enzyme activity detection, carrying out secondary screening on the high-yield positively mutated strains, and finally carrying out 6-batch genetic stability experiment to obtain the final high-yield mutagenized strain F6-C7.

Example 2ARTP and UV Complex mutagenesis

Taking 0.01ml of original strain F6 bacterial suspension in the center of a sterilized mutagenesis slide (8 slides are added at the same time), putting the slide into MPMS (ARTP + Uv) for mutagenesis treatment, wherein the total mutagenesis time is respectively 0s, 10s, 20s, 30s, 40s, 50s, 60s, 70s and 80s, firstly carrying out ultraviolet mutagenesis and then carrying out plasma mutagenesis, the two mutagenesis time are half respectively, and the MPMS is opened for ultraviolet sterilization for 20min in advance; taking 0.2ml of normal saline into a mutagenic slide, blowing and suspending thalli on the slide by using a gun, completely sucking out and placing the slide into a sterilized EP tube, adding 0.8ml of normal saline into the EP tube to prepare bacterial suspension diluted by 100 times, and after all slides are washed, uniformly diluting original bacteria and mutagenic bacteria to 1000 times in a gradient manner; 0.2ml of bacterial suspension diluted by 1000 times is taken and coated in a developing plate by an applicator, each mutagenesis gradient is coated with 3 plates, the developing plate is placed at 30 ℃ for inverted culture for 48 hours, the growth condition of plate bacterial colonies is observed, the plate bacterial colonies are counted, and a lethality curve is drawn. The optimal mutagenesis conditions were determined as: 25s UV mutagenesis +25s plasma mutagenesis.

EXAMPLE 3 rescreening of mutagenized high producing strains

Numbering all strains on a flat plate for re-screening, taking 1 24 deep-hole plate, adding 2ml of sterilized LB culture medium into each hole, sequentially inoculating the numbered strains on the flat plate into the deep-hole plates in sequence, and inoculating original bacteria into the last hole. Placing the deep-well plate in a 30-degree shaking table for culturing for 24 hours at 200r/min, transferring the deep-well plate to a new 24-deep-well plate added with 2ml of sterilized LB culture medium, inoculating 0.2ml of bacterial liquid to each well, culturing at 37 ℃ and 200r/min, after culturing for 4 hours, sampling 0.1ml of the deep-well plate in each well, directly placing the deep-well plate in a 96-well plate, adding 0.1ml of p-nitroacetanilide, placing the deep-well plate in a water bath at 37 ℃ for reacting for 30 minutes, taking out and measuring the light absorption value at 400 nm. The strains B4, B24, B39, C7, C26, C35, C41, C59 and C63 are subjected to 6 batches of genetic stability experiments to obtain the final high-yield mutant strain C7, and the final high-yield mutant strain is named as F6-C7.

Example 4 Rhodococcus equi mutant Strain F6-C7 enzyme production by fermentation

The Rhodococcus F6-C7 obtained in example 3 was subjected to fermentation culture in multiple batches.

(1) Seed culture

The culture conditions are as follows: stirring at 180rpm at 35 deg.C, and fermenting for 18 h;

the seed culture medium is as follows: 10g/L of peptone, 5g/L of yeast extract powder, 10g/L of sodium chloride and pH 6.0-7.0;

(2) fermentation culture

Fermentation conditions are as follows: the inoculation amount is 10 percent, the stirring speed is 180rpm, and the temperature is 35 ℃;

fermentation medium: 5g/L of yeast extract powder, 0.5g/L of glucose, 1.0g/L of magnesium sulfate, 0.3g/L of monopotassium phosphate, 1.0g/L of dipotassium phosphate, 0.5g/L of sodium chloride and pH of 6.0-7.0;

the inoculation amount is 10%, the stirring speed is 200rpm, the temperature is 37 ℃, the fermentation is carried out for 48 hours, and the fermentation liquor is taken regularly for the enzymatic activity detection of the chitin deacetylase.

The enzyme activity detection method comprises the following steps:

the fermentation liquor is centrifuged at 12000r/min for 5min, and then the bacteria are washed by phosphate buffer solution with pH 7.0 and crushed by an ultrasonic crusher under the conditions that: the power is 30%, the time is 55min after the power is started for 3s and stopped for 5 s. Then, the crude enzyme solution is obtained by centrifugation for 5min at 12000 r/min. Adding 0.3mL of crude enzyme solution into 0.3mL of 200mg/L paranitroacetanilide solution and 0.9mL of phosphate buffer solution with the pH value of 7.0, reacting at 45 ℃ for 15min, detecting the absorbance value of 400nm, and calculating the enzyme activity through a standard curve.

Definition of enzyme activity unit: the amount of enzyme required to produce 1. mu.g of p-nitroaniline per hour is defined as one unit of enzyme activity.

The fermentation enzyme production curve is shown in figure 4, the maximum enzyme activity is reached when the fermentation lasts for 24 hours, and the enzyme activity is 3836.6U/mL through determination.

Example 5 Rhodococcus equi mutant Strain F6-C7 enzyme production by fermentation

(1) Seed culture

The culture conditions are as follows: stirring at 160rpm at 30 deg.C, and fermenting for 12 hr;

seed culture medium: 5g/L of peptone, 3g/L of yeast extract powder, 5g/L of sodium chloride and pH 6.0-7.0;

(2) fermentation culture

Fermentation conditions are as follows: the inoculation amount is 2 percent, the stirring speed is 160rpm, the temperature is 30 ℃, and the fermentation is carried out for 18 hours;

fermentation medium: 7g/L of yeast extract powder, 1.0g/L of glucose, 1.0g/L of magnesium sulfate, 0.3g/L of monopotassium phosphate, 1.0g/L of dipotassium phosphate, 1.0g/L of sodium chloride and pH of 6.0-7.0;

after fermenting for 18h, the content of chitin deacetylase in the fermentation liquor reaches 3417.5U/mL.

Example 6 Rhodococcus equi mutant Strain F6-C7 enzyme production by fermentation

(1) Seed culture

The culture conditions are as follows: stirring at 200rpm at 37 deg.C, and fermenting for 24 hr;

seed culture medium: 10g/L of peptone, 8g/L of yeast extract powder, 10g/L of sodium chloride and pH 6.0-7.0;

(2) fermentation culture

Fermentation conditions are as follows: the inoculation amount is 5 percent, the stirring speed is 200rpm, the temperature is 38 ℃, and the fermentation is carried out for 20 hours;

fermentation medium: 10g/L of yeast extract powder, 2.0g/L of glucose, 1.0g/L of magnesium sulfate, 0.3g/L of monopotassium phosphate, 1.0g/L of dipotassium phosphate, 2.0g/L of sodium chloride and pH of 6.0-7.0;

after fermenting for 20h, the content of chitin deacetylase in the fermentation liquor reaches 3726.2U/mL.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the patent. It should be noted that, for those skilled in the art, various changes, combinations and improvements can be made in the above embodiments without departing from the patent concept, and all of them belong to the protection scope of the patent. Therefore, the protection scope of this patent shall be subject to the claims.

Claims (3)

1. The Rhodococcus equi for high yield of the chitin deacetylase is characterized in that the Rhodococcus equi is Rhodococcus equi (Rhodococcus equi) F6-C7 with the preservation number of CGMCC No. 15723.

2. Use of the Rhodococcus equi for producing chitin deacetylase in high yield according to claim 1 for producing chitin deacetylase.

3. The use of Rhodococcus equi for the production of chitin deacetylase according to claim 2, wherein the method for the fermentative production of chitin deacetylase comprises the following steps:

(1) seed culture

The culture conditions are as follows: the stirring speed is 160-200rpm, the temperature is 30-37 ℃, and the fermentation is carried out for 12-24 h;

seed culture medium: 5-10g/L of peptone, 3-8g/L of yeast extract powder, 5-10g/L of sodium chloride and 6.0-7.0 pHs;

(2) fermentation culture

Fermentation conditions are as follows: the inoculation amount is 2-10%, the stirring speed is 160-200rpm, the temperature is 30-40 ℃, and the fermentation is carried out for 18-24 h;

fermentation medium: 5-10g/L of yeast extract powder, 0.5-2.0g/L of glucose, 1.0g/L of magnesium sulfate, 0.3g/L of monopotassium phosphate, 1.0g/L of dipotassium phosphate, 0.5-2.0g/L of sodium chloride and pH 6.0-7.0.

Images