CN111705014B

CN111705014B - Arthrobacter protoformiae CDA2-2-2 and method for producing chitin deacetylase by using same

Info

Publication number: CN111705014B
Application number: CN202010505809.5A
Authority: CN
Inventors: 房耀维; 张晓彤; 刘姝; 杨杰; 陈佳雨; 李泽信; 卢静; 焦豫良
Original assignee: Jiangsu Ocean University
Current assignee: Jiangsu Ocean University
Priority date: 2020-06-05
Filing date: 2020-06-05
Publication date: 2022-07-08
Anticipated expiration: 2040-06-05
Also published as: CN111705014A

Abstract

The invention discloses an arthrobacter protoformis CDA2-2-2 and a method for producing chitin deacetylase by the arthrobacter protoformis CDA2-2-2, wherein the bacterial strain is gram-positive brevibacterium, is stored in China general microbiological culture Collection center (CGMCC) 12.12.2018, and has the storage number of CGMCC NO.16932, the GenBank accession number of a 16S rDNA sequence of the chitinase is MH978647, and discloses a method for producing the chitin deacetylase by the bacterial strain CDA 2-2-2.

Description

Arthrobacter protoformiae CDA2-2-2 and method for producing chitin deacetylase by using same

Technical Field

The invention relates to the field of microorganisms, in particular to arthrobacter vitronectici CDA2-2-2 and a method for producing chitin deacetylase by using the same.

Background

Chitin is a renewable polysaccharide next to cellulose on earth, is generally present in exoskeletons, cuticles and cell walls of fungi of invertebrates, is a product obtained by partially or completely removing acetyl groups in chitin molecules, has greatly improved solubility compared with chitin due to a large number of free amino groups in the molecules, has biodegradability, and is widely applied to the fields of food, medicine, light industry, printing and dyeing, environmental protection, agriculture and the like. At present, the method for industrially preparing chitosan mainly removes acetyl by hot concentrated alkali, so that the problem of serious environmental pollution exists, and the removal degree of acetyl in the obtained chitosan product is inconsistent.

The chitosan produced by the enzyme method has mild reaction conditions and relatively low energy consumption value, and can obtain products with uniform and stable deacetylation degree, so that the production of chitosan by using chitin deacetylase is a future development direction of the industry. Most of currently disclosed chitin deacetylases are terrestrial fungi, the enzyme production level is low, the optimum catalysis temperature is high, and few reported chitin deacetylases derived from bacteria can only catalyze chitosan oligosaccharide deacetylation but cannot catalyze crystal chitin deacetylation; in addition, the bacteria have more advantages than the fungi in the aspect of fermentation enzyme production, so the obtained bacteria with higher fermentation level and chitin deacetylase production lay the foundation for the industrial catalytic preparation of chitosan by using an enzyme method.

Disclosure of Invention

The invention provides a marine Arthrobacter protovitriolus (Arthrobacter protophorae) CDA2-2-2 and a method for producing chitin deacetylase by using the strain, aiming at the defects of the prior art, the Arthrobacter protovitriolus (Arthrobacter protophorae) CDA2-2-2 is obtained by screening sea mud from the Hakko island sea area in the Cauchun city in 2017 in 9 and 5 days in 2018, the strain is preserved in the common microorganism center of China Committee for culture Collection of microorganisms in 12 and 12 days in 2018, and the preservation number is CGMCC NO.16932, the unit address of the strain is No. 3 of the Xilu No.1 of Beijing sunward region in Beijing, the GenBank accession number of the 16S rDNA sequence is MH978647, and the specific technical scheme is as follows:

an Arthrobacter protoformiae (Arthrobacter protophorae) CDA2-2-2, characterized in that: the strain is gram-positive brevibacterium, has no spore, grows on 2216E culture medium, has a round colony, is yellow and semitransparent, has a wet surface, regular edges, no halo, a central bulge and a diameter of 0.1-0.5mm, and is easy to pick; the strain is positive in oxygen, gram stain, gelatin hydrolysis, catalase, indole test and glucose fermentation, is negative in spore, flagellum stain, starch hydrolysis, nitrate reduction and H2S production, and is preserved in the general microbiological center of China general microbiological culture Collection center in 12 months and 12 days in 2018 by the CDA2-2-2 of Arthrobacter protoformis (Arthrobacter protophorae) with the preservation number of CGMCC NO.16932 and the GenBank accession number of the 16S rDNA sequence of the strain is MH 978647.

A method for producing chitin deacetylase from Arthrobacter protoformiae (Arthrobacter protophorae) CDA2-2-2 is characterized in that: the method comprises the following steps:

(1) preparing a seed culture medium and a fermentation culture medium;

(2) inoculating Arthrobacter protozogenes (Arthrobacter protophorae) CDA2-2-2 into a seed culture medium for culturing to obtain a seed solution;

(3) inoculating the seed liquid into a fermentation culture medium in an inoculation amount of 3%, and culturing to obtain chitin deacetylase.

Further, the seed culture medium is prepared by using aged seawater, the pH value is 7.0, and the seed culture medium also comprises 0.1% of yeast powder and 0.5% of peptone.

Further, the fermentation medium is prepared from aged seawater, has pH of 7.0, and also comprises chitin 0.5%, yeast powder 1%, glucose 0.5%, and MgSO₄ 0.01％，K₂HPO₄ 0.1％，KH₂PO₄ 0.03％。

Further, in the step (2), the solution volume is 30%, and the culture is performed at 180rpm and 30 ℃ for 24 hours.

Further, in the step (3), the culture is carried out for 48h at 140rpm and 30 ℃, and after the culture is finished, the chitin deacetylase crude solution is obtained by centrifugation for 10min at 12000 Xg and 4 ℃.

Compared with the prior art, the invention has the following advantages:

the culture scheme of fermentation culture of chitin deacetylase adopted by the invention can ferment to obtain chitin deacetylase with higher fermentation level, is prepared by using oceanic Arthrobacter protoformiae, has higher enzyme production level and mild catalysis temperature compared with the existing terrestrial fungi, has higher economic value and social value, and is worthy of popularization.

Drawings

FIG. 1 shows gram stain (. times.1000) of strain CDA2-2-2(Arthrobacter protophoriae).

FIG. 2 shows the color change circle formed by strain CDA2-2-2(Arthrobacter protophoriae) on the primary screening plate.

FIG. 3 is a phylogenetic tree of strain CDA2-2-2(Arthrobacter protophoriae).

FIG. 4 shows the effect of carbon source on the enzyme production of strain CDA2-2-2(Arthrobacter protophoriae).

FIG. 5 shows the effect of nitrogen source on the enzyme production of strain CDA2-2-2(Arthrobacter protophoriae).

FIG. 6 shows the effect of metal ions on the enzyme production of strain CDA2-2-2(Arthrobacter protophoriae).

FIG. 7 shows the effect of inoculum size on the enzyme production by fermentation of strain CDA2-2-2(Arthrobacter protophoriae).

FIG. 8 shows the effect of liquid loading on enzyme production by fermentation of strain CDA2-2-2(Arthrobacter protophoriae).

FIG. 9 shows the effect of fermentation temperature on the enzyme production by fermentation of strain CDA2-2-2(Arthrobacter protophoriae).

FIG. 10 shows the effect of the initial pH of the medium on the enzyme production by fermentation of strain CDA2-2-2(Arthrobacter protophoriae).

FIG. 11 shows the effect of rotation speed on the enzyme production by fermentation of strain CDA2-2-2(Arthrobacter protophoriae).

FIG. 12 shows the effect of fermentation time on the enzyme production by fermentation of strain CDA2-2-2(Arthrobacter protophoriae).

FIG. 13 is MgSO₄The effect of the interaction with the fermentation temperature on the enzymatic activity of chitin deacetylase.

FIG. 14 is MgSO₄The effect of interaction with glucose on the enzymatic activity of chitin deacetylase.

FIG. 15 shows the effect of fermentation temperature interaction with glucose on chitin deacetylase enzymatic activity.

Detailed Description

The present invention will be described in detail with reference to the accompanying drawings.

Arthrobacter protoformiae (Arthrobacter protophorae) CDA 2-2-2:

the morphological characteristics are as follows: the strain is gram-positive brevibacterium without spores, as shown in figure 1; the strain grows for 3 days at 25 ℃ on a 2216E culture medium, the bacterial colony is circular, yellow and semitransparent, the surface is wet, the edge is regular, no halo is generated, the center is protruded, the diameter is 0.1-0.5mm, and the strain is easy to pick; a yellow transparent circle can be generated on the screening medium containing the p-nitro-N-acetanilide and the powdered chitin, as shown in FIG. 2;

the physiological and biochemical characteristics are as follows: the strain is positive in oxygen, gram stain, gelatin hydrolysis, catalase, indole test and glucose fermentation, and negative in spore and flagellum stain, starch hydrolysis, nitrate reduction and H2S production. The results of the physiological and biochemical tests are shown in table 1, wherein in table 1, +: positive, -: negative;

TABLE 1 results of physiological and biochemical tests of CDA2-2-2 Strain

(III) amplification and analysis of 16S rDNA sequence: extracting the genome of CDA2-2-2 by using an Axygen kit, and selecting a universal primer for amplifying a prokaryotic microorganism 16S rDNA sequence to react in a PCR mix system, wherein the universal primer for the PCR reaction comprises 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' and 1492R: 5'-GGTTACCTTGTTACGACTT-3', respectively; the reaction system (50 μ L) was: premix (25. mu.L), ddH2O (22. mu.L), upstream and downstream primers (1. mu.L each), DNA template (1. mu.L); reaction procedures are as follows: denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 30s, annealing at 54 ℃ for 30s, extension at 72 ℃ for 90s, 35 cycles, and final extension at 72 ℃ for 5 min; GenBank (accession number MH978647) is submitted after sequence sequencing; homology alignment of this sequence with sequences in the GenBank database revealed 99% similarity to the 16S rDNA of the strain Arthrobacter protophoriae (accession No.: EU294415.1), phylogenetic tree showing the closest relationship between strain CDA2-2-2 and Arthrobacter protophoriae, as shown in FIG. 3.

A method for producing chitin deacetylase by fermenting Arthrobacter protoformiae (Arthrobacter protophorae) CDA2-2-2 comprises the following steps:

preparing a seed culture medium: preparing aged seawater, 0.1% of yeast powder and 0.5% of peptone, and controlling the pH value to be 7.0;

(II) preparing a fermentation medium: adopts aged seawater, chitin 0.5%, yeast powder 1%, glucose 0.5%, and MgSO 0.5%₄0.01％，K₂HPO₄ 0.1％，KH₂PO₄0.03 percent of the preparation, and the pH value is controlled to be 7.0;

(III) preparing a seed solution: inoculating a single colony of Arthrobacter protoformiae (Arthrobacter protophorae) CDA2-2-2 to a seed culture medium by 30% liquid loading amount, and culturing at 30 ℃ and 140rpm for 24h to obtain a seed solution;

(IV) Arthrobacter protoformiae (Arthrobacter protophorae) CDA2-2-2 and optimization of single factor for chitin deacetylase production by Arthrobacter protoformiae (Arthrobacter protophorae) CDA2-2-2 fermentation:

(1) influence of carbon Source on the enzyme production by the strain CDA2-2-2(Arthrobacter protophoriae): inoculating the seed solution to a fermentation medium with 3 percent of inoculation amount, wherein the initial pH value of the medium is 7.0, the liquid loading amount is 30 percent, respectively adding different carbon sources to ferment for 48 hours under the conditions of 30 ℃ and 180rpm, sampling and measuring the enzyme activity, researching the influence of the carbon sources on the enzyme production of the strain CDA2-2-2(Arthrobacter protophorae), and showing that the enzyme production is highest when glucose is added, as shown in figure 4.

(2) Effect of nitrogen source on the enzyme production of the strain CDA2-2-2(Arthrobacter protophorae): inoculating the seed solution to a fermentation medium with 3 percent of inoculation amount, wherein the initial pH value of the medium is 7.0, the liquid loading amount is 30 percent, respectively adding different nitrogen sources to ferment for 48 hours under the conditions of 30 ℃ and 180rpm, sampling and measuring the enzyme activity, and researching the influence of the nitrogen sources on the enzyme production of the strain CDA2-2-2(Arthrobacter protophorae), wherein the result shows that the enzyme production is highest when yeast powder is added, as shown in figure 5.

(3) Inorganic salt couple strain CDA2-2-2(Arthrobacter protophorm)iae) effects of enzyme production: inoculating the seed liquid to a fermentation culture medium with 3 percent of inoculation amount, wherein the initial pH of the culture medium is 7.0, the liquid loading amount is 30 percent, respectively adding different metal ions at 30 ℃ and 180rpm, fermenting for 48 hours, sampling, determining enzyme activity, researching the influence of inorganic salt on the enzyme production of a strain CDA2-2-2(Arthrobacter protophorae), and indicating that the result shows that MgSO (MgSO) is added₄The enzyme production was highest at that time, as shown in FIG. 6.

(4) Effect of the inoculum size on the enzyme production of the strain CDA2-2-2(Arthrobacter protophoriae): inoculating the seed liquid to a fermentation culture medium with different inoculation amounts, wherein the initial pH of the culture medium is 7.0, the liquid loading amount is 30%, and the enzyme activity is measured after fermentation is carried out for 48 hours at 30 ℃ and 180rpm, and the result shows that the enzyme yield is highest when the inoculation amount is 2%, as shown in figure 7.

(5) Effect of the liquid charge on the enzyme production of the strain CDA2-2-2(Arthrobacter protophoriae): the seed solution was inoculated to the fermentation medium at 20%, 30%, 40%, 50%, 60% inoculum size, the enzyme activity was measured after fermentation at 180rpm for 48h at medium initial ph7.0, 30 ℃, the results showed that the enzyme production was highest at 40% liquid loading, as shown in fig. 8.

(6) Influence of the fermentation temperature on the enzyme production of the strain CDA2-2-2(Arthrobacter protophoriae): the seed solution was inoculated to the fermentation medium at 3% inoculum size, the medium had an initial ph of 7.0 and a liquid loading of 30%, and the fermentation was carried out at 180rpm for 48h at different temperatures, showing that the highest enzyme production was achieved at 35 ℃ and a higher enzyme production was achieved at 30 ℃ to 90% of the highest enzyme production, as shown in fig. 9.

(7) Effect of the initial pH of the medium on the enzyme production of the strain CDA2-2-2(Arthrobacter protophoriae): inoculating the seed solution to a fermentation medium by 3 percent of inoculation amount, measuring the liquid loading amount to be 30 percent, adjusting different initial pH values of the fermentation medium under the conditions of 30 ℃ and 180rpm, and measuring the enzyme activity after fermenting for 48 hours, wherein the result shows that the highest enzyme yield is achieved when the pH value is 7.0, and the result is shown in figure 10.

(8) Effect of the rotational speed on the enzyme production of the strain CDA2-2-2(Arthrobacter protophoriae): inoculating the seed liquid to a fermentation culture medium by 3 percent of inoculation amount, measuring the enzyme activity after fermenting the seed liquid for 48 hours at different rotating speeds under the condition of 30 ℃ and the initial pH value of the culture medium of 7.0 and the liquid loading amount of 30 percent, and the result shows that the enzyme yield is the highest when the rotating speed is 140rpm, as shown in figure 11.

(9) Influence of fermentation time on the enzyme production of the strain CDA2-2-2(Arthrobacter protophoriae): inoculating the seed liquid to a fermentation culture medium by 3 percent of inoculation amount, wherein the initial pH value of the culture medium is 7.0, the liquid loading amount is 30 percent, fermenting the seed liquid at the conditions of 30 ℃ and 180rpm, sampling every 12 hours and measuring the enzyme activity, and the result shows that the enzyme yield is the highest in 84 hours, as shown in figure 12.

(10) The method for measuring the activity of chitin deacetylase comprises the following steps: 3mL of 0.05mol/L pH7.0 phosphate buffer solution pre-insulated at 30 ℃, 1mL of 200mg/L p-nitroacetanilide aqueous solution and 1mL of enzyme solution are added into the test tube, the reaction is carried out in 30 ℃ water bath for 15min, the enzymatic reaction is stopped in boiling water bath, the mixture is centrifuged at 3000 Xg for 10min, and the absorbance of the supernatant is measured. As a control, 1mL of an enzyme solution inactivated in a boiling water bath of the same concentration for 15min was added. Definition of enzyme activity unit (U): the amount of enzyme required to produce 1. mu.g of p-nitroaniline per hour under the above reaction conditions was defined as one unit of enzyme activity.

(V) the PB experiment design preferably selects significant factors influencing the enzyme activity: selecting glucose, soybean meal and MgSO (MgSO) on the basis of single-factor test₄The fermentation time, the fermentation temperature, the initial pH of a culture medium, the liquid loading amount, the inoculation amount and the rotating speed are considered factors, each factor is designed to have high (1) and low (-1)2 levels (table 1), Design-Expert V8.0.6 software is used for carrying out PB test Design for 12 times (see table 2), data analysis is carried out on 9 factors respectively, the significant factors influencing the relative enzyme activity are determined, and the results are shown in tables 2 to 4:

TABLE 2 Plackett-Burman test factor levels

TABLE 3Plackett-Burman test design and results

Code	Variables of	t value	p value	Ranking of importance
					X₁	Glucose (100%)	3.47	0.0152	3
X₂	Soybean flour (100%)	-0.11	0.9256	8
					X₃	MgSO4(100％)	4.65	0.0069	1
X₄	Fermentation time (h)	1.07	0.2599	9
					X₅	Fermentation temperature (. degree.C.)	3.65	0.013	2
X₆	Initial pH of the culture Medium	-1.05	0.2701	6
					X₇	Liquid loading amount (100%)	0.2	0.8566	7
X₈	Inoculum (100%)	-1.46	0.1475	4
					X₉	Rotating speed (rpm)	-0.04	0.9744	5

TABLE 4 Plackett-Burman test design analysis of variance

The data in Table 3 were subjected to significance testing using Design-Expert V8.0.6 software, and the results are shown in Table 4. As can be seen from Table 4, MgSO₄The fermentation temperature and the glucose have obvious influence on the relative enzyme activity, and the significance is MgSO₄(X₃)>Fermentation temperature (X)₅)>Glucose (X)₁) (ii) a Other factors have no significant influence on the relative enzyme activity. Therefore MgSO was chosen₄(X₃) Fermentation temperature (X)₅) Glucose (X)₁) As a factor in the investigation of the response surface model.

(VI) optimizing the fermentation condition of producing chitin deacetylase by the strain CDA2-2-2 by a response surface method: determination of preferred MgSO based on PB testing results₄(A) 3 factors of fermentation temperature (B) and glucose (C) are used as variables, enzyme activity is used as a response value, Design of Experiments is used for designing Box-Behnken response surface analysis tests of 17 test points with 3 factors and 3 levels, as shown in Table 5, regression analysis is carried out on test data to obtain a quadratic polynomial equation:

Y＝12.8+0.35A+2.03B+1.73C-0.55AB-0.41AC+0.85BC-2.16A²-1.44B²-1.91C²

TABLE 5 Box-Behnken test design and results

TABLE 6 regression analysis of variance table for chitin deacetylase enzymatic activity

Note: -is not significant, -is very significant

As can be seen from Table 6: the F value can be obtained, and the influence sequence of all factors in the first term on the enzymatic activity of the chitin deacetylase isA>C>B. Analysis of variance of chitin deacetylase enzymatic activity yielded model P<0.01, showing that the equation model is extremely obvious, and the difference among different treatments is extremely obvious; missimilitude term P>0.05, showing that the model distortion item is not significant; r of the model²And the value is 0.965, which shows that the model can explain the change of 96.5% response value, so that the model has good fitting degree and small experimental error and can be used for predicting the optimal fermentation condition parameters.

The three-dimensional figures 12-14 of the response surface show that the response surface is open downward, and the response values increase and then decrease with the value of the respective variable, indicating that the model has a prominent stable point, and that the stable point is the maximum value of the model. The concrete embodiment is as follows: glucose 0.5%, when MgSO₄The enzyme activity is maximum when the fermentation temperature is between 35.5 and 37.5 ℃ and between 0.015 and 0.022 percent, as shown in figure 12; at a fermentation temperature of 35 ℃ in MgSO₄0.015% -0.023% of the total weight of the enzyme, 0.53% -0.59% of glucose and the maximum enzyme activity are shown in figure 13; MgSO (MgSO)₄When the concentration is 0.1%, the fermentation temperature is 35.5-38 ℃, the glucose is 0.53-0.59%, and the enzyme activity is the maximum, as shown in figure 14.

TABLE 7 normalized analysis of the stabilization points

The fermentation process conditions were optimized using the response surface method, and as shown in table 7, the optimal media composition was obtained as follows: MgSO (MgSO) in vitro₄0.01 percent, the fermentation temperature is 37.76 ℃, the glucose is 0.57 percent, and the predicted value of the enzyme activity produced by fermentation is 14.29U/mL. In order to ensure the convenience and feasibility of the test, a little correction is made on each condition, MgSO₄The results of 0.01 percent, 38 ℃ of fermentation temperature and 0.57 percent of glucose in a verification test show that the fermentation level of the chitin deacetylase is 14.58U/mL, and the error is relative to the theoretical value<5%, indicating that the equation fits well.

Therefore, the optimal culture medium formula of the strain CDA2-2-2 for producing the chitin deacetylase is as follows: prepared by using aged seawater, and controlling the initial pH to be 7.0, the chitin to be 0.5 percent, the yeast powder to be 1 percent, the glucose to be 0.57 percent and the MgSO₄ 0.01％，K₂HPO₄ 0.1％，KH₂PO₄0.03%, and the optimal culture conditions are as follows: the inoculation amount is 3 percent, the temperature is 30 ℃, and the fermentation is carried out for 48 hours at 140 rpm.

The above examples of the present invention are merely examples for clearly illustrating the present invention and are not intended to limit the embodiments of the present invention. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. Not all embodiments are exhaustive. All obvious changes and modifications which are obvious to the technical scheme of the invention are covered by the protection scope of the invention.

Claims

1. Arthrobacter protoformiae (A. glaber)Arthrobacter protophormiae) CDA2-2-2, characterized by: the strain is gram-positive brevibacterium, has no spore, grows on 2216E culture medium, has a round colony, is yellow and semitransparent, has a wet surface, regular edges, no halo, a central bulge and a diameter of 0.1-0.5mm, and is easy to pick; can produce yellow transparent circles on a screening culture medium containing p-nitro-N-acetanilide and powdered chitin, and the strain is positive in oxygen, gram stain, gelatin hydrolysis, catalase, indole test and glucose fermentation, and negative in spore, flagella stain, starch hydrolysis, nitrate reduction and H2S production, and the Arthrobacter protoformiae (Arthrobacter protoformiae) (A. the strain is a strain with no toxicity, No. one or No. oneArthrobacter protophormiae) The CDA2-2-2 is preserved in the China general microbiological culture Collection center in 2018, 12 months and 12 days, the preservation number is CGMCC No.16932, and the GenBank accession number of the 16S rDNA sequence is MH 978647.

2. The Arthrobacter protoformiae of claim 1, (b)Arthrobacter protophormiae) A method for producing chitin deacetylase from CDA2-2-2 is characterized by comprising the following steps: the method comprises the following steps:

(1) preparing a seed culture medium and a fermentation culture medium;

(2) inoculating Arthrobacter protoformiae CDA2-2-2 into a seed culture medium for culture to obtain a seed solution;

(3) inoculating the seed liquid into a fermentation culture medium in an inoculation amount of 3%, and culturing to obtain the chitin deacetylase.

3. Arthrobacter protoformiae (A) according to claim 2 (A)Arthrobacter protophormiae) A method for producing chitin deacetylase from CDA2-2-2 is characterized by comprising the following steps: the seed culture medium is prepared from aged seawater, the pH value is 7.0, and the seed culture medium also comprises 0.1% of yeast powder and 0.5% of peptone.

4. Arthrobacter protoformiae (A) according to claim 2 (A)Arthrobacter protophormiae) A method for producing chitin deacetylase from CDA2-2-2 is characterized by comprising the following steps: the fermentation culture medium is prepared from aged seawater with pH of 7.0, and also comprises chitin 0.5%, yeast powder 1%, glucose 0.5%, and MgSO 0.5%₄0.01%，K₂HPO₄0.1%，KH₂PO₄0.03%。

5. Arthrobacter protoformiae (A) according to claim 2 (A)Arthrobacter protophormiae) A method for producing chitin deacetylase from CDA2-2-2 is characterized by comprising the following steps: the liquid loading amount in the step (2) is 30%, and the culture is carried out for 24h under the conditions of 180rpm and 30 ℃.

6. Arthrobacter protoformiae (A.vitronella) of claim 2 (A.vitronella), (B.vitronella) and (C.vitronella) aArthrobacter protophormiae) A method for producing chitin deacetylase from CDA2-2-2 is characterized by comprising the following steps: culturing for 48h at 140rpm and 30 ℃, and centrifuging for 10min at 12000 Xg and 4 ℃ after the culture is finished to obtain chitin deacetylase crude liquid.