A strain of Yarrowia lipolytica producing chitin deacetylase and its application

Abstract

The invention discloses a chitin deacetylase-producing Yarrowia lipolytica and its application, belonging to the field of genetic engineering technology. The chitin deacetylase-producing Yarrowia lipolytica is classified and named as Yarriwia lipolytica OUC‑DAC‑LAn(S)XAr(S)3‑4, deposited in China Center for Type Culture Collection, with a deposit number of CCTCC NO: M 20242369 and a deposit date of October 29, 2024. The use of the chitosan deacetylase-producing Yarrowia lipolytica in chitosan deacetylation. The chitosan deacetylase-producing Yarrowia lipolytica of the present invention has a high deacetylation effect on chitosan. Experiments have shown that after the strain is treated with a dual-carbon fermentation system, α The acetyl content of chitin can be reduced by up to 19.95%, which is an extremely significant effect.

Description

Technical Field

[0001] The invention relates to a chitin deacetylase-producing Yarrowia lipolytica and application thereof, belonging to the technical field of genetic engineering. Background Art

[0002] Chitin is composed of many N -Acetylamino-D-glucose and very small amounts of amino-D-glucose β -(1,4)-Long-chain polymers connected by glycosidic bonds, there are three subtypes: α -Chitosan, β -Chitosan and γ -Chitin. In the structure of chitin N The CN single bond of the -acetylamino group can be broken to undergo deacetylation reaction. Generally, when the degree of deacetylation (DD) is greater than 50%, it is considered to be chitosan. The activity and solubility of chitosan are better than those of chitin.

[0003] Chitosan is rarely distributed in nature and mainly comes from the deacetylation of chitin. Therefore, a green and efficient chitin deacetylation method is urgently needed. Microbial fermentation is a process that uses the metabolic activities of microorganisms to convert organic matter into useful substances. Using microbial fermentation to produce chitin deacetylase and deacetylate chitin is a feasible and effective way. lipolytica ) is a representative unconventional yeast, a generally recognized as safe (GRAS) microorganism, and has been proven to be able to grow using acetic acid as a carbon source. Therefore, Yarrowia lipolytica can be used to construct a genetically engineered bacterium that produces chitin deacetylase. Summary of the invention

[0004] In view of the above-mentioned prior art, the present invention provides a strain of Yarrowia lipolytica producing chitin deacetylase and application thereof, belonging to the technical field of genetic engineering.

[0005] The present invention is achieved through the following technical solutions:

[0006] A strain of Yarrowia lipolytica producing chitin deacetylase, taxonomically named Yarriwia lipolytica OUC-DAC-LAn(S)XAr(S)3-4 is deposited in the China Center for Type Culture Collection, with the deposit number being CCTCC NO: M20242369 and the deposit date being October 29, 2024.

[0007] The biological characteristics of the chitin deacetylase-producing Yarrowia lipolytica are as follows: the aerobic yeast is cultured on a YPD agar medium, the colonies are red, wrinkled, and have hairy edges; the cells have three forms: yeast type, hyphae type, and pseudohyphae type.

[0008] The chitin deacetylase-producing Yarrowia lipolytica is used in chitin deacetylation.

[0009] Furthermore, the chitosan is α -Chitosan ( α -chitin) or colloidal chitin.

[0010] Furthermore, in a specific application, the deacetylation method of chitosan is as follows: inoculating Yarrowia lipolytica producing chitosan deacetylase into a culture medium containing chitosan, fermenting and culturing the culture medium to obtain a fermentation broth, and centrifuging the culture medium to obtain deacetylated chitosan.

[0011] Furthermore, the specific method can be: the seed liquid of Yarrowia lipolytica producing chitin deacetylase is inoculated at an inoculation rate of 2% to 5% (volume percentage) α -chitosan-acetic acid double carbon fermentation medium, cultured at 30°C and 220 rpm for 5 days; the fermentation culture solution was taken, centrifuged and the supernatant was discarded, and the obtained solid was the deacetylated chitosan.

[0012] Said α -Chitosan-acetic acid double carbon fermentation medium preparation method is: based on the preparation of 100 mL, take 2 g α -Chitosan, 0.5 g ammonium sulfate, 0.319 g dipotassium hydrogen phosphate and 0.4 g potassium dihydrogen phosphate, mix, add 96 mL deionized water, sterilize; add 0.44 mL acetic acid, and adjust the pH to 6 with sodium hydroxide, and then add trace element solution and vitamin solution to obtain.

[0013] The chitin deacetylase-producing Yarrowia lipolytica of the present invention is constructed by using Yarrowia lipolytica PO1h (a host strain suitable for expressing foreign proteins in the prior art) as a starting strain, using pMT015 plasmid as an expression vector, and transferring chitin deacetylase into An CDA and chitin deacetylase Ar CE4, then increased the copy number of chitin deacetylase gene and overexpressed the expansion protein ScExlx1, and finally screened a chitin deacetylase-producing Yarrowia lipolytica. The experiment showed that it had a high deacetylation effect on chitin. After the strain was treated with a dual-carbon fermentation system, α -The acetyl content of chitin can be reduced by up to 19.95%, which is an extremely significant effect.

[0014] The present invention establishes a new chitosan deacetylation method based on microorganisms, and provides a method and idea for the preparation of high-quality chitosan products and environmentally friendly industrial chitosan deacetylation technology. The Yarrowia lipolytica producing chitosan deacetylase of the present invention has the potential for industrial application and has good application prospects.

[0015] Various terms and phrases used herein have the general meanings that are well known to those skilled in the art. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The Yarrowia lipolytica producing chitin deacetylase of the present invention is classified and named as Yarriwia lipolytica OUC-DAC-LAn(S)XAr(S)3-4 is deposited in the China Center for Type Culture Collection with the deposit number CCTCC NO: M 20242369. The deposit date is October 29, 2024. The deposit address is: Wuhan University, Bayi Road, Wuchang District, Wuhan City, Hubei Province, Postal Code: 430072.

[0017] Figure 1 : OD of each transformant in Example 1 600 Comparison chart.

[0018] Figure 2 : OD of each transformant in Example 2 600 Comparison chart.

[0019] Figure 3 : Comparison of chitin deacetylase activity of each transformant in Example 3.

[0020] Figure 4 : Schematic diagram of the results of deacetylation degree determination in Example 4.

[0021] Figure 5 : Schematic diagram of the results of deacetylation degree determination in Example 5. DETAILED DESCRIPTION

[0022] The present invention will be further described below in conjunction with the embodiments. However, the scope of the present invention is not limited to the following embodiments. It will be appreciated by those skilled in the art that various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention.

[0023] The instruments, reagents, and materials involved in the following examples, unless otherwise specified, are all conventional instruments, reagents, and materials available in the prior art and can be obtained through regular commercial channels. The experimental methods, detection methods, etc. involved in the following examples, unless otherwise specified, are all conventional experimental methods and detection methods available in the prior art.

[0024] The present invention adoptsα -Chitin, purchased from Sigma-Aldrich (USA).

[0025] The shake flask fermentation method used in the present invention is: the genetically engineered bacteria seed liquid is inoculated with 2% to 5% of the inoculation amount into 50 mL of α -Chitosan-acetic acid double carbon fermentation medium was placed in a 250 mL shake flask and cultured at 30°C and 220 rpm for 5 days.

[0026] Said α -Chitosan-acetic acid double carbon fermentation medium preparation method is: based on the preparation of 100 mL, weigh 2 g α -chitoxin, 0.5 g ammonium sulfate, 0.319 g dipotassium hydrogen phosphate and 0.4 g potassium dihydrogen phosphate, mix, add 92 mL deionized water, sterilize (high temperature and high pressure 115°C, 30 min); after cooling slightly, add 0.44 mL acetic acid, adjust the pH to 6 with sodium hydroxide (Note: acetic acid and sodium hydroxide are mixed to prepare a solution, pass through a 0.22 μm aqueous phase filter membrane, and then add it to the culture medium); then add trace element solution and vitamin solution to obtain.

[0027] The preparation method of the trace element solution is as follows: based on the preparation of 100 mL, 0.45 g of calcium chloride dihydrate, 0.45 g of zinc sulfate heptahydrate, 0.3 g of ferrous sulfate heptahydrate, 0.1 g of manganese chloride tetrahydrate, 0.04 g of sodium molybdate, 0.03 g of cobalt chloride hexahydrate and 0.01 g of copper sulfate pentahydrate are added into deionized water, and the volume is adjusted to 100 mL to obtain the solution.

[0028] The vitamin solution is prepared as follows: based on the preparation of 100 mL, 0.005 g of biotin is dissolved in 2 mL of 0.1 mol / L sodium hydroxide solution, 90 mL of deionized water is added, the pH is adjusted to 6.5 with hydrochloric acid, 0.1 g of calcium pantothenate, 0.1 g of nicotinic acid, 0.1 g of nicotinic acid ammonium sulfate, 0.1 g of nicotinic acid pyridoxine and 0.02 g of p-aminobenzoic acid are added, the pH is adjusted to 6.5, 2.5 g of inositol is added, the pH is adjusted to 6.5, the volume is fixed to 100 mL, and the solution is stored at 4°C.

[0029] The preparation method of the inorganic nitrogen source-acetic acid carbon source culture medium adopted by the present invention is as follows: based on the preparation of 100 mL, 0.5 g of ammonium sulfate, 0.319 g of dipotassium hydrogen phosphate and 0.4 g of potassium dihydrogen phosphate are weighed, mixed, 92.5 mL of deionized water is added, and sterilized (high temperature and high pressure 115°C, 30 min); after slightly cooling, 2 mL of acetic acid is added, and the pH is adjusted to 6 with sodium hydroxide (Note: acetic acid and sodium hydroxide are mixed to prepare a solution, passed through a 0.22 μm aqueous phase filter membrane, and then added to the culture medium); then a trace element solution and a vitamin solution are added to obtain the culture medium.

[0030] The method for recovering chitosan from fermentation culture broth of the present invention comprises the following steps: taking the fermentation culture broth, centrifuging it at 8000 g for 2 min, discarding the supernatant, and obtaining the chitosan as a solid.

[0031] The method for determining the enzymatic activity of chitin deacetylase of the present invention is as follows: using p-nitroacetanilide as a substrate, determining the activity of chitin deacetylase. During the reaction, 3 mL of 50 mM Tris-HCl with a pH of 8 is first added as a reaction buffer, 1 mL of the fermentation broth supernatant and 1 mL of a 200 mg / L p-nitroacetanilide solution are added, the mixture is shaken and mixed, incubated in a 37°C water bath for 15 min, and the reaction is terminated in a boiling water bath. Deionized water is added to make the volume 10 mL, centrifuged at 8000 g for 10 min, the supernatant is aspirated, and its absorbance is determined at 400 nm. The blank control is to inactivate the crude enzyme and then add it to the reaction system.

[0032] The calculation formula of extracellular enzyme activity was as follows: (1), and one unit of CDA was defined as the activity that catalyzes the release of 1 μg of p-nitroaniline per hour from p-nitroacetanilide under standard assay conditions.

[0033] Formula (1);

[0034] Where, T is the enzyme-catalyzed substrate reaction time, h; k is the linear coefficient of the standard curve (0.4494); A 400 is the absorbance value after the crude enzyme catalyzes the substrate; A0 is the absorbance value of the blank control group.

[0035] The method for determining the degree of deacetylation adopted by the present invention is: substrate α -Chitosan was washed with ultrapure water for 2-3 times, placed in a 40℃ oven for 24-48 hours, the dry solid was weighed, dissolved with excess hydrochloric acid, and titrated with 0.5 mol / L sodium hydroxide solution. The pH was recorded every 0.25 mL. The interval was shortened near the pH jump point, and the pH was recorded every 0.1 mL added. A pH-V potentiometric titration curve was made, and two jump points were found. The degree of deacetylation was calculated according to formula (2).

[0036] 100% Formula (2);

[0037] Wherein, ∆V is the volume of sodium hydroxide standard titration solution consumed between the two “jump points”, mL; c1 is the concentration of sodium hydroxide standard titration solution, M; 16 is the molar mass of amino group, g / mol; m1 is the mass of the sample after fermentation and drying, g.

[0038] Example 1 Construction of genetically engineered Yarrowia lipolytica expressing chitin deacetylase

[0039] Using Yarrowia lipolytica PO1h as the starting strain, chitin deacetylase was introduced An CDA and chitin deacetylase Ar CE4. For specific operations, see below.

[0040] (1) Chitin deacetylase from Aspergillus nidulans An CDA, the nucleotide sequence of its coding gene after codon optimization is shown in SEQ ID NO.1. Chitin deacetylase from marine bacteria Ar CE4, the nucleotide sequence of its coding gene after codon optimization is shown in SEQ ID NO.2.

[0041] (2) Signal peptide prediction was performed using SignalP-5.0, and it was found that both enzymes carry signal peptides, among which chitin deacetylase An The gene encoding the signal peptide of CDA is shown in SEQ ID NO.3, chitin deacetylase Ar The gene encoding the signal peptide of CE4 is shown in SEQ ID NO.4.

[0042] To improve the secretion of chitin deacetylase, the endogenous signal peptides of the two enzymes were removed and An The exogenous signal peptide Lip2, which is commonly used for secretion in Yarrowia lipolytica, was added to CDA to construct the recombinant gene Lip2-AnCDA , in chitin deacetylase Ar The exogenous signal peptide XPR2, which is commonly used for secretion in Yarrowia lipolytica, was added to CE4 to construct the recombinant gene XPR2-ArCE4 The nucleotide sequence of the gene encoding the signal peptide Lip2 is shown in SEQ ID NO.5, and the nucleotide sequence of the gene encoding the signal peptide XPR2 is shown in SEQ ID NO.6.

[0043] (3) The pMT015 plasmid was used as the expression vector. Its two expression cassettes with promoter and terminator were P GPD -T LIP1 and P TEF -T XPR2The promoter is followed by the kozak sequence "GCCACC". The selection marker of the pMT015 plasmid is URA3, and the promoter and terminator are P LEU2 and T LEU2 , with LoxP sites at both ends. Lip2-AnCDA and recombinant genes XPR2-ArCE4 The P TEF -T XPR2 and P GPD -T LIP1 The expression cassette was cloned and the pMT015-LAn(S)-ura3-XAr(S) plasmid was obtained.

[0044] (4) The LAn(S)-ura3-XAr(S) expression cassette in the pMT015-LAn(S)-ura3-XAr(S) plasmid was linearized by PCR and transferred into Yarrowia lipolytica PO1h by PEG / LiAc chemical transformation to obtain a genetically engineered Yarrowia lipolytica strain expressing chitin deacetylase.

[0045] Pick a single colony with good growth, mix it with 10 μL of water, take out 1 μL as a template for PCR verification, and prepare seed solution for the successful transformants. Inoculate it into 50 mL of inorganic nitrogen source-acetic acid carbon source medium and culture it at 30℃ and 220 rpm for 5 days. Select 10 successful transformants and number them 1 to 10 of the DAC-LAn(S)XAr(S)1 series strains. Compare the OD values ​​of each transformant on the 5th day of culture. 600 , the results are as follows Figure 1 As shown in the figure, the transformants numbered DAC-LAn(S)XAr(S)1-4 had an OD of 0.0447 on the 5th day of cultivation. 600 The value is the largest, which is significantly better than other transformants, indicating that this strain has the best growth effect in acetic acid as the carbon source medium and has a strong ability to utilize acetic acid. Therefore, the transformant DAC-LAn(S)XAr(S)1-4 was selected as the optimal strain in the DAC-LAn(S)XAr(S)1 series.

[0046] Example 2 Construction of a genetically engineered strain of Yarrowia lipolytica with increased chitin deacetylase gene copy number

[0047] On the basis of the genetically engineered bacteria DAC-LAn(S)XAr(S)1-4 screened in Example 1, the copy number of the chitin deacetylase gene was increased. The specific operation is as follows.

[0048] (1) Using pMT015 plasmid as expression vector, the recombinant gene Lip2-AnCDA and recombinant genes XPR2-ArCE4The P GPD -T LIP1 and P TEF -T XPR2 The expression cassette was cloned and the pMT015-XAr(S)-ura3-LAn(S) plasmid was obtained.

[0049] (2) The pUB4-CRE plasmid was transferred into the genetically engineered bacteria DAC-LAn(S)XAr(S)1-4 using the PEG / LiAc chemical transformation method to remove the URA3 selection marker so that it can be reused repeatedly.

[0050] (3) The XAr(S)-ura3-LAn(S) expression cassette in the pMT015-XAr(S)-ura3-LAn(S) plasmid is linearized by PCR and transferred into the genetically engineered bacteria obtained in step (2) by PEG / LiAc chemical transformation to obtain a genetically engineered Yarrowia lipolytica bacteria with an increased copy number of the chitin deacetylase gene.

[0051] Pick a single colony with good growth, mix it with 10 μL of water, take out 1 μL as a template for PCR verification, and prepare seed solution for the successful transformant. Inoculate it into 50 mL of inorganic nitrogen source-acetic acid carbon source medium and culture it at 30℃ and 220 rpm for 5 days. Select 10 successful transformants and number them 1 to 10 of the DAC-LAn(S)XAr(S)2 series strains. Compare the OD values ​​of each transformant on the 5th day of culture. 600 , the results are as follows Figure 2 As shown in the figure, the transformant numbered DAC-LAn(S)XAr(S)2-4 had an OD of 0.0447 on the 5th day of cultivation. 600 The value is the largest, indicating that this strain has the best growth effect in the medium with acetic acid as the carbon source and has a strong ability to utilize acetic acid. Therefore, the transformant DAC-LAn(S)XAr(S)2-4 was selected as the optimal strain in the DAC-LAn(S)XAr(S)2 series.

[0052] Example 3 Construction of genetically engineered Yarrowia lipolytica strains overexpressing expansin

[0053] Based on the genetically engineered bacteria DAC-LAn(S)XAr(S)2-4 screened in Example 2, the expansin was overexpressed. The specific operation is as follows.

[0054] (1) Derived from Schizophyllum Schizophyllum commune )of ScExlx1 The gene was obtained by searching the NCBI website. According to the preference of Yarrowia lipolytica, it was commissioned to Jiutian Gene Technology (Tianjin) Co., Ltd. for codon optimization, and the nucleotide sequence is shown in SEQ ID NO.7.

[0055] (2) Using pMT015 plasmid as expression vector, ScExlx1 The gene was constructed into the pMT015 plasmid by seamless cloning. TEF -T XPR2 The expression cassette was cloned and the pMT015-XPR2-ScExlx1-ura3 plasmid was obtained.

[0056] (3) The pUB4-CRE plasmid was transformed into the genetically engineered bacterium DAC-LAn(S)XAr(S)2-4 using the PEG / LiAc chemical transformation method to remove the URA3 selection marker so that it can be reused.

[0057] (4) The XPR2-ScExlx1 expression cassette in the pMT015-XPR2-ScExlx1-ura3 plasmid is linearized by PCR, and the expression cassette is transferred into the genetically engineered bacteria obtained in step (3) by PEG / LiAc chemical transformation to obtain genetically engineered Yarrowia lipolytica bacteria that overexpress expansin.

[0058] The 5 successful transformants were selected by shake flask fermentation and numbered as 1 to 5 of the DAC-LAn(S)XAr(S)3 series strains. The chitin deacetylase activities of the transformants were compared. The results are as follows: Figure 3 As shown, it can be seen that the transformant DAC-LAn(S)XAr(S)3-4 has the best effect, which is significantly better than other transformants.

[0059] Example 4 Treatment with genetically engineered bacteria α -Chitosan

[0060] The genetically engineered bacteria screened in Examples 1, 2, and 3 were subjected to double-carbon fermentation treatment and the degree of deacetylation was determined. The specific operation is as follows.

[0061] (1) Ferment the genetically engineered bacteria DAC-LAn(S)XAr(S)1-4 in a shake flask and recover α -Chitin, the degree of deacetylation was determined, and the results were as follows Figure 4 The results showed that after being treated with the dual-carbon fermentation system of genetically engineered bacteria DAC-LAn(S)XAr(S)1-4, α -The acetyl content of chitin changed from 69.82% to 54.46%, and the acetyl content decreased by 15.36%.

[0062] (2) Ferment the genetically engineered bacteria DAC-LAn(S)XAr(S)2-4 in a shake flask and recover α -Chitin, the degree of deacetylation was determined, and the results were as follows Figure 4As shown, the results showed that after being treated with the genetically engineered bacteria DAC-LAn(S)XAr(S)2-4 dual-carbon fermentation system, α -The acetyl content of chitin changed from 69.82% to 51.99%, and the acetyl content decreased by 17.83%.

[0063] (3) Ferment the genetically engineered bacteria DAC-LAn(S)XAr(S)3-4 in a shake flask and recover α -Chitin, the degree of deacetylation was determined, and the results were as follows Figure 4 As shown, the results showed that after being treated with the genetically engineered bacteria DAC-LAn(S)XAr(S)3-4 dual-carbon fermentation system, α -The acetyl content of chitin changed from 69.82% to 49.87%, and the acetyl content decreased by 19.95%.

[0064] By comparison, it can be seen that the deacetylation effect of the genetically engineered bacteria DAC-LAn(S)XAr(S)3-4 is the best.

[0065] The genetically engineered bacteria DAC-LAn(S)XAr(S)3-4 were preserved in the China Center for Type Culture Collection and named Yarriwia lipolytica OUC-DAC-LAn(S)XAr(S)3-4, the deposit number is CCTCC NO: M20242369, the deposit date is October 29, 2024, and the deposit address is: Wuhan University, Bayi Road, Wuchang District, Wuhan City, Hubei Province, Postal Code: 430072.

[0066] Example 5 Treatment of colloid chitin by genetically engineered bacteria DAC-LAn(S)XAr(S)3-4

[0067] The chitin gel was treated with the genetically engineered bacteria DAC-LAn(S)XAr(S)3-4 to determine the degree of deacetylation. The specific operation is as follows.

[0068] (1) Preparation of chitosan: weigh 2.5 g α -Chitosan was dissolved in 100 mL of hydrochloric acid (concentration 12 mol / L) and stirred thoroughly at 37°C for 45 min. Then 1 L of water was added, mixed evenly, and allowed to stand at 4°C for 12 hours. The solid was separated by vacuum filtration and the filtrate was discarded to obtain colloidal chitosan. The colloidal chitosan was washed with distilled water 5 times, dissolved in an appropriate amount of distilled water, and the pH was adjusted to neutral with 0.2 mol / L sodium hydroxide solution to prepare a colloidal chitosan solution with a concentration of 10% (w / v, g / mL). The 10% colloidal chitosan solution was stored at 4°C for later use.

[0069] (2) Preparation of colloidal chitosan-acetic acid dual-carbon fermentation medium: Based on the preparation of 100 mL, take 20 mL of 10% colloidal chitosan solution, 0.5 g of ammonium sulfate, 0.319 g of potassium dihydrogen phosphate and 0.4 g of potassium dihydrogen phosphate, mix, add 77 mL of deionized water, and sterilize: high temperature and high pressure at 115°C for 30 min. After cooling slightly, add 0.44 mL of acetic acid, adjust the pH to 6 with sodium hydroxide (Note: acetic acid and sodium hydroxide are mixed to prepare a solution, filtered through a 0.22 μm aqueous phase filter, and then added to the culture medium), and then add trace element solution and vitamin solution to obtain the product.

[0070] (3) The genetically engineered bacteria DAC-LAn(S)XAr(S)3-4 were inoculated into the above-prepared gelatin chitosan-acetic acid double carbon fermentation medium and cultured at 30°C and 220 rpm for 5 days. The gelatin chitosan was recovered and its deacetylation degree was determined. The results were as follows: Figure 5 As shown, the results showed that after being treated with the genetically engineered bacteria DAC-LAn(S)XAr(S)3-4 dual-carbon fermentation system, the acetyl content of gelatin chitin changed from 59.76% to 37.41%, and the acetyl content decreased by 22.35%.

[0071] The above examples are provided to those skilled in the art to fully disclose and describe how to make and use the claimed embodiments, and are not intended to limit the scope of the disclosure herein. Modifications that are obvious to those skilled in the art will fall within the scope of the appended claims.

Claims

1. A strain of Yarrowia lipolytica producing chitin deacetylase, characterized in that: Classification name: Yarriwia lipolytica OUC-DAC-LAn(S)XAr(S)3-4 is deposited in the China Center for Type Culture Collection, with the deposit number being CCTCC NO: M 20242369 and the deposit date being October 29, 2024.

2. Use of the Yarrowia lipolytica producing chitin deacetylase according to claim 1 in chitin deacetylation.

3. The use according to claim 2, characterized in that: The chitosan is α -Chitosan or chitosan gel.

4. The use according to claim 2, characterized in that: The chitosan deacetylation method comprises the following steps: inoculating Yarrowia lipolytica producing chitosan deacetylase into a chitosan-containing culture medium, fermenting and culturing to obtain a fermentation culture solution, and centrifuging to obtain deacetylated chitosan.

5. The use according to claim 4, characterized in that: The seed liquid of Yarrowia lipolytica producing chitin deacetylase was inoculated at a rate of 2% to 5%. α -Chitosan-acetic acid double carbon fermentation medium, cultured at 30°C and 220 rpm for 5 days; the fermentation culture was taken, centrifuged and the supernatant was discarded, and the obtained solid was the deacetylated chitosan; Said α -Chitosan-acetic acid double carbon fermentation medium preparation method is: based on the preparation of 100 mL, take 2 g α -Chitosan, 0.5 g ammonium sulfate, 0.319 g dipotassium hydrogen phosphate and 0.4 g potassium dihydrogen phosphate, mix, add 96 mL deionized water, sterilize; add 0.44 mL acetic acid, and adjust the pH to 6 with sodium hydroxide, and then add trace element solution and vitamin solution to obtain.

Images