Strain for producing chitin deacetylase and application thereof

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to a bacterial strain for producing chitin deacetylase and application of the bacterial strain. The invention relates to a bacterial strain for producing chitin deacetylase, which is bacillus firmus, and is preserved in China General Microbiological Culture Collection Center (CGMCC) on October 30, 2024, and the preservation number is CGMCC NO.32397. The invention further discloses a method for producing the chitin deacetylase. According to the present invention, the chitin deacetylase is produced by using the chitin deacetylase producing strain screened from mangrove forest soil, the chitin deacetylase is applied to the biological preparation of chitosan, and compared with the currently researched chitin deacetylase, the chitin deacetylase produced by the strain has advantages of high activity, wide application range and industrial production potential.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to a strain producing chitin deacetylase and application thereof. Background Art

[0002] Chitin is a polysaccharide substance that is very abundant in nature. It is widely found in shrimp and crab shells and is a common waste from seafood processing. After the chitin is deacetylated, when the deacetylation degree reaches more than 50%, it is chitosan, which has good biocompatibility, safety and many biological activities, such as anti-inflammatory, antibacterial, hypoglycemic, anti-tumor, etc., and has great application value in the fields of food, medicine, materials, etc. The traditional method of preparing chitosan generally uses shrimp and crab shells as raw materials and then treats them with concentrated alkali to obtain the target product. The specific process is as follows: shrimp shell, crab shell → crushing → acid leaching demineralization → water washing and filtration → dilute alkali deproteinization → oxidation decolorization → washing and filtration → concentrated alkali deacetylation → water washing to neutral → drying → chitosan.

[0003] The traditional concentrated alkali removal conditions are relatively harsh, generally using 40%-50% (w / v) sodium hydroxide solution at 80-100°C. This process will cause serious environmental pollution and has great production safety risks. The development of environmentally friendly, safe, and easy-to-industrial bioenzymatic chitosan preparation technology is the main direction to break through the bottleneck of chitosan preparation.

[0004] In the past 30 years, the research on chitin deacetylase has gradually become a hot topic at home and abroad. Enzymatic deacetylation has many advantages over traditional hot alkaline deacetylation. Its reaction conditions are mild and the amount of alkali used can be greatly reduced, avoiding serious environmental pollution and production safety hazards. Every year in my country, a large amount of shrimp and crab shell waste is not fully utilized, resulting in a waste of resources. Therefore, the development of bio-enzyme method to prepare chitosan is of great significance for the high-value utilization of waste. With the deepening of research on chitin deacetylase, its application in industry can also be gradually realized. Summary of the invention

[0005] The purpose of the present invention is to provide a strain producing chitin deacetylase and application thereof.

[0006] To achieve the above object, the present invention provides a technical solution: a chitin deacetylase-producing strain, the strain being Bacillus firmus Cytobacillus firmus , and was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on October 30, 2024, with the deposit number CGMCC NO.32397.

[0007] The invention also provides an application of a strain producing chitin deacetylase, wherein the strain is used for preparing chitin deacetylase.

[0008] The present invention also provides another application of a strain producing chitin deacetylase, wherein the strain uses chitin as a substrate to prepare chitosan by enzymatic method.

[0009] The present invention further provides a chitin deacetylase, comprising the strain or its fermentation liquid.

[0010] The present invention further provides a method for preparing chitin deacetylase, comprising inoculating the strain into an LB liquid culture medium to obtain a strain seed culture solution; inoculating the seed culture solution into a chitin deacetylase producing culture medium at a ratio of 1% to 3% V / V, and fermenting and culturing at 30 to 40° C. for 24 to 72 hours to obtain a fermentation solution rich in chitin deacetylase.

[0011] Preferably, the chitin deacetylase production medium is as follows: carbon source addition amount 1%-3% w / v, nitrogen source addition amount 1%-3% w / v, potassium dihydrogen phosphate addition amount 0.01%-0.2% w / v, dipotassium hydrogen phosphate addition amount 0.01%-0.2% w / v, magnesium sulfate addition amount 0.1%-0.3% w / v, sodium chloride addition amount 0.05%-0.2% w / v, the balance is water, pH value 6-7.5, sterilized at 121°C for 15 minutes, and cooled to room temperature.

[0012] Preferably, the nitrogen source is one or a mixture of beef extract, yeast extract, ammonium sulfate, and peptone; and the carbon source is one or a mixture of colloidal chitin, glucose, sucrose, and glycerol.

[0013] The present invention further provides a method for preparing chitosan by enzymatic method, wherein colloidal chitin is added to the fermentation broth obtained by the above method in an amount of 2%-5% W / V, mixed and reacted in a water bath at 25-45° C. for 48-72 hours to obtain chitosan from which the chitin acetyl group has been removed.

[0014] Preferably, the colloidal chitin is chitin treated with a deep eutectic solvent, concentrated hydrochloric acid, sodium hydroxide and urea solution; the added amount of chitin is 2%-4% W / V.

[0015] Preferably, the deep eutectic solvent is a solution prepared by mixing choline chloride and urea in a molar ratio of 1:2.

[0016] Compared with the prior art, the present invention has the following advantages: 1. The Bacillus firmus obtained in the present invention is isolated from mangrove soil, has simple culture conditions, and has strong adaptability and survival ability. After fermentation culture, the bacteria in the fermentation liquid can be removed by centrifugation; 2. The preparation process of the chitin deacetylase-containing Bacillus firmus fermentation broth obtained by the present invention is simple, the energy consumption is extremely low, the required raw materials are simple and easy to obtain, and the culture and fermentation can be carried out under mild conditions; 3. The chitin deacetylase-containing fermentation liquid obtained by the present invention has good chitin deacetylase activity and stability, can be stored in a low temperature environment (4°C~-20°C), has obvious deacetylase activity, and is active for chitin after various pretreatments. The process conditions are mild, the required energy consumption is low, and the pollution level is low. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Agarose gel electrophoresis of 16S rDNA of the screening strain provided in Example 1 of the present invention; Figure 2 The infrared spectrum of chitin after deacetylation treated with a mixture of choline chloride and urea provided in Example 2 of the present invention; Figure 3 The infrared spectrum of chitin after deacetylation after being treated with concentrated hydrochloric acid provided in Example 3 of the present invention; Figure 4 The infrared spectrum of chitin after deacetylation treated with a mixed solution of sodium hydroxide and urea provided in Example 4 of the present invention; Figure 5 The chitin deacetylated after being treated with a mixture of choline chloride and urea provided in Example 2 of the present invention 13 CNMR spectrum; Figure 6 The chitin deacetylated after being treated with concentrated hydrochloric acid provided in Example 3 of the present invention 13 CNMR spectrum; Figure 7 The chitin deacetylated after being treated with a mixed solution of sodium hydroxide and urea provided in Example 4 of the present invention 13 CNMR spectrum; Figure 8 Chitin used as a reference in Examples 2, 3 and 4 of the present invention 13 CNMR spectrum. DETAILED DESCRIPTION

[0018] The present invention is further described below in conjunction with specific embodiments and the accompanying drawings. More details are set forth in the following description to facilitate a full understanding of the present invention. However, the present invention is obviously capable of being implemented in a variety of other ways different from the description herein. For those skilled in the art, any substitution, improvement or change made to the embodiments of the present invention is within the protection scope of the present invention, and the protection scope of the present invention should not be limited by the content of this specific embodiment.

[0019] Example 1 Isolation and identification of strains 1. Isolation and purification of strains Soil filtrate from mangrove Kandelia candel was diluted 10 -4 After 2 times, the strain was spread on the screening medium to obtain a single colony of the strain, and the single colony of the strain was inoculated in LB liquid medium to obtain the strain seed culture solution; the seed culture solution was inoculated in the re-screening acetylase medium at a ratio of 1% (v / v), and the fermentation culture was carried out at 35°C for 48 hours before centrifugation (10000rpm, 10 minutes), and the precipitate was washed with 2% glacial acetic acid solution, and the pH of the washing solution was adjusted to neutral alkaline with 1mol / L sodium hydroxide solution to observe whether there was precipitation. The strain with chitin deacetylase activity is CDA-QQ, and the colony morphology of this strain is white round protrusions, and yellow circles are produced on the primary screening medium. In the re-screening medium, precipitation can be precipitated after adjusting the pH.

[0020] The screening medium was 10 g / L ammonium sulfate, 0.5 g / L potassium dihydrogen phosphate, 0.5 g / L potassium hydrogen phosphate, 0.1 g / L magnesium sulfate, 10 g / L colloidal chitin, 0.4 g / L p-nitroacetanilide, 15 g / L agar, and pH was 7; the rescreening medium was 10 g / L ammonium sulfate, 0.5 g / L potassium dihydrogen phosphate, 0.5 g / L potassium hydrogen phosphate, 0.1 g / L magnesium sulfate, 10 g / L colloidal chitin, and pH was 7. The above culture media were sterilized at 121°C for 15 min and then cooled to room temperature.

[0021] 2. Molecular Biological Identification The strain CDA-QQ was subjected to 16s rDNA sequencing, and the sequence is shown in the sequence listing SEQ ID NO.1.

[0022] The strain was identified as Bacillus firmus by 16S rDNA full sequence analysis Cytobacillus firmus , was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on October 30, 2024. The address of the deposit unit is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. The deposit number is CGMCC NO.32397.

[0023] The above 16s rDNA complete sequence was compared with other Bacillus firmus bacteria, see Table 1.

[0024] Table 1 Comparison of 16s rDNA sequences with those of similar Bacillus bacteria Description Max Score Total Score Query Cover Evalue Per.Ident Accession Cytobacillus firmus strainNBRC 15306 16S ribosomalRNA, partial sequence 2593 2593 98% 0 98.77% NR_112635.1 Cytobacillus firmus strainIAM 12464 16S ribosomalRNA, partial sequence 2577 2577 98% 0 98.70% NR_025842.1 Cytobacillusoceanisediminis strain H216S ribosomal RNA, partialsequence 2490 2490 94% 0 98.92% NR_117285.1 Cytobacillus firmus strainNCIMB 9366 16S ribosomalRNA, partial sequence 2459 2459 95% 0 98.36% NR_118955.1 Cytobacillus gottheiliistrain WCC 4585 16Sribosomal RNA, partialsequence 2449 2449 98% 0 97.04% NR_108491.1 Cytobacillus depressusstrain BZ1 16S ribosomalRNA, partial sequence 2423 2423 98% 0 96.96% NR_146034.1 Bacillus dakarensis strainMarseille-P3515 16Sribosomal RNA, partialsequence 2422 2422 98% 0 96.70% NR_147382.1 Cytobacilluspurgationiresistens strainDS22 16S ribosomal RNA,partial sequence 2422 2422 96% 0 97.40% NR_108492.1 Mesobacillus subterraneusstrain COOI3B 16Sribosomal RNA, partialsequence 2412 2412 98% 0 96.64% NR_104749.1 Mesobacillus thioparansstrain BMP-1 16S ribosomalRNA, partial sequence 2407 2407 98% 0 96.57% NR_043762.1 .

[0025] By comparison, the strain was identified as Bacillus firmus Cytobacillus firmus . Its taxonomic units are: cellular organisms; Bacteria; Terrabacteria group; Bacillota; Bacilli; Bacilles; Bacillaceae; Cytobacillus .

[0026] Example 2 Preparation of chitosan by enzymatic method using strain CDA-QQ The strain CDA-QQ was cultured on LB solid culture medium and placed in a constant temperature incubator at 28°C for 24 hours, and then the strain was inoculated into LB liquid culture medium and cultured in a constant temperature incubator at 28°C for 24 hours to obtain a strain seed culture solution; the seed culture solution was inoculated into a chitin deacetylase-producing culture medium at a ratio of 1% (v / v) at 28°C for 48 hours of fermentation. After the fermentation was completed, the bacterial precipitate was removed by centrifugation (10000 rpm, 10 minutes) to obtain a fermentation solution rich in chitin deacetylase.

[0027] The chitin deacetylase production culture medium is composed of 10 g / L ammonium sulfate, 0.5 g / L potassium dihydrogen phosphate, 0.5 g / L dipotassium hydrogen phosphate, 0.1 g / L magnesium sulfate, 10 g / L glucose, and the balance is water. The pH value is 7.0. After sterilization at 121°C for 15 minutes, it is cooled to room temperature.

[0028] Prepare colloidal chitin: choline chloride and urea in a molar ratio of 1:2 (choline chloride 13.96g, urea 12g) and stir at 60°C until melted and transparent to obtain a deep eutectic solvent. Add chitin at a mass fraction of 3% to the deep eutectic solvent, stir at 100°C for 12 hours, cool to room temperature, and wash repeatedly with water.

[0029] The colloidal chitin was added at a rate of 50 g / L and mixed with the fermentation supernatant, incubated in a water bath at 35°C for 48 hours, centrifuged (10000 rpm, 10 minutes), the precipitate was fully stirred and dissolved with 2% (v / v) acetic acid solution, centrifuged (5000 rpm, 5 minutes) to remove the precipitate, the pH value of the supernatant was adjusted to neutral to alkaline with 1 mol / L sodium hydroxide solution, centrifuged (5000, 5 minutes) to obtain chitosan, and the deacetylation degree of the product was determined by Fourier transform infrared spectroscopy, see Table 2, Figure 2 , and use 13 Characterization by CNMR, see Figure 5 , Figure 8 .

[0030] Table 2 Deacetylation degree of colloidal chitin after deacetylation prepared by deep eutectic reagent sample Deacetylation degree (%) Chitin 25% Fermentation enzymatic hydrolysis products 71% .

[0031] Example 3 Enzymatic Preparation of Chitosan Using Strain CDA-QQ The strain CDA-QQ was cultured on LB solid culture medium and placed in a constant temperature incubator at 28°C for 24 hours, and then the strain was inoculated into LB liquid culture medium and cultured in a constant temperature incubator at 28°C for 24 hours to obtain the strain seed culture solution; the seed culture solution was inoculated into a chitin deacetylase-producing culture medium at a ratio of 1% (v / v) and fermented at 28°C for 48 hours to obtain a fermentation solution rich in chitin deacetylase.

[0032] The chitin deacetylase production culture medium is composed of 10 g / L ammonium sulfate, 0.5 g / L potassium dihydrogen phosphate, 0.5 g / L dipotassium hydrogen phosphate, 0.1 g / L magnesium sulfate, 10 g / L colloidal chitin, and the balance is water. The pH value is 7.0. After sterilization at 121°C for 15 minutes, it is cooled to room temperature.

[0033] Preparation of colloidal chitin: Add 2 g chitin to 50 mL concentrated hydrochloric acid, stir for 2 hours, cool at 4 °C for 1 hour, add 200 mL ethanol solution (50%, v / v), stir thoroughly and centrifuge (8000 rpm, 10 minutes), discard the supernatant, wash with water and centrifuge repeatedly until the supernatant has a pH of 7.

[0034] After the fermentation, centrifuge (10000rpm, 10 minutes) to remove the bacterial precipitate, add 50 g / L of colloidal chitin to the fermentation supernatant, incubate in a 35°C water bath for 48 hours, centrifuge (10000rpm, 10 minutes), stir and dissolve the precipitate with 2% (v / v) acetic acid solution, centrifuge (5000rpm, 5 minutes) to remove the precipitate, adjust the pH value of the supernatant to neutral to alkaline with 1 mol / L sodium hydroxide solution, centrifuge (5000, 5 minutes) to obtain chitosan, and determine the deacetylation degree of the product by Fourier transform infrared spectroscopy scanning, see Table 3, Figure 3 , and use 13 Characterization by CNMR, see Figure 6 , Figure 8 .

[0035] Table 3 Deacetylation degree of colloidal chitin after deacetylation prepared by concentrated hydrochloric acid sample Deacetylation degree (%) Chitin 25% Fermentation enzymatic hydrolysis products 73% .

[0036] Example 4 Enzymatic Preparation of Chitosan Using Strain CDA-QQ The strain CDA-QQ was cultured on LB solid culture medium and placed in a constant temperature incubator at 28°C for 24 hours, and then the strain was inoculated into LB liquid culture medium and cultured in a constant temperature incubator at 28°C for 24 hours to obtain the strain seed culture solution; the seed culture solution was inoculated into a chitin deacetylase-producing culture medium at a ratio of 1% (v / v) and fermented at 28°C for 48 hours to obtain a fermentation solution rich in chitin deacetylase.

[0037] The chitin deacetylase production culture medium is composed of 10 g / L ammonium sulfate, 0.45 g / L potassium dihydrogen phosphate, 0.5 g / L potassium hydrogen phosphate, 0.1 g / L magnesium sulfate, 10 g / L colloidal chitin, and the balance is water. The pH value is 7.0. After sterilization at 121°C for 15 minutes, it is cooled to room temperature.

[0038] Preparation of colloidal chitin: chitin was added to a sodium hydroxide / urea solution at a concentration of 20 g / L, wherein the amount of sodium hydroxide added was 200 g / L and the amount of urea added was 20 g / L. After mixing, the mixture was stirred at -25°C for 2 hours. After warming to room temperature, the mixture was washed with water and centrifuged until the pH value reached 7.

[0039] After the fermentation, centrifuge (10000rpm, 10 minutes) to remove the bacterial precipitate, add 50 g / L of colloidal chitin to the fermentation supernatant, incubate in a 35°C water bath for 48 hours, centrifuge (10000rpm, 10 minutes), stir and dissolve the precipitate with 2% (v / v) acetic acid solution, centrifuge (5000rpm, 5 minutes) to remove the precipitate, adjust the pH value of the supernatant to neutral to alkaline with 1 mol / L sodium hydroxide solution, centrifuge (5000, 5 minutes) to obtain chitosan, and determine the degree of deacetylation of the product by Fourier transform infrared spectroscopy, see Table 4, Figure 4 , and characterized by 13CNMR, see Figure 7 , Figure 8 .

[0040] Table 4 Deacetylation degree of chitin after deacetylation by sodium hydroxide and urea mixture sample Deacetylation degree (%) Chitin 25% Fermentation enzymatic hydrolysis products 68% .

Claims

1. A chitin deacetylase producing strain, characterized in that: Bacillus firmus Cytobacillus firmus , and was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on October 30, 2024, with the deposit number CGMCC NO.32397.

2. The use of the chitin deacetylase producing strain according to claim 1, characterized in that: The strain is used for preparing chitin deacetylase.

3. The use of the chitin deacetylase producing strain according to claim 1, characterized in that: The strain uses chitin as a substrate to prepare chitosan by enzymatic method.

4. A chitin deacetylase, characterized in that: The method comprises the strain according to claim 1 or a fermentation broth thereof.

5. A method for preparing chitin deacetylase, characterized in that: The strain according to claim 1 is inoculated into LB liquid culture medium to obtain a strain seed culture solution; the seed culture solution is inoculated into a chitin deacetylase producing culture medium at a ratio of 1%-3% V / V, and fermented at 30-40° C. for 24-72 hours to obtain a fermentation solution rich in chitin deacetylase.

6. The method for preparing chitin deacetylase according to claim 5, wherein: The chitin deacetylase production culture medium comprises: a carbon source addition amount of 1%-3% (w / v), a nitrogen source addition amount of 1%-3% (w / v), a potassium dihydrogen phosphate addition amount of 0.01%-0.2% (w / v), a dipotassium hydrogen phosphate addition amount of 0.01%-0.2% (w / v), a magnesium sulfate addition amount of 0.1%-0.3% (w / v), a sodium chloride addition amount of 0.05%-0.2% (w / v), the remainder being water, a pH value of 6-7.5, sterilization at 121° C. for 15 minutes, and cooling to room temperature.

7. The method for preparing chitin deacetylase according to claim 6, wherein: The nitrogen source is one of beef extract, yeast extract, ammonium sulfate, and peptone, or a mixture of several of them; the carbon source is one of colloidal chitin, glucose, sucrose, and glycerol, or a mixture of several of them.

8. A method for preparing chitosan by enzymatic method, characterized in that: The chitosan from which the acetyl group of chitin is removed is obtained by adding colloidal chitin in an amount of 2%-5% W / V to the fermentation liquid obtained by the method of claim 5, mixing and reacting in a water bath at 25-45° C. for 48-72 hours.

9. The method for preparing chitosan by enzymatic method according to claim 8, characterized in that: The colloidal chitin is chitin treated with a deep eutectic solvent, concentrated hydrochloric acid, sodium hydroxide and urea solution; the added amount of chitin is 2%-4% W / V.

10. The method for preparing chitosan by enzymatic method according to claim 9, characterized in that: The deep eutectic solvent is a solution prepared by mixing choline chloride and urea in a molar ratio of 1:2.

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