Chitinilytic bacterium FCG-7 and application thereof in preparation of n-acetylglucosamine

Abstract

This invention provides a *Chitozobacterium mangroveense* FCG-7 strain and its application in the preparation of N-acetylglucosamine, belonging to the field of microbial technology. The *Chitozobacterium mangroveense* FCG-7 strain provided by this invention is deposited at the Guangdong Provincial Microbial Culture Collection Center, with accession number GDMCC No: 63488. This strain exhibits good low-temperature resistance and the ability to produce extracellular chitinase, enabling it to efficiently degrade chitin under low-temperature conditions to produce N-acetylglucosamine with excellent biological effects. Furthermore, this strain remains stable after subculturing and holds promise for application in the production of high-value-added N-acetylglucosamine from chitin-based biomass under low-temperature conditions.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, and in particular relates to a mangrove chitosan strain FCG-7 and its application in the preparation of N-acetylglucosamine. Background Technology

[0002] The rapid development of aquaculture and processing industries has led to the accumulation of large amounts of shrimp and crab shell waste. Currently, the processing of these chitin-rich byproducts results in resource waste and environmental pollution, hindering the healthy and sustainable development of aquaculture.

[0003] N-acetylglucosamine (GlcNAc), as a monomer of chitin, plays a crucial role in biomass synthesis and is a key intermediate in the synthesis of nitrogen-containing chemicals. In addition, GlcNAc is widely used in the preparation of heterocyclic compounds, acids, alcohols, and amino sugars. GlcNAc has extensive applications in the food, health, cosmetics, and biopharmaceutical industries. In the food and health industry, GlcNAc is often used as an ingredient; its supplementation can significantly reduce the incidence of colon and lung cancer by more than 25% and reduce cancer mortality by 13%. In the cosmetics industry, GlcNAc is used as an excellent substrate for the synthesis of hyaluronic acid, helping to stimulate collagen synthesis. Furthermore, GlcNAc has significant therapeutic effects in treating inflammatory bowel disease. In the plant field, GlcNAc can promote seed germination and enhance plant resistance, and is often used in plant seed and foliar fertilizers.

[0004] Currently, the main method for preparing GlcNAc is chemical extraction. However, due to the problems of violent reactions, high costs, and environmental pollution associated with chemical extraction, researchers have turned their attention to microbial methods, specifically the green degradation of chitin by microorganisms to produce GlcNAc, which has become a current research hotspot. Compared with traditional chemical methods, the microbial degradation of shrimp and crab shells to produce GlcNAc has the advantages of mild reaction conditions and low energy consumption. Furthermore, cryogenic microorganisms can degrade chitin waste at lower temperatures, accelerating the degradation process and improving treatment efficiency. This method not only saves energy consumption and reduces production costs but also reduces environmental pollution. Therefore, finding cryogenic microorganisms with efficient shrimp and crab shell degradation capabilities is of great significance for the comprehensive utilization of chitin waste to produce GlcNAc and for environmental protection. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a mangrove chitinobacterium FCG-7 and its application in the preparation of N-acetylglucosamine; the strain provided by the present invention has good low temperature resistance and the ability to produce extracellular chitinase, and can efficiently degrade chitin under low temperature conditions to produce N-acetylglucosamine with excellent biological effects.

[0006] This invention provides a Chitinibacter mangrovi FCG-7 strain, which is deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No: 63488.

[0007] The present invention also provides a method for producing extracellular chitinase using the aforementioned mangrove chitinobacterium FCG-7, comprising the following steps:

[0008] 1) Inoculate mangrove chitosan FCG-7 into seed culture medium to obtain seed solution;

[0009] 2) The seed culture was inoculated into chitinase fermentation medium for fermentation culture to obtain fermentation broth;

[0010] 3) The fermentation broth is separated into solid and liquid components, and the supernatant is collected as chitinase solution.

[0011] Preferably, the chitinase fermentation medium described in step 2) uses water as a solvent and comprises the following components at the following concentrations:

[0012] The concentrations of KH₂PO₄ (0.25–0.35 g / L), K₂HPO₄ (0.6–0.8 g / L), FeSO₄ (0.08–0.12 g / L), CaCl₂ (0.08–0.12 g / L), MgSO₄ (0.45–0.55 g / L), NaCl (0.8–1.2 g / L), powdered chitin (8–12 g / L), and urea (8–12 g / L) in the chitinase fermentation medium are as follows: pH 8–9.

[0013] Preferably, the fermentation temperature in step 2) is 20-30°C, the fermentation speed is 190-220 rpm, and the fermentation time is 48-96 h.

[0014] Preferably, the inoculation amount of the seed liquid in step 2) is 4-5% (v / v).

[0015] The present invention provides a chitinase solution prepared by the method described above.

[0016] This invention provides the application of the aforementioned *Chitozobacterium mangrove* FCG-7 or the aforementioned chitinase solution in the degradation of chitin, and the use of the aforementioned *Chitozobacterium mangrove* FCG-7 or chitinase solution to degrade chitin to prepare N-acetylglucosamine.

[0017] Preferably, the chitinase solution is mixed with the chitin substrate and reacted at 4–30°C for 4–24 hours. After the reaction is terminated, the supernatant is collected to obtain N-acetylglucosamine.

[0018] Preferably, the volume ratio of the chitinase solution to the chitin substrate is (1-2):(1-2).

[0019] Preferably, the chitin substrate is colloidal chitin, powdered chitin, or waste shrimp and crab shells.

[0020] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a mangrove chitosan strain FCG-7 and its application in the preparation of N-acetylglucosamine. Compared with the strains known in the prior art, the mangrove chitosan strain FCG-7 of the present invention can grow well under low temperature conditions, and has a good extracellular enzyme production ability. It has improved the performance of chitin degradation under low temperature conditions and has the characteristic of passage stability. It is expected to be applied to the production of high-value-added N-acetylglucosamine from chitin-based biomass (such as shrimp and crab shells) under low temperature conditions. Attached Figure Description

[0021] Figure 1 The image shows the clear zone formed by *Chitozobacterium mangroveii* FCG-7.

[0022] Figure 2 PCR electrophoresis image for identifying the 16S rDNA strain of *Chitozobacterium mangroves* FCG-7.

[0023] Figure 3 Phylogenetic tree of the whole genome of Chitinoblastoma f.C.-7.

[0024] Figure 4 Image showing the Gram staining results of FCG-7 chitosan from mangroves.

[0025] Figure 5 Scanning electron microscope image of mangrove chitosan FCG-7.

[0026] Figure 6 This is the standard curve for N-acetylglucosamine.

[0027] Figure 7 The growth curve of *Chitozobacterium mangroveii* FCG-7.

[0028] Figure 8 Analysis of carbon source for chitinase produced by fermentation of *Chitinobacterium mangrove* FCG-7.

[0029] Figure 9 Analysis of nitrogen source for chitinase produced by fermentation of mangrove chitinobacterium FCG-7.

[0030] Figure 10 Initial pH analysis for chitinase production by fermentation of mangrove chitinobacterium FCG-7.

[0031] Figure 11Analysis of culture temperature for chitinase production by fermentation of *Chitinobacterium mangrove* FCG-7.

[0032] Figure 12 Analysis of culture rotation speed for chitinase production by fermentation of *Chitinobacterium mangrove* FCG-7.

[0033] Figure 13 Analysis of inoculum size for chitinase production by fermentation of *Chitinobacterium mangrove* FCG-7.

[0034] Figure 14 Analysis of the culture time for chitinase production by fermentation of *Chitinobacterium mangrove* FCG-7.

[0035] Figure 15 HPLC analysis of colloidal chitin products treated with crude enzyme of *Chitosporum tobira* FCG-7 at different temperatures. A, B, C, and D are HPLC analyses of colloidal chitin products treated with crude enzyme at 4℃, 10℃, 20℃, and 30℃, respectively.

[0036] Figure 16 HPLC analysis of powdered chitin products treated with crude enzyme of *Chitosporum tobira* FCG-7 at different temperatures. A, B, C, and D are HPLC analyses of powdered chitin products treated with crude enzyme at 4℃, 10℃, 20℃, and 30℃, respectively.

[0037] Biological Preservation Instructions

[0038] Chitinibacter mangrovi FCG-7 is deposited at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, on May 19, 2023, with accession number GDMCC No: 63488. Detailed Implementation

[0039] This invention provides a Chitinibacter mangrovi FCG-7 strain, which is deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No: 63488.

[0040] In this invention, the mangrove chitosan FCG-7 was isolated from mangrove soil sediment samples and has the ability to hydrolyze chitin; the 16S rDNA sequencing results of the mangrove chitosan FCG-7 are shown in SEQ ID No. 1.

[0041] The present invention also provides a method for producing extracellular chitinase using the aforementioned mangrove chitinobacter FCG-7, comprising the following steps: 1) inoculating mangrove chitinobacter FCG-7 into a seed culture medium to obtain a seed liquid; 2) inoculating the seed liquid into a chitinase fermentation medium for fermentation culture to obtain a fermentation broth; 3) separating the fermentation broth into solid and liquid phases, and collecting the supernatant as the chitinase solution.

[0042] In this invention, *Chitozobacterium mangroveii* FCG-7 is inoculated into a seed culture medium to obtain a seed solution. In this invention, the seed culture medium is preferably R2A liquid medium, the culture temperature is preferably 20–30°C, the culture rotation speed is preferably 190–220 rpm, and the culture time is preferably 6–20 h, more preferably 15–19 h, and even more preferably 17–18 h. In this invention, the OD of the seed solution... 600 The preferred value is 0.28-0.29, and it is concentrated to OD. 600 The concentration is 2.0-2.5; the inoculation amount of the seed solution is preferably 4-5% (v / v).

[0043] After obtaining the seed culture, this invention inoculates the seed culture into a chitinase fermentation medium for fermentation culture to obtain the fermentation broth. In this invention, the chitinase fermentation medium uses water as a solvent and preferably comprises the following components at concentrations: KH₂PO₄ 0.25–0.35 g / L, K₂HPO₄ 0.6–0.8 g / L, FeSO₄ 0.08–0.12 g / L, CaCl₂ 0.08–0.12 g / L, MgSO₄ 0.45–0.55 g / L, NaCl 0.8–1.2 g / L, powdered chitin 8–12 g / L, and urea 8–12 g / L; more preferably, it comprises the following components at concentrations: KH₂PO₄ 0.28–0.32 g / L, K₂HPO₄ 0.65–0.75 g / L, FeSO₄ The preferred composition is as follows: KH₂PO₄ 0.3 g / L, K₂HPO₄ 0.7 g / L, FeSO₄ 0.1 g / L, CaCl₂ 0.1 g / L, MgSO₄ 0.5 g / L, NaCl 1.0 g / L, powdered chitin 9 g / L, and urea 8 g / L. In this invention, the pH of the chitinase fermentation medium is preferably 8-9. In this invention, the fermentation medium is preferably used after sterilization, and the sterilization is preferably high-temperature moist heat sterilization, with the sterilization conditions preferably being sterilization at 121°C for 20 min.

[0044] In this invention, the fermentation temperature is preferably 20-30℃, and can be selected as 20-21℃, 22-24℃, 25-26℃, 27-28℃ or 29-30℃; the fermentation speed is preferably 190-220 rpm, more preferably 200-210 rpm; and the fermentation time is preferably 48-96 h, more preferably 60-84 h.

[0045] After the fermentation culture is completed, the fermentation broth is separated into solid and liquid components, and the supernatant is collected as chitinase solution. In this invention, the solid-liquid separation is preferably performed by centrifugation, the centrifugation speed is preferably 6000-8000 rpm, the centrifugation time is preferably 25-35 min, more preferably 28-32 min, and most preferably 30 min; the centrifugation temperature is preferably 3-5℃, and more preferably 4℃.

[0046] The present invention also provides a chitinase solution prepared by the method, wherein the chitinase solution has a maximum activity of 12.43 U, and the chitinase solution is preferably stored at a low temperature, preferably 3 to 5°C.

[0047] This invention provides the application of the aforementioned *Chitozobacterium mangrove* FCG-7 or the aforementioned chitinase solution in the degradation of chitin, and the use of the aforementioned *Chitozobacterium mangrove* FCG-7 or chitinase solution to degrade chitin to prepare N-acetylglucosamine.

[0048] In this invention, the chitinase solution is mixed with the chitin substrate and reacted at 4–30°C for 1–24 h. After the reaction is terminated, the supernatant is collected to obtain N-acetylglucosamine.

[0049] In this invention, the preferred volume ratio of the chitinase solution to the chitin substrate is (1-2):(1-2), more preferably 1:1; the preferred chitin substrate is colloidal chitin or powdered chitin. The preferred chitin substrate is derived from shrimp shells, crab shells, insect exoskeletons, or fungal mycelium; the shrimp shell is from marine shrimp or farmed shrimp. In this invention, degradation can be achieved within a temperature range of 4-30°C, and the preferred reaction time is 4-12 hours, more preferably 6-8 hours. The reaction is terminated after completion, preferably by a boiling water bath, with a boiling water bath time of 5-10 minutes. After the boiling water bath, the mixture is centrifuged, and the supernatant is collected; the preferred centrifugation speed is 6000-8000 rpm, and the preferred centrifugation time is 10-15 minutes.

[0050] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0051] The enrichment medium used water as a solvent and consisted of the following: potassium dihydrogen phosphate 0.7 g / L, dipotassium hydrogen phosphate 0.3 g / L, magnesium sulfate heptahydrate 0.6 g / L, ferric sulfate heptahydrate 0.01 g / L, powdered chitin 2 g / L, and colloidal chitin 12 g / L; the initial pH was 7.0.

[0052] The screening medium used water as a solvent and consisted of the following: potassium dihydrogen phosphate 0.7 g / L, dipotassium hydrogen phosphate 0.3 g / L, magnesium sulfate heptahydrate 0.6 g / L, ferric sulfate heptahydrate 0.01 g / L, powdered chitin 1 g / L, colloidal chitin 10 g / L, and agar 20 g / L; the initial pH was 7.0.

[0053] Seed culture medium: R2A medium powder (Haibo Biotechnology Co., Ltd.) 3.2g / L, NaCl 5g / L, distilled water to a final volume of 1000mL, sterilized at 121℃ for 20min.

[0054] Fermentation medium: KH2PO4 0.3 g / L, K2HPO4 0.7 g / L, FeSO4 0.1 g / L, CaCl2 0.1 g / L, MgSO4 0.5 g / L, NaCl 1.0 g / L, powdered chitin 10 g / L, urea 10 g / L, pH 8-9, distilled water to a final volume of 1000 mL, sterilized at 121℃ for 20 min.

[0055] Example 1

[0056] Isolation of strains

[0057] Soil sediment samples were collected from mangrove forests in Beibu Gulf Marine Culture Park, Fangchenggang City, Guangxi Zhuang Autonomous Region, and the Pearl Bay Coastal Ecological Experimental Station of Guangxi Mangrove Research Center, Guangxi Academy of Sciences, with 10 soil samples taken from each area, for a total of 20 samples. These 20 soil samples were then screened for chitin-degrading bacteria.

[0058] Take 1g of soil sample and add it to 25mL of sterile water. Incubate at 30℃ and 200rpm for 4h. After thorough mixing, take 1mL of the supernatant and place it in 100mL of enrichment medium. Incubate at 30℃ and 150rpm for 3d to obtain the enriched bacteria.

[0059] The enrichment medium used water as a solvent and consisted of the following: potassium dihydrogen phosphate 0.7 g / L, dipotassium hydrogen phosphate 0.3 g / L, magnesium sulfate heptahydrate 0.6 g / L, ferric sulfate heptahydrate 0.01 g / L, powdered chitin 2 g / L, and colloidal chitin 12 g / L; the initial pH was 7.0.

[0060] Pipette 100 μL of the culture medium for enriched bacteria into a 2 mL centrifuge tube containing 900 μL of sterile water and mix thoroughly. Then, transfer 100 μL of this culture medium to another centrifuge tube containing 900 μL of sterile water, mix thoroughly by pipetting, and press 10... -3 ~10 -4 After serial dilution, 100 μL of the bacterial culture from each dilution was pipetted onto the selection medium and evenly spread. The medium was then incubated upside down in a 30°C incubator. Colonies with a clear zone around them were selected and subjected to four streak purifications, resulting in 15 strains producing clear zones. One strain, designated FCG-7, was selected because it had the largest clear zone diameter ratio. The ratio of the diameter of the clear zone to the colony diameter of this strain over time was measured, and the results are shown in Table 1.

[0061] The screening medium used water as a solvent and consisted of the following: potassium dihydrogen phosphate 0.7 g / L, dipotassium hydrogen phosphate 0.3 g / L, magnesium sulfate heptahydrate 0.6 g / L, ferric sulfate heptahydrate 0.01 g / L, powdered chitin 1 g / L, colloidal chitin 10 g / L, and agar 20 g / L; the initial pH was 7.0.

[0062] Table 1. FCG-7 diameter ratio as a function of time

[0063] Days / d D / mm d / mm D / d 1 0.65 0.2 3.25 2 1 0.25 4.00 3 1.25 0.3 4.17 4 1.45 0.35 4.14 5 1.7 0.4 4.25 6 1.9 0.42 4.52

[0064] D represents the diameter of the transparent zone, and d represents the diameter of the colony.

[0065] The clear zone formed by strain FCG-7 is like Figure 1 As shown.

[0066] Example 2

[0067] Identification of strain FCG-7

[0068] A small number of bacterial cells from the selection plate of strain FCG-7 were selected using a picker for PCR amplification. Universal primers for the 16S rDNA gene (upstream primer 27F and downstream primer 1492R) were used, and a 25 μL system was prepared for PCR amplification. A 1% agarose gel was prepared for electrophoresis. The amplified products are shown below. Figure 2 As shown. M represents Marker DL2000, and 1 represents strain FCG-7.

[0069] Samples containing the target fragment from the PCR product were sent to Sangon Biotech (Shanghai) Co., Ltd. for 16S rDNA sequencing. The 16S rDNA sequence of strain FCG-7 is shown in SEQ ID NO.1. The sequencing results were submitted to NCBI (National Center for Biotechnology Information) for BLAST analysis of the 16S rDNA sequence to confirm the taxonomic position of the strain.

[0070] SEQ ID NO.1 is:

[0071]

[0072] Therefore, the 16S rDNA sequence length of FCG-7 is 1379 bp. BLAST alignment analysis revealed that strain FCG-7 shared the highest homology (99.71%) with Chitinibacter bivalviorum 2T18. Whole-genome sequencing and sequence alignment analysis were then performed, and the phylogenetic tree is shown below. Figure 3 As shown, the similarity with Chitinibacter fontanus STM-7 was the highest at 91%, and below 95%, indicating that it was identified as a new bacterium and named Chitinibacter mangrovi FCG-7.

[0073] After culturing the bacterial strain in R2A liquid medium at 25°C and 200 rpm for 18 hours, Gram staining was performed as follows: Figure 4 As shown, Gram staining was negative, and scanning electron microscopy revealed that the strain was rod-shaped (e.g., ...). Figure 5 (As shown in the table). The physiological and biochemical identification results are shown in Table 2.

[0074] Table 2 Physiological and biochemical identification results of FCG-7

[0075] Testing items result Testing items result Chitin + Acid phosphatase w Nitrate reduction + Naphthol AS-BI phosphate hydrolase w D-glucose fermentation + α-Mannosidase – Aesculin hydrolysis experiment + D-ribose – Gelatin hydrolysis – D-mannose – D-mannose assimilation – α-D-glucose + α-glucosidase – D-mannose – β-glucosidase – 1% Sodium Lactate + alkaline phosphatase w D-glucose-6-phosphate – Esterase (C4) w D-fructose-6-phosphate + Esterase / Lipase (C8) w Lincomycin w Lipase (C14) w Guanidine hydrochloride w Leucine aramidase + glucuronamide + Valine aramidase w Acetic acid – Cystine arylamidinase w Aztreonam –

[0076] + indicates a positive reaction; – indicates a negative reaction; W indicates a weak positive reaction.

[0077] Example 3: Low Temperature Resistance Test of Strains

[0078] After activating the strain of *Chitosanthes mangrove*, a bacterial concentration of 10 was prepared. 9 A bacterial suspension of CFU / mL was inoculated at a 1% inoculum into 10 mL of R2A liquid medium. After incubation at 4℃, 10℃, 15℃, 20℃, 25℃, 30℃, 37℃, and 45℃ for 12 h and 24 h respectively, the absorbance was measured at 600 nm. The results were averaged in triplicate. Table 3 shows that the strain can grow at a low temperature of 10℃, and exhibits good growth at 15℃. The optimal growth temperature is 30℃. Strain FCG-7 is a cryogenic bacterium, and it functions better in low-temperature environments.

[0079] Table 3. Results of FCG-7 Low Temperature Resistance Test (OD) 600 )

[0080]

[0081] Example 4: Stability Study in Subculture

[0082] The selected *Chitosporum tobira* FCG-7 strain was used as the first generation. The first generation strain was then inoculated into a selection solid medium to obtain the second generation strain. The second generation strain was then inoculated into the solid selection medium to obtain the third generation strain, and so on until the tenth generation strain was obtained. These selection solid media inoculated with FCG-7 were placed in an incubator at 30℃ and cultured upside down. The diameter ratio of different generations of FCG-7 strains on day 6 was observed, and the results are shown in Table 4.

[0083] Table 4. Chitin degradation capacity of different generations of *Chitobacter moniliforme* FCG-7 strains from mangrove forests.

[0084]

[0085]

[0086] Example 5

[0087] Methods for determining the activity of crude extracellular enzymes

[0088] Weigh 0.2212 g of N-acetylglucosamine powder and dissolve it in 10 mL of double-distilled water to obtain a 100 mM N-acetylglucosamine solution. Add 1 mL of the 100 mM N-acetylglucosamine solution to 9 mL of double-distilled water to obtain a 10 mM N-acetylglucosamine solution. Prepare N-acetylglucosamine solutions of different concentrations by adding double-distilled water, and determine the OD using DNS colorimetric assay. 540 .

[0089] Table 5. Preparation of the N-acetylglucosamine standard curve

[0090]

[0091]

[0092] Plotting N-acetylglucosamine concentration on the x-axis, OD 540 The value is used as the ordinate. A graph is plotted, and the resulting standard curve of N-acetylglucosamine is shown below. Figure 6 As shown, the linear equation of the standard curve is y = 0.5653x - 0.1202, and the linear correlation coefficient R0 is... 2 =0.9996. This indicates that the absorbance values ​​of N-acetylglucosamine solutions of different concentrations have a good linear relationship at 540 nm, which conforms to Beer's Law, and the amount of N-acetylglucosamine can be quantitatively determined by spectrophotometry.

[0093] Preparation of crude enzyme solution: Centrifuge the fermentation broth at 6000-8000 rpm for 10 min and collect the supernatant.

[0094] Enzyme activity assay: The reducing sugar content was determined using the dinitrosalicylic acid (DNS) method, and the chitinase activity was measured. The principle is that 3,5-dinitrosalicylic acid reacts with reducing sugar to form a reddish-brown reaction product, 3-amino-5-nitrosalicylic acid. Its absorbance can be measured at a wavelength of 540 nm using a UV spectrophotometer. The chitinase activity was then calculated based on a previously prepared standard curve.

[0095] Enzyme activity unit (U) is defined as the amount of enzyme required to hydrolyze chitin to produce 1 μmol of N-acetylglucosamine per minute under appropriate temperature conditions.

[0096] Optimal conditions for extracellular chitinase production by the strain were optimized (see above for the method of determining the activity of the crude extracellular enzyme).

[0097] The growth curve of the strain serves as the basis for subsequent optimization of enzyme production conditions. For example... Figure 7 As shown, the strain is in the lag phase from 0 to 6 hours, during which its growth is relatively slow. From 6 to 20 hours, it enters the exponential growth phase, during which the strain grows rapidly and exhibits strong growth viability. Selecting the bacterial culture during this period as the seed culture ensures a high viable cell count; therefore, this invention selects 15 to 20 hours as the seed culture time. After 24 hours, the strain grows steadily, with a high and stable cell count. Based on this growth curve, in subsequent experiments, the enzyme activity of the fermentation broth after 48 hours of culture was measured.

[0098] The initial fermentation medium composition is as follows:

[0099] KH₂PO₄ 0.3 g / L, K₂HPO₄ 0.7 g / L, FeSO₄ 0.1 g / L, CaCl₂ 0.1 g / L, MgSO₄ 0.5 g / L, NaCl 1.0 g / L, colloidal chitin 10 g / L, peptone 2.5 g / L, beef extract 2.5 g / L, balance water, pH 7.5.

[0100] Powdered chitin, colloidal chitin, lactose, glucose, and sucrose were selected as the carbon source for the initial fermentation medium. After 48 hours of fermentation, the enzyme activity of the fermentation broth was tested. The results showed that powdered chitin produced the highest crude enzyme activity, followed by glucose. Therefore, in this invention, powdered chitin is preferred as the carbon source in the fermentation medium, with an optimal addition amount of 8–12 g / L. Figure 8 As shown.

[0101] Based on the initial fermentation medium, powdered chitin was used as the carbon source, and peptone, yeast extract, beef extract, urea, and ammonium sulfate were used as nitrogen sources, respectively. The results showed that peptone was the nitrogen source with the highest crude enzyme activity in the fermentation broth, and urea also performed well. Considering the high cost of peptone and yeast extract, this invention preferentially selects urea as the nitrogen source for the fermentation medium, with the preferred urea addition amount being 8–12 g / L. Figure 9 As shown.

[0102] Using the optimized culture medium formulation, different initial pH values ​​(6–11) were selected. Results showed that the crude enzyme activity of the fermentation broth was highest at an initial pH of 8–9. Therefore, the preferred initial pH of the fermentation medium in this invention is 8–9. Figure 10 As shown.

[0103] Using the optimized culture medium formulation, fermentation was carried out at different culture temperatures (20.0, 25.0, 30.0, 35.0, and 40.0℃) at an initial pH of 8–9. The results showed that the crude enzyme activity of the fermentation broth was highest at a culture temperature of 20–30℃. Therefore, the preferred fermentation culture temperature for this invention is 20–30℃. Figure 11 As shown.

[0104] Using the optimized culture medium formulation and at an initial pH of 8–9 and a culture temperature of 20–30°C, fermentation was carried out at different fermentation speeds of 140–220 rpm. The results showed that the crude enzyme activity of the fermentation broth was highest at a speed of 190–220 rpm. Therefore, the preferred fermentation speed in this invention is 190–220 rpm. Figure 12 As shown.

[0105] Fermentation was conducted using an optimized culture medium formulation at an initial pH of 8–9, a culture temperature of 20–30°C, and different inoculum sizes of 190–220 rpm, with inoculum sizes of 2%, 3%, 4%, 5%, and 6% (V / V). The results showed that the highest crude enzyme activity was observed in the fermentation broth at inoculum sizes of 4% and 5%. Therefore, the preferred inoculum size in this invention is 4–5%. Figure 13 As shown.

[0106] Using an optimized culture medium formulation and under the following conditions: initial pH 8–9, culture temperature 20–30℃, different fermentation speeds (190–220 rpm), and inoculum size of 4–5%, fermentation was carried out for different times: 24 h, 48 h, 72 h, 96 h, 120 h, 144 h, and 168 h. The results showed that the crude enzyme activity of the fermentation broth was highest when the fermentation time was 48–96 h. Therefore, the preferred fermentation time in this invention is 48–96 h. Figure 14 As shown.

[0107] The final optimized enzyme production conditions were as follows: *Chitozobacterium mangroveense* FCG-7 was inoculated into 100 mL of R2A liquid medium and cultured on a shaker at 20–30°C and 190–220 rpm for 18 h. The culture was then concentrated 6–8 times to obtain the seed culture. The seed culture was inoculated at a rate of 4–5% into a 500 mL Erlenmeyer flask containing 100 mL of chitozobacterase fermentation medium at pH 8–9 and cultured on a shaker at 20–30°C and 190–220 rpm for 48–96 h. The fermentation broth was centrifuged at 4°C and 6000–8000 rpm for 30 min, and the supernatant was collected as the crude enzyme solution. The highest activity was 12.43 U, which is 29.2 times that before optimization. The solution was stored at 4°C.

[0108] Example 7

[0109] Obtaining N-acetylglucosamine

[0110] The high-performance liquid chromatography (HPLC) conditions are as follows: 5 μm NH2 column (LC column 250×4.6 mm); HPLC system with differential refractive index detector (RID) (UltiMate 3000 series); mobile phase: 70% acetonitrile, flow rate: 0.5 mL / min.

[0111] Chitin powder: purchased from Aladdin Reagent (Shanghai) Co., Ltd.

[0112] Preparation of colloidal chitin: 20g of powdered chitin was added to 200mL of concentrated hydrochloric acid, stirred evenly at room temperature, and allowed to stand at 4℃ for 24h. Then, it was stirred evenly with 2L of pre-cooled 95% ethanol, allowed to stand for another 24h, centrifuged at 7000rpm for 15min to collect the precipitate, and washed repeatedly with deionized water until neutral.

[0113] The crude enzyme solution was mixed with 1% colloidal chitin at a 1:1 ratio and reacted at 4℃, 10℃, 20℃, and 30℃ for 12h, 10h, 8h, and 6h, respectively. After boiling in a water bath for 5min, the mixture was centrifuged, and the supernatant was analyzed by HPLC. The results are as follows: Figure 15 As shown, the main product is N-acetylglucosamine.

[0114] The crude enzyme solution was mixed with 1% powdered chitin at a 1:1 ratio and reacted at 4℃, 10℃, 20℃ and 30℃ for 12h, 12h, 10h and 4h respectively. After boiling in a water bath for 5min, the mixture was centrifuged, and the supernatant was analyzed by HPLC. The results are as follows. Figure 16 As shown, the main product is N-acetylglucosamine.

[0115] The products from different temperature treatments were all N-acetylglucosamine, and the substrate could be completely degraded into N-acetylglucosamine even at low temperatures.

[0116] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A type of mangrove chitinobacillus ( Chitinibacter mangrovi FCG-7, characterized in that, It is deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No: 63488.

2. The method for producing extracellular chitinase using *Chitozobacterium mangroveense* FCG-7 according to claim 1, characterized in that, Includes the following steps: 1) Inoculate mangrove chitosan FCG-7 into seed culture medium to obtain seed solution; 2) The seed culture was inoculated into chitinase fermentation medium for fermentation culture to obtain fermentation broth; 3) The fermentation broth is separated into solid and liquid components, and the supernatant is collected as chitinase solution.

3. The method according to claim 2, characterized in that, Step 2) describes a chitinase fermentation medium that uses water as a solvent and comprises the following components at the following concentrations: The concentrations of KH₂PO₄ are 0.25–0.35 g / L, K₂HPO₄ are 0.6–0.8 g / L, FeSO₄ is 0.08–0.12 g / L, CaCl₂ is 0.08–0.12 g / L, MgSO₄ is 0.45–0.55 g / L, NaCl is 0.8–1.2 g / L, powdered chitin is 8–12 g / L, and urea is 8–12 g / L; the pH of the chitinase fermentation medium is 8–9.

4. The method according to claim 3, characterized in that, Step 2) The fermentation temperature is 20~30°C, the fermentation speed is 190~220 rpm, and the fermentation time is 48~96h.

5. The method according to claim 3 or 4, characterized in that, The inoculation amount of the seed liquid in step 2) is 4~5% (v / v).

6. The application of the mangrove chitosan strain FCG-7 as described in claim 1 in the degradation of chitin, characterized in that, N-acetylglucosamine was prepared by degrading chitin using the aforementioned mangrove chitinobacillus FCG-7.

7. The application according to claim 6, characterized in that, The chitin is colloidal chitin, powdered chitin, or waste from shrimp and crab shells.

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