Mangrove chitin bacillus FCG-7 and application of mangrove chitin bacillus FCG-7 in preparation of N-acetylglucosamine

Abstract

The invention provides mangrove chitin bacillus FCG-7 and application of the mangrove chitin bacillus FCG-7 in preparation of N-acetylglucosamine, and belongs to the technical field of microorganisms, the mangrove chitin bacillus FCG-7 provided by the invention is preserved in Guangdong Microbial Culture Collection Center, and the preservation number is GDMCC No: 63488. The strain provided by the invention has good low-temperature resistance and extracellular chitinase production capability, and can efficiently degrade chitin under a low-temperature condition to produce N-acetylglucosamine with an excellent biological effect; and the strain is still stable after passage, and is expected to be applied to the production of high-added-value N-acetylglucosamine by using chitin biomass as a raw material under a low-temperature condition.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a mangrove chitinobacterium FCG-7 and an application thereof in preparing N-acetylglucosamine. Background Art

[0002] The rapid development of aquaculture and processing industries has resulted in the accumulation of a large amount of shrimp and crab shell waste. The current treatment of these chitin-rich byproducts causes resource waste and environmental pollution, affecting the healthy and sustainable development of aquaculture.

[0003] As a monomer of chitin, N-acetylglucosamine (GlcNAc) plays an important role in biomass synthesis and is a key intermediate for the synthesis of nitrogen-containing chemicals. In addition, GlcNAc is also widely used in the preparation of nitrogen-containing chemicals such as heterocyclic compounds, acids, alcohols and amino sugars. In the fields of food health, cosmetics and biomedicine, GlcNAc is widely used. In the field of food health, GlcNAc is often used as an ingredient, and its supplementation can significantly reduce the incidence of colon cancer and lung cancer by more than 25%, and reduce cancer mortality by 13%. In the cosmetics industry, GlcNAc is used as an excellent synthetic substrate for hyaluronic acid, which helps stimulate the synthesis of collagen. In addition, GlcNAc has a significant effect on the treatment of inflammatory bowel disease. In the field of plants, GlcNAc can promote seed germination and enhance plant resistance, and is often used in fertilizers for plant seeds and leaves.

[0004] At present, the main preparation method of GlcNAc is chemical extraction. However, due to the problems of violent reaction, high cost and environmental pollution in chemical extraction, researchers have turned their attention to microbial method, that is, the method of using microorganisms to greenly degrade chitin to produce GlcNAc, which has become a current research hotspot. Compared with traditional chemical methods, the method of producing GlcNAc by microbial degradation of shrimp and crab shells has the advantages of mild reaction conditions and low energy consumption. In addition, low-temperature resistant microorganisms can degrade chitin waste at lower temperatures, accelerate the degradation process of chitin, and improve treatment efficiency. This method can not only save energy consumption and reduce production costs, but also reduce environmental pollution. Therefore, finding low-temperature resistant microorganisms with the ability to efficiently degrade shrimp and crab shells is of great significance for the comprehensive utilization of chitin waste to produce GlcNAc and environmental protection. Summary of the invention

[0005] In view of this, the object 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] The invention provides a mangrove chitinobacterium (Chitinibacter mangrovi) FCG-7, which is preserved in Guangdong Province Microbiological Culture Collection Center with a preservation number of GDMCC No: 63488.

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

[0008] 1) Inoculating mangrove chitinobacterium FCG-7 into a seed culture medium to obtain a seed solution;

[0009] 2) inoculating the seed liquid into a chitinase fermentation medium for fermentation to obtain a fermentation liquid;

[0010] 3) solid-liquid separation of the fermentation broth, and collecting the supernatant as the chitinase solution.

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

[0012] 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, urea 8-12 g / L; the pH value of the chitinase fermentation medium is 8-9.

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

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

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

[0016] The invention provides the use of the mangrove chitinobacterium FCG-7 or the chitinase liquid in degrading chitin, and the mangrove chitinobacterium FCG-7 or the chitinase liquid is used to degrade chitin to prepare N-acetylglucosamine.

[0017] Preferably, the chitinase solution is mixed with the chitin substrate, reacted at 4-30° C. for 4-24 hours, and the supernatant is collected after the reaction is terminated, which is 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 shrimp and crab shell waste.

[0020] Compared with the prior art, the present invention has the following beneficial effects: the present invention provides a mangrove chitinobacterium FCG-7 and its application in the preparation of N-acetylglucosamine. Compared with the strains known in the prior art, the mangrove chitinobacterium FCG-7 of the present invention can grow well under low temperature conditions, and has good extracellular enzyme production ability, improves the performance of chitin degradation under low temperature conditions, and has the characteristics of stability in passage; it is expected to be applied to the production of high-value-added N-acetylglucosamine under low temperature conditions using chitin biomass as raw materials (such as shrimp and crab shells, etc.). BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is the transparent zone formed by mangrove chitinobacterium FCG-7.

[0022] Figure 2 This is the PCR electrophoresis diagram for the 16S rDNA species identification of mangrove chitinobacterium FCG-7.

[0023] Figure 3 This is the phylogenetic tree of the complete genome of mangrove chitinobacterium FCG-7.

[0024] Figure 4 This is the Gram staining result of mangrove chitinobacterium FCG-7.

[0025] Figure 5 This is a scanning electron micrograph of mangrove chitinobacterium FCG-7.

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

[0027] Figure 7 This is the growth curve of mangrove chitinobacterium FCG-7.

[0028] Figure 8 Analysis of carbon sources for chitinase production by fermentation of mangrove Chitinobacterium FCG-7.

[0029] Fig. 9 Analysis of nitrogen sources for chitinase production by fermentation of mangrove Chitinobacterium FCG-7.

[0030] Fig.10 Initial pH analysis of chitinase production by fermentation of mangrove Chitinobacterium FCG-7.

[0031] Fig.11This is the culture temperature analysis of chitinase production by fermentation of mangrove Chitinobacterium FCG-7.

[0032] Fig.12 Analysis of the culture speed for the fermentation of chitinase by mangrove Chitinobacterium FCG-7.

[0033] Fig.13 This is an analysis of the inoculum dosage for the fermentation of chitinase by mangrove Chitinobacterium FCG-7.

[0034] Fig.14 This is an analysis of the cultivation time for the fermentation of chitinase by mangrove Chitinobacterium FCG-7.

[0035] Fig.15 This is the HPLC analysis of the colloidal chitin products treated by the crude enzyme of Chitinobacterium mangroveenum FCG-7 at different temperatures, where a, b, c, and d are the HPLC analyses of the colloidal chitin products treated by the crude enzyme at 4°C, 10°C, 20°C, and 30°C, respectively.

[0036] Fig.16 This is the HPLC analysis of the powdered chitin products treated by the crude enzyme of mangrove Chitinobacterium FCG-7 at different temperatures, where a, b, c, and d are the HPLC analyses of the powdered chitin products treated by the crude enzyme at 4°C, 10°C, 20°C, and 30°C, respectively.

[0037] Biological Deposit Description

[0038] Chitinibacter mangrovi FCG-7 is deposited in Guangdong Microbial Culture Collection Center, with the deposit address at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. The deposit date is May 19, 2023, and the deposit number is GDMCC No: 63488. DETAILED DESCRIPTION

[0039] The invention provides a mangrove chitinobacterium (Chitinibacter mangrovi) FCG-7, which is preserved in Guangdong Province Microbiological Culture Collection Center with a preservation number of GDMCC No: 63488.

[0040] In the present invention, the mangrove chitinobacterium FCG-7 is separated from a mangrove soil sediment sample and has the ability to hydrolyze chitin; the 16S rDNA sequencing result of the mangrove chitinobacterium FCG-7 is shown in SEQ ID No.1.

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

[0042] In the present invention, mangrove chitinobacterium FCG-7 is inoculated into a seed culture medium to obtain a seed solution. In the present invention, the seed culture medium is preferably R2A liquid culture medium, the culture temperature is preferably 20 to 30°C, the culture speed is preferably 190 to 220 rpm, the culture time is preferably 6 to 20 hours, more preferably 15 to 19 hours, and more preferably 17 to 18 hours. In the present invention, the OD of the seed solution is 600 Preferably 0.28-0.29, and concentrated to OD 600 is 2.0-2.5; the inoculation amount of the seed solution is preferably 4-5% (v / v).

[0043] After obtaining the seed liquid, the present invention inoculates the seed liquid into a chitinase fermentation medium for fermentation culture to obtain a fermentation liquid; in the present invention, the chitinase fermentation medium uses water as a solvent and preferably includes the following components in 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, urea 8-12 g / L; more preferably includes the following components in concentrations: KH2PO4 0.28-0.32 g / L, K2HPO4 0.65-0.75 g / L, FeSO4 0.09~0.11g / L, CaCl20.09~0.11g / L, MgSO40.48~0.52g / L, NaCl 0.9~1.1g / L, powdered chitin 8.5~11.5g / L, urea 8.5~11.5g / L; the most preferred composition is as follows: KH2PO4 0.3g / L, K2HPO40.7 g / L, FeSO40.1g / , CaCl20.1 g / L, MgSO40.5g / L, NaCl 1.0g / L, powdered chitin 9g / L, urea 8g / L. In the present invention, the pH value of the chitinase fermentation medium is preferably 8-9. In the present invention, the fermentation medium is preferably sterilized before use, and the sterilization is preferably high temperature and moist heat sterilization, and the sterilization conditions are preferably sterilized at 121°C for 20min.

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

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

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

[0047] The invention provides the use of the mangrove chitinobacterium FCG-7 or the chitinase liquid in degrading chitin, and the mangrove chitinobacterium FCG-7 or the chitinase liquid is used to degrade chitin to prepare N-acetylglucosamine.

[0048] In the present invention, the chitinase solution is mixed with the chitin substrate, reacted at 4-30° C. for 1-24 hours, and the supernatant is collected after the reaction is terminated, which is N-acetylglucosamine.

[0049] In the present invention, the volume ratio of the chitinase solution and the chitin substrate is preferably (1-2): (1-2), more preferably 1:1; the chitin substrate is preferably colloidal chitin or powdered chitin. In the present invention, the chitin substrate is preferably derived from shrimp shells, crab shells, insect shells or fungal mycelium; the shrimp shells are shrimp shells of seawater shrimps or shrimp shells of farmed shrimps. In the present invention, degradation can be achieved within the temperature range of 4-30°C, and the reaction time is preferably 4-12h, more preferably 6-8h. The present invention terminates the reaction after the reaction is completed, and the termination reaction is preferably carried out in a boiling water bath, and the boiling water bath time is preferably 5-10min. After the boiling water bath, the present invention centrifuges and collects the supernatant; the centrifugal speed is preferably 6000-8000rpm, and the centrifugal time is preferably 10-15min.

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

[0051] The enrichment culture medium uses water as solvent and has the following composition: 0.7 g / L potassium dihydrogen phosphate, 0.3 g / L potassium hydrogen phosphate, 0.6 g / L magnesium sulfate heptahydrate, 0.01 g / L iron sulfate heptahydrate, 2 g / L powdered chitin, and 12 g / L colloidal chitin; the initial pH is 7.0.

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

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

[0054] Fermentation medium: KH2PO4 0.3 g / L, K2HPO4 0.7 g / L, FeSO4 0.1 g / , 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, made up to 1000 mL with distilled water, sterilized at 121°C for 20 min.

[0055] Example 1

[0056] Isolation of strains

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

[0058] Take 1g of soil sample and add it to 25mL of sterile water, culture it at 30℃, 200rpm, and mix it thoroughly for 4h. Then take 1mL of the supernatant and place it in 100mL of enrichment medium. Culture it at 30℃, 150rpm in a constant temperature shaker for 3d to obtain enriched bacteria.

[0059] The enrichment culture medium uses water as solvent and has the following composition: 0.7 g / L potassium dihydrogen phosphate, 0.3 g / L potassium hydrogen phosphate, 0.6 g / L magnesium sulfate heptahydrate, 0.01 g / L iron sulfate heptahydrate, 2 g / L powdered chitin, and 12 g / L colloidal chitin; the initial pH is 7.0.

[0060] Pipette 100 μL of the culture medium of the enriched bacteria into a 2 mL centrifuge tube with 900 μL of sterile water and mix thoroughly. Then, pipette 100 μL from the centrifuge tube and transfer it to another centrifuge tube with 900 μL of sterile water. Pipette and mix thoroughly. -3 ~10 -4 After gradient dilution, 100 μL of bacterial solution of each dilution gradient was pipetted and evenly coated on the screening medium, and then placed in a 30°C constant temperature incubator for inversion. The colonies with transparent circles around the colonies were selected for 4 streaking purifications, and a total of 15 strains with transparent circles were screened. Among them, one strain numbered FCG-7 was selected, which had the largest diameter ratio of the colony circle, and the ratio of the diameter of the transparent circle produced by this strain over time to the colony diameter was measured. The results are shown in Table 1.

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

[0062] Table 1 Changes of FCG-7 circle diameter ratio over 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 transparent zone formed by strain FCG-7 is as follows Figure 1 shown.

[0066] Example 2

[0067] Identification of strain FCG-7

[0068] A small amount of bacteria was selected from the screening plate of strain FCG-7 using a bacterial pick stick for PCR amplification. Universal primers for 16S rDNA gene (upstream primer 27F and downstream primer 1492R) were used to prepare a 25 μL system for PCR amplification. A 1% agarose gel was prepared for electrophoresis. The amplified product was as follows: Figure 2 As shown. M represents Marker DL2000, and 1 represents strain FCG-7.

[0069] The samples with the target fragments of the PCR products were selected and sent to Shanghai Shenggong Biotechnology Co., Ltd. for 16S rDNA sequencing. The sequence of 16S rDNA of strain FCG-7 obtained by sequencing is shown in SEQ ID NO. 1. The sequencing results were submitted to BLAST on the official website of NCBI (National Center for Biotechnology Information) to analyze the 16S rDNA sequence and confirm the taxonomic status of the strain.

[0070] SEQ ID NO.1 is:

[0071]

[0072] It can be seen that the length of the 16S rDNA sequence of FCG-7 is 1379bp. After BLAST comparison analysis, it was found that the highest homology between strain FCG-7 and Chitinibacter bivalviorum 2T18 was 99.71%. Then the whole genome sequencing was performed and sequence comparison analysis was performed. The evolutionary tree is as follows Figure 3 As shown, the highest similarity with Chitinibacter fontanus STM-7 was 91%, and the lowest was lower than 95%, so it was identified as a new bacterium and named Chitinibacter mangrovi FCG-7.

[0073] The strain was cultured in R2A liquid medium at 25°C and 200 rpm for 18 h and then Gram staining was performed. Figure 4 As shown, Gram staining was negative, and scanning electron microscopy results showed that the strain was rod-shaped (such as Figure 5 The physiological and biochemical identification results are shown in Table 2.

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

[0075] Test items result Test 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 Aroamidase + Glucuronamide + Valine Aroamidase w Acetic acid – Cysteine ​​Aroamidase w Aztreonam –

[0076] +, positive reaction; –, negative reaction; W, weakly positive.

[0077] Example 3 strain low temperature resistance test

[0078] The strain of mangrove chitinobacterium was activated and made into a bacterial cell concentration of 10 9 cfu / mL of bacterial suspension, inoculated into 10mL R2A liquid culture medium at 1% inoculation amount, and cultured at 4℃, 10℃, 15℃, 20℃, 25℃, 30℃, 37℃, 45℃ for 12h and 24h, and then measured the absorbance at 600nm wavelength, and averaged the results by 3 parallels. The results are shown in Table 3. The strain can grow at a low temperature of 10℃, and the bacterial growth is good at 15℃. The optimal growth temperature is 30℃. The strain FCG-7 is a low-temperature bacterium, which can play a better role in low-temperature environment.

[0079] Table 3 FCG-7 low temperature resistance test results (OD 600 )

[0080]

[0081] Example 4: Study on the stability of passage

[0082] The mangrove chitinobacterium FCG-7 obtained by screening was used as the first generation strain, and the first generation strain was inoculated into the screening solid medium to obtain the second generation strain, and the second generation strain was inoculated into the solid screening medium to obtain the third generation strain, and so on until the tenth generation strain was obtained. The screening solid culture medium inoculated with FCG-7 bacteria was placed in a 30°C constant temperature incubator for inverted culture. The circle diameter ratio of different generations of FCG-7 bacteria on the 6th day was observed, and the results are shown in Table 4.

[0083] Table 4 Chitin degradation ability of different generations of mangrove Chitinobacterium FCG-7 strains

[0084]

[0085]

[0086] Example 5

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

[0088] Weigh 0.2212g N-acetylglucosamine powder and dissolve it in 10mL double distilled water to obtain a 100mM N-acetylglucosamine solution; take 1mL of 100mM N-acetylglucosamine solution and add 9mL double distilled water to obtain a 10mM N-acetylglucosamine solution. Add double distilled water to prepare N-acetylglucosamine solutions of different concentrations, add DNS for color development, and measure OD 540 .

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

[0090]

[0091]

[0092] The concentration of N-acetylglucosamine is taken as the horizontal axis, OD 540 The value is the ordinate, and the standard curve of N-acetylglucosamine is obtained as follows Figure 6 As shown, the linear equation of the standard curve is y = 0.5653x-0.1202, and the linear correlation coefficient R 2 =0.9996. This indicates that the absorbance values ​​of N-acetylglucosamine solutions of different concentrations at 540 nm have a good linear relationship and conform 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 take the supernatant.

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

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

[0096] Optimization of the best enzyme production conditions for extracellular chitinase produced by the strain (the method for determining the activity of extracellular crude enzyme is described above)

[0097] The growth curve of the strain is the basis for optimizing the subsequent enzyme production conditions. Figure 7 As shown, at 0 to 6 hours, the strain is in the lag phase, and the strain grows slowly. At 6 to 20 hours, it enters the exponential growth phase, and the strain grows rapidly and has strong growth vitality. Selecting the bacterial liquid in this time period as the seed liquid for inoculation can ensure a higher amount of viable bacteria, so the present invention selects 15 to 20 hours as the seed liquid culture time. After 24 hours, the strain grows steadily, and the bacterial amount is high and remains stable. According to this growth curve, in subsequent experiments, the fermentation liquid after 48 hours of culture is selected to measure its enzyme activity.

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

[0099] KH2PO40.3 g / L, K2HPO40.7 g / L, FeSO4 0.1 g / L, CaCl20.1 g / L, MgSO40.5 g / L, NaCl1.0 g / L, colloidal chitin 10 g / L, peptone 2.5 g / L, beef extract 2.5 g / L, and the rest is water, pH value is 7.5.

[0100] Powdered chitin, colloidal chitin, lactose, glucose and sucrose were selected as carbon sources for the initial fermentation medium, respectively. The fermentation was carried out for 48 hours, and the enzyme activity of the fermentation broth was tested. The results showed that the crude enzyme activity of the fermentation broth was the highest when powdered chitin was used as the carbon source, followed by glucose. Therefore, the preferred carbon source in the fermentation medium of the present invention is powdered chitin, and the addition amount is preferably 8-12 g / L. Figure 8 shown.

[0101] On the basis of the initial fermentation medium, powdered chitin was used as the carbon source, and peptone, yeast powder, beef extract, urea and ammonium sulfate were used as nitrogen sources. The results showed that the crude enzyme activity of the fermentation broth was the highest when peptone was used as the nitrogen source, and urea also performed well. Considering that peptone and yeast powder are expensive, the present invention preferably selects urea as the nitrogen source of the fermentation medium, and the addition amount of urea is preferably 8-12 g / L, such as Fig. 9 shown.

[0102] The optimized medium formula was used to select different initial pH values ​​of 6 to 11. The results showed that the crude enzyme activity of the fermentation broth was the highest when the initial pH was 8 to 9. Therefore, the preferred initial pH value of the fermentation medium of the present invention was 8 to 9. Fig.10 shown.

[0103] The optimized culture medium formula was used, and fermentation was performed at different culture temperatures of 20.0, 25.0, 30.0, 35.0, and 40.0°C at an initial pH of 8 to 9. The results showed that the crude enzyme activity of the fermentation broth was the highest at a culture temperature of 20 to 30°C. Therefore, the preferred fermentation culture temperature of the present invention is 20 to 30°C. Fig.11 shown.

[0104] The optimized culture medium formula was used for fermentation at an initial pH of 8 to 9 and a culture temperature of 20 to 30°C, and different rotation speeds of 140 to 220 rpm were selected. The results showed that the crude enzyme activity of the fermentation broth was the highest at a rotation speed of 190 to 220 rpm. Therefore, the preferred fermentation culture speed of the present invention is 190 to 220 rpm. Fig.12 shown.

[0105] The optimized culture medium formula was used for fermentation at an initial pH of 8 to 9, a culture temperature of 20 to 30°C, and a rotation speed of 190 to 220 rpm. The inoculation amounts of 2%, 3%, 4%, 5%, and 6% (V / V) were selected. The results showed that the crude enzyme activity of the fermentation broth was the highest when the inoculation amount was 4% and 5%. Therefore, the preferred inoculation amount of the present invention is 4 to 5%. Fig.13 shown.

[0106] The optimized culture medium formula was used for fermentation at an initial pH of 8 to 9, a culture temperature of 20 to 30°C, a rotation speed of 190 to 220 rpm, and an inoculation amount of 4 to 5%. The fermentation was carried out at different fermentation times of 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 the highest when the fermentation time was 48 to 96 h. Therefore, the preferred fermentation time of the present invention is 48 to 96 h. Fig.14 shown.

[0107] The final optimized enzyme production conditions are as follows: inoculate mangrove chitin bacteria FCG-7 into 100mL R2A liquid culture medium, culture on a shaker at a temperature of 20-30°C and a speed of 190-220rpm for 18h, and concentrate 6-8 times the volume to form the seed liquid. Inoculate the seed liquid at a 4-5% inoculation rate into a 500mL triangular flask containing 100mL pH 8-9 chitinase fermentation medium, and culture on a shaker at a temperature of 20-30°C and a speed of 190-220rpm for 48-96h. Centrifuge the fermentation broth at 4°C and 6000-8000rpm for 30min, collect the supernatant, and the crude enzyme liquid has the highest activity value of 12.43U, which is 29.2 times that before optimization, and store it in a refrigerator at 4°C.

[0108] Example 7

[0109] Obtaining N-acetylglucosamine

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

[0111] Powdered chitin: purchased from Aladdin Reagent (Shanghai) Co., Ltd.

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

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

[0114] The crude enzyme solution was mixed with 1% powdered chitin substrate in a 1:1 ratio, reacted at 4°C, 10°C, 20°C and 30°C for 12h, 12h, 10h and 4h, boiled in a water bath for 5min and centrifuged, the supernatant was taken for HPLC analysis, the results were as follows Fig.16 As shown, the major product is N-acetylglucosamine.

[0115] The products of treatment at different temperatures are all N-acetylglucosamine, and the substrate can be completely degraded into N-acetylglucosamine under low temperature conditions.

[0116] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A mangrove chitinobacterium (Chitinibacter mangrovi) FCG-7, characterized in that It is deposited in Guangdong Microbiological Culture Collection Center with the deposit number GDMCC No: 63488.

2. The method for producing extracellular chitinase using the mangrove chitinobacterium FCG-7 according to claim 1, characterized in that: The following steps are involved: 1) Inoculating mangrove chitinobacterium FCG-7 into a seed culture medium to obtain a seed solution; 2) inoculating the seed liquid into a chitinase fermentation medium for fermentation to obtain a fermentation liquid; 3) solid-liquid separation of the fermentation broth, and collecting the supernatant as the chitinase solution.

3. The method according to claim 2, characterized in that The chitinase fermentation medium described in step 2) uses water as solvent and includes the following components in the following 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, urea 8-12 g / L; the pH value 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 48-96 hours.

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

6. The chitinase solution prepared by the method according to any one of claims 2 to 5.

7. Use of the mangrove chitinobacterium FCG-7 according to claim 1 or the chitinase solution according to claim 6 in degrading chitin, characterized in that: The mangrove chitinobacterium FCG-7 or chitinase solution is used to degrade chitin to prepare N-acetylglucosamine.

8. The use according to claim 7, characterized in that: The chitinase solution of claim 6 is mixed with chitin substrate, reacted at 4-30° C. for 4-24 hours, and after terminating the reaction, the supernatant is collected to obtain N-acetylglucosamine.

9. The use according to claim 8, characterized in that: The volume ratio of the chitinase solution and the chitin substrate is (1-2):(1-2).

10. The use according to claim 9, characterized in that: The chitin substrate is colloidal chitin, powdered chitin or shrimp and crab shell waste.

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