Method for improving activity of chitin deacetylase through mixed fermentation

Abstract

The invention belongs to the technical field of microorganisms, and discloses a method for improving the activity of chitin deacetylase through mixed fermentation, which is characterized in that rhodococcus qingshengii with the preservation number of CGMCC (China General Microbiological Culture Collection Center) No.34439 is used as an enzyme producing bacterium, bacillus zhangzhouensis with the preservation number of CGMCC No.34440 is used as a companion bacterium, and mixed fermentation is carried out to produce the chitin deacetylase. The chitin deacetylase obtained by adopting the method disclosed by the invention is high in activity and high in deacetylation efficiency, a potential production strain is provided for chitin biodegradation, and a theoretical basis is provided for application of the CDA enzyme generated by the strain.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of microorganisms, and particularly relates to a method for improving chitin deacetylase activity through mixed bacterial fermentation. BACKGROUND

[0002] Chitin is the second most abundant polysaccharide in nature after cellulose, and is widely sourced, but is difficult to dissolve in conventional chemical solvents, thus greatly limiting its application. Chitosan generated after chitin deacetylation has good biocompatibility, antibacterial activity, biodegradability and other excellent properties, and plays an important role in the fields of medicine, food, environmental protection and the like. At present, the method for chitin deacetylation in industry is to use high temperature, strong acid and strong alkali chemical methods, which consumes high energy and pollutes the environment. The enzymatic conversion using chitin deacetylase has the advantages of mild reaction conditions, uniform deacetylation degree and environmental friendliness, and is more in line with the needs of green and sustainable development. However, the application of biological conversion method is hindered by the low deacetylation efficiency of chitin deacetylase CDA.

[0003] Therefore, there is an urgent need for a method for improving chitin deacetylase activity to improve the deacetylation efficiency, so as to realize industrial production as soon as possible. SUMMARY

[0004] Therefore, the purpose of the present application is to provide a method for improving chitin deacetylase activity through mixed bacterial fermentation. The present application aims to solve the problems of low chitin deacetylase activity and low deacetylation efficiency of the chitin deacetylase cultivated by the existing method.

[0005] The present application provides a method for improving chitin deacetylase activity through mixed bacterial fermentation, which uses Rhodococcus qingshengii with the preservation number of CGMCC No. 34439 as the enzyme-producing bacteria and Bacillus zhangzhouensis with the preservation number of CGMCC No. 34440 as the associated bacteria for mixed bacterial fermentation production.

[0006] Further, the method for improving chitin deacetylase activity through mixed bacterial fermentation specifically comprises the following steps:

[0007] S1. Seed culture;

[0008] The enzyme-producing bacteria and the associated bacteria are respectively subjected to seed culture under the following conditions:

[0009] Culture conditions: stirring speed of 180 r / min, temperature of 30℃, and culture time of 24h;

[0010] Seed culture medium: 1% proteose peptone, 1% sodium chloride and 0.5% yeast extract powder;

[0011] S2. Fermentation culture

[0012] The fermentation conditions are as follows: the seed liquid of the enzyme-producing bacteria and the companion bacteria is mixed in a ratio of 1:9, inoculated into the fermentation, the inoculation amount is 2-4%, the stirring speed is 160 r / min, the temperature is 30°C, and the fermentation time is 12-48 h.

[0013] Further, in step S2, the fermentation medium components required for fermentation are as follows: maltose 0.2%, peptone 0.5%, and yeast extract 0.2%.

[0014] Further, in step S2, the mixed bacteria ratio of the enzyme-producing bacteria and the companion bacteria seed liquid is 7:3.

[0015] Further, in step S2, the fermentation time is 36 h.

[0016] Further, in step S2, the inoculation amount is 3%.

[0017] Further, in step S2, the mixed bacteria fermentation is carried out by using a shake flask culture method, and the liquid loading amount is 50 mL / 250 mL.

[0018] Further, in the mixed bacteria fermentation process of step S2, the initial pH is 6.0.

[0019] Beneficial effects:

[0020] Through shake flask culture, the mixed bacteria fermentation medium and culture conditions of the two strains are optimized through single factor experiment and response surface experiment. The results show that the optimal ratio of HP-3-1 and HP-3-2 is 7:3, the optimal medium formula for chitin deacetylase production is maltose 0.2%, peptone 0.5%, and yeast extract 0.2%; after optimization, the enzyme activity reaches 175.32 U / mL, which is increased by 125.8%; the optimal culture conditions are as follows: fermentation time 36 h, temperature 30°C, stirring speed 160 r / min, liquid loading amount 50 mL / 250 mL; inoculation amount 3%, initial pH 6.0; after optimization, the enzyme activity reaches 232.41 U / mL, which is increased by 32.6%.

[0021] The protein is purified by ammonium sulfate precipitation, Q Sepharose ion exchange chromatography, and Sephadex G-75 gel filtration chromatography. The SDS-PAGE detection shows that CDA is a single band, and the molecular weight is about 60 kDa. The enzymatic property of the purified CDA is studied, and the results show that the optimal reaction temperature of CDA is 50°C, the optimal pH is 7, the CDA enzyme activity is relatively stable at 30°C, the enzyme is most stable under neutral conditions, and Zn 2+ , K + , Mg 2+ , Mn2+ Ca 2+ and Na + ions promote the enzyme activity of CDA, while Cr 2+ and Fe 2+ have obvious inhibitory effect; phenylmethylsulfonyl fluoride (PMSF) almost completely inhibits the enzyme activity of CDA, and high concentrations of sodium dodecyl sulfate (SDS), Tween 80 and Tween 20 have obvious inhibitory effect on CDA.

[0022] The scanning electron microscope analysis and infrared spectrum determination of the product after CDA treatment show that the chitin after CDA treatment has obvious reticular structure and pores, which indicates that a certain deacetylation effect is achieved; the infrared spectrum result shows that the deacetylation degree of the enzyme product is increased.

[0023] In conclusion, the application provides a method for improving the chitin deacetylase activity by mixed fermentation, and the chitin deacetylase activity is high and the deacetylation efficiency is high by using the method, which provides potential production strains for chitin biodegradation and provides a theoretical basis for the application of the CDA enzyme generated by the strain.

[0024] Other advantages, objects, and features of the present application will be better understood from the following specification and drawings. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 Morphological characteristics of strains HP-3-1 and HP-3-2;

[0026] Figure 2 Enzyme activity of supernatant and precipitate under different ammonium sulfate saturation;

[0027] Figure 3 Q Sepharose ion exchange chromatogram;

[0028] Figure 4 Sephadex G-75 gel filtration chromatogram

[0029] Figure 5 SDS-PAGE electrophoresis map of chitin deacetylase;

[0030] Figure 6 Effect of temperature on CDA enzyme activity;

[0031] Figure 7 Effect of pH on CDA enzyme activity

[0032] Figure 8 Effect of temperature on CDA enzyme stability

[0033] Figure 9 Effect of pH on CDA enzyme stability

[0034] Figure 10 Effect of metal ions on CDA enzyme activity

[0035] Figure 11 Effect of compounds on CDA enzyme activity

[0036] Figure 12 Scanning electron microscope results

[0037] Figure 13 Infrared spectrum curve. DETAILED DESCRIPTION

[0038] In order to make the technical solutions, advantages and purposes of the present application clearer, the technical solutions of the embodiments of the present application will be described clearly and completely below in combination with the drawings of the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. Based on the described embodiments of the present application, all other embodiments obtained by a person of ordinary skill in the art without any creative effort fall within the protection scope of the present application.

[0039] The present application provides a method for improving chitin deacetylase activity by mixed fermentation, which is produced by mixed fermentation of Rhodococcus qingshengii with preservation number of CGMCC No. 34439 as enzyme-producing bacteria and Bacillus zhangzhouensis with preservation number of CGMCC No. 34440 as associated bacteria.

[0040] The strain of enzyme-producing bacteria (Rhodococcus qingshengii) is named as HP-3-1, and the strain of associated bacteria (Bacillus zhangzhouensis) is named as HP-3-2. The morphological characteristics of the strains of HP-3-1 and HP-3-2 are shown in Table 1. Figure 1 The strain HP-3-1 grows slowly, and forms visible single colony after about 48 h of culture in LB medium. The colony is circular, smooth, white, opaque, raised, and has a diameter of about 1.2-2.5 mm (A). The result of gram staining is positive, and the morphology is short rod-shaped (B). Figure 1 Figure 1 ​B, C); the strain HP-3-2 grows faster on the LB medium, and forms visible single colonies after 24 hours, the colony surface is smooth, transparent, flat, and the diameter is about 2-5mm Figure 1 D), and after continuous culture, the HP-3-2 has a wrinkling phenomenon, and the gram staining result is positive, and the morphology is rod-shaped Figure 1 E, F),

[0041] The method specifically comprises the following steps:

[0042] S1. Seed culture;

[0043] The enzyme-producing bacteria and the companion bacteria are respectively subjected to seed culture under the following conditions:

[0044] The culture conditions are as follows: the stirring speed is 180 r / min, the temperature is 30 DEG C, and the culture time is 24h;

[0045] The seed culture medium comprises 1% of proteose peptone, 1% of sodium chloride, 0.5% of yeast extract powder, and the rest is water;

[0046] S2. Fermentation culture

[0047] The fermentation conditions are as follows: the seed liquid of the enzyme-producing bacteria and the companion bacteria which are respectively cultured is mixed in a mixing ratio of 7:3, and then inoculated for fermentation; the inoculation amount is 3%, the stirring speed is 160 r / min, the temperature is 30 DEG C, and the fermentation time is 36h;

[0048] The fermentation medium comprises 0.2% of maltose, 0.5% of proteose peptone, 0.2% of yeast extract powder, and the rest is water.

[0049] As a preferred embodiment of the present embodiment, the mixed fermentation is carried out in the way of shake flask culture, and the liquid loading amount is 50mL / 250mL.

[0050] As a preferred embodiment of the present embodiment, the initial pH in the mixed fermentation process is 6.0.

[0051] Protein purification and enzymatic property research of CDA enzyme

[0052] 1. Preparation of CDA crude enzyme solution

[0053] A large amount of co-fermentation is carried out on the two strains under the optimal fermentation conditions, the bacterial cells are collected by centrifugation of the fermentation liquid at 4500 rpm for 20 min, the bacterial cells are washed to avoid interference of other substances, about 100ml of bacterial cells of the fermentation liquid in each centrifuge tube are suspended with 5mL of 0.05mol / L phosphate buffer with pH of 7, and then ultrasonic crushing is carried out, the crushing power is 400w, the ultrasonic time is 3s, the interval is 6s, and the ultrasonic time is 30min; after the ultrasonic crushing, the crushed liquid is centrifuged at 12000 rpm for 10min, and the supernatant is collected, which is the crude enzyme solution.

[0054] 2. Ammonium sulfate fractionation of CDA crude enzyme solution

[0055] Prepare saturated ammonium sulfate solution at a specific temperature (until no more ammonium sulfate particles dissolve in the solution), and filter to remove undissolved solid particles. Take equal amounts of crude enzyme solution and add different volumes of saturated ammonium sulfate solution to obtain final concentrations of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100% ammonium sulfate. Mix well and let stand at 4°C overnight, then centrifuge at 8000 rpm for 20 min at 4°C. Measure the enzyme activity of the supernatant and precipitate at each saturation level, and plot the ammonium sulfate salting-out curve of CDA using the ammonium sulfate saturation as the horizontal coordinate and the relative enzyme activity of the supernatant and precipitate as the vertical coordinate. Determine the optimal ammonium sulfate saturation range for CDA fractionation based on the curve characteristics, and obtain the target CDA crude enzyme component through fractionation.

[0056] After ammonium sulfate precipitation, dialyze the precipitate sample using PBS, and do not exceed 2 / 3 of the dialysis bag with the dialysate. Dialyze three times, with an interval of 8 hours each time, and use saturated barium chloride to detect whether it contains sulfate ions. Until there are no sulfate ions, the next step can be performed.

[0057] Ammonium sulfate salting-out is a classic purification technique based on the difference in protein solubility, which achieves selective precipitation of target proteins by adjusting the ionic strength of the solution. In this study, the precipitation efficiency of chitin deacetylase (CDA) at different saturation levels was systematically evaluated by drawing the ammonium sulfate salting-out curve. The experimental results are shown in Figure 2 , CDA target protein began to precipitate when the ammonium sulfate saturation reached 40%, and the precipitation amount increased significantly with increasing saturation. In the range of 60%-80% saturation, the precipitation efficiency of CDA reached a peak, with a target protein recovery rate of over 95%. Based on the above results, the subsequent experiment selected 60%-80% ammonium sulfate saturation as the optimal condition for fractionation.

[0058] 3. Q Sepharose anion exchange chromatography of CDA crude enzyme

[0059] Centrifuge the crude enzyme solution after ammonium sulfate fractionation at 12000 rpm for 10 min to remove insoluble precipitate, and filter the supernatant after salting-out using a 0.22 μm filter to obtain a clear enzyme solution. Use pure protein purification system to load the sample into a Q Sepharose anion exchange chromatography column pre-equilibrated with 0.02 mol / L sodium phosphate buffer (pH 7.0) at a flow rate of 1.0 mL / min, with a sample volume of 1 mL. The equilibration buffer volume is 5 times the column volume to ensure stable conductivity and UV-280.

[0060] The elution phase was performed in a linear gradient mode with a gradient of ionic strength from 0 to 1.0 mol / L NaCl, and the flow rate was maintained at 1.0 mL / min. The elution peaks were monitored in real time by a UV detector (280 nm) and the conductivity changes (tracer ionic strength). When the peaks appeared, the eluted peak components were collected into different 15 mL volume centrifuge tubes. The enzyme activity in each collected tube was tested, and the components with measurable enzyme activity were used for subsequent experiments.

[0061] The Q Sepharose anion exchange chromatogram is shown in Fig. 3. During the chromatographic elution process, by collecting the eluate in sections, three protein elution peaks, I, II, and III, appeared in turn. In the experiment, the corresponding effluent of each elution peak was collected separately, and the activity was detected according to the pre-established CDA enzyme activity detection method. The detection results showed that only elution peak I showed CDA enzyme activity, while peaks II and III did not show the target enzyme activity. Based on the above enzyme activity detection results, it is clear that peak I is the target enzyme protein peak, and peaks II and III are impurity protein peaks without target enzyme. Subsequently, the collected liquid of peak I was transferred to a concentration tube with a molecular weight cutoff of 10 kDa, and concentrated by centrifugation at 4°C. After completion, the concentrated enzyme solution was properly stored for use.

[0062] 4. Superdex 75 gel filtration chromatography of CDA crude enzyme solution

[0063] The active eluate obtained by anion exchange chromatography was concentrated by a ultrafiltration centrifuge tube with a molecular weight cutoff of 10 kDa. The centrifugation conditions were set at 4°C, 3500 x g for 10 min, and the concentrated sample was collected. The concentrated sample was filtered through a 0.22 μm filter membrane. The concentrated sample was loaded into a Superdex 75 gel filtration chromatography column at a flow rate of 0.8 mL / min using a pure protein purification system, and the column bed was pre-equilibrated to the baseline with a gel filtration equilibration buffer (0.05 mol / L sodium phosphate buffer containing 0.15 M NaCl, pH 7.0).

[0064] The elution phase maintained the same flow rate, and the UV detector (280 nm) recorded the elution curve in real time, and the conductivity was monitored synchronously (to verify the stability of the salt concentration), and the elution peak components were collected into different 15 ml volume centrifuge tubes. After testing the CDA enzyme activity by colorimetry, the collected solution with CDA activity was concentrated and subjected to SDS-PAGE detection.

[0065] The Sephadex G-75 gel filtration chromatogram is shown in Fig. 4. During the chromatographic elution process, by collecting the eluate in sections, three protein elution peaks, I, II, and III, appeared in turn. In the experiment, the corresponding effluent of each elution peak was collected separately, and the activity was detected according to the pre-established CDA enzyme activity detection method. The detection results showed that only elution peak I showed CDA enzyme activity, while peaks II and III did not show the target enzyme activity. Based on the above enzyme activity detection results, it is clear that peak I is the target enzyme protein peak, and peaks II and III are impurity protein peaks without target enzyme. Subsequently, the collected liquid of peak I was transferred to a concentration tube with a molecular weight cutoff of 10 kDa, and concentrated by centrifugation at 4°C. After completion, the concentrated enzyme solution was properly stored for use. Figure 4The elution peaks of three proteins, I, II and III, appeared in turn on the chromatogram after elution. The eluate was detected for enzyme activity. The results showed that only the eluate of peak I had significant CDA enzyme activity, while the eluate of peaks II and III had no target enzyme activity. Based on the above enzyme activity detection results, peak I was the target CDA enzyme protein peak, and peaks II and III were impurity protein peaks without target enzyme. Then, the collected liquid of peak I was transferred to a concentration tube with a molecular weight cutoff of 10 kDa, and concentrated by centrifugation at 4°C. After completion, the concentrated enzyme solution was properly stored for subsequent experimental analysis.

[0066] 5. SDS-PAGE detection of CDA enzyme solution

[0067] The gel was prepared using SERVA SDS-PAGE kit, and 40 μl of sample was used for sample preparation, 10 μl of protein loading buffer (5x) was added, inactivated for 10 min, 20 μl was added, and then run at 90 v for 30 min and at 120 v for 90 min. After staining for 20-30 min, the color was removed.

[0068] The purity and molecular weight of the purified CDA were determined by SDS-PAGE gel electrophoresis, and the electrophoresis results are shown in Figure 5 The 6 bands in the figure are 1: Marker; 2: supernatant of broken liquid; 3: after dialysis; 4: after anion exchange chromatography; 5: after concentration; 6: gel filtration chromatography. As can be seen from the figure, the crude enzyme solution obtained after cell disruption was subjected to a series of chromatographic purification such as ammonium sulfate precipitation, dialysis, Q Sepharose anion exchange chromatography and Superdex 75 gel filtration chromatography. The enzyme protein showed a single band on the electrophoretogram, i.e. electrophoretically pure chitin deacetylase was obtained. The supernatant of the broken liquid had more impurities (2nd lane), and some impurities were removed by ammonium sulfate precipitation, but impurities still existed (3rd lane). Then, the pure protein of single band was separated by anion exchange chromatography and gel filtration chromatography. Protein electrophoresis showed that the molecular weight of CDA produced by fermentation of the strain was about 60 kDa, and almost existed as a single component.

[0069] 6. Analysis of CDA enzymatic properties

[0070] 6.1 Effect of temperature on CDA

[0071] CDA 200 μL and 200 μL 0.02 mol / L p-nitroacetanilide were mixed, 600 μL of 0.05 mol / L phosphate buffer (pH 7.0) was added, and the mixture was reacted at 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, and 70°C for 15 min, inactivated for 10 min, and the enzyme activity of CDA was determined. The highest enzyme activity was defined as 100%, and the relative enzyme activity at different temperatures was calculated. Each group had three replicates.

[0072] As shown in Figure 6 , the enzyme activity of CDA increased at 25-50°C, reached the highest value at 50°C, which was defined as 100%, and then rapidly decreased after 50°C, and basically lost the enzyme activity at 70°C. The high temperature may have caused protein denaturation, so 50°C was the optimum temperature for CDA catalysis.

[0073] 6.2 Effect of pH on CDA

[0074] CDA 200 μL and 200 μL 0.02 mol / L p-nitroacetanilide were mixed, 600 μL of 0.05 mol / L phosphate buffer (pH 7.0) was added, and the mixture was reacted at 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, and 70°C for 15 min, inactivated for 10 min, and the enzyme activity of CDA was determined. The highest enzyme activity was defined as 100%, and the relative enzyme activity at different temperatures was calculated. Each group had three replicates.

[0075] As shown in Figure 7 , the enzyme activity of CDA increased at 25-50°C, reached the highest value at 50°C, which was defined as 100%, and then rapidly decreased after 50°C, and basically lost the enzyme activity at 70°C. The high temperature may have caused protein denaturation, so 50°C was the optimum temperature for CDA catalysis.

[0076] 6.3 Optimum temperature stability of CDA

[0077] CDA enzyme solution was placed in a water bath at 30°C, 35°C, 40°C, 45°C, and 50°C for 3 h, and samples were taken every half hour, cooled, and then 200 μL of p-nitroacetanilide and 600 μL of 0.05 mol / L phosphate buffer were added. The mixture was reacted at 50°C for 15 min, inactivated for 10 min, and the enzyme activity of CDA was determined. The highest enzyme activity was defined as 100%, and the relative enzyme activity at different temperatures was calculated. Each group had three replicates.

[0078] As shown in Figure 8As shown, CDA enzyme activity is relatively stable at 30℃, with virtually no change after 3 hours of incubation. At 35-40℃, CDA enzyme activity also remains above 60%. However, at 45-50℃, enzyme activity decreases rapidly with longer incubation times, and after 3 hours, enzyme activity decreases or is lost. Therefore, this CDA enzyme is a low-temperature enzyme, and higher temperatures have a greater impact on it. Once the optimal temperature range is exceeded, enzyme activity gradually decreases.

[0079] Optimal pH stability of 6.4CDA

[0080] CDA enzyme solutions were incubated in phosphate buffer solutions at pH 3, 4, 5, 6, 7, 8, 9, and 10 for 2 h and 20 h, respectively. 200 μL of p-nitroacetanilide was added, and the mixture was reacted at 50 °C for 15 min, followed by inactivation for 10 min. The enzyme activity of CDA was then measured, with the highest enzyme activity defined as 100%. The relative enzyme activity of the strains at different pH values ​​was calculated. Each group was tested in triplicate.

[0081] like Figure 9 As shown, the activity and stability of chitin deacetylase varied significantly under different pH values ​​(pH 6–8) and incubation times (2 h and 20 h). During short-term incubation (2 h), the enzyme activity remained above 80% of its initial activity within the pH range of 6–8. After 20 h of incubation, the enzyme activity was still highest at pH 7. Therefore, the optimal pH for the enzyme's activity is around 7, and it exhibits good stability under neutral conditions.

[0082] 6.5 Effects of Metal Ions on CDA

[0083] CDA enzyme solution was reacted with 1 mmol / L and 5 mmol / L Cu, respectively. 2+ Cr 2+ Zn 2+ K + Mn 2+ Fe 2+ Ca 2+ Mg 2+ Na + The mixture was incubated at room temperature for 2 hours, then centrifuged at 12,000 rpm for 5 minutes. 200 μL of the supernatant was mixed with 200 μL of 0.02 mol / L p-nitroacetanilide, and 600 μL of 0.05 mol / L phosphate buffer (pH 7.0) was added. The mixture was incubated at 50°C for 15 minutes. After inactivation for 10 minutes, the enzyme activity of CDA was measured. The untreated enzyme solution was defined as 100%. The relative enzyme activity under different metal ions was calculated. Each group was tested in triplicate.

[0084] Metals act as cofactors in enzyme catalysis, enhancing the activity of specific enzymes. For example...Figure 10 As shown, at low concentrations (1 mM), Cu 2+ Zn 2+ K + Mg 2+ Mn 2+ Ca 2+ and Na + Ions stimulate the enzyme activity of CDA, among which Mg 2+ and Na + It has a significant activating effect. However, regardless of whether it is 1mM or 5mM, Cr 2+ and Fe 2+ It has a significant inhibitory effect on enzyme activity, and at high concentrations (5mM), Cu 2+ It also significantly inhibited the enzyme activity of CDA.

[0085] 6.6 Effects of Compounds on CDA

[0086] CDA enzyme solution was mixed with 1 mmol / L and 5 mmol / L EDTA, SDS, PSMF, Tween 80, and Tween 20, respectively, and incubated at room temperature for 2 h. The mixture was then centrifuged at 12000 rpm for 5 min. 200 μL of the supernatant was mixed with 200 μL of 0.02 mol / L p-nitroacetanilide, and 600 μL of 0.05 mol / L phosphate buffer (pH 7.0) was added. The mixture was incubated at 50 °C for 15 min, followed by 10 min of inactivation. The enzyme activity of CDA was measured, with the untreated enzyme solution defined as 100%. The relative enzyme activity of different compounds was calculated. Each group was tested in triplicate.

[0087] like Figure 11 As shown, regardless of the concentration, PMSF has a significant inhibitory effect on enzyme activity. At high concentration (5mM), SDS, Tween 20, and Tween 80 inhibit CDA activity, while Tween 20 slightly inhibits CDA activity. At low concentration (1mM), Tween 80 promotes CDA activity.

[0088] 7. Scanning electron microscopy analysis

[0089] 0.2 g of chitin was dissolved in 10 mL of CDA enzyme solution and placed in a 50 mL centrifuge tube. The mixture was reacted at 30 °C and 240 r / min for 48 h. Afterward, the mixture was centrifuged at 8000 rpm for 10 min using a pre-cooled refrigerated centrifuge. The precipitate at the bottom of the tube was carefully collected and washed repeatedly with sterile water until its pH was neutral. The washed precipitate was then transferred to a 50 °C drying oven and dried until its mass was constant. Three samples were respectively adhered to a metal sample stage with conductive adhesive. A metal film was deposited onto the sample surface using a vacuum evaporator. The microstructure of the sample surface was photographed using scanning electron microscopes with magnifications of 200, 2000, 5000, and 20000.

[0090] Scanning electron microscopy (SEM) was used to characterize chitin, CDA-treated chitin, and chitosan, as shown in the results. Figure 12 As shown in the figure, a: chitin; b: CDA-treated chitin; c: chitosan. Untreated chitin exhibits a large, dense, layered structure with a surface composed of tightly packed crystalline microfibrils. These structures are dense, rough, and textured (see Figure 1). Figure 12 a). After CDA treatment of chitin, the originally dense layered structure was severely damaged, resulting in many irregular pits, pores and dense cracks on the surface, while its fibers became dispersed and blurred, and the interface with the separated flakes was not very obvious (see...). Figure 12 (b) Because CDA deacetylation reduces the acetyl content on the chitinous polysaccharide chains, the hydrogen bonds that maintain the intramolecular and intermolecular structural stability of the polysaccharide chains are interrupted or weakened, resulting in changes in the microstructure of chitin. The particle state of CDA-treated chitin is similar to that of chitosan, but significantly different from chitin itself, indicating that CDA has a good deacetylation effect and can be used to prepare chitosan products with a high degree of deacetylation.

[0091] 8. Infrared spectroscopy determination

[0092] 0.2 g of chitin was dissolved in 10 mL of CDA enzyme solution and placed in a 50 mL centrifuge tube. The mixture was reacted at 30 °C and 240 r / min for 48 h. After centrifugation at 8000 rpm for 10 min using a pre-cooled refrigerated centrifuge, the precipitate at the bottom of the tube was carefully collected. The precipitate was washed repeatedly with sterile water until its pH was neutral. The washed precipitate was then transferred to a 50 °C drying oven and dried until its mass was constant. Dry potassium bromide and the sample were weighed at a ratio of 50:1, ground and mixed. 0.2 g of the mixture was weighed and manually compressed into thin sheets. FT-IR spectroscopy was used to scan the wavelength range of 400-4000 nm. The intensity ratio of the characteristic absorption peaks at wavenumbers of 1655 cm⁻¹ (amide I) and 3450 cm⁻¹ (OH group) showed a linear correlation with the degree of deacetylation of the chitin sample, calculated using the following formula:

[0093]

[0094] Fourier transform infrared spectroscopy (FT-IR) was used to compare the CDA enzymatic hydrolysis products with those of chitin and chitosan. The results are as follows. Figure 13 As shown, a: chitin; b: CDA-treated chitin; c: chitosan. At 3450 cm⁻¹ -1 A significant broad absorption peak was observed at 2928 cm⁻¹, attributed to the overlap of the stretching vibration peaks of -OH and -NH, indicating the formation of hydrogen bonds and enhanced intermolecular interactions during enzymatic hydrolysis. -1 The peak, attributed to the -CH stretching vibration, shifts towards lower frequencies, which is related to the high crystallinity of chitin. The weakening of this peak shift in the CDA-treated sample indicates that enzymatic hydrolysis reduces the crystallinity of chitin. (1635 cm⁻¹) -1 With 1558cm -1 These correspond to the amide I band (C=O stretching vibration) and the amide II band (coupling of NH bending vibration and CN stretching vibration), respectively, indicating the presence of residual acetyl groups in the sample. 1381 cm⁻¹ -1 With 1316cm -1 These correspond to the -CH2 oscillating vibration and the amide III band (CN stretching and NH bending vibrations), respectively, further verifying the degree of deacetylation. 1158 cm⁻¹ -1 1073cm -1 With 1027cm -1 The degrees of deacetylation (DD) are attributed to the asymmetric oxygen-bridge stretching vibration (COC) and CO stretching vibration of the sugar ring, respectively, reflecting the structural characteristics of the chitin polysaccharide backbone. The degree of deacetylation (DD) is based on the amide I band (1655 cm⁻¹). -1 ), Amide III band (1320cm) -1 ) and -NH vibration (3269cm) -1The intensity change of the peak was calculated by the peak area integration method. The results showed that the DD of chitin was 23.21%, while the DD of the CDA enzymatic product was about 41.6%. This indicated that the CDA produced by the screened strain had the ability to catalyze the deacetylation of chitin, which helped to convert chitin into chitosan.

[0095] In this statement, the above examples are only used to illustrate the technical solutions of the present application and are not limited. Although the present application has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solutions of the present application can be modified or equivalently replaced without departing from the purpose and scope of the present application, which should be covered in the scope of the claims of the present application.

Claims

1. A method for improving chitin deacetylase activity through mixed-culture fermentation, characterized in that: The enzyme was produced by mixed fermentation using Rhodococcus qingshengii (CGMCC No. 34439) as the enzyme-producing bacterium and Bacillus zhangzhouensis (CGMCC No. 34440) as the concomitant bacterium.

2. The method for improving chitin deacetylase activity through mixed-culture fermentation according to claim 1, characterized in that, Specifically, the following steps are included: S1. Seed culture; The enzyme-producing bacteria and associated bacteria were cultured under the following conditions: Culture conditions: stirring speed 180 r / min, temperature 30℃, culture time 24 h; Seed culture medium: 1% peptone, 1% sodium chloride, 0.5% yeast extract; S2. Fermentation culture; Fermentation conditions: The seed cultures of enzyme-producing bacteria and associated bacteria that have been cultured separately are mixed at a ratio of 1 to 9:1 and then inoculated for fermentation; the inoculation amount is 2 to 4%, the stirring speed is 160 r / min, the temperature is 30℃, and the fermentation time is 12 to 48 h.

3. The method for improving chitin deacetylase activity through mixed-culture fermentation according to claim 2, characterized in that: In step S2, the fermentation medium components required for fermentation are: 0.2% maltose, 0.5% peptone, and 0.2% yeast extract.

4. The method for improving chitin deacetylase activity through mixed-culture fermentation according to claim 3, characterized in that: In step S2, the ratio of the mixed culture of enzyme-producing bacteria and associated bacteria seed liquid is 7:

3.

5. The method for improving chitin deacetylase activity through mixed-culture fermentation according to claim 4, characterized in that: In step S2, the fermentation time is 36 hours.

6. The method for improving chitin deacetylase activity through mixed-culture fermentation according to claim 5, characterized in that: In step S2, the inoculation amount is 3%.

7. The method for improving chitin deacetylase activity through mixed-culture fermentation according to claim 6, characterized in that: In step S2, mixed-culture fermentation is carried out using a shake flask culture method, with a liquid volume of 50mL / 250mL.

8. The method for improving chitin deacetylase activity through mixed-culture fermentation according to claim 7, characterized in that: In the mixed-culture fermentation process of step S2, the initial pH is 6.0.

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