A kind of chitin deacetylase efficient production method

Abstract

The invention discloses a chitosan deacetylase efficient production method, and relates to the field of microbial fermentation technology. The method comprises the following steps: (1) inoculating Bacillus equilensis in a fermentation medium for cultivation; (2) adding solid chitosan and inducer α-pentacetyl glucose to the fermentation medium; (3) separating the fermentation liquid and taking the supernatant; (4) separating and purifying the protein. During the cultivation process, chitosan is used as an inducer, and α-pentacetyl glucose is added to the culture fluid, which has a great promoting effect on the production of chitosan deacetylase. After about 72 hours of cultivation, the crude enzyme activity of the culture fluid can reach 100-400 U / mL, and the enzyme production activity is close to that of engineering bacteria.

Description

Technical Field

[0001] The invention relates to the technical field of microbial fermentation, and more particularly to a method for efficiently producing chitin deacetylase. Background Art

[0002] Chitosan, also known as chitosan, is made of N-acetylglucosamine condensed with β-1,4 glycosidic bonds. Natural chitosan has poor solubility and is difficult to dissolve in water, acid, alkali and general organic solvents, which greatly limits its application. Chitosan, as the product of chitosan after removing the N-acetyl group, has greatly improved solubility and is widely used in medicine and health, food industry, textile industry and agriculture.

[0003] The premise of chitosan application is to dissolve it. At present, the methods of chitosan dissolution in industry and research mainly use strong acids and alkalis, which not only consume a lot of reagents, but also cause serious pollution to the environment and increase the cost of subsequent pollutant treatment. The use of biological enzymatic hydrolysis of chitosan to obtain chitosan can obtain high-quality chitosan products while avoiding the use of strong acids and alkalis, achieving the goal of environmental friendliness.

[0004] Chitin deacetylase (chitin deacetylase, referred to as CDA) (EC3.5.1.41), specifically removes N-acetyl in chitin, and chitin is converted into chitosan. At present, utilizing bioenzymatic method to prepare chitosan has become one of the most promising technologies for chitin industrial application. But chitin deacetylase is expensive at present, and output is limited, and it is difficult to carry out industrial application. The inducer of current literature report is all colloidal chitin basically, is the product after natural chitin is treated with strong acid. Strong acid treatment can destroy the crystalline structure of chitin, and is convenient to the effect of deacetylase. Due to the similar crystalline structure of chitin, the chitin deacetylase currently produced or studied is difficult to contact the action site of natural chitin, and the deacetylation effect of natural chitin is very low.

[0005] Therefore, providing a method for efficiently producing chitin deacetylase is a problem that those skilled in the art need to solve urgently. Summary of the invention

[0006] In view of this, the present invention provides a method for efficiently producing chitin deacetylase;

[0007] In order to achieve the above object, the present invention adopts the following technical solution:

[0008] A method for efficiently producing chitin deacetylase comprises the following steps:

[0009] (1) inoculating Bacillus tequilensis into a fermentation medium, wherein the medium comprises: 0-5.0 g / 100 mL of glucose, 0-5.0 g / 100 mL of solid chitosan, and 0.2-1.5 g / 100 mL of an inorganic nitrogen source or an organic nitrogen source, and culturing for 12-48 hours;

[0010] (2) adding 0.1-5.0 g / 100 mL of solid chitosan and 0.1-5.0 g / 100 mL of inducer α-pentaacetyl glucose to the fermentation medium, and fermenting for 24-118 hours to obtain a fermentation liquid;

[0011] (3) centrifuging the fermentation liquid in step (2), and the supernatant is the crude chitin deacetylase enzyme solution;

[0012] (4) The crude enzyme solution is separated and purified by protein separation and purification methods to obtain liquid enzyme or solid enzyme with higher purity.

[0013] Furthermore, the inoculation amount in step (1) is 10% of the volume fraction of the fermentation medium.

[0014] Furthermore, the inorganic nitrogen source or organic nitrogen source in the fermentation medium in step (1) is any one of yeast extract, peptone or ammonium sulfate.

[0015] Furthermore, the pH of the fermentation medium in step (1) is 7.0-7.5.

[0016] Furthermore, the fermentation medium in step (1) includes: NaCl 0.05 g, KH2PO4 0.1 g, MgSO4 0.02 g, NaHPO4 0.6 g, and deionized water 100 mL.

[0017] Furthermore, the protein separation and purification method in step (4) is ammonium sulfate precipitation or ultrafiltration concentration.

[0018] It can be seen from the above technical solution that, compared with the prior art, the beneficial effects of the present invention are:

[0019] 1. This application uses Bacillus tequilensis, which has high enzyme production activity;

[0020] 2. During the culture process, natural chitin was used as an inducer, and α-pentacetyl glucose was added to the culture medium, which greatly promoted the production of chitin deacetylase. After about 72 hours of culture, the crude enzyme activity of the culture medium could reach 100-400U / mL, and the enzyme activity was close to that of the engineered bacteria.

[0021] 3. The substrate used in the present invention is natural chitin, which does not need acid treatment, simplifies the operation on the one hand, and avoids the use of strong acid in addition, which has environmental advantages. The induced chitin deacetylase can be used for the degradation of natural chitin, solving the bottleneck problem of the current chitin enzymatic degradation. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0023] Figure 1 The accompanying drawing shows the effect of the culture temperature on the enzyme production of the present invention;

[0024] Figure 2 The accompanying drawing shows the effect of the initial pH on enzyme production according to the present invention. DETAILED DESCRIPTION

[0025] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0026] Source of experimental materials: Bacillus tequilensis and other experimental materials were commercially available.

[0027] Embodiment 1:

[0028] Bacillus tequilensis was inoculated into a fermentation medium at a 10% inoculation rate: 3.0 g / 100 mL of glucose, 1.0 g / 100 mL of solid chitin, and 1.0 g / 100 mL of yeast extract as a nitrogen source, with a pH of 7.2, and cultured for 24 hours, and the chitin deacetylase activity in the fermentation broth was 4.2 U / mL; at this time, 1.0 g / 100 mL of α-pentacetyl glucose was added to the culture broth, and cultured for 48 hours, and the chitin deacetylase activity in the fermentation broth reached 322 U / mL, and the deacetylation degree of chitin reached 32.5%;

[0029] The supernatant was collected by centrifugation and concentrated by ultrafiltration to obtain chitin deacetylase.

[0030] Embodiment 2:

[0031] Bacillus tequilensis was inoculated into a fermentation medium at a 10% inoculum amount: 3.5 g / 100 mL glucose, 1.5 g / 100 mL peptone as a nitrogen source, pH 7.0, and cultured for 12 hours; solid chitin 3.0 g / 100 mL, α-pentaacetyl glucose 1.5 g / 100 mL were added, and cultured for 72 hours, and the chitin deacetylase activity in the fermentation broth reached 415 U / mL, and the deacetylation degree of chitin was 43.2%;

[0032] The supernatant was collected by centrifugation, and the protein was precipitated with ammonium sulfate to obtain chitin deacetylase.

[0033] Embodiment 3:

[0034] Bacillus tequilensis was inoculated into a fermentation medium at a 10% inoculum amount: glucose 5.0 g / 100 mL, solid chitosan 2.0 g / 100 mL, ammonium sulfate 1.5 g / 100 mL, α-pentaacetyl glucose 3.0 g / 100 mL, pH 7.3, and cultured for 72 hours. The chitosan deacetylase activity in the fermentation broth reached 434 U / mL, and the deacetylation degree of chitosan reached 51.3%;

[0035] The supernatant was collected by centrifugation and concentrated by ultrafiltration to obtain chitin deacetylase.

[0036] Optimization experiment:

[0037] 1. Effect of inducers on enzyme production

[0038] Fermentation medium (g / 100 mL): chitosan 1.0 g, glucose 1.0 g, peptone 1.0 g, NaCl 0.5 g, initial pH 7.0.

[0039] Different types of inducers were added to the culture medium, including 0.5 g each of α-pentacetyl glucose, β-pentacetyl glucose, acetylglucosamine, and glucosamine, and the culture medium without the addition of the inducer was used as a blank control.

[0040] The strain was cultured in seed solution until 1.03×10 8 cfu·mL -1 , inoculated into the fermentation medium at 10mL / 100mL, cultured at 28℃, and measured intracellular and extracellular enzyme activities every 24h. The results are shown in Table 1:

[0041] Table 1 Effect of adding inducers on enzyme production Enzyme activity U / mL

[0042] Inducers 24h 48h 72h 96h 120h α-Pentaacetyl Glucose 4.2 10.2 25.3 49.6 47.3 β-Pentaacetyl Glucose 1.1 2.6 4.9 11.3 10.2 Acetyl Glucosamine 0.9 2.1 3.9 4.2 4.3 Glucosamine 0.8 1.1 1.5 2.2 2.0 Blank control 1.0 2.3 4.5 10.1 9.2

[0043] As can be seen from Table 1, after adding the inducer α-acetylglucose, the enzyme production capacity of the strain has been greatly improved. β-pentacetylglucose has a certain promoting effect on the enzyme production capacity of the strain, and other additives have basically no promoting effect on the enzyme production capacity of the strain, and even have a certain inhibitory effect.

[0044] 2. Effect of chitin type on enzyme production

[0045] Fermentation medium (g / 100 mL): 0.5 g of α-pentacetyl glucose, 1.0 g of glucose, 1.0 g of peptone, 0.5 g of NaCl, initial pH 7.0.

[0046] 1.0 g of natural chitin, colloidal chitin, and chitosan were added to the culture medium to compare the effects of different types of chitin on enzyme production. The culture medium without any type of chitin was used as a blank control.

[0047] The strain was cultured in seed solution until 1.03×10 8 cfu·mL -1 , inoculated into the fermentation medium at 10mL / 100mL, cultured at 28℃, and measured intracellular and extracellular enzyme activities every 24h. The results are shown in Table 2:

[0048] Table 2 Effect of chitin type on enzyme production, enzyme activity (U / mL)

[0049] Cultivation time 24h 48h 72h 96h 120h Natural Chitosan 4.2 10.2 25.3 49.6 47.3 Colloidal Chitosan 3.5 7.2 20.1 32.3 30.6 Chitosan 1.5 2.6 3.1 3.4 3.2 Blank control 0.1 0.2 0.3 0.3 0.3

[0050] As shown in Table 2, adding natural chitosan to the culture medium promotes the enzyme production activity of the strain the most, followed by colloidal chitosan. Colloidal chitosan is the product of the reaction between natural chitosan and strong acid, and is treated with strong acid to remove part of the acetyl group. The present invention has a stronger utilization capacity for natural chitosan, which can also avoid the introduction of a large amount of strong acid in production, indicating that the present invention has a stronger environmental advantage.

[0051] 3. Effect of chitosan addition on enzyme production

[0052] Fermentation medium (g / 100 mL): chitin 0.1-10.0 g, α-pentaacetyl glucose 0.5 g, glucose 1.0 g, peptone 1.0 g, NaCl 0.5 g, initial pH 7.0.

[0053] The strain was cultured in seed solution until 1.03×10 8 cfu·mL -1 , inoculated into the fermentation medium at 10mL / 100mL, cultured at 28℃, and measured intracellular and extracellular enzyme activities every 24h. The results are shown in Table 3:

[0054] Table 3 Effect of chitin addition on enzyme production and enzyme activity (U / mL)

[0055] Chitosan addition amount 24h 48h 72h 96h 120h 0.1g / 100ml 2.3 4.2 7.3 10.6 10.9 1.0g / 100ml 4.2 10.2 25.3 49.6 47.3 3.0g / 100ml 4.1 11.4 25.3 52.6 51.7 5.0g / 100ml 4.1 11.9 24.2 48.1 44.6 8.0g / 100ml 3.2 8.1 20.3 32.1 30.6 10.0g / 100ml 3.0 7.6 15.6 22.4 20.6

[0056] With the increase of chitosan addition, the enzyme production capacity of the strain gradually increased, reaching a relatively high enzyme activity at 1.0-5.0g / 100ml. After that, the enzyme production capacity decreased to a greater extent when the chitosan dosage was increased.

[0057] 4. Effect of α-pentacetyl glucose addition on enzyme production

[0058] Fermentation medium (g / 100 mL): chitin 1.0 g, α-pentaacetyl glucose 0.1-5.0 g, glucose 1.0 g, peptone 1.0 g, NaCl 0.5 g, initial pH 7.0.

[0059] The strain was cultured in seed solution until 1.03×10 8 cfu·mL -1 , inoculated into the fermentation medium at 10mL / 100mL, cultured at 28℃, and measured intracellular and extracellular enzyme activities every 24h. The results are shown in Table 3:

[0060] Table 4 Effect of α-pentacetyl glucose addition on enzyme production, enzyme activity (U / mL)

[0061] α-pentaacetyl glucose added 24h 48h 72h 96h 120h 0.1g / 100ml 2.1 3.2 6.3 7.6 7.9 0.5g / 100ml 4.2 10.2 25.3 49.6 47.3 1.0g / 100ml 7.2 15.6 37.9 96.3 95.2 1.5g / 100ml 10.3 26.9 47.8 125.6 122.4 3.0g / 100ml 9.2 20.4 32.7 86.5 80.2 5.0g / 100ml 2.1 4.6 10.6 20.4 20.1

[0062] With the increase of α-pentacetyl glucose added, the enzyme production capacity of the strain gradually increased, reaching a relatively high enzyme activity at 0.5-3.0g / 100ml. After that, the enzyme production capacity decreased to a greater extent when the amount of α-pentacetyl glucose was increased.

[0063] 5. Effect of culture temperature on enzyme production

[0064] Fermentation medium (g / 100 mL): chitin 3.0 g, α-pentaacetyl glucose 1.5 g, glucose 1.0 g, peptone 1.0 g, NaCl 0.5 g, initial pH 7.0.

[0065] The strain was cultured in seed solution until 1.03×10 8 cfu·mL -1 , inoculated into the fermentation medium at 10mL / 100mL, the culture temperature was set at 25℃, 28℃, 30℃, 35℃, 40℃, cultured for 72h, and the intracellular and extracellular enzyme activities were measured. Figure 1 As shown, with the increase of temperature, the enzyme production capacity of the strain gradually increased, 35°C was the optimal enzyme production temperature, and the enzyme activity could reach 412U / mL (relative enzyme activity 100%).

[0066] 6. Effect of initial pH on enzyme production

[0067] Fermentation medium (g / 100 mL): chitin 3.0 g, α-pentaacetyl glucose 1.5 g, glucose 1.0 g, peptone 1.0 g, NaCl 0.5 g, initial pH set to 4, 5, 6, 7, 8.

[0068] The strain was cultured in seed solution until 1.03×10 8 cfu·mL -1 , inoculated into the fermentation medium at 10 mL / 100 mL, cultured at 35 °C for 72 h, and measured the intracellular and extracellular enzyme activities. Figure 2 As shown, with the increase of pH, the enzyme production capacity of the strain gradually increased, about 7.0 was the optimal pH for enzyme production, and the enzyme activity was higher than 400 U / mL (relative enzyme activity 100%).

[0069] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for efficiently producing chitin deacetylase, characterized in that: The following steps are involved: (1) Bacillus tequila Bacillus tequilensis Inoculate in a fermentation medium for culturing, wherein the medium includes: 1.0-5.0 g / 100 mL of glucose, 0.1-5.0 g / 100 mL of solid chitin, and 0.2-1.5 g / 100 mL of an inorganic nitrogen source or an organic nitrogen source, and culture for 12-48 hours; (2) adding 0.1-5.0 g / 100 mL of solid chitosan and 0.1-5.0 g / 100 mL of inducer α-pentaacetyl glucose to the fermentation medium, and fermenting for 24-118 hours to obtain a fermentation liquid; (3) centrifuging the fermentation liquid in step (2), and obtaining a supernatant which is a crude chitin deacetylase enzyme solution; (4) The crude enzyme solution is separated and purified by protein separation and purification methods to obtain liquid enzyme or solid enzyme with higher purity.

2. A chitin deacetylase efficient production method according to claim 1, characterized in that, The inoculation amount in step (1) is 10% of the volume fraction of the fermentation medium.

3. A chitin deacetylase efficient production method according to claim 1, characterized in that, The inorganic nitrogen source or organic nitrogen source in the fermentation medium in step (1) is any one of yeast extract, peptone or ammonium sulfate.

4. A chitin deacetylase efficient production method according to claim 1, characterized in that, The pH of the fermentation medium in step (1) is 7.0-7.

5.

5. A chitin deacetylase efficient production method according to claim 1, characterized in that, Step (1) The fermentation medium includes: NaCl 0.05 g, KH2PO4 0.1 g, MgSO4 0.02 g, NaHPO4 0.6 g, and deionized water 100 mL.

6. A method for efficiently producing chitin deacetylase according to claim 1, characterized in that: The protein separation and purification method in step (4) is ammonium sulfate precipitation or ultrafiltration concentration.

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