Low-product inhibition chitosanase mutant and application thereof

Abstract

The invention relates to a low-product-inhibition chitosanase mutant and application thereof.The amino acid sequence of the chitosanase mutant is shown as SEQ ID NO.1. On the basis that the function of wild enzyme (Csn168 chitosanase) is kept, the mutant enzyme greatly improves the enzyme activity compared with the wild enzyme, the product inhibition phenomenon is reduced, the enzyme catalysis efficiency is remarkably improved, and the low-product-inhibition chitosanase mutant can be used for preparing the low-product-inhibition chitosanase mutant. And high-concentration chitosan oligosaccharide can be accumulated in single-batch production.

Description

Technical Field

[0001] This invention belongs to the field of enzyme design, specifically relating to a low-product chitosanase inhibitor mutant and its applications. Background Technology

[0002] Chitosanase is a class of hydrolytic enzymes that can efficiently catalyze the hydrolysis of chitosan into chitosan oligosaccharides, and has broad application prospects in food, medicine, agriculture and environmental engineering.

[0003] In practical catalytic reactions, chitosanase commonly suffers from product inhibition, where the generated hydrolysis product, chitosan oligosaccharide, competitively binds to the enzyme's active site or binding pocket, hindering further substrate entry and hydrolysis. This phenomenon leads to a decrease in reaction rate and catalytic efficiency, severely impacting the industrial application value of chitosanase. The root cause of product inhibition lies primarily in the strong hydrogen bonds, hydrophobic interactions, and electrostatic interactions between chitosan oligosaccharides and the enzyme. In particular, the molecular structure of oligosaccharides is similar to that of the substrate chain, making them more likely to occupy key binding sites on the enzyme. Furthermore, the accumulation of chitosan oligosaccharide concentration in solution alters the enzyme's microenvironment, further exacerbating the inhibitory effect. Summary of the Invention

[0004] The purpose of this invention is to overcome the problems existing in the prior art and provide a low-product chitosanase inhibitor mutant.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] A low-product-inhibiting chitosanase mutant, the amino acid sequence of which is shown in SEQ ID NO: 1.

[0007] The present invention further provides a gene encoding the above-mentioned mutant.

[0008] The present invention further provides a recombinant vector containing the above-mentioned genes.

[0009] In some embodiments of the present invention, the recombinant vector uses pET-22b as the expression vector.

[0010] The present invention further provides recombinant engineered bacteria containing the above-mentioned recombinant vector.

[0011] In some embodiments of the present invention, the recombinant engineered bacteria are constructed using Escherichia coli as the host bacterium.

[0012] The present invention further provides a method for purifying the above-mentioned chitosanase mutant, comprising:

[0013] The above-mentioned recombinant engineered bacteria were cultured to OD200. 600=0.5-0.8, then IPTG was added to induce expression;

[0014] After induction, the bacterial culture was centrifuged and the bacterial cells were collected, resuspended in phosphate buffer, and then sonicated.

[0015] After the lysate is centrifuged at ultraspeed, the supernatant is collected and purified by nickel column chromatography to obtain purified protein.

[0016] The present invention further provides the application of the above-mentioned chitosanase mutant in the enzymatic hydrolysis of chitosan.

[0017] In some embodiments of the present invention, the chitosanase mutant is used to enzymatically hydrolyze chitosan at 45-50°C.

[0018] In some embodiments of the present invention, the chitosanase mutant enzymatically hydrolyzes chitosan under acidic conditions.

[0019] This invention introduces four mutation sites without altering the function of the Csn168 enzyme to obtain the M8 mutant. Compared with the wild-type enzyme (Csn168 enzyme), the M8 mutant significantly increases enzyme activity and reduces product inhibition, thereby significantly improving enzyme catalytic efficiency and enabling it to accumulate high concentrations of chitosan oligosaccharides in a single batch of production. Attached Figure Description

[0020] Figure 1 This is an amino acid sequence alignment diagram of Csn168 and M8.

[0021] Figure 2 This is a diagram of the enzymatic properties of M8, where a is pH; b is reaction temperature; c is enzyme concentration; and d is specific enzyme activity.

[0022] Figure 3 This is the product distribution of M8.

[0023] Figure 4 This is the time-varying curve of chitosan oligosaccharide production using M8 feed catalysis.

[0024] Figure 5 This is a time-varying curve of chitosan oligosaccharide production using Csn168 feed catalysis. Detailed Implementation

[0025] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0026] The main sources of the biomaterials and reagents involved in the examples are as follows:

[0027] The wild-type enzyme (chitosanase Csn168) is derived from Bacillus subtilis 168 (ATCC23857).

[0028] Chitosan was purchased from Shanghai Maclean Biochemical Co., Ltd.

[0029] The chitosan oligosaccharide standard was purchased from Beijing Solarbio Technology Co., Ltd.

[0030] In this embodiment, the substrate was prepared as follows: 15 g of chitosan was dissolved in an appropriate amount of 200 mM acetic acid and stirred magnetically until a colloidal solution was formed. Subsequently, 200 mM sodium acetate solution was added to adjust the pH to the required experimental value. Finally, the mixture was diluted to a total volume of 1 L to prepare a 1.5% colloidal chitosan solution (15 g / L chitosan solution). When using, dilute according to the required substrate concentration.

[0031] Chitosanase activity was determined using the DNS method, in which an appropriate amount of enzyme was mixed with 5 g / L chitosan at pH 6.0 and 50 °C for 10 minutes. The chitosan oligosaccharide content was quantitatively determined using the 3,5-dinitrosalicylic acid (DNS) method. Enzyme activity units (U) were defined as the amount of enzyme required to synthesize 1 µmol of chitosan oligosaccharide per minute.

[0032] Example 1

[0033] This embodiment specifically illustrates the method for constructing mutant M8.

[0034] The wild-type enzyme (chitosanase Csn168) was derived from Bacillus subtilis 168. Four mutation sites were introduced into chitosanase Csn168, namely T23A, V59E, S88N and N128D, to obtain mutant M8.

[0035] The M8 gene was synthesized in its entirety at Sangon Biotech (Shanghai) Co., Ltd., and cloned into the pET-22b vector to obtain the recombinant plasmid pET-22b-M8. The restriction enzyme sites were Nde I and Xho I. Recombinant Escherichia coli BL21(DE3)-pET-22b-M8 containing the recombinant plasmid pET-22b-M8 was constructed using Escherichia coli BL21(DE3) as the host bacterium. Recombinant plasmid pET-22b-Csn168 containing the chitosanase Csn168 gene and recombinant Escherichia coli BL21(DE3)-pET-22b-Csn168 were constructed using the same method.

[0036] Example 2

[0037] This embodiment specifically illustrates the purification method of mutant M8.

[0038] Recombinant Escherichia coli BL21(DE3)-pET-22b-M8 and BL21(DE3)-pET-22b-Csn168 were cultured in LB medium at 37 °C and 200 rpm until OD600 = 0.5-0.8. 1 M IPTG was added to a final concentration of 0.1 mM, and the mixture was induced at 20 °C and 200 rpm for 12 h.

[0039] After induction, the cells were centrifuged at 10,000 × g for 10 min, the supernatant was discarded, and the cells were collected. The cells were washed twice with 3 mL of PBS (pH 7.2-7.4) and resuspended in 5 mL of the same buffer solution. The cells were then vortexed and sonicated at 400 W for 10 min.

[0040] The lysate was centrifuged at 12,000 × g at 4 °C for 10 min. The supernatant was collected and purified into protein using Ni-NTA resin. After loading, the column was washed with 5 mL of buffer A to remove non-specifically bound proteins. Then, the target protein was eluted with 3 mL of buffer B. The protein concentration was determined using the BCA kit (Novizan).

[0041] Buffer A: 20 mM Na + - Phosphate buffer (pH 8.0) containing 20 mM imidazole and 500 mM sodium chloride.

[0042] Buffer B: 20 ​​mM Na + - Phosphate buffer (pH 8.0) containing 300 mM imidazole and 500 mM NaCl.

[0043] Example 3

[0044] This embodiment systematically evaluated the enzymatic properties of M8.

[0045] The reaction system is as follows:

[0046] Optimal temperature: Add 1.5% colloidal chitosan solution to 1 mL of the reaction system to make the final concentration 0.5%, add 100 µL of crude enzyme solution, add MnCl2 with a final concentration of 1 mM, and react for 10 min at different temperatures.

[0047] Optimal pH: Add 1.5% colloidal chitosan solution to 1 mL of the reaction system to make the final concentration 0.5%. Adjust the pH using 5M Tris or 5M hydrochloric acid. Then add 100 µL of crude enzyme solution and add MnCl2 to a final concentration of 1 mM. React at 50℃ for 10 min.

[0048] The results are as follows Figure 2 As shown, its optimal pH is 5.0. Figure 2 (a) The optimal temperature is between 40 and 50°C, and the catalytic efficiency remains above 90% within the range of 30-50°C. Figure 2 (b)

[0049] Chitosanase activity was determined using the DNS method. Comparative activity assays showed that, over a wide concentration range (5 µM–50 nM), M8 consistently exhibited significantly higher activity than the wild-type enzyme Csn168. Figure 2 (c). Notably, the difference between Csn168 and M8 became more pronounced when the enzyme concentration dropped below 100 nM, indicating that Csn168 requires a minimum concentration threshold to maintain effective hydrolysis of chitosan. Specific activity assays further showed that M8 had a specific activity of 350 U / mg (…). Figure 2 (d), which is 10 times higher than Csn168.

[0050] The product distribution of the M8 enzymatic hydrolysis products was detected using LC-MS, and the results are as follows: Figure 3 As shown, the main hydrolysis products of M8 are (GlcN)2-(GlcN)4, and trace amounts of higher oligomers (GlcN)5-(GlcN)7 were also detected. These results indicate that M8 is conducive to the generation of short-chain chitosan oligosaccharides, which are generally considered to be ideal end products for industrial applications.

[0051] Example 4

[0052] This embodiment provides a comparison of the fed-batch fermentation effects of wild enzymes and M8.

[0053] The following system was used to catalyze the chitosanase reaction:

[0054] 5% colloidal chitosan, 10 µM M8, and 1 mM MnCl2 were added, and the reaction was carried out at 50 °C and pH 5.0 for 30 min. Subsequently, 5% colloidal chitosan, 10 µM M8, and 1 mM MnCl2 were added again, and the reaction was continued for another 30 min. After the enzymatic reaction, 5% colloidal chitosan, 10 µM M8, and 1 mM MnCl2 were added again, and the reaction was continued for another 60 min. Finally, 5% colloidal chitosan, 10 µM M8, and 1 mM MnCl2 were added, and the reaction was continued for another 60 min.

[0055] The above process was followed for two more batches of catalytic reaction, with a total reaction time of 3 hours and a total chitosan concentration of 20%.

[0056] Samples were taken every 30 minutes during the reaction. After the reaction was completed, the concentration of chitosan oligosaccharides was determined using the DNS method.

[0057] The fed-batch fermentation results of M8 and Csn168 are as follows: Figure 4 , Figure 5As shown, after 2 hours, with continued substrate and enzyme supplementation, the increase in chitosan oligosaccharide content in Csn168 slowed down, reaching a peak concentration of 67 g / L at approximately 2.5 hours. In contrast, after 2 hours, with continued substrate and enzyme supplementation, M8's yield continued to increase steadily, reaching 142 g / L after 3 hours without reaching a peak. This indicates that after mutation, the product inhibition of Csn168 was significantly reduced.

[0058] The various embodiments of the present invention have been described above. These descriptions are exemplary and not exhaustive, nor are they limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments.

Claims

1. A low-product chitosanase inhibitor mutant, characterized in that, The amino acid sequence of the chitosanase mutant is shown in SEQ ID NO:

1.

2. The gene encoding the mutant of claim 1.

3. A recombinant vector containing the gene of claim 2.

4. The recombinant vector according to claim 3, characterized in that, The recombinant vector uses pET-22b as the expression vector.

5. Recombinant engineered bacteria containing the recombinant vector as described in claim 3 or 4.

6. The recombinant engineered bacteria according to claim 5, characterized in that, The recombinant engineered bacteria were constructed using Escherichia coli as the host bacterium.

7. The purification method of the chitosanase mutant according to claim 1, characterized in that, include: The recombinant engineered bacteria described in claim 5 were cultured to OD200. 600 =0.5-0.8, then IPTG was added to induce expression; After induction, the bacterial culture was centrifuged and the bacterial cells were collected, resuspended in phosphate buffer, and then sonicated. After the lysate is centrifuged at ultraspeed, the supernatant is collected and purified by nickel column chromatography to obtain purified protein.

8. The application of the chitosanase mutant of claim 1 in the enzymatic hydrolysis of chitosan.

9. The method according to claim 8, characterized in that, The chitosanase mutant was used to enzymatically hydrolyze chitosan at 45-50°C.

10. The method according to claim 8, characterized in that, The chitosanase mutant enzymatically hydrolyzes chitosan under acidic conditions.

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