Chitosanase mutant WP-E80A, plasmid, recombinant bacterium and application of chitosanase mutant WP-E80A

Abstract

The invention relates to a chitosanase mutant WP-E80A, a plasmid, a recombinant bacterium and application of the chitosanase mutant WP-E80A, and belongs to the technical field of enzyme engineering, the amino acid sequence of the chitosanase mutant is shown as SEQ ID NO: 1, and the amino acid sequence of wild chitosanase CsnWP is shown as SEQ ID NO: 2. Compared with wild chitosanase CsnWP, the chitosanase CsnWP has the advantage that site-specific mutagenesis is carried out on the 80th amino acid. The invention also provides a plasmid and a recombinant engineering bacterium containing the gene of the chitosanase mutant WP-E80A, and the chitosanase mutant WP-E80A can be used for preparing and generating chitopentaose by taking chitobiose as a substrate.

Description

Technical Field

[0001] The invention belongs to the technical field of enzyme engineering, and specifically relates to a chitosanase mutant WP-E80A, a plasmid, a recombinant bacterium and applications thereof. Background Art

[0002] Chitosan is the product of chitin deacetylation. It is the only alkaline cationic polysaccharide in nature. It has good adsorption and viscosity and can be used in food, materials and other fields. Compared with chitosan, its degradation product, chitosan oligosaccharide, has a variety of biological activities, such as anti-tumor, antibacterial, antioxidant and cholesterol-lowering, and is widely used in the fields of medicine, food, agriculture and cosmetics. At the same time, studies have shown that the biological activity of chitosan oligosaccharide depends to a large extent on its molecular weight, that is, the degree of polymerization. Chitosan oligosaccharides with a degree of polymerization of 4-7 have the effect of inhibiting the growth and metastasis of cancer cells. As an auxiliary drug for tumor radiotherapy, they can effectively improve the anti-tumor immune function of the human body; chitosan oligosaccharides with a degree of polymerization of 3-6 have anti-infection effects, and the activity increases with the increase of the degree of polymerization. Therefore, the production of chitosan oligosaccharides with a specific degree of polymerization, especially chitosan oligosaccharides with a higher degree of polymerization, has aroused widespread interest among researchers.

[0003] At present, the methods for preparing chitosan oligosaccharides from chitosan mainly include physical method, chemical method and bioenzyme method. Among them, the physical method has problems such as low product yield, uncontrollable product and high equipment requirements; the chemical method has drastic reaction conditions, easy to cause environmental pollution, and the product polymerization degree is widely distributed. The bioenzyme method has the advantages of mild reaction conditions, strong product selectivity and easy preparation. Chitosanase (EC 3.2.1.132) is an enzyme that specifically degrades chitosan. It can hydrolyze the β-1,4-glycosidic bond of chitosan and plays an important role in the preparation of chitosan oligosaccharides. Although there are many reports of chitosanase, most of its products are mixtures of chitosan oligosaccharides with lower polymerization degree, such as chitobiose / chitotriose. At present, the types of chitosanase that can prepare high-polymerization degree chitosan oligosaccharides are scarce, and this deficiency seriously restricts the development of bioenzymatic production of chitosan oligosaccharides. Research in recent years has found that some chitosanases not only have hydrolysis activity, but also have transglycosylation activity. When producing high-polymerization degree oligosaccharides by transglycosylation reaction, they show the advantages of easy access to substrates and low production cost. Therefore, it is urgent to explore chitosanase genes with transglycosylation activity, identify the key amino acid sites that affect their hydrolysis / transglycosylation activity, and carry out targeted modification to achieve efficient enzymatic preparation of high-polymerization degree chitosan oligosaccharides. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a chitosanase with transglycosylation activity, which can convert low-polymerization-degree chitosan oligosaccharides into high-polymerization-degree chitosan oligosaccharides.

[0005] In view of the deficiencies of the prior art, the present invention provides a chitosanase mutant WP-E80A, a plasmid, a recombinant bacterium and its application, which effectively inhibits its hydrolysis activity by implementing site-directed mutation on the active center of chitosanase CsnWP, thereby making its transglycosylation more significant, thereby realizing the directional synthesis from low-polymerization-degree chitosan oligosaccharides to high-polymerization-degree products. This technological breakthrough not only provides an innovative solution for the industrial-scale preparation of high-polymerization-degree chitosan oligosaccharides, but also lays a foundation for the iterative upgrading of my country's chitosan oligosaccharide production technology and the high-value utilization of chitosan resources.

[0006] The present invention is achieved through the following technical solutions:

[0007] A chitosanase mutant WP-E80A, the amino acid sequence of the chitosanase mutant is shown in SEQ ID NO: 1, and the amino acid sequence of the wild-type chitosanase CsnWP is shown in SEQ ID NO: 2. Compared with the wild-type chitosanase CsnWP, the 80th amino acid is subjected to site-directed mutation.

[0008] Furthermore, the wild-type chitosanase CsnWP of the chitosanase WP-E80A is derived from Paenibacillus anaericanus (GenBank ID: WP_127192817.1).

[0009] The present invention also provides a gene encoding the chitosanase mutant WP-E80A, whose nucleotide sequence is shown in SEQ ID NO.3, and the nucleotide sequence of the wild-type chitosanase CsnWP is shown in SEQ ID NO:4.

[0010] The present invention also provides a recombinant plasmid carrying the gene shown in SEQ ID NO.3, and the expression vector is preferably pET-28a(+).

[0011] The present invention also provides a recombinant engineering strain transformed with the above recombinant plasmid, and the expression host is preferably E. coli BL21 (DE3).

[0012] The present invention also provides an enzyme preparation, which contains the chitosanase mutant WP-E80A.

[0013] The present invention also provides the use of the chitosanase mutant WP-E80A in preparing chitopentaose using chitobiose as a substrate.

[0014] The beneficial effects of the present invention compared with the prior art are as follows:

[0015] The present invention aims at the problem that the existing chitosan enzymes have low polymerization degree in preparing chitosan products. The present invention uses CsnWP as a starting point and carries out site-directed mutagenesis on its active center to obtain a mutant WP-E80A (the 80th glutamic acid is mutated to alanine). The results show that CsnWP can convert chitobiose into chitotriose through transglycosylation, while WP-E80A can convert chitobiose into chitopentaose. This discovery of the present invention lays a foundation for utilizing the transglycosylation activity of chitosanase to convert low-polymerization-degree chitosan oligosaccharides into high-polymerization-degree chitosan oligosaccharides. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The product analysis diagram of wild-type chitosanase CsnWP catalyzing glucosamine, chitosan oligosaccharides (polymerization degree of 2-6) and chitosan; a is the product analysis of wild-type chitosanase CsnWP catalyzing glucosamine, chitosan oligosaccharides (polymerization degree of 2-6) and chitosan; b is the product analysis of wild-type chitosanase CsnWP catalyzing chitosan at different time periods;

[0017] Figure 2 This is the product analysis diagram of the mutant chitosanase WP-E80A catalyzing glucosamine, chitooligosaccharides (polymerization degree of 2-6) and chitosan. DETAILED DESCRIPTION

[0018] The method of the present invention is further described below by way of examples in conjunction with the accompanying drawings. However, the experimental conditions used in the examples can be selected according to the prior art. For experimental methods in which specific conditions are not indicated in the examples, they can usually be operated under conventional conditions or under conditions recommended by the manufacturer.

[0019] The present invention will be further described below in conjunction with specific examples. It should be understood that the following examples are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0020] Example 1. Preparation of chitosanase CsnWP recombinant bacteria

[0021] In order to explore chitosanase with transglycosylation specificity, the present invention screened out a chitosanase (GenBank ID: WP_127192817.1) from Paenibacillus anaericanus with possible transglycosylation activity through NCBI search and comparison, and its sequence is shown in SEQ ID NO.2, named chitosanase Csn-WP, and the full-length gene was obtained by whole gene synthesis, and connected with expression vector pET-28a (+) to obtain a recombinant plasmid. The plasmid was transformed into host E. coli BL21 (DE3) competent cells to obtain recombinant bacteria of chitosanase CsnWP.

[0022] Example 2: Construction of chitosanase mutant WP-E80A

[0023] Chitosanase CsnWP was used as a template, and its active center was E80 and D98, and the amino acid at position 80 was selected for mutation. E80A-F and E80A-R in Table 1 were used as primers for PCR amplification, and the product was digested with DpnI enzyme and transformed into E. coli BL21 (DE3) to construct the chitosanase mutant WP-E80A, in which the glutamic acid at position 80 was mutated to alanine.

[0024] Table 1 Primer sequences

[0025]

[0026] Example 3: Induced expression and purification of chitosanase CsnWP and mutant WP-E80A

[0027] The wild-type chitosanase CsnWP and the mutant WP-E80A recombinant bacteria were inoculated into LB liquid medium (containing 100 μg / mL kanamycin) and cultured at 37°C until OD 600 After reaching 0.6-0.8, IPTG was added at a final concentration of 0.1 mM, and the cells were cultured in a 20°C incubator for 18 h. The cells were collected by centrifugation at 4°C, and after ultrasonic disruption, the protein was purified using a Ni-NTA affinity column, the impurities were eluted with NPI-20, and the target protein was eluted with NPI-100, and then ultrafiltration, desalting and concentration were performed to obtain purified chitosanase CsnWP and mutant WP-E80A.

[0028] The amino acid sequence of chitosanase mutant WP-E80A is SEQ ID NO: 1:

[0029] MLNTKQNSGSKRLKFSLLVLLSFVITISFGLLSNPFQSKSYAATNPDSNFSPATLKFLKDNTGLDGEQWNNIMKLVNKPAQDDLNWTNYYGYCEKLTDKRGFTIGIFGATTGGANDTGPDGPDLFKEYDKVKGA TNPSVSGALKRLGINGSMSGSILVIKDSDSAFIKKINALQNDPAWREAMWKTFYNVYIKYSVQQANQRGFKTALTIGSFVDAALNHGATGDSNSLQGLLKKSGTSTNEKTFMTSFYAKRTLIVDTNDYNQAPNG KNRVKQWSTLLSQGETDLKGADAAIVKVTNWEMQ

[0030] The amino acid sequence of chitosanase CsnWP is SEQ ID NO: 2:

[0031] MLNTKQNSGSKRLKFSLLVLLSFVITISFGLLSNPFQSKSYAATNPDSNFSPATLKFLKDNTGLDGEQWNNIMKLVNKPEQDDLNWTNYYGYCEKLTDKRGFTIGIFGATTGGANDTGPDGPDLFKEYDKVKGATNPSVSGALKRLGINGSMSGSILVIKDSDSAFIKKINALQNDPAWREAMWKTFYNVYIKYSVQQANQRGFKTALTIGSFVDAALNHGATGDSNSLQGLLKKSGTSTNEKTFMTSFYAKRTLIVDTNDYNQAPNGKNRVKQWSTLLSQGETDLKGADAAIVKVTNWEMQ

[0032] Nucleotide sequence of chitosanase mutant WP-E80A: SEQ ID NO: 3

[0033] ATGCTGAACACCAAACAGAACTCTGGTTCTAAACGTCTGAAATTCTCTCTGCTGGTTCTGCTGTCTTTCGTTATCACCATCTCTTTCGGTCTGCTGTCTAACCCGTTCCAGTCTAAATCTTACGCTGCTACCAACCCGGACTCTAACTTCTCTCCGGCTACCCTGAAATTCCTGAAAGACAACACCGGTCTGGACGGTGAACAGTGGAACAACATCATGAAACTGGTTAACAAACCGGCACAGGACGACCTGAACTGGACCAACTACTACGGTTACTGCGAAAAACTGACCGACAAACGTGGTTTCACCATCGGTATCTTCGGTGCTACCACCGGTGGTGCTAACGACACCGGTCCGGACGGTCCGGACCTGTTCAAAGAATACGACAAAGTTAAAGGTGCTACCAACCCGTCTGTTTCTGGTGCTCTGAAACGTCTGGGTATCAACGGTTCTATGTCTGGTTCTATCCTGGTTATCAAAGACTCTGACTCTGCTTTCATCAAAAAAATCAACGCTCTGCA GAACGACCCGGCTTGGCGTGAAGCTATGTGGAAAACCTTCTACAACGTTTACATCAAATACTCTGTTCAGCAGGCTAACCAGCGTGGTTTCAAAACCGCTCTGACCATCGGTTCTTTCGTTGACGCTGCTCTGAACCACGGTGCTACCGGTGACTCTAACTCTCTGCAGGGTCTGCTGAAAAAATCTGGTACCTCTACCAACGAAAAAACCTTCATGACCTCTTTCTACGCTAAACGTACCCTGATCGTTGACACCAACGACTACAACCAGGCTCCGAACGGTAAAAACCGTGTTAAACAGTGGTCTACCCTGCTGTCTCAGGGTGAAACCGACCTGAAAGGTGCTGACGCTGCTATCGTTAAAGTTACCAACTGGGAAATGCAG

[0034] Nucleotide sequence of chitosanase CsnWP SEQ ID NO:4:

[0035] ATGCTGAACACCAAACAGAACTCTGGTTCTAAACGTCTGAAATTCTCTCTGCTGGTTCTGCTGTCTTTCGTTATCACCATCTCTTTCGGTCTGCTGTCTAACCCGTTCCAGTCTAAATCTTACGCTGCTACCAACCCGGACTCTAACTTCTCTCCGGCTACCCTGAAATTCCTGAAAGACAACACCGGTCTGGACGGTGAACAGTGGAACAACATCATGAAACTGGTTAACAAACCGGAACAGGACGACCTGAACTGGACCAACTACTACGGTTACTGCGAAAAACTGACCGACAAACGTGGTTTCACCATCGGTATCTTCGGTGCTACCACCGGTGGTGCTAACGACACCGGTCCGGACGGTCCGGACCTGTTCAAAGAATACGACAAAGTTAAAGGTGCTACCAACCCGTCTGTTTCTGGTGCTCTGAAACGTCTGGGTATCAACGGTTCTATGTCTGGTTCTATCCTGGTTATCAAAGACTCTGACTCTGCTTTCATCAAAAAAATCAACGCTCTGC AGAACGACCCGGCTTGGCGTGAAGCTATGTGGAAAACCTTCTACAACGTTTACATCAAATACTCTGTTCAGCAGGCTAACCAGCGTGGTTTCAAAACCGCTCTGACCATCGGTTCTTTCGTTGACGCTGCTCTGAACCACGGTGCTACCGGTGACTCTAACTCTCTGCAGGGTCTGCTGAAAAAATCTGGTACCTCTACCAACGAAAAAACCTTCATGACCTCTTTCTACGCTAAACGTACCCTGATCGTTGACACCAACGACTACAACCAGGCTCCGAACGGTAAAAACCGTGTTAAACAGTGGTCTACCCTGCTGTCTCAGGGTGAAACCGACCTGAAAGGTGCTGACGCTGCTATCGTTAAAGTTACCAACTGGGAAATGCAG

[0036] Example 4: Activity Detection of Chitosanase CsnWP and Mutant WP-E80A

[0037] The chitosanase CsnWP obtained in Example 3 was tested for enzyme activity using the DNS method. The specific operation was as follows: 350 μL sodium phosphate buffer (50 mM, pH 8), 100 μL colloidal chitosan (1%), and then 50 μL chitosanase CsnWP and mutant WP-E80A were added respectively. Incubate at 37°C for 30 min, add 300 μL DNS reagent, then boil in water for 5 min, and measure the absorbance at 520 nm after cooling to room temperature. Enzyme activity (U) definition: the amount of enzyme required to produce 1 μmol of reducing sugar per minute under standard conditions. The enzyme activity of wild-type chitosanase CsnWP was measured to be 75.59 U / mg, and the enzyme activity of mutant WP-E80A was 0.91 U / mg.

[0038] Example 5. Product analysis of chitosanase CsnWP and mutant WP-E80A

[0039] Chitosanase CsnWP and mutant WP-E80A were reacted with colloidal chitosan (1%, w / v), glucosamine and chitosan oligosaccharides (polymerization degree of 2-6, 10 mg / mL), respectively. After the reaction, an equal volume of ethanol was added and centrifuged. An appropriate amount of sample was spotted on a silica gel plate, with n-propanol-water-ammonia water (8:3:1, v / v / v) as the mobile phase and aniline-diphenylamine solution as the color developer. An appropriate amount of sample was spotted on a silica gel plate. The results showed that the final products of the reaction between wild-type chitosanase CsnWP and chitosan were chitobiose and chitotriose, and chitotriose could be generated using chitobiose as a substrate, indicating that chitosanase CsnWP has transglycosylation activity ( Figure 1 The chitosanase mutant WP-E80A can no longer degrade chitosan, but can convert chitobiose into chitopentaose, and its transglycosylation activity is more significant ( Figure 2 ). Compared with the wild-type chitosanase CsnWP, the degree of polymerization of the product of the mutant WP-E80A changed significantly, and chitosan pentose with a high degree of polymerization was obtained.

[0040] The above embodiments are only for illustrating the technical concept and features of the present invention, and cannot be used to limit the protection scope of the present invention. Those skilled in the art can make various changes or modifications according to the present invention, and these equivalent forms are also included in the scope defined by the claims attached to this application.

Claims

1. A chitosanase mutant WP-E80A, characterized in that: The amino acid sequence of the chitosanase mutant WP-E80A is shown in SEQ ID NO:

1.

2. A recombinant plasmid, characterized in that: The recombinant plasmid carries the gene encoding the chitosanase mutant WP-E80A according to claim 1, the nucleotide sequence of the gene is shown in SEQ ID NO.3, and the expression vector is pET-28a(+).

3. A recombinant engineering strain, characterized in that: The strain contains the gene encoding the chitosanase mutant WP-E80A according to claim 1, and the expression host is E.coli BL21 (DE3).

4. An enzyme preparation, characterized in that The enzyme preparation contains the chitosanase mutant WP-E80A according to claim 1.

5. The use of the chitosanase mutant WP-E80A according to claim 1, characterized in that: The application is to use chitobiose as a substrate and utilize the chitosanase mutant WP-E80A described in claim 1 to prepare chitopentaose.

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