阅读说明：本技术 一种壳聚糖酶突变体及其构建方法与应用 (Chitosanase mutant and construction method and application thereof ) 是由 郭静 满在伟 杜昌阳 蔡志强 于 2021-08-30 设计创作，主要内容包括：本发明公开了一种壳聚糖酶突变体和工程菌及应用,属于酶工程技术领域。其氨基酸序列如SEQ ID NO:1所示的壳聚糖酶第121位脯氨酸突变为天冬酰胺或半胱氨酸或缬氨酸。本发明壳聚糖酶突变体与野生型壳聚糖酶相比,三种突变体催化胶体壳聚糖获得壳寡糖的比酶活分别提高1.69、1.97及2.15倍,特别是P121N突变体,提高催化活性的同时,没有损失热稳定性,具备良好的应用前景；本发明还提供一种上述突变体的构建方法,通过分析该酶蛋白结构上的高去折叠自由能位点,以此选取突变位点,采用基因工程方法对该位点进行饱和突变,然后筛选目的蛋白。(The invention discloses a chitosanase mutant, engineering bacteria and application, and belongs to the technical field of enzyme engineering. The amino acid sequence of the chitosanase is shown in SEQ ID NO. 1, proline at position 121 is mutated into asparagine, cysteine or valine. Compared with the wild type chitosanase, the chitosanase mutant of the invention has the advantages that the specific enzyme activities of the chitosanase mutant for catalyzing the colloidal chitosan to obtain the chitooligosaccharide are respectively improved by 1.69, 1.97 and 2.15 times, particularly the P121N mutant, the catalytic activity is improved, meanwhile, the thermal stability is not lost, and the invention has good application prospect; the invention also provides a construction method of the mutant, which comprises the steps of analyzing the high unfolding free energy site on the structure of the zymoprotein, selecting a mutation site, carrying out saturation mutation on the site by adopting a genetic engineering method, and then screening the target protein.)

1. The chitosanase mutant is characterized in that the chitosanase mutant is a mutant which is formed by point mutation of bacillus subtilis chitosanase and has the function of catalyzing and producing chitosan oligosaccharide, and the mutation position is that the 121 th proline on the nucleotide sequence of the coding bacillus subtilis chitosanase is subjected to site-specific mutation; the amino acid sequence of the bacillus subtilis chitosanase is SEQ ID NO. 1.

2. The gene of claim 1, wherein the nucleotide sequence encoding the bacillus subtilis chitosanase is SEQ ID No. 2.

3. The chitosanase mutant according to claim 1, wherein when proline at position 121 is mutated to asparagine, the chitosanase mutant is designated as P121N; when the proline at the 121 st position is mutated into cysteine, the chitosanase mutant is marked as P121C; when the proline at position 121 is mutated into valine, the chitosanase mutant is marked as P121V.

4. The chitosanase mutant according to claim 1, wherein the amino acid sequence of chitosanase mutant P121N is shown in SEQ ID NO. 3; the amino acid sequence of the chitosanase mutant P121C is shown in SEQ ID NO. 4; the amino acid sequence of the chitosanase mutant P121V is shown in SEQ ID NO. 5.

5. A gene encoding the chitosanase mutant of claim 3.

6. A recombinant vector carrying the chitosanase mutant of claim 3 or the gene of claim 5.

7. A recombinant bacterium which is a host bacterium transformed/transfected with the recombinant vector according to claim 6.

8. The construction method of chitosanase mutant according to claim 1, comprising the following steps:

taking a recombinant plasmid of a chitosan enzyme gene carried by an escherichia coli host capable of being methylated as a template, taking an oligonucleotide sequence with a mutation site as a primer, and carrying out reverse PCR (polymerase chain reaction) to amplify the full length of the mutant plasmid; digesting the template plasmid by using DpnI restriction endonuclease; transformation of DpnI restriction endonuclease-treated PCR product intoE. coliDH5 alpha competent cells, spread on solid LB plate containing kanamycin resistance and cultured overnight; selecting a single colony, inoculating the single colony to an LB liquid culture medium containing kanamycin resistance, performing overnight culture, extracting plasmids and performing sequencing verification; the plasmid with correct sequencing result is transformed into Escherichia coliE.coliBL21(DE3) in competent cells to obtain chitosanase mutants.

9. Use of a chitosanase mutant according to any of claims 1-4 or claim 8 for the catalytic production of chitooligosaccharides.

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to a chitosanase mutant and a construction method and application thereof.

Background

Chitosanase (EC.3.2.1.132) is a kind of glycosidase capable of hydrolyzing chitosan to produce chitosan oligosaccharide or glucosamine. According to the difference of characteristic amino acid sequences in glycosidase databases, chitosanases are mainly distributed in GH8, GH46, GH75 and GH80 families, wherein the GH46 family chitosanases are the most deeply researched. The GH46 family chitosanase is dumbbell-shaped, and is connected with two spheres with different sizes by an alpha helix, the active center has two catalytic residues Asp and Glu, one amino acid residue is used as a nucleophilic reagent, and the other is used as a generalized acid/base.

Chitosan, also known as chitosan, is a linear high polymer formed by connecting glucosamine as a monomer through beta-1.4-glycosidic bonds, and widely exists in shells of various arthropods (shrimps and crabs) and cell walls of certain fungi and algae. Each year, chitosan biosynthesized is approximately 100 million tons, and is a renewable polysaccharide with the content next to cellulose. Chitosan is degraded to produce chitosan oligosaccharide (degree of polymerization of 2-10), and if completely degraded, glucosamine is produced. The products have the advantages of high water solubility, easy absorption and utilization by organisms and the like which are not possessed by chitosan. The research reports indicate that the chitosan oligosaccharide has the functions of resisting tumor, inflammation and bacteria, improving the immunity of the organism, promoting the growth of lactic acid bacteria and the like, so the chitosan oligosaccharide has wide application prospect in the fields of medicine, food, agriculture, cosmetics and the like.

At present, the preparation method of the chitosan oligosaccharide mainly comprises a physical degradation method, a chemical degradation method and an enzymatic hydrolysis method. The physical degradation method has the defects of overlarge molecular weight of the product, difficulty in obtaining bioactive and soluble oligosaccharide and the like. The chemical method has the defects of severe reaction conditions, poor selectivity, difficult separation and purification and the like, and meanwhile, a large amount of acid, oxidant and the like are introduced in the chemical reaction process to cause serious pollution to the environment. The enzymatic hydrolysis method has the advantages of strong selectivity, mild reaction conditions, environmental friendliness, easiness in preparation and the like, and is a research hotspot in recent years rapidly.

Obtaining enzymes with high catalytic activity is a prerequisite for their industrial application. There are two main methods for obtaining enzymes with excellent catalytic properties: strain screening and protein engineering. The strain screening has the defects of time and labor waste, low efficiency and the like, and the existing enzymes are improved through protein engineering, so that the strain screening has the advantages of high efficiency, time saving, obvious effect taking and the like, and therefore, the strain screening becomes the first choice for screening excellent enzymes in recent years. At present, protein engineering is combined with rational design based on a computer-simulated protein model to successfully obtain a plurality of mutant enzymes with remarkably improved catalytic properties.

Although some point mutations are applied in the prior art, most of published articles are directed to research the catalytic function of a specific amino acid on an enzyme by the site-directed mutation, but not to improve the catalytic activity of the enzyme, such as the influence of site-directed mutation on the enzyme activity of Microbacterium sp.OU01 chitosanase Glu51 and Asp69 published in food industry science and technology in 2016, although the specific activity of the mutant enzyme (Glu51 → Gln51) is about 10% of that of a wild type, Glu51 is determined as a key amino acid residue related to the catalytic function of the enzyme.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a chitosanase mutant and a construction method and application thereof.

The Bacillus subtilis chitosanase BsCsn46A is GH46 family chitosanase obtained by early cloning in a laboratory, can hydrolyze colloidal chitosan to produce chitobiose and chitotriose, and has the advantages of high catalytic activity, strong stability and the like, so the Bacillus subtilis chitosanase has better application prospect. In order to improve the catalytic activity, the chitosan oligosaccharide is better applied to the production of chitosan oligosaccharide. The invention utilizes a site-directed mutagenesis technology based on computer simulation to carry out molecular modification on BsCsn46A based on an obtained expression platform of chitosanase in escherichia coli, so as to obtain a chitosanase mutant with improved catalytic activity.

In order to solve the problems of the prior art, the invention adopts the technical scheme that:

a chitosanase mutant is a mutant which is formed by point mutation of bacillus subtilis chitosanase and has the function of catalyzing and producing chitosan oligosaccharide, and the mutation is that the 121 th proline is subjected to site-directed mutation on a nucleotide sequence of the coding bacillus subtilis chitosanase; the amino acid sequence of the bacillus subtilis chitosanase is SEQ ID NO. 1.

SEQ ID NO:1

1 AGLNKDQKRRAEQLTSIFEN

21 GTTEIQYGYVERLDDGRGYT

41 CGRAGFTTATGDALEVVEVY

61 TKAVPNSKLKKYLPELRRLA

81 KEESDDTSNLKGFASAWKSL

101 ANDKEFRAAQDKVNDHLYYQ

121 PAMKRSDNAGLKTALARAVM

141 YDTVIQHGDGDDPDSFYALI

161 KRTNKKAGGSPKDGIDEKKW

181 LNKFLDVRYDDLMNPANHDT

201 RDEWRESVARVDVLRSIAKE

221 NNYNLNGPIHVRSNEYGNFV

241 IK

The amino acid site with mutation is positioned in an alpha-helical structure on the back of the active center of the chitosanase, and the mutation can increase the flexibility of protein so as to improve the catalytic activity of the protein.

In an improvement, the nucleotide sequence for coding the bacillus subtilis chitosanase is SEQ ID NO. 2.

SEQ ID NO:2

1GCGGGACTGAATAAAGATCAAAAGCGCCGGGCGGAACAGCTGACAAGTATCTTTGAAAAC

61GGCACAACGGAGATCCAATATGGATATGTAGAGCGATTGGATGACGGGCGAGGCTATACA

121TGCGGTCGGGCAGGCTTTACAACGGCTACCGGGGATGCATTGGAAGTAGTGGAAGTATAC

181ACAAAGGCAGTTCCGAATAGCAAACTGAAAAAGTATCTGCCTGAATTGCGCCGTCTGGCC

241AAGGAAGAAAGCGATGATACAAGCAATCTCAAGGGATTCGCTTCTGCCTGGAAGTCGCTT

301GCAAATGATAAGGAATTTCGCGCCGCTCAAGACAAAGTAAATGACCATTTGTATTATCAG

361CCTGCCATGAAACGATCGGATAATGCCGGACTAAAAACAGCATTGGCAAGAGCTGTGATG

421TACGATACGGTTATTCAGCATGGCGATGGTGATGACCCTGACTCTTTTTATGCCTTGATT

481AAACGTACGAACAAAAAAGCGGGCGGATCACCTAAAGACGGAATAGACGAGAAGAAGTGG

541TTGAATAAATTCTTGGACGTACGCTATGAGATCTGATGAATCCGGCCAATCATGACACC

601CGTGACGAATGGAGAGAATCAGTTGCCCGTGTGGACGTGCTTCGCTCTATCGCCAAGGAG

661AACAACTATAATCTAAACGGACCGATTCATGTTCGTTCAAACGAGTACGGTAATTTTGTA

721ATCAAATAA

The improvement is that when the proline at position 121 is mutated into asparagine, the chitosanase mutant is marked as P121N; when the proline at the 121 st position is mutated into cysteine, the chitosanase mutant is marked as P121C; when the proline at position 121 is mutated into valine, the chitosanase mutant is marked as P121V.

In further improvement, the amino acid sequence of the chitosanase mutant P121N is shown in SEQ ID NO. 3; the amino acid sequence of the chitosanase mutant P121C is shown in SEQ ID NO. 4; the amino acid sequence of the chitosanase mutant P121V is shown in SEQ ID NO. 5.

SEQ ID NO:3

1 AGLNKDQKRRAEQLTSIFEN

21 GTTEIQYGYVERLDDGRGYT

41 CGRAGFTTATGDALEVVEVY

61 TKAVPNSKLKKYLPELRRLA

81 KEESDDTSNLKGFASAWKSL

101 ANDKEFRAAQDKVNDHLYYQ

121 NAMKRSDNAGLKTALARAVM

141 YDTVIQHGDGDDPDSFYALI

161 KRTNKKAGGSPKDGIDEKKW

181 LNKFLDVRYDDLMNPANHDT

201 RDEWRESVARVDVLRSIAKE

221 NNYNLNGPIHVRSNEYGNFV

241 IK

The corresponding nucleotide sequence is shown in SEQ ID NO. 6:

1GCGGGACTGAATAAAGATCAAAAGCGCCGGGCGGAACAGCTGACAAGTATCTTTGAAAAC

61GGCACAACGGAGATCCAATATGGATATGTAGAGCGATTGGATGACGGGCGAGGCTATACA

121TGCGGTCGGGCAGGCTTTACAACGGCTACCGGGGATGCATTGGAAGTAGTGGAAGTATAC

181ACAAAGGCAGTTCCGAATAGCAAACTGAAAAAGTATCTGCCTGAATTGCGCCGTCTGGCC

241AAGGAAGAAAGCGATGATACAAGCAATCTCAAGGGATTCGCTTCTGCCTGGAAGTCGCTT

301GCAAATGATAAGGAATTTCGCGCCGCTCAAGACAAAGTAAATGACCATTTGTATTATCAG

361AATGCCATGAAACGATCGGATAATGCCGGACTAAAAACAGCATTGGCAAGAGCTGTGATG

421TACGATACGGTTATTCAGCATGGCGATGGTGATGACCCTGACTCTTTTTATGCCTTGATT

481AAACGTACGAACAAAAAAGCGGGCGGATCACCTAAAGACGGAATAGACGAGAAGAAGTGG

541TTGAATAAATTCTTGGACGTACGCTATGAGATCTGATGAATCCGGCCAATCATGACACC

601CGTGACGAATGGAGAGAATCAGTTGCCCGTGTGGACGTGCTTCGCTCTATCGCCAAGGAG

661AACAACTATAATCTAAACGGACCGATTCATGTTCGTTCAAACGAGTACGGTAATTTTGTA

721ATCAAATAA

SEQ ID NO:4

1 AGLNKDQKRRAEQLTSIFEN

21 GTTEIQYGYVERLDDGRGYT

41 CGRAGFTTATGDALEVVEVY

61 TKAVPNSKLKKYLPELRRLA

81 KEESDDTSNLKGFASAWKSL

101 ANDKEFRAAQDKVNDHLYYQ

121 CAMKRSDNAGLKTALARAVM

141 YDTVIQHGDGDDPDSFYALI

161 KRTNKKAGGSPKDGIDEKKW

181 LNKFLDVRYDDLMNPANHDT

201 RDEWRESVARVDVLRSIAKE

221 NNYNLNGPIHVRSNEYGNFV

241 IK

the corresponding nucleotide sequence is shown in SEQ ID NO: 7:

1GCGGGACTGAATAAAGATCAAAAGCGCCGGGCGGAACAGCTGACAAGTATCTTTGAAAAC

61GGCACAACGGAGATCCAATATGGATATGTAGAGCGATTGGATGACGGGCGAGGCTATACA

121TGCGGTCGGGCAGGCTTTACAACGGCTACCGGGGATGCATTGGAAGTAGTGGAAGTATAC

181ACAAAGGCAGTTCCGAATAGCAAACTGAAAAAGTATCTGCCTGAATTGCGCCGTCTGGCC

241AAGGAAGAAAGCGATGATACAAGCAATCTCAAGGGATTCGCTTCTGCCTGGAAGTCGCTT

301GCAAATGATAAGGAATTTCGCGCCGCTCAAGACAAAGTAAATGACCATTTGTATTATCAG

361TGCGCCATGAAACGATCGGATAATGCCGGACTAAAAACAGCATTGGCAAGAGCTGTGATG

421TACGATACGGTTATTCAGCATGGCGATGGTGATGACCCTGACTCTTTTTATGCCTTGATT

481AAACGTACGAACAAAAAAGCGGGCGGATCACCTAAAGACGGAATAGACGAGAAGAAGTGG

541TTGAATAAATTCTTGGACGTACGCTATGAGATCTGATGAATCCGGCCAATCATGACACC

601CGTGACGAATGGAGAGAATCAGTTGCCCGTGTGGACGTGCTTCGCTCTATCGCCAAGGAG

661AACAACTATAATCTAAACGGACCGATTCATGTTCGTTCAAACGAGTACGGTAATTTTGTA

721ATCAAATAA

SEQ ID NO:5

1 AGLNKDQKRRAEQLTSIFEN

21 GTTEIQYGYVERLDDGRGYT

41 CGRAGFTTATGDALEVVEVY

61 TKAVPNSKLKKYLPELRRLA

81 KEESDDTSNLKGFASAWKSL

101 ANDKEFRAAQDKVNDHLYYQ

121 VAMKRSDNAGLKTALARAVM

141 YDTVIQHGDGDDPDSFYALI

161 KRTNKKAGGSPKDGIDEKKW

181 LNKFLDVRYDDLMNPANHDT

201 RDEWRESVARVDVLRSIAKE

221 NNYNLNGPIHVRSNEYGNFV

241 IK

the corresponding nucleotide sequence is shown in SEQ ID NO: 8:

1GCGGGACTGAATAAAGATCAAAAGCGCCGGGCGGAACAGCTGACAAGTATCTTTGAAAAC

61GGCACAACGGAGATCCAATATGGATATGTAGAGCGATTGGATGACGGGCGAGGCTATACA

121TGCGGTCGGGCAGGCTTTACAACGGCTACCGGGGATGCATTGGAAGTAGTGGAAGTATAC

181ACAAAGGCAGTTCCGAATAGCAAACTGAAAAAGTATCTGCCTGAATTGCGCCGTCTGGCC

241AAGGAAGAAAGCGATGATACAAGCAATCTCAAGGGATTCGCTTCTGCCTGGAAGTCGCTT

301GCAAATGATAAGGAATTTCGCGCCGCTCAAGACAAAGTAAATGACCATTTGTATTATCAG

361GTTGCCATGAAACGATCGGATAATGCCGGACTAAAAACAGCATTGGCAAGAGCTGTGATG

421TACGATACGGTTATTCAGCATGGCGATGGTGATGACCCTGACTCTTTTTATGCCTTGATT

481AAACGTACGAACAAAAAAGCGGGCGGATCACCTAAAGACGGAATAGACGAGAAGAAGTGG

541TTGAATAAATTCTTGGACGTACGCTATGAGATCTGATGAATCCGGCCAATCATGACACC

601CGTGACGAATGGAGAGAATCAGTTGCCCGTGTGGACGTGCTTCGCTCTATCGCCAAGGAG

661AACAACTATAATCTAAACGGACCGATTCATGTTCGTTCAAACGAGTACGGTAATTTTGTA

721ATCAAATAA

a gene encoding the chitosanase mutant described above.

A recombinant vector carrying a gene encoding a chitosanase mutant.

A recombinant bacterium is a host bacterium for transforming/transfecting the recombinant vector.

The construction method of the chitosanase mutant comprises the steps of taking a recombinant plasmid of a chitosanase gene carried by a methylated escherichia coli host as a template, taking an oligonucleotide sequence with a mutation site as a primer, and performing reverse PCR to amplify the full length of the mutation plasmid; digesting the template plasmid by using DpnI restriction endonuclease; transforming the PCR product treated by the DpnI restriction endonuclease into E.coli DH5 alpha competent cells, and coating the competent cells on a solid LB plate containing kanamycin resistance for overnight culture; selecting a single colony, inoculating the single colony to an LB liquid culture medium containing kanamycin resistance, performing overnight culture, extracting plasmids and performing sequencing verification; and (3) transforming the plasmid with the correct sequencing result into an escherichia coli E.coli BL21(DE3) competent cell to obtain the chitosanase mutant.

The method comprises the following specific steps:

step 1, cloning a gene sequence SEQ ID No.2 into a plasmid pET-28a, and constructing a recombinant plasmid pET-BsCsn 46A;

step 2, simulating Bacillus subtilis chitosanase BsCsn46A by using Swiss-Model online software to obtain a space structure of the chitosanase;

step 3, submitting the space structure of the chitosanase to PoPMuSiC online prediction software, and determining a P121 site as a saturated mutation site;

step 4, designing a site-directed mutagenesis primer, and carrying out site-directed mutagenesis on the chitosan enzyme gene sequence through reverse PCR to obtain a recombinant vector containing the mutated chitosan enzyme gene sequence;

and 5, thermally shocking the mutated recombinant vector into Escherichia coli E.coli BL21(DE3), carrying out induction expression, centrifugally collecting thalli, breaking cells by ultrasonic waves, and purifying proteins by using Ni-NTA to obtain the chitosanase mutant.

The application of any one of the chitosanase mutants in catalyzing the production of chitosan oligosaccharide.

Has the advantages that:

compared with the prior art, the chitosanase mutant and the construction method and application thereof have the advantages that the chitosanase is subjected to molecular modification, the mutated amino acid site is positioned in an alpha-helical structure on the back of the active center of the chitosanase, the mutation can increase the flexibility of protein, and the enzyme activity of the obtained chitosanase mutant is remarkably changed, wherein the enzyme activities of mutants P121N, P121C and P121V are remarkably improved, particularly the P121N mutant is improved, and the enzyme activity is improved while the thermal stability of the enzyme is not influenced. The chitosanase mutant has wide application prospect in improving the yield of chitosan oligosaccharide.

Drawings

FIG. 1 shows the temperature stability of Wild-type chitosanase and its mutants, wherein Wild-type is Bacillus subtilis chitosanase BsCsn 46A;

FIG. 2 is a block diagram of the chitosanase protein.

Detailed Description

The present invention will be described in detail with reference to examples.

EXAMPLE 1 construction of mutants

Coli DH5 α, purchased from Wuhan vast Ling Biotech Co., Ltd;

bacillus subtilis chitosanase BsCsn46A, purchased from Wuhan vast Ling Biotech, Inc.

1. Determination of chitosanase mutation sites

The method comprises the steps of simulating chitosanase BsCsn46A by using Swiss-Model online software to obtain a space structure of the chitosanase, submitting a protein structure obtained by Swiss-Model simulation to a PoPMuSiC online server to calculate the unfolding free energy change (delta G) of each mutant amino acid of the chitosanase so as to assist in searching for amino acids which have great influence on the catalysis of the chitosanase, selecting the amino acid with the highest delta G as the mutant amino acid, and mutating the amino acid into other 19 amino acids. Reverse PCR amplification with the primers of table 1 gave the corresponding mutant plasmid template from e.coli DH5 α (this strain was able to be methylated at the GATC site).

TABLE 1 primer sequences

The reverse PCR system is as follows:

name of reagent	Volume (μ L)
		Form panel	2
PCR Buffer	5
		dNTPs(10mM)	1
Up/down stream primer (100mM)	0.3 each
		PfuDNA polymerase	1.5
ddH2O	40
		Total volume	50

Reverse PCR amplification conditions: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 50s, annealing at 58 ℃ for 30s, extension at 68 ℃ for 12.5min, and 12 cycles; keeping the temperature at 4 ℃.

DpnI digestion template plasmid

mu.L of PCR product was taken and 1. mu.L of DpnI restriction enzyme was directly added to the PCR product.

3. Transformation of Escherichia coli

10. mu.L of the enzyme-cleaved product was directly transformed into E.coli DH 5. alpha. competent cells. The recombinant cells carrying the mutated plasmids were sent to Shanghai bioengineering, Inc. for sequencing. Coli BL21 was transformed with the correctly sequenced mutant plasmid.

4. Protein purification

The crude enzyme solution obtained by disrupting the cells by ultrasonic waves was applied to a Ni-NTA affinity chromatography column, unbound proteins were sufficiently eluted using the application buffer, and finally the recombinant protein having a histidine tag was eluted with an elution buffer (50mM Tris-HCl, 0.5mM NaCl, 0.1M imidazole, pH 8.0), and the recombinant protein having a histidine tag was stored at-20 ℃ for future use. The protein content in the enzyme solution was determined by the Broadford method.

5. Enzyme activity assay

1475. mu.L of pH 6.2 phosphate buffer, 500. mu.L of 1% colloidal chitosan solution, 18. mu.L of 100mM Mn were added to the cuvette²⁺Finally, 25 mu L of purified chitosanase obtained by purification is added, water bath at 55 ℃ is carried out for 5min immediately after uniform mixing, 1.5mL of DNS reagent is added immediately after the water bath is finished, the reaction is stopped, and a sample without enzyme solution is used as a blank control; after boiling in a water bath for 5min, water was added to 25mL and the absorbance at 520nm was measured.

6. Definition of enzyme Activity

Under these conditions, the amount of enzyme catalyzing the production of 1. mu.M of reducing sugar per minute is defined as one enzyme activity unit (U).

7. Enzymological Properties

(1) Optimum pH

The enzymatic activity of the chitosanase was determined at different pH (phosphate buffered saline) at a temperature of 50 ℃. The highest point of enzyme activity is taken as 100 percent.

(2) Stability of pH

The chitosanase is placed in phosphate buffer solution with pH 6.2 and stored for 2h at 4 ℃, and the enzyme activity of the chitosanase is determined as 100% at 0 h.

(3) Optimum temperature

Under the condition of the most suitable pH, the reaction systems are respectively placed at 40-75 ℃ for reaction, and the enzyme activity of the chitosanase is measured. The highest point of enzyme activity is taken as 100 percent.

(4) Temperature stability

The chitosanase is stored for 2 hours at the temperature of 55 ℃, and the enzyme activity of the chitosanase measured at 0 hour is 100 percent.

8. Enzymological properties of Bacillus subtilis chitosanase BsCsn46A and mutants thereof

The enzymological properties of the Bacillus subtilis BsCsn46A and the mutant thereof are shown in fig. 1 and table 2, the catalytic activities of the mutants P121N, P121C, P121V, P121S and P121R are obviously higher than those of the Bacillus subtilis BsCsn46A, but the temperature stability of the mutants P121S and P121R is obviously reduced.

TABLE 2 enzymological Properties of Bacillus subtilis chitosanase BsCsn46A and mutants thereof

From the results, the chitosanase mutant and the construction method and the application thereof can obtain six chitosanase mutants with obviously improved catalytic activity, wherein the P121N mutant improves the catalytic activity on the premise of not influencing the enzyme stability, and the activity is the highest activity reported by the current chitosanase.

The above description is only a preferred embodiment of the present invention, and the scope of the present invention is not limited thereto, and any simple modifications or equivalent substitutions of the technical solutions that can be obviously obtained by those skilled in the art within the technical scope of the present invention are within the scope of the present invention.

Sequence listing

<110> university of Changzhou

<120> chitosanase mutant and construction method and application thereof

<160> 48

<170> SIPOSequenceListing 1.0

<210> 1

<211> 242

<212> PRT

<213> Amino acid Sequence (Amino acid Sequence)

<400> 1

Ala Gly Leu Asn Lys Asp Gln Lys Arg Arg Ala Glu Gln Leu Thr Ser

1 5 10 15

Ile Phe Glu Asn Gly Thr Thr Glu Ile Gln Tyr Gly Tyr Val Glu Arg

20 25 30

Leu Asp Asp Gly Arg Gly Tyr Thr Cys Gly Arg Ala Gly Phe Thr Thr

35 40 45

Ala Thr Gly Asp Ala Leu Glu Val Val Glu Val Tyr Thr Lys Ala Val

50 55 60

Pro Asn Ser Lys Leu Lys Lys Tyr Leu Pro Glu Leu Arg Arg Leu Ala

65 70 75 80

Lys Glu Glu Ser Asp Asp Thr Ser Asn Leu Lys Gly Phe Ala Ser Ala

85 90 95

Trp Lys Ser Leu Ala Asn Asp Lys Glu Phe Arg Ala Ala Gln Asp Lys

100 105 110

Val Asn Asp His Leu Tyr Tyr Gln Pro Ala Met Lys Arg Ser Asp Asn

115 120 125

Ala Gly Leu Lys Thr Ala Leu Ala Arg Ala Val Met Tyr Asp Thr Val

130 135 140

Ile Gln His Gly Asp Gly Asp Asp Pro Asp Ser Phe Tyr Ala Leu Ile

145 150 155 160

Lys Arg Thr Asn Lys Lys Ala Gly Gly Ser Pro Lys Asp Gly Ile Asp

165 170 175

Glu Lys Lys Trp Leu Asn Lys Phe Leu Asp Val Arg Tyr Asp Asp Leu

180 185 190

Met Asn Pro Ala Asn His Asp Thr Arg Asp Glu Trp Arg Glu Ser Val

195 200 205

Ala Arg Val Asp Val Leu Arg Ser Ile Ala Lys Glu Asn Asn Tyr Asn

210 215 220

Leu Asn Gly Pro Ile His Val Arg Ser Asn Glu Tyr Gly Asn Phe Val

225 230 235 240

Ile Lys

<210> 45

<211> 728

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 45

gcgggactga ataaagatca aaagcgccgg gcggaacagc tgacaagtat ctttgaaaac 60

ggcacaacgg agatccaata tggatatgta gagcgattgg atgacgggcg aggctataca 120

tgcggtcggg caggctttac aacggctacc ggggatgcat tggaagtagt ggaagtatac 180

acaaaggcag ttccgaatag caaactgaaa aagtatctgc ctgaattgcg ccgtctggcc 240

aaggaagaaa gcgatgatac aagcaatctc aagggattcg cttctgcctg gaagtcgctt 300

gcaaatgata aggaatttcg cgccgctcaa gacaaagtaa atgaccattt gtattatcag 360

cctgccatga aacgatcgga taatgccgga ctaaaaacag cattggcaag agctgtgatg 420

tacgatacgg ttattcagca tggcgatggt gatgaccctg actcttttta tgccttgatt 480

aaacgtacga acaaaaaagc gggcggatca cctaaagacg gaatagacga gaagaagtgg 540

ttgaataaat tcttggacgt acgctatgag atctgatgaa tccggccaat catgacaccc 600

gtgacgaatg gagagaatca gttgcccgtg tggacgtgct tcgctctatc gccaaggaga 660

acaactataa tctaaacgga ccgattcatg ttcgttcaaa cgagtacggt aattttgtaa 720

tcaaataa 728

<210> 2

<211> 242

<212> PRT

<213> Amino acid Sequence (Amino acid Sequence)

<400> 2

Ala Gly Leu Asn Lys Asp Gln Lys Arg Arg Ala Glu Gln Leu Thr Ser

1 5 10 15

Ile Phe Glu Asn Gly Thr Thr Glu Ile Gln Tyr Gly Tyr Val Glu Arg

20 25 30

Leu Asp Asp Gly Arg Gly Tyr Thr Cys Gly Arg Ala Gly Phe Thr Thr

35 40 45

Ala Thr Gly Asp Ala Leu Glu Val Val Glu Val Tyr Thr Lys Ala Val

50 55 60

Pro Asn Ser Lys Leu Lys Lys Tyr Leu Pro Glu Leu Arg Arg Leu Ala

65 70 75 80

Lys Glu Glu Ser Asp Asp Thr Ser Asn Leu Lys Gly Phe Ala Ser Ala

85 90 95

Trp Lys Ser Leu Ala Asn Asp Lys Glu Phe Arg Ala Ala Gln Asp Lys

100 105 110

Val Asn Asp His Leu Tyr Tyr Gln Asn Ala Met Lys Arg Ser Asp Asn

115 120 125

Ala Gly Leu Lys Thr Ala Leu Ala Arg Ala Val Met Tyr Asp Thr Val

130 135 140

Ile Gln His Gly Asp Gly Asp Asp Pro Asp Ser Phe Tyr Ala Leu Ile

145 150 155 160

Lys Arg Thr Asn Lys Lys Ala Gly Gly Ser Pro Lys Asp Gly Ile Asp

165 170 175

Glu Lys Lys Trp Leu Asn Lys Phe Leu Asp Val Arg Tyr Asp Asp Leu

180 185 190

Met Asn Pro Ala Asn His Asp Thr Arg Asp Glu Trp Arg Glu Ser Val

195 200 205

Ala Arg Val Asp Val Leu Arg Ser Ile Ala Lys Glu Asn Asn Tyr Asn

210 215 220

Leu Asn Gly Pro Ile His Val Arg Ser Asn Glu Tyr Gly Asn Phe Val

225 230 235 240

Ile Lys

<210> 46

<211> 728

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 46

gcgggactga ataaagatca aaagcgccgg gcggaacagc tgacaagtat ctttgaaaac 60

ggcacaacgg agatccaata tggatatgta gagcgattgg atgacgggcg aggctataca 120

tgcggtcggg caggctttac aacggctacc ggggatgcat tggaagtagt ggaagtatac 180

acaaaggcag ttccgaatag caaactgaaa aagtatctgc ctgaattgcg ccgtctggcc 240

aaggaagaaa gcgatgatac aagcaatctc aagggattcg cttctgcctg gaagtcgctt 300

gcaaatgata aggaatttcg cgccgctcaa gacaaagtaa atgaccattt gtattatcag 360

aatgccatga aacgatcgga taatgccgga ctaaaaacag cattggcaag agctgtgatg 420

tacgatacgg ttattcagca tggcgatggt gatgaccctg actcttttta tgccttgatt 480

aaacgtacga acaaaaaagc gggcggatca cctaaagacg gaatagacga gaagaagtgg 540

ttgaataaat tcttggacgt acgctatgag atctgatgaa tccggccaat catgacaccc 600

gtgacgaatg gagagaatca gttgcccgtg tggacgtgct tcgctctatc gccaaggaga 660

acaactataa tctaaacgga ccgattcatg ttcgttcaaa cgagtacggt aattttgtaa 720

tcaaataa 728

<210> 3

<211> 242

<212> PRT

<213> Amino acid Sequence (Amino acid Sequence)

<400> 3

Ala Gly Leu Asn Lys Asp Gln Lys Arg Arg Ala Glu Gln Leu Thr Ser

1 5 10 15

Ile Phe Glu Asn Gly Thr Thr Glu Ile Gln Tyr Gly Tyr Val Glu Arg

20 25 30

Leu Asp Asp Gly Arg Gly Tyr Thr Cys Gly Arg Ala Gly Phe Thr Thr

35 40 45

Ala Thr Gly Asp Ala Leu Glu Val Val Glu Val Tyr Thr Lys Ala Val

50 55 60

Pro Asn Ser Lys Leu Lys Lys Tyr Leu Pro Glu Leu Arg Arg Leu Ala

65 70 75 80

Lys Glu Glu Ser Asp Asp Thr Ser Asn Leu Lys Gly Phe Ala Ser Ala

85 90 95

Trp Lys Ser Leu Ala Asn Asp Lys Glu Phe Arg Ala Ala Gln Asp Lys

100 105 110

Val Asn Asp His Leu Tyr Tyr Gln Cys Ala Met Lys Arg Ser Asp Asn

115 120 125

Ala Gly Leu Lys Thr Ala Leu Ala Arg Ala Val Met Tyr Asp Thr Val

130 135 140

Ile Gln His Gly Asp Gly Asp Asp Pro Asp Ser Phe Tyr Ala Leu Ile

145 150 155 160

Lys Arg Thr Asn Lys Lys Ala Gly Gly Ser Pro Lys Asp Gly Ile Asp

165 170 175

Glu Lys Lys Trp Leu Asn Lys Phe Leu Asp Val Arg Tyr Asp Asp Leu

180 185 190

Met Asn Pro Ala Asn His Asp Thr Arg Asp Glu Trp Arg Glu Ser Val

195 200 205

Ala Arg Val Asp Val Leu Arg Ser Ile Ala Lys Glu Asn Asn Tyr Asn

210 215 220

Leu Asn Gly Pro Ile His Val Arg Ser Asn Glu Tyr Gly Asn Phe Val

225 230 235 240

Ile Lys

<210> 47

<211> 728

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 47

gcgggactga ataaagatca aaagcgccgg gcggaacagc tgacaagtat ctttgaaaac 60

ggcacaacgg agatccaata tggatatgta gagcgattgg atgacgggcg aggctataca 120

tgcggtcggg caggctttac aacggctacc ggggatgcat tggaagtagt ggaagtatac 180

acaaaggcag ttccgaatag caaactgaaa aagtatctgc ctgaattgcg ccgtctggcc 240

aaggaagaaa gcgatgatac aagcaatctc aagggattcg cttctgcctg gaagtcgctt 300

gcaaatgata aggaatttcg cgccgctcaa gacaaagtaa atgaccattt gtattatcag 360

tgcgccatga aacgatcgga taatgccgga ctaaaaacag cattggcaag agctgtgatg 420

tacgatacgg ttattcagca tggcgatggt gatgaccctg actcttttta tgccttgatt 480

aaacgtacga acaaaaaagc gggcggatca cctaaagacg gaatagacga gaagaagtgg 540

ttgaataaat tcttggacgt acgctatgag atctgatgaa tccggccaat catgacaccc 600

gtgacgaatg gagagaatca gttgcccgtg tggacgtgct tcgctctatc gccaaggaga 660

acaactataa tctaaacgga ccgattcatg ttcgttcaaa cgagtacggt aattttgtaa 720

tcaaataa 728

<210> 4

<211> 242

<212> PRT

<213> Amino acid Sequence (Amino acid Sequence)

<400> 4

Ala Gly Leu Asn Lys Asp Gln Lys Arg Arg Ala Glu Gln Leu Thr Ser

1 5 10 15

Ile Phe Glu Asn Gly Thr Thr Glu Ile Gln Tyr Gly Tyr Val Glu Arg

20 25 30

Leu Asp Asp Gly Arg Gly Tyr Thr Cys Gly Arg Ala Gly Phe Thr Thr

35 40 45

Ala Thr Gly Asp Ala Leu Glu Val Val Glu Val Tyr Thr Lys Ala Val

50 55 60

Pro Asn Ser Lys Leu Lys Lys Tyr Leu Pro Glu Leu Arg Arg Leu Ala

65 70 75 80

Lys Glu Glu Ser Asp Asp Thr Ser Asn Leu Lys Gly Phe Ala Ser Ala

85 90 95

Trp Lys Ser Leu Ala Asn Asp Lys Glu Phe Arg Ala Ala Gln Asp Lys

100 105 110

Val Asn Asp His Leu Tyr Tyr Gln Val Ala Met Lys Arg Ser Asp Asn

115 120 125

Ala Gly Leu Lys Thr Ala Leu Ala Arg Ala Val Met Tyr Asp Thr Val

130 135 140

Ile Gln His Gly Asp Gly Asp Asp Pro Asp Ser Phe Tyr Ala Leu Ile

145 150 155 160

Lys Arg Thr Asn Lys Lys Ala Gly Gly Ser Pro Lys Asp Gly Ile Asp

165 170 175

Glu Lys Lys Trp Leu Asn Lys Phe Leu Asp Val Arg Tyr Asp Asp Leu

180 185 190

Met Asn Pro Ala Asn His Asp Thr Arg Asp Glu Trp Arg Glu Ser Val

195 200 205

Ala Arg Val Asp Val Leu Arg Ser Ile Ala Lys Glu Asn Asn Tyr Asn

210 215 220

Leu Asn Gly Pro Ile His Val Arg Ser Asn Glu Tyr Gly Asn Phe Val

225 230 235 240

Ile Lys

<210> 48

<211> 728

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 48

gcgggactga ataaagatca aaagcgccgg gcggaacagc tgacaagtat ctttgaaaac 60

ggcacaacgg agatccaata tggatatgta gagcgattgg atgacgggcg aggctataca 120

tgcggtcggg caggctttac aacggctacc ggggatgcat tggaagtagt ggaagtatac 180

acaaaggcag ttccgaatag caaactgaaa aagtatctgc ctgaattgcg ccgtctggcc 240

aaggaagaaa gcgatgatac aagcaatctc aagggattcg cttctgcctg gaagtcgctt 300

gcaaatgata aggaatttcg cgccgctcaa gacaaagtaa atgaccattt gtattatcag 360

gttgccatga aacgatcgga taatgccgga ctaaaaacag cattggcaag agctgtgatg 420

tacgatacgg ttattcagca tggcgatggt gatgaccctg actcttttta tgccttgatt 480

aaacgtacga acaaaaaagc gggcggatca cctaaagacg gaatagacga gaagaagtgg 540

ttgaataaat tcttggacgt acgctatgag atctgatgaa tccggccaat catgacaccc 600

gtgacgaatg gagagaatca gttgcccgtg tggacgtgct tcgctctatc gccaaggaga 660

acaactataa tctaaacgga ccgattcatg ttcgttcaaa cgagtacggt aattttgtaa 720

tcaaataa 728

<210> 5

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

cgggatccgc gggactgaat aaagatc 27

<210> 6

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

cccaagcttt tatttgatta caaaattacc 30

<210> 7

<211> 35

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

ccatttgtat tatcagggtg ccatgaaacg atcgg 35

<210> 8

<211> 35

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

ccgatcgttt catggcaccc tgataataca aatgg 35

<210> 9

<211> 33

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

ccatttgtat tatcaggctg ccatgaaacg atc 33

<210> 10

<211> 33

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

gatcgtttca tggcagcctg ataatacaaa tgg 33

<210> 11

<211> 37

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

gaccatttgt attatcagat tgccatgaaa cgatcgg 37

<210> 12

<211> 37

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

ccgatcgttt catggcaatc tgataataca aatggtc 37

<210> 13

<211> 35

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

ccatttgtat tatcagctgg ccatgaaacg atcgg 35

<210> 14

<211> 35

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

ccgatcgttt catggccagc tgataataca aatgg 35

<210> 15

<211> 37

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

gaccatttgt attatcaggt tgccatgaaa cgatcgg 37

<210> 16

<211> 37

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

ccgatcgttt catggcaacc tgataataca aatggtc 37

<210> 17

<211> 37

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

gaccatttgt attatcagta tgccatgaaa cgatcgg 37

<210> 18

<211> 37

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

ccgatcgttt catggcatac tgataataca aatggtc 37

<210> 19

<211> 37

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

gaccatttgt attatcagtg ggccatgaaa cgatcgg 37

<210> 20

<211> 37

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

ccgatcgttt catggcccac tgataataca aatggtc 37

<210> 21

<211> 37

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

gaccatttgt attatcagtt tgccatgaaa cgatcgg 37

<210> 22

<211> 37

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 22

ccgatcgttt catggcaaac tgataataca aatggtc 37

<210> 23

<211> 37

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 23

gaccatttgt attatcagtg cgccatgaaa cgatcgg 37

<210> 24

<211> 37

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 24

ccgatcgttt catggcgcac tgataataca aatggtc 37

<210> 25

<211> 37

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 25

gaccatttgt attatcagat ggccatgaaa cgatcgg 37

<210> 26

<211> 37

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 26

ccgatcgttt catggccatc tgataataca aatggtc 37

<210> 27

<211> 34

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 27

ccatttgtat tatcagtctg ccatgaaacg atcg 34

<210> 28

<211> 34

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 28

cgatcgtttc atggcagact gataatacaa atgg 34

<210> 29

<211> 34

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 29

ccatttgtat tatcagactg ccatgaaacg atcg 34

<210> 30

<211> 34

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 30

cgatcgtttc atggcagtct gataatacaa atgg 34

<210> 31

<211> 35

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 31

ccatttgtat tatcagcagg ccatgaaacg atcgg 35

<210> 32

<211> 35

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 32

ccgatcgttt catggcctgc tgataataca aatgg 35

<210> 33

<211> 37

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 33

gaccatttgt attatcagaa tgccatgaaa cgatcgg 37

<210> 34

<211> 37

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 34

ccgatcgttt catggcattc tgataataca aatggtc 37

<210> 35

<211> 37

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 35

gaccatttgt attatcagga tgccatgaaa cgatcgg 37

<210> 36

<211> 37

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 36

ccgatcgttt catggcatcc tgataataca aatggtc 37

<210> 37

<211> 37

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 37

gaccatttgt attatcagga agccatgaaa cgatcgg 37

<210> 38

<211> 37

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 38

ccgatcgttt catggcttcc tgataataca aatggtc 37

<210> 39

<211> 35

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 39

ccatttgtat tatcagcgag ccatgaaacg atcgg 35

<210> 40

<211> 35

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 40

ccgatcgttt catggctcgc tgataataca aatgg 35

<210> 41

<211> 37

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 41

gaccatttgt attatcagaa agccatgaaa cgatcgg 37

<210> 42

<211> 37

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 42

ccgatcgttt catggctttc tgataataca aatggtc 37

<210> 43

<211> 34

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 43

catttgtatt atcagcatgc catgaaacga tcgg 34

<210> 44

<211> 34

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 44

ccgatcgttt catggcatgc tgataataca aatg 34