CN113493781B - Chitosan enzyme and application thereof - Google Patents
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- CN113493781B CN113493781B CN202110714063.3A CN202110714063A CN113493781B CN 113493781 B CN113493781 B CN 113493781B CN 202110714063 A CN202110714063 A CN 202110714063A CN 113493781 B CN113493781 B CN 113493781B
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- C12P19/26—Preparation of nitrogen-containing carbohydrates
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- C12Y402/00—Carbon-oxygen lyases (4.2)
- C12Y402/02—Carbon-oxygen lyases (4.2) acting on polysaccharides (4.2.2)
- C12Y402/02011—Poly(alpha-L-guluronate) lyase (4.2.2.11), i.e. alginase II
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Abstract
The invention discloses a coding gene of chitosan enzyme CsnH and application thereof. The amino acid sequence of the chitosan enzyme CsnH is shown as SEQ ID NO. 1. The sequence similarity with the chitosan enzyme reported by the prior property is only 67.64 percent. After recombinant expression in escherichia coli, the output of the chitosan enzyme can reach 665.3U/mL, the optimal reaction temperature of the chitosan enzyme CsnH is 70 ℃, the chitosan enzyme has the characteristic of high temperature resistance, 30.25% of activity can be kept after the chitosan enzyme is incubated for 1 hour at the high temperature of 80 ℃, the action mode is inscription, and the degradation product is chitosan disaccharide. Can be widely applied to industries such as food, cultivation, medicine and health care, etc.
Description
Technical Field
The invention relates to a coding gene of endo-chitosanase CsnH with heat-resistant property and application thereof, belonging to the technical field of biology.
Background
The chitosan oligosaccharide is an oligosaccharide with the polymerization degree of less than 20 and the molecular weight of less than 3900 after the chitosan is hydrolyzed. It is formed by connecting N-acetyl-D-glucosamine (GLcNAc) and D-glucosamine (GLcN) through beta-l, 4-glycosidic bond. Chitosan oligosaccharides are widely paid attention to for their various biological activities, and they are not only water-soluble, but also nontoxic, having anticoagulant, antioxidant, inflammatory response, immunostimulating, antibacterial, anticancer functions, etc. The chitosan oligosaccharide has greater application value in the fields of food, cultivation, medicine, health care and the like.
Chitosan oligosaccharides can be prepared by physical methods (ultrasonic treatment, ultraviolet radiation and microwave treatment), chemical methods (acid hydrolysis, hydrogen peroxide oxidation) and enzymatic methods (specific and non-specific enzymes). The enzymatic degradation method has the advantages of mild reaction conditions, easy control of the reaction process and the like, and the product purity is higher, and the engineering bacteria for constructing the high-yield chitosanase by utilizing genetic engineering are effective ways for industrially preparing the chitosan oligosaccharide. At present, reported chitosan enzyme has low activity and is not high-temperature resistant, so that the industrial application prospect is severely limited.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a novel chitosan enzyme CsnH and a preparation method thereof. The optimal reaction temperature of the chitosan enzyme CsnH is 70 ℃, and the optimal reaction pH is 5.5; the catalyst has the characteristic of high temperature resistance, can still keep 30.25% of activity after being incubated for 1 hour at the high temperature of 80 ℃, and is beneficial to solving the problems of enzyme stability and enzymolysis efficiency in the catalytic process. The enzyme degradation mode is inscribed, and the main enzymolysis product is chitosan.
In one aspect, the invention provides a novel chitosan enzyme CsnH, the amino acid sequence of which is shown as SEQ ID NO. 1.
SEQ ID NO.1：
MVYLSARTLALVACVLSLLELGAPVPNLRTRTVAVAVGGLLIASGAIAGTAAQANAVSSLAPAITAVSAASTGDLSAPAKKEIAMQLVCSAEGNGQKLRCHNLVWHNQLPNWVTSGSWTNETLLAAQYGYIEDIDDDRGYTGGIIGFTSGTGDMLELVQNYANTKPDNNVLKPGAKIDGVGLQSHFIVGSTPSQSAQAQKYVDAWHQAAKDSVFLKEQDKLRDSMYFNPAVSQGKDSNMSNLGQFMYYDAIFMHGPGDSSDSFGGIRKSAMKNAYTAAAQGGDEKTYLQAFATARKKIMKQENAHSDTSRVDDAQLKFAVAQHWGQCGGQGWTGATSCATGYICTFVNDWYYEIK。
On the other hand, the invention also provides a novel nucleic acid sequence corresponding to the chitosan enzyme CsnH, which is shown as SEQ ID NO. 2.
SEQ ID NO.2：
Atggtgtatctgagcgcgcgcaccctggcgctggtggcgtgcgtgctgagcctgctggaactgggcgcgccggtgccgaacctgcgcacccgcaccgtggcggtggcggtgggcggcctgctgattgcgagcggcgcgattgcgggcaccgcggcgcaggcgaacgcggtgagcagcctggcgccggcgattaccgcggtgagcgcggcgagcaccggcgatctgagcgcgccggcgaaaaaagaaattgcgatgcagctggtgtgcagcgcggaaggcaacggccagaaactgcgctgccataacctggtgtggcataaccagctgccgaactgggtgaccagcggcagctggaccaacgaaaccctgctggcggcgcagtatggctatattgaagatattgatgatgatcgcggctataccggcggcattattggctttaccagcggcaccggcgatatgctggaactggtgcagaactatgcgaacaccaaaccggataacaacgtgctgaaaccgggcgcgaaaattgatggcgtgggcctgcagagccattttattgtgggcagcaccccgagccagagcgcgcaggcgcagaaatatgtggatgcgtggcatcaggcggcgaaagatagcgtgtttctgaaagaacaggataaactgcgcgatagcatgtattttaacccggcggtgagccagggcaaagatagcaacatgagcaacctgggccagtttatgtattatgatgcgatttttatgcatggcccgggcgatagcagcgatagctttggcggcattcgcaaaagcgcgatgaaaaacgcgtataccgcggcggcgcagggcggcgatgaaaaaacctatctgcaggcgtttgcgaccgcgcgcaaaaaaattatgaaacaggaaaacgcgcatagcgataccagccgcgtggatgatgcgcagctgaaatttgcggtggcgcagcattggggccagtgcggcggccagggctggaccggcgcgaccagctgcgcgaccggctatatttgcacctttgtgaacgattggtattatgaaattaaa。
On the other hand, the invention also provides a preparation and purification method of the chitosan enzyme CsnH.
On the other hand, the invention also provides application of the chitosan enzyme CsnH in degrading chitosan.
In another aspect, a method of degrading chitosan is provided wherein the selected chitosanase is CsnH.
Preferably: the reaction temperature in the degradation condition is 0-90 ℃. The optimum reaction temperature was 70 ℃.
Preferably: the reaction pH in the degradation condition is 4.9-7.1. The optimal reaction pH was 5.5.
The beneficial effects are that:
1. the chitosan enzyme CsnH is an algin lyase with novel structure and function, and the similarity of the amino acid sequence of the chitosan enzyme CsnH and the sequence of the chitosan enzyme reported by the prior property is only 67.64 percent.
2. The invention provides a method for preparing chitosan enzyme CsnH, namely, by utilizing a genetic engineering technical method, the gene sequence heterologous recombination of CsnH is expressed to escherichia coli, and after fermentation, the enzyme activity of fermentation liquor is as high as 665.3U/mL, so that the method has the potential of industrial production. The enzyme purification method is simple, the nickel column is utilized to carry out one-step affinity purification, the recovery rate is up to 88.3%, and the protein purity is up to 95.1%.
3. The chitosan enzyme CsnH has excellent physicochemical property, the optimal reaction pH of the enzyme is 5.5, the enzyme has the characteristic of high temperature resistance, the optimal reaction temperature is 70 ℃, and the activity of 30.25% can be maintained after the enzyme is incubated for 1 hour at the temperature of 80 ℃. The degradation product analysis is carried out by utilizing the recombinase, and the main degradation product of the enzyme is chitosan oligosaccharide disaccharide. The chitosan enzyme CsnH has good industrial application prospect.
Drawings
FIG. 1 is a diagram showing separation and purification of the CsnH protein of the chitosan enzyme of the present invention (M, protein standard; 1, purified resulting CsnH protein);
FIG. 2 shows the temperature and pH adaptability analysis of the chitosan enzyme CsnH of the present invention (A, optimum reaction temperature of the chitosan enzyme CsnH; B, temperature stability of the chitosan enzyme CsnH; C, optimum reaction pH of the chitosan enzyme CsnH; D, pH stability of the chitosan enzyme CsnH);
FIG. 3 shows the detection of the enzymatic hydrolysis product of the chitosan enzyme CsnH of the present invention by Thin Layer Chromatography (TLC) (M, chitosan oligosaccharide DP=2-4 standard; 0-2 is the enzymatic degradation of chitosan for 0min, 60min and 360min in sequence);
Detailed Description
Example 1 sequence analysis and recombinant expression of the Chitosan CsnH
The enzyme producing gene csnH of the chitosan enzyme csnH is derived from marine bacterium Pseudoalteromonase sp.SY52, comprises 1065 base sequences and codes 355 amino acid sequences. Analysis of the Conserved domain in National Center for Biotechnology Information (NCBI) for a Conserved domain (CDD) and multiple sequence alignment Basic Local Alignment Search Tool (Blast) revealed that the sequence contains a segment of the chitinase Conserved region of the polysaccharide hydrolase GH family. Among the chitosanase enzymes, the chitosanase enzyme (Genbank ABY 24857.1) having the highest similarity to CsnH amino acid sequence is the polysaccharide hydrolase 46 family (GH 46), and the amino acid sequence similarity (Identity) between the two is 67.64%. The chitosan enzyme CsnH of the invention belongs to the family of polysaccharide hydrolases (GH 46).
The enzyme-producing sequence of CsnH takes restriction enzymes Nco I and Xho I as enzyme cutting sites, and recombinant primers are designed as follows (underlined as restriction enzyme sites, italics as restriction enzyme protecting bases):
forward primer: SEQ ID NO.3: pcsnh-F:
5’-CATGCCATGGAAGTTGTCTTGTATCGCT-3’(Nco I)
reverse primer: SEQ ID NO.4: pcsnh-R:
5’-CCGCTCGAGCTTGATTTCGTATGGGTCA-3’(Xho I)
the PCR amplification conditions were: pre-denaturation at 94℃for 3min; denaturation at 94℃for 30 seconds, annealing at 55℃for 30 seconds, extension at 72℃for 1min, 30 cycles total; extending at 72 ℃ for 5min; stable at 4 ℃ for 15min. The DNA polymerase used for the PCR reaction was Primerstar HS available from Dalianbao Biotechnology.
The PCR product was digested with restriction enzymes Nco I and Xho I, and the digested PCR product was recovered by agarose gel electrophoresis. pET22b (+) plasmid DNA (Invitrogen, USA) was digested simultaneously with restriction enzymes Nco I and Xho I, subjected to agarose gel electrophoresis and the digested product fragments were recovered. The enzyme and substrate reaction systems (temperature, time, DNA amount, etc.) used for the enzyme digestion are described with reference to the products provided by Dalianbao organisms.
The PCR product treated by double enzyme digestion and pET-22b (+) plasmid vector are subjected to ligation reaction according to the specification of DNA ligase (Dalianbao biological company); the ligation product was transformed into E.coli DH 5. Alpha. Strain (Invitrogen Co., U.S.A.), spread on a solid plate of Luria-Bertani (LB) medium (containing 50. Mu.g/mL ampicillin), and after incubation in a 37℃incubator for 12-16 hours, the monoclonal was picked up; the monoclonal was transferred to LB liquid medium (containing 50. Mu.g/mL ampicillin) and incubated overnight in a shaker at 37℃at 180 rpm. The monoclonal was sequenced, positive clones were selected and designated pET22b-csnH. The recombinant plasmid was transformed into E.coli BL21 (DE 3) (purchased from Dalianbao biological Co.), and the recombinant E.coli strain was designated BL21 (DE 3)/pET 22b-csnH and stored at-80℃for use.
It should be noted that, on the premise that the amino acid sequence and the corresponding nucleotide sequence of the chitosan enzyme CsnH are disclosed in the present invention, a person of ordinary skill in the art may use artificial synthesis or other technical means to obtain the amino acid sequence and the corresponding nucleotide sequence of the chitosan enzyme CsnH. On the premise of known amino acid sequences, various nucleotide sequences capable of being expressed can be deduced, and all the nucleotide sequences are understood to be within the scope of the present invention.
Example 2 preparation and purification method of Chitosan CsnH
Will weighGroup strain BL21 (DE 3)/pET 22b-csnH was shake-cultured in 100mL of LB liquid medium (50. Mu.g/mL ampicillin) at 180rpm in a shaker at 37℃to OD 600 =0.6, isopropyl- β -D-thiogalactoside (IPTG), an inducer, was added at a final concentration of 0.1mM, and induction was performed for 20h at 20 ℃. The method for measuring the activity of the chitosan enzyme comprises the following steps: mu.L of the enzyme solution was added to 900. Mu.L of 0.3% chitosan substrate (20 mM acetic acid-sodium acetate, pH=5.9), reacted at 40℃for 10 minutes, and 750. Mu.L of DNS reagent was added thereto, reacted in boiling water for 10 minutes to develop a color, and the absorbance was measured at OD 520. The enzyme activity is defined as the amount of enzyme required for 1U to produce 1. Mu.M reducing sugar per minute. Through detection, the activity of the chitosan enzyme in the fermentation broth can reach 665.3U/mL.
After fermentation was stopped, the mixture was centrifuged at 12000rpm for 10min, and the cells were discarded to collect the supernatant; loading the fermentation supernatant onto a 10mL nickel ion affinity chromatography column at a loading flow rate of 5mL/min, eluting with 10mM imidazole to remove the impurity proteins, eluting with 150mM imidazole, and collecting the eluting components. Dialyzing the active ingredient to remove imidazole, and storing at-20deg.C. Through one-step affinity purification of nickel ions, the protein recovery rate reaches 88.3 percent. The purified chitosan enzyme was subjected to polyacrylamide gel electrophoresis (SDS-PAGE), as shown in FIG. 1, and the molecular weight of CsnH of the purified chitosan enzyme was about 38kDa, which was consistent with the protein size predicted in the sequence analysis. Gel analysis shows that the protein purity of the purified chitosan enzyme CsnH reaches more than 95.1 percent.
Example 3 Effect of temperature and pH on the Chitosan CsnH
The enzyme activity of the chitosan enzyme CsnH purified in the example 2 was measured under different conditions, and the influence of different temperatures and pH values on the enzyme activity was detected. And (3) reacting for 10min at different temperatures (0-90 ℃), detecting the influence of different reaction temperatures on the enzyme activity, and calculating the relative enzyme activity of the CsnH at different temperatures by taking the highest enzyme activity as 100%. As shown in FIG. 2A, the optimum reaction temperature for the chitosan enzyme CsnH was 70 ℃.
The purified chitosan enzyme CsnH obtained in the example 2 is incubated for 1h at different temperatures (0-90 ℃), and after the purified chitosan enzyme CsnH is taken out, the enzyme activity is immediately detected at the optimal reaction temperature (70 ℃) and is taken as 100%, as shown in FIG. 2B, the temperature stability of the chitosan enzyme CsnH is better, and the enzyme activity is still kept at 30.25% after the purified chitosan enzyme CsnH is incubated for 1h at 80 ℃.
The purified chitosan enzyme CsnH of example 2 was reacted with chitosan substrates formulated at different pH using different buffer systems, sodium acetate buffer (50 mM, pH 4.49-5.9), phosphate buffer (50 mM, pH 5.88-7.12) and Tris-HCl buffer (50 mM, pH 6.22-7.11), respectively. The activity was measured at the optimum temperature, and the highest value of the enzyme activity was 100%. As shown in FIG. 2C, the optimum reaction pH for the chitosan enzyme CsnH was 5.5.
The purified chitosan enzyme CsnH of example 2 was incubated for 24h at different pH conditions (4.7-10.8), and immediately after removal, its enzyme activity was examined at its optimal reaction pH (5.5) with activity before incubation as 100%, as shown in fig. 2D.
EXAMPLE 4 thin layer chromatography analysis of the enzymatic hydrolysis product of Chitosan CsnH
The purified chitosan enzyme from example 2, csnH pure enzyme, was incubated with 0.3% chitosan at 70 ℃ for 0min and 60min, respectively, and then detected by High Performance Thin Layer Chromatography (HPTLC). The method comprises the following steps: the HPTLC chromatographic plate is cut into samples with a proper size and a width of 7cm, samples before and after incubation are spotted at an origin, placed in a spreading cylinder with a spreading agent (n-butanol: glacial acetic acid: water=2:2:1) for 30min, the chromatographic plate is dried by blowing, immersed in a color developing agent (0.5% ninhydrin ethanol solution) for 2s, taken out, dried by blowing, and baked at 80 ℃ until the samples appear. As shown in FIG. 3, compared with the migration rate of the standard, the main product of the enzymolysis of the chitosan enzyme CsnH is chitosan (DP 2).
Sequence listing
<110> university of Qingdao
<120> a chitosan enzyme and use thereof
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 355
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 1
Met Val Tyr Leu Ser Ala Arg Thr Leu Ala Leu Val Ala Cys Val Leu
1 5 10 15
Ser Leu Leu Glu Leu Gly Ala Pro Val Pro Asn Leu Arg Thr Arg Thr
20 25 30
Val Ala Val Ala Val Gly Gly Leu Leu Ile Ala Ser Gly Ala Ile Ala
35 40 45
Gly Thr Ala Ala Gln Ala Asn Ala Val Ser Ser Leu Ala Pro Ala Ile
50 55 60
Thr Ala Val Ser Ala Ala Ser Thr Gly Asp Leu Ser Ala Pro Ala Lys
65 70 75 80
Lys Glu Ile Ala Met Gln Leu Val Cys Ser Ala Glu Gly Asn Gly Gln
85 90 95
Lys Leu Arg Cys His Asn Leu Val Trp His Asn Gln Leu Pro Asn Trp
100 105 110
Val Thr Ser Gly Ser Trp Thr Asn Glu Thr Leu Leu Ala Ala Gln Tyr
115 120 125
Gly Tyr Ile Glu Asp Ile Asp Asp Asp Arg Gly Tyr Thr Gly Gly Ile
130 135 140
Ile Gly Phe Thr Ser Gly Thr Gly Asp Met Leu Glu Leu Val Gln Asn
145 150 155 160
Tyr Ala Asn Thr Lys Pro Asp Asn Asn Val Leu Lys Pro Gly Ala Lys
165 170 175
Ile Asp Gly Val Gly Leu Gln Ser His Phe Ile Val Gly Ser Thr Pro
180 185 190
Ser Gln Ser Ala Gln Ala Gln Lys Tyr Val Asp Ala Trp His Gln Ala
195 200 205
Ala Lys Asp Ser Val Phe Leu Lys Glu Gln Asp Lys Leu Arg Asp Ser
210 215 220
Met Tyr Phe Asn Pro Ala Val Ser Gln Gly Lys Asp Ser Asn Met Ser
225 230 235 240
Asn Leu Gly Gln Phe Met Tyr Tyr Asp Ala Ile Phe Met His Gly Pro
245 250 255
Gly Asp Ser Ser Asp Ser Phe Gly Gly Ile Arg Lys Ser Ala Met Lys
260 265 270
Asn Ala Tyr Thr Ala Ala Ala Gln Gly Gly Asp Glu Lys Thr Tyr Leu
275 280 285
Gln Ala Phe Ala Thr Ala Arg Lys Lys Ile Met Lys Gln Glu Asn Ala
290 295 300
His Ser Asp Thr Ser Arg Val Asp Asp Ala Gln Leu Lys Phe Ala Val
305 310 315 320
Ala Gln His Trp Gly Gln Cys Gly Gly Gln Gly Trp Thr Gly Ala Thr
325 330 335
Ser Cys Ala Thr Gly Tyr Ile Cys Thr Phe Val Asn Asp Trp Tyr Tyr
340 345 350
Glu Ile Lys
355
<210> 2
<211> 1065
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 2
atggtgtatc tgagcgcgcg caccctggcg ctggtggcgt gcgtgctgag cctgctggaa 60
ctgggcgcgc cggtgccgaa cctgcgcacc cgcaccgtgg cggtggcggt gggcggcctg 120
ctgattgcga gcggcgcgat tgcgggcacc gcggcgcagg cgaacgcggt gagcagcctg 180
gcgccggcga ttaccgcggt gagcgcggcg agcaccggcg atctgagcgc gccggcgaaa 240
aaagaaattg cgatgcagct ggtgtgcagc gcggaaggca acggccagaa actgcgctgc 300
cataacctgg tgtggcataa ccagctgccg aactgggtga ccagcggcag ctggaccaac 360
gaaaccctgc tggcggcgca gtatggctat attgaagata ttgatgatga tcgcggctat 420
accggcggca ttattggctt taccagcggc accggcgata tgctggaact ggtgcagaac 480
tatgcgaaca ccaaaccgga taacaacgtg ctgaaaccgg gcgcgaaaat tgatggcgtg 540
ggcctgcaga gccattttat tgtgggcagc accccgagcc agagcgcgca ggcgcagaaa 600
tatgtggatg cgtggcatca ggcggcgaaa gatagcgtgt ttctgaaaga acaggataaa 660
ctgcgcgata gcatgtattt taacccggcg gtgagccagg gcaaagatag caacatgagc 720
aacctgggcc agtttatgta ttatgatgcg atttttatgc atggcccggg cgatagcagc 780
gatagctttg gcggcattcg caaaagcgcg atgaaaaacg cgtataccgc ggcggcgcag 840
ggcggcgatg aaaaaaccta tctgcaggcg tttgcgaccg cgcgcaaaaa aattatgaaa 900
caggaaaacg cgcatagcga taccagccgc gtggatgatg cgcagctgaa atttgcggtg 960
gcgcagcatt ggggccagtg cggcggccag ggctggaccg gcgcgaccag ctgcgcgacc 1020
ggctatattt gcacctttgt gaacgattgg tattatgaaa ttaaa 1065
<210> 3
<211> 28
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 3
catgccatgg aagttgtctt gtatcgct 28
<210> 4
<211> 28
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 4
ccgctcgagc ttgatttcgt atgggtca 28
Claims (8)
1. A chitosanase has an amino acid sequence shown in SEQ ID NO. 1.
2. The chitosanase of claim 1, wherein the nucleotide encoding the chitosanase is shown in SEQ ID No. 2.
3. Use of the chitosanase of claim 1 for degrading chitosan.
4. A method for degrading chitosan, wherein the chitosan enzyme selected is the chitosan enzyme of claim 1.
5. The method of claim 4, wherein the reaction temperature in the degradation condition is 0-90 ℃.
6. The method of claim 4, wherein the reaction temperature in the degradation conditions is 70 ℃.
7. The method of claim 4, wherein the reaction pH is 4.9 to 7.1 under degradation conditions.
8. The method of claim 4, wherein the reaction pH in the degradation conditions is 5.5.
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