Chitosanase CsnQ and application thereof
Technical Field
The invention relates to a chitosanase CsnQ and application thereof, belonging to the technical field of biology.
Background
Chitosan oligosaccharide has important biological activities of antibiosis, antitumor, anti-inflammation and the like, and is known as the sixth element of life. The chitosan oligosaccharide is formed by connecting N-acetyl-D-glucosamine (GLcNAc) and D-glucosamine (GLcN) through beta-l, 4-glycosidic bond, has molecular weight less than 3900 and polymerization degree less than 20, is very easy to dissolve in water, and has higher application value than macromolecular chitosan in the fields of medicine, health care products, food, culture and the like.
The chitosan oligosaccharide is formed by degrading macromolecular chitosan. In general, degradation methods are classified into three types, physical, chemical and biological enzymatic degradation. The biological enzymatic degradation has the advantages of mild reaction conditions, single degradation product, easy control of the reaction process, environmental friendliness and the like. The method for degrading macromolecular chitosan by using the chitosan enzyme method gradually becomes a mainstream method in the preparation process of chitosan oligosaccharide. However, the chitosanase sold in the market at present is expensive, low in activity and poor in stability, and the application prospect of the chitosanase is severely limited.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides chitosanase CsnQ and a preparation method thereof. The yield of the chitosanase CsnQ is 1058.2U/mL, the optimal reaction temperature is 60 ℃, the stability is kept between pH4.0 and 6.0, the activity is still kept at 61.2 percent after 1h of incubation at 40 ℃, and various metal ions have promotion effect on the enzyme activity of the chitosanase CsnQ. The degradation mode is internal cutting, and the main degradation products are chitobiose and chitotriose. The chitosanase CsnQ has high yield, good stability and good industrial application potential.
On one hand, the invention provides chitosanase CsnQ, and the amino acid sequence of the chitosanase CsnQ is shown in SEQ ID NO. 1.
SEQ ID NO.1:
MKYLLPTAAAGLLLLAAQPAMAMGVGGTGATHAGAAGSGVNLTDPHKKEIAMELVSSAENSSLDWKAQYKYIEDIGDGRGYTGGIIGFCSGTGDMLELVQHYTDLEPGNILAKYLPALKKVNGSASHSGLGTPFTKDWATAAKDTVFQQAQNDERDRVYFDPAVSQAKADGLRALGQFAYYDAIVMHGPGNDPTSFGGIRKTAMKKARTPAQGGDETTYLNAFLDARKAAMLTEAAHDDTSRVDTEQRVFLKAGNLDLNPPLHWKTYGDSYSINSLEHTTTPLRSGC
On the other hand, the invention also provides a nucleic acid sequence corresponding to the chitosanase CsnQ, which is shown as SEQ ID NO. 2.
SEQ ID NO.2:
ATGAAATACTTATTACCAACAGCAGCAGCAGGTTTATTATTATTAGCAGCACAACCAGCAATGGCAATGGGTGTAGGTGGTACAGGTGCAACACATGCAGGTGCAGCAGGTTCTGGTGTAAACTTAACAGATCCACATAAAAAAGAAATTGCAATGGAATTAGTATCTTCTGCAGAAAACTCTTCTTTAGATTGGAAAGCACAATACAAATACATTGAAGATATTGGTGATGGTCGTGGTTACACAGGTGGTATTATTGGTTTCTGTTCTGGTACAGGTGATATGTTAGAATTAGTACAACATTACACAGATTTAGAACCAGGTAACATTTTAGCAAAATACTTACCAGCATTAAAAAAAGTAAACGGTTCTGCATCTCATTCTGGTTTAGGTACACCATTCACAAAAGATTGGGCAACAGCAGCAAAAGATACAGTATTCCAACAAGCACAAAACGATGAACGTGATCGTGTATACTTCGATCCAGCAGTATCTCAAGCAAAAGCAGATGGTTTACGTGCATTAGGTCAATTCGCATACTACGATGCAATTGTAATGCATGGTCCAGGTAACGATCCAACATCTTTCGGTGGTATTCGTAAAACAGCAATGAAAAAAGCACGTACACCAGCACAAGGTGGTGATGAAACAACATACTTAAACGCATTCTTAGATGCACGTAAAGCAGCAATGTTAACAGAAGCAGCACATGATGATACATCTCGTGTAGATACAGAACAACGTGTATTCTTAAAAGCAGGTAACTTAGATTTAAACCCACCATTACATTGGAAAACATACGGTGATTCTTACTCTATTAACTCTTTAGAACATACAACAACACCATTACGTTCTGGTTGT
On the other hand, the invention also provides a preparation and purification method of the chitosanase CsnQ.
On the other hand, the invention also provides application of the chitosanase CsnQ in degrading chitosan.
On the other hand, the chitosan degrading method adopts CsnQ as chitosanase.
Preferably: the reaction temperature in the degradation condition is 0-80 ℃. The optimum reaction temperature is 60 ℃.
Preferably: the reaction pH value in the degradation condition is 4.0-9.6. The optimum reaction pH was 5.0.
Has the advantages that:
1. the chitosanase CsnQ is novel,the similarity of the amino acid sequence of a conserved region of the xylanase and the chitosanase (PDB: 1CHK) reported by the prior structure is 96.63 percent and is basically consistent, so that the xylanase keeps good catalytic activity (the specific activity reaches 690.5U/mg); the sequence difference between the chitosanase CsnQ and the chitosanase (PDB: 1CHK) reported by the prior structure is mainly shown as follows: the chitosanase CsnQ contains 287 amino acid sequences, while the chitosanase (PDB: 1CHK) contains only 238 amino acid sequences. The N end of the chitosanase CsnQ comprises a section of 39 amino acids (Met)1-Gly39) Composed of N-trunk structure, the C section of which comprises C-tail structure (His) of 9 amino acids278-Cys287). The two contain a plurality of rigid amino acids, which increases the rigidity of the three-dimensional structure of the chitosanase and is the structural basis with better stability.
2. The invention provides a method for preparing chitosanase CsnQ, namely, a gene sequence of the chitosanase CsnQ is heterologously recombined and expressed to escherichia coli by utilizing a technical method of genetic engineering, and after fermentation, the enzyme activity of a fermentation liquid is up to 1058.2U/mL, so that the chitosanase CsnQ has the potential of industrial production. The enzyme purification method is simple, and the recovery rate is up to 79.6 percent and the protein purity is up to 95 percent by one-step affinity purification by utilizing a nickel column.
3. The chitosanase CsnQ has excellent physicochemical property, the most suitable pH value is 5.0, the chitosanase CsnQ can keep stable within the range of pH4.0-6.0, the activity of 61.2 percent can still be kept after 1h of incubation at 40 ℃, and various metal ions have promotion effect on the enzyme activity of the chitosanase CsnQ. The degradation mode is internal cutting, and the main products of chitobiose and chitotriose are degraded. The chitosanase CsnQ has high yield, good stability and good industrial application potential.
Drawings
FIG. 1 shows the result of the evolutionary relationship between the sequences of chitosanase CsnQ of the present invention and known chitosanase;
FIG. 2 is a protein isolation and purification diagram of the chitosanase CsnQ of the present invention (M, protein standard; 1, sample before purification, 2, purified chitosanase CsnQ);
FIG. 3 shows the temperature and pH adaptation analysis of the chitosanase CsnQ of the present invention (A, the optimum reaction temperature of the chitosanase CsnQ; B, the temperature stability of the chitosanase CsnQ; C, the optimum reaction pH of the chitosanase CsnQ; D, the pH stability of the chitosanase CsnQ);
FIG. 4 shows the Thin Layer Chromatography (TLC) method for detecting the degradation mode and the enzymolysis products (M, chitooligosaccharide DP1-4, chitooligosaccharide 1-4 sugar standard) of the chitosanase CsnQ of the present invention;
FIG. 5 shows that anion first-order mass spectrometry is used for detecting the degradation products of the chitosanase CsnQ of the invention.
Detailed Description
Example 1 sequence analysis of chitosanase CsnQ
The enzyme producing gene csnQ of the chitosanase CsnQ is an artificial synthetic sequence. In the previous research, the marine bacterium Vibrio sp.Q108 has high chitosan enzyme activity, and when the marine bacterium Vibrio sp.Q108 is subjected to whole-gene sequencing, the marine bacterium Vibrio sp.Q108 is found to contain a predicted chitosanase sequence. Under the condition of no change of amino acid sequence, the gene sequence is optimized according to the codon preference of a host (of escherichia coli), and efficient expression of the gene sequence in the escherichia coli is facilitated. Sequence synthesis was performed at Huada Gene. The chitosanase CsnQ comprises 861 base sequences and encodes 287 amino acid sequences. Conserved domain analysis (CDD) and multiple sequence Alignment of Basic Local Alignment Search Tool (Blast) using the Conserved domain in the National Center for Biotechnology Information (NCBI) revealed that this sequence contains a Conserved region of family 46 (GH-46) of polysaccharide hydrolases. The similarity between the amino acid sequence of the CSnQ conserved region of the chitosanase and the sequence of the chitosanase (PDB: 1CHK) reported by the prior crystal structure is 96.63 percent, and the sequence is basically consistent, so that the chitosanase keeps good catalytic activity (the specific activity reaches 690.5U/mg); the sequence difference between the chitosanase CsnQ and the chitosanase (PDB: 1CHK) reported by the existing crystal structure is mainly shown as follows: the chitosanase CsnQ contains 287 amino acid sequences, while the chitosanase (PDB: 1CHK) contains only 238 amino acid sequences. The N-terminal of the chitosanase CsnQ comprises a section of 39 amino acids (Met)1-Gly39) Composed of N-trunk structure, wherein the C section of the N-trunk structure comprises a C-tail structure (His) with 9 amino acids278-Cys287) The two types of amino acids contain various rigid amino acids, so that the rigidity of the three-dimensional structure of the chitosanase is increased, and the structural basis of the chitosanase CsnQ with better stability is provided.
The chitosanase CsnQ and an amino acid sequence belonging to the same family (GH46) are subjected to Blast analysis, multiple sequence comparison is carried out by using ClustalX software, and evolutionary tree analysis is carried out by using Mega 7.1 software. As shown in figure 1, the chitosanase CsnQ has a far-away relationship with other reported chitosanases, and is a novel chitosanase.
The nucleotide sequence of the chitosanase CsnQ takes restriction enzymes Nco I and Xho I as enzyme cutting sites, and recombinant primers are designed as follows (restriction enzyme sites are underlined, and restriction enzyme protecting bases are italicized):
a forward primer: SEQ ID NO. 3: PcssnQ-F:
5’-CATGCCATGGATGAAATACTTATTACCAAC-3’(Nco I)
reverse primer: SEQ ID NO. 4: PcssnQ-R:
5’-CCGCTCGAGACAACCAGAACGTAATGGT-3’(Xho I)
the PCR amplification conditions were: pre-denaturation at 94 ℃ for 3 min; denaturation at 94 ℃ for 30 seconds, annealing at 55 ℃ for 30 seconds, and extension at 72 ℃ for 1min for 30 cycles; extending for 5min at 72 ℃; stabilizing at 4 deg.C for 15 min. The DNA polymerase used for the PCR reaction was Primerstar HS, available from Dalibao Bio.
The PCR product was double-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 also double-digested with restriction enzymes Nco I and Xho I, subjected to agarose gel electrophoresis, and the product fragment after the digestion was recovered. The enzyme and substrate reaction system (temperature, time, DNA dosage and the like) used in the enzyme digestion are operated according to the product instruction provided by the Dalianbao organism.
Performing ligation reaction on the PCR product subjected to double digestion treatment and a pET-22b (+) plasmid vector according to the instruction of DNA ligase (Dalibao biological Co., Ltd.); the ligation product was transformed into E.coli DH5 alpha strain (Invitrogen, USA), spread on Luria-Bertani (LB) medium solid plate (containing 50. mu.g/mL ampicillin), cultured in an incubator at 37 ℃ for 12-16 hours, and then single clone was picked; the single clones were transferred to LB liquid medium (containing 50. mu.g/mL ampicillin) and cultured overnight in a shaker at 37 ℃ at 180 rpm. The single clone was sequenced, and a positive clone was selected and named pET22 b-csnQ. The recombinant plasmid was transformed into E.coli BL21(DE3) (purchased from Dalibao Bio Inc.), and the recombinant Escherichia coli strain was named BL21(DE3)/pET22b-csnQ and stored at-80 ℃ for further use.
Example 2 preparation and purification methods of chitosanase CsnQ
The recombinant strain BL21(DE3)/pET22b-csnQ was shake-cultured in 100mL LB liquid medium (50. mu.g/mL ampicillin) at 180rpm in a shaker at 37 ℃ to OD6000.6, isopropyl- β -D-thiogalactoside (IPTG) was added at a final concentration of 0.1mM and induced at 20 ℃ for 20 h. The standard assay for the activity of chitosanase CsnQ was: mu.L of the enzyme solution was added to 900. mu.L of 0.3% chitosan substrate (20mM acetic acid-sodium acetate, pH 5.9), reacted at 60 ℃ for 10min, 750. mu.L of DNS reagent was added, and the mixture was reacted in boiling water for 10min to develop color, and the absorbance was measured at OD 520. Enzyme activity was defined as the amount of enzyme required to produce 1. mu.M reducing sugar per min at 1U. Through detection, the activity of the chitosan enzyme in the fermentation liquor can reach 1058.2U/mL.
After fermentation is stopped, centrifuging at 12000rpm for 10min, discarding thalli, and collecting supernatant; and (3) loading the fermentation supernatant into a 10mL nickel ion affinity chromatography column at the loading flow rate of 5mL/min, eluting by using 10mM imidazole to remove impure proteins, eluting by using 150mM imidazole, and collecting the eluted components. Dialyzing the active ingredient to remove imidazole, packaging and storing at-20 deg.C for use. Through one-step affinity purification of nickel ions, the recovery rate of protein reaches 79.6 percent.
The purified chitosanase CsnQ was subjected to polyacrylamide gel electrophoresis (SDS-PAGE), and as shown in FIG. 2, the molecular weight of the chitosanase CsnQ was 27kDa, which was consistent with the predicted protein size in the sequence analysis. Gel analysis shows that the protein purity of the purified chitosanase CsnQ reaches more than 95%.
Example 3 Effect of temperature and pH on chitosanase CsnQ
The chitosanase CsnQ purified in the example 2 is subjected to enzyme activity determination under different conditions, and the influence of different temperatures and pH values on the enzyme activity is detected. Reacting for 10min at different temperatures (0-80 ℃), detecting the influence of different reaction temperatures on the enzyme activity, and calculating the relative enzyme activity of the xylanase CsnQ at different temperatures by taking the highest enzyme activity as 100%. As shown in FIG. 3A, the optimum reaction temperature for the chitosanase CsnQ was 60 ℃.
The purified chitosanase CsnQ obtained in the example 2 is incubated for 1h at different temperatures (0-80 ℃), after being taken out, the enzyme activity of the chitosanase CsnQ is detected at the optimal reaction temperature (60 ℃), the activity before incubation is taken as 100%, as shown in figure 3B, the chitosanase CsnQ has good temperature stability, and the enzyme activity can reach 61.2% before incubation after incubation for 1h at 40 ℃.
The activity of the purified chitosanase CsnQ obtained in example 2 was measured in three different buffers (50mM, Sodium acetate buffer, Phosphate buffer, Gly-NaOH buffer) at different pH values, and the highest value of the enzyme activity was 100%. As shown in FIG. 3C, the optimum reaction pH for the chitosanase CsnQ was 5.0.
The chitosanase CsnQ purified in example 2 is incubated for 24h under different pH conditions, and immediately after being taken out, the enzyme activity of the chitosanase CsnQ is detected under the optimum reaction pH (5.0), and the activity before incubation is taken as 100%, as shown in FIG. 3D, the activity of the chitosanase CsnQ can be maintained more than 80% within the range of pH 4.0-6.0.
Example 4 Effect of different Metal ions on chitosanase CsnQ
Adding 5mM of different metal ions into the purified chitosanase CsnQ obtained in the example 2 in the enzyme activity determination process, incubating for 24h, detecting the influence of the different metal ions on the activity of the chitosanase CsnQ under the standard activity determination condition of the chitosanase CsnQ, taking an enzyme solution without the added metal ions as a reference, and as shown in table 1, a plurality of metal ions have certain promotion effect on the activity of the chitosanase CsnQ.
TABLE 1 Effect of different Metal ions on the enzymatic Activity of the chitosanase CsnQ of the present invention
Ion(s)
|
Concentration (mM)
|
Relative enzyme activity (%)
|
None
|
--
|
100
|
Aluminum ion
|
5
|
48±10.1
|
Ferric ion
|
5
|
88±2.6
|
Potassium ion
|
5
|
101±4.3
|
Sodium ion
|
5
|
104±3.9
|
Magnesium ion
|
5
|
106±0.7
|
Lithium ion
|
5
|
106±3.3
|
Ammonium ion
|
5
|
107±3.1
|
Ferrous iron ion
|
5
|
108±2.6
|
Copper ion
|
5
|
108±3.3
|
Barium ion
|
5
|
109±4.2
|
Cobalt ion
|
5
|
110±2.4
|
Calcium ion
|
5
|
112±3.4
|
Zinc ion
|
5
|
114±3.8
|
Manganese ion
|
5
|
121±3.1 |
Example 5 thin layer chromatography analysis of chitosanase CsnQ enzymatic hydrolysate
The purified chitosanase CsnQ of example 2 was incubated with 0.3% chitosan at 60 ℃ for different periods of time, and then detected on a high performance thin layer chromatography plate (HPTLC). The method specifically comprises the following steps: cutting the HPTLC chromatographic plate into samples with the width of 7cm and the proper size, spotting the samples before and after incubation at the origin, placing the samples in a developing tank with a developing agent (n-butyl alcohol: glacial acetic acid: water: 2:1) for 30min, drying the chromatographic plate, immersing the plate in a color developing agent (0.5% ninhydrin ethanol solution) for 2s, taking out the plate, drying the plate, and baking the plate at 80 ℃ until the samples appear. As shown in figure 4, compared with the standard product migration rate, the chitosanase CsnQ enzymolysis main product is chitobiose (DP2) and chitotriose (DP 3).
As shown in FIG. 4, in the initial stage of the enzymatic hydrolysis (0-10min), a large amount of large-fragment oligosaccharides are produced, and as the enzymatic hydrolysis proceeds, the large-fragment oligosaccharides are gradually converted into small-fragment oligosaccharides, which indicates that the mode of action of the enzyme is to cut from the interior of chitosan molecules in an endo-mode.
Example 6 chitosanase Csn Q enzymatic hydrolysate Mass Spectrometry assay
The purified chitosanase CsnQ of example 2 was incubated with 0.3% chitosan substrate for 24 hours, the degradation product was centrifuged at 12,000g for 10 minutes, and the pellet was discarded. The obtained sample is mixed with acetonitrile in equal volume,
and (4) carrying out primary anion mass spectrum detection to determine the molecular weight of the enzymolysis product. The result of anion mode primary mass spectrometry shows (fig. 5) that the main degradation products are chitobiose and chitotriose, which is consistent with the TLC detection result in example 5.
Sequence listing
<110> Shandong Hao Yue medicine science and technology Co., Ltd
<120> novel chitosanase CsnQ and application thereof
<160>4
<170>SIPOSequenceListing 1.0
<210>1
<211>287
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>1
Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala
1 5 10 15
Ala Gln Pro Ala Met Ala Met Gly Val Gly Gly Thr Gly Ala Thr His
20 25 30
Ala Gly Ala Ala Gly Ser Gly Val Asn Leu Thr Asp Pro His Lys Lys
35 40 45
Glu Ile Ala Met Glu Leu Val Ser Ser Ala Glu Asn Ser Ser Leu Asp
50 55 60
Trp Lys Ala Gln Tyr Lys Tyr Ile Glu Asp Ile Gly Asp Gly Arg Gly
65 70 75 80
Tyr Thr Gly Gly Ile Ile Gly Phe Cys Ser Gly Thr Gly Asp Met Leu
85 90 95
Glu Leu Val Gln His Tyr Thr Asp Leu Glu Pro Gly Asn Ile Leu Ala
100 105 110
Lys Tyr Leu Pro Ala Leu Lys Lys Val Asn Gly Ser Ala Ser His Ser
115 120 125
Gly Leu Gly Thr Pro Phe Thr Lys Asp Trp Ala Thr Ala Ala Lys Asp
130 135 140
Thr Val Phe Gln Gln Ala Gln Asn Asp Glu Arg Asp Arg Val Tyr Phe
145 150 155 160
Asp Pro Ala Val Ser Gln Ala Lys Ala Asp Gly Leu Arg Ala Leu Gly
165 170 175
Gln Phe Ala Tyr Tyr Asp Ala Ile Val Met His Gly Pro Gly Asn Asp
180 185 190
Pro Thr Ser Phe Gly Gly Ile Arg Lys Thr Ala Met Lys Lys Ala Arg
195 200 205
Thr Pro Ala Gln Gly Gly Asp Glu Thr Thr Tyr Leu Asn Ala Phe Leu
210 215 220
Asp Ala Arg Lys Ala Ala Met Leu Thr Glu Ala Ala His Asp Asp Thr
225 230 235 240
Ser Arg Val Asp Thr Glu Gln Arg Val Phe Leu Lys Ala Gly Asn Leu
245 250 255
Asp Leu Asn Pro Pro Leu His Trp Lys Thr Tyr Gly Asp Ser Tyr Ser
260 265 270
Ile Asn Ser Leu Glu His Thr Thr Thr Pro Leu Arg Ser Gly Cys
275 280 285
<210>2
<211>861
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>2
atgaaatact tattaccaac agcagcagca ggtttattat tattagcagc acaaccagca 60
atggcaatgg gtgtaggtgg tacaggtgca acacatgcag gtgcagcagg ttctggtgta 120
aacttaacag atccacataa aaaagaaatt gcaatggaat tagtatcttc tgcagaaaac 180
tcttctttag attggaaagc acaatacaaa tacattgaag atattggtga tggtcgtggt 240
tacacaggtg gtattattgg tttctgttct ggtacaggtg atatgttaga attagtacaa 300
cattacacag atttagaacc aggtaacatt ttagcaaaat acttaccagc attaaaaaaa 360
gtaaacggtt ctgcatctca ttctggttta ggtacaccat tcacaaaaga ttgggcaaca 420
gcagcaaaag atacagtatt ccaacaagca caaaacgatg aacgtgatcg tgtatacttc 480
gatccagcag tatctcaagc aaaagcagat ggtttacgtg cattaggtca attcgcatac 540
tacgatgcaa ttgtaatgca tggtccaggt aacgatccaa catctttcgg tggtattcgt 600
aaaacagcaa tgaaaaaagc acgtacacca gcacaaggtg gtgatgaaac aacatactta 660
aacgcattct tagatgcacg taaagcagca atgttaacag aagcagcaca tgatgataca 720
tctcgtgtag atacagaaca acgtgtattc ttaaaagcag gtaacttaga tttaaaccca 780
ccattacatt ggaaaacata cggtgattct tactctatta actctttaga acatacaaca 840
acaccattac gttctggttg t 861
<210>3
<211>30
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>3
catgccatgg atgaaatact tattaccaac 30
<210>4
<211>28
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>4
ccgctcgaga caaccagaac gtaatggt 28