CN114292835B - Chitin deacetylase, encoding gene, preparation method and application - Google Patents

Abstract

The invention relates to the technical field of bioengineering, in particular to chitin deacetylase, a coding gene, a preparation method and application thereof, and provides a novel chitin deacetylase, the amino acid sequence of which is shown as SEQ ID NO.1, and the coding gene and an expression method thereof, and further finds the application thereof in preparing chitosan or nanofiber by a bioenzyme method. The chitin deacetylase of the invention catalyzes the extremely high enzymatic activity of long-chain chitin.

Description

Chitin deacetylase, encoding gene, preparation method and application

Technical Field

The invention relates to the technical field of bioengineering, in particular to chitin deacetylase, a coding gene, a preparation method and application.

Background

Chitin is a renewable resource next to cellulose, the second largest natural macromolecule. The high degree of polymerization and strong intramolecular and intermolecular hydrogen bonding of natural crystalline chitin render it insoluble in common solvents, greatly limiting its development and commercial use.

Chitin deacetylase (Chitin deacetylase, CDA, EC 3.5.1.41) can catalyze chitin deacetylation to prepare chitosan and chitin nanofiber with high added value and large market demand, and the chitosan and chitin nanofiber have the characteristics of safety, no toxicity, biodegradability, good biocompatibility and the like, and are widely applied to the fields of medicine, cosmetics, bioremediation and the like. Compared with the traditional physical method and chemical method for preparing chitosan and chitin nanofiber, the chitosan prepared by CDA has the advantages of large molecular weight, narrow molecular distribution range, consistent deacetylation position, thorough deacetylation degree and the like, the mechanical properties, nanofiber length, width and the like of the prepared chitin nanofiber are remarkably improved, and the CDA enzymatic production process has the advantages of safety, controllability, greenness, high efficiency and the like. However, only a few of the CDAs reported at present have lower catalytic activity on crystalline chitin, and the requirements of the CDA on high-activity catalytic crystalline chitin CDA are not met, so that the CDA becomes a technical bottleneck for industrially preparing high-added-value products such as chitosan, chitin nanofibers and the like.

Disclosure of Invention

The invention aims to provide a novel chitin deacetylase which has higher enzyme activity so as to meet the industrial requirement.

The invention adopts the following technical scheme:

a chitin deacetylase has an amino acid sequence shown in SEQ ID NO. 1.

A gene encoding the aforementioned chitin deacetylase.

Preferably, the coding gene sequence is shown as SEQ ID NO. 2.

The preparation method of the chitin deacetylase comprises the following steps: cloning the coding gene of any one of the above to an expression vector pET-28a to obtain a recombinant vector PET-28a-AsCDA, transferring the recombinant vector PET-28a-AsCDA into BL21 escherichia coli competent cells, culturing the escherichia coli until the OD600 is 0.55-0.65, and performing IPTG induced expression to obtain the recombinant vector.

Preferably, the IPTG induction concentration is 0.35 mM-0.45 mM, and the BL21 E.coli culture temperature is 36-38 ℃.

The use of the chitin deacetylase described above in deacetylation of acetyl-containing polysaccharides.

Preferably, the acetyl-containing polysaccharide is selected from the group consisting of long chain chitins.

Preferably, the long chain chitin is selected from the group consisting of alpha-chitin, beta-chitin, colloidal chitin and gamma-chitin.

The application of the chitin deacetylase in preparing chitosan or nanofiber by using a chitin biological enzyme method.

Preferably, the pH optimum for the application is between 6 and 9, and the temperature optimum is between 25 and 35 ℃.

Preferably, the application optimum pH is 7 and the optimum temperature is 30 ℃.

Preferably, in the application, one or more of the following metal ions are added to promote the reactionThe method comprises the following steps: zn (zinc) ²⁺ 、Mg ²⁺ 、Ca ²⁺ 、Ni ⁺ 、 Mn ²⁺ 、Ag ⁺ 、Sr ²⁺ 、Fe ³⁺ 。

Preferably, the final concentration of the metal ions is 0.5-1.5 mmol/L.

Preferably, the final concentration of the metal ions is 1mmol/L.

Advantageous effects

The Acinetobacter shen chitin deacetylase AsCDA recombinase prepared by the invention has higher chitin deacetylase activity, in particular to the chitosan prepared by catalyzing acetyl of chitin in nature, wherein the specific enzyme activity 478.96U/mg of the specific enzyme activity catalyzing long-chain chitin is the highest activity published in the prior art.

The chitin deacetylase AsCDA can meet the requirement of preparing chitosan by enzymatic catalysis in industry, can be widely applied to the aspects of food science, biological medicine, chemical materials and the like, and has good application prospect.

Drawings

Fig. 1: electrophoresis patterns of related gene fragments.

Fig. 2: control group and related supernatant.

Fig. 3: enzyme activity assay of recombinant AsCDA.

Fig. 4: enzymatic characterization of recombinant AsCDA.

Fig. 5: recombinant AsCDA catalyzed long chain chitin deacetylation electron microscopy scans.

Fig. 6: and (3) measuring the deacetylation infrared spectrogram of the recombinant AsCDA catalytic long-chain chitin.

Detailed Description

The technical solutions of the present invention will be clearly and completely described in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments. The following description of at least one exemplary embodiment is merely exemplary in nature and is in no way intended to limit the invention, its application, or uses. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Term definition

Long chain chitin: the long-chain chitin in the present invention means (C) ₈ H ₁₃ NO ₅ ) _n N is greater than 20. It includes natural chitin, colloidal chitin, etc., such as alpha-chitin, beta-chitin, colloidal chitin, or gamma-chitin, etc., as referred to herein. Natural chitin has a high degree of polymerization, a compact and ordered crystalline structure and strong intramolecular and intermolecular hydrogen bonds, which make it insoluble in common solvents, and is also called crystalline chitin.

By looking up the literature, we found that none of the other chitin deacetylases were high in deacetylation capacity, as shown in the following table.

TABLE 1 enzymatic Properties of CDA with catalytic Crystal chitin deacetylation Activity

1.Z.Liu,L.M.Gay,T.R.Tuveng,J.W.Agger,B.Westereng,G.Mathiesen,S.J.Horn,G. Vaaje-Kolstad,D.van Aalten and V.Eijsink,Sci Rep,2017,7,1746.

2.X.Y.Zhu,Y.Zhao,H.D.Zhang,W.X.Wang,H.H.Cong and H.Yin,MAR DRUGS, 2019,17.

3.T.R.Tuveng,U.Rothweiler,G.Udatha,G.Vaaje-Kolstad,A.Smalas and V.Eijsink, PLOS ONE,2017,12,e187544.

4.J.Cai,J.Yang,Y.Du,L.Fan,Y.Qiu,J.Li and J.F.Kennedy,CARBOHYD POLYM, 2006,65,211-217.

5.B.Shrestha,K.Blondeau,W.F.Stevens and F.L.Hegarat,Protein Expr Purif,2004,38, 196-204.

6.J.Chai,J.Hang,C.Zhang,J.Yang,S.Wang,S.Liu and Y.Fang,INT J BIOL MACROMOL,2020,152,922-929.

The AsCDA consists of 324 amino acids, and the amino acid sequence of the AsCDA is as follows (SEQ ID NO. 1):

VTTAYLRGKQLIETIQKQEYSRDLIGYHGKPPHAQWPNAARIAVQFVLNYEEGGENHV EHGDSSSEQFLSEIVGAASFPAVHRSMDSMYEYGSRAGFWRIHEEFQKRGWPMTIFGVGM ALARNPYIVEAIKAADYDVVSHGQRWLHYQDMEIETERQHMDQALSVLTELFGETPIGWY TGRDSPNTRQLLAEFSQIKYDSDYYGDDLPFWSTLTEPNGQKRPHLIIPYTLECNDMKFSSP GGFNSGEQFYQYLKDAFDVLYSEGETAPKMMSIGMHCRLLGRPGRFKALQRFMDYVQSH DKVWICCRNDIAQHWYTHHFPAHQD

the coding gene of the AsCDA consists of 975 bases, and the base sequence of the coding gene is as follows (SEQ ID NO. 2):

GTGACAACTGCTTACTTACGCGGAAAACAATTGATTGAAACGATACAAAA GCAAGAATATTCACGCGATTTAATCGGCTATCACGGCAAACCACCCCATG CACAGTGGCCCAATGCTGCCCGGATCGCGGTACAGTTTGTATTGAACTAT GAAGAAGGCGGTGAAAATCATGTCGAACATGGTGATAGCAGTTCCGAGCA GTTTCTGTCCGAAATCGTGGGTGCTGCCAGTTTTCCAGCAGTACACCGCT CTATGGATTCAATGTATGAATATGGTTCACGTGCTGGCTTCTGGCGTATT CATGAGGAGTTTCAAAAACGTGGCTGGCCTATGACAATTTTTGGAGTCGG TATGGCACTGGCTCGTAATCCTTATATCGTCGAGGCCATTAAGGCGGCTG ATTATGATGTGGTCTCCCATGGTCAGCGCTGGTTGCATTATCAGGATATG GAGATTGAAACTGAACGTCAGCATATGGATCAGGCCTTAAGTGTGCTGAC TGAACTCTTTGGTGAAACGCCAATTGGCTGGTATACCGGACGCGACAGTC CCAATACACGTCAATTATTAGCTGAATTTTCTCAGATTAAATATGACTCT GATTATTATGGTGATGACCTCCCATTTTGGAGCACCTTAACTGAACCAAA TGGTCAAAAACGCCCTCATTTGATCATCCCCTATACATTAGAGTGCAATG ATATGAAGTTTAGCTCTCCGGGTGGTTTCAATTCAGGTGAGCAATTTTAC CAATATTTAAAAGATGCCTTCGATGTGCTCTACAGTGAAGGTGAAACTGC ACCAAAAATGATGTCAATTGGCATGCACTGTCGCCTGCTCGGACGTCCGG GACGCTTCAAAGCCTTGCAGCGCTTTATGGATTACGTACAGAGCCATGAC AAAGTATGGATCTGTTGTCGTAACGATATTGCACAACATTGGTACACCCA CCATTTTCCTGCGCACCAGGACTAA

1. the preparation method of the chitin deacetylase is as follows

(1) Genome extraction: extracting Acinetobacter schel MCDA01 (Acinetobacter schindleri MCDA 01) by using a bacterial genome extraction kit (Shanghai biological Co., ltd.) and submitting the strain to a national center for collection of bacterial strains, wherein the collection number of the strain is CGMCC NO.13539, and reserving the strain at-80 ℃;

(2) Primer design: different primers AsCDA1-F (enzyme cleavage site underlined as BamHI) were designed based on the gene sequence of chitin deacetylase AsCDA and the purpose of the experiment:GGATCCACAACTGCTTACTTACGCGGA) andAsCDA1-R (HindIII underlined cleavage site:AAGCCTGTCCTGGTGCGCAGGAAAATG) primers were synthesized by Shanghai Biotechnology Co., ltd;

(3) Amplification of the target Gene: the genome of the gentleman acinetobacter is used as a template, and ASCDA1-F/R is used for PCP amplification, and the reaction system is as follows:

PCR amplification conditions: pre-denaturation at 94℃for 3min; denaturation at 94℃for 30s, annealing at 60℃for 30s, extension at 72℃for 2min for a total of 30 cycles; extending at 72 ℃ for 5min;

(4) And (3) sequencing and verifying a target gene sequence: detecting PCR amplified products by agarose gel electrophoresis, recovering and purifying target bands by a DNA recovery kit, and respectively inserting the target bands into pGM-simple-T fast vectors, wherein a connection system is as follows:

e.coli DH 5. Alpha. Transformation was performed after 30min of water bath ligation at 22 ℃.

White colonies were picked according to the blue-white screening principle, inoculated into LB liquid medium containing 100. Mu.g/mL Amp, and cultured at 37℃and 180rpm for 6-8 hours. Colony PCR verification is carried out by using a universal primer T7/SP6, and the PCR reaction system is as follows:

PCR amplification conditions: pre-denaturation at 94℃for 3min; denaturation at 94℃for 30s, annealing at 60℃for 30s, extension at 72℃for 2min for a total of 25 cycles; extending at 72℃for 5min.

(5) Recombinant vector construction: and (3) preserving the bacterial liquid of the obtained positive transformant, and extracting plasmids for later use by using a GeneMark plasmid small extraction kit. According to the designed enzyme cutting site, the extracted plasmid and pET-28a empty vector are subjected to enzyme cutting, and the enzyme cutting system is as follows:

double digestion with BamHI and HindIII at 37℃for 2h, and recovery of the desired fragment by gel followed by T ₄ The DNA ligase connects the two, and the connection system is as follows:

e.coli DH5 alpha conversion is carried out after water bath connection for 4 hours at the temperature of 22 ℃, positive transformants are screened for amplification, and plasmids are extracted to obtain recombinant vector pET-28a-AsCDA.

(6) Recombinant strain construction: to a tube of 100. Mu.l BL21 E.coli competent cells was added 0.2ng (1. Mu.l) of recombinant plasmid pET-28a-AsCDA, mixed with gentle shaking, and the tube was then reset in ice; ice bath for 5 minutes; water bath at 42 ℃ for 30 seconds, and can not shake; placing on ice for 2 minutes; then adding 800 μl of liquid LB, and incubating at 37deg.C; 5-50. Mu.l of the transformation product was spread on LB plates with the appropriate antibiotics.

In the above steps, PCR amplification verification was performed on the AsCDA gene fragment (i.e., the target gene amplified in step (3)), the BamHI and HindIII double cleavage products of pET-28a-AsCDA, and the recombinant strain obtained in step (6), and the results are shown in FIG. 1, in FIG. 1:

a: clone electrophoresis diagram of AsCDA gene fragment, 1 is target fragment, M is D2000 DNA ladder Marker, and from top to bottom is 2Kb, 1Kb, 750bp, 500bp, 250bp and 100bp in sequence;

b: pET-28a double enzyme digestion electrophoresis diagram, 1 is pET-28a double enzyme digestion fragment, M is D2000 DNA ladder Marker, and 2Kb, 1Kb, 750bp, 500bp, 250bp and 100bp are arranged from top to bottom in sequence;

c: verification electrophoresis patterns of recombinant strains, wherein 1, 2, 3 and 4 are positive transformants respectively, M is D2000 DNA ladder Marker, and 2Kb, 1Kb, 750bp, 500bp, 250bp and 100bp are arranged from top to bottom.

As can be seen from FIG. 1, the recombinant vector pET-28a-AsCDA was constructed successfully and has been introduced successfully into E.coli BL21, and can be used for the following experiments.

(7) Induction expression of recombinant strains: and (3) carrying out PCR identification on the grown recombinant strain (as in step (5)), and culturing the identified recombinant strain to a certain extent and then carrying out IPTG induction expression. The method comprises the following steps:

shaking the recombinant strain at 37℃to an OD600 of about 0.6, which is reached after about 3 hours; some samples were taken as uninduced control groups; adding 100mM IPTG to the rest sample to a final concentration of 0.4mM, and culturing for 2-3 hours; placing the shake flask on ice for 5 minutes, centrifuging at 8000rpm/min and 4 ℃ for 5 minutes, and collecting thalli; the cells were resuspended in 0.25 volumes of pre-chilled 20mM Tris-HCl (pH 8.0) and centrifuged; separating the supernatant and the thalli, and storing the thalli in a refrigerator at the temperature of-70 ℃ or continuing purifying.

(8) Nickel column purification of recombinase: washing the thalli obtained in the step (7) for 2-3 times by using a balance buffer solution, carrying out vacuum cell disruption after the volume of the washed thalli reaches 5mL, centrifuging the disruption solution at 12000rpm/min and 4 ℃ for 10 minutes, collecting supernatant (intracellular disruption supernatant) and sediment (inclusion body protein), and refrigerating at 4 ℃ for standby. The resin nickel column was removed, washed with 10mL equilibration buffer added horizontally, 10mL of AsCDA crude enzyme solution (i.e., intracellular disruption supernatant) was added to the nickel column, and the nickel column was slowly inverted at 4 ℃ for 1 hour to allow the enzyme to bind to the nickel column. The column was placed vertically, the resin was allowed to settle, the bottom screen was opened, the fraction (i.e., bound liquid) was collected, the nickel column was washed with 10mL of equilibration buffer, 10mL of wash buffer was added, the washed effluent liquid was collected, 10mL of elution buffer was added, the eluate was collected, about 1mL of eluate per tube, and SDS-PAGE protein electrophoresis verification was performed on the collected liquid.

The balancing buffer solution comprises the following components: 20mM phosphate buffer; 500Mm NaCl;20mM imidazole; ph7.4;

the washing buffer is: 20mM phosphate buffer; 500Mm NaCl;100mM imidazole; ph7.4;

the elution buffer was: 20mM phosphate buffer; 500Mm NaCl;280mM imidazole; pH7.4.

(9) SDS-PAGE protein electrophoresis verification: taking and centrifuging, respectively adding 5 mu L of 5 Xprotein loading buffer solution, crude enzyme solution, purifying enzyme solution, shaking and mixing uniformly, and then carrying out boiling water bath for 10min. The gel plate was placed in an electrophoresis tank, SDS-PAGE running buffer was added, and samples were added to the spotting wells, respectively, and 3. Mu.L of protein Marker was added. The initial voltage is 90V, after the strip crosses the separation line between the separation glue and the concentrated glue, the voltage is adjusted to 120V, and after the strip reaches the bottom of the glue plate, the power supply is turned off. And opening the glue plate after glue running, cutting out glue, dyeing with protein dyeing liquid at 40 ℃ for 1 hour, recovering the dyeing liquid and washing off residual dyeing liquid on the surface. Adding the dyed glue into a decoloring solution, decoloring in a shaker at 60rpm, changing the decoloring solution every 1 hour, stopping decoloring until the strip is clearly visible, placing the decolored glue into an imager, and photographing. The results are shown in FIG. 2. The upper symbols in fig. 2 have the following meanings:

m: standard molecular weight proteins; 1: a control group; 2: the final supernatant obtained in step (7) of the above example; 3: the inclusion body protein obtained in the step (8) of the above example; 4: the intracellular disruption supernatant obtained in step (8) of the above example; 5: the combined solution of step (8) of the above-mentioned embodiment; 6: the effluent after washing with 100mM imidazole washing solution of step (8) of the above example; 7: the effluent from the 280mM imidazole eluent wash of step (8) of the above example.

And when the control group is recombinant strain fermentation expression, no IPTG is added, other fermentation induction conditions are the same, and finally, SDS-PAGE protein electrophoresis detection is carried out on the expressed fermentation broth (containing thalli).

As can be seen from FIG. 2, asCDA was successfully expressed in E.coli BL21, recombinant protein was not detected in the fermentation supernatant, a small amount of recombinant protein was found in inclusion bodies, and most of recombinant protein was soluble expressed in cytoplasm, and was purified by Ni-IDA-affinity chromatography to form a single band with a size of about 38.7kDa.

2. Measurement of enzyme Activity

The determination of the acetic acid content to characterize the enzymatic activity of chitin deacetylase was performed using the method of Megazyme acetate Kit K-ACETRM Kit, the specific assay method is as follows.

The preparation method of the colloidal chitin in the following examples is as follows: 15g of alpha-chitin was added to a 150mL beaker, 100mL of concentrated HCl was slowly poured into an ice-water bath and gently stirred to a paste with a glass rod, the beaker was sealed with a preservative film and placed in a refrigerator at 4℃overnight. The overnight mixture was added to 500mL of 95% glacial ethanol, after stirring thoroughly, sealed with a preservative film, and after standing overnight at room temperature, the mixture was centrifuged at 4℃for 20min at 5,000r/min, and the precipitate was taken. Repeatedly washing the precipitate with distilled water until the pH is neutral, and adding 300mL distilled water to obtain colloidal chitin. Sealing the prepared colloid chitin with a preservative film, and storing in a refrigerator at 4 ℃ for standby.

Measurement of enzyme Activity:

(1) Drawing an acetic acid standard curve:

according to the acetic acid detection Kit K-ACETRM Kit (available from Megazyme company), 0.1mg/ml acetic acid standard solution was diluted with sterile water to different concentrations (1, 1.5, 2, 2.5, 3, 4, 5, 10), a standard curve was prepared, distilled water was used as a blank, and acetic acid standard solution A of different concentrations was measured ₃₄₀ Values, with acetic acid standard solutions of different concentrations as abscissa, A ₃₄₀ Values are plotted as ordinate, and a standard curve is plotted.

(2) Measurement of acetic acid and enzyme activity calculation:

the enzyme activity of recombinant chitin deacetylase is characterized by detecting acetic acid which is another product of the catalytic reaction, wherein a 100-mu L reaction system comprises 50 mu L of enzyme solution, 50 mu L of chitin substrate (colloidal chitin, alpha-chitin and beta-chitin) and 100 mu L of 0.05mol/L pH 7.0 phosphate buffer solution, after the reaction is uniformly mixed, the reaction is incubated for 30min at 30 ℃, the reaction is boiled for 1min at 100 ℃, the reaction is stopped, 12000g of the reaction is centrifuged for 5min, the supernatant is collected, the amount of acetic acid in the supernatant is measured, the enzyme activity is calculated, and detailed steps are shown in the specification of an acetic acid detection kit.

Definition of enzyme activity: the amount of acetic acid produced per minute was characterized by 1. Mu. Mol of enzyme in. Mu. Mol/min/. Mu. Mol.

As shown in FIG. 3, the results of the enzyme activities of AsCDA are shown, and it can be seen from the graph that the specific enzyme activities of AsCDA catalytic colloid chitin, alpha-chitin and beta-chitin are 478.96U/mg,397.07U/mg and 133.27U/mg, respectively.

3. Enzymatic properties:

(1) Determination of optimum temperature

Selecting 20 ℃,25 ℃,30 ℃,35 ℃,40 ℃,45 ℃ and 50 ℃ as reaction temperatures, wherein the pH is 7, and the purified recombinant AsCDA enzyme solution and the alpha-chitin with the mass fraction of 1% react for 20min according to the volume ratio of 1:1, so as to determine the enzyme activity. The relative enzyme activities were determined by incubating for 2 hours under the above conditions, with the highest enzyme activity obtained at 30℃being 100%.

(2) Determination of optimum pH

Preparing an alpha-chitin solution with the mass fraction of 1% by using a buffer solution, respectively adjusting the pH values to 3.0,4.0,5.0,6.0,7.0 and 8.0,9.0, and mixing the alpha-chitin solution and a crude enzyme solution according to the volume ratio of 1:1 at 30 ℃ to determine the enzyme activity. The respective incubation was carried out at the above pH for 2 hours, and the relative enzyme activities were measured with the highest enzyme activity at pH7 being 100%.

(3) Influence of Metal ions on recombinant AsCDA enzyme Activity

Respectively select Ba ²⁺ ，Mn ²⁺ ，Co ²⁺ ，Ni ²⁺ ，Na ⁺ ，Mg ²⁺ ，K ⁺ ，Fe ²⁺ ，Ca ²⁺ ，Cu ²⁺ ，Zn ²⁺ Preparing 20mmol/L solution by plasma, adding crude enzyme solution and 1% alpha-chitin solution according to the volume ratio of 1:1, respectively enabling the final concentration of metal ions to reach 1mmol/L and 5mmol/L, and reacting for 20min at 30 ℃, and determining the enzyme activity of a reaction system. A blank group without metal ions was also set.

As shown in FIG. 4, the results of the above reaction measurement are shown in the figure, and it is clear that the preferable conditions for the enzyme activity are the pH of 6 to 9, the optimal temperature of 25 to 35℃and the more preferable conditions are the pH of 7 and the temperature of 30 ℃. As can be seen from FIG. 4C, zn has a low concentration ²⁺ 、Mg ²⁺ 、Ca ²⁺ 、Ni ⁺ 、Mn ²⁺ 、Ag ⁺ 、Sr ²⁺ 、Fe ³⁺ Has promoting effect on the enzyme activity of the invention, in particular to adding Ni ⁺ The subsequent increase in enzyme activity was 2.39 times higher, which was not expected by the person skilled in the art.

4. Deacetylation electron microscope observation of recombinant AsCDA catalyzed chitin

The morphology of the samples was observed using a scanning electron microscope.

Mixing 50mL of crude enzyme solution with 50mmol/L PBS buffer solution with pH of 7.0 at 30deg.C, and treating with enzyme solution after 12 hrαChitin was centrifuged at 10000rpm/min for 10min and dried to constant weight in an oven at 100 ℃. And fixing the samples to be detected on the metal sample table by using conductive adhesive respectively, spraying a metal film on the samples to be detected, and observing the samples.

As a result, FIG. 5 shows a scanning image of chitin before enzyme solution treatment, FIG. 5 shows a scanning image of chitin after enzyme solution treatment of AsCDA (AsCDA-Ch), and FIG. 5 shows a scanning image of chitosan.

As can be seen from the figure, the chitin is a large-particle compact particle, a layered structure, and compact, coarse and compact crystalline microfibers are closely arranged on the surface; the whole structure of chitin treated by AsCDA is severely damaged, holes and compact cracks appear on the surface of AsCDA-Ch fiber, the fiber becomes blurred, and interface separation is not obvious. The granular state of AsCDA-Ch is similar to that of chitosan, and is obviously different from that of chitin. The results indicate that the microstructure of chitin after treatment with AsCDA is altered due to reduced acetyl content, resulting in destruction or weakening of the intramolecular and molecular structure.

5. Deacetylation detection of recombinant AsCDA-catalyzed chitin

Potassium bromide was dried to constant at 100 ℃. And (3) putting a certain amount of dried potassium bromide into a mortar, fully grinding, then putting a recombinant AsCDA treated alpha-chitin sample (AsCDA-Ch), and continuously grinding, wherein the mass ratio of the potassium bromide to the sample is about 100/1-50/2. The well-ground mixture is then compressed into tablets with a tablet press and measured at 4 to 4000cm with an infrared spectrometer ^-1 Is analyzed for wavenumber regions. By the same methodThe deacetylation degree of the chitin and chitosan is determined.

The results are shown in FIG. 6, wherein FIG. 6A shows a deacetylation infrared spectrum, and FIG. 6B shows quantitative data of deacetylation degree, wherein the chitosan, the AsCDA enzymatic hydrolysate and the alpha-chitin are sequentially arranged from top to bottom.

As can be seen from the figure, 3450.13cm ^-1 Multiple absorption peaks are formed, and the stretching vibration peak of-OH and stretching vibration peak of-NH are overlapped to widen 1630cm ^-1 The position is provided with a stronger amide I characteristic peak; 1630cm ^-1 Absorption peak at 3450cm ^-1 Ratio of absorption peaks (A ₁₆₃₀ /A ₃₄₅₀ ) Linear to the deacetylation degree of chitin, so it is commonly used to calculate the deacetylation degree of samples. The deacetylation degree of the crystalline chitin and the AsCDA treated crystalline chitin and the commercial chitosan were 25.44%,88.49% and 97.54%, respectively. The infrared spectrum measurement result shows that AsCDA can catalyze the deacetylation degree of crystalline chitin to be reduced by 63.05 percent.

The foregoing description of the preferred embodiments of the invention is not intended to limit the invention to the precise form disclosed, and any such modifications, equivalents, and alternatives falling within the spirit and scope of the invention are intended to be included within the scope of the invention.

SEQUENCE LISTING

<110> university of Jiangsu ocean

<120> chitin deacetylase, coding gene, preparation method and application

<130> 20211224

<160> 2

<170> PatentIn version 3.3

<210> 1

<211> 324

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 1

Val Thr Thr Ala Tyr Leu Arg Gly Lys Gln Leu Ile Glu Thr Ile Gln

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Lys Gln Glu Tyr Ser Arg Asp Leu Ile Gly Tyr His Gly Lys Pro Pro

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His Ala Gln Trp Pro Asn Ala Ala Arg Ile Ala Val Gln Phe Val Leu

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Asn Tyr Glu Glu Gly Gly Glu Asn His Val Glu His Gly Asp Ser Ser

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Ser Glu Gln Phe Leu Ser Glu Ile Val Gly Ala Ala Ser Phe Pro Ala

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Val His Arg Ser Met Asp Ser Met Tyr Glu Tyr Gly Ser Arg Ala Gly

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Phe Trp Arg Ile His Glu Glu Phe Gln Lys Arg Gly Trp Pro Met Thr

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Ile Phe Gly Val Gly Met Ala Leu Ala Arg Asn Pro Tyr Ile Val Glu

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Ala Ile Lys Ala Ala Asp Tyr Asp Val Val Ser His Gly Gln Arg Trp

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Leu His Tyr Gln Asp Met Glu Ile Glu Thr Glu Arg Gln His Met Asp

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Gln Ala Leu Ser Val Leu Thr Glu Leu Phe Gly Glu Thr Pro Ile Gly

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Trp Tyr Thr Gly Arg Asp Ser Pro Asn Thr Arg Gln Leu Leu Ala Glu

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Gly Gly Phe Asn Ser Gly Glu Gln Phe Tyr Gln Tyr Leu Lys Asp Ala

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Phe Asp Val Leu Tyr Ser Glu Gly Glu Thr Ala Pro Lys Met Met Ser

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Ile Gly Met His Cys Arg Leu Leu Gly Arg Pro Gly Arg Phe Lys Ala

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Leu Gln Arg Phe Met Asp Tyr Val Gln Ser His Asp Lys Val Trp Ile

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Cys Cys Arg Asn Asp Ile Ala Gln His Trp Tyr Thr His His Phe Pro

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gtgacaactg cttacttacg cggaaaacaa ttgattgaaa cgatacaaaa gcaagaatat 60

tcacgcgatt taatcggcta tcacggcaaa ccaccccatg cacagtggcc caatgctgcc 120

cggatcgcgg tacagtttgt attgaactat gaagaaggcg gtgaaaatca tgtcgaacat 180

ggtgatagca gttccgagca gtttctgtcc gaaatcgtgg gtgctgccag ttttccagca 240

gtacaccgct ctatggattc aatgtatgaa tatggttcac gtgctggctt ctggcgtatt 300

catgaggagt ttcaaaaacg tggctggcct atgacaattt ttggagtcgg tatggcactg 360

gctcgtaatc cttatatcgt cgaggccatt aaggcggctg attatgatgt ggtctcccat 420

ggtcagcgct ggttgcatta tcaggatatg gagattgaaa ctgaacgtca gcatatggat 480

caggccttaa gtgtgctgac tgaactcttt ggtgaaacgc caattggctg gtataccgga 540

cgcgacagtc ccaatacacg tcaattatta gctgaatttt ctcagattaa atatgactct 600

gattattatg gtgatgacct cccattttgg agcaccttaa ctgaaccaaa tggtcaaaaa 660

cgccctcatt tgatcatccc ctatacatta gagtgcaatg atatgaagtt tagctctccg 720

ggtggtttca attcaggtga gcaattttac caatatttaa aagatgcctt cgatgtgctc 780

tacagtgaag gtgaaactgc accaaaaatg atgtcaattg gcatgcactg tcgcctgctc 840

ggacgtccgg gacgcttcaa agccttgcag cgctttatgg attacgtaca gagccatgac 900

aaagtatgga tctgttgtcg taacgatatt gcacaacatt ggtacaccca ccattttcct 960

gcgcaccagg actaa 975

Claims (12)

1. A coding gene of chitin deacetylase is characterized in that the coding gene sequence is shown as SEQ ID NO. 2.

2. The method for preparing chitin deacetylase according to claim 1, comprising the steps of: cloning the coding gene of claim 1 onto an expression vector pET-28a to obtain a recombinant vector PET-28a-AsCDA, transferring the recombinant vector PE T-28a-AsCDA into BL21 escherichia coli competent cells, culturing the escherichia coli until the OD600 is 0.55-0.65, and performing IPTG induction expression to obtain the recombinant vector.

3. The method according to claim 2, wherein the IPTG induction concentration is 0.35 mM-0.45 mM, and the BL21 E.coli culture temperature is 36-38 ℃.

4. Use of the chitin deacetylase of claim 1, for deacetylation of acetyl-containing polysaccharides.

5. The use according to claim 4, wherein the acetyl-containing polysaccharide is selected from the group consisting of long chain chitins.

6. The use according to claim 5, wherein the long chain chitin is selected from the group consisting of alpha-chitin, beta-chitin, colloidal chitin and gamma-chitin.

7. Use of the chitin deacetylase of claim 1 in the preparation of chitosan or nanofibers by a chitin bioenzyme process.

8. Use according to any one of claims 4 to 7, wherein the pH optimum for the use is between 6 and 9 and the temperature optimum is between 25 ℃ and 35 ℃.

9. The use according to claim 8, wherein the optimal pH for the use is 7 and the optimal temperature is 30 ℃.

10. Use according to any one of claims 4 to 7, wherein, in the use, one or more of the following metal ions are added to promote the reaction: zn (zinc) ²⁺ 、Mg ²⁺ 、Ca ²⁺ 、Ni ⁺ 、Mn ²⁺ 、Ag ⁺ 、Sr ²⁺ 、Fe ³⁺ 。

11. The use according to claim 10, characterized in that the final concentration of metal ions is 0.5-1.5 mmol/L.

12. The use according to claim 11, characterized in that the final concentration of metal ions is 1mmol/L.

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