CN113388598B

CN113388598B - Chitinase Chi3002 and application thereof

Info

Publication number: CN113388598B
Application number: CN202110666791.1A
Authority: CN
Inventors: 于敏; 张晓华; 李肸; 冉凌蔓
Original assignee: Ocean University of China
Current assignee: Ocean University of China
Priority date: 2021-06-16
Filing date: 2021-06-16
Publication date: 2022-05-10
Anticipated expiration: 2041-06-16
Also published as: CN113388598A

Abstract

The invention provides a novel chitinase, the amino acid sequence of which is SEQ ID NO 1. The chitinase provided by the invention is used for preparation. The novel vibrio chitinase provided by the invention has good thermal stability, and the residual enzyme activity is kept above 80% at 80 ℃. The pH tolerance range is wide, the residual enzyme activity is kept above 80% under the condition of pH 4-12, and the application value is high. Moreover, when it degrades chitin, the degradation product is mainly chitobiose.

Description

Chitinase Chi3002 and application thereof

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to chitinase Chi3002 and application thereof.

Background

Chitin is the most abundant renewable carbohydrate polymer in marine environments, is a chain compound formed by connecting beta-N-acetyl-D-glucosamine through beta-1, 4 glycosidic bonds, is produced in various marine organisms such as mollusks, coelenterates, protozoa, fungi, crustaceans and the like, and is the main source of carbon and nitrogen in marine organisms. Today, most marine waste products produced after marine processing are rich in chitin. It is estimated that about 10 is generated from marine waste annually¹¹Ton of chitin. Because chitin is not easily degraded and may cause harm to the environment, rapid degradation of chitin is critical to the circulation of ocean carbon and nitrogen.

The biological technology for treating the marine waste is widely applied due to the advantages of green, high efficiency, environmental protection and the like. In 1938 scientists Zobell and Rittenberg suggested that many marine bacteria could utilize chitin as an energy source for carbon and nitrogen. The microorganisms degrade chitin by secreting chitinase out and utilize the degraded products to meet the requirements of self-growth metabolism. In addition, the chitinase derived from bacteria shows strong activity in a large range of salinity, pH value and temperature, and has great significance for the industrial process applied to most of harsh physical and chemical industries. In addition, in the fields of medicine, biotechnology, agriculture, industrial application, waste management and the like, the application value of chitinase in the aspects of killing insects, resisting fungi, resisting malaria, reducing cholesterol, reducing blood pressure, improving food quality and the like/the application value of chitinase-345 bacteria in the aspects of degrading chitin waste, generated chitinase and degradation products thereof and the like in a marine ecosystem attracts great attention of researchers.

Disclosure of Invention

The invention aims to provide chitinase Chi3002 and application thereof, namely the chitinase Chi3002 with higher thermal stability,

the chitinase provided by the invention comprises:

1) a protease having the amino acid sequence of SEQ ID NO. 1;

MKQKAIYLAVALGLGGLTNVASANEMVNPDGGVVVGYWHNWCDGAGYKGGNAPCVTLEDVDPMYNVVNVSFMKVFDTSEGRIPTFKLDSNIGLSEQQLIDQIEALNKQGRAVLIALGGADAHVELKTGDEQAFADEIIRLTDKFGFDGLDIDLEQSAVTAENNQTVIPAALRLVKEHYQQQGKNFLITMAPEFPYLTEGGKYVPYITGLEGYYDWINPQFYNQGGDGIWVDGVGWIAQNNDALKQEFIYYISDSLSNGTRGFHKIPHDKLVFGIPSNIDAAATGFVQDPQDLYDAFEQLKAQGQALRGVMTWSVNWDMGTNKDGQAYGEKFVKDYGPFVHGQTPPPSSEGEPVFNGINDVRVQHGSSFDPHAGVTASDKEDGDLTNSINVEGSVDVNTVGTYVLVYSVKDSDNNETKQTRTVVVYSLVPEFEGVANTTIQLGEAFDPMAGVKAIDAEDGDLTGQVKVEGSVDVNTLGVYNLVYRVTDSANQTATAQRAVTVSDGSGYPAYEAGKAYEAGEIVTGSDGNLYQCKPWPYTGWCANPSYAPGETVYWSDAWDKL

2) the protease which is obtained by substituting, deleting and adding one or more amino acids on the protein in the step 1) and has the protease efficacy in the step 1);

the invention also protects the gene for coding the chitinase, and a specific nucleotide sequence of the gene is as follows:

atgaagcaaaaagcgatatatctagcagtggcattagggttgggcggtcttacgaacgttgcatccgctaacgaaatggtcaacccagatggtggtgtcgtcgtcggttattggcataactggtgtgatggcgctggttacaaaggtggtaacgcaccatgtgtaaccttggaagacgttgatccaatgtacaatgtggttaacgtttcttttatgaaagtcttcgacaccagtgaaggacgcattccaacatttaaacttgactcaaatattggtctctcagaacagcaactgattgatcaaattgaagcgttgaacaaacaaggtcgtgcagtgcttattgcactcggcggcgctgatgctcatgttgagttgaaaaccggtgatgagcaagcattcgctgatgagatcattcgcttaactgataaatttggttttgatggtctagatatcgacttggagcaatcggcggtaacagcagagaataaccaaacggtgattccagcagcacttcgtcttgtgaaagagcattaccagcagcaaggtaaaaacttcctgatcacgatggcgcctgaattcccatacctaacagagggtggtaaatacgtcccttacattacgggtttagaagggtattacgattggatcaacccacagttctacaaccaaggtggcgatggtatttgggttgacggcgtaggttggattgctcaaaacaatgatgcgctaaaacaagaattcatctattacatttctgattctctatcgaatggtactcgtggtttccacaaaatccctcacgataaattggtgtttggtattccatcaaacatcgacgctgcagcaacaggttttgttcaggatcctcaagatctttacgatgctttcgagcagctaaaagcgcaaggtcaggcacttcgcggcgtaatgacatggtcagtaaactgggacatgggcacaaacaaagacggtcaggcgtatggtgaaaaattcgtcaaagattacggtccgtttgttcatggtcagacaccaccaccgtcaagcgaaggtgagccagtattcaatggcatcaatgacgtgcgtgtgcaacatggcagttcattcgaccctcacgcaggtgtgaccgcttcagacaaagaagatggcgacttaactaatagcatcaatgtagaaggttctgttgatgtaaacactgtcggtacctatgttttggtttacagtgtaaaagacagcgacaacaatgaaaccaagcaaacaagaaccgttgtagtatacagtcttgttccagaattcgaaggtgtcgcgaacacgactatccaacttggtgaagcatttgatccaatggctggagtgaaagctattgacgcagaagacggtgacttgactggccaagtaaaggtagaaggtagcgtagatgttaatacacttggcgtttacaacctagtttaccgtgtaaccgatagcgcgaaccagactgcaacagctcaacgagcagtgacagtatctgatggcagtggctaccctgcgtatgaagccggaaaagcctatgaagctggtgagattgttacgggctcagacggtaacttgtatcaatgtaaaccgtggccttacacagggtggtgtgcaaacccttcttatgcgccaggtgaaaccgtttattggtcggacgcatgggataagttgtaa(SEQ ID NO:2)。

the invention also provides a recombinant expression vector, wherein a nucleotide fragment for coding the chitinase gene chi3002 is inserted into the recombinant expression vector;

the recombinant expression vector is specifically described as an escherichia coli plasmid vector pUCm-T-chi 3002;

in another aspect, the invention also provides a genetically engineered host cell carrying the recombinant expression vector.

The invention also provides a method for preparing the chitinase Chi3002, which is prepared by fermenting the host cell and purifying a fermentation product;

the invention also provides the application of the chitinase Chi3002 in the preparation of chitosan oligosaccharide;

the chitooligosaccharide is preferably chitobiose.

The invention also provides a method for preparing the chitobiose, which is to use the chitinase Chi3002 to enzymolyze chitin to prepare the chitobiose;

the enzymolysis temperature is 50 ℃, and the pH value is 6.0.

The vibrio chitinase Chi3002 provided by the invention has good thermal stability, and the residual enzyme activity of the Chi3002 is kept above 80% at 80 ℃. Chi3002 has wide pH tolerance range, and has high application value, and the residual enzyme activity is kept above 80% under the condition of pH 4-12. Furthermore, when it degrades chitin again, the degradation product is mainly chitobiose.

Drawings

FIG. 1: the effect of different temperatures and pH on the chitinase production of the strain SIR5 used in the present invention. A is the effect of different temperatures on chitinase production by strain SIR 5; b is the effect of different pH on chitinase production by strain SIR 5.

FIG. 2 is a schematic diagram: agarose gel electrophoresis picture of PCR amplification product of chitinase gene chi3002 used in the invention.

FIG. 3: SDS-PAGE electrophoresis of purified chitinase Chi 3002. M: protein Maker, a: coli BL21/pET-24a (+) disruption supernatant, B: coli BL21/pET-24a (+)/chi3002 disruption supernatant, C: chitinase Chi3002 eluted by imidazole.

FIG. 4: chitinase Chi3002 is subjected to an optimal temperature and temperature stability experiment and an optimal pH and pH stability experiment. A: optimum temperature experiment of Chi3002, B: temperature stability experiment of Chi3002, C: optimal pH experiment of Chi3002, D: pH stability experiment of Chi 3002.

FIG. 5: the effect of high or low concentrations of metal ions and reducing agents on chitinase Chi 3002.

FIG. 6: kinetic analysis of the enzymatic reaction of chitinase Chi 3002.

FIG. 7: TLC analysis of chitinase Chi3002 hydrocolloid chitin products, where M is a chitooligosaccharide standard.

Detailed Description

The strain Vibrio thalassae SIR5 used in the present invention was isolated from the biological sample of Alwen shrimp intestine at 1165m of the hydrothermal area of the Tang stamp of Okinawa Hai channel. Extracellular enzyme biopsy is carried out on the recombinant protein Chi3002, and the recombinant protein Chi3002 has strong activity of degrading chitin, and then the potential chitinase gene Chi3002 in the strain is subjected to heterologous expression, so that the obtained recombinant protein Chi3002 has obvious chitinase activity.

The present invention will be described in detail below with reference to examples and the accompanying drawings.

Example 1: cloning transformation of chitinase Chi3002

Screening chitinase gene

The inventor obtains a bacterial SIR5 with strong chitin degrading capability through screening. The strain is separated from the TVG11-2 station of the Tang-stamp hydrothermal area of Shanghai Hai. 16S rRNA gene sequencing analysis shows that the strain belongs to the genus Vibrio, and the strain is deposited in the marine microorganism preservation center of the third Marine research institute of the national Marine agency, at the accession number MCCC 1K04105, Ministry of thought of Ministry of mansion, Fujian province.

Extracting V.thalassae SIR5 genome by phenol-chloroform method

1. The strain SIR5 was removed from the freezer at-80 ℃ and inoculated on 2216E medium and cultured at 28 ℃ for two days. The activated strain was then inoculated onto a 1% chitin plate (pH 7), and placed in incubators at different temperatures, respectively, and after 7 days of incubation, the results were observed (fig. 1A). Appropriate amounts of the cells were scraped off and inoculated on chitin plates (pH 6-11) of different pH, and the cells were cultured at the optimum temperature for 7 days, followed by observation (fig. 1B).

Inoculating the strain on an MA inclined plane, culturing for 24h, adding 2-3mL of physiological saline, and performing vortex oscillation to elute the thallus on the inclined plane to obtain a bacterial suspension. The bacterial suspension is centrifuged at 12000rpm for 10min, and the supernatant is discarded to obtain the thallus.

2. 475. mu.L of 1 XTE buffer was added to an EP tube in which the cells were collected, and the cells were resuspended. And 10. mu.L of lysozyme (20mg/mL) was added and incubated at 37 ℃ for 1h, and the samples were mixed by gentle inversion every 20 min.

3. mu.L of SDS solution (10%) and 5. mu.L of proteinase K (20mg/mL) were added sequentially and incubated at 55 ℃ for 1.5-2h, and the samples were mixed by gentle inversion every 20 min.

4. Adding 15 μ L RNase A, mixing by gentle inversion, incubating in a 37 deg.C water bath for 1h, and mixing by gentle inversion every 20 min.

5. Add 250. mu.L Tris-equilibrated phenol and 250. mu.L chloroform in sequence: isoamyl alcohol (24:1, v/v), mixing by gentle inversion, centrifuging at 12000rpm for 10min at room temperature, and transferring the supernatant to a new 1.5mL sterile EP tube;

6. equal volumes of chloroform: adding isoamyl alcohol into the supernatant obtained in the step (5), slightly reversing, uniformly mixing, centrifuging at 12000rpm for 10min, and transferring the supernatant into a new 1.5mL EP tube;

7. repeating the step (6) until the middle layer has no white precipitate;

8. sequentially adding pre-cooled 1/10 volumes of 3M sodium acetate (pH 5.2) and 2 volumes of anhydrous ethanol into all the supernatants, slowly reversing, mixing, and settling at-20 deg.C for 2h or overnight;

9. after sedimentation, taking out a sample, centrifuging at 12000rpm for 10min at 4 ℃, then discarding the supernatant, washing the precipitate with 70% ethanol for 2-3 times, and inverting and drying;

10. the pellet was dissolved in 50. mu.L of 1 XTE buffer and stored in a freezer at-20 ℃.

Genome sequencing is carried out on the extracted genome DNA of the strain SIR5 by Shenzhen HuaDagenescience and technology Limited. And filtering the original data obtained by sequencing, and assembling by using software such as SPAdes and the like to obtain the whole genome sequence of the strain. RAST annotation is carried out on the genome sequence to obtain a gene chi3002 for coding chitinase, wherein the amino acid sequence of the protein is SEQ ID NO. 1, and the nucleotide sequence of the coding gene is SEQ ID NO. 2.

Secondly, amplifying chitinase gene chi3002 sequence

Designing a primer according to the gene sequence with the nucleotide sequence of SEQ ID NO. 2 and an expression vector pET-24a (+), and adding proper enzyme cutting sites on the upstream and downstream of a target gene. The restriction sites are Sac I and Hind III respectively. Chi3002 was amplified using DNA of strain SIR5 as a template.

The primer sequences are as follows:

chi3002-F 5’-CGAGCTCATGAACGAAATGGTCAAC-3’

chi3002-R 5’-CCCAAGCTTCAACTTATCCCATGCG-3’

the reaction system of PCR is shown in Table 1, and the reaction procedure of PCR is shown in Table 2.

Table 1: reaction System Table of PCR

Table 2: reaction schedule of PCR

The PCR product was subjected to agarose gel electrophoresis to verify that the band of interest was correct in size (FIG. 2). And removing the band of non-specific amplification by using a gel cutting recovery kit, and purifying and recovering the PCR product to obtain a purified target gene fragment.

Thirdly, cloning and transforming chitin gene chi3002

The desired gene fragment was ligated with a polyA tail (Table 3) and subsequently with pUCm-T overnight at 16 ℃ (Table 5) to construct a cloning plasmid pUCm-T-chi 3002. Coli JM109 was transformed with a heat shock method, spread on an LB plate containing ampicillin, X-gal and IPTG, and cultured overnight at 37 ℃. The single colony was inoculated into 2mL of LB liquid medium containing ampicillin at a final concentration of 50. mu.g/mL, and cultured at 37 ℃ and 200rpm for 8 to 12 hours. When the bacterial liquid is turbid, the positive recombinants are verified by a bacterial liquid PCR method, PCR products are detected by 1.0% (w/v) agarose gel electrophoresis, and a target band is about 1700bp and is consistent with the target gene chi3002 in size. And (4) testing the positive recombinant bacteria liquid, and selecting strains with correct sequences for subsequent experiments.

Table 3: adding polyA tail system table

Example 2 inducible expression of the chitinase-encoding Gene chi3002

The cloning plasmid and pET-24a (+) plasmid obtained in example 1 were subjected to Sac I and HindIII double digestion, respectively, and the digestion systems are shown in Table 4. Performing double enzyme digestion for 20min at 37 ℃ in a metal bath, detecting by agarose electrophoresis, and recovering the target gene and the vector in the agarose gel.

Table 4: list of enzyme digestion systems

The double-digested target gene was ligated with pET-24a (+) vector overnight at 16 ℃. The ligation system is shown in Table 5. The ligated plasmid was transformed into E.coli BL21(DE3) competent by heat shock. Adding 800 μ L LB liquid culture medium pre-iced in the system, culturing at 37 deg.C under 150rpm for 1-2 h; taking out, centrifuging at 4000rpm for 10min, sucking 800 μ L of supernatant, and re-suspending the rest; spreading 200 μ L bacterial liquid on resistant plate (kanamycin with final concentration of 50 μ g/mL), drying, and culturing in 37 deg.C incubator for 12-16 h; and (3) picking the recombinants to an LB culture medium containing kanamycin with the final concentration of 50 mu g/mL, verifying positive recombinants by using a bacteria liquid PCR method when the bacteria liquid is turbid, and detecting a PCR product by using 1.0% (w/v) agarose gel electrophoresis to obtain a target band of about 1700bp which is consistent with the target gene chi3002 in size. And (5) sending the bacterial liquid identified as the positive recombinant to test, and further verifying the correctness of the target gene sequence and the insertion site.

Table 5: connection system watch

The recombinant E.coli strain BL21(DE3) with the correct gene insert was taken in 5mL of LB liquid medium (50. mu.g/mL kanamycin), 37 ℃ at 170rpm,shaking and culturing to obtain seed liquid. The seed solution was inoculated in 300mL of LB medium (50. mu.g/mL kanamycin) at an inoculum size of 1%, cultured at 37 ℃ with shaking at 150rpm to OD₆₀₀0.6-0.8, then IPTG (final concentration of 0.1mM) is added, and the mixture is cultured for 12-16h at 16 ℃ and 150rpm in a shaking way for induction expression.

Collecting thalli at low temperature, and reserving part of fermentation broth supernatant for activity test; adding Binding Buffer into the thallus to resuspend the thallus, concentrating the thallus by 10-20 times, and blowing and uniformly mixing; carrying out ultrasonic disruption on the thalli, wherein the conditions of disruption are as follows: the working voltage is 300V, the crushing time is 5s, the gap time is 10s, the crushing is carried out for 200 times and 300 times, and the operation is carried out on ice; after the completion of the disruption, the mixture is centrifuged at 12000rpm for 15min at 4 ℃, and the disrupted supernatant, i.e., the crude enzyme solution, is collected, and meanwhile, the disrupted precipitate is resuspended in Binding Buffer and retained for subsequent enzyme activity test.

Adding the crude enzyme solution into a balanced nickel column, and repeatedly loading for 2-3 times; washing the nickel column with imidazole of different concentrations in sequence, and collecting effluent liquid for SDS-PAGE electrophoresis detection; putting the obtained high-purity protein into a dialysis bag, soaking in dialysate, dialyzing at 4 deg.C to remove salt ions, replacing dialysate every 12h, and dialyzing for 48h to obtain high-purity target protein (figure 3).

Example 3: enzymatic Properties of chitinase

Determination of optimum temperature and temperature stability of chitinase

Optimum temperature: incubating 1% chitin colloid at different temperatures for 30min, reacting 20 μ L purified chitinase with 180 μ L1% chitin colloid incubated at different temperatures for 1h, and taking the sample without chitinase solution as a control. The enzyme activity of the chitinase is measured by using a DNS method, the enzyme activity at the optimal reaction temperature is taken as 100 percent, and the relative enzyme activity of the chitinase at different reaction temperatures is calculated. From FIG. 4A, it is clear that the optimum temperature of chitinase Chi3002 is 50 ℃.

And (3) measuring the temperature stability: and (3) incubating the purified chitinase solution for 1h at different temperatures, reacting 20 mu L of treated chitinase solution with 180 mu L of 1% chitin colloid at the optimal reaction temperature for 1h, taking a sample without the chitinase solution as a negative control, and determining the residual enzyme activity of the chitinase by using a DNS method, wherein the enzyme activity with the highest residual chitinase activity is taken as 100%, so as to determine the stability of the chitinase at different temperatures. As can be seen from FIG. 4B, Chi3002 has good thermal stability, Chi3002 remains stable at 0-60 deg.C, and the enzyme activity is substantially unchanged, while at 60-80 deg.C, the enzyme activity can be maintained at more than 80%. Therefore, Chi3002 is a thermostable enzyme and has high application value.

Determination of optimum pH and pH stability of chitinase

Optimum pH: buffer solutions with different pH values were prepared according to Table 6, 1% chitin colloid was prepared from the buffer solutions with different pH values, and incubated at the optimum reaction temperature for 30 min. 20 mu L of chitinase solution is respectively reacted with 180 mu L of 1% chitin colloid treated with different pH values for 1h at the optimum reaction temperature, a sample without the chitinase solution is taken as a negative control, and the other conditions are consistent. The enzyme activity of the chitinase is measured by using a DNS method, so that the optimal reaction pH of the chitinase is determined. The enzyme activity under the optimum reaction pH is taken as 100%, and the relative enzyme activity of the chitinase under different reaction pH is calculated. From FIG. 4C, Chi3002 can be seen to have an optimum pH of 6.0.

pH stability: the chitinase solution was absorbed in an appropriate amount and treated with buffer solutions (Table 6) of different pH values, and left at 4 ℃ for 1 hour. And (3) reacting 20 mu L of treated chitinase solution with 180 mu L of 1% chitin colloid at the optimal reaction temperature for 1h, taking a sample without the chitinase solution as a negative control, and determining the residual enzyme activity of the chitinase by using a DNS (domain name system) method, wherein the enzyme activity with the highest residual chitinase activity is taken as 100%, thereby determining the stability of the chitinase under different pH values. As shown in FIG. 4D, Chi3002 was treated with buffers having different pH values, and the enzyme activity was changed. When the pH is 2-3, the enzyme activity of Chi3002 is not high (below 70%), and when the pH is between 4-12, the enzyme activity of Chi3002 is almost unchanged. Therefore, the chitinase Chi3002 has a wide pH tolerance range.

Table 6: different pH buffer system table

Thirdly, the influence of chemical reagents such as metal ions, reducing agents and the like on the enzyme activity of the recombinant chitinase Chi3002

To 2% chitin colloid, Ca2+, Fe3+, Mn2+, Cu2+ and EDTA, SDS, etc. were added at a final concentration of 1mM and 10mM, respectively, to give a final concentration of 1%. Uniformly mixing, placing the treated chitin colloid at the optimal reaction temperature for incubation for 30min, sucking 20 mu L of chitinase solution, adding into 180 mu L of 1% treated chitin colloid, uniformly mixing, reacting for 1h at the optimal reaction temperature, taking a sample without the chitinase solution as a negative control, and determining the enzyme activity of the chitinase by using a DNS method, wherein the other conditions are the same. The enzyme activity corresponding to the sample without any reagent is taken as 100 percent, so that the influence of metal ions, reducing agents and the like on the enzyme activity of the chitinase is determined. Among them, ions such as Fe3+, Ni2+, Zn2+, Co2+, Cu2+ have a significant inhibitory effect on Chi3002 activity, and the inhibitory effect is more significant with the increase (10 mM) of ion concentration. While Na +, Mg2+, Ca2+ and K + have obvious promoting effect on Chi3002 enzyme activity, and the higher the concentration is, the more obvious the promoting effect is (FIG. 5).

Enzymatic kinetic assay for chitinase

Chitin colloid with the concentration of 0.05-2% is prepared. And placing the chitin colloids with different concentrations at the optimal reaction temperature for incubation for 30min, sucking 180 mu L of incubated chitin colloids with different concentrations to react with 20 mu L of chitinase for 1h at the optimal reaction temperature, and determining the initial reaction speed. The data obtained were processed with Lineweaver-Burke mapping to determine the V of the chitinase_maxValue sum K_mThe value is obtained. V of chitinase Chi3002_maxA value of 0.45mg/U, K_mThe value was 3.04mg/mL (FIG. 6).

Analysis of chitinase degradation products

Under the optimal reaction conditions (optimal temperature 50 ℃, optimal pH 6.0), 1% chitin colloid reacts with excessive enzyme differentlyAfter the time, the reaction mixture was taken out and quenched by boiling water bath. The products of different reaction times were checked by TLC plates, and the main product was (GlcNAc) in the early and middle stages of the degradation reaction₂(FIG. 7), it can be seen that chitinase Chi3002 is an exo-chitinase.

Sequence listing

<110> China oceanic university

<120> novel chitinase Chi3002 and application thereof

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 561

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Met Lys Gln Lys Ala Ile Tyr Leu Ala Val Ala Leu Gly Leu Gly Gly

1 5 10 15

Leu Thr Asn Val Ala Ser Ala Asn Glu Met Val Asn Pro Asp Gly Gly

20 25 30

Val Val Val Gly Tyr Trp His Asn Trp Cys Asp Gly Ala Gly Tyr Lys

35 40 45

Gly Gly Asn Ala Pro Cys Val Thr Leu Glu Asp Val Asp Pro Met Tyr

50 55 60

Asn Val Val Asn Val Ser Phe Met Lys Val Phe Asp Thr Ser Glu Gly

65 70 75 80

Arg Ile Pro Thr Phe Lys Leu Asp Ser Asn Ile Gly Leu Ser Glu Gln

85 90 95

Gln Leu Ile Asp Gln Ile Glu Ala Leu Asn Lys Gln Gly Arg Ala Val

100 105 110

Leu Ile Ala Leu Gly Gly Ala Asp Ala His Val Glu Leu Lys Thr Gly

115 120 125

Asp Glu Gln Ala Phe Ala Asp Glu Ile Ile Arg Leu Thr Asp Lys Phe

130 135 140

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

145 150 155 160

Glu Asn Asn Gln Thr Val Ile Pro Ala Ala Leu Arg Leu Val Lys Glu

165 170 175

His Tyr Gln Gln Gln Gly Lys Asn Phe Leu Ile Thr Met Ala Pro Glu

180 185 190

Phe Pro Tyr Leu Thr Glu Gly Gly Lys Tyr Val Pro Tyr Ile Thr Gly

195 200 205

Leu Glu Gly Tyr Tyr Asp Trp Ile Asn Pro Gln Phe Tyr Asn Gln Gly

210 215 220

Gly Asp Gly Ile Trp Val Asp Gly Val Gly Trp Ile Ala Gln Asn Asn

225 230 235 240

Asp Ala Leu Lys Gln Glu Phe Ile Tyr Tyr Ile Ser Asp Ser Leu Ser

245 250 255

Asn Gly Thr Arg Gly Phe His Lys Ile Pro His Asp Lys Leu Val Phe

260 265 270

Gly Ile Pro Ser Asn Ile Asp Ala Ala Ala Thr Gly Phe Val Gln Asp

275 280 285

Pro Gln Asp Leu Tyr Asp Ala Phe Glu Gln Leu Lys Ala Gln Gly Gln

290 295 300

Ala Leu Arg Gly Val Met Thr Trp Ser Val Asn Trp Asp Met Gly Thr

305 310 315 320

Asn Lys Asp Gly Gln Ala Tyr Gly Glu Lys Phe Val Lys Asp Tyr Gly

325 330 335

Pro Phe Val His Gly Gln Thr Pro Pro Pro Ser Ser Glu Gly Glu Pro

340 345 350

Val Phe Asn Gly Ile Asn Asp Val Arg Val Gln His Gly Ser Ser Phe

355 360 365

Asp Pro His Ala Gly Val Thr Ala Ser Asp Lys Glu Asp Gly Asp Leu

370 375 380

Thr Asn Ser Ile Asn Val Glu Gly Ser Val Asp Val Asn Thr Val Gly

385 390 395 400

Thr Tyr Val Leu Val Tyr Ser Val Lys Asp Ser Asp Asn Asn Glu Thr

405 410 415

Lys Gln Thr Arg Thr Val Val Val Tyr Ser Leu Val Pro Glu Phe Glu

420 425 430

Gly Val Ala Asn Thr Thr Ile Gln Leu Gly Glu Ala Phe Asp Pro Met

435 440 445

Ala Gly Val Lys Ala Ile Asp Ala Glu Asp Gly Asp Leu Thr Gly Gln

450 455 460

Val Lys Val Glu Gly Ser Val Asp Val Asn Thr Leu Gly Val Tyr Asn

465 470 475 480

Leu Val Tyr Arg Val Thr Asp Ser Ala Asn Gln Thr Ala Thr Ala Gln

485 490 495

Arg Ala Val Thr Val Ser Asp Gly Ser Gly Tyr Pro Ala Tyr Glu Ala

500 505 510

Gly Lys Ala Tyr Glu Ala Gly Glu Ile Val Thr Gly Ser Asp Gly Asn

515 520 525

Leu Tyr Gln Cys Lys Pro Trp Pro Tyr Thr Gly Trp Cys Ala Asn Pro

530 535 540

Ser Tyr Ala Pro Gly Glu Thr Val Tyr Trp Ser Asp Ala Trp Asp Lys

545 550 555 560

Leu

<210> 2

<211> 1686

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

atgaagcaaa aagcgatata tctagcagtg gcattagggt tgggcggtct tacgaacgtt 60

gcatccgcta acgaaatggt caacccagat ggtggtgtcg tcgtcggtta ttggcataac 120

tggtgtgatg gcgctggtta caaaggtggt aacgcaccat gtgtaacctt ggaagacgtt 180

gatccaatgt acaatgtggt taacgtttct tttatgaaag tcttcgacac cagtgaagga 240

cgcattccaa catttaaact tgactcaaat attggtctct cagaacagca actgattgat 300

caaattgaag cgttgaacaa acaaggtcgt gcagtgctta ttgcactcgg cggcgctgat 360

gctcatgttg agttgaaaac cggtgatgag caagcattcg ctgatgagat cattcgctta 420

actgataaat ttggttttga tggtctagat atcgacttgg agcaatcggc ggtaacagca 480

gagaataacc aaacggtgat tccagcagca cttcgtcttg tgaaagagca ttaccagcag 540

caaggtaaaa acttcctgat cacgatggcg cctgaattcc catacctaac agagggtggt 600

aaatacgtcc cttacattac gggtttagaa gggtattacg attggatcaa cccacagttc 660

tacaaccaag gtggcgatgg tatttgggtt gacggcgtag gttggattgc tcaaaacaat 720

gatgcgctaa aacaagaatt catctattac atttctgatt ctctatcgaa tggtactcgt 780

ggtttccaca aaatccctca cgataaattg gtgtttggta ttccatcaaa catcgacgct 840

gcagcaacag gttttgttca ggatcctcaa gatctttacg atgctttcga gcagctaaaa 900

gcgcaaggtc aggcacttcg cggcgtaatg acatggtcag taaactggga catgggcaca 960

aacaaagacg gtcaggcgta tggtgaaaaa ttcgtcaaag attacggtcc gtttgttcat 1020

ggtcagacac caccaccgtc aagcgaaggt gagccagtat tcaatggcat caatgacgtg 1080

cgtgtgcaac atggcagttc attcgaccct cacgcaggtg tgaccgcttc agacaaagaa 1140

gatggcgact taactaatag catcaatgta gaaggttctg ttgatgtaaa cactgtcggt 1200

acctatgttt tggtttacag tgtaaaagac agcgacaaca atgaaaccaa gcaaacaaga 1260

accgttgtag tatacagtct tgttccagaa ttcgaaggtg tcgcgaacac gactatccaa 1320

cttggtgaag catttgatcc aatggctgga gtgaaagcta ttgacgcaga agacggtgac 1380

ttgactggcc aagtaaaggt agaaggtagc gtagatgtta atacacttgg cgtttacaac 1440

ctagtttacc gtgtaaccga tagcgcgaac cagactgcaa cagctcaacg agcagtgaca 1500

gtatctgatg gcagtggcta ccctgcgtat gaagccggaa aagcctatga agctggtgag 1560

attgttacgg gctcagacgg taacttgtat caatgtaaac cgtggcctta cacagggtgg 1620

tgtgcaaacc cttcttatgc gccaggtgaa accgtttatt ggtcggacgc atgggataag 1680

ttgtaa 1686

Claims

1. The chitinase is characterized in that the amino acid sequence of the chitinase is SEQ ID NO. 1.

2. A gene encoding the chitinase of claim 1.

3. The gene of claim 2, wherein the nucleotide sequence of the gene is SEQ ID NO 2.

4. A recombinant expression vector comprising a nucleic acid fragment encoding the chitinase of claim 1 inserted therein.

5. The recombinant expression vector of claim 4, wherein the expression vector is an E.coli plasmid vector pUCm-T-chi 3002.

6. A genetically engineered host cell carrying the recombinant expression vector of claim 4.

7. Use of the chitinase of claim 1 in the preparation of chitobiose.

8. A method for preparing chitobiose, wherein the method comprises hydrolyzing chitin with the chitinase of claim 1 or the host cell of claim 6 to prepare chitobiose.

9. The method of claim 8, wherein said enzymatic hydrolysis is carried out at a temperature of 50 ℃ and a pH of 6.0.