Glycoside hydrolase CmChi3 and application thereof in degradation of hydrocolloid chitin
阅读说明：本技术 一种糖苷水解酶CmChi3及其在降解水解胶体几丁质上的应用 (Glycoside hydrolase CmChi3 and application thereof in degradation of hydrocolloid chitin ) 是由 陈可泉 王成勇 张阿磊 周宁 王莹莹 陈燕 陈雪曼 欧阳平凯 于 2020-12-18 设计创作，主要内容包括：本发明公开了一种糖苷水解酶CmChi3及其在降解胶体几丁质上的应用。将从土壤中筛选到的菌株Chitinolyticbacter meiyuanensis SYBC-H1进行基因组测序分析、克隆CmChi3、构建重组菌株、表达CmChi3蛋白、纯化HIS-TAG、并鉴定重组蛋白具有两个催化结构域,两个几丁质结合域,具有内切酶和糖苷水解酶两种酶的性质,能高效水解几丁质并以GlcNAc为终产物。此研究预期为单酶法高效生产GlcNAc提供理论依据和基础。(The invention discloses a glycoside hydrolase Cm Chi3 and its application in degrading colloidal chitin. Strains screened from soil Chitinolyticbacter meiyuanensis SYBC-H1 for genome sequencing analysis and cloning Cm Chi3, construction of recombinant strain and expression Cm Chi3 protein, purified HIS-TAG, and identified recombinant protein having two catalytic domains, two chitin binding domains, and two enzymes, endonuclease and glycoside hydrolaseThe chitin can be efficiently hydrolyzed and GlcNAc is used as a final product. This study is expected to provide theoretical basis and foundation for efficient production of GlcNAc by a single enzyme process.)
1. Glycoside hydrolaseCmChi3, characterized in that its amino acid sequence is as given in SEN ID NO: 2, respectively.
2. Glycoside hydrolase according to claim 1CmChi3, characterized in that the nucleotide sequence encoding the amino acid sequence is as defined in SEN ID NO: 1 is shown.
3. A recombinant expression vector comprising the glycoside hydrolase-encoding gene of claim 2CmA recombinant expression vector of the nucleotide sequence of Chi3 gene.
4. A recombinant bacterium which expresses a strain containing the recombinant expression vector according to claim 3.
5. Glycoside hydrolase according to claim 1CmThe cloning expression of the Chi3 gene is characterized by comprising the following steps:
step 1, taking SYBC-H1 strain whole genome DNA as a template, designing a primer and amplifying the primer by PCR to obtain the DNA with a His tagCmThe full-length sequence of Chi3 gene;
step 2, constructing recombinant plasmid pCold I-CmChi3
The PCR-amplified DNA sequence and pCold I vector were ligated using the same restriction enzymeNdeIAndEcoRIperforming double enzyme digestion, recovering and purifying the enzyme digestion product, and performing digestion by using T4The enzyme cleavage product purified by DNA ligase obtains the plasmid vector pCold I-CmChi3；
Step 3, recombinant plasmid pCold I-CmChi3Introduced into Escherichia coli BL21(DE3) to obtain a recombinant plasmid containing pCold I-CmChi3The Escherichia coli BL21(DE3) was named recombinant strain;
step 4, selecting a monoclonal of the recombinant bacteria, inoculating the seed solution into an LB culture medium with the inoculum size of 1-5% by volume fraction after overnight culture in a shaking table, and culturing at 37 ℃ until the bacterial density OD600Adding inducer for inducing for 12-14h, collecting fermentation liquor, centrifuging at 4 deg.C for 6000-8000g, and collecting strain;
and 5, adding a buffer solution into the precipitate to resuspend the strain, centrifuging after ultrasonic lysis, collecting supernatant, and freezing for later use.
6. Glycoside hydrolase according to claim 5CmThe cloning expression of Chi3 gene is characterized in that the primers used for PCR amplification in the step 1 are as follows:
CmChi3-F：5’-GGGAATTCCATATGACCGAGATCGCCCCGTAC-3’；
CmChi3-R：5’-CCGGAATTCGCGGCCGTTGCCCAGCAC-3’ 。
7. glycoside hydrolase obtained according to claim 5CmUse of Chi3 for the hydrolysis of colloidal chitin, wherein said colloidal chitin is degraded to N-acetylglucosamine.
8. Use according to claim 7, characterized in that the glycoside hydrolaseCmChi3 hydrocolloid chitin has a temperature of 25-55 deg.C and a pH of 5.0-8.0.
Technical Field
The invention belongs to the field of genetic engineering, and particularly relates to glycoside hydrolaseCmChi3 and its application in degrading hydrocolloid chitin.
Background
Chitin (Chitin) is a natural high-molecular polysaccharide polymerized from N-acetylglucosamine through beta-1, 4-glycosidic bond, is the second largest carbohydrate polymer with the next to cellulose in earth, and has a molecular formula of (C)8H13O5N) N, the relative molecular weight is more than 100 ten thousand. Chitin is widely present in the cell wall of fungi, insect carapace, shrimp and crab shell in nature. The chitin is mainly derived from shrimp and crab shells, and is deproteinized and decalcified by using 10% sodium hydroxide and concentrated hydrochloric acid to obtain the chitin with higher purity. With the development of the shrimp and crab breeding industry, more and more leftovers such as shrimp and crab shells are generated, the leftovers are mostly discarded as solid wastes, and the leftovers are decomposed and emit unpleasant odor after being stacked for several days, so that not only is the resources wasted, but also the environmental burden is greatly increased. Therefore, the chitin waste is utilized to produce high value-added chemicals, so that the environmental problem can be solved, and huge economic benefits can be brought.
Chitin is a main component in shrimp and crab shells and can be used for producing various nitrogenous chemicals with high added values, such as N-acetylglucosamine (GlcNAc), chitosan oligosaccharide and derivatives thereof. Among them, GlcNAc belongs to a functional amino monosaccharide, and has an important physiological function. There are many reports on the use of the physiological activity of GlcNAc, for example, GlcNAc has been shown to enhance the therapeutic effect of arthrosis on arthritis, rheumatoid arthritis, cartilage contusion, joint injury and degenerative joint disease treatment; the GlcNAc contains amino with positive charge and reacts with acidic groups to regulate the pH around the cells, so as to achieve the effect of inhibiting the growth of cancer cells; GlcNAc as a component of hyaluronic acid has a good moisturizing effect when used in skin care products, and the amount of GlcNAc added is usually 0.1-0.5%. GlcNAc has also been reported to have wide application in the food additive industry.
The current methods for producing GlcNAc by using chitin mainly include chemical methods, physical methods and biological enzyme methods. The chemical conversion method mainly comprises the steps of decomposing chitin by concentrated hydrochloric acid to obtain glucosamine hydrochloride, and then further performing acetylation to synthesize GlcNAc. The method is low in cost and is a main means for industrially producing the GlcNAc at present, but the product of the method is not considered as natural GlcNAc and cannot be used as a food additive, and the method can produce a large amount of industrial wastewater and cause certain pollution to the environment. The microbial fermentation method is a method which is popular at present, and GlcNAc is produced by genetic engineering strains of Escherichia coli. Although the microbial fermentation synthesis method has mild conditions, the method has high separation cost and difficulty in the later period, and can also cause a large amount of shrimp and crab shells to accumulate. The biological enzyme method is used for carrying out enzymolysis on chitin through enzyme catalysis reaction, is green in process, low in energy consumption and strong in specificity, is an optimal way for converting the chitin into GlcNAc, and has become a hotspot of chitin degradation research in recent years.
In the literature "Production of N-Acetyl-d-glucosamine from Bacterial waters by a Combination of Bacterial Chitinases and an Insect N-Acetyl-d-glucosamine [ J ]. Journal of Agricultural & Food Chemistry, 2016, 64(35): 6738" report Zhu et al, which uses three Bacterial-derived recombinant Chitinases (SmChiA, SmChiB, SmChiC) and one Insect-derived recombinant N-acetylglucosaminidase to synergistically degrade crystalline chitin, the ratio of GlcNAc to chitobiose reaches 93:7 after 24 h. The method for obtaining GlcNAc from degraded chitin needs the combined action of multiple enzymes, has high cost and long conversion time, and cannot be applied to large-scale production.
Application No. 201910242877.4 discloses a polypeptide derived fromChitinolyticbacter meiyuanensis Chitinase of SYBC-H1 strain. When the chitinase uses colloidal chitin as a substrate, the main hydrolysate is chitobiose, and GlcNAc cannot be directly obtained.
Application No. 201410785604.1 discloses a method for directly converting chitin to N-acetylglucosamine by crude enzyme solution produced by microbial fermentation. The method needs the synergistic effect of a plurality of chitinases in the process of degrading chitin, the product is not easy to separate, and the degradation process is easy to contaminate bacteria.
In combination with some literatures and patent reports, enzymatic preparation of GlcNAc requires synergistic action of multiple chitinases such as endo-chitinase (EC 3.2.1.14), exo-chitinase (EC 3.2.1.20) and (EC 3.2.1.201) and N-acetylglucosaminidase (NAGase, EC 3.2.1.52), and at present, industrial production of GlcNAc has not been realized due to high cost of extraction and purification of multiple enzymes and low conversion rate. Therefore, the method for preparing the GlcNAc by converting chitin by a single enzyme method by digging chitinase with multiple catalytic activities is an effective method capable of reducing the production cost.
Disclosure of Invention
Enzymatic degradation of chitin to produce GlcNAc requires the synergistic participation of multiple enzymes, and industrial production has not been achieved due to the high cost of extraction and purification of multiple enzymes. The invention provides a glycoside hydrolaseCmChi3 and application thereof in degrading colloidal chitin, and aims to realize a method for preparing GlcNAc by efficiently hydrolyzing chitin with single enzyme. The method can effectively reduce the enzyme extraction cost and simplify the production process, and is an effective way for preparing GlcNAc by using chitin as a raw material in a biological enzyme method.
The results of comparison of amino acid sequence homology showedCmChi3 belongs to the glycoside hydrolase GH18 family (http:// www.cazy.org /).
Glycoside hydrolaseCmChi3, the amino acid sequence of which is as given in ID NO: 2 is shown in the specification;
the SEN ID NO: 2 specifically, the following:
MLKQTRAPYGAKPAFALKTLSAALLLASSVASATEIAPYFEMWYGGASGYPAPTLASAQQQLGLKSVTLAFTIASNGQCKISNDGGGNDLLNGAMKGDIASFRQAGGRVIMSFGGAAGTYIEAVCSVDQMVSLIEGMILNHGIRALDFDVEGGQLSNTQLNNTRNAALKALQAKYPDLYVSFTLPVLPSGLTSPGVAVVRSAAQAGVRVDLVNVMAMDYGSSISSGKKMGDLAVQAAQSLFNQIKPIFPTKTDAELWSMVGVTPMIGQNDVQGEVFTLADAQTLTDFAKQKGLGLIAWWSFQRDRTGTGSYGEYSQVNKANFDFYNIFKAAGTPGSSPTPSPVTPTPVTPVPVTPAPVTPTPVAGQYPEWSASGVYTGGQRVTYQGAVYEAKWWTQGDNPGQSGEWGVWKKVGGAPSPVTPAPVTPAPVTPAPVTPAPVTPAPVTPAPVTPVPVTPAPSGCYAAWAEGKTYAAGTQVTYNNRNYQALVAHTAYAGTGWNPAASPTLWKDIGACSGSPSPVTPAPVTPAPVTPAPVTPAPVTPVPVTPAPVTPAPVTPVPAGDRVVGSYFTQWGVYGRGYEVADVVSSGSASQLTFINYAFGNIYQKNGGYECAAGIDKLESGATNPSDPSAGTGGDAWADYGRTPGRLVDPSKPYTWESPLAGNFGELKNLKAKYPQLKVLISLGGWTWSKWFSAASKTDALRKQLVSSCVDVFIKGNLPSYSGRGGPGSAKGVFDGIDIDWEYPGVIGQPYNTIDAADKQNFTLLLAEFRRQLDALNDGHKLLTVAIGSGKDKIDQTEPAKYSQYLDWINVMTYDFHGGWEATGPTNFQSHLYPDPADPSQGTARTYNVDDAIKNMIAAGTPAKKLVVGVPFYGRGWTGVAAGGNNGLYQAATGPARGTYEAGIEDYKVLKNAAGTVYVHPVTKQSYKYDGTNWWSYDTPAVIQTKIDYVKSQGLGGVFSWSLDGDSNGELAKVLGNGR。
nucleotide sequences encoding the above amino acid sequences such as SEN ID NO: 1 is shown in the specification;
the SEN ID NO: 1 specifically the following:
ATGTTGAAGC AAACCCGGGC GCCTTACGGC GCCAAGCCGG CTTTTGCGCT GAAAACGCTG 60
AGCGCGGCCC TGCTGCTGGC ATCGAGCGTT GCCAGCGCCA CCGAGATCGC CCCGTACTTC 120
GAGATGTGGT ACGGCGGCGC GAGCGGCTAT CCCGCCCCCA CCCTCGCTTC GGCCCAGCAG 180
CAGCTCGGCC TCAAGAGCGT GACACTGGCG TTCACCATCG CCAGCAATGG CCAGTGCAAG 240
ATCTCCAATG ACGGCGGTGG CAACGATCTG CTGAATGGCG CGATGAAGGG CGATATCGCC 300
AGCTTCCGCC AAGCCGGTGG TCGCGTGATC ATGTCGTTCG GCGGCGCCGC TGGTACCTAT 360
ATCGAGGCGG TGTGTTCGGT CGACCAGATG GTCAGCCTGA TCGAGGGCAT GATCCTCAAC 420
CACGGCATCC GCGCGCTGGA CTTCGACGTC GAGGGCGGCC AGCTTTCCAA CACCCAGCTC 480
AACAACACCC GCAATGCCGC ACTGAAGGCA CTGCAGGCCA AGTATCCGGA TCTGTATGTG 540
TCGTTCACGC TGCCGGTGCT GCCGAGCGGC CTGACCAGCC CGGGCGTGGC CGTGGTGCGT 600
TCCGCTGCGC AAGCTGGCGT GCGCGTTGAC CTCGTCAACG TGATGGCGAT GGATTACGGC 660
TCCAGCATCT CCAGCGGCAA GAAGATGGGC GACCTCGCTG TCCAGGCCGC GCAATCGCTG 720
TTCAACCAGA TCAAGCCGAT CTTCCCGACC AAGACCGATG CCGAATTGTG GTCCATGGTC 780
GGCGTGACGC CGATGATTGG CCAGAACGAC GTGCAGGGTG AAGTCTTCAC CCTGGCCGAT 840
GCGCAGACGC TGACCGACTT CGCCAAGCAG AAGGGCCTTG GCCTGATCGC CTGGTGGTCG 900
TTCCAGCGCG ACCGCACTGG TACGGGCAGC TACGGCGAAT ACAGCCAAGT CAATAAGGCC 960
AACTTCGACT TCTACAACAT CTTCAAGGCT GCCGGCACGC CGGGCAGCTC GCCGACGCCG 1020
TCGCCGGTGA CCCCTACCCC GGTCACCCCG GTACCGGTGA CCCCAGCGCC TGTGACGCCG 1080
ACCCCGGTGG CCGGCCAGTA TCCGGAATGG AGCGCGAGCG GCGTCTACAC CGGTGGCCAG 1140
CGCGTGACCT ATCAAGGTGC CGTGTACGAA GCCAAGTGGT GGACCCAGGG CGACAACCCG 1200
GGCCAATCCG GCGAATGGGG CGTATGGAAG AAGGTTGGCG GCGCGCCGTC CCCGGTGACC 1260
CCGGCTCCCG TGACACCCGC TCCGGTCACC CCGGCTCCGG TCACACCTGC CCCGGTGACT 1320
CCGGCCCCGG TGACTCCGGC CCCTGTGACG CCCGTTCCGG TCACCCCGGC GCCGTCCGGC 1380
TGCTATGCGG CCTGGGCTGA AGGCAAGACC TACGCCGCCG GCACCCAGGT CACCTACAAC 1440
AACCGCAATT ACCAGGCGCT GGTGGCGCAC ACCGCCTACG CCGGTACTGG CTGGAACCCT 1500
GCCGCGAGCC CGACGCTGTG GAAGGACATT GGCGCCTGCA GCGGCTCGCC GAGCCCGGTC 1560
ACCCCGGCGC CGGTGACACC CGCTCCCGTA ACGCCTGCGC CGGTCACTCC GGCCCCTGTC 1620
ACCCCGGTTC CGGTCACCCC GGCTCCCGTG ACGCCCGCTC CGGTCACCCC GGTCCCGGCC 1680
GGTGATCGCG TCGTCGGCTC CTACTTCACC CAGTGGGGCG TGTACGGCCG TGGCTACGAA 1740
GTGGCCGATG TGGTCAGCAG CGGCAGCGCG TCGCAGCTCA CCTTCATCAA CTACGCCTTC 1800
GGCAATATCT ACCAGAAGAA TGGCGGCTAC GAGTGCGCGG CCGGCATCGA CAAGCTGGAA 1860
TCGGGCGCGA CCAACCCGAG CGATCCGTCC GCCGGCACCG GTGGTGACGC CTGGGCCGAC 1920
TACGGCCGCA CACCGGGCCG CCTGGTCGAT CCGAGCAAGC CCTACACCTG GGAATCGCCG 1980
CTGGCCGGTA ACTTCGGCGA GCTGAAGAAC CTGAAGGCCA AGTATCCTCA GCTCAAGGTG 2040
CTGATCTCGC TGGGCGGCTG GACTTGGTCG AAGTGGTTCT CGGCAGCCAG CAAGACCGAC 2100
GCACTGAGGA AGCAGTTGGT GAGCTCGTGC GTGGACGTGT TCATCAAGGG CAACCTGCCG 2160
AGCTACAGCG GTCGCGGTGG TCCTGGTTCG GCCAAGGGCG TGTTCGACGG CATCGACATC 2220
GACTGGGAAT ACCCGGGCGT GATCGGCCAG CCGTACAACA CCATCGACGC TGCCGACAAG 2280
CAGAACTTCA CGCTGCTGCT GGCCGAGTTC CGCCGCCAGC TCGACGCGCT GAACGACGGC 2340
CACAAGCTCC TGACCGTGGC CATCGGCTCG GGCAAGGACA AGATCGACCA GACCGAGCCG 2400
GCCAAGTACA GCCAGTACCT CGACTGGATC AACGTGATGA CCTACGACTT CCACGGTGGT 2460
TGGGAAGCGA CTGGTCCGAC CAACTTCCAG TCGCACCTGT ACCCGGATCC GGCCGATCCG 2520
TCGCAAGGCA CCGCGCGGAC CTACAACGTC GACGATGCGA TCAAGAACAT GATCGCCGCC 2580
GGTACACCGG CCAAGAAGCT GGTGGTGGGC GTGCCGTTCT ACGGCCGTGG CTGGACCGGT 2640
GTCGCCGCAG GTGGCAATAA CGGTCTGTAC CAGGCTGCGA CTGGCCCGGC CCGTGGCACG 2700
TACGAAGCCG GCATCGAGGA CTACAAGGTA CTGAAGAACG CCGCCGGTAC CGTGTATGTC 2760
CACCCGGTGA CCAAGCAGTC GTACAAGTAC GATGGCACCA ACTGGTGGTC GTACGATACG 2820
CCGGCCGTGA TCCAAACCAA GATCGACTAC GTGAAGTCGC AAGGCCTGGG TGGCGTGTTC 2880
AGCTGGTCGC TCGATGGCGA CTCCAACGGC GAACTTGCCA AGGTGCTGGG CAACGGCCGC 2940
TAA 2943。
a recombinant expression vector comprising the glycoside hydrolase-encoding gene of claim 2CmA recombinant expression vector of the nucleotide sequence of Chi3 gene.
A recombinant bacterium which expresses a strain containing the recombinant expression vector according to claim 3.
Based on the glycoside hydrolaseCmThe cloning expression of Chi3 gene comprises the following steps:
step 1, amplifying a nucleotide sequence shown as SEQ ID No.1 by PCR;
step 2, constructing recombinant plasmid pCold I-CmChi3
Carrying out double enzyme digestion on the DNA sequence amplified by the PCR and the pCold I vector by using the same restriction enzyme, recovering and purifying enzyme digestion products, and connecting the purified enzyme digestion products by using T4 DNA ligase to obtain recombinant plasmids; pCold I-CmChi3；
Step 3, recombinant plasmid pCold I-CmChi3Introduced into Escherichia coli BL21(DE3) to obtain a recombinant plasmid containing pCold I-CmChi3The Escherichia coli BL21(DE3) was named recombinant strain;
step 4, selecting a monoclonal of the recombinant bacteria, inoculating the seed solution into an LB culture medium with the inoculum size of 1-5% by volume fraction after overnight culture in a shaking table, and culturing at 37 ℃ until the bacterial density OD600When the concentration is 0.4-0.6, adding an inducer to induce for 12-14h, collecting fermentation liquor, performing centrifugation at 6000-;
and 5, adding a buffer solution into the precipitate to resuspend the strain, centrifuging after ultrasonic lysis, collecting supernatant, and freezing for later use.
As an improvement, the primers used for PCR amplification in step 1 are:
CmChi3-F：5’-GGGAATTCCATATGACCGAGATCGCCCCGTAC-3’；
CmChi3-R：5’-CCGGAATTCGCGGCCGTTGCCCAGCAC-3’。
the glycoside hydrolaseCmUse of Chi3 for the hydrolysis of colloidal chitin, which is degraded to N-acetylglucosamine.
As an improvement, glycoside hydrolaseCmChi3 hydrocolloid chitin has a temperature of 25-55 deg.C and a pH of 5.0-8.0.
Has the advantages that:
compared with the prior art, the glycoside hydrolase of the inventionCmChi3 and the application thereof in degrading hydrocolloid chitin have the following advantages:
the bifunctional chitinase of the inventionCmChi3 has the properties of both endo-chitinase and N-acetylglucosaminidase (NAGase), and can efficiently degrade chitin and GlcNAc as a final product. The optimum temperature is 50 ℃, and the optimum pH is 6.0; incubating for 2h below 50 ℃ to keep the activity of more than 80 percent; can keep higher activity under the pH value of 5.0-8.0. Chitinase according to the inventionCmChi3 belongs to a novel multifunctional chitinase with double catalytic domainsCmChi3, can realize the high-efficiency conversion of chitin into a single product GlcNAc by a single enzyme.
Drawings
FIG. 1 is chitinaseCmConserved domain schematic of Chi 3;
FIG. 2 is chitinaseCmAn SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) picture of Chi3, wherein M is a protein marker which is respectively 180 kDa, 130 kDa, 95 kDa, 70 kDa, 53 kDa, 40 kDa and 33kDa from top to bottom, 1 is a purified band, and 2 is a crude enzyme band after induction expression of a recombinant strain;
FIG. 3 is a graph of temperature vs. chitinase in accordance with the present inventionCmEffect of Chi3 activity;
FIG. 4 is a graph of pH vs. chitinase in accordance with the present inventionCmEffect of Chi3 activity;
FIG. 5 is chitinaseCmChi3 shows the hydrolysis pattern of chitin substrate.
Detailed description of the preferred embodiments
The present invention is further described by the following examples, which are not intended to limit the scope of the present invention, and the experimental methods in the examples are conventional methods unless otherwise specified.
Example 1
Bacterial source
Strains used in the inventionChitinolyticbacter meiyuanensis SYBC-H1 screened by the laboratory and preserved in China general microbiological culture Collection center (CGMCC 3438) and the American type culture Collection (ATCC BAA-2140).
Example 2
ChitinaseCmThe construction of Chi3 recombinant strains comprises the following specific steps:
(1) obtaining chitinaseCmChi3, the nucleotide sequence of which is shown as SEN ID NO: 1 is shown.
(2) Designing PCR amplification primers:
CmChi3-F：5’-GGGAATTCCATATGACCGAGATCGCCCCGTAC-3’，
Cmchi 3-R: 5'-CCGGAATTCGCGGCCGTTGCCCAGCAC-3' amplifying the gene to its full length;
(3) PCR amplification procedure:
preheating at 95 ℃ for 5 min; denaturation at 94 ℃ for 45s, annealing at 55 ℃ for 45s, extension at 72 ℃ for 1.5 min, 30 cycles; extending for 10min at 72 ℃; storing at 4 deg.C;
(4) target gene after PCR amplificationCmBoth Chi3 and plasmid Pcoled I use restriction enzymesNdeI and EcoR I, incubating for 1h at 37 ℃, purifying the target gene after enzyme digestion by using a common DNA purification kit, storing the purified target gene at-20 ℃ for later use, carrying out agarose gel electrophoresis on the vector after enzyme digestion, recovering the vector by using an agarose gel recovery kit, and storing the recovered vector at-20 ℃ for later use;
(5) the target gene segments recovered from the gel are respectively connected with the corresponding carriers after enzyme digestion by T4 ligase; converting the linked liquid toE.coliDH5 α, transformation step: competent cellE.coliTaking out DH5 alpha from-80 ℃, and rapidly placing on ice for melting; adding connecting liquid into the thawed competent cells, and placing on ice for 30 min; carrying out water bath heat shock for 45s at 42 ℃, and then rapidly placing on ice for 2-3 min; adding the liquid after heat shock into 800 mu L LB liquid culture medium, culturing for 1h at 37 ℃; after 1h of culture, the bacterial solution was spread on LB solid medium containing 0.05g/L ampicillin (Amp), and cultured overnight at 37 ℃;
(6) clones on the transformation plate were verified by colony PCR, and positive clones were screened. And selecting the colony with correct colony PCR verification to 5mL of liquid LB culture medium containing 0.05g/L Amp, culturing at 37 ℃ for 12h, extracting the plasmid by using a plasmid extraction kit, and sending to Nanjing engine biotechnology Limited for sequencing. And comparing the sequencing result with the gene, wherein the plasmid with the correct comparison result is the successfully constructed recombinant plasmid, and meanwhile, the successfully constructed recombinant plasmid is stored at the temperature of-20 ℃ for later use.
Example 3
Recombinant chitinaseCmChi3 expression and purification
(1) After the preserved recombinant expression strain is revived on a plate, a single colony is selected and inoculated in 5mL LB liquid culture medium containing 0.05g/L Amp, and cultured for 8-10 h at the temperature of 37 ℃ and the rpm of 200. The activated bacterial liquid was transferred to a 500 mL Erlenmeyer flask containing 100 mL LB medium (0.05 g/L Amp) at an inoculum size of 1% by volume, and cultured at 37 ℃ and 200rpm to OD600And adding an inducer IPTG (isopropyl-beta-D-thiogalactoside) for induction at the temperature of between 0.6 and 0.8, wherein the induction temperature is 18 ℃ and the induction time is 16 hours.
(2) After the culture, the cells were centrifuged at 6000 rpm at 4 ℃ for 10min to collect the cells. After disruption with an ultrasonic disruptor, centrifugation was carried out at 8000 rpm for 20 min at 4 ℃ and the supernatant was collected. The supernatant is the crude enzyme solution containing recombinant chitinase, and is stored at 4 ℃ for later use.
(3) The collected supernatant crude enzyme solution utilizes Ni2+And (3) purifying the protein by using an NTA chromatographic column and an AKTA protein purification system, carrying out SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) electrophoretic analysis on the purified recombinant protein solution, wherein the percentage of the upper concentrated gel is 5%, the lower separated gel is 8%, dyeing the gel by using a protein dyeing solution after the electrophoresis is finished, and then carrying out 3 times of decolorization by using a decolorizing solution. A protein band of 103kDa was obtained (as shown in FIG. 2),
the enzyme activity unit (U) is defined as: the amount of enzyme required to produce 1. mu. mol of reducing sugar per minute at 37 ℃. According to the enzyme activity calculation method, the enzyme activity is 4.36U/mg (colloidal chitin is taken as a substrate).
Example 4
ChitinaseCmChi3 enzyme Activity identification
The activity of the glycosidase can be qualitatively detected by using 4-methylumbelliferone-N-acetyl-D glucosamine, which has fluorescence under ultraviolet. The purified enzyme was reacted with 4-methylumbelliferone-N-acetyl-D-glucosamine at a volume ratio of 1:10, and the 4-methylumbelliferone showed fluorescence at a wavelength of 450 nm. Whether it is active or not can be determined by whether it has fluorescence at 450nm or not.
Example 5
Recombinant chitinaseCmDetermination of optimum reaction temperature of Chi3
(1) Determination of recombinase by using 1% colloidal chitin as substrateCmThe enzyme activity of Chi3 is as follows: adding 450 μ L PBS (pH7.0, 50 mM) into 500 μ L substrate, preheating for 10min, adding 50 μ L diluted enzyme solution, reacting for 30min, boiling in boiling water bath for 5min to inactivate enzyme, adding 1ml DNS solution, and boiling at 100 deg.C for 5 min. After cooling to room temperature, centrifugation was carried out at 12000rpm for 10min, and the supernatant was taken to measure its absorbance at an absorbance value of 540 nm, and an inactivated equivalent enzyme solution was used as a blank.
The enzyme activity unit (U) is defined as: the amount of enzyme required to produce 1. mu. mol of reducing sugar per minute at 37 ℃.
(2) Protein content determination with reference to Coomassie Brilliant blue method, protein concentration was determined to be 0.66 g/L.
(3) The reaction temperatures were: measuring at 25 deg.C, 30 deg.C, 35 deg.C, 40 deg.C, 45 deg.C, 50 deg.C, 55 deg.C, 60 deg.CCmChi3, the results of which were averaged over 3 replicates.
(4) The results are shown in FIG. 3, where the enzyme activity was highest at a reaction temperature of 50 ℃ and the highest enzyme reaction rate was 100%, and the remaining enzyme activities were calculated and plotted.
Example 6
Recombinant chitinaseCmDetermination of temperature stability of Chi3
(1) Will be provided withCmChi3 protein solution is incubated at 25 deg.C, 30 deg.C, 35 deg.C, 40 deg.C, 45 deg.C, 50 deg.C, 55 deg.C, and 60 deg.C for 2 h.
(2) Determination of recombinase by using 1% colloidal chitin as substrateCmEnzyme activity of Chi 3. Adding 450 μ L PBS (pH 7.0, 50 mM) into 500 μ L substrate, preheating for 10min, adding 50 μ L diluted enzyme solution, reacting for 30min, boiling in boiling water bath for 5min to inactivate enzyme, and boiling for 5min1mL of DNS solution was added and boiled at 100 ℃ for 5 min. After cooling to room temperature, centrifugation was carried out at 12000rpm for 10min, and the supernatant was taken to measure its absorbance at an absorbance value of 540 nm, and an inactivated equivalent enzyme solution was used as a blank. The enzyme activity unit (U) is defined as: the amount of enzyme required to produce 1. mu. mol of reducing sugar per minute at 37 ℃.
(3) Protein content determination with reference to Coomassie Brilliant blue method, protein concentration was determined to be 0.66 g/L.
(4) Will not be incubatedCmThe enzyme activity of Chi3 protein solution is 100%, and the relative enzyme activity (%) of each temperature pretreatment is calculated, and the result is shown in FIG. 3.
(5) The results show that:Cmchi3 shows that after 2 hours of treatment, the reaction rate of enzyme is 100%, 98%, 95.4%, 91.55% of the reaction rate of enzyme which is not pretreated at 25 ℃, 30 ℃, 35 ℃, 40 ℃, 45 ℃, 50 ℃, 55 ℃ and 60 ℃ respectively,
85.7%, 53.6%, 20.9% and 18.4%. The stability decreases sharply at a temperature of 45 ℃ or higher.
Example 7
Determination of the optimum pH of the recombinant chitinase CmChi3
(1) Determination of recombinase by using 1% colloidal chitin as substrateCmEnzyme activity of Chi 3. mu.L of substrate was taken and 850. mu.L of different pH buffers were added: citric acid-sodium citrate buffer solution (pH 3.5-6.0), phosphate buffer solution (pH 6.0-8.0), Tis-HCl (pH 8.0-9.0), glycine-sodium hydroxide (pH 9.0-10.0). Preheating for 10min, adding 50 μ L of diluted enzyme solution, reacting for 30min, boiling in boiling water bath for 5min to inactivate enzyme, adding 1mL of DNS solution, and boiling at 100 deg.C for 5 min. After cooling to room temperature, centrifugation was carried out at 12000rpm for 10min, and the supernatant was taken to measure its absorbance at an absorbance value of 540 nm, and an inactivated equivalent enzyme solution was used as a blank.
(2) The enzyme activity unit (U) is defined as: the amount of enzyme required to produce 1. mu. mol of reducing sugar per minute at 37 ℃.
(3) Protein content determination with reference to Coomassie Brilliant blue method, protein concentration was determined to be 0.66 g/L.
(4) The enzyme activity was highest at pH6.0, the highest enzyme reaction rate was taken as 100%, and the reaction rates at other pH were divided by the highest reaction rate to give the corresponding relative reaction rates, the results are shown in fig. 4.
(5) The results show thatCmChi3 has an optimum pH of 6.0 as a glycoside hydrolase and a high activity at a pH of 5.5 to 8.0.
Example 8
Recombinant chitinaseCmpH stability test of Chi3
(1) Will be provided withCmChi3 protein solution and buffer solution with different pH are mixed according to the equal volume, and incubated for 2 hours at 25 ℃; when the buffer solution is citric acid-sodium citrate buffer solution (pH 3.5-6.0), phosphate buffer solution (pH 6.0-8.0), Tis-HCl (pH 8.0-9.0), and glycine-sodium hydroxide (pH 9.0-10.0), respectively.
(2) Determining recombinase with 1% colloidal chitin as substrate by the pretreated protein solutionCmEnzyme activity of Chi 3. Adding 850 μ L of buffer solution with different pH values into 100 μ L of substrate, reacting at 37 deg.C and pH6.0 for 30min with the same enzyme amount of 50 μ L, boiling in boiling water bath for 5min to inactivate enzyme, adding 1mL of DNS solution, and boiling at 100 deg.C for 5 min. After cooling to room temperature, centrifugation was carried out at 12000rpm for 10min, and the supernatant was taken to measure its absorbance at an absorbance value of 540 nm, and an inactivated equivalent enzyme solution was used as a blank.
(3) The enzyme activity unit (U) is defined as: the amount of enzyme required to produce 1. mu. mol of reducing sugar per minute at 37 ℃.
(4) Protein content determination is carried out by referring to a Coomassie brilliant blue method, and the protein concentration is determined to be 0.66 g/L;
(5) the results are shown in figure 4 of the drawings,Cmthe relative viability of Chi3 protein fluid when stored at pH5.0, 6.0, 7.0, 8.0 (calculated as in example 6) is as follows: 67.5%, 96.7%, 96.3%, 95.5%;
(6) the results show thatCmChi3 protein solution is stable at pH5.0-8.0, which indicates that it has better pH stability under neutral condition.
Example 9
Recombinant chitinaseCmDetermination of hydrolysis Pattern of Chi3
(1) The chitin oligosaccharide (GlcNAc) to be purchased2-6 (Qingdao)Bozhihui biotechnology limited) standard substance is prepared into 10g/L aqueous solution;
(2) the results were analyzed by High Performance Liquid Chromatography (HPLC) using 1% colloidal chitin as a substrate, using a Prevail Carbohydrate ES 5u chromatographic column of ALLTECH;
(3) the mobile phase comprises acetonitrile and methanol, and the gradient elution conditions are as follows: 0min, 75% acetonitrile, 7min, 75% acetonitrile; 8min, 65% acetonitrile; 15min, 65% acetonitrile; 16min, 75% acetonitrile; 22min, 75% acetonitrile. The column oven temperature was 40 ℃ and the flow rate was 1 mL/min.
(4) The results are shown in FIG. 5, the enzymeCmChi3 cleaves colloidal chitin into monosaccharides, disaccharides and trisaccharides at the very beginning and exhibits endoenzyme activity, with the monosaccharide content in the product becoming higher with increasing cleavage time, and to the end, with the presence of only monosaccharides, exhibiting glycosidase activity. Thus, the enzyme of the invention has two distinct chitinase activities, an endonuclease activity and a glycosidase activity, respectively, which are capable of specifically hydrolyzing chitin to N-acetylglucosamine.
The above-mentioned embodiments, objects, technical solutions and advantages of the present invention are further described in detail, it should be understood that the above-mentioned embodiments are only illustrative of the present invention and are not intended to limit the present invention, and any modifications, equivalents, improvements and the like made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Sequence listing
<110> Nanjing university of industry
<120> glycoside hydrolase CmChi3 and application thereof in degradation of hydrocolloid chitin
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 32
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
gggaattcca tatgaccgag atcgccccgt ac 32
<210> 2
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
ccggaattcg cggccgttgc ccagcac 27
<210> 3
<211> 2943
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
atgttgaagc aaacccgggc gccttacggc gccaagccgg cttttgcgct gaaaacgctg 60
agcgcggccc tgctgctggc atcgagcgtt gccagcgcca ccgagatcgc cccgtacttc 120
gagatgtggt acggcggcgc gagcggctat cccgccccca ccctcgcttc ggcccagcag 180
cagctcggcc tcaagagcgt gacactggcg ttcaccatcg ccagcaatgg ccagtgcaag 240
atctccaatg acggcggtgg caacgatctg ctgaatggcg cgatgaaggg cgatatcgcc 300
agcttccgcc aagccggtgg tcgcgtgatc atgtcgttcg gcggcgccgc tggtacctat 360
atcgaggcgg tgtgttcggt cgaccagatg gtcagcctga tcgagggcat gatcctcaac 420
cacggcatcc gcgcgctgga cttcgacgtc gagggcggcc agctttccaa cacccagctc 480
aacaacaccc gcaatgccgc actgaaggca ctgcaggcca agtatccgga tctgtatgtg 540
tcgttcacgc tgccggtgct gccgagcggc ctgaccagcc cgggcgtggc cgtggtgcgt 600
tccgctgcgc aagctggcgt gcgcgttgac ctcgtcaacg tgatggcgat ggattacggc 660
tccagcatct ccagcggcaa gaagatgggc gacctcgctg tccaggccgc gcaatcgctg 720
ttcaaccaga tcaagccgat cttcccgacc aagaccgatg ccgaattgtg gtccatggtc 780
ggcgtgacgc cgatgattgg ccagaacgac gtgcagggtg aagtcttcac cctggccgat 840
gcgcagacgc tgaccgactt cgccaagcag aagggccttg gcctgatcgc ctggtggtcg 900
ttccagcgcg accgcactgg tacgggcagc tacggcgaat acagccaagt caataaggcc 960
aacttcgact tctacaacat cttcaaggct gccggcacgc cgggcagctc gccgacgccg 1020
tcgccggtga cccctacccc ggtcaccccg gtaccggtga ccccagcgcc tgtgacgccg 1080
accccggtgg ccggccagta tccggaatgg agcgcgagcg gcgtctacac cggtggccag 1140
cgcgtgacct atcaaggtgc cgtgtacgaa gccaagtggt ggacccaggg cgacaacccg 1200
ggccaatccg gcgaatgggg cgtatggaag aaggttggcg gcgcgccgtc cccggtgacc 1260
ccggctcccg tgacacccgc tccggtcacc ccggctccgg tcacacctgc cccggtgact 1320
ccggccccgg tgactccggc ccctgtgacg cccgttccgg tcaccccggc gccgtccggc 1380
tgctatgcgg cctgggctga aggcaagacc tacgccgccg gcacccaggt cacctacaac 1440
aaccgcaatt accaggcgct ggtggcgcac accgcctacg ccggtactgg ctggaaccct 1500
gccgcgagcc cgacgctgtg gaaggacatt ggcgcctgca gcggctcgcc gagcccggtc 1560
accccggcgc cggtgacacc cgctcccgta acgcctgcgc cggtcactcc ggcccctgtc 1620
accccggttc cggtcacccc ggctcccgtg acgcccgctc cggtcacccc ggtcccggcc 1680
ggtgatcgcg tcgtcggctc ctacttcacc cagtggggcg tgtacggccg tggctacgaa 1740
gtggccgatg tggtcagcag cggcagcgcg tcgcagctca ccttcatcaa ctacgccttc 1800
ggcaatatct accagaagaa tggcggctac gagtgcgcgg ccggcatcga caagctggaa 1860
tcgggcgcga ccaacccgag cgatccgtcc gccggcaccg gtggtgacgc ctgggccgac 1920
tacggccgca caccgggccg cctggtcgat ccgagcaagc cctacacctg ggaatcgccg 1980
ctggccggta acttcggcga gctgaagaac ctgaaggcca agtatcctca gctcaaggtg 2040
ctgatctcgc tgggcggctg gacttggtcg aagtggttct cggcagccag caagaccgac 2100
gcactgagga agcagttggt gagctcgtgc gtggacgtgt tcatcaaggg caacctgccg 2160
agctacagcg gtcgcggtgg tcctggttcg gccaagggcg tgttcgacgg catcgacatc 2220
gactgggaat acccgggcgt gatcggccag ccgtacaaca ccatcgacgc tgccgacaag 2280
cagaacttca cgctgctgct ggccgagttc cgccgccagc tcgacgcgct gaacgacggc 2340
cacaagctcc tgaccgtggc catcggctcg ggcaaggaca agatcgacca gaccgagccg 2400
gccaagtaca gccagtacct cgactggatc aacgtgatga cctacgactt ccacggtggt 2460
tgggaagcga ctggtccgac caacttccag tcgcacctgt acccggatcc ggccgatccg 2520
tcgcaaggca ccgcgcggac ctacaacgtc gacgatgcga tcaagaacat gatcgccgcc 2580
ggtacaccgg ccaagaagct ggtggtgggc gtgccgttct acggccgtgg ctggaccggt 2640
gtcgccgcag gtggcaataa cggtctgtac caggctgcga ctggcccggc ccgtggcacg 2700
tacgaagccg gcatcgagga ctacaaggta ctgaagaacg ccgccggtac cgtgtatgtc 2760
cacccggtga ccaagcagtc gtacaagtac gatggcacca actggtggtc gtacgatacg 2820
ccggccgtga tccaaaccaa gatcgactac gtgaagtcgc aaggcctggg tggcgtgttc 2880
agctggtcgc tcgatggcga ctccaacggc gaacttgcca aggtgctggg caacggccgc 2940
taa 2943
<210> 4
<211> 980
<212> PRT
<213> Amino acid (Amino acid)
<400> 4
Met Leu Lys Gln Thr Arg Ala Pro Tyr Gly Ala Lys Pro Ala Phe Ala
1 5 10 15
Leu Lys Thr Leu Ser Ala Ala Leu Leu Leu Ala Ser Ser Val Ala Ser
20 25 30
Ala Thr Glu Ile Ala Pro Tyr Phe Glu Met Trp Tyr Gly Gly Ala Ser
35 40 45
Gly Tyr Pro Ala Pro Thr Leu Ala Ser Ala Gln Gln Gln Leu Gly Leu
50 55 60
Lys Ser Val Thr Leu Ala Phe Thr Ile Ala Ser Asn Gly Gln Cys Lys
65 70 75 80
Ile Ser Asn Asp Gly Gly Gly Asn Asp Leu Leu Asn Gly Ala Met Lys
85 90 95
Gly Asp Ile Ala Ser Phe Arg Gln Ala Gly Gly Arg Val Ile Met Ser
100 105 110
Phe Gly Gly Ala Ala Gly Thr Tyr Ile Glu Ala Val Cys Ser Val Asp
115 120 125
Gln Met Val Ser Leu Ile Glu Gly Met Ile Leu Asn His Gly Ile Arg
130 135 140
Ala Leu Asp Phe Asp Val Glu Gly Gly Gln Leu Ser Asn Thr Gln Leu
145 150 155 160
Asn Asn Thr Arg Asn Ala Ala Leu Lys Ala Leu Gln Ala Lys Tyr Pro
165 170 175
Asp Leu Tyr Val Ser Phe Thr Leu Pro Val Leu Pro Ser Gly Leu Thr
180 185 190
Ser Pro Gly Val Ala Val Val Arg Ser Ala Ala Gln Ala Gly Val Arg
195 200 205
Val Asp Leu Val Asn Val Met Ala Met Asp Tyr Gly Ser Ser Ile Ser
210 215 220
Ser Gly Lys Lys Met Gly Asp Leu Ala Val Gln Ala Ala Gln Ser Leu
225 230 235 240
Phe Asn Gln Ile Lys Pro Ile Phe Pro Thr Lys Thr Asp Ala Glu Leu
245 250 255
Trp Ser Met Val Gly Val Thr Pro Met Ile Gly Gln Asn Asp Val Gln
260 265 270
Gly Glu Val Phe Thr Leu Ala Asp Ala Gln Thr Leu Thr Asp Phe Ala
275 280 285
Lys Gln Lys Gly Leu Gly Leu Ile Ala Trp Trp Ser Phe Gln Arg Asp
290 295 300
Arg Thr Gly Thr Gly Ser Tyr Gly Glu Tyr Ser Gln Val Asn Lys Ala
305 310 315 320
Asn Phe Asp Phe Tyr Asn Ile Phe Lys Ala Ala Gly Thr Pro Gly Ser
325 330 335
Ser Pro Thr Pro Ser Pro Val Thr Pro Thr Pro Val Thr Pro Val Pro
340 345 350
Val Thr Pro Ala Pro Val Thr Pro Thr Pro Val Ala Gly Gln Tyr Pro
355 360 365
Glu Trp Ser Ala Ser Gly Val Tyr Thr Gly Gly Gln Arg Val Thr Tyr
370 375 380
Gln Gly Ala Val Tyr Glu Ala Lys Trp Trp Thr Gln Gly Asp Asn Pro
385 390 395 400
Gly Gln Ser Gly Glu Trp Gly Val Trp Lys Lys Val Gly Gly Ala Pro
405 410 415
Ser Pro Val Thr Pro Ala Pro Val Thr Pro Ala Pro Val Thr Pro Ala
420 425 430
Pro Val Thr Pro Ala Pro Val Thr Pro Ala Pro Val Thr Pro Ala Pro
435 440 445
Val Thr Pro Val Pro Val Thr Pro Ala Pro Ser Gly Cys Tyr Ala Ala
450 455 460
Trp Ala Glu Gly Lys Thr Tyr Ala Ala Gly Thr Gln Val Thr Tyr Asn
465 470 475 480
Asn Arg Asn Tyr Gln Ala Leu Val Ala His Thr Ala Tyr Ala Gly Thr
485 490 495
Gly Trp Asn Pro Ala Ala Ser Pro Thr Leu Trp Lys Asp Ile Gly Ala
500 505 510
Cys Ser Gly Ser Pro Ser Pro Val Thr Pro Ala Pro Val Thr Pro Ala
515 520 525
Pro Val Thr Pro Ala Pro Val Thr Pro Ala Pro Val Thr Pro Val Pro
530 535 540
Val Thr Pro Ala Pro Val Thr Pro Ala Pro Val Thr Pro Val Pro Ala
545 550 555 560
Gly Asp Arg Val Val Gly Ser Tyr Phe Thr Gln Trp Gly Val Tyr Gly
565 570 575
Arg Gly Tyr Glu Val Ala Asp Val Val Ser Ser Gly Ser Ala Ser Gln
580 585 590
Leu Thr Phe Ile Asn Tyr Ala Phe Gly Asn Ile Tyr Gln Lys Asn Gly
595 600 605
Gly Tyr Glu Cys Ala Ala Gly Ile Asp Lys Leu Glu Ser Gly Ala Thr
610 615 620
Asn Pro Ser Asp Pro Ser Ala Gly Thr Gly Gly Asp Ala Trp Ala Asp
625 630 635 640
Tyr Gly Arg Thr Pro Gly Arg Leu Val Asp Pro Ser Lys Pro Tyr Thr
645 650 655
Trp Glu Ser Pro Leu Ala Gly Asn Phe Gly Glu Leu Lys Asn Leu Lys
660 665 670
Ala Lys Tyr Pro Gln Leu Lys Val Leu Ile Ser Leu Gly Gly Trp Thr
675 680 685
Trp Ser Lys Trp Phe Ser Ala Ala Ser Lys Thr Asp Ala Leu Arg Lys
690 695 700
Gln Leu Val Ser Ser Cys Val Asp Val Phe Ile Lys Gly Asn Leu Pro
705 710 715 720
Ser Tyr Ser Gly Arg Gly Gly Pro Gly Ser Ala Lys Gly Val Phe Asp
725 730 735
Gly Ile Asp Ile Asp Trp Glu Tyr Pro Gly Val Ile Gly Gln Pro Tyr
740 745 750
Asn Thr Ile Asp Ala Ala Asp Lys Gln Asn Phe Thr Leu Leu Leu Ala
755 760 765
Glu Phe Arg Arg Gln Leu Asp Ala Leu Asn Asp Gly His Lys Leu Leu
770 775 780
Thr Val Ala Ile Gly Ser Gly Lys Asp Lys Ile Asp Gln Thr Glu Pro
785 790 795 800
Ala Lys Tyr Ser Gln Tyr Leu Asp Trp Ile Asn Val Met Thr Tyr Asp
805 810 815
Phe His Gly Gly Trp Glu Ala Thr Gly Pro Thr Asn Phe Gln Ser His
820 825 830
Leu Tyr Pro Asp Pro Ala Asp Pro Ser Gln Gly Thr Ala Arg Thr Tyr
835 840 845
Asn Val Asp Asp Ala Ile Lys Asn Met Ile Ala Ala Gly Thr Pro Ala
850 855 860
Lys Lys Leu Val Val Gly Val Pro Phe Tyr Gly Arg Gly Trp Thr Gly
865 870 875 880
Val Ala Ala Gly Gly Asn Asn Gly Leu Tyr Gln Ala Ala Thr Gly Pro
885 890 895
Ala Arg Gly Thr Tyr Glu Ala Gly Ile Glu Asp Tyr Lys Val Leu Lys
900 905 910
Asn Ala Ala Gly Thr Val Tyr Val His Pro Val Thr Lys Gln Ser Tyr
915 920 925
Lys Tyr Asp Gly Thr Asn Trp Trp Ser Tyr Asp Thr Pro Ala Val Ile
930 935 940
Gln Thr Lys Ile Asp Tyr Val Lys Ser Gln Gly Leu Gly Gly Val Phe
945 950 955 960
Ser Trp Ser Leu Asp Gly Asp Ser Asn Gly Glu Leu Ala Lys Val Leu
965 970 975
Gly Asn Gly Arg
980