CN111218464A - Kelp mannuronic acid C5-isomerase gene and application thereof - Google Patents

Kelp mannuronic acid C5-isomerase gene and application thereof Download PDF

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CN111218464A
CN111218464A CN201910584945.5A CN201910584945A CN111218464A CN 111218464 A CN111218464 A CN 111218464A CN 201910584945 A CN201910584945 A CN 201910584945A CN 111218464 A CN111218464 A CN 111218464A
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段德麟
张朋艳
邵展茹
卢畅
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Abstract

The invention belongs to the technical field of molecular biology, and relates to clone, expression and enzymatic analysis of a kelp MC5E gene sequence. In particular to a kelp mannuronic acid C5-isomerase gene and application thereof. The gene has a base sequence in a sequence table SED IQ No. 1. The protein coded by the MC5E gene has an amino acid sequence in a sequence table SED IQ No. 2. The recombinant protein of the invention is in Ca2+Under the existing condition, mannuronic acid (M) can be converted into guluronic acid (G), the G content in the algin is increased, the M/G of the algin is reduced, the viscosity of the algin can be increased, and the physicochemical properties of the algin are improved.

Description

Kelp mannuronic acid C5-isomerase gene and application thereof
The technical field is as follows:
the invention belongs to the technical field of molecular biology, and relates to clone, expression and enzymatic analysis of a kelp MC5E gene sequence. In particular to a kelp mannuronic acid C5-isomerase gene and application thereof.
Background art:
alginic acid (C)6H8O6)nis heteropolysaccharide formed by β -D-mannuronic acid (M) and α -L-guluronic acid (G) through β -1,4 glycosidic bond linear polymerization, alginic acid can be mixed with metal with valence more than 2The ionic bonding, the forms of water-insoluble alginic acid and water-soluble alkali metal salts such as sodium alginate and potassium alginate are collectively called as algin. The M, G ratio and sequence are different among different algin molecules, which results in the difference of physicochemical properties of alginic acid: the high M/G ratio alginate has low gel strength, while the low M/G ratio alginate has high gel strength. At present, algin produced industrially is prepared from large-scale brown algae, and mainly comprises kelp, kelp and the like. In brown algae, alginic acid accounts for 10-40% of the dry cell weight. In industrial production, alginic acid of different structures and properties is required. Especially, the demand for alginic acid with high G content is larger, and at present, the M/G ratio is higher<The alginic acid of 1 accounts for 25% of the alginic acid market, and the annual growth rate is 10%, but the high-G-content algin directly extracted cannot meet the requirements in quality and quantity.
Compared with physical and chemical methods, biological modification, namely, the property of the alginic acid is changed by an enzyme method, so that the fine control of the alginic acid structure can be achieved, the limitation of brown algae source species is broken, the problem of secondary pollution cannot be caused, and the method has great advantages. In brown algae, mannuronic acid C5-isomerase can specifically catalyze the conversion of M residue to G residue, at present, the mannuronic acid C5-isomerase of brown algae has been analyzed, but the problems of low expression level, insolubility of expressed protein and the like exist in the in vitro production process of protein, and at present, the high-activity kelp mannuronic acid C5-isomerase in vitro is not obtained. Therefore, the directional development of the soluble mannuronic acid C5-isomerase can provide a good tool for industrial precise preparation of alginic acid.
The invention content is as follows:
the invention aims to provide a gene (MC5E gene) for cloning and obtaining high-expression kelp mannuronic acid C5-isomerase from kelp and application thereof.
In order to achieve the purpose, the invention adopts the technical scheme that:
a kelp mannuronic acid C5-isomerase gene has the base sequence in SEDIQ No. 1.
The protein coded by the MC5E gene has an amino acid sequence in a sequence table SEDIQ No. 2.
Further, kelp transcriptome data is searched, candidate MC5E genes are screened, the transcription levels of all MC5E genes are analyzed, and the MC5E gene with the highest transcription level is screened for research; on the basis, a transcription level higher than that of the kelp is cloned from the kelp
The Open Reading Frame (ORF) of the cDNA sequence of the mannuronic acid C5-isomerase (MC5E) gene is 1458bp, and encodes a protein sequence containing 485 amino acids (namely, the protein sequence is shown by the amino acids in the SEDIQ No.2 of the sequence table). And successfully obtaining soluble recombinant protein expression by using an in vitro prokaryotic expression system, and purifying to obtain the high-purity MC5E protein.
An application of kelp mannuronic acid C5-isomerase gene, and an application of protein coded by MC5E gene in catalyzing conversion from mannuronic acid to guluronic acid.
The protein encoded by the MC5E gene catalyzes the conversion of mannuronic acid to guluronic acid at 30 ℃ and pH 7.5.
The protein coded by the MC5E gene is used as an enzyme preparation for increasing the content of guluronic acid (G) in algin and reducing the M/G ratio of the algin.
The invention has the following outstanding advantages:
according to the invention, on the basis of kelp transcriptome data, an MC5E gene sequence with high kelp expression is screened, a soluble MC5E protein is obtained in vitro through a prokaryotic expression technology, and the enzyme activity of the recombinant protein is identified, so that a relevant result can provide a theoretical basis for biological modification of alginic acid and development and utilization of seaweed active substances.
The MC5E protein of the invention is proved by in vitro enzymatic reaction that the MC5E protein can catalyze the conversion from M to G in alginic acid, and is a key enzyme for catalyzing the reduction of M/G ratio of alginic acid. Furthermore, the kelp MC5E protein can be expressed in a prokaryotic expression system in a soluble way and can be expressed in Ca2+Effectively catalyze the conversion of M to G in alginic acid.
Drawings
FIG. 1 is a gel electrophoresis diagram of MC5E gene and protein provided by the embodiments of the present invention; wherein, a, MC5E gene amplification; and b, detecting the result of affinity chromatography, namely 1: crude protein extract, and 2 and 3: purified protein.
The specific implementation mode is as follows:
the invention is further illustrated in the following examples, without being limited thereto.
The mannuronic acid C5-isomerase (MC5E) gene sequence and its soluble recombinant expression in colibacillus are disclosed. The invention screens candidate MC5E genes by searching kelp transcriptome data, analyzes the transcription level of all MC5E genes, screens the MC5E gene with the highest transcription level for research; on the basis, a cDNA sequence of mannuronic acid C5-isomerase (MC5E) gene with higher transcription level is cloned from the kelp, the Open Reading Frame (ORF) of the cDNA sequence is 1458bp, and the cDNA sequence codes protein sequence containing 485 amino acids. And successfully obtaining soluble recombinant protein expression by using an in vitro prokaryotic expression system, and purifying to obtain the high-purity MC5E protein. The recombinant protein is in Ca2+Under the existing condition, mannuronic acid (M) can be converted into guluronic acid (G), the G content in the algin is increased, the M/G of the algin is reduced, the viscosity of the algin can be increased, and the physicochemical properties of the algin are improved.
Examples 1
The method for cloning the kelp MC5E gene mainly comprises the following steps:
1. extracting total RNA of the kelp juvenile sporophyte and synthesizing cDNA;
synthesizing MC5E gene specific primer and sequencing PCR amplification product;
MC5E protein structure prediction analysis.
The specific operation is as follows:
1. extracting total RNA of kelp juvenile sporophyte: taking a proper amount of kelp sample (less than or equal to 100mg), grinding in a mortar precooled by liquid nitrogen, repeating for 3-5 times, and taking the ground powder for RNA extraction. Total RNA from Laminaria japonica was extracted according to the procedure of Qiagen Plant Mini Kit (RNeasy Plant Mini Kit). cDNA was synthesized by reverse transcription using Transcriptor first Strand cDNA Synthesis Kit of Roche for gene amplification. See the kit instructions for detailed procedures.
MC5E GeneSynthesizing specific primers and sequencing PCR amplification products: on the basis of transcription data, the highly expressed MC5E gene in kelp is obtained by screening, and specific Primer sequences MC5E-F1 and MC5E-R1 (Table 1) for amplifying the ORF of the MC5E gene are designed by using Primer Premier 5 software. The amplification system is as follows: mu.l cDNA, 2. mu.l MC5E-F1, 2. mu.l MC5E-R1, 25. mu.l PrimeSTAR Max Premix (2X), 20. mu.l ddH2And O. The reaction procedure is as follows: pre-denaturation at 94 ℃ for 5 min; then 35 cycles were entered: denaturation at 94 ℃ for 30s, annealing at 55-50 ℃ for 30s, and extension at 72 ℃ for 1.5min, wherein the annealing temperatures are 55, 53 and 50 in sequence, and the cycle numbers are 5, 5 and 25 respectively; finally, extension was carried out at 72 ℃ for 10 min. And carrying out DNA electrophoresis detection, gel recovery, connection and transformation on the PCR product, and then carrying out sequencing verification.
MC5E protein structure prediction analysis: the obtained MC5E gene sequence of interest is predicted for its open reading frame by ORF finder tool and translated into an amino acid sequence by the translation tool MyHits. The Molecular Weight (MW) and isoelectric point (pI) of the protein were predicted with the ProtParam online tool, while the signal peptide sequence of the protein was predicted with SignalP 4.1 and whether the protein contained transmembrane structure was predicted with TMHMMServer v.2.0. The prediction shows that the total length of ORF of the MC5E gene is 1458bp, 485 aa sequences are encoded, the molecular weight MW of the encoded protein is 52.89kDa, and the theoretical isoelectric point is 4.36. It is predicted that MC5E contains a signal peptide sequence 22 aa in length, contains no transmembrane structure, and is predicted to be located outside the cell membrane (see SEDIQ No.1 and SEDIQNo.2 for sequences).
TABLE 1 primers and nucleotide sequences used in this protocol
Figure BDA0002114107660000031
SEDIQ No.1 kelp mannuronic acid C5-isomerase (MC5E) gene base sequence
Figure BDA0002114107660000032
Figure BDA0002114107660000041
SEDIQ No.2 kelp mannitol dehydrogenase (MC5E) gene coding protein amino base sequence
Figure BDA0002114107660000042
EXAMPLES example 2
The kelp MC5E recombinant protein expression and purification method mainly comprises the following steps:
construction and transformation of MC5E recombinant plasmid;
MC5E recombinant protein expression;
purification of MC5E recombinant protein.
The specific operation is as follows:
construction and transformation of MC5E recombinant plasmid: in order to realize the soluble expression of the target protein, a cold shock protein expression vector pCold II is selected for expressing the target protein, and the N end of the protein expressed by the vector is provided with a His tag, so that the cold shock protein expression vector can be used for purifying subsequent fusion protein. According to the sequence information of the target gene, amplification primers are designed, the expression amplification sequence of the gene does not contain a signal peptide sequence, Nde I and EcoRI are selected as double enzyme cutting sites, and the terminal sequences of linearized cloning vectors, namely MC5E-F2 and MC5E-R2 (Table 1), are added at the two ends of the primers. The PCR amplification conditions are the same as the ORF conditions, and the target band is obtained by amplification, and the electrophoretogram is shown in FIG. 1 a. Construction of expression vector
Figure BDA0002114107660000051
II One Step Cloning Kit instructions, recombinant plasmid transformed into Trans1-T1 competent cells. And (3) carrying out amplification culture on the strains which are positive in sequencing, extracting plasmids, transforming pG-Tf2/BL21 competent cells, and carrying out sequencing verification.
MC5E recombinant protein expression: taking a proper amount of positive bacteria liquid, and shaking the bacteria overnight at 37 ℃; when the culture medium is distributed into 4L LB culture medium (containing ampicillin, chloramphenicol and tetracycline) and the OD600 is about 0.6, the culture bottle is precooled for 30min in ice bath at 15 ℃, 0.1mM IPTG is added, and the bacteria are shaken for 24h at the temperature of 15 ℃ and the rpm.
MC5E recombinant protein purification: the expressed fusion protein contains a 6 XHis tag at the N-terminal, and can be compatible with a Ni column, thereby achieving the purpose of purifying the target protein, and the used affinity chromatography column is His Trip HP. Centrifuging the bacterial solution at 8000rpm and 4 deg.C for 15min, collecting thallus precipitate, suspending in sodium phosphate buffer (20mM sodium phosphate, pH 8.0, 500mM NaCl, 20mM imidazole), ultrasonic crushing under ice bath condition for 5s at an interval of 10s, and crushing the bacterial solution to be clear; the disruption solution was centrifuged at 13000rpm for 40min at 4 ℃ and the supernatant obtained by centrifugation was filtered through a 0.45 μm filter. The fusion protein was affinity chromatographed using the AKTA avant system as follows: cleaning the chromatographic column by ultrapure water with 5 times of the volume of the column bed; 10 times of the volume of sodium phosphate buffer solution, 5ml/min flow rate balance chromatographic column; feeding the crushed supernatant into the column at a flow rate of 2.5 ml/min; washing the chromatographic column with 10 times of column volume of sodium phosphate buffer solution at the flow rate of 5 ml/min; the protein was eluted with 10 column volumes of elution buffer containing 500mM (20mM sodium phosphate, pH 8.0, 500mM NaCl, 500mM imidazole) at a flow rate of 2 ml/min. According to the change of the absorption peak at 280nm shown by the detector, the component with higher concentration of the fusion protein is collected, and the protein is concentrated by an Amicon Ultra-15 Ultra-filtration tube (MWCO 10 kDa). Further purification of the protein was carried out using a gel filtration chromatography column with purification buffer composition: 20mM Tris-HCl (1M, pH 7.4); 150mM NaCl, program: equilibrating the column with 1 bed volume of buffer (120ml) at a flow rate of 1 ml/min; after loading, the protein was eluted at a flow rate of 1ml/min, and the target protein was collected by a collector (see SEDIQNo.2 for sequence). The content and purity of the protein are detected by gel electrophoresis, the obtained protein is used for subsequent enzyme kinetic analysis, and the electrophoresis result is shown in figure 1.
EXAMPLE 3
The method for analyzing the activity of the kelp MC5E recombinant protease mainly comprises the following steps:
1. enzymatic reaction: a10 ml reaction system was prepared with the following ingredients: 10mM MOPS, 0.1M NaCl, 4mM CaCl25 μ g of MC5E, 0.25% polyM. Meanwhile, a control reaction system is prepared, wherein MC5E is not added in the reaction system, and other components are the same.
2. And (3) determining the ratio of alginic acid M/G before and after the reaction: heating 3ml of reaction solution and the same control reaction solution at 95 deg.C for 10minHigh speed centrifugation (12000rpm) to remove denatured proteins; adding 4 times volume of anhydrous ethanol into the supernatant, uniformly mixing by vortex, centrifuging at 12000rpm for 15min, and removing the supernatant; washing the precipitate with anhydrous ethanol for 3 times according to the above steps; the precipitate obtained is dried and 1ml D is added2O, repeated freeze drying for 3 times, and dissolving in 1ml D2O; the sample was subjected to a nuclear magnetic resonance hydrogen spectrometer (Bruker Avance III 600MHz) at a detection temperature of 25 ℃. The results are shown in table 2: kelp MC5E from the above results, it was found that the reaction group decreased FMM and increased FMG and FG compared to the control group, resulting in a decrease of the M/G ratio from pre-reaction 3.5893 to 2.6697, indicating that MC5E was able to exert mannuronic acid C5-isomerase catalytic activity.
TABLE 2 distribution of 2M, G
Figure BDA0002114107660000061
。
Sequence listing
<110> oceanographic institute of Chinese academy of sciences
<120> gene of kelp mannuronic acid C5-isomerase and application thereof
<160>2
<170>SIPOSequenceListing 1.0
<210>1
<211>1458
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>1
atggcgggca aggcactctc ggcgcttgcg gcgacgctca gcctctgtgc agtcaacgcg 60
tgcggtgacc accactgctg gggggtgaac atttgcaccg gggatgatgt ttgcggcccc 120
ccgagcgact gcgatgcgtc ctcgatcacg gttgaggatg acgtgatcca aatcagaggc 180
tcgcgatgcc gtgtgaccat ggaggccatc cacctcgcgc ttccggacgc tgtaacaaag 240
gacgggactg agtacaccct caacaacaag ctggtggttc gcgatggctg tatccttgag 300
attcacggcg cttccaaggc ctcttcgccc gacgcagtcg tgacgctcct aaagctaaag 360
agcgatgaga cgtcctacgc acccatcatc gcgatggcgg gccagatctc catcctcgac 420
acggccatca cgtcttacga cgagtccaag gggggcagcg acgaaagact tgaaggcagg 480
gcgtacatcg cggctctgag cttggaggac gaaacgaccg gcgaggcctg gatctctcgc 540
atggacgtcg aggacagcga gatctcctac ctcggcaacg agggcgacta ccacaatgac 600
atcaacaacg actacggcct ggtgtggaag gtagaaggtt acgacgaccg tttcccggga 660
gacctgtccc tcttctccag agtcggcgtc tacggcacgc tgaagcgcaa caagatccac 720
gacatgttca tcggcgcata ctgctacggg atgaaaggaa acacgctctg gaccggcaac 780
gagatttacg acaactacct gaacggcatg aaccctcaag acaacagcga cggcatgacc 840
atctccgaca accacgtgca cgacaacgga tggcacggca tcgaatacag caagcgctgc 900
aaaggggcgc ttgtgttcga caacctcgtg gaggacaacg ctcgcgccgg tatcttcttc 960
caccgctcca cggactacgc ggaagcttac ggcaacacgt gcaggcgtaa cttggagggc 1020
gacttcggca tcgtggagtc cacgggcgtc acggtgcacg ataacgtcat ggaggggggc 1080
aagtacggca tccgattgag ccttggggca caggagtgcg acgtgtacga caacgtgatg 1140
tcggacaact tccgttacgg cgtcttcttc taccgcggca gcgacgaggc agaggcttcc 1200
gcggattggg acggacgccc tcgcctgaac tacatcagga acaacaaaat ctccgacccc 1260
gtggagggca agggcgtcgc cgtcacgggg tcggacgaca acttcatcat cgacaacgag 1320
ttcatcggta tcgactctct gcgcttcgac gacgctcaga acaccctcgt cacgggcaac 1380
atcctgccgg acggggtgga gttcacgctg gacgacgggg cgactcttgc cgacgggtcc 1440
cagtcggata ccgactag 1458
<210>2
<211>485
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>2
Met AlaGly Lys Ala Leu Ser Ala Leu Ala Ala Thr Leu Ser Leu Cys
1 5 10 15
Ala Val Asn Ala Cys Gly Asp His His Cys Trp Gly Val Asn Ile Cys
20 25 30
Thr Gly Asp Asp Val Cys Gly Pro Pro Ser Asp Cys Asp Ala Ser Ser
35 40 45
Ile Thr Val Glu Asp Asp Val Ile Gln Ile Arg Gly Ser Arg Cys Arg
50 55 60
Val Thr Met Glu Ala Ile His Leu Ala Leu Pro Asp Ala Val Thr Lys
65 70 75 80
Asp Gly Thr Glu Tyr Thr Leu Asn Asn Lys Leu Val Val Arg Asp Gly
85 90 95
Cys Ile Leu Glu Ile His Gly Ala Ser Lys Ala Ser Ser Pro Asp Ala
100 105 110
Val Val Thr Leu Leu Lys Leu Lys Ser Asp Glu Thr Ser Tyr Ala Pro
115 120 125
Ile Ile Ala Met Ala Gly Gln Ile Ser Ile Leu Asp Thr Ala Ile Thr
130 135 140
Ser Tyr Asp Glu Ser Lys Gly Gly Ser Asp Glu Arg Leu Glu Gly Arg
145 150 155 160
Ala Tyr Ile Ala Ala Leu Ser Leu Glu Asp Glu Thr Thr Gly Glu Ala
165 170 175
Trp Ile Ser Arg Met Asp Val Glu Asp Ser Glu Ile Ser Tyr Leu Gly
180 185 190
Asn Glu Gly Asp Tyr His Asn Asp Ile Asn Asn Asp Tyr Gly Leu Val
195 200 205
Trp Lys Val Glu Gly Tyr Asp Asp Arg Phe Pro Gly Asp Leu Ser Leu
210 215 220
Phe Ser Arg Val Gly Val Tyr Gly Thr Leu Lys Arg Asn Lys Ile His
225 230 235 240
Asp Met Phe Ile Gly Ala Tyr Cys Tyr Gly Met Lys Gly Asn Thr Leu
245 250 255
Trp Thr Gly Asn Glu Ile Tyr Asp Asn Tyr Leu Asn Gly Met Asn Pro
260 265 270
Gln Asp Asn Ser Asp Gly Met Thr Ile Ser Asp Asn His Val His Asp
275 280 285
Asn Gly Trp His Gly Ile Glu Tyr Ser Lys Arg Cys Lys Gly Ala Leu
290 295 300
Val Phe Asp Asn Leu Val Glu Asp Asn Ala Arg Ala Gly Ile Phe Phe
305 310 315 320
His Arg Ser Thr Asp Tyr Ala Glu Ala Tyr Gly Asn Thr Cys Arg Arg
325 330 335
Asn Leu Glu Gly Asp Phe Gly Ile Val Glu Ser Thr Gly Val Thr Val
340 345 350
His Asp Asn Val Met Glu Gly Gly Lys Tyr Gly Ile Arg Leu Ser Leu
355 360 365
Gly Ala Gln Glu Cys Asp Val Tyr Asp Asn Val Met Ser Asp Asn Phe
370 375 380
Arg Tyr Gly Val Phe Phe Tyr Arg Gly Ser Asp Glu Ala Glu Ala Ser
385 390 395 400
Ala Asp Trp Asp Gly Arg Pro Arg Leu Asn Tyr Ile Arg Asn Asn Lys
405 410 415
Ile Ser Asp Pro Val Glu Gly Lys Gly Val Ala Val Thr Gly Ser Asp
420 425 430
Asp Asn Phe Ile Ile Asp Asn Glu Phe Ile Gly Ile Asp Ser Leu Arg
435 440 445
Phe Asp Asp Ala Gln Asn Thr Leu Val Thr Gly Asn Ile Leu Pro Asp
450 455 460
Gly Val Glu Phe Thr Leu Asp Asp Gly Ala Thr Leu Ala Asp Gly Ser
465 470 475 480
Gln Ser Asp Thr Asp
485

Claims (5)

1. A kelp mannuronic acid C5-isomerase gene is characterized in that: the gene has a base sequence in a sequence table SEDIQ No. 1.
2. The protein encoded by the gene for laminarmnuronic acid C5-isomerase of claim 1, wherein: the protein coded by the MC5E gene has an amino acid sequence in a sequence table SEDIQ No. 2.
3. The use of the kelp mannuronic acid C5-isomerase gene as claimed in claim 1, wherein the gene comprises: the MC5E gene is used for catalyzing the conversion of mannuronic acid into guluronic acid.
4. Use of the laminarin mannuronic acid C5-isomerase gene according to claim 3 wherein: the protein encoded by the MC5E gene catalyzes the conversion of mannuronic acid to guluronic acid at 30 ℃ and pH 7.5.
5. The application of the kelp mannuronic acid C5-isomerase gene in 1 is characterized in that: the protein coded by the MC5E gene is used as an enzyme preparation for increasing the content of guluronic acid (G) in algin and reducing the M/G ratio of the algin.
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WO2004065594A2 (en) * 2002-12-19 2004-08-05 Centre National De La Recherche Scientifique (C.N.R.S.) Mannuronan c5-epimerases of brown algae, methods of obtaining same and use thereof
CN102497883A (en) * 2009-06-03 2012-06-13 阿尔吉法玛公司 Alginate oligomers for use in overcoming multidrug resistance in bacteria
WO2014147209A1 (en) * 2013-03-20 2014-09-25 Norwegian University Of Science And Technology (Ntnu) Mannuronan c-5 epimerase variants and methods for their identification

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WO2004065594A2 (en) * 2002-12-19 2004-08-05 Centre National De La Recherche Scientifique (C.N.R.S.) Mannuronan c5-epimerases of brown algae, methods of obtaining same and use thereof
CN102497883A (en) * 2009-06-03 2012-06-13 阿尔吉法玛公司 Alginate oligomers for use in overcoming multidrug resistance in bacteria
WO2014147209A1 (en) * 2013-03-20 2014-09-25 Norwegian University Of Science And Technology (Ntnu) Mannuronan c-5 epimerase variants and methods for their identification

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张朋艳: "海带褐藻酸合成途径关键基因的转录谱及功能研究", 《中国优秀博硕士学位论文全文数据库(博士) 基础科学辑》 *

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Application publication date: 20200602