CN109554382B - Heat-resistant esterase gene, engineering bacterium containing gene, encoding protein and application thereof - Google Patents
Heat-resistant esterase gene, engineering bacterium containing gene, encoding protein and application thereof Download PDFInfo
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Abstract
The invention discloses a heat-resistant esterase gene, engineering bacteria containing the gene, a coding protein and application thereof, wherein the base sequence of the esterase gene estA 'is SEQ ID NO.1, the gene estA' is connected with plasmid to construct a recombinant expression vector pET28b-estA ', and the expression plasmid is transformed into escherichia coli to construct recombinant engineering bacteria Rosetta (DE3) pLysS/pET28 b-estA'; the esterase obtains heterologous expression in escherichia coli, the enzymology property of the esterase EstA is identified by adopting a p-nitrophenol method, and the esterase has high thermal stability.
Description
Technical Field
The invention belongs to the technical field of biological engineering, relates to the technical field of gene and encoded protein engineering, and particularly relates to a heat-resistant esterase gene, an engineering bacterium containing the gene, an encoded protein of the gene and application of the gene.
Background
Esterases (Esterases, EC 3.1.1.X) are a class of enzymes that catalyze the hydrolysis and formation of ester bonds, widely distributed in animals, plants, and microorganisms. As an important industrial enzyme, esterase has been widely used in the fields of pharmaceutical and chemical industry, food industry, fine chemical industry, bioenergy, agriculture, environmental control and the like. With the continuous widening of the application range of esterase, the esterase is now the third industrial enzyme behind protease and amylase in the world industrial enzyme preparation market. However, the conditions for industrial production of esterases are relatively extensive, and therefore, in order to save production cost, the search for esterases with high thermal stability and high catalytic efficiency is always the object of efforts.
Streptomyces (Streptomyces) belongs to Streptomyces of Actinomycetales, and is an aerobic, filamentous, spore-forming gram-positive bacterium existing in soil. The streptomyces has rich secondary metabolic pathways, can generate a large amount of secondary metabolites with important economic values, such as antibiotics, hydrolytic enzymes, enzyme inhibitors, herbicides, antitumor drugs and the like, and is an extremely important industrial production bacterium. The size of the streptomycete genome is 5.5Mb-8.6Mb, which is about 2 times of the Escherichia coli genome, and the G + C content is up to 70-74%. Streptomycete contains rich esterase genes, and the sequence information provides researchers with rich resources, but the research on the esterase genes is less at present. Researchers can clone corresponding esterase genes according to the sequence information, and carry out heterologous expression and enzymological property research on the esterase genes. However, due to the high GC content of Streptomyces genes and the relatively complex protein translation system, expression of Streptomyces genes in heterologous hosts is often limited. Currently, only a few kinds of esterase genes are well expressed in heterologous expression systems such as Escherichia coli (Escherichia coli), Streptomyces lividans, Saccharomyces cerevisiae (Saccharomyces erevisiae), and Pichia pastoris (Pichia pastoris). The non-translated region of esterase gene, the existence of esterase propeptide and signal peptide, the difference of codons, glycosylation of recombinant esterase expressed by different expression systems and the like all influence the heterologous expression of esterase gene. In particular, Escherichia coli, which is a lower prokaryotic organism, lacks a complete post-translational modification system in which esterases are often not expressed or exist in the form of inclusion bodies, and rarely obtains active proteins.
The nucleotide sequence of a Complete Genome (Complete Genome) of Streptomyces lividans TK24 is in a GenBank database, the Accession number of the nucleotide sequence is CP009124, the Genome size is 8.3Mb, and the content of G + C reaches 72.2%. The gene researched by the invention is a Locus (Locus tag) SLIV _ RS27090 on a chromosome (NZ _ CP009124), the Start site (Start) of a nucleotide sequence on the chromosome is 6057885, and the Stop site (Stop) is 6058688. Interpretation of the nucleotide sequence according to the genome sequence, the encoded Protein product is esterase, Protein product (Protein product) number (Accession) is WP-003976692, and Protein Accession number in GenBank is AIJ 16329. The sequence has the full length of 804bp, the Initiation codon (TCA), the termination codon (Stop codon) and GC content of 72.5 percent, and embodies the characteristic of high GC content of the streptomycete genome.
Disclosure of Invention
According to the defects of the prior art, the technical problem to be solved by the invention is to provide a heat-resistant esterase gene, engineering bacteria containing the gene, and a coding protein and application thereof, and in order to solve the technical problem, the technical scheme adopted by the invention is as follows:
a heat-resistant esterase gene has a base sequence of SEQ ID No.1 and can be successfully expressed in heterologous host escherichia coli.
A recombinant plasmid contains pET-28b (+) recombinant plasmid of the heat-resistant esterase gene.
A heat-resistant esterase gene recombinant engineering bacterium is Escherichia coli and contains the recombinant plasmid expression vector.
Preferably, the conditions for expressing the heat-resistant esterase protein by the engineering bacteria are 0.01-0.5mM of IPTG induction concentration and 16-30 ℃.
The coding protein of a heat-resistant esterase gene has an amino acid sequence of SEQ ID NO. 2.
Preferably, the reaction temperature of the coded protein is 30-60 ℃, the reaction pH is 7-9, and the activity half-life period is more than or equal to 6h at 100 ℃.
Preferably, the encoded protein has a reaction temperature of 55 ℃, a reaction pH of 8.5, and an activity half-life of 6h at 100 ℃.
An application of the coded protein of heat-resistant esterase gene in the fields of medicine, food and fine chemical engineering.
Compared with the prior art, the invention has the beneficial effects that:
1. the invention optimizes the nucleotide sequence of the gene locus SLIV _ RS27090 on the chromosome of the streptomyces lividans TK24, the amino acid coded by the obtained nucleotide sequence is kept unchanged, 609 nucleotides in 804 nucleotide sequences are changed and account for 75.7 percent of the whole gene, and the GC content is reduced to 53.4 percent from 72.5 percent of the original nucleotides in the strain, thereby being beneficial to optimizing the expression of the gene in escherichia coli.
2. The optimized gene estA 'is connected with pET28b plasmid, and the recombinant expression vector pET28b-estA' with Kan resistance is successfully constructed.
3. The recombinant plasmid is transformed into an escherichia coli competent cell, and a genetically engineered bacterium Rosetta (DE3) pLysS/pET28b-estA 'for expression of estA' is successfully constructed, wherein the recombinant engineered bacterium has Kan and Cam resistance.
4. By using Co in the invention 2+ The highly purified esterase protein EstA is obtained by an ion affinity chromatography and SDS-PAGE electrophoresis, and the obtained esterase protein EstA has high thermal stability and can be applied to the fields of medicines, foods, fine chemicals and the like.
Drawings
1. FIG. 1 is a sequence alignment of esterase estA gene and optimized estA';
2. FIG. 2 is SDS-PAGE analyzing the expression of EstA;
3. FIG. 3 is a graph of EstA activity versus reaction pH trend;
4. FIG. 4 is a graph of EstA activity versus reaction temperature trend;
5. FIG. 5 is a graph of the thermal stability trend of EstA at 55 ℃;
6. FIG. 6 is a graph of the tendency of thermal stability of EstA at 100 ℃.
M, protein Marker, 1, IPTG induced crushing liquid, 2, IPTG induced crushing liquid supernatant, 3, purified EstA protein, ■, Na-citrate buffer with pH6.0-7.0, ●, Tris-HCl buffer with pH7.0-9.0, tangle-solidup and KH with pH9.0-10.5 2 PO 4 -NaOH buffer。
Detailed Description
The following embodiments are described in further detail to help those skilled in the art to more fully, accurately and deeply understand the inventive concept and technical solutions of the present invention.
What needs to be clarified is: the gene Locus (Locus tag) SLIV _ RS27090 coded esterase gene is named as estA, the optimized esterase gene is named as estA ', and the amino acid composition of the protein coded by the estA and the estA' is the same, so the coded amino acid is uniformly named as EstA.
The standard assay system used in the examples was 1mL and consisted of 980. mu.L of 50mM Tris-HCl pH 8.0, 10. mu.L of 0.5mM p-nitrophenol butyrate, and 10. mu.L of enzyme solution.
Example 1
1. Optimizing and synthesizing streptomyces lividans TK24 esterase gene estA
Theoretical guidance: the whole length of streptomyces lividans TK24 esterase estA gene is 804bp, the initiation codon is TCA, and the termination codon is CAC; 122A bases account for 15.2%; 99T bases account for 12.3%; 297C bases account for 36.9%; 286G bases account for 35.6%; the GC content is 72.5 percent, which embodies the high GC content characteristic of the streptomyces lividans genome. Because the high GC content of the estA gene seriously affects the expression of the protein in escherichia coli, and streptomyces lividans esterase is secondary metabolic protein, molecular chaperones are possibly needed to assist the folding of the protein in the expression process, so that the expression of the gene in the escherichia coli is obstructed, and the heterologous expression in the escherichia coli cannot be completed. Synthesized by the biosciences of Kinry.
2. Sequence analysis of optimized esterase genes
The full length of the optimized esterase gene estA' is still 804bp, but the optimized esterase gene estA is represented as an initiation codon ATG and a termination codon TAA in the aspect of nucleotide characteristics; 162A bases, accounting for 20.1%; 208T bases accounting for 25.9%; 201C bases, accounting for 25.0%; 233G bases accounted for 29.0%. The nucleotide sequence of Streptomyces lividans TK24 esterase estA gene is compared with the optimized nucleotide sequence estA' of the gene, as shown in figure 1, only 195 bases in 804 bases are unchanged and only occupy 24.3 percent of the whole amino acid sequence. In the aspect of GC content, compared with an original sequence, the GC content of the optimized base is reduced to 53.4 percent, so that the defect that the esterase cannot be expressed due to overhigh GC content is overcome, and the guarantee is provided for the heterologous expression of the optimized target gene in escherichia coli.
Example 2
Constructing expression plasmids and expression engineering bacteria: and (3) carrying out Nde I and Xho I corresponding enzyme digestion on the optimized gene estA' sequence and the optimized vector pET-28b (+) respectively, wherein the enzyme digestion condition is 37 ℃, the reaction is carried out for 6 hours, and the enzyme digestion system is as follows:
the double-restriction enzyme gene estA 'is connected with a vector pET-28b (+), so as to construct an expression plasmid pET28 b-estA'. The ligation was carried out overnight at 16 ℃ and the linker system:
mixing expression plasmid pET28b-estA' with E.coli Rosetta (DE3) competent cells, ice-bathing for 30min, shaking gently for several times to prevent thallus precipitation, heat-shocking for 90s at 42 deg.C to avoid shaking, and constructing recombinant engineering bacteria
Rosetta (DE3) pLysS/pET28b-estA' has Kan and Cam resistance, and recombinant engineering bacteria can be screened on LB culture medium with Kan and Cam resistance.
Example 3
Expression of EstA
Selecting recombinant engineering bacteria in a test tube containing 5mL of LB liquid culture medium, carrying out amplification culture at 37 ℃ and 225rpm overnight; inoculating the culture at a ratio of 1:100 into a conical flask containing 50mL LB liquid medium, culturing at 37 deg.C and 225rpm under shaking to OD600 of 0.4-1.0 (preferably 0.6); adding IPTG with low concentration of 0.01-0.5mM, inducing expression for 24h at the temperature of 16-30 ℃ and the rpm of 180; and centrifuging at 4 ℃ and 5000rpm for 5min, collecting thalli, and carrying out ultrasonic disruption to obtain esterase EstA.
2. Purification of esterase EstA
Esterase EstA had a total of 267 amino acids, a Molecular Weight (Molecular Weight) of 28.5kDa, an isoelectric point (isoelectric point) of 6.9 and an Aliphatic amino acid index (Aliphatic index) of 96.8%, and was prepared using Co 2+ Purification by ion affinity chromatography column, Co 2+ Ion affinity column purification Co from Clontech was used 2+ Purifying and collecting fusion protein EstA with 6 His-tags by an ion affinity column, identifying the purified protein by 12% SDS-PAGE, and obtaining the proteinThe protein has a band with high specificity at the position corresponding to esterase EstA molecular weight as shown in figure 2, which shows that the protein successfully obtains heterologous expression in Escherichia coli and passes through Co 2+ And (3) purifying by using an ion affinity chromatography column to obtain a protein with higher purity, and detecting the protein concentration of the purified protein, wherein bovine serum albumin is taken as a standard, and the concentration of the purified esterase protein is 0.807 mg/mL.
Example 4
Detection of the optimum pH of EstA
The enzyme activity detection of the EstA adopts a standard system, a Cary 300UV spectrophotometer capable of automatically controlling the temperature is used, the detection wavelength is 410nm, a mixed solution without enzyme is used as a reference, the reaction temperature is 25 ℃, the reaction time is 5min, a corresponding buffer solution is used in a corresponding pH range, the pH value is 6.0-7.0, and 50mM sodium citrate buffer is used; 50mM Tris-HCl buffer, pH 7.0-9.0; pH9.0-10.5, 50mM K 2 HPO 4 NaOH buffer, with a detection wavelength of OD410, and the assay was repeated 3 times independently, and the results showed that the optimum pH of the enzyme was 8.5, and the trend was as shown in FIG. 3.
Detection of the optimum temperature of EstA
A standard determination system is adopted, the reaction pH is 8.5, the reaction temperature is set to be 10-60 ℃, the test is independently repeated for 3 times, the average value is taken, the result shows that the optimal temperature of the enzyme is 55 ℃, and the trend is shown in figure 4.
3. Esterase EstA optimum substrate assay
Detecting the specific activity of the p-nitrophenol ester substrates with different chain lengths C4-C16 by adopting a standard determination system at the temperature of 25 ℃ and the pH value of 8.5, wherein the recombinant enzyme activity is calculated by a p-nitrophenol standard curve, one enzyme activity unit (U) is defined as the enzyme amount required for catalyzing and generating 1 mu moL of p-nitrophenol from the p-nitrophenol ester per minute at the temperature of 25 ℃, and the enzyme specific activity unit is U/mg and represents the enzyme activity per mg of enzyme protein; the results show that the specific activity of the enzyme is the highest when the substrate is p-nitrophenol acetate, and reaches 48.0U/mg, as shown in the following table 1:
TABLE 1 specific Activity of esterase EstA
| Compounds | Specific activity(U/mg)±SD |
| pNPA 2 | 48.0±0.7 |
| pNPB 4 | 31.7±0.8 |
| pNPC 6 | 16.1±0.2 |
| pNPC 8 | 12.1±0.6 |
| pNPC 10 | 8.4±0.4 |
| pNPL 12 | 7.9±0.3 |
| pNPM 14 | 7.4±0.2 |
| pNPP 16 | 7.0±0.3 |
4. Esterase EstA thermostability assay
Respectively incubating EstA pure enzyme at 55 ℃ and 100 ℃ for a certain time, detecting residual enzyme activity at 25 ℃ by adopting a standard determination system, and determining thermal stability, wherein the activity of the untreated pure enzyme is set as 100%. The results show that the enzyme activity is not obviously changed after the enzyme is placed at 55 ℃ for 337h as shown in figure 5, the enzyme activity is still over 80 percent after the enzyme is placed at 100 ℃ for 5.5h, and the half-life period is gradually reached after 6h as shown in figure 6.
Example 5
1. Effect of Metal ions, EDTA and PMSF on the Activity of EstA
Under the conditions of 25 ℃, pH8.5 and adopting a standard determination system, metal ions, EDTA and PMSF are respectively added to detect the activity of the EstA enzyme, the final concentration of the used buffer solution is 50mM Tris-HCl, the enzyme activity without adding a chemical reagent is set as 100 percent, the experiment is independently repeated for 3 times, and as shown in Table 2, 1mM K + 、Fe 2+ 、Mn 2+ 、Ca 2+ And Na + Has strong activating effect on enzyme, can improve the enzyme activity by more than 20 percent, especially K + 、Fe 2+ And Mn 2 + can increase the enzyme activity by 53.3%, 48.6% and 44.2% respectively; zn 2+ And Mg 2+ Has certain activation effect on enzyme activity, and can respectively improve the enzyme activity by 13.7 percent and 11.3 percent; k of 10mM + 、Fe 2+ 、Mn 2+ 、Ca 2+ And Na + Has some activating effect on enzyme, but removes Ca 2+ And Na + The activation effect on the enzyme is basically unchanged, and other three types of ions are reduced to a certain extent along with the increase of the concentration; 10mM Mg 2+ Activating effect on enzyme relative to 1mM Mg 2+ No obvious change, and the relative enzyme activity is 109.1 percent; 10mM Zn 2 +、Cu 2+ And Ni 2+ Strongly inhibiting enzyme activity, and respectively reducing the enzyme activity to 35.2%, 34.4% and 10.2%; EDTA and PMSF with the concentration of 1mM have no influence on the esterase enzyme activity, and when the concentration is increased to 10mM, the enzyme activity is reduced to 86.0 percent and 72.6 percent respectively.
TABLE 2 Effect of Metal ions, EDTA and PMSF on EstA Activity
2. Effect of organic solvents on EstA enzymatic Activity
Under the conditions of 25 ℃ and pH8.5, respectively detecting the activity of the EstA enzyme by adopting different organic solvents by adopting a standard determination system, wherein the final concentration of a used buffer solution is 50mM Tris-HCl, the enzyme activity without adding a chemical reagent is set as 100%, and the experiment is independently repeated for 3 times, as shown in Table 3, in all the detected organic solvents, dimethyl sulfoxide with the concentration of 15% and 30% and isopropanol with the concentration of 15% have no obvious influence on the enzyme activity of the esterase, but when the concentration of the isopropanol reaches 30%, a strong inhibition effect on the enzyme activity of the esterase is generated, so that the enzyme activity is reduced to 51.6%; 15 percent and 30 percent of ethanol, methanol and dimethyl amide can inhibit the enzyme activity to a certain extent, and the enzyme activity inhibition effect is enhanced along with the increase of the concentration of the ethanol, the methanol and the dimethyl amide, but the increase is not large; 15 percent and 30 percent of acetonitrile and acetone have stronger inhibition effect on enzyme activity, and the inhibition effect on the enzyme activity is also enhanced along with the increase of the concentration of the organic solvent.
TABLE 3 Effect of organic solvents on EstA Activity
3. Effect of detergents on EstA enzyme Activity
Under the conditions of 25 ℃ and pH8.5, respectively detecting the activity of EstA enzyme by adopting different organic solvents by adopting a standard determination system, wherein the final concentration of a used buffer solution is 50mM Tris-HCl, the enzyme activity without adding a chemical reagent is set as 100%, and the experiment is independently repeated for 3 times, as shown in Table 4, in all detected 0.1% detergents, Span-20 has a strong activating effect on the enzyme activity of esterase, so that the enzyme activity is increased by 37.2%; tween-20 improves the esterase activity to a certain extent, but the action effect is not obvious; SDS and Triton X-100 have no influence on the activity of esterase, and in 1% of detergents, Span-20, SDS and CTAB have strong inhibition on the enzyme activity of esterase, so that the enzyme activity is respectively reduced to 30.8%, 35.6% and 44.2%.
TABLE 4 Effect of detergents on EstA Activity
The present invention has been described in connection with the embodiments, and it is to be understood that the invention is not limited to the specific embodiments described above, and that various insubstantial modifications of the inventive concepts and solutions, or their direct application to other applications without modification, are intended to be covered by the scope of the invention. The protection scope of the present invention shall be subject to the protection scope defined by the claims.
SEQUENCE LISTING
<110> university of teacher's university in Anhui
<120> heat-resistant esterase gene, engineering bacterium containing gene, and coding protein and application thereof
<130> 2018.04.12
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 804
<212> DNA
<213> Streptomyces lividans (Streptomyces lividands)
<400> 1
atgtcagtgt taccgggtgc tgaaccattt cgtcatgaag gcggagatgt gggcgtttta 60
ctgtgtcatg gctttacggg ttctccacag tctctgcgtc cttgggctcg ctacttagcc 120
gcacgcggcc tgacagtttc actgccatta ttaccaggcc acggtacacg ttggcaggat 180
atgcaggtga ccggttggca ggattggtat gcagaagtgg atcgcgaact tagagccctg 240
cgcgaacgct gcgaacgagt gtttgttgcg ggtctgtcaa tgggcggcgc actggccctg 300
cgcttagcag ctaaacatgg ggatgccgtt tcaggcgtgg ttgtggttaa tccggctaac 360
aagatgcatg gcgttgctca acacgccctg cctgtgctgc gccatttggt tccagcgacc 420
aaaggcatcg caagcgatat tgcaaaacct ctgagtacgg aactgggcta tgatcgtgtt 480
ccgttacata gcgcacatag cttacgcgcc ttctttcgtc tggccgatgg ggatctccct 540
caggtgacac agccactgtt attattgcgc agtcctcagg atcatgttgt tcctccagcc 600
gatagtgctc gtatcttagg tcgcgtgtct tctacagatg tgacggaaat tctgctggaa 660
cagagctatc atgtggcaac cttagatcat gatgcagatc gtatctttgc tgaatcagtt 720
gccttcatcg gtcgcttagc tccgggtagt gtgggtgaac cagaaagcgg cttaggcaaa 780
gaaggaaccg ccgccggcgg ttaa 804
<210> 2
<211> 267
<212> PRT
<213> Escherichia coli (Escherichia coli)
<400> 2
Met Ser Val Leu Pro Gly Ala Glu Pro Phe Arg His Glu Gly Gly Asp
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Val Gly Val Leu Leu Cys His Gly Phe Thr Gly Ser Pro Gln Ser Leu
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Arg Pro Trp Ala Arg Tyr Leu Ala Ala Arg Gly Leu Thr Val Ser Leu
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Pro Leu Leu Pro Gly His Gly Thr Arg Trp Gln Asp Met Gln Val Thr
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Gly Trp Gln Asp Trp Tyr Ala Glu Val Asp Arg Glu Leu Arg Ala Leu
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Arg Glu Arg Cys Glu Arg Val Phe Val Ala Gly Leu Ser Met Gly Gly
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Ala Leu Ala Leu Arg Leu Ala Ala Lys His Gly Asp Ala Val Ser Gly
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Val Val Val Val Asn Pro Ala Asn Lys Met His Gly Val Ala Gln His
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Ala Leu Pro Val Leu Arg His Leu Val Pro Ala Thr Lys Gly Ile Ala
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Ser Asp Ile Ala Lys Pro Leu Ser Thr Glu Leu Gly Tyr Asp Arg Val
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Pro Leu His Ser Ala His Ser Leu Arg Ala Phe Phe Arg Leu Ala Asp
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Gly Asp Leu Pro Gln Val Thr Gln Pro Leu Leu Leu Leu Arg Ser Pro
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Gln Asp His Val Val Pro Pro Ala Asp Ser Ala Arg Ile Leu Gly Arg
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Val Ser Ser Thr Asp Val Thr Glu Ile Leu Leu Glu Gln Ser Tyr His
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Val Ala Thr Leu Asp His Asp Ala Asp Arg Ile Phe Ala Glu Ser Val
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Ala Phe Ile Gly Arg Leu Ala Pro Gly Ser Val Gly Glu Pro Glu Ser
245 250 255
Gly Leu Gly Lys Glu Gly Thr Ala Ala Gly Gly
260 265
Claims (4)
1. A heat-resistant esterase gene, characterized in that the heat-resistant esterase gene has a base sequence of SEQ ID No.1 and can be successfully expressed in heterologous host Escherichia coli.
2. A recombinant plasmid comprising pET-28b (+) recombinant plasmid of the thermotolerant esterase gene of claim 1.
3. A thermotolerant esterase gene recombinant engineering bacterium which is Escherichia coli and contains the recombinant plasmid expression vector of claim 2.
4. The thermotolerant esterase gene recombinant engineering bacterium according to claim 3, wherein the conditions for expressing the thermotolerant esterase protein are 0.01-0.5mM of IPTG induction concentration and 16-30 ℃.
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| CN101603037A (en) * | 2009-07-23 | 2009-12-16 | 安徽师范大学 | A kind of thermostable carboxylesterase gene, proteins encoded and application thereof |
| CN102286441A (en) * | 2011-07-24 | 2011-12-21 | 国家海洋局第二海洋研究所 | Low-temperature esterase and coding gene and use thereof |
| CN105164260A (en) * | 2013-03-13 | 2015-12-16 | 自动显示生物技术有限公司 | Improved surface display of functional proteins in a broad range of gram negative bacteria |
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| CN109554382A (en) | 2019-04-02 |
| CN108546695A (en) | 2018-09-18 |
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