CN103409443B - Gene try1A for encoding peptidase and application of gene - Google Patents

Abstract

A gene try1A encoding peptidase, its nucleotide sequence is shown in Sequence Listing No.1; amino acid residues are shown in Sequence Listing No.2. The DNA in Sequence Listing No.1 starts from the first nucleotide at the 5' end with the start codon ATG to the 1221st nucleotide and ends with the stop codon TAA, and there is a complete ORF (nucleotides 1-1221 ), the 1st-3rd nucleotide from the 5' end is the start codon ATG of the try1A gene, and the 1219-1221st nucleotide from the 5' end is the stop codon TAA of the try1A gene. The protein coded by the gene provided by the invention has wide applications in hydrolyzing protein macromolecular substrates, making it widely used in food technology, life science research, protein waste treatment and the like.

Description

A gene try1A encoding peptidase and its application

technical field

The invention relates to a gene try1A encoding peptidase and its application.

Background technique

Peptidase is a general term for a class of enzymes that can hydrolyze peptide bonds, and can also be called protease or proteolytic enzyme. Peptidases are a large class of enzymes. According to reports, about 2% of proteins in all organisms belong to peptidases or enzymes homologous to peptidases. Peptidase is an important industrial enzyme. It is reported that the sales volume of peptidase accounts for 50% of the industrial enzyme market and is widely used in food, textile, medicine and environmental protection. Peptidases are divided into serine-type peptidases, cysteine-type, threonine-type, aspartic acid-type, glutamic acid-type, metal ion-type, and some Several categories of peptidases whose catalytic mechanism is unknown.

There are 1,382 known peptidases and their zymogen structures. The conserved structural features of this family member are: (1) Contains a conserved catalytic triplet structure consisting of His, Asp and Ser, and several conserved structures near the catalytic site (2) Conserved amino acid Asp189 at the bottom of the substrate binding pocket; (3) Gly216 and Gly226 at the opening of the substrate binding pocket; (4) Trypsin in humans and animals has 3-4 disulfides Bonds, most insects only have 3 disulfide bonds. Trypsin also has two barrel domains composed of six antiparallel β-sheets. The two barrel domains are connected by a disulfide bond, and the active center is located between the two barrel domains. Trypsin selectively hydrolyzes the carboxyl-terminal peptide bonds of lysine or arginine in proteins to form a product with a C-terminus of a basic amino acid. His and Asp in the catalytic center are located in the N-terminal domain, and the most important residue Ser and other important functional sites are located in the C-terminal domain. They act as regulators by activating or inhibiting proteases , and two other very important conserved disulfide bonds are also located at the C-terminus. Therefore, the C-terminal domain mainly performs the catalytic function of trypsin, while the main function of the N-terminal domain may be to cooperate with the C-terminal domain, thereby ensuring the activity and structural stability of the catalytic center triplet.

The many biological functions of peptidase make it have important applications in food technology, life science research and environmental protection, mainly listed as follows.

In food technology, it hydrolyzes the hydrophobic amino acids at both ends of the bitter peptide, increases the concentration of free amino acids, debitterizes food, and inhibits cold turbidity of beer, etc. Yang Jie et al. used trypsin and a compound protease containing exonuclease to enzymatically hydrolyze desugared rice dregs to obtain a depicroprotein peptide with a protein content of more than 88% and a degree of hydrolysis of 19%. After Wen Yanmei treated beer with immobilized trypsin, the turbidity of beer decreased by 0.08EBC, and the original flavor was still maintained. After 100 days in the refrigerator at 4°C, no obvious cold turbidity occurred.

In life science research, trypsin can be used to digest tissue blocks, isolate cells, and stain tissue-specifically. In 2012, Bai Yanhui and others successfully cultured and identified human umbilical vein endothelial cells in vitro by using the trypsin digestion method. The research results of Wang Guoyun et al. showed that trypsin has many advantages in reticular fiber staining, such as easy operation and clear color background. In the treatment of diseases, it is used to remove necrotic tissue, repair burns, treat snake venom, etc. In 2012, the research results of Zhang Peng and others showed that Antarctic krill trypsin can significantly promote the healing of animal wounds and shorten the healing time of wounds. Duan Zuowei and others studied the trypsin and chymotrypsin first aid kit to treat poisonous snake bites. The results showed that 256 cases of poisonous snake bite patients were all cured, and the cure rate reached 100%.

In terms of environmental protection, it is used to treat protein waste such as poultry hair, leather and shrimp heads, and to degrade organic pesticides. In 2012, Feng Chengli and others found that under the conditions of 48°C, pH 7.5, enzyme-to-pigskin ratio of 40mg/g, substrate concentration of 25%, and time of 2h, the degree of hydrolysis of trypsin on pigskin reached 16. %. Sun Lixin (2006), Xiong Chunrong (2009) and others have confirmed through research that the trypsin gene is a gene related to deltamethrin resistance in Culex pipiens, and it can directly degrade the organic insecticide deltamethrin.

At present, the main ways to obtain peptidase are: (1) directly extracting, separating and purifying from animal and plant tissues; this method requires a large amount of biological tissue to obtain a large amount of trypsin, and the pre-treatment of biological tissue is relatively difficult. Trouble, the price is more expensive. (2) Production of peptidase by microbial fermentation. This method has many advantages, such as low cost, high yield, and easier purification through secreted expression system. Peptidase-producing strains can be screened by pure culture technology, but due to the limitations of various conditions, about 99% of microorganisms in nature cannot be achieved by pure culture technology, and there are certain limitations in the discovery of new genes. Therefore, macro More and more scientists pay attention to genome technology in discovering new genes. Metagenome refers to the sum of the genetic material of all tiny organisms in the habitat. At present, it mainly refers to the genome sum of bacteria and fungi in environmental samples. The bottleneck of pure culture technology can be bypassed through metagenomic technology.

Contents of the invention

The object of the present invention is to provide a peptidase-encoding gene try1A and its application, a peptidase gene obtained by constructing a compost soil environment metagenomic library and adopting an activity screening strategy.

Another object of the present invention is to provide a method for cloning a gene encoding peptidase, comprising the following steps:

(1) Extract uncultured microbial metagenomic DNA from compost soil environmental samples in Nanning, Guangxi, and construct an environmental metagenomic library;

(2) The activity screening strategy was used to isolate peptidase clones from the metagenomic library.

In the present invention, by constructing an environmental metagenomic library and using an activity screening strategy, a new gene encoding peptidase is obtained, which can be expressed in large quantities in host cells, and the produced peptidase has better affinity and hydrolysis for casein substrates ability.

The recombinant plasmid strain E.coli BL21(DE3)pLysS/pGXTRY1A is preserved in the General Microbiology Center of China Committee for Culture Collection of Microorganisms, with the preservation number CGMCC No.7472, and the classification name is Escherichia coli, and the preservation date is 2013 On April 15, 2010, the preservation address was: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing.

The DNA of Sequence List No.1 is the partial DNA sequence of the cloning vector pGEM-3Zf(+) and the DNA of the exogenous uncultivated microorganism cloned on the vector, including the complete novel peptidase gene try1A. The ORF of the gene is located between the 1st and 1221st nucleotides of the clone, starting with the start codon ATG of the 1st-3rd nucleotides, and ending with the stop codon TAA of the 1219th-1221st nucleotides . A total of 1221 nucleotides in the ORF can encode a polypeptide consisting of 406 amino acids. At the amino acid level, it has 96% and 80% identity with Trypsin-like serine protease with C-terminal PDZdomain of Brevibacillus sp.CF112 and Brevibacillus sp.BC25, respectively; it has 79% identity with Serineprotease do-like protein precursor of Brevibacillus NBRC100599 Consistency; 48% concordance with Serine protease do-like protein precursor of Anoxybacillus flavithermus; these sequences are sourced from whole genome sequencing, and there is no report on enzymatic property identification.

The protein coded by the gene provided by the invention has wide applications in hydrolyzing protein macromolecular substrates, making it widely used in food technology, life science research, protein waste treatment and the like.

Description of drawings

Figure 1 shows metagenomic DNA extracted from compost soil samples in Nanning, Guangxi.

Fig. 2 is an electrophoresis image of metagenomic DNA after double enzyme digestion.

Figure 3 shows the restriction endonuclease digestion analysis of the clones of the compost soil metagene library to judge the quality of the library.

Figure 4 is the screening of clones expressing peptidase activity

Figure 5 is the result of enzyme digestion detection of the original clone pGEMA7.

Fig. 6 is the result of PCR amplification of gene try1A.

Fig. 7 is the result of enzyme digestion detection of the expression recombinant plasmid pGXTRY1A.

Fig. 8 is the sequencing result of expressing recombinant plasmid pGXTRY1A.

Figure 9 is the SDS-PAGE analysis result of the induced expression after the recombinant plasmid pGXTRY1A was transformed into E.coli BL21(DE3)pLysS.

Figure 10 is the result of identification of Try1A protein activity.

Fig. 11 Effects of different temperatures on the activity of Try1A recombinant protease.

Fig. 12 Effects of different pH on the activity of Try1A recombinant protease.

Detailed ways

The main materials used in the embodiments of the present invention include: restriction endonucleases (EcoRI, PstI and HindIII) and T4DNA ligase were purchased from Fermentas company, cloning vector pGEM-3Zf(+) and expression vector pET-32a (+) were purchased from Promega, PVPP (crosslinked polyvinylpyrrolidone) was purchased from Sigma, and CTAB (cetyltrimethylammonium bromide) was purchased from Sigma.

Example 1

A method for cloning the gene try1A encoding peptidase, comprising the steps of:

Step 1: Extract and purify metagenomic DNA from compost soil, the method steps are as follows.

(1) First prepare the DNA extraction solution: 100mM sodium phosphate buffer (pH8.0), 1% (w/v) CTAB, 100mM EDTA (pH8.0), 0.3M NaCl, 0.01mM Tris-HCl (pH8.0 );

(2) PVPP pickling treatment: Weigh 10g PVPP, add 3M HCl, soak for 12h, filter with filter paper, wash and stir PVPP with 20mM potassium phosphate buffer (pH7.4), repeat several times until the suspension reaches neutrality, put PVPP filter and dry at 50°C;

(3) Weigh 1 g of the sample into a 15 mL centrifuge tube, add 0.25 g of acidified PVPP, 4 mL of extraction buffer, mix well, freeze and thaw at 70°C and liquid nitrogen three times, each time for 30 min. After the last dissolution, add SDS to a final concentration of 1%, mix by inversion, place at room temperature for 20 minutes, centrifuge at 5000rpm for 8 minutes, and take the supernatant for use;

(4) Add 2 mL of extract solution to resuspend the precipitate, centrifuge to get the supernatant, repeat this step twice, and combine all supernatants;

(5) Add proteinase K to a final concentration of 0.2mg/mL, bathe in water at 65°C for 1 hour, add 0.5g of acidified PVPP and 0.5g of Sephadex G-200, mix well, combine at room temperature for 30 minutes, centrifuge at 5000rpm for 8 minutes, and take the supernatant for use;

(6) Add 2 mL of extract solution to resuspend the precipitate, centrifuge to get the supernatant, repeat this step twice, and combine all supernatants;

(7) Add PEG-8000 to make the final concentration 10%, add NaCl to make the final concentration 1.1M, let it rest at 4°C for 2 hours, divide into 2mL centrifuge tubes, centrifuge at 13000rpm at 4°C for 10min, discard clear;

(8) Wash twice with 75% ethanol and once with absolute ethanol, concentrate in vacuo until ethanol volatilizes completely, add TE buffer to dissolve, and store at -20°C.

Take an appropriate amount of metagenomic DNA for agarose gel electrophoresis detection, and the obtained metagenomic DNA fragments have good integrity (see Figure 1), and form diffuse bands after double digestion with HindⅢ and PstⅠ (see Figure 2), confirming their purity Comply with molecular experiment requirements.

In contrast to Figure 1, it is the electrophoresis image of the extracted metagenomic DNA. Lane 1 is λ/HindⅢMarker, and the fragment sizes from large to small are: 23.13kb, 9.4kb, 6.6kb, 4.4kb, 2.3kb, 2.0kb; Lanes 2-5 are metagenomes extracted and purified from compost soil environment DNA.

Contrasting with Figure 2, it is the electrophoresis image after digestion of the metagenomic DNA. Lane 1 is λ/HindⅢMarker, and the fragment sizes from large to small are: 23.13kb, 9.4kb, 6.6kb, 4.4kb, 2.3kb, 2.0kb; Lane 2 is the result of double digestion of metagenomic DNA with HindⅢ and PstⅠ.

Step 2: Construction of the compost soil metagenomic library, the detailed method is as follows.

Firstly, the metagenomic DNA was extracted manually, and the metagenomic DNA was double-digested with two restriction enzymes, HindⅢ and PstⅠ. The 3-10kb fragment was recovered by agarose gel electrophoresis, connected with the cloning vector pGEM-3Zf(+) that had been double-digested and purified by the same two restriction endonucleases, introduced into the E.coli DH5α host, and coated Blue-white selection was performed on LA plates containing 40 μg/mL X-gal, 40 μg/mL IPTG, and 50 μg/mL Amp. Pick the white clones onto an LA plate containing 50 μg/mL Amp, and culture it upside down at 37°C until the colony is 2-3mm in size, then pick the plate again, and pick the transformants into the wells of a 96-well culture plate based on library preservation and culture, at 37°C Cultivate at a constant temperature for about 24 hours, and then store in a -80°C low-temperature refrigerator. Finally, 32,100 white transformants were obtained. 14 transformants were randomly selected to extract plasmids, and HindⅢ and PstⅠ were used for enzyme digestion detection, and 10 foreign DNA fragments with random insertions were found, with an average length of 3.5kb. The results are shown in Figure 3.

Comparing with Figure 3, it is the quality detection of the compost soil metagenomic library. Lane 1 is 1kb Marker, and the fragment sizes from top to bottom are: 10.0kb, 8.0kb, 6.0kb, 5.0kb, 4.0kb, 3.5kb, 3.0kb, 2.5kb, 2.0kb, 1.5kb, 1.0kb, 750bp, 500bp, 250bp; lanes 2-15 are the results of double digestion of randomly selected recombinant plasmids with HindⅢ and PstⅠ.

Step 3: The activity screening strategy is used to isolate the peptidase gene from the metagenomic library, and the detailed method is as follows.

In order to obtain the peptidase gene, the activity screening strategy was adopted in this study, and the metagenomic library was screened with skimmed milk powder as the substrate. Copy the clones in the library onto an LA plate with 50 μg/mL Amp and 1% (w/v) skimmed milk powder, and incubate upside down at 37°C for 24-72 hours to obtain a positive clone with a transparent circle around it, which is named as pGEMA7. Its plasmid was extracted for double enzyme digestion and it was found that it carried a foreign fragment of about 8kb.

In contrast to Figure 4, it is the screening of clones expressing peptidase activity.

Compared with Figure 5, it is the enzyme digestion map of positive clone pGEMA7, lane 1 is 1kb Ladder Marker, and the fragment sizes from top to bottom are: 10.0kb, 8.0kb, 6.0kb, 5.0kb, 4.0kb, 3.5kb, 3.0kb, 2.5 kb, 2.0kb, 1.5kb, 1.0kb, 750bp, 500bp, 250bp; Lane 2 is the double digestion result of clone pGEMA7.

Step 4: Clone pGEMA7 for sequencing and bioinformatics analysis, the detailed method is as follows.

The nucleotide sequence of cloned pGEMA7 was determined on the ABI377 DNA automatic sequencer by the dideoxynucleotide method, and the sequence obtained by the sequencing was spliced with the software DNA Star. Using the ORF Finder tool to analyze the sequencing results, it was found that the possible new gene encoding the peptidase carried in the exogenous DNA fragment was 1221bp in size. The gene was named try1A, and its sequence is shown in Sequence Table 1. From the start codon ATG of the first nucleotide at the 5' end to the stop codon TAG at 1221 bases, there is a complete ORF, and the 1st-3rd nucleotide at the 5' end is the start of the try1A gene The start codon ATG, the 1219-1221 nucleotides from the 5' end is the stop codon TAA of the try1A gene, encoding a polypeptide consisting of 406 amino acids.

According to the results of Blastp, it can be seen that the gene try1A has 96% and 80% concordance with the Trypsin-like serine protease with C-terminal PDZ domain of Brevibacillus sp. 31); with 79% concordance with the Serine protease do-like protein precursor of Brevibacillus NBRC100599 (data submitted on October 28, 2011), and 48% concordance with the Serine protease do-like protein precursor of Anoxybacillus flavithermus, The sources of these sequences are whole-genome sequencing, and there is no report on their functional identification. After SMART analysis, it was found that the 20th to 42nd amino acid has a transmembrane helical structure, there is a low-complexity region structure from the 82nd amino acid to the 95th amino acid region, and the 118th to 290th amino acid region exists A Pfam Trypsin (trypsin family catalytic domain) structure; a PDZ structure of unknown function exists from amino acids 306 to 396. Through multiple sequence alignments combined with MEROPS Blast Server analysis, it was found that the conserved three-way catalyst structure of Try1A protein was His134-Asp164-Ser247, which contained a GDSGG conserved motif, and it was speculated that it belonged to the S1C peptidase subfamily.

Example 2

Construction of try1A Gene Expression Recombinant Plasmid

The plasmid pET-32a(+) provided by Novagen, USA was used as the expression vector and E.coli BL21(DE3)pLysS was used as the host cell for heterologous high-efficiency expression of the target gene.

According to the sequence of the try1A gene, primers were designed using relevant software. Forward amplification primer F1: 5'-CCG GAATTC ATGGGGTTTTACGATGATTTG-3', introducing EcoRI restriction site; reverse amplification primer R1: 5'-CCC AAGCTT CTCCGGCGTTTTAGCCAGCT-3', adding HindⅢ restriction site. Using pGEMA7 as a template, Pfu DNA polymerase was used to perform a PCR reaction to amplify the try1A gene, as shown in FIG. 6 . The expression vector pET-32(a)+ plasmid was extracted, digested with EcoRI and HindⅢ, purified and ligated with the amplified product of try1A that had also undergone double digestion and purification, and the expression recombinant plasmid pGXTRY1A was constructed, and transformed into E.coli DH5α was sequenced after digestion and verification (see Figure 7), and the sequence results are shown in Figure 8. The Theoretical pI/Mw of pGXTRY1A was analyzed by the related software of ExPASy website, and it was found that its theoretical isoelectric point was 6.03 and its molecular weight was about 63.9kDa.

Embodiment 2 Please refer to FIG. 7 . Figure 7 shows the detection results of the expression recombinant plasmid pGXTRY1A, 1 is 1kb Ladder Maker, and the fragment sizes from top to bottom are: 10.0kb, 8.0kb, 6.0kb, 5.0kb, 4.0kb, 3.5kb, 3.0kb, 2.5kb, 2.0kb, 1.5kb, 1.0kb, 750bp, 500bp, 250bp; 2 is the result after digesting pGXTRY1A with EcoRI and HindIII.

Fig. 8 is the partial sequence of pET-32a(+) vector, the DNA sequence of pGXTRY1A and the amino acid sequence of the target ORF.

Example 3

Induced expression and purification of try1A gene in Escherichia coli

The recombinant plasmid pGXTRY1A with correct sequence was extracted and transformed into E.coli BL21(DE3)pLysS expression host cell. The recombinant expressing bacteria with verified peptidase activity were transferred to 10 mL of LB medium containing Amp and Cm, and cultured overnight at 37°C and 200 rpm. Take 200 μL of 1% inoculum and add it to 10 mL of LB medium (containing Amp and Cm), 37 ° C, 200 rpm shaking culture for 2-3 h to OD ₆₀₀ to 0.6-0.8, add IPTG with a final concentration of 0.8 mM, 28 ° C, After induction at 200rpm for 12h, the crude enzyme solution was prepared; the recombinant protein was isolated and purified using the His-Bind Purification Kit from Qiagen. The prepared control sample and the recombinant protein obtained by separation and purification were detected by SDS-PAGE for protein expression.

Embodiment 3 Please refer to FIG. 9 . Fig. 9 is the result of SDS-PAGE analysis after the recombinant plasmid pGXTRY1A was transformed into E. coli E. coliBL21(DE3) pLysS to induce expression. 1 is the protein standard sample band, the size from top to bottom is: 116.0kDa, 66.2kDa, 45.0kDa, 35.0kDa, 25.0kDa, 18.4kDa, 14.4kDa; 2 is E.coli BL21(DE3)pLysS/pET-32a (+) Whole protein; 3 is the whole protein of E.coli BL21(DE3)pLysS/pGXTRY1A; 4 is the Try1A protein separated and purified by nickel column affinity chromatography.

Example 4

Activity identification of Try1A recombinant protein

Mix the sample with SDS loading buffer, and directly load the sample for electrophoresis without boiling. The electrophoresis process of zymography analysis is the same as that of ordinary SDS-PAGE gel electrophoresis; after electrophoresis, use 50mM Tris- Wash twice with HCl (pH 8.0) on a decolorizing shaker, 30 min each time; add 50 mM Tris-HCl (pH 8.0), incubate overnight at 40 ° C; stain with Coomassie brilliant blue for 30 min, add decolorizing solution, and decolorize until white stripes appear band appears.

Please refer to Figure 10 for Embodiment 4. Figure 10 shows the results of Try1A recombinant protein zymogram analysis. Swimming lane 1 is the protein standard sample band. E.coli BL21(DE3)pLysS/pET-32a(+) whole protein; Lane 3 is E.coli BL21(DE3)pLysS/pGXTRY1A whole protein.

Example 5

Effects of Temperature and pH on Enzyme Activity of Try1A Protein

Different temperatures were set to measure the enzyme activity of the recombinant protein Try1A. The OD value was measured at a wavelength of 660 nm using a microplate reader from Thermo Company.

The results are shown in Figure 11, Try1A protein has the highest enzyme activity at 50°C.

Try1A protein and substrate were tested for the enzymatic activity of Try1A protein in buffers with different pH values at the optimal temperature: disodium hydrogen phosphate-citric acid buffer (6.0-8.0); Tirs-HCl buffer (7.5-8.9) ; Gly-NaOH buffer (8.6-10.6).

The results are shown in Figure 12, the Try1A protein has the highest enzyme activity when the pH value is 9.0.

The expression vector, cell line and peptidase product containing the open reading frame of the gene of the present invention and its partial sequence all belong to the protection scope of the present invention.

Claims (3)

1. the gene of the peptase of encoding try1A, it is characterized in that, its nucleotide sequence is as shown in sequence table No.1.

2. the protein of the coded by said gene of claim 1, is characterized in that, its aminoacid sequence is as shown in sequence table No.2.

3. the application of protein claimed in claim 2 in preparing peptase.