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

Abstract

The invention relates to a gene try1A for encoding a peptidase. The gene has the nucleotide sequence as shown in a sequence table No.1 and an amino acid residue as shown in a sequence table No.2. An integral ORF (Open Reading Frame) (nucleotides 1-1221) exists in a DNA (Deoxyribonucleic Acid) in the sequence table No.1 which is started by the first nucleotide at the 5'end, i.e., one of initiation codons ATG (Anti-Thymocyte Gl) and ended by the 1221st nucleotide, i.e., one of termination codons TAA (Tumor Associated Antigen), wherein the nucleotides from the first position to the third position at the 5'end are used as the initiation codons ATG of the gene try1A, and the nucleotides from the 1219th position to the 1221st position at the 5'end are used as the termination codons TAA of the gene try1A. A protein encoded by using the gene provided by the invention can be used for preparing macromolecular hydrolyzed protein substrates so as to have wide purposes in the aspects such as food processes, life science research, protein waste treatment and the like.

Description

A kind of gene try1A and application thereof of the peptase of encoding

Technical field

The present invention relates to a kind of gene try1A and application thereof of the peptase of encoding.

Background technology

Peptase is the general name of the enzyme of a class energy hydrolysising peptide key, also can be referred to as proteolytic enzyme or proteolytic ferment.Peptase is the enzyme of a class enormous amount, it is reported, the protein nearly 2% in all organisms belong to peptase or with the enzyme of peptase homology.Peptase is a kind of important industrial enzyme, it is reported, the sales volume of peptase accounts for 50% of industrial enzyme market, is widely used in the each side such as food, weaving, medicine and environmental protection.Peptase is the amino-acid residue type of catalytic site according to catalytic chemistry mechanism, be divided into serine-type peptase, halfcystine type, Threonine type, asparagine acid type, L-glutamic acid type, metal ion type, and several large classes such as peptase of some unknown its catalyst mechanisms.

Existing known peptase and proenzyme structure thereof has 1382, and this family member's conserved structure feature is: (1) contains a conservative catalysis triplet configuration being comprised of His, Asp and Ser, also has several conservative amino-acid residues near catalytic site; (2) the conserved amino acid Asp189 of Binding Capacity pocket bottom; (3) be positioned at Gly216 and the Gly226 at Binding Capacity pocket openings place; (4) trypsinase in people and animal body has 3-4 disulfide linkage, and insect only has 3 disulfide linkage mostly.Trypsinase structurally also has two barrel-like structure territories that consist of six antiparallel beta sheets.These two barrel-like structure territories are connected by disulfide linkage, and active centre is between two barrel-like structure territories.Trypsinase is Methionin or arginic carboxy-terminal peptide bond in protein hydrolysate optionally, forms the product with basic aminoacids C-terminal.The His of its catalytic center and Asp are positioned at N end structure territory, most important residue Ser and other important functional sites are all positioned on C end structure territory, they,, by the activation of pepsinogen or inhibition being played to the effect of regulatory factor, also have two very important conservative disulfide linkage to be also positioned at C end in addition.Therefore tryptic catalysis is mainly carried out in C end structure territory, and the structural domain major function of N end may be to coordinate C end structure territory, thereby guarantees activity and the structural stability of catalytic center triplet.

Many biological functions of peptase make it at aspects such as food technology and life science and environmental protection, have important application, are mainly listed below.

On food technology, the hydrophobic amino acid at hydrolysis bitter peptides two ends, the concentration of increase total free aminoacids, makes food debitterize; Suppress beer cold turbid phenomenon etc.The employing trypsinase such as Yang Jie and the compound protease that contains excision enzyme carry out enzymolysis to desugar rice slag, have made debitterize protein peptide, and protein content is greater than 88%, and degree of hydrolysis reaches 19%.Wen Yanmei etc. are with after immobilized trypsin treatment beer, and the turbidity decline 0.08EBC of beer, still keeps original local flavor, are placed on 4 ℃ of refrigerators after 100 days, without obvious cold turbid phenomenon, occur.

In life science, trypsinase can be used for digesting tissue block, isolated cell, tissue specificity dyeing.2012, Bai Yanhui etc. application tryptic digestion method in vitro successful culture & identification human umbilical vein endothelial cell.The people's such as kingdom's cloud result of study shows, trypsinase has multiple advantage in reticular fiber staining, as easy and simple to handle, colour developing background is clear etc.In disease treatment, for removing necrotic tissue, repair burn, treating snake venom etc.2012, the people's such as Zhang Peng result of study demonstration, South Pole large krill trypsinase can obviously promote the healing of the animal surface of a wound and shorten wound healing time.Duan Zuowei etc. have studied trypsinase and Chymotrypsin First-aid medicine box treatment venomous snake bite, result demonstration, and 256 routine Bit by Vipers are all cured, and curative ratio reaches 100%.

Aspect environmental protection, for the treatment of the first-class albumen refuse of poultry hair, leather and shrimp, degraded organic pesticide etc.2012, the people such as Feng Chengli studied discovery, trypsinase at 48 ℃, pH7.5, enzyme with pigskin than being 40mg/g, concentration of substrate 25%, under the condition such as the time is 2h, reaches 16% to the degree of hydrolysis of pigskin.Sun Lixin (2006), Xiong Chunrong (2009) etc., by research, confirm that trypsase gene is culex pipiens pollens deltamethrin resistance genes involved, and organic insecticide Deltamethrin is had to direct Degradation.

At present, the major way that obtains peptase has: directly extract from animal vegetable tissue (1), and separation and purification obtains; This method will obtain a large amount of trypsinase, needs a large amount of bio-tissues, and processes trouble the early stage of biological tissue, and price is more expensive.(2) utilize Production by Microorganism Fermentation peptase.This method has many advantages, and as low in cost, acquisition amount is large, by secreting, expressing system, make purifying easier etc.The bacterial strain that produces peptase can be screened and be obtained by pure culture technigne, but the restriction due to each side condition, nature boundary treaty has 99% microorganism not realize by pure culture technigne, aspect the new gene of excavation, there iing certain limitation, therefore, technique of metagenome is obtaining increasing scientist's attention aspect the new gene of excavation.Grand genome refers to the summation of whole tiny organism genetic material in habitat, and the bacterium in current mainly finger environmental sample and the genome summation of fungi, can walk around pure culture technigne bottleneck by technique of metagenome.

Summary of the invention

The object of this invention is to provide a kind of gene try1A and application thereof of the peptase of encoding, by building compost soil environment macro genomic library, a kind of peptidase genes that adopts screening active ingredients strategy to obtain.

The cloning process of the gene of a kind of peptase of encoding that another object of the present invention is to provide, comprises the following steps:

(1) from the compost soil environmental sample of Nanning, extract not culturing micro-organisms macro genome DNA, the grand genomic library of constructing environment;

(2) from this grand genomic library, utilize the clone of the separated peptase of screening active ingredients strategy.

The present invention by the grand genomic library of constructing environment, utilizes screening active ingredients strategy, has obtained a kind of gene of new coding peptase, can be in host cell great expression, the peptase of generation has good avidity and hydrolysis ability to casein substrate.

Recombinant plasmid bacterial strain E.coli BL21 (DE3) pLysS/pGXTRY1A is kept at China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation is numbered CGMCC No.7472, Classification And Nomenclature is: colon bacillus Escherichia coli, date saved is on April 15th, 2013, and preservation address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City.

The DNA of sequence table No.1 is cloning vector pGEM-3Zf (+) partial dna sequence and is cloned in the not DNA of culturing micro-organisms of external source on carrier, comprises complete peptidase gene try1A.The ORF of this gene is between the 1st to the 1221st Nucleotide of clone, initial by the initiator codon ATG of 1-3 position Nucleotide, and the terminator codon TAA of 1219-1221 position Nucleotide stops.This ORF is 1221 polypeptide that Nucleotide codified is comprised of 406 amino acid altogether.On amino acid levels, there is respectively 96% and 80% consistence with the Trypsin-like serine protease with C-terminal PDZ domain of Brevibacillus sp.CF112, Brevibacillus sp.BC25; There is 79% consistence with the Serine protease do-like protein precursor of Brevibacillus NBRC100599; There is 48% consistence with the Serine protease do-like protein precursor of Anoxybacillus flavithermus; These sequence sources are all genome sequencings, and the relevant report that does not have zymologic property to identify.

The protein of coded by said gene provided by the present invention at protein hydrolysate class macromolecule substrate, it is had been widely used at aspect tools such as food technology and life science and the processing of albumen refuse.

Accompanying drawing explanation

The macro genome DNA of Fig. 1 for extracting from the compost pedotheque of Nanning.

Fig. 2 is the electrophorogram after macro genome DNA double digestion.

Fig. 3 is that the grand gene library of compost soil clone's restriction analysis is with judgement library quality.

Fig. 4 is expression of peptides enzymic activity clone's screening

Fig. 5 is that the enzyme of original clone pGEMA7 is cut detected result.

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

Fig. 7 is that the enzyme of recombinant expression pGXTRY1A is cut detected result.

Fig. 8 is the sequencing result of recombinant expression pGXTRY1A.

Fig. 9 is the SDS-PAGE analytical results of abduction delivering after recombinant expression pGXTRY1A Transformed E .coli BL21 (DE3) pLysS.

Figure 10 is Try1A protein-active qualification result.

The impact that Figure 11 differing temps is lived on Try1A recombinant protein enzyme.

The impact that the different pH of Figure 12 live on Try1A recombinant protein enzyme.

Embodiment

Main raw used comprises in an embodiment of the present invention: restriction enzyme (EcoR I, Pst I and Hind III) and T4DNA ligase enzyme all purchased from Fermentas company, cloning vector pGEM-3Zf (+) and expression vector pET-32a (+) all purchased from Promega company, PVPP(cross-linked polyvinylpyrrolidone) purchased from Sigma company, CTAB(cetyl trimethylammonium bromide) purchased from Sigma company.

Embodiment 1

The encode cloning process of gene try1A of peptase, comprises the steps:

Step 1: extract and purifying macro genome DNA from compost soil, method steps is as follows.

(1) first prepare DNA extraction liquid: 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 cleanup acid treatment: take 10g PVPP, add 3M HCl, soak 12h, have filter paper filtering, clean and stir PVPP with 20mM potassium phosphate buffer (pH7.4), repeat several times, until suspension reaches neutral, PVPP to be filtered, in 50 ℃ of oven dry;

(3) take 1g sample in 15mL centrifuge tube, add the PVPP of 0.25g acidifying, 4mL Extraction buffer, mixes, 70 ℃ and liquid nitrogen multigelation 3 times, each 30min.After last dissolving, add SDS to final concentration be 1%, put upside down and mix, place room temperature 20min, the centrifugal 8min of 5000rpm, gets supernatant stand-by;

(4) add the resuspended precipitation of 2mL extracting solution, centrifuging and taking supernatant, repeats this step twice, merges all supernatants;

(5) adding Proteinase K to final concentration is 0.2mg/mL, and 65 ℃ of water-bath 1h add 0.5g acidifying PVPP and 0.5g Sephadex G-200 to mix, and room temperature is in conjunction with 30min, and the centrifugal 8min of 5000rpm, gets supernatant stand-by;

(6) add the resuspended precipitation of 2mL extracting solution, centrifuging and taking supernatant, repeats this step twice, merges all supernatants;

(7) add PEG-8000 to make its final concentration reach 10%, adding NaCl to make its final concentration is 1.1M, puts to 4 ℃ of static 2h, is sub-packed in 2mL centrifuge tube, and 4 ℃, the centrifugal 10min of 13000rpm, abandons supernatant;

(8) by 75% washing with alcohol 2 times, absolute ethanol washing 1 time, vacuum concentration to ethanol volatilization completely, add TE damping fluid to dissolve ,-20 ℃ of preservations.

Get appropriate macro genome DNA and carry out agarose gel electrophoresis detection, the better (see figure 1) of macro genome DNA complete segment of gained becomes smear (see figure 2) through Hind III after Pst I double digestion, confirms that its purity meets molecule requirement of experiment.

Contrast Fig. 1 is the macro genome DNA electrophorogram extracting.Swimming lane 1 is λ/Hind III Marker, and clip size is followed successively by from big to small: 23.13kb, 9.4kb, 6.6kb, 4.4kb, 2.3kb, 2.0kb; Swimming lane 2-5 extracts and the macro genome DNA of purifying from compost edatope.

Contrast Fig. 2, for macro genome DNA enzyme is cut rear electrophoresis figure.Swimming lane 1 is λ/Hind III Marker, and clip size is followed successively by from big to small: 23.13kb, 9.4kb, 6.6kb, 4.4kb, 2.3kb, 2.0kb; Swimming lane 2 carries out double digestion result by Hind III and Pst I for macro genome DNA.

Step 2: the structure of the grand genomic library of compost soil, detailed method is as follows.

First by manual method, extract macro genome DNA, adopt Hind III and two kinds of restriction enzymes of Pst I to carry out double digestion to macro genome DNA.Through agarose gel electrophoresis, reclaim the fragment of 3-10kb, be connected with the cloning vector pGEM-3Zf (+) through same two kinds of restriction enzymes double zyme cuttings and purifying, import in E.coli DH5 α host, coat and on the LA flat board that contains 40 μ g/mL X-gal, 40 μ g/mL IPTG and 50 μ g/mLAmp, carry out indigo plant and screen in vain.White clone is chosen to the LA flat board that contains 50 μ g/mL Amp, after 37 ℃ of inversions are cultured to bacterium colony and are 2-3mm, bridging piece again, by transformant picking to library Storaged media in 96 well culture plate holes, 37 ℃ of constant temperature culture 24h left and right, are then stored in-80 ℃ of cryogenic refrigerators.32,100 white transformants have finally been obtained.14 transformants of random picking extract plasmid, carry out enzyme cut detection by Hind III and Pst I, find 10 exogenous dna fragments with radom insertion, and mean length is 3.5kb, the results are shown in Figure 3.

Contrast Fig. 3 is the quality examination of the grand genomic library of compost soil.Swimming lane 1 is 1kb Marker, and clip size is followed successively by from top to bottom: 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; Swimming lane 2-15 is that the recombinant plasmid of random picking carries out double digestion result by Hind III and Pst I.

Step 3: adopt screening active ingredients strategy separated peptidase genes from grand genomic library, detailed method is as follows.

In order to obtain peptidase genes, this research adopts screening active ingredients strategy, and the skim-milk of take screens this grand genomic library as substrate.By clone photocopy in library to being added with 50 μ g/mL Amp and 1%(w/v) on the LA flat board of skim-milk, be inverted for 37 ℃ and cultivate 24-72h, obtain a positive colony that produces transparent circle around, by its called after pGEMA7.Extract its plasmid and carry out double digestion detection, find that it carries the external source fragment of an about 8kb.

Contrast Fig. 4 is expression of peptides enzymic activity clone's screening.

Contrast Fig. 5, positive clone pGEMA7 cleavage map, swimming lane 1 is 1kb Ladder Marker, clip size is followed successively by from top to bottom: 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; Swimming lane 2 is clone pGEMA7 double digestion result.

Step 4: clone pGEMA7 order-checking and bioinformatic analysis, detailed method is as follows.

Adopt dideoxyribonucleoside acid system on ABI377DNA automatic sequencer, to measure the nucleotide sequence of clone pGEMA7, with software DNA Star, the resulting sequence that checks order is spliced.Use its sequencing result of ORF Finder tool analysis, the new gene size of the possible coding peptase carrying in discovery exogenous dna fragment is 1221bp, by this unnamed gene, is try1A, and its sequence is shown in sequence table 1.From ATG to 1221 base of 5 ' the 1st Nucleotide initiator codon of end, with terminator codon TAG, finish, there is a complete ORF, 1-3 position Nucleotide from 5 ' end is the initiator codon ATG of try1A gene, 1219-1221 position Nucleotide from 5 ' end is the terminator codon TAA of try1A gene, the polypeptide being comprised of 406 amino acid of encoding.

According to Blastp result, can on amino acid levels, there is respectively 96% and 80% consistence (data are filed on July 31st, 2012) with the Trypsin-like serine protease with C-terminal PDZ domain of Brevibacillus sp.CF112, Brevibacillus sp.BC25 by major gene try1A; There is 79% consistence (data are filed on October 28th, 2011) with the Serine protease do-like protein precursor of Brevibacillus NBRC100599, there is 48% consistence with the Serine protease do-like protein precursor of Anoxybacillus flavithermus, these sequence sources are all genome sequencings, and the temporary relevant report without its Function Identification.After SMART analyzes, find that its 20th to 42 amino acid have the spirane structure of cross-film, from 95 amino acid regions of the 82nd amino acid to the, having the domain structure of a sequence low complex degree, there is Pfam Trypsin(trypsinase family's catalysis territory in the 118th to the 290th amino acid) structure; There is the PDZ structure of a Unknown Function in the 306th to the 396th amino acid.Through multiple sequence comparison and in conjunction with MEROPS Blast Server analysis, find that the conservative three-element catalytic body structure of Try1A albumen is His134-Asp164-Ser247, containing the conservative motif of a GDSGG, infer and belong to S1C peptase subfamily.

Embodiment 2

The structure of try1A genetic expression recombinant plasmid

Utilize the plasmid pET-32a (+) that U.S. Novagen company provides as host cell, to carry out the high efficient expression of allos of target gene as expression vector and E.coli BL21 (DE3) pLysS.

According to try1A gene order, utilize related software to carry out design of primers.Forward amplimer F1:5 '-CCG gAATTCaTGGGGTTTTACGATGATTTG-3 ', introduces EcoR I restriction enzyme site; Reverse amplimer R1:5 '-CCC aAGCTTcTCCGGCGTTTTAGCCAGCT-3 ', adds Hind III restriction enzyme site.Take pGEMA7 as template, adopt Pfu archaeal dna polymerase to carry out PCR reaction, amplification try1A gene, as shown in Figure 6.Extract expression vector pET-32 (a)+plasmid, by EcoR I and Hind III, carry out double digestion, after purifying, be connected with the same try1A amplified production through double digestion purifying, construction expression recombinant plasmid pGXTRY1A, adopt chemical transformation to proceed to E.coli DH5 α, after enzyme is cut checking, (see figure 7) checks order, and sequence results is shown in Fig. 8.Utilize ExPASy website related software to analyze the Theoretical pI/Mw of pGXTRY1A, find that its theoretical iso-electric point is 6.03, molecular weight is about 63.9kDa.

Embodiment 2 please refer to Fig. 7.Fig. 7 is the detected result of recombinant expression pGXTRY1A, and 1 is 1kb Ladder Maker, and clip size is followed successively by from top to bottom: 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 for cutting pGXTRY1A result afterwards with EcoR I and Hind III enzyme.

Fig. 8 is pET-32a (+) carrier part sequence, the DNA sequence dna of pGXTRY1A and the aminoacid sequence of target ORF.

Embodiment 3

The expression and purity of try1A gene in intestinal bacteria

The recombinant plasmid pGXTRY1A that abstraction sequence is correct, proceeds to E.coli BL21 (DE3) pLysS expression host cell.To verify that the recombinant expressed bacterium with peptidase activity transfers in the LB substratum that contains Amp and Cm in 10mL, 37 ℃, 200rpm overnight incubation.By 1% inoculation, measure 200 μ L and add in the LB substratum (containing Amp and Cm) of 10mL, 37 ℃, 200rpm shaking culture 2-3h to OD ₆₀₀to 0.6-0.8, adding final concentration is the IPTG of 0.8mM, and 28 ℃, 200rpm induces 12h, prepares crude enzyme liquid; Adopt the His-Bind Purification Kit of Qiagen company to carry out separation and purification to recombinant protein.The recombinant protein that the control sample of preparation and separation and purification obtain detects protein expression situation by SDS-PAGE.

Embodiment 3 please refer to Fig. 9.Fig. 9 is that recombinant plasmid pGXTRY1A transforms the SDS-PAGE analytical results after intestinal bacteria E.coliBL21 (DE3) pLysS abduction delivering.1 is protein standard model band, and size is followed successively by from top to bottom: 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 E.coli BL21 (DE3) pLysS/pGXTRY1A whole protein; 4 is the Try1A albumen that adopts the separation and purification of affinity chromatography technology to obtain.

Embodiment 4

The activity identification of Try1A recombinant protein

Sample and SDS sample-loading buffer mix, and without boiling direct loading electrophoresis, the electrophoresis process of enzyme spectrum analysis is the same with common SDS-PAGE gel electrophoresis; After electrophoresis finishes, with the 50mM Tris-HCl(pH8.0 containing 2%TritionX-100) washed twice on decolorization swinging table, each 30min; Add 50mMTris-HCl(pH8.0), 40 ℃ are incubated overnight; Use coomassie brilliant blue staining 30min, add destainer, decolour to adularescent band and occur.

Embodiment 4 refers to Figure 10.Figure 10 is Try1A recombinant protein enzyme spectrum analysis result, and swimming lane 1 is protein standard model band, and size is followed successively by from top to bottom: 116.0kDa, 66.2kDa, 45.0kDa, 35.0kDa, 25.0kDa, 18.4kDa, 14.4kDa; Swimming lane 2 is E.coli BL21 (DE3) pLysS/pET-32a (+) whole protein; Swimming lane 3 is E.coli BL21 (DE3) pLysS/pGXTRY1A whole protein.

Embodiment 5

Temperature and pH live and affect the enzyme of Try1A albumen

Different temperature is set, measures the enzyme of recombinant protein Try1A and live.Use the microplate reader of Thermo company, at 660nm wavelength place, measure OD value.

Result is referring to Figure 11, and in the time of 50 ℃, Try1A albumen has the highest enzyme work.

Try1A albumen from substrate under optimum temperuture, the enzyme of measuring Try1A albumen in the damping fluid of different pH values lives: Sodium phosphate dibasic-citrate buffer solution (6.0-8.0); Tirs-HCl damping fluid (7.5-8.9); Gly-NaOH damping fluid (8.6-10.6).

Result is referring to Figure 12, and pH value is that 9.0 o'clock Try1A albumen has the highest enzyme work.

The open reading frame that contains gene of the present invention and the expression vector of partial sequence thereof, clone and peptase product all belong to 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.