CN110305806A - A kind of the candida utili single-gene expression bacterial strain and its construction method of protein degradation matter - Google Patents
A kind of the candida utili single-gene expression bacterial strain and its construction method of protein degradation matter Download PDFInfo
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Abstract
The present invention relates to genetic engineerings and field of fermentation engineering, a kind of candida utili single-gene expression bacterial strain of protein degradation matter is specifically disclosed, the candida utili single-gene expression bacterial strain is built-up on candida utili genome for carboxypeptidase gene to be integrated by candida utilis expression vector.The present invention handles kitchen fermentation raffinate, organic matter therein is further utilized, for compared with the existing technology, breaks down proteins a large amount of in kitchen fermentation liquid are transformed into amino acid, it not only can solve the protein contamination problem of remaining, can also produce amino acid, amino acid be pharmacy, biological feedstuff, organic fertilizer raw material, there is certain economic value.
Description
Technical field
The present invention relates to genetic engineerings and field of fermentation engineering, false more particularly, to a kind of production protein of protein degradation matter
Silk yeast single-gene expression bacterial strain and its construction method.
Background technique
Kitchen garbage is commonly called as swill, is the solid waste that resident is formed in life, the quantity of food waste with
Population quickly increase and economic fast development and increase rapidly, remaining meat, vegetables, grease, rice is kitchen rubbish
Main matter in rubbish.In chemical component, mainly contain cellulose family, protein-based, carbohydrate, the organic matters such as fats at
Point, have water content high, organic matter ratio is high, the high feature of salt content.Since kitchen garbage yield is huge, Staphylococcus aureus
Bacterium, a variety of poisonous and hazardous pathogenic microorganisms such as salmonella are often grown in the kitchen garbage of nutriment rich in
It is raw.If can also rot without timely working process, NH is generated3And H2S etc. is largely with gas frowzy and toxin
Deng, not only cause serious air problem, while the illegal waste oil extracted using kitchen garbage the health of people has been generated it is tight
The harm of weight.
According to statistics, just have about 400,000,000 tons of kitchen garbages every year in catering industry, caused by waste total value be up to
750000000000 dollars, but also ever-increasing trend is being presented.All urgent needs will find the effective method of one kind to kitchen
Rubbish is comprehensively utilized, the unreasonable waste that serious resource is not only resulted in using kitchen garbage, also will cause serious
Environmental pollution.Burning disposal and be traditional processing method to kitchen garbage as feed；It burns not only to kitchen garbage
Utilization efficiency it is low, and a large amount of carbon dioxide can be discharged, aggravate global warming, a large amount of nitrogenous, sulfur-bearing, two
The generation for disliking the compounds such as English, not only pollutes air, and greatly harm can be also generated to the body of people.It is done after kitchen garbage processing
Livestock is fed at feed, a large amount of poisonous and harmful microorganism is contained in feed and is difficult to the toxin degraded, after raise livestock, not only
Livestock can be easy to cause to cause a disease, also toxin can be made to be enriched in livestock body, eventually affect the health of people, and wherein containing not
With a large amount of bioproteins in source, potential bio-safety risk existing for unprocessed feeding livestock, institute's development one in need
The effective method of kind, comprehensive utilization is carried out to kitchen garbage, both can protect environment, subtrahend nocuousness gas to avoid garbage-surrounded city
Body, the discharge of greenhouse gases also can achieve the utility value for promoting waste product, reach comprehensive utilization to resource.
Microbial fermentation is paid attention to by domestic and international industry, and fermentation technique is becoming a kind of pair of kitchen garbage comprehensive utilization
Effective means, be really achieved resource-effective and environmental-friendly ground purpose.Ferment raffinate, generally refers in industrial production application
In, the general name of raw material generated waste residue or waste liquid after the fermentation of microorganism.And kitchen alcohol fermentation raffinate, refer to original
The kitchen garbage of beginning has passed through grease removal, and the series of process process such as saccharomycete alcoholic fermentation and alcohol distillation extraction obtains
The unused waste of microorganism.By the grease removal and alcoholic fermentation in previous process, original oil in kitchen garbage
Lipid and saccharide compound have been utilized substantially, and the protein-based compound in kitchen garbage only has seldom a part micro-
Biological growth is utilized, and most of protein is remained in the form of waste in fermentation raffinate, and with industrially to meal
The further research and development of kitchen rubbish fermentation, the raffinate that fermentation will necessarily be made to generate is more and more, if without effective
Processing is arbitrarily discharged, and a large amount of protein-based organic matter, not only results in the mass propagation of poisonous and harmful microorganism in raffinate,
And serious harm can be caused to environment, so effective processing mode must be taken, kitchen fermentation raffinate is handled,
And the key handled is further to utilize organic matter therein, a large amount of breaks down proteins, which are transformed into amino acid, just becomes one
The feasible method of item not only can solve the protein contamination problem of remaining, amino acid or pharmacy, biological feedstuff, organic fertilizer
Raw material, have certain economic value.
Summary of the invention
The present invention is directed to overcome at least one of the above-mentioned prior art insufficient, a kind of Candida utilis of protein degradation matter is provided
Yeast single-gene expresses bacterial strain and its construction method, for solving the problems, such as protein in degradation kitchen fermentation liquid.
The technical solution adopted by the present invention is that providing a kind of candida utili single-gene expression bacterium of protein degradation matter
Strain, the candida utili single-gene expression bacterial strain is to be integrated into carboxypeptidase gene by candida utilis expression vector
It is built-up on candida utili genome.
Of the invention focuses on constructing a kind of candida utili single-gene expression bacterial strain for capableing of protein degradation matter,
Carboxypeptidase gene is transferred to candida utili and makes its secreting, expressing, selected tool is that candida utili expression carries
Body.
Wherein, carboxypeptidase selectively can generate a kind of corresponding protease of amino acid from the hydrolysis of the C-terminal of protein,
The zymogen forms of carboxypeptidase enzyme molecule can be found in a variety of organisms.Carboxypeptidase takes part in the new of multiple organs in body
Old metabolism.In addition, in medical applications, the undesirable substance generated in many bodies all relies on the hydrolysis of carboxypeptidase.It is now raw
The main source for producing carboxypeptidase is the carboxypeptidase that microorganism own metabolism generates or the carboxylic peptide with microorganism for host's overexpression
Enzyme.
Candida utili is widely used in production and life, is United States Food and Drag Administration certification
Can be considered safe microbial strains, candida utili has a series of excellent characteristics；Condition of culture is simple, Ke Yi
It can be carried out growing under some simple condition of culture, and there are five hexose disaccharide symport systems, can be realized penta
Sugar is shared with hexose, has widened application range；Because itself not secreting extracellular protease, candida utili can be applied to
The expression of plurality of target albumen, itself as a kind of biological safety biology, it is only necessary to by simply isolating and purifying program
The separation of target product, is greatly saved production cost.
Further, the candida utilis expression vector is candida utili single-gene expression vector.
Further, the candida utilis expression vector is pcGAPGA.
A kind of construction method of the candida utili single-gene expression bacterial strain of protein degradation matter, which is characterized in that including
Following steps:
S1: the carboxypeptidase gene in clone's aspergillus niger；
S2: above-mentioned carboxypeptidase gene is accessed in candida utilis expression vector, building recombination single-gene expression vector；
S3: the recombination single-gene expression vector that above-mentioned building obtains is cut with restriction endonuclease, after being allowed to linearisation
Candida utili is converted, building genetic recombination candida utili single-gene expresses bacterial strain.
Further, include the following steps: in the step S2
S21: it is purified the carboxypeptidase gene that clone obtains to obtain pMD19T-pepF cloning vector；
S22: cloning vector pMD19T-pepF and expression vector pcGAPGA is subjected to double digestion；
S23: it is attached the pcGAPGA skeleton after the carboxypeptidase target gene fragment being recovered to and digestion to form new plasmid
pcGAPGA-pepF。
Further, the restriction endonuclease in the step S3 is ScaI.
Further, conversion described in step S3 is converted using electrotransformation, freezing or chemical-agent technique.
Further, in the step S22, cloning vector pMD19T-pepF and expression vector pGAPGA are used simultaneously
Two kinds of restriction endonucleases of XhoI and XbaI carry out double digestion.
Further, the carboxypeptidase gene in the step S1 is the carboxypeptidase gene pepF of aspergillus niger.
Further, the candida utilis expression vector is the production containing CuGAP promoter or ScTEF1 promoter
Protein Candida expression vector pGAPGA.
Compared with prior art, the invention has the benefit that
Contain a large amount of nutriment in kitchen garbage, wherein original grease type and saccharide compound are utilized substantially
It is complete, and the protein-based compound in kitchen garbage only has seldom a part to be utilized by microorganism growth, most of albumen
Matter is remained in the form of waste in fermentation raffinate.In consideration of it, the present invention provides a kind of Candida utilis of protein degradation matter
Yeast single-gene expresses bacterial strain, handles kitchen fermentation raffinate, organic matter therein is further utilized, by a large amount of egg
White matter decomposition is transformed into amino acid, not only can solve the protein contamination problem of remaining, and amino acid or pharmacy, biology are raised
The raw material of material, organic fertilizer have certain economic value.
Difficult point of the invention is carboxypeptidase gene being transferred to Candida utilis together by candida utilis expression vector
Yeast realizes secreting, expressing simultaneously.
Detailed description of the invention
Fig. 1 is the building flow chart of carboxypeptidase expression vector pcGAPGA-pepF of the present invention.
Fig. 2 is the measurement tyrosine standard curve of recombinant protease enzyme activity.
Fig. 3 is protaminase c-S1 and Bc-S2 gene electrophoresis.
Fig. 4 is overlapping PCR amplification pepF gene electrophoresis.
Fig. 5 is carboxypeptidase positive clone identification.
Fig. 6 is all kinds of carboxypeptidase bacterium solution PCR electrophoresis.
Fig. 7 is all kinds of carboxypeptidase recombination yeast Genomic PCR electrophoresis.
Fig. 8 is different types of proteinase activity.
Specific embodiment
Attached drawing of the present invention only for illustration, is not considered as limiting the invention.It is following in order to more preferably illustrate
Embodiment, the certain components of attached drawing have omission, zoom in or out, and do not represent the size of actual product；For art technology
For personnel, the omitting of some known structures and their instructions in the attached drawings are understandable.Production protein in following embodiment is false
Silk yeast rDNA sequence is as shown in SEQ ID No.1, and pepF sequence is as shown in the SEQ ID No.2 in sequence table, Bc-S1 sequence
As shown in SEQ ID No.3 in sequence table, Bc-S2 sequence is as shown in sequence table SEQ ID No.4.
The clone of 1 carboxypeptidase of embodiment.
This clone carboxypeptidase gene Bc-S1 and Bc-S2 of 12714 bacterial strain of bacillus coagulans NBRC, (reference
Bacillus coagulans S-lac sequence, GenBank accession number: CP011939.1), clone the carboxylic in aspergillus niger N400
Peptidase genes pepF, referring to (A.niger (N400) pepF gene order, GenBank accession number: X79541.1), and in primer
XhoI and XbaI enzyme cutting site are added in the 5 ' ends and 3 ' ends of sequence respectively.Primer sequence sees attached list one.
(1) high fidelity enzyme PCR amplification carboxypeptidase gene Bc-S1 and Bc-S2.
The solidifying bacillus gene group extracted is taken to expand several carboxylic peptides with TaKaRa high-fidelity DNA polymerase for template
Enzyme gene segment, reaction system are as follows:
PCR reaction condition are as follows:
After the completion of PCR, 5 μ L PCR products are subjected to detected through gel electrophoresis as follows: weighing 0.25g agarose,
It after 1 × TAE of 25mL buffer is added, is made it dissolve using microwave stove heating, when being cooled to 55 DEG C or so, 2 μ L EB is added and replace
For dyestuff.Agarose solution is poured into plastic plate after mixing, is inserted into suitable comb, is stood at room temperature until glue is completely solidifying
Gu.Comb is taken out, Ago-Gel is transferred in electrophoresis tank, pours into 1 × TAE electrophoretic buffer to no mistake gel.Successively by 5
μ L DNA maker and the PCR product for being mixed with sample-loading buffer are added in gel pore.Electrophoresis 15min is left under 120V constant voltage
Behind the right side, gel is transferred in gel imager, result is observed under ultraviolet lamp and is saved.
Purified pcr product is carried out later: after agarose gel electrophoresis, gel being placed under ultraviolet lamp, it is then fast
Speed cuts the gel containing target DNA fragment, and removes extra gel as far as possible；The gel piece cut is transferred to 1.5mL's
In centrifuge tube, the weight of gel is weighed.It is calculated by 1 μ L volume of 1mg gel piece, isometric Buffer GDP is added.50 DEG C of water
20min is bathed, during which turns upside down centrifuge tube for several times, makes gel piece dissolution complete；It is of short duration liquid is collected by centrifugation after, DNA is adsorbed
Column sleeve is in 2mL collecting pipe.≤ 700 μ L sol solutions are transferred in pillar.12,000 × g is centrifuged 2min；300 μ L are added
Buffer GDP stands 2min into pillar.12,000 × g is centrifuged 1min, abandons filtrate；600 μ L Buffer DW2 are added extremely
In pillar.12,000 × g is centrifuged 1min, abandons filtrate；Pillar is put back in collecting pipe.12,000 × g is centrifuged 3min；Pillar
It covers in 1.5mL centrifuge tube, the sterile water of 50 DEG C of preheatings of 25 μ L is added to pillar film center, places 3min.12,000 × g from
Heart 2min abandons pillar, after surveying its concentration with micro-spectrophotometer, DNA fragmentation is stored in -20 DEG C.
Since the product of PrimeSTAR HS DNA Polymerase reaction is flat end, T-vector cannot connect to
On, it is therefore desirable to add A to react, reaction system is as follows:
It is purified, the DNA fragmentation after 72 DEG C of reaction 15min afterwards with micro light splitting light according to PCR product purification step
Degree meter measurement DNA concentration, -20 DEG C of preservations.
Bacillus coagulans protaminase c-S1, Bc-S2 genetic fragment size is respectively 915bp, 1524bp, such as Fig. 3 institute
Show, purpose band size is consistent with expected band length, can be used for subsequent experimental.M:DNAMarker 2000；1-2:Bc-S1 base
Because of segment；3-4:Bc-S2 genetic fragment.
(2) pepF carboxypeptidase genetic fragment over-lap PCR expands.
Aspergillus niger is eucaryote, containing there are three intrones in carboxypeptidase pepF gene, there is 1-199bp, 200-
Tetra- sections of exons of 482bp, 483-709bp, 710-1596bp, this research is by the way of over-lap PCR, first respectively by carboxypeptidase
Four fragment amplifications of pepF gene go out, then respectively by 1 and 2 segments, 3 and 4 segments are attached, finally carry out 1-4 segment
Connection, obtains complete pepF genetic fragment, after the completion of PCR, 5 μ L carboxypeptidase pepF segments is taken to carry out electrophoresis detection analysis,
Remaining is stored in -20 DEG C.
Four independent segments are respectively obtained first, and segment 1 and segment 2 are attached acquisition clip size by first time PCR
For 482bp；It is 1114bp that segment 3 and 4 is attached and obtains clip size by second of PCR；Third time PCR is by preceding twice PCR
Product be attached, primer size is 1,596bp after over-lap PCR, as a result as shown in figure 4, each clip size and expected consistent,
It can be used for subsequent experimental.M:DNA Marker 2000；The connection product of 1-2:pepF segment 1 and segment 2；3-4:pepF segment 3
With the connection product of segment 4；The connection product of 5-6:pepF segment 1,2 and segment 3,4.
(3) the T-Vector connection of carboxypeptidase gene
After the carboxypeptidase pepF fragment purification that (2) are obtained, it is connect with pMD19-T carrier, under the reaction system of connection:
After 22 DEG C of reaction 1h, the carrier molecule connected is subjected to Escherichia coli conversion, in 37 DEG C of inversion constant temperature after coated plate
Overnight incubation.
As a result as shown in figure 5, each recon amplifies single band, and size is consistent, and shows each carboxypeptidase gene piece
Section is successfully cloned into carrier T.M:DNA Marker 2000；1-3:Bc-S1 bacterium solution PCR result；4-6:Bc-S2 bacterium solution PCR
As a result；7-10:pepF bacterium solution PCR result.
The building of 2 expression vector of embodiment
(1) escherichia coli plasmid extracts
It will be obtained by embodiment 1, and the Escherichia coli with pMD19T- target gene fragment after sequence verification exist
37 DEG C, overnight incubation under the conditions of 200rpm collects the bacterium solution that 5mL is incubated overnight, referring to Tiangeng biochemical technology Co., Ltd
TIANprep Mini Plasmid Kit kit extracts plasmid, the specific steps are as follows:
10,000rpm centrifugation 2min, abandon supernatant, collect thallus.300 μ L Buffer P1/RNaseA mixing is added
Cell is thoroughly resuspended in liquid.300 μ L Buffer P2 are added, mixing gently 8 times cracks thallus sufficiently, stands 1min.It is added
400 μ L Buffer P3, gently turn upside down 8 times immediately, mix well.10,000rpm is centrifuged 5min.By the supernatant of previous step
Liquid pours into collecting pipe adsorption column, 10,000rpm centrifugation 1min.Filtrate is abandoned, adsorption column is put into collecting pipe.To adsorption column
It is middle that 600 μ L Buffer PD, 10,000rpm centrifugation 1min are added.Filtrate is abandoned, adsorption column is placed back in collecting pipe, to
600 μ LBufferPW (being diluted with dehydrated alcohol), 10,000rpm centrifugation 1min are added in adsorption column.Filtrate is abandoned, will be inhaled
Attached column is put into collecting pipe, and 10,000rpm centrifugation 2min are placed at room temperature for 8min and dry pillar.Adsorption column is placed in clean
In 1.5mL centrifuge tube, the H2O that 80 μ L50 DEG C are preheated is added dropwise to the intermediate position of adsorbed film, is placed at room temperature for 3min, 10,000rpm
It is centrifuged 3min, abandons pillar, after measuring its concentration with micro-spectrophotometer, plasmid is stored in -20 DEG C.
(2) the building process of carboxypeptidase expression vector pcGAPGA-pepF
As shown in Figure 1.
(3) pMD19T- target gene and pcGAPGA double digestion
Reaction system is as follows:
After reaction system is prepared, 1h is reacted under the conditions of being placed on 37 DEG C, total overall reaction product is subjected to gel electrophoresis
After detection, gel recycling is carried out to target DNA fragment.
(4) DNA gel recycles
DNA gel recycling is said referring to Mei Ji Biotechnology Co., Ltd HiPure Gel Pure Micro Kit kit
Bright book is purified.
(5) target gene fragment is connect with pcGAPGA skeleton
PcGAPGA skeleton after target gene fragment and digestion that (3) agarose gel electrophoresis purifies is connected
It connects to form new plasmid pcGAPGA-X (X- represents target gene), the reaction system of connection is as follows:
It is attached reaction under the conditions of 22 DEG C, by the product after connection after 1h, converts Escherichia coli.
(6) the Escherichia coli conversion of connection product
Heat-shock transformed E. coli DH5 α concrete operations are referring to " Molecular Cloning:A Laboratory guide (third edition) ".
(7) identification of positive colony bacterial strain
Corresponding PCR reaction system is configured as follows:
After above-mentioned PCR reaction solution is prepared, dispenses according to the 25 every pipes of μ L into PCR pipe, dip a small amount of recombinant bacterium with pipette tips
Body is put into PCR pipe, is centrifuged 1min, carries out PCR reaction, and reaction condition is as follows:
After PCR reaction is completed, the PCR product of 5 μ L is subjected to electrophoretic analysis.It identifies target gene and successfully connects with expression vector
The positive recombinant connect.
Cloning vector pMD19T- target gene and expression vector pGAPGA that each identification is completed are used into XhoI and XbaI simultaneously
Two kinds of restriction endonucleases carry out double digestion, and the carboxypeptidase genetic fragment being recovered to is attached with pGAPGA skeleton, thermal excitation conversion
Escherichia coli, the recon grown on picking plate, identify, such as Fig. 6 through bacterium colony PCR, have single and clearly purpose band, say
Bright each carboxypeptidase gene has been successively inserted into expression vector pGAPGA.
11-12:Bc-S1 bacterium solution PCR result；13-14:Bc-S2 bacterium solution PCR result；15-16:pepF bacterium solution PCR result.
3 expression vector pcGAPGA-X linearization for enzyme restriction of embodiment and electroporated candida utili.
(1) preparation of candida utili competent cell
(2) electroporated
It is separately added into the recombinant plasmid of 20 μ L (about 5 μ g) into every pipe candida utili competent cell, mixes gently,
It is transferred to after ice bath 15min in the electric revolving cup of pre-cooling；Voltage is 1.8kv, and after the 5ms that shocks by electricity, the 1M sorb of 1mL pre-cooling is added immediately
Alcoholic solution simultaneously mixes, and room temperature stands 15min；Aseptically by the bacterium solution in electric revolving cup be fully transferred to sterile 4mL from
In heart pipe, 2mL YPD fluid nutrient medium is added and simultaneously mixes, in 30 DEG C, 175rpm recovery 4h；After 4,000 × g is centrifuged 8min,
Most of supernatant is abandoned, thallus is resuspended in 200 μ L of residue；Thallus re-suspension liquid is all applied on the YPD solid medium containing G418,
Culture 3-5 days is inverted in 30 DEG C of incubators.
(3) candida utili positive recombinant is identified
Recon is grown with dipping on plate for pipette tips, is put into the YPD culture medium with G418 resistance, 30
DEG C, cultivated under 200rpm constant temperature, after twenty four hours, to each recon by 1% inoculum concentration by yeast-inoculated extremely
5ml YPD fluid nutrient medium after cultivating for 24 hours under the conditions of 200rpm, is extracted using U.S. base salting out method Yeast genome and is tried at 30 DEG C
Agent box solPure Yeast DNA Kit extracts Yeast genome, then carries out PCR identification with corresponding primer.Extract yeast
Genome method is as follows:
1mL Yeast Cultivation liquid is drawn to 1.5mL centrifuge tube, 12,000 × g is centrifuged 2min and collects thallus；300 μ l are added
Buffer SE, 20 μ L Lyticase (5U/ μ l) and 20 μ L (1M) DTT are into yeast cells.Yeast cells is resuspended with liquid-transfering gun
Afterwards, 37 DEG C of water-bath 1h；12,000 × g is centrifuged 2min and collects Yeast Protoplast, carefully discards supernatant liquid；400 μ L are added
Buffer WLB and 2 μ L RNase Solution are into protoplast, after high speed vortex 30s, 65 DEG C of water-bath 45min；It puts on ice
After setting 3min, 100 μ L Buffer PPS are added into lysate, vortex 30s；12,000 × g is centrifuged 5min, collects supernatant
It pours into new 1.5mL centrifuge tube.
After pGAPGA- target gene carrier restriction endonuclease ScdI linearization process, electricity turns wild type and produces protein
Candida under 30 DEG C of constant temperatures, is cultivated 2-3 days, the recon that picking is grown is transferred to liquid in the plate containing G418
Expand culture in body culture medium, extracts the genome of recon later.With corresponding primer, each recombination subgenom is carried out
PCR identification, can amplify single and clearly purpose band, illustrate carboxypeptidase gene successful conversion into candida utili.
As shown in fig. 7,16-17:Bc-S2 bacterium solution PCR result；18-19:pepF bacterium solution PCR result；20-21:Bc-S1 bacterium
Liquid PCR result.
The measurement of 4 recombinant protease enzyme activity of embodiment
Each positive recombinant is chosen, is inoculated in YPD culture medium according to the initial inoculum of OD600=0.1,60h is cultivated,
3mL culture solution is taken, supernatant is taken after 12000rpm centrifugation 5min, is measured with enzyme activity of the Forint phenol method to protease.
(1) drafting of tyrosine standard curve
The configuration of 1 tyrosine directrix curve of table
(2) good corresponding solution is configured by table 1, after mixing, develop the color 20min in 40 DEG C of thermostat water baths, it takes out,
It adjusts spectrophotometer and measures the light absorption value of other each pipes respectively using No. 0 pipe without amino acid as blank to wavelength 680nm.
Using absorbance A as ordinate, the concentration c of tyrosine is abscissa, draws standard curve.As shown in Figure 2.
Casein: being first placed in 40 DEG C of thermostat water baths by the measurement of sample enzyme activity, preheats 5min；Test tube A (blank) adds
Enter enzyme solution 1mL, 40 DEG C of warm bath 2min add trichloroacetic acid 2mL to shake up, and 40 DEG C of warm bath 10min add casein solution 1mL, after taking-up
10min is stood, filtrate is collected in slow filter paper filtering；Test tube B (need to make three samples) in parallel, add enzyme solution 1mL, 40 DEG C of warm bath
2min adds casein solution 1mL, 40 DEG C of warm bath 10min to add trichloroacetic acid 2mL to shake up, 10min, slow filter paper are stood after taking-up
Filtrate is collected in filtering；The filtrate that two steps are collected into is separately added into the sodium carbonate liquor of 5mL, forint phenol 1mL, 40 DEG C of colour developings
20min is compareed under 680nm wavelength with A pipe, carries out the measurement of absorbance.
It calculates: reading corresponding tyrosine content after the hydrolysis of enzyme sample dilution from standard curve, according to the following formula
Calculate the enzyme activity of sample: proteinase activity (U/mL)=A × K × 4/10 × n
A in formula: the mean OD value of parallel sample；
K: extinction constant (when OD=1, the amount of corresponding tyrosine)
N: the extension rate of enzyme solution.
As shown in Figure 8 in carboxypeptidase, the enzymatic activity highest of pepF, highest enzyme activity be 7.66U/mL, be respectively Bc-S1 and
1.8 times and 1.4 times of two kinds of carboxypeptidases of Bc-S2, so selection carboxypeptidase pepF.
The primer used during above-described embodiment 1~4 is as shown in table 2, wherein letter F represents forward primer, R is represented
Reverse primer, serial number represent different primer combinations.
Table 2
Obviously, the above embodiment of the present invention is only intended to clearly illustrate technical solution of the present invention example, and
It is not the restriction to a specific embodiment of the invention.It is all made within the spirit and principle of claims of the present invention
Any modifications, equivalent replacements, and improvements etc., should all be included in the scope of protection of the claims of the present invention.
Sequence table
<110>Guangdong Li Shikang low-carbon Science and Technology Ltd.
<120>the candida utili single-gene expression bacterial strain and its construction method of a kind of protein degradation matter
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 444
<212> DNA
<213>unknown (Unknown)
<400> 1
acatgtgtag tgacaataaa taacgataca gggcccttct gggtcttgta attggaatga 60
gtacaatgta aataccttaa cgaggaacaa ttggagggca agtctggtgc cagcagccgc 120
ggtaattcca gctccaatag cgtatattaa agttgttgca gttaaaaagc tcgtagttga 180
actttgggcc tggcaggccg gtccgctttt tggcgagtac tgaccctgcc gggcctttcc 240
ttctggctac cctcccctct ggagaggcga accaggactt ttactttgaa aaaattagag 300
tgttcaaagc aggcctttgc tcgaatatat tagcatggaa taatagaata ggacgtttgg 360
ttctattttg ttggtttcta ggaccatcgt aatgattaat agggacggtc gggggcatca 420
gtattcagtt gtcagaggac atgt 444
<210> 2
<211> 1608
<212> DNA
<213>unknown (Unknown)
<400> 2
ctcgagatgc tgtttcgcag tctgttgtcg acggctgtcc tagccgtctc gctgtgcacg 60
gataatgctt cagctgctaa acatggtcga tttggccaaa aagctcgcga cgccatgaac 120
atcgcgaagc gttccgctaa cgccgtgaaa cactcgttga agatccctgt cgaggactat 180
cagttcttga acaacaagac taagccttac cgcgtggaaa gcctgcctga tgttcacttc 240
gatctgggcg agatgtattc cggcttggtc cctattgaga agggcaacgt gtcacggtcc 300
cttttctttg tcttccagcc cactattggc gagcctgtgg atgagatcac catctggctg 360
aatggtggcc ctggttgcag ttcccttgag gcctttctcc aggagaatgg tagattcgtg 420
tggcagcctg gaacctacca gcctgttgag aacccatact cgtgggtgaa tctcaccaat 480
gttctgtggg ttgaccaacc tgtgggaacg ggattctctc tgggtgtccc aaccgctacg 540
tccgaggagg agattgctga agactttgtg aagttcttca agaactggca gcagatcttt 600
gggatcaaaa acttcaagat ctatgttact ggagaaagtt atgcgggccg ttatgttcct 660
tacatatccg ctgctttcct agatcagaat gatacagaac acttcaacct aaaaggtgca 720
ctggcatatg atccctgtat tggtcagttt gactacgtgc aggaggaagc acctgttgtt 780
ccctttgtcc agaagaacaa tgccctcttc aatttcaatg caagcttttt ggcggaacta 840
gagagcatcc atgagcaatg tggatacaag gatttcatcg accagtatct agtcttccca 900
gcatccggtg tccagccgcc aaaggctatg aactggagcg atcccacctg tgatgtttat 960
gacatcgtta ataacgccgt cctggatccc aacccgtgct tcaaccccta cgaaatcaac 1020
gagatgtgcc ccattctctg ggacgttctt ggattcccca ccgaagtcga ctatctccct 1080
gcgggcgcca gcatctactt tgaccgcgct gatgttaagc gtgccatgca cgctcctaac 1140
atcacctggt ccgagtgctc ggtggagagc gtctttgtcg ggggcgacgg cggtcccgag 1200
caggagggcg actactcggc caaccccatc gagcatgtct tgccccaggt catcgaaggc 1260
accaaccgag ttctgatcgg taacggtgat tatgacatgg tcatccttac caacggcacc 1320
cttctctcga tccagaacat gacatggaat ggaaagcttg gattcgacac ggcccccagc 1380
acccccatca acatcgacat ccctgacctg atgtacaatg aagtgttcat tgagaacggc 1440
tatgacccac aaggtggtca gggtgtcatg ggcatccagc actatgagcg tggtcttatg 1500
tgggctgaga ccttccagag cggacacatg cagccccaat tccaacccag agtgtcatac 1560
cgtcaccttg agtggctgct tggccggcgt gataccctgt aatctaga 1608
<210> 3
<211> 927
<212> DNA
<213>unknown (Unknown)
<400> 3
ctcgagatga gaaaagggaa acgattacag gcgggagata cgatcgggct ggtcgcgcct 60
gcaagcccgg tctcaatcga aaggcttcaa actgctgttg cgaaattgcg gcggctcggt 120
ttccgggtaa aagtgcgaga aaatgcggag aaagtatacg gtgggtatat ggccggtacg 180
ccgcttgaac gggcagccga tctgcatgcg atgtttgcag acgatgacgt gcgggcaatt 240
ctttgtttgc gcggcgggta cgggacaccg caaattttac cgtatctcga cgttgaatgg 300
atccgggaga atccgaaatt atttatcggt tacagcgata ttacagcact acacatcgtt 360
ttccagcaaa aatgcggcct cgccacgatc catggcccga tggcggctgt cgaatttatc 420
acagacgatg cgctttcggt acaatcattg ctcaagctgg cgtcaaccgg tgttcctacc 480
ggcagttaca cgggggaagc gtggggtgaa ggggtcatga cagcgccgct tacaggcggg 540
aatctgtcat taatctgcgc actgatggga acgccgtttg aaatcgatac aagcggaaaa 600
attctttttc ttgaagaagt gcatgaagag ccatatgcga tcgaccggat gctgacccag 660
cttcagcttt ccggaaaact ggatgacgcc tccgggtttg tgctcgggca atggacggac 720
tgtgagccgg aagcatacgc ggatgggttt acggcacagg atgtcctgaa aaagtttttt 780
tacgggagcg gtaagccggt gcttgccggc atacagtccg ggcacggtac cccgaatctg 840
gcgcttccgc tcggcatccc ggtcacactg gatacggtag gccaacgcct gcatttttgc 900
gaaaccattg tcgttgaata atctaga 927
<210> 4
<211> 1536
<212> DNA
<213>unknown (Unknown)
<400> 4
ctcgagatga tgggaacaga gtcgattgaa aaactggagc aggacttttt ggcatatgtc 60
aaaaaaatca aagcttatga agaagcgctc gccctcatat actgggattt gcggaccggc 120
gcgccgaaaa aaggcgtcgg gcagcgcgcg gaagtgatcg gcattttatc gggtgaagtc 180
tttgacatgc aaacttcaaa agaaatggcc gaatttttgg agaagctttt gccgcacaag 240
cagcaattgc cggaacggac agccggtaca ttggaagaat gccggaaaac atacgaacgc 300
agcaaaaaaa tcccgcctga cgaataccag gcttatacgg tgctcgtctc gaaagcggaa 360
aatgtttggg aagaagcaaa ggaaaaagca gacttctcct tgttcaagcc ttatctcgaa 420
caaatcgtgg cattcaaaaa gaaatttgtc cgctactggg ggtatgaagg ccatccgtat 480
aatcccctgc tcgatctgta tgaaccgggc atgacggtcg atgtgctcga ccgtgtgttc 540
aaacaagtga aggatgcgat tgtgccgctt gtccagaaaa tcagtatgtc ggaagaaaaa 600
ccggatacgg catttctttt tcacccgttc ccgaaagaaa agcaaaaagc gtttacgctt 660
gaaattctaa agcaaatggg ctttgacttt gaggcgggac ggctggatga aacggtgcat 720
ccgtttgcaa ccggcatcaa ccagggggat gtgcggatta cgacacgtta taatgaaaac 780
gactggcgcg tcgatgtgtt cggtgtcatg catgaaggcg ggcacgcttt gtatgagcag 840
catatttcaa aagatctgtc gggtacgccg cttgcagaag gcgcgtcaat ggggatacac 900
gaatcacaat ccctgttttt tgaaaacatc atcggccgtt cttttccgtt ctggaagaaa 960
aattatccgc ttttgaaaca gtttgcatcc gggcagtttg atgacgtgcc gcttgaagca 1020
ttttaccgtg cagtgaacga ggcgaaacca tccctgatcc ggattgaagc cgatatgctg 1080
acgtatccgc ttcatattat ggtgcggtat gaaattgaaa aagcgctcct caatgatgaa 1140
atcgaaatcg gggacctccc ggaaatctgg aatgcgaaat atgaggacta tcttggcatc 1200
cggccggcac atgacgggga aggcgtcctc caggatgtgc attgggccgg cggggatttc 1260
ggctattttc cgtcttatgc gctcggcctg atgtacgccg cccagttcaa acatgcgatg 1320
ttaaaatcgc ttccagattt tgacaagctt gtggaatcgg gcaatctgga gccggtccga 1380
gaatggctca cggcgcatgt tcaccggttc ggcaaattga aaaaacctgc cgatattttg 1440
cgcgatgcca ctggtgaagc gctcaacccg aaatacttga ttgattactt aaccgaaaaa 1500
tataccgatg tataccggat caaagattaa tctaga 1536
Claims (10)
1. a kind of candida utili single-gene of protein degradation matter expresses bacterial strain, which is characterized in that the candida utili
Single-gene expression bacterial strain is that carboxypeptidase gene is integrated into candida utili genome by candida utilis expression vector
It is upper built-up.
2. a kind of candida utili single-gene expression vector of protein degradation matter according to claim 1, feature exist
In the candida utilis expression vector is candida utili single-gene expression vector.
3. a kind of building side of the candida utili single-gene expression bacterial strain of protein degradation matter according to claim 1
Method, which is characterized in that the candida utilis expression vector is pcGAPGA.
4. a kind of candida utili single-gene of protein degradation matter according to any one of claims 1 to 3 expresses bacterial strain
Construction method, which comprises the following steps:
S1: the carboxypeptidase gene in clone's aspergillus niger；
S2: above-mentioned carboxypeptidase gene is accessed in candida utilis expression vector, building recombination single-gene expression vector；
S3: the recombination single-gene expression vector that above-mentioned building obtains is cut with restriction endonuclease, after being allowed to linearisation
Candida utili is converted, building genetic recombination candida utili single-gene expresses bacterial strain.
5. a kind of building side of the candida utili single-gene expression bacterial strain of protein degradation matter according to claim 4
Method, which is characterized in that include the following steps: in the step S2
S21: it is purified the carboxypeptidase gene that clone obtains to obtain pMD19T-pepF cloning vector；
S22: cloning vector pMD19T-pepF and expression vector pcGAPGA is subjected to double digestion；
S23: it is attached the pcGAPGA skeleton after the carboxypeptidase target gene fragment being recovered to and digestion to form new plasmid
pcGAPGA-pepF。
6. a kind of building side of the candida utili single-gene expression bacterial strain of protein degradation matter according to claim 4
Method, which is characterized in that the restriction endonuclease in the step S3 is ScaI.
7. a kind of building side of the candida utili single-gene expression bacterial strain of protein degradation matter according to claim 4
Method, which is characterized in that conversion described in step S3 is converted using electrotransformation, freezing or chemical-agent technique.
8. a kind of building side of the candida utili single-gene expression bacterial strain of protein degradation matter according to claim 5
Method, which is characterized in that in the step S22, by cloning vector pMD19T-pepF and expression vector pGAPGA use simultaneously XhoI and
Two kinds of restriction endonucleases of XbaI carry out double digestion.
9. a kind of building side of the candida utili single-gene expression bacterial strain of protein degradation matter according to claim 4
Method, which is characterized in that the carboxypeptidase gene in the step S1 is the carboxypeptidase gene pepF of aspergillus niger.
10. a kind of building side of the candida utili single-gene expression bacterial strain of protein degradation matter according to claim 3
Method, which is characterized in that the candida utilis expression vector is that the production protein containing CuGAP promoter or ScTEF1 promoter is false
Silk Yeast expression carrier pGAPGA.
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Application publication date: 20191008 |