CN108949726A - A kind of reorganization cellulose enzyme gene and its expression vector and application - Google Patents
A kind of reorganization cellulose enzyme gene and its expression vector and application Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
- C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
- C12N9/2437—Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
Abstract
The invention discloses reorganization cellulose enzyme gene and its expression vector and applications, a kind of protein of the invention, and for following (a) or (b): (a) the amino acid composition sequence of the protein is as shown in SEQ.1；(b) by (a) by the substitution and/or deletion and/or addition of one or several amino acid residues and with the protein as derived from SEQ.1 of cellulase activity.Recombinant plant Bacillus acidi lactici of the invention not only increases the palatability of fermented stalk, and also improves the nutritive value of stalk, while also avoiding adding cellulase in stalk treatment process, greatly reduces production cost.
Description
Technical field
The present invention relates to gene engineering technology fields, and in particular to a kind of reorganization cellulose enzyme gene and its expression vector and
Using.
Background technique
Crude fibre is the main component of crop material, including macromolecular polysaccharide cellulose, monosaccharide isomery polymer hemicellulose
Element and armaticity high polymer lignin.Cellulose has been arranged to form tiny vascular bundle knot according to certain mode in space
Structure, complicated vascular bundle constitute the cell wall of netted form.Contain mass fraction 70%-in common crop material
85% crude fibre, in addition to this, agricultural crop straw also contain a small amount of other materials, and (ingredient is mainly titanium dioxide to such as 5% ash content
Silicon, dense distribution is on stalk surface layer), the crude protein of 2-8%, the lipid of 1-2%, 10% moisture etc..
Cellulose is natural high-molecular compound, polymerize a large amount of monosaccharide, constitute the Major Nutrient of crop material at
Point, it is common to be made of 1000 or so C6H10O5 structural unit.Its molecular structure is with a large amount of β-D- glucopyranosyl (1-
4)-β-glycosidic bond links are formed.Cellulosic molecule usually exists with coherent condition, there is crystal region and noncrystalline domain,
Two regions are gradually transition, without apparent boundary.Crystal region is characterized in that the molecule between cellulose and cellulose takes
Tropism is good, compact-sized, inside solid, and integral strength is big；Noncrystalline domain molecules align is irregular, and spacing is big, and consistency is not
Height, intermolecular hydrogen bond number are less.Due to " network-like " structure of cellulose molecular chain, cellulose has and organic solvent and water
Incompatible characteristic.
And crude fibre is the main component of crop material, stalk micro storage is used as common problem during animal feed
Though being that biofermentation improves the palatability of stalk, digestibility improves without apparent, and this is mainly due to bacterium and enzymes
Caused by concertedness difference, the enzymatic activity that most of microbe generates in the strong acidic environment of stalk micro storage is lower.In addition, in stalk
It is directly added into cellulase in treatment process and although improves the utilization rate of stalk, but greatly improves cost, in production
It is difficult to promote and apply again.
In conclusion utilization rate is lower when the cellulose in stalk is as feed, straw can not be improved at a lower cost
The utilization rate of stalk, and enzymatic activity is lower under acidic environment, it would be highly desirable to it is further improved.
Summary of the invention
The present invention provides a kind of protein, for following (a) or (b):
(a) the amino acid composition sequence of the protein is as shown in SEQ.1；
(b) (a) by the substitution and/or deletion and/or addition of one or several amino acid residues and had into cellulose
The protein as derived from SEQ.1 of enzymatic activity.
Preferably, the amino acid composition sequence of the protein is as shown in SEQ.2.
The gene for encoding protein shown in SEQ.1 is also protection scope of the present invention.
The primer pair of gene described above is expanded, the forward primer of the primer pair is shown in SEQ.5, and reverse primer is such as
Shown in SEQ.6；The forward primer of the primer pair is shown in SEQ.7, and reverse primer is as shown in SEQ.8.
Gene described above is following (1) or (1) or (3):
(1) code area DNA molecular as shown in SEQ.3；
(2) hybridize under strict conditions with the DNA sequence dna that (1) limits and encode with cellulase activity protein
DNA molecular；
(3) DNA sequence dna limited with (1) or (2) at least have 80%, at least have 85%, at least with 90%, at least
With 95%, at least with 98% or at least with 99% homology and coding has DNA points of cellulase activity protein
Son.
Preferably, the base sequence of gene sequence described above is as shown in SEQ.4.
Recombinant vector, expression cassette, transgenic cell line or recombinant bacterium containing gene described above.
Preferably, the recombinant vector is pMD19- carrier, pMG36e carrier.
Preferably, the recombinant bacterium is lactobacillus plantarum body.
The present invention also provides a kind of methods for constructing lactobacillus plantarum described above comprising
It is expanded using the cellulose enzyme gene in koning trichoderma or aspergillus niger as template, by the way that amplification gene is carried out piece
Duan Hua, primer free PCR, there is primer PCR to reorganize cellulase, and the cellulose enzyme gene of reorganization is constructed into recombination and is carried
Body converts the recombinant vector in lactobacillus plantarum, forms reorganization cellulose enzyme gene recombinant plant Bacillus acidi lactici.
The present invention has the advantage that
The present invention reorganizes enzyme gene using the method for directed evolution, and obtains the higher cellulase base of enzyme activity
Cause is transferred to cellulose enzyme gene in Bacillus acidi lactici, when Bacillus acidi lactici raised growth and breeding in stalk of transgenosis
Meanwhile a large amount of cellulase can be secreted, to achieve the purpose that cellulose in decomposing straw.Recombinant plant lactic acid of the invention
Bacillus not only increases the palatability of fermented stalk, and also improves the nutritive value of stalk, while also avoiding in stalk
Cellulase is added in treatment process, greatly reduces production cost.
Detailed description of the invention
Fig. 1 is the electrophoretogram of the cellulose enzyme gene of koning trichoderma and aspergillus niger that PCR amplification of the invention obtains,
In, 1: aspergillus niger primer amplified result；2: koning trichoderma primer amplified result.
Fig. 2 is the blue hickie the selection result photo figure of pMD19-T carrier recombinant clone of the invention.
Fig. 3 is the cellulose enzyme gene segment that DNase I of the invention is handled, wherein 1: cellulase mixes segment F1；
2: cellulase mixes segment F2.
Fig. 4 has primer PCR for cellulose enzyme gene of the invention, wherein 1,3: aspergillus niger primer amplified knot
Fruit；2,4: koning trichoderma primer amplified result.
Fig. 5 is the map of expression plasmid pMG36e of the invention.
Fig. 6 is expression vector pMG36e double digestion result of the invention.
Fig. 7 is I plasmid identification of recombinant expression carrier pMG36e-CBH of the invention, wherein 1,2: reorganization I；3,4: reorganization
Ⅱ；5:pMG36e.
Fig. 8 is glucose standard curve figure of the invention.
Fig. 9 is that Filter paperlyase enzyme activity of the invention varies with temperature curve graph.
Figure 10 is Filter paperlyase enzyme activity of the invention with pH change curve.
Figure 11 is in the present invention, primordial plant Bacillus acidi lactici and recombinant plant Bacillus acidi lactici culture supernatant of the invention
SDS-PAGE electrophoresis result figure, wherein M: albumen Maker；1,3: recombinant plant Bacillus acidi lactici culture supernatant of the invention；2,
4: primordial plant Bacillus acidi lactici culture supernatant.
Specific embodiment
The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention..
Embodiment 1
The present embodiment uses aspergillus niger and koning trichoderma to save strain, bacillus coli DH 5 alpha and cloned plasmids for laboratory
PMD19-T is purchased from TAKARA company.PCR amplification design of primers checks in the gene total order of aspergillus niger and koning trichoderma from NCBI
Column, and the downstream of the code area signal peptide sequence in I gene of CBH starts design primer, and the I digestion position Xba is added in forward primer
I restriction enzyme site of Pst is added in point, reverse primer, and plus protectiveness bases G C, CCG, (this primer is by Shanghai respectively for the end of reverse primer 5 '
Sheng Gong bioengineering limited liability company synthesis).
Koning trichoderma primer:
Forward primer K1 is as shown in SEQ NO.5: 5'-GCT CTA GAA TGT ATC GGA AGT TGG CCG TC Xba Ⅰ
Reverse primer K2 is as shown in SEQ NO.6: 5'-CCGCTC GAG TTA CAG GCA CTG AGA GTA GTA Pst Ⅰ
Aspergillus niger primer:
Forward primer H1 is as shown in SEQ NO.7: 5'-GCT CTA GAA TGC ATC AAC GTG CCC TTC TCT TTT Xba Ⅰ
Reverse primer H2 is as shown in SEQ NO.8: 5'-CCGCTC GAG TTA TGC GGA AGC GCT GAA GGT CGA GT Pst Ⅰ
The PCR amplification of 2 cellulose enzyme gene of embodiment
Koning trichoderma, aspergillus niger are inoculated in PDA culture medium respectively, 120rpm shaking table culture 48h, use are sterile at 30 DEG C
Filter paper filters thallus and suck dry moisture, and the total serum IgE of koning trichoderma, aspergillus niger is extracted using RNA extracts kit.
Wherein, PDA culture medium is prepared, and weighs MgSO40.15g, soluble starch 3g, glucose 10g, KH2PO41g, ferment
Mother soaks powder 1g, and tryptone 2.5g is dissolved in the distilled water of 400mL, dissolution is sufficiently stirred, is settled to 500mL with distilled water.
121℃、1.05×105Pa high pressure steam sterilization 20min, it is spare.
The cDNA library of cellulose enzyme gene is established, and handles total serum IgE with DNase I, 37 DEG C of reaction 20-30min, after in PCR
80 DEG C of inactivation 10min in instrument；Following system is configured by table 1 in micro-pipe.
1 reverse transcription reaction system of table (μ L)
It is anxious on ice rapidly after (3) 70 DEG C of heat preservation 10min to freeze 2min or more；
(4) following solution is configured by table 2 in above-mentioned micro-pipe after being centrifuged the several seconds；
2 reverse transcription reaction system of table (μ L)
(5) 42 DEG C of heat preservation 1h；
Cooled on ice after (6) 70 DEG C of heat preservation 15min, obtained cDNA are used for PCR amplification.
Using above-mentioned cDNA as template, K1, K2/H1, H2 are respectively primer, and RNase free water and 2 × Es is added
Taq MasterMix is mixed with vortex oscillation instrument and is carried out PCR amplification.Reaction system is 50 μ L, and composition is shown in Table 3.
Table 3 is two kinds of cellulose enzyme gene PCR reaction systems (μ L)
PCR reaction condition is as follows: 94 DEG C of initial denaturation 5min；94 DEG C of denaturation 30s, 60-63 DEG C (not according to cellulose enzyme gene
With difference) 30s, 72 DEG C of extension 80s, 35 circulations；72 DEG C of extension 10min.After reaction with 1% Ago-Gel
Electrophoresis is detected, as shown in Figure 1, the cellulase band of koning trichoderma and aspergillus niger that PCR amplification obtains is clear, position and
Size is consistent with corresponding cellulose enzyme gene, selects PCR product stripe size to meet and expected carries out lower step test.
The connection of 3 cellulose enzyme gene of embodiment converts
The connection of cellulose enzyme gene and pMD19- carrier configures the company of cellulose enzyme gene PCR recovery product according to table 4
Junctor system, 4 DEG C of connections are overnight.
4 PCR product of table and pMD19-T carrier coupled reaction system (μ L)
Well-grown E. coli plate is placed in 4 DEG C of refrigerators, is developed the color a few hours.As shown in Fig. 2, without recombination
The Escherichia coli of plasmid have the activity of beta galactosidase, and bacterium colony center is light blue, periphery presentation navy blue；And contain recombination
Activity of the Escherichia coli of plasmid without beta galactosidase, milky is presented in bacterium colony, to carry out preliminary screening.
Gene prepares, and carries out PCR amplification to the cellulose enzyme gene that completion has been sequenced, and recycle using Sepharose Purification
Kit recycles PCR product, obtains cellulose enzyme gene.
The fragmentation and primer free PCR of 4 cellulose enzyme gene of embodiment
The fragmentation of cellulose enzyme gene, the determination of I restriction endonuclease reaction condition of Dnase sterilize I restriction endonuclease of Dnase
Deionized water afterwards is diluted to 0.07U/ μ L, configures reaction system according to table 5,15 DEG C respectively reaction 5,10,15, after 20min,
80 DEG C of inactivation 10min.Genetic fragment size is detected by 1% agarose gel electrophoresis.
5 Dnase I endonuclease reaction system (μ L) of table
Genetic fragment prepares cellulose enzyme gene fragmentation reaction system according to table 5, passes through 2% Ago-Gel electricity
Swimming detection fragmentation effect, digestion result is as shown in figure 3, utilize agarose gel purification QIAquick Gel Extraction Kit recycled fiber element enzyme base
Because of segment.
6 Dnase I endonuclease reaction system (μ L) of table
Primer free PCR configures reaction system by table 7, carries out primer free using obtained cellulose enzyme gene segment as template
PCR, reaction condition are as follows: 94 DEG C of 5min, later 94 DEG C of 30s, 63 DEG C of 30s, and 72 DEG C of 80s carry out 45 circulations, and last 72 DEG C are prolonged
Stretch 10min.Then it is detected by 1% agarose gel electrophoresis, and recovery product.
7 cellulose enzyme gene segment primer free PCR reaction system (μ L) of table
There is primer PCR, using PCR reaction product as template, be separately added into specific primer, configures reaction system by table 8, into
Row has primer PCR amplification, response procedures are as follows: 94 DEG C of initial denaturation 5min；94 DEG C of denaturation 30s, 60-63 DEG C (according to cellulase base
Because of primer difference difference) 30s, 72 DEG C of extension 80s, 35 circulations；72 DEG C of extension 10min.Then with 1% Ago-Gel
Electrophoresis detection, and recycle the product for having specificity, as shown in figure 4, cellulose enzyme gene has a primer PCR, it is available with it is black
The identical single-minded band of aspergillus cellulose enzyme gene size (reorganization I reorganizes No. II), without with koning trichoderma cellulase
The identical single-minded band of gene size occurs.
8 cellulose enzyme gene of table has primer PCR reaction system (μ L)
The PCR product of recycling is configured linked system by table 9 by the connection for reorganizing cellulose enzyme gene and pMD19-T carrier,
4 DEG C of connections are overnight.
9 PCR product of table and pMD19-T carrier coupled reaction system (μ L)
The building of 5 cellulase CBH of embodiment, I shuffled gene carrier for expression of eukaryon
The present embodiment constructs carrier for expression of eukaryon using expression plasmid pMG36e, as shown in figure 5, reorganization cellulose enzyme gene
And the purification and recovery of carrier for expression of eukaryon pMG36e plasmid extract two kinds of shuffled genes using plasmid extraction kit, and use
1% agarose gel electrophoresis detects recombinant plasmid vector.
As shown in fig. 6, expression vector pMG36e double digestion is as a result, carry recombinant plasmid using corresponding restriction enzyme
Body and pMG36e empty plasmid vector carry out double digestion respectively.37 DEG C of digestions are stayed overnight, and by 1% agarose gel electrophoresis to digestion
As a result it is detected, then with the two kinds of shuffled genes and pMG36e after agarose gel purification QIAquick Gel Extraction Kit recycling double digestion
Plasmid.
The connection for reorganizing cellulose enzyme gene and carrier for expression of eukaryon pMG36e plasmid, by reorganization cellulose enzyme gene and very
Nuclear expression carrier pMG36e plasmid is attached, 16 DEG C of reaction 16-18h.
Connection product converts the screening of bacillus coli DH 5 alpha and recombinant conversion, and connection product is transformed into Escherichia coli sense
It is the LB culture medium containing 200 μ l/mL erythromycin by the culture medium in state cell, used.In carrier for expression of eukaryon pMG36e plasmid
Genetic fragment containing anti-erythromycin, if target gene and expression vector successful connection and being transferred to Escherichia coli, so that it may
It is grown in culture medium containing erythromycin, carries out preliminary screening.It is mentioned using a small amount of extraction agent boxes of SanPrep pillar Plasmid DNA
Recombinant plasmid is taken, and is detected with 1% agarose gel electrophoresis.
Using the lactic acid bacteria of bacterium solution PCR detection conversion, with the well-grown lactobacillus plantarum list of aseptic inoculation ring picking
Bacterium colony is inoculated into the sterile eppendorf tubes containing 8 μ L sterile waters of the MRS culture medium containing erythromycin and reference numeral, preceding
37 DEG C of stationary culture 48h of person；The latter is after 95 DEG C of processing 10min, then adds premix enzyme and reverse primer progress PCR amplification, then
Agarose gel electrophoresis detection is carried out, whether Preliminary Determination lactobacillus plantarum bacterium colony contains target gene, will amplify purpose item
The PCR product of band is sent to the raw work in Shanghai and is sequenced, and the cellulase gene sequence of reorganization I is shown in SEQ.3, and the gene is complete
Whole open reading frame 1365bp encodes 454 amino acid, and for amino acid sequence as shown in SEQ.1, No. II fiber is reorganized in reorganization
For plain enzyme gene sequence as shown in SEQ.4, the complete open reading frame 1365bp of the gene encodes 454 amino acid, amino acid sequence
Column are as shown in SEQ.2.As shown in fig. 7, I plasmid identification of recombinant expression carrier pMG36e-CBH.
6 SDS-PAGE testing goal albumen of embodiment
(1) prepared by supernatant samples
Picking recombinant plant Bacillus acidi lactici single colonie is inoculated in 10mL MRS fluid nutrient medium, 37 DEG C of stationary culture mistakes
Night.Take the switching of 100 μ L overnight cultures in 100mL MRS fluid nutrient medium, 37 DEG C stationary culture 3 days, collect fermentation culture medium,
12000rpm is centrifuged 5min, takes supernatant as protein sample.
(2) preparation of PAGE gel
It needs thoroughly to clean electrophoresis tank with tap water and deionized water before glue, until the very clean nothing of glass plate
Any spot.Once there are the dirts such as grease on glass, bubble is easily generated during glue.The specific method is as follows: amount
It takes 100mL methanol to dissolve 5g sodium hydroxide, glass plate merging is wherein impregnated, is then cleaned one time with the detergent of heat, then divide
It is not washed with tap water and deionized water, is finally cleaned with ethyl alcohol.Disposable glove need to be worn when assembling electrophoresis tank.By 10 institute of table
Arrange into assignment system gel.
10 PAGE gel of table prepares ingredient (mL)
The separation gel of Fresh is slowly injected between two pieces of glass plates with liquid-transfering gun along glass plate edge, has to keep away
Exempt to generate bubble.Stopping injection glue at the about 2cm of upper edge.Distilled water is gently carefully injected into glue with 1mL syringe
On face, for obstructing air.Then 30min or so is stood at room temperature so that glue polymerize, between glue surface to be separated and the water surface
When a high-visible waterline is presented, indicate that polymerization is completed.After separation gel polymerization, topples over the water layer of top, inhaled with filter paper
Do the water of remaining.The concentration glue of Fresh is gently first injected on a small quantity in separation gel upper layer with liquid-transfering gun again, is inserted into electrophoresis
Comb (is eluted with water in advance, is cleaned with cotton ball soaked in alcohol, dried), and comb lower edge pays attention to placing comb away from edge about 1cm on separation gel
When not generate bubble.Then concentration glue is slowly supplemented with syringe, until flooding comb completely, is carefully pulled out after polymerization
Comb is removed, electrophoretic buffer is added.Power on.
(3) processing of electrophoresis Sample and deposition condition
Crude enzyme liquid sample after concentration is mixed with sample buffer equivalent, 100 DEG C are boiled 3-5min, it is cooled to room temperature,
1500rpm is centrifuged 2min, takes 15 μ L point samples.
Voltage is first set as 80V, after passing through concentration glue completely to sample, voltage is turned up to 8-15V/cm glue until electricity
Swimming terminates, and during which needs that electrophoretic buffer is continuously replenished.
(4) it dyes and decolourizes
The glue run is removed from electrophoresis glass plate from glass plate carefully, is put into colouration box, about 5 times of volumes are added
Dyeing liquor impregnate, be placed on room temperature stained over night in the shaking table gently shaken.Dyeing liquor is outwelled, after being rinsed with destainer,
Gentle to shake by soak in destainer, every 3h changes a destainer, until decoloration is completely, there is apparent protein band
Until.
Protein electrophoresis testing result, as shown in figure 11, original and recombinant plant Bacillus acidi lactici culture supernatant SDS-
PAGE electrophoresis result, the protein band that molecular weight is about 49.8kDa as we can see from the figure are consistent with theoretical size, and compare
Group does not have then, this shows that I gene of cellulase CBH of reorganization is successfully transferred in lactobacillus plantarum, and has obtained secreting type
Expression.
The measurement of 6 recombinant plant lactobacillus cellulase activity of embodiment
The measurement of recombinant plant lactobacillus cellulase activity, the preparation of crude enzyme liquid, picking recombinant plant lactobacillus single bacterium
It falls and is inoculated in 10mL MRS fluid nutrient medium, 37 DEG C of stationary culture 48h.(2) above-mentioned culture is connect respectively by 2% inoculum concentration
Kind in low sugar MRS and sugar-free MRS fluid nutrient medium containing 5% stalk, 37 DEG C of stationary cultures, per sampling for 24 hours, 12000rpm from
Heart 5min takes supernatant as crude enzyme liquid, continuously collects 7d.
Cellulase activity measurement, the production of (1) glucose standard curve weigh the anhydrous Portugal to dry to constant weight through 105 DEG C
Grape sugar 1.0000g, adding citric acid salt buffer dissolution, is settled to 100mL, mixes well, be configured to the glucose mark of 10mg/mL
Quasi- solution.Respectively measure 0.00,1.00,2.00,3.00,4.00,5.00,6.00,7.00, the Glucose standards of 8.00mL it is molten
Liquid, and it is settled to 50mL respectively with citrate buffer, the Glucose standards system that concentration is 0.00-1.600mg/mL is made
Column.
Each 1.00mL of above-mentioned glucose standards solution is drawn respectively in 25mL scale test tube, respectively plus 2.00mL distilled water and
2.00mL DNS reagent, boiling water bath 5min are cooled to room temperature and are settled to 25mL, measure OD value under 540nm wavelength.With
Absorbance is abscissa, and milligram number of the corresponding glucose standards solution containing sugar is ordinate, draws out glucose standard curve,
See Fig. 8.
The measurement of 7 recombinant plant lactobacillus cellulase activity of embodiment
Recombinant plant lactobacillus cellulase activity measurement result, Filter paperlyase measurement result, filter paper enzyme activity measurement result is such as
Table 10.Primordial plant Bacillus acidi lactici enzymatic activity is 0.00U/L.The filter paper in low glucose MRS culture medium as can be seen from the table
The lactobacillus plantarum enzyme activity that enzyme activity is transferred to reorganization I cellulose enzyme gene reaches as high as 4.83U/L, is transferred to reorganization II cellulose
The lactobacillus plantarum enzyme activity of enzyme gene reaches as high as 4.19U/L.
The cellulose enzyme activity (U/L) of 10 low glucose MRS of table culture Bacillus acidi lactici
Note: same column indicates significant difference (P < 0.05) that colleague's lowercase difference indicates that difference is aobvious with capitalization difference
It writes (P < 0.05).
Reorganize cellulase zymetology property analysis as a result, recombinant plant lactobacillus cellulase activity is influenced by temperature result
As shown in Figure 9, by finding out in figure, No. 2 reorganization 1, reorganization supernatant cellulase optimal reactive temperatures are 50 DEG C.?
Between 30-50 DEG C, enzyme activity is increased as temperature increases, and when temperature continues to increase, enzyme activity all declines, and temperature rises to 60
After DEG C, the comparison of enzyme activity decline is slow, it may be possible to which temperature drift causes part enzyme to inactivate.
Recombinant plant lactobacillus cellulase activity is shown in Figure 10 with the change curve of pH, reorganizes No. 1 as can be seen from the figure
Supernatant cellulase optimal reaction pH is 5.0, and reorganizing No. 2 supernatant cellulase optimal reaction pH is 5.5.
The present invention carries out cellulose enzyme gene therein from High Cellulase Production bacterium koning trichoderma and aspergillus niger
DNA reorganization, and gene after reorganization is electroporated into lactobacillus plantarum by expression vector pMG36e, successfully construct plant
Object Bacillus acidi lactici recombinant bacterium, to recombination lactobacillus plantarum cellulase-producing property analysis the results show that its optimal reactive temperature
At 50 DEG C, optimal reaction pH is in 5.0-5.5.
Although above having used general explanation and specific embodiment, the present invention is described in detail, at this
On the basis of invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Therefore,
These modifications or improvements without departing from theon the basis of the spirit of the present invention are fallen within the scope of the claimed invention.
Sequence table
<110>Agricultural University Of He'nan
Henan De Lin biological products Co., Ltd
<120>a kind of reorganization cellulose enzyme gene and its expression vector and application
<130> 20180605
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Met His Gln Arg Ala Leu Leu Phe Ser Ala Leu Leu Thr Ala Val Arg
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Ala Gln Gln Ala Gly Thr Leu Thr Glu Glu Val His Pro Ser Leu Thr
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Trp Gln Lys Cys Thr Ser Glu Gly Ser Cys Thr Glu Gln Ser Gly Ser
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Val Val Ile Asp Ser Asn Trp Arg Trp Thr His Ser Val Asn Asp Ser
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Thr Asn Cys Tyr Thr Gly Asn Thr Trp Asp Ala Thr Leu Cys Pro Asp
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Asp Glu Thr Cys Ala Ala Asn Cys Ala Leu Asp Gly Ala Asp Tyr Glu
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Ser Thr Tyr Gly Ile Thr Thr Asp Gly Asp Ser Leu Thr Leu Lys Phe
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Val Thr Gly Ser Asn Val Gly Ser Arg Leu Tyr Leu Met Asp Thr Ser
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Asp Glu Gly Tyr Gln Thr Phe Asn Leu Leu Asp Ala Glu Phe Thr Phe
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Asp Val Asp Val Ser Asn Leu Pro Cys Gly Leu Asn Gly Ala Leu Tyr
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Phe Thr Ala Met Asp Ala Asp Gly Arg Ala Ser Lys Tyr Pro Ala Asn
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Lys Ala Gly Ala Lys Tyr Gly Thr Gly Tyr Cys Asp Ser Gln Cys Pro
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Arg Asp Leu Lys Phe Ile Asp Gly Gln Glu Ala Asn Val Asp Gly Trp
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Glu Pro Ser Ser Asn Asn Asp Asn Thr Gly Ile Gly Asn His Gly Ser
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Cys Cys Pro Glu Met Asp Ile Trp Glu Ala Asn Lys Ile Ser Thr Ala
225 230 235 240
Leu Thr Pro His Pro Cys Asp Ser Ser Glu Gln Thr Met Cys Glu Gly
245 250 255
Asn Asp Cys Gly Gly Thr Tyr Ser Asp Asp Arg Tyr Gly Gly Gly Thr
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Cys Asp Pro Asp Gly Cys Asp Phe Asn Pro Tyr Arg Met Gly Asn Asp
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Ser Phe Tyr Gly Pro Gly Lys Thr Ile Asp Thr Gly Ser Lys Met Thr
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Val Val Thr Gln Phe Ile Thr Asp Gly Ser Gly Ser Leu Ser Glu Ile
305 310 315 320
Lys Arg His Tyr Val Gln Asn Gly Asn Val Ile Ala Asn Ala Asp Ser
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Asn Ile Ser Gly Val Thr Gly Asn Ser Ile Thr Thr Asp Phe Cys Thr
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Gly Leu Ala Gly Ile Ser Asp Ala Met Ser Ser Met Val Leu Ile Leu
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Ser Leu Trp Asp Asp Tyr Tyr Ala Ser Met Glu Trp Leu Asp Ser Asp
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Tyr Pro Glu Asn Ala Thr Ala Thr Asp Pro Gly Val Ala Arg Gly Thr
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20 25 30
Trp Gln Lys Cys Thr Ser Glu Gly Ser Cys Thr Glu Gln Ser Gly Ser
35 40 45
Val Val Ile Asp Ser Asn Trp Arg Trp Thr His Ser Val Asn Asp Ser
50 55 60
Thr Asn Cys Tyr Thr Gly Asn Thr Trp Asp Ala Thr Leu Cys Pro Asp
65 70 75 80
Asp Glu Thr Cys Ala Ala Asn Cys Ala Leu Asp Gly Ala Asp Tyr Glu
85 90 95
Ser Thr Tyr Gly Ile Thr Thr Asp Gly Asp Ser Leu Thr Leu Lys Phe
100 105 110
Val Thr Gly Ser Asn Val Gly Ser Arg Leu Tyr Leu Met Asp Thr Ser
115 120 125
Asp Glu Gly Tyr Gln Thr Phe Asn Leu Leu Asp Ala Glu Phe Thr Phe
130 135 140
Asp Val Asp Val Ser Asn Leu Pro Cys Gly Leu Asn Gly Ala Leu Tyr
145 150 155 160
Phe Thr Ala Met Asp Ala Asp Gly Gly Ala Ser Lys Tyr Pro Ala Asn
165 170 175
Lys Ala Gly Ala Lys Tyr Gly Thr Gly Tyr Cys Asp Ser Gln Cys Pro
180 185 190
Arg Asp Leu Arg Phe Ile Asp Gly Gln Glu Ala Asn Val Asp Gly Trp
195 200 205
Glu Pro Ser Ser Asn Asn Asp Asn Thr Gly Ile Gly Asn His Gly Ser
210 215 220
Cys Cys Pro Glu Met Asp Ile Trp Glu Ala Asn Lys Ile Ser Thr Ala
225 230 235 240
Leu Thr Pro His Pro Cys Asp Ser Ser Glu Gln Thr Met Cys Glu Gly
245 250 255
Asn Asp Cys Gly Gly Thr Tyr Ser Asp Asp Arg Tyr Gly Gly Gly Thr
260 265 270
Cys Asp Pro Asp Gly Cys Asp Phe Asn Pro Tyr Arg Met Gly Asn Asp
275 280 285
Ser Phe Tyr Gly Pro Gly Lys Thr Ile Asp Thr Gly Ser Lys Met Thr
290 295 300
Val Val Thr Gln Phe Ile Thr Asp Gly Ser Gly Ser Leu Ser Glu Ile
305 310 315 320
Lys Arg Tyr Tyr Val Gln Asn Gly Asn Val Ile Ala Asn Ala Asp Ser
325 330 335
Asn Ile Ser Gly Val Thr Gly Asn Ser Ile Thr Thr Asp Phe Cys Thr
340 345 350
Ala Gln Lys Lys Ala Phe Gly Asp Asp Asp Ile Phe Ala Glu His Asn
355 360 365
Gly Leu Ala Gly Ile Ser Asp Ala Met Ser Ser Met Val Leu Ile Leu
370 375 380
Ser Leu Trp Asp Asp Tyr Tyr Ala Ser Met Glu Trp Leu Asp Ser Asp
385 390 395 400
Tyr Pro Glu Asn Ala Thr Ala Thr Asp Pro Gly Val Ala Arg Gly Thr
405 410 415
Cys Asp Ser Glu Ser Gly Val Pro Ala Thr Val Glu Gly Ala His Pro
420 425 430
Asp Ser Ser Val Thr Phe Ser Asn Ile Lys Phe Gly Pro Ile Asn Ser
435 440 445
Thr Phe Ser Ala Ser Ala
450
<210> 3
<211> 1365
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
atgcatcaac gtgcccttct cttttcagcc ctgctgacgg ctgttcgcgc ccagcaagcc 60
ggaacgctta cggaggaagt ccatccttcc ttgacctggc agaaatgcac ttctgaaggc 120
agctgcactg aacagagtgg ctcagttgtc attgactcga actggcgctg gacccattct 180
gtcaatgaca gcaccaattg ctacactggc aacacctggg atgcaactct ctgccctgat 240
gatgagacct gtgcggccaa ctgcgccctg gacggagcgg actacgagtc cacctacggt 300
atcaccactg acggtgattc attgacactg aaattcgtca ctggctccaa tgttggctcg 360
cggttgtatc tcatggacac gagtgacgag ggataccaga cgttcaactt gcttgacgca 420
gagttcactt tcgacgttga tgtgtctaac ctcccatgtg ggctaaacgg cgcgttgtac 480
ttcactgcaa tggacgccga tggtagagcc tcaaaatacc ctgccaacaa ggctggagcc 540
aagtatggaa caggatactg tgactcccaa tgcccccggg acctgaagtt catcgacgga 600
caagaggcca acgtcgatgg ctgggaacct tctagcaaca atgacaacac aggtatcggc 660
aatcacggtt cttgctgccc tgaaatggat atctgggagg caaacaagat ctcgaccgca 720
ttgacacccc atccctgtga cagcagcgaa cagaccatgt gtgagggtaa cgactgcggt 780
ggaacctact cggatgatcg ctacggagga ggaacctgcg accctgacgg ctgcgacttc 840
aacccttatc gcatgggcaa cgactctttc tacggtcctg gcaagaccat cgacaccgga 900
tccaagatga cggttgtgac ccagttcatc actgatggct ctggctccct cagcgagatc 960
aagcgtcact acgtgcagaa cggaaatgtc atagcgaacg ctgattccaa catctctgga 1020
gtgactggaa actcgatcac aacggacttc tgcactgcgc agaagaaggc ctttggcgac 1080
gacgatatat tcgctgagca caatggactt gctggaatca gtgatgccat gtcttccatg 1140
gttctcatct tgagcttgtg ggatgattac tatgccagca tggagtggct cgacagcgac 1200
tatcccgaga acgctaccgc taccgaccca ggtgttgcac gcggaacatg cgactcggaa 1260
tcaggcgtcc ctgcgacagt cgagggggcg catcccgatt cttcggtgac cttctcaaac 1320
atcaagttcg gacccatcaa ctcgaccttc agcgcttccg cataa 1365
<210> 4
<211> 1365
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
atgcatcaac gtgcccttct cttttcagcc ctgctgacgg ctgttcgcgc ccagcaagcc 60
ggaacgctta cggaggaagt ccatccttcc ttgacctggc agaaatgcac ttctgaaggc 120
agctgcactg aacagagtgg ctcagttgtc attgactcga actggcgctg gacccattct 180
gtcaatgaca gcaccaattg ctacactggc aacacctggg atgcaactct ctgccctgat 240
gatgagacct gtgcggccaa ctgcgccctg gacggagcgg actacgagtc cacctacggt 300
atcaccactg acggtgattc attgacactg aaattcgtca ctggctccaa tgttggctcg 360
cggttgtatc tcatggacac gagtgacgag ggataccaga cgttcaactt gcttgacgca 420
gagttcactt tcgacgttga tgtgtctaac ctcccatgtg ggctaaacgg cgcgttgtac 480
ttcactgcaa tggacgccga tggtggagcc tcaaaatacc ctgccaacaa ggctggagcc 540
aagtatggaa caggatactg tgactcccaa tgcccccggg acctgaggtt catcgacgga 600
caagaggcca acgtcgatgg ctgggaacct tctagcaaca atgacaacac aggtatcggc 660
aatcacggtt cttgctgccc tgaaatggat atctgggagg caaacaagat ctcgaccgca 720
ttgacacccc atccctgtga cagcagcgaa cagaccatgt gtgagggtaa cgactgcggt 780
ggaacctact cggatgatcg ctacggagga ggaacctgcg accctgacgg ctgcgacttc 840
aacccttatc gcatgggcaa cgactctttc tacggtcctg gcaagaccat cgacaccgga 900
tccaagatga cggttgtgac ccagttcatc actgatggct ctggctccct cagcgagatc 960
aagcgttact acgtgcagaa cggaaatgtc atagcgaacg ctgattccaa catctctgga 1020
gtgactggaa actcgatcac aacggacttc tgcactgcgc agaagaaggc ctttggcgac 1080
gacgatatat tcgctgagca caatggactt gctggaatca gtgatgccat gtcttccatg 1140
gttctcatct tgagcttgtg ggatgattac tatgccagca tggagtggct cgacagcgac 1200
tatcccgaga acgctaccgc taccgaccca ggtgttgcac gcggaacatg cgactcggaa 1260
tcaggcgtcc ctgcgacagt cgagggggcg catcccgatt cttcggtgac cttctcaaac 1320
atcaagttcg gacccatcaa ctcgaccttc agcgcttccg cataa 1365
<210> 5
<211> 29
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 5
gctctagaat gtatcggaag ttggccgtc 29
<210> 6
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 6
ccgctcgagt tacaggcact gagagtagta 30
<210> 7
<211> 33
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 7
gctctagaat gcatcaacgt gcccttctct ttt 33
<210> 8
<211> 35
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 8
ccgctcgagt tatgcggaag cgctgaaggt cgagt 35
Claims (10)
1. a kind of protein, for following (a) or (b):
(a) the amino acid composition sequence of the protein is as shown in SEQ.1；
(b) (a) by the substitution and/or deletion and/or addition of one or several amino acid residues and had into cellulase activity
The protein as derived from SEQ.1 of property.
2. protein described in claim 1, which is characterized in that the amino acid composition sequence of the protein is as shown in SEQ.2.
3. encoding the gene of protein described in claim 1.
4. expanding the primer pair of gene described in claim 3, which is characterized in that
The forward primer of the primer pair is shown in SEQ.5, and reverse primer is as shown in SEQ.6；
The forward primer of the primer pair is shown in SEQ.7, and reverse primer is as shown in SEQ.8.
5. gene as claimed in claim 3, which is characterized in that the gene is following (1) or (1) or (3):
(1) code area DNA molecular as shown in SEQ.3；
(2) hybridize under strict conditions with the DNA sequence dna that (1) limits and encode DNA points with cellulase activity protein
Son；
(3) DNA sequence dna limited with (1) or (2) at least has 80%, at least has 85%, at least having with 90%, at least
95%, at least with 98% or at least with 99% homology and coding have cellulase activity protein DNA molecular.
6. gene described in claim 5, which is characterized in that the base sequence of the gene sequence is as shown in SEQ.4.
7. containing recombinant vector, expression cassette, transgenic cell line or the recombinant bacterium of gene described in claim 5 or 6.
8. recombinant vector as claimed in claim 7 is pMD19- carrier, pMG36e carrier.
9. recombinant bacterium as claimed in claim 7 is lactobacillus plantarum body.
10. a kind of method for constructing lactobacillus plantarum according to any one of claims 8 comprising
It is expanded using the cellulose enzyme gene in koning trichoderma or aspergillus niger as template, by the way that amplification gene is carried out segment
Change, primer free PCR, there is primer PCR to reorganize cellulase, and the cellulose enzyme gene of reorganization is constructed into recombinant vector,
The recombinant vector is converted in lactobacillus plantarum, reorganization cellulose enzyme gene recombinant plant Bacillus acidi lactici is formed.
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