CN103725707A - Genetic engineering strain for recombinant expression of phytase - Google Patents
Genetic engineering strain for recombinant expression of phytase Download PDFInfo
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- CN103725707A CN103725707A CN201310608963.5A CN201310608963A CN103725707A CN 103725707 A CN103725707 A CN 103725707A CN 201310608963 A CN201310608963 A CN 201310608963A CN 103725707 A CN103725707 A CN 103725707A
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- phytase
- engineering strain
- genetic engineering
- recombinant expression
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Abstract
The invention relates to the technical field of genetic engineering, particularly provides a genetic engineering strain for recombinant expression of phytase. Phytase genes are inserted into trichoderma reesei, so that the trichoderma reesei engineering strain capable of realizing the recombinant expression of the phytase is established. The phytase can be expressed efficiently by the engineering strain, and the shake-flask fermentation enzyme activity is 880 U/mL. The recombinant phytase prepared from the genetic engineering strain can be widely applied to feed, increase the average daily gain and the feed conversion ratio in each period of bred animals greatly and reduce the excretion of phosphorus by 13.3%, so that increase of the breeding benefit and reduction of environmental pollution are facilitated.
Description
Technical field
The invention belongs to microbial engineering field, be specifically related to a kind of genetic engineering bacterium and application thereof of recombinant expressed phytase.
Background technology
Phytase (Phytase) is phytinic acid lytic enzyme (Myo-i-nositol hexaphosphate phosphohydrotase), is the general name that catalysis phytic acid and phytate are hydrolyzed into the class of enzymes of inositol and phosphoric acid (or phosphoric acid salt).Phytase has special space structure, and the phosphorus in separated phytic acid molecule, is degraded to inorganic phosphorus and inositol by phytate successively, discharges other nutritive substances of phytate combination simultaneously.Can be widely used as fodder additives.At present, utilize the feed effect of monogastric animal of phytase to obtain checking.In feed, add after phytase, can reduce the consumption of 5-70% inorganic phosphorus, more than in ight soil, the quantity discharged of phosphorus has reduced 30-40%, not only greatly reduce the anti-oxidant action of phytate, increase productivity effect, can also effectively reduce environmental pollution, therefore the research of phytase be had great importance.
In natural materials, the expression level of phytase is lower at present, zymetology performance can not meet the application of feed processing, adopt engineered means, not only can improve the enzyme activity of phytase, can also, by building the some properties of engineering strain transformation enzyme, make it more to adapt to the condition of suitability for industrialized production.
Summary of the invention
The present invention, for solving prior art problem, provides a kind of genetic engineering bacterium of recombinant expressed phytase.The present invention, by the phytase gene in Aspergillus fumigatus source is transformed in Trichodermareesei, has built the Trichodermareesei engineering strain of high this phytase of efficient expression of energy, thereby has made up the deficiencies in the prior art.
One aspect of the present invention provides a kind of expression vector, and it carries the gene of coding phytase.
Described phytase, its aminoacid sequence is SEQ ID NO:1.
Described phytase, the nucleotides sequence of its encoding gene is classified SEQ ID NO:2 as.
The present invention provides a kind of host cell on the other hand, and it carries above-mentioned expression vector.
Described host cell be Trichodermareesei (
trichoderma reesie).
The application of described host cell in producing phytase.
The present invention also provides the application of above-mentioned phytase in feed.
beneficial effect
The phytase in the high efficiency recombinant expressed Aspergillus fumigatus of the Trichodermareesei engineering strain energy source that the present invention builds, the work of shake flask fermentation enzyme is up to 880U/mL.The phytase that the present invention prepares can be widely used in feed, can greatly improve cultivated animals each phase average daily gain and feedstuff-meat ratio, and make the excretion of phosphorus reduce by 13.3%, thereby be conducive to increase culture benefit, reduces the pollution to environment.
Embodiment
The present invention has used routine techniques and the method for genetic engineering and biology field use, MOLECULAR CLONING:A LABORATORY MANUAL for example, 3nd Ed. (Sambrook, 2001) and the method for recording in CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Ausubel, 2003).These general reference provide definition well known by persons skilled in the art and method.But this does not also mean that any concrete grammar, experimental program and the reagent described in limiting the invention to, because they can change.
Unless be separately construed as limiting in this article, whole technical terms used herein and scientific terminology have common the understood identical meanings of common counting personnel in the affiliated field of the present invention.DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 3nd Ed. (Singleton et al., 2006) and COLLINS DICTIONARY BIOLOGY (Hale et al., 2003) for technician provides the generality of the many terms that use in the present invention, explain.
the clone of embodiment 1 Phytase Gene from Aspergillus fumigatus
With Aspergillus fumigatus (
aspergillus fumigatus) genome DNA is template, utilizes upstream and downstream primer to increase.
Pcr amplification condition is 95 ℃ of 4min; 94 ℃ of 30S; 55 ℃ of 40S, 30 circulations of 72 ℃ of 1min; 72 ℃ of 7min.Utilize gel to reclaim test kit and reclaim pcr amplification product, carry out sequencing analysis.Result demonstration, the nucleotides sequence of amplified production is classified SEQ ID NO:2 as, and the aminoacid sequence of its coding is SEQ ID NO:1.Through NCBI Blast, comparison is found, the phytase sequence similarity of this aminoacid sequence and Aspergillus fumigatus is 89%, is a new allelotrope.
the structure of embodiment 2 expression vectors
The amplified production reclaiming in embodiment 1 is connected to pMD18-T carrier, obtains cloning vector pMD-TR plasmid, then with NcoI and KpnI, carry out double digestion, reclaim TR fragment; Get 2 μ l recovery products and be connected with KpnI double digestion pKDN-5 carrier the importing bacillus coli DH 5 alpha that spends the night with NcoI, obtain recombinant expression plasmid pKDN-TR.
embodiment 3 transforms and screening
3.1 protoplastis preparations
Inoculation Trichodermareesei (
trichoderma reesei) mycelia grows 4 days on PDA flat board; The bacterium colony that cuts diameter 3 cm is placed in approximately 60 mlYEG(0.5% yeast powders, 1% glucose) liquid nutrient medium, 30 ℃, 200 rpm shaking culture spend the night; Multilayer filtered through gauze is collected mycelia; Mycelia is placed in and fills 10-20 ml lyase liquid (Sigma L1412) enzymolysis 2-3 hour; Take out enzymolysis solution, add 0.7 M NaCl solution, jiggle, fall and filter in three layers of sterilizing lens wiping paper, collect filtrate, 3000 rpm, centrifugal 10 min; Abandon supernatant, add 10-20 ml STC liquid (20% sucrose, 50mM Tris-Cl, 50mM CaCl
2) suspend, 3000 rpm, centrifugal 10 min; Add appropriate STC suspension packing (150 μ l/ pipes, 10
8individual/ml).
transform and checking
Get 2 μ g pKDN-TR DNA and join in 150 μ l protoplastiss, then add 500 μ l 25%PEG to mix gently, standing 25 min of room temperature; Then divide 2-3 time and add 1ml 25%PEG again, mix gently, the standing 25min of room temperature, is added to protoplastis after melt 50 ml left and right and is cooled to the upper strata semisolid medium (0.1%MgSO of 45-55 ℃
4, 1%KH
2pO4,0.6% (NH
4)
2sO
4, 1% glucose, 18.3% sorbyl alcohol, 0.35% agarose), after mixing gently, pour into containing 100 μ g/ml Totomycin subfoundation culture medium flat plate (2% glucose, 0.5% (NH4)
2sO
4, 1.5%KH
2pO
4, 0.06%MgSO
4, 0.06%CaCl
2, 1.5% agar), 28 ℃ of dark culturing a couple of days to transformants grow.
Extracting transformant genomic dna is template, utilizes primer amplification hygromycin gene checking transformant.Pcr amplification condition is 95 ℃ of 4min; 94 ℃ of 30S; 55 ℃ of 40S, 30 circulations of 72 ℃ of 1min; 72 ℃ of 7min.Utilizing gel to reclaim test kit reclaims pcr amplification product and carries out sequencing analysis.To a wherein strain positive transformant called after Trichodermareesei TR-01(
trichoderma reeseitR-01).
embodiment 4 fermentation and enzyme activity determinations
4.1 fermentation checkings
By positive transformant
trichoderma reeseitR-01 is inoculated in MM fermention medium (1.5% glucose, 1.7% lactose, 2.5% corn steep liquor, 0.44% (NH
4)
2sO
4, 0.09%MgSO
4, 2%KH
2pO
4, 0.04%CaCl
2, 0.018% tween-80,0.018% trace element, 0.018% polypropylene glycol-2000) cultivate, cultivate 48 hours for 28 ℃, then cultivate 48 hours for 25 ℃; 8 layers of filtered through gauze for gained fermented liquid, filtrate is centrifugal 10 min under 14000 g conditions, collect supernatant liquor; On the SDS-PAGE glue that is 12% in concentration by supernatant liquor, carry out electrophoresis detection, in 23kDa left and right there is a band in place, is recombinant expressed phytase.
enzyme activity determination
The enzyme definition of living: be that under 37 ℃, the pH condition that is 5.0, per minute is to discharge 1 μ mol inorganic phosphorus 5.0mmol/L sodium phytate from concentration, is a phytase activity unit, represents with U in temperature.
Measuring method: accurately take 0.6804g at 105 ℃ of benchmark potassium primary phosphates (5.9) that dry to constant weight in 100ml volumetric flask, with acetate buffer (5.1), dissolve, and be settled to 100ml, concentration is 50.0mmol/L.Acetate buffer for ratio (5.2) in table 1 is diluted to different concns, reaction assay together with sample to be tested.Take inorganic phosphorus concentration as X-coordinate, and light absorption value is ordinate zou, lists linear regression equation (y=ax+b).
Reacted sample is standing 10min in water-bath, upper with the centrifugal 10min of 4000r/min at whizzer (6.7), and supernatant liquor is with the blank zeroing of typical curve, at the blank (A of spectrophotometer (6.3) 415nm wavelength place working sample
0) and the light absorption value of sample solution (A), A-A
0for actual measurement light absorption value.With linear regression equation, calculate the activity of phytase.
Phytase activity is calculated as follows
U=F×C /（m×30）
In formula:
The activity of phytase in U-sample, U/g
The enzymic activity of C-calculated by linear regression equation according to the light absorption value of actual sample liquid, U.
Total extension rate before the reaction of F-sample solution.
M-sample mass, g.
30-reaction times, min.
Adopt aforesaid method to record the work of Trichodermareesei engineering bacteria shake flask fermentation supernatant liquor enzyme and be about 880U/mL.
The Trichodermareesei engineering bacteria that the present invention builds can high efficiency recombinant expressed Aspergillus fumigatus phytase, and after repeatedly going down to posterity, still can keep genetic stability.
the experiment of feeding of embodiment 5 cultivated animals
Purchase the white plumage meat cock of 1 age in days Roche 308, be divided into control group and experimental group, 16 repetitions of each treatment group, each repeats 8 plumage chickens, the production test phase is 35 days, and wherein control group is the general goods daily ration of feeding, and experimental group is to feed to add the general goods daily ration of recombinant phytase of the present invention, food consumption in experiment with measuring process, material anharmonic ratio, the variation of body weight and the emission behaviour of phosphorus, experimental result as shown in Table 1 and Table 2:
Table 1 meat chicken production performance statistics
| Group | Day weight gain (g) | Feedstuff-meat ratio | Fatten index |
| Control group | 70.05±2.16 | 1.59±0.15 b | 341.33±30.64 |
| Experimental group | 72.32±2.81 | 1.49±0.14 a | 350.47±29.75 |
As shown in Table 1, the feedstuff-meat ratio of experimental group broiler chicken has reduced by 9% than control group, with contrast difference significantly (P < 0.05), day weight gain, fattens index and you can well imagine high 3.2%, 2.7% than contrast component.This shows by adding in feed after the recombinant expressed phytase of the present invention, and the production performance of broiler chicken has obtained generally improving, and has not only reduced the production cost of broiler chicken, has also improved the production efficiency of broiler chicken.
Table 2 adds the impact of the recombinant expressed phytase of the present invention on phosphorus excretion
| Index | Control group | Experimental group |
| Phosphorus excretion (g/kg) | 3.0±0.4 a | 2.6±0.4 b |
As can be seen from Table 2, with control group comparison, add after recombinant expressed phytase of the present invention, the phosphorus amount of broiler chicken excretion reduces by 13.3% (P < 0.05).Illustrate that phytase of the present invention can effectively improve the absorption rate of broiler chicken to phosphorus, reduce the discharge of nitrogenouz wastes, be conducive to improve efficiency of feed utilization, and alleviate the pollution to environment.
SEQUENCE LISTING
<110> Qingdao Weilan Biology Group Co., Ltd.
The genetic engineering bacterium of a <120> recombinant expressed phytase
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 207
<212> PRT
<213> Phytase protein
<400> 1
Met Asp Met Phe Thr Leu Glu Thr Ile Ser Thr Ser Thr Val Asp Thr
1 5 10 15
Lys Leu Ser Pro Phe Cys Asp Leu Phe Thr His Glu Gly Trp Ile Asn
20 25 30
Tyr Asp Tyr Leu Gln Ser Leu Asn Lys Tyr Tyr Gly His Gly Ala Gly
35 40 45
Asn Pro Leu Gly Pro Thr Gln Gly Val Gly Tyr Ala Asn Glu Leu Ile
50 55 60
Ala Arg Leu Thr His Leu Pro Val Leu Asp Asp Thr Ser Ser Lys Pro
65 70 75 80
Thr Phe Glu Ser Asn Pro Ala Thr Phe Pro Leu Asn Ser Thr Leu Tyr
85 90 95
Ala Asp Phe Ser His Glu Lys Gly Ile Ile Ser Ile Leu Phe Ala Leu
100 105 110
Gly Leu Tyr Asn Gly Thr Lys Pro Leu Ser Ser Thr Thr Ala Glu Asn
115 120 125
Ile Thr Gln Thr Asp Gly Phe Ser Ser Ala Trp Thr Val Pro Phe Ala
130 135 140
Ser Arg Met Tyr Val Glu Met Met Gln Cys Gln Ser Glu Gln Glu Pro
145 150 155 160
Leu Val Arg Val Leu Val Asn Glu Leu Val Val Pro Leu His Gly Ser
165 170 175
Pro Val Glu Pro Leu Gly Arg Cys Thr Arg Asp Ser Phe Gln Lys Gly
180 185 190
Leu Ser Phe Ala Lys Ser Gly Gly Asp Trp Val Lys Ser Phe Pro
195 200 205
<210> 2
<211> 624
<212> DNA
<213> Phytase nucleotide
<400> 2
atggacatgt tcaccttgga gaccatctcc accagcaccg tcgacaccaa gctgtccccc
60
ttctgtgacc tgttcaccca tgaaggatgg atcaactacg actacctcca gtccctgaac
120
aaatactacg gccatggcgc aggtaacccg ctcggcccga cccagggcgt cggctacgct
180
aacgagctca tcgcccgtct cacccacttg cccgtgctcg atgacaccag ctctaaaccc
240
acattcgaat ccaacccggc tactttcccg ctcaactcca ctctctatgc ggacttttcg
300
catgaaaaag gcatcatctc tatcctcttt gctttgggtc tgtacaacgg caccaagccg
360
ctgtcttcca cgaccgcgga gaatatcacc cagaccgatg ggttctcatc tgcctggacg
420
gttcctttcg cgtcgcgcat gtacgtcgag atgatgcaat gccagtccga gcaggagcct
480
ttggtccgtg tcttggttaa tgaacttgtt gttccgctgc atggttctcc ggttgaacct
540
ttgggaagat gtacccggga tagcttccag aaggggttga gctttgctaa atctggtggt
600
gattgggtaa agtcttttcc ttag
624
Claims (7)
1. an expression vector, it carries the gene of coding phytase.
2. expression vector as claimed in claim 1, is characterized in that, described phytase, and its aminoacid sequence is SEQ ID NO:1.
3. expression vector as claimed in claim 2, is characterized in that, described phytase, and the nucleotides sequence of its encoding gene is classified SEQ ID NO:2 as.
4. a host cell, it carries expression vector described in claim 1.
Host cell claimed in claim 4 be Trichodermareesei (
trichoderma reesie).
6. the application of host cell in producing phytase described in claim 5.
7. the application of phytase in feed described in claim 6.
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|---|---|---|---|
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|---|---|---|---|
| CN201310608963.5A CN103725707B (en) | 2013-11-27 | 2013-11-27 | Genetic engineering strain for recombinant expression of phytase |
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| Publication Number | Publication Date |
|---|---|
| CN103725707A true CN103725707A (en) | 2014-04-16 |
| CN103725707B CN103725707B (en) | 2015-05-13 |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110484455A (en) * | 2019-06-10 | 2019-11-22 | 青岛蔚蓝生物集团有限公司 | A kind of trichoderma mutant strain of stable, high-yielding phytase |
| CN111235043A (en) * | 2018-11-28 | 2020-06-05 | 青岛蔚蓝生物集团有限公司 | Temperature-resistant phytase producing strain and application thereof |
| CN112779169A (en) * | 2019-11-08 | 2021-05-11 | 青岛蔚蓝生物集团有限公司 | Mutant strain for high-yield phytase and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101955957A (en) * | 2009-08-21 | 2011-01-26 | 青岛康地恩生物科技有限公司 | New phytase gene and expression vector thereof |
-
2013
- 2013-11-27 CN CN201310608963.5A patent/CN103725707B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101955957A (en) * | 2009-08-21 | 2011-01-26 | 青岛康地恩生物科技有限公司 | New phytase gene and expression vector thereof |
Non-Patent Citations (2)
| Title |
|---|
| MISHRA,I.G等: "登录号：AFJ79737.1", 《GENBANK》, 19 May 2012 (2012-05-19), pages 1 * |
| 冮洁等: "基因工程菌里氏木霉染色体DNA的提取方法", 《生物技术》, vol. 14, no. 2, 31 December 2004 (2004-12-31), pages 24 - 26 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111235043A (en) * | 2018-11-28 | 2020-06-05 | 青岛蔚蓝生物集团有限公司 | Temperature-resistant phytase producing strain and application thereof |
| CN111235043B (en) * | 2018-11-28 | 2022-05-31 | 青岛蔚蓝生物集团有限公司 | Temperature-resistant phytase producing strain and application thereof |
| CN110484455A (en) * | 2019-06-10 | 2019-11-22 | 青岛蔚蓝生物集团有限公司 | A kind of trichoderma mutant strain of stable, high-yielding phytase |
| CN112779169A (en) * | 2019-11-08 | 2021-05-11 | 青岛蔚蓝生物集团有限公司 | Mutant strain for high-yield phytase and application thereof |
| CN112779169B (en) * | 2019-11-08 | 2022-10-28 | 青岛蔚蓝生物集团有限公司 | Mutant strain for producing phytase and application thereof |
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| Publication number | Publication date |
|---|---|
| CN103725707B (en) | 2015-05-13 |
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