CN103205443A - High heat resisting phytase yeast engineering bacteria and constructing method thereof - Google Patents

Abstract

The invention relates to high heat resisting phytase yeast engineering bacteria and a constructing method thereof. Reported phytase AppA gene is analyzed by a biological information method to find a potential thermal stable associated locus. Site-specific mutagenesis is performed to the key locus of the gene by a site-specific mutagenesis technology to obtain a mutant gene Appa-M2. The mutant gene is constructed to a yeast shuttle vector pPIC9 and converted to a pichia pastoris GS115 so as to screen heat-resisting phytase strains with efficient expression. The heat resisting performance of phytase expressed by recombinant yeast engineering bacteria screened is good. The residual enzyme activity of the yeast engineering bacteria insulated for 10 minutes at 85 DEGC can reach 60%. The yeast engineering bacteria has wide application prospect in feed and food industry.

Description

A kind of high heat resistance phytase Yeast engineering bacteria and construction process thereof

Technical field

The present invention relates to bioengineering field, specifically belong to a kind of high than high heat resistance phytase Yeast engineering bacteria alive and construction process thereof.

Background technology

Phytase is that catalysis phytic acid and saline hydrolysis thereof are the general name of the class of enzymes of inositol and phosphoric acid (salt), belongs to orthophosphoric ester monohydrolase.Phytase has special space structure, can separate the phosphorus in the phytic acid molecule successively, and phytic acid (salt) is degraded to inositol and inorganic phosphorus, discharges other nutritive substance with phytic acid (salt) combination simultaneously.Phytic acid is one of main antinutritional factor that exists in the plant feed, and mineral ion, protein etc. is all had sequestering action, thereby influences animal to the absorption of nutritive substance.Plant feed as major ingredient in the animal-feed contains a considerable amount of phosphorus, wherein be that the form with phytate phosphorus exists more than 50%, and monogastric animal for want of decomposes the necessary enzyme of phytic acid and can not effectively utilize phytate phosphorus.Will inevitably cause the phosphor resource waste if in the feed of monogastric animal, directly add phosphorus, form high phosphorus ight soil and cause environmental pollution; If do not add phosphorus in feed, this certainly will will have influence on growth of animal, growth and breeding.At present, the suitability for industrialized production of phytase has worldwide formed a huge industry.Not only bring considerable economic for manufacturing enterprise, also fundamentally solved in the present monogastric animal ight soil phosphorus to the pollution of environment, reduce the pressure that uses inorganic phosphate to bring for China's mineral substance resource as fodder additives, shown great economic results in society.Therefore, phytase is as novel fodder additive, can improve the utilization ratio of phosphorus and other compositions in the feed and alleviate environmental pollution, more and more paid attention to and use (Ding Qiang etc., Chinese agriculture science and technology Leader, 2010,12 (3); 27～33).

The strongest phytase (Lei, et al., Appl Microbiol Biotechnol, 2001,57 (4): 474-481) of decomposition phytic acid ability known to deriving from colibacillary phytase (appA phytase) and be so far.Since nineteen fifty-two found in the intestinal bacteria phytase activity is arranged, people had separated intestinal bacteria phytase (appA) gene, in prokaryotic expression system (Golovan, et al., Can J Microbiol, 2000,46 (1): 59～71; Luo Huiying, biotechnology journal, 2004,20 (1): 78～84; Chen Yin etc., Microbiol, 2004,31 (3) 74～78; 371～375) and eukaryotic expression system (Rodriguez, et al., Arch Biochem Biophys, 2000,382 (1): 105～112 Huang Min etc. use and the environmental organism journal 2009,15 (3):; Luo Huiying etc., biotechnology journal, 2006,22 (4); 528～533; Gold-tinted is auspicious etc., Hubei University's journal, 2007,29 (3); 290～293) expressed the appA phytase in, and its zymologic property has been carried out systematic study.That uses in production practice at present derives from colibacillary phytase appA, its pH characteristic is better, specific activity also is that report is the highest at present, but its thermostability is relatively poor, to lose more than 70% by its activity after the granulation pyroprocess, be difficult in main particulate material, use (Ding Qiang etc., Chinese agriculture science and technology Leader, 2010,12 (3); 27～33).In recent years, utilize genetic engineering technique directional transformation appA phytase to improve the thermostability of enzyme, become a direction transforming phytase.

Summary of the invention

The object of the present invention is to provide a kind of high heat resistance phytase Yeast engineering bacteria and construction process thereof.

A kind of high heat resistance phytase gene (Appa-M provided by the invention ₂), its nucleotides sequence is classified SEQ ID No.1 as.

This gene obtains by the following method:

According to the AppA sequence of reporting among the GenBank (DQ513832), entrust biotech company to synthesize phytase gene Appa; Add EcoRI and NotI restriction enzyme site at 5 ' and 3 ' end respectively;

By bioinformatic analysis, found two can introduce glycosylated potential glycosylation site to the Appa sequence, by introducing the thermostability that glycosylation improves Expressing Recombinant Phytase in yeast;

Utilize complementary primer that above-mentioned two sites are suddenlyd change by rite-directed mutagenesis, introduce glycosylation, synthetic rite-directed mutagenesis primer, underscore is labeled as the mutational site;

Q258N primer: (CAG--AAC)

F _Q258N5 '-CTACTTGCTG AACIts nucleotides sequence of AGAACTCCAGAGG-3 ' is classified SEQ ID No.3 as

R _Q258N5 '-CCTCTGGAGTTCT GTTIts nucleotides sequence of CAGCAAGTAG-3 ' is SEQ ID No.4

Q349N primer: (CAG--AAC)

F _Q349N5 '-CTCTCAATGGATT AACIts nucleotides sequence of GTTTCGTTGG-3 ' is classified SEQ ID No.5 as

R _Q349N5 '-CCAACGAAAC GTTIts nucleotides sequence of AATCCATTGAGAG-3 ' is classified SEQ ID No.6 as

Phytase gene (Appa-M after the improvement that acquisition can efficiently express in yeast ₂), its nucleotides sequence is classified SEQ ID No.1 as.

Compare the phytase gene (Appa-M after the improvement with the phytase gene Appa that has synthesized ₂)+772 ,+774 ,+change taken place in 1045 and 1047 base.Cause coded aminoacid sequence+259 ,+change taken place in 350 amino acid, make+259Q ,+350Q changes into+259N ,+350N, its aminoacid sequence are SEQ ID No.2.

The invention provides a kind of high heat resistance phytase Yeast engineering bacteria, contain said gene.

The invention provides a kind of construction process of high heat resistance phytase Yeast engineering bacteria, comprise the steps:

1) with the phytase gene (Appa-M after the above-mentioned improvement ₂) by restriction enzyme EcoRI and NotI digestion, be building up on the yeast shuttle vector pPIC9, obtain recombinant vectors pPIC9-Appa-M ₂

2) prepare pPIC9-Appa-M in a large number ₂Granulate, carry out linearizing by the BglII enzyme then;

3) transform pichia spp GS115 by the lithium chloride conversion method;

4) screening efficiently expresses phytase Appa-M ₂Restructuring yeast strains.

Abduction delivering purifying Appa-M ₂Phytase is analyzed the phytase zymologic property that purifying obtains, and the result shows that the phytase resistance toheat of the recombination yeast engineering bacterium expression of this research and establishment screening is good, and 85 ℃ are incubated 10 minutes, and remnant enzyme activity can reach 60%.

Recombination yeast engineering strain provided by the present invention further can be used for large-scale industrial production phytase through after the pilot scale fermentation, the genetic modification that the present invention sets up, and yeast expression system can provide new thinking for the transformation of phytase.In feed and foodstuffs industry, have wide practical use.

Description of drawings

Fig. 1: the Appa sequencer map that suddenlys change

Fig. 2: pPIC9-Appa-M ₂Enzyme is cut evaluation figure

Fig. 3: pPIC9-Appa-M ₂BglII restriction endonuclease linearizing electrophorogram

Fig. 4: pPIC9-Appa-M ₂Transformed yeast RDB substratum transforms figure

Fig. 5: MD, MM substratum screening figure

Fig. 6: the stability cultivation figure that goes down to posterity

Fig. 7: extract yeast genes group PCR and identify Appa-M ₂Figure

Fig. 8: purifying SDS-PAGE electrophorogram

Fig. 9: phytase optimum temperuture figure

Figure 10: phytase optimal pH figure

Figure 11: the phytase temperature stability detects figure

Embodiment

Embodiment 1

One, the acquisition of phytase gene Appa

According to the AppA sequence of reporting among the GenBank (DQ513832), entrust the synthetic phytase gene Appa of biotech company, be cloned on the T-Easy carrier, obtain recombinant plasmid T-Easy-Appa.

Two, Appa sequence glycosylation site is analyzed

When protein contains consensus sequence Asn-X-Ser/Thr(N-X-S/T) when (X represents any amino acid), pichia spp can carry out glycosylation to the amide nitrogen on the aspartoyl residue wherein, produces the N-glycosylation.According to the glycosylation of pertinent literature report the thermostability of enzyme is had significant effects, sequential analysis shows, by the phytase of Appa genes encoding itself three glycosylation sites arranged, and is respectively: N139, N204, N317 site.Analyze to find that this gene also has two potential can introduce glycosylated site by transforming, and is respectively: Q259, Q350, l-asparagine N codon has: AAT, AAC, AAC is preference codon in the yeast.So the design mutant primer is replaced into l-asparagine (N) codon (AAC) to glutamine codon (CAG).

Three, introduce glycosylation site by quick rite-directed mutagenesis

Utilize two pairs of complementary primers (SEQ ID No.3-6), use the recombinant plasmid T-Easy-Appa that has synthetic phytase gene Appa as template, increase the recombinant plasmid T-Easy-Appa-M after obtaining suddenling change by round pcr ₂Process DpnI digests the template plasmid T-Easy-Appa in the PCR system again, and digestion product changes bacillus coli DH 5 а over to and increases in a large number, extracts the plasmid order-checking, and the result shows Appa-M ₂Suddenly change successfully the Appa-M that obtains ₂Nucleotide sequence SEQ ID No.1, aminoacid sequence SEQ ID No.2, sudden change the results are shown in Fig. 1.

Four, make up yeast recombinant vectors pPIC9-Appa-M ₂

Respectively to Appa-M ₂Carry out EcoRI and NotI digestion with shuttle vectors pPIC9, enzyme is cut product and is reclaimed, the Appa-M after cutting with enzyme ₂And pPIC9, be mixed in proportion, with the connection of spending the night of 16 ℃ of T4 ligase enzymes, connect product transformed into escherichia coli DH5 а, be coated with the LB solid medium flat board that contains ammonia benzyl mycin, 37 ℃ of incubated overnight, picking list bacterium colony is cultivated in the LB liquid nutrient medium in a large number, plasmid purification is done EcoRI and NotI enzyme and is cut and identify and see Fig. 2, pPIC9-Appa-M ₂Fragment after enzyme is cut is respectively 8kb and 1.236kb.

Five, pPIC9-Appa-M ₂Granulate a large amount of the preparation and linearizing

Use 500 μ l reaction systems, 15 μ l BglII restriction endonucleases, the damping fluid 400 μ l pPIC9-Appa-M of 50 μ l enzymes ₂, supply 500 μ l with sterilized water, digested overnight is passed through the extracting of phenol chloroform again, and 20% ethanol sedimentation dissolves with 20 μ l sterilized waters at last.Fig. 3 is seen in digestion electrophorogram as a result.

Six, the lithium chloride conversion method transforms pichia spp

A. competent cell is prepared:

1. in 50mlYPD, cultivate pichia spp to about 108 of OD600=0.8-1.0(/ml).

2. collecting cell washs the centrifugal 10min of room temperature 1500g with the 25ml aqua sterilisa.

3. removal water is used 1ml100mM LiCl re-suspended cell.

4. cell suspension is gone in the 1.5ml centrifuge tube.

5. maximum speed of revolution sedimentation cell 15s removes LiCl with the suction nozzle suction.

6. suspension cell in 400 μ l100mM LiCl.

7. 50 μ l cell suspending liquids are moved on in the 1.5ml centrifuge tube, with a pipe, now do existing usefulness at every turn, do not place on ice or-20 degree preservations.

B. transform:

1. boil single stranded DNA 5min, be put in fast to make in the frozen water and turn cold, be placed on ice.Attention: carrier DNA does not need.Will each with before all boil, at-20 degree preservation equal portions small amount of sample, the pipe that at every turn thaws boils after 3-4 time again.

2. get cell in top the 7th step, centrifugal removal LiCl.

3. each transforms sample, is sequentially added into following reagent, and PEG can protect cell to avoid the deleterious effect of high density LiCl.

240 μ l50%PEG, 36 μ l1M LiCl, 25 μ l2mg/ml single stranded DNAs are dissolved in the plasmid DNA (5-10 μ g) in the 50 μ l aqua sterilisas.

4. violent vortex cell precipitation is to complete mixing (1min).

5. hatching 30min(at 30 ℃ does not shake).

6. at 42 ℃ of water-bath thermal shock 20-25min.

7.6000-8000rpm centrifugal, will transform solution and move away.

8. with 1ml aqua sterilisa re-suspended cell precipitation.

9. be coated with 25-100 μ l at RDB or MD flat board, hatched 2-4 days at 30 ℃, transformation result is seen Fig. 4.

Seven, efficiently express phytase Appa-M2 yeast strain

Screening method one (conventional screening method):

Plate screening:

Distinguish the dull and stereotyped and MD flat board of dibbling MM with the recon that aseptic toothpick picking screens above.

Consistent with the MD growth at MM, its phenotype is Mut+.

Normal in MD growth, poor growth or long on MM, its phenotype is MutS, sees Fig. 5.Cultivating through going down to posterity makes the phytase gene stable integration see Fig. 6 on the yeast genes group again.

PCR identifies:

The positive colony that the picking flat screen is selected extracts the total DNA of yeast.

1. use 10ml MD respectively, MDH cultivates reorganization bacterium and recipient bacterium (GS115) to about 12 hours of OD600=5-10(for 30 ℃), 5000rpm room temperature 10min centrifugal collecting cell, with 10ml sterilized water washing thalline once.

2. cell precipitation is resuspended in the 2ml SCED pH7.5 damping fluid of new preparation [SCED:1M sorbyl alcohol, 10mM Trisodium Citrate (pH7.5), 10mM EDTA, 10mM DTT], adds 37 ℃ of incubation 50min of 0.3mg zymolase mixing.

3. add 2ml1%SDS and will manage inversion mixing content for several times, place 5min on ice, operation should be gentle as far as possible careful, adds 1.5ml5M potassium acetate (pH8.9), softly mixing.12000rpm4 ℃ of 10min is centrifugal.Abandon precipitation and get supernatant.

4. add 2 times of volume dehydrated alcohols in the supernatant, room temperature leaves standstill 15min, 12000rpm4 ℃ of 20min centrifuging and taking precipitation.

5. precipitation is resuspended in 0.7ml TE(pH7.4), go in the 1.5ml centrifuge tube after the soft dissolving.

6. phenol: chloroform (1:1), chloroform: centrifuging and taking water of each extracting of primary isoamyl alcohol (24:1).

7. the water equal portions are transferred in two 1.5ml centrifuge tubes.The dehydrated alcohol that adds 1/2 volume 7.5M ammonium acetate (pH7.5) and 2 times of volumes, mixing-20 ℃ 60min precipitation.

8.12000rpm4 the centrifugal recovery nucleic acid of ℃ 20min is abandoned supernatant with 70% washing with alcohol throw out, the centrifugal supernatant of abandoning dries residual ethanol, and every pipe precipitation is resuspended in 50 μ l TE(pH7.5) in ,-20 ℃ of preservations are stand-by.

The yeast genes group of extracting utilizes the phytase special primer to carry out pcr amplification, the results are shown in Figure 7.Choose the order-checking of PCR positive colony, sequencing result shows that improved phytase gene successfully is incorporated on the yeast genes group.

Eight, to being integrated with Appa-M2 recombinant chou yeast abduction delivering phytase and purifying

A. abduction delivering

Recombination yeast 500mL BMGY[1% yeast extract, 2% peptone, 1.34%YNB, 0.00004%Biotin, 1% glycerine (volume ratio)] in, 30 ℃ of violent joltings make cell grow to state of saturation (A600=10～20), centrifugal collection thalline, add 250mL inducing culture BMMY (with the glycerine among the methyl alcohol replacement BM GY), 30 ℃ were continued inducing culture 2 days down.

B. the purifying of enzyme

To the above-mentioned centrifugal 30min of fermented liquid 8000rpm that obtains, with supernatant 80% ammonium sulfate precipitation, centrifugal removal supernatant, again with the dissolving of 40mM pH4.5 sodium acetate buffer, use same buffer dialysis three days, use the resin cation (R.C.) purifying, obtain the phytase of purity 98%, the sodium-chlor of sloughing in the elution buffer through Hiprep26/10desalting obtains pure enzyme again.The SDS-PAGE detected result is seen Fig. 8.

Nine, Appa-M ₂Zymologic property is measured

Purified phytase carries out enzymatic reaction to measure its optimal pH and optimum temperuture under different pH and condition of different temperatures, the results are shown in Figure 9-10.Enzyme liquid was handled respectively 10 minutes under differing temps (40,50,60,70,80,85,90,95,100 ℃), under 37 ℃, the condition of pH4.5, measured enzymic activity respectively to measure the thermostability of enzyme, the results are shown in Figure 11.The result shows that the phytase Appa-M2 resistance toheat of recombination yeast engineering bacterium expression is good, and 85 ℃ are incubated 10 minutes, and remnant enzyme activity can reach 60%.

Claims (3)

1. high heat resistance phytase gene Appa-M ₂, its nucleotides sequence is classified SEQ ID No.1 as.

2. a high heat resistance phytase Yeast engineering bacteria contains the described Gene A ppa-M of claim 1 ₂

3. the construction process of a high heat resistance phytase Yeast engineering bacteria is characterized in that, comprises the steps:

1) with the described phytase gene Appa-M of claim 1 ₂By restriction enzyme EcoRI and NotI digestion, be building up on the yeast shuttle vector pPIC9, obtain recombinant vectors pPIC9-Appa-M ₂

2) prepare pPIC9-Appa-M in a large number ₂Granulate, carry out linearizing by the BglII enzyme then;

3) transform pichia spp GS115 by the lithium chloride conversion method;

4) screening efficiently expresses phytase Appa-M ₂Restructuring yeast strains.

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