CN102559632A - Optimized and improved escherichia coli phytase APPA-M with enhanced catalytic activity in acidic range, and gene and application of optimized and improved escherichia coli phytase APPA-M - Google Patents
Optimized and improved escherichia coli phytase APPA-M with enhanced catalytic activity in acidic range, and gene and application of optimized and improved escherichia coli phytase APPA-M Download PDFInfo
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Abstract
The invention relates to the field of gene engineering, in particular to an optimized and improved escherichia coli phytase APPA-M with enhanced catalytic activity in acidic range, and gene and application of the optimized and improved escherichia coli phytase APPA-M. For improving the catalytic capability and the specific activity of the escherichia coli phytase APPA in acidic range, 22 key amino acids in active center of the escherichia coli phytase APPA are subjected to saturation mutagenesis by utilizing gene engineering means. When mutational sites are M216C and N306D, the catalytic activity of the optimized and improved escherichia coli phytase APPA-M in acidic range of pH2-5 is greatly improved, and a huge application potential can be showed in application.
Description
Technical field
The present invention relates to the genetically engineered field, particularly, the present invention relates to a kind of intestinal bacteria Sumizyme PHY APPA-M and gene and application of the optimization that catalytic activity improves in acid range improvement.
Background technology
(Phytate, Phytic acid IP6) claim phytinic acid again to phytic acid, contain 6 phosphate groups, have abundant phosphorus, are the important storage forms of phosphorus in the feed.Phosphorus is the essential mineral element of animal body; Lack the Sumizyme PHY (Phytase that decomposes phytic acid in the monogastric animal body; The enzyme of catalysis phytic acid and phytate hydrolysis), cause the utilization ratio of phosphorus in the feed only to have 1/3 or lower, in order to replenish the deficiency of available phosphorus; Must in feed, add inorganic phosphate, commonly used is secondary calcium phosphate and bone meal.So not only increased the cost of feed greatly, and a large amount of phytate phosphorus can not be utilized and directly excrete, cause the waste and the serious environmental problem in phosphorus source.
A large amount of experiment both at home and abroad all proves; In feed, add Sumizyme PHY and can improve the utilization ratio of phosphorus and other mineral elements in the plant feed; Raising is to starch, fat, the utilization ratio of nutritive substances such as protein; Thereby promote that animal health increases fast, thereby reduce the pollution that phosphorus content alleviates environment in the ight soil.It is generally acknowledged, in feed, add Sumizyme PHY and can make the utilization ratio of phosphorus improve 20-60%.Simons et al. (1990) adds microbial phytase in corn-dregs of beans daily ration, the utilization ratio of phosphorus improves 60%; Waldroup et al. (2000) adds Sumizyme PHY in the broiler fodder that with the soya-bean cake is basal diet, nearly 50% phytate phosphorus is released.
Along with development of biology, the particularly application of DNA recombinant technology makes the extensive expression of various microbe-derived phytase genes become possibility.Derive from Escherichia coli at present, the Sumizyme PHY of mikrobes such as Aspergillus niger, Aspergillus fumigatus has been efficiently expressed in pichia pastoris phaff, and successful in feed, using.Wherein deriving from intestinal bacteria Sumizyme PHY APPA and have the advantage of high specific acitivity, is the Sumizyme PHY of using at present the most widely.This full length gene 1299bp, 432 amino acid of encoding, 22 amino acid of N end are signal peptide.Its optimum temperuture is 55 to 60 ℃, ph optimum 4.5 to 5, and pH 2 to 3 o'clock, the activity ratio was lower, has only the 30-40% under the ph optimum condition, was unfavorable for that this Sumizyme PHY brings into play the hydrolysis phytic acid in gastric environment.
The animal rearing test-results shows that the main position of the Sumizyme PHY hydrolysis phytic acid that adds in the feed is different because of different animals, pig mainly under one's belt, chicken is mainly medium at crop.Improving the hydrolytic activity of Sumizyme PHY in the pig feed, mainly is will improve pig with the hydrolysis of phytic acid ability of Sumizyme PHY under gastric environment, and wherein improving stability and the catalytic activity of Sumizyme PHY under acidic conditions is emphasis.Therefore, optimize intestinal bacteria Sumizyme PHY APPA, make it in whole acid range, have high catalytic activity, bigger application potential and industrial value are arranged.
Summary of the invention
The objective of the invention is through transformation, make improved Sumizyme PHY APPA-M in whole acid range, have high catalytic activity, make it can better in the animal stomach, bring into play the effect of hydrolysis phytic acid intestinal bacteria Sumizyme PHY APPA.
The purpose of this invention is to provide a kind of Sumizyme PHY APPA-M and gene thereof of optimizing improvement.
A purpose more of the present invention provides the recombinant vectors that comprises above-mentioned phytase gene.
A purpose more of the present invention provides the recombinant bacterial strain that comprises above-mentioned phytase gene.
A purpose more of the present invention provides the application of above-mentioned Sumizyme PHY APPA-M.
The present invention preferably adopts the method for the saturated transgenation of fixed point that 22 key amino acids in the Sumizyme PHY APPA active site in intestinal bacteria source are transformed, and obtains in whole acid range, having the Sumizyme PHY APPA-M of high catalytic activity through the method screening of high-throughput screen mutation.Sumizyme PHY APPA-M of the present invention compares with original intestinal bacteria Sumizyme PHY APPA, has two amino acid that sudden change has taken place, and the mutational site is respectively M216C and N306D.Aminoacid sequence after the sudden change (not comprising signal peptide sequence) is shown in SEQ ID NO.1:
qsepelklesvvivsrhgvraptkatqlmqdvtpdawptwpvklgwltprggeliaylghyqrqrlvadgllakkgcpqsgqvaiiadvdertrktgeafaaglapdcaitvhtqadtsspdplfnplktgvcqldnanvtdailsraggsiadftghrqtafrelervlnfpqsnlclkrekqdescsltqalpselkvsadnvsltgavslasClteifllqqaqgmpepgwgritdshqwntllslhnaqfyllqrtpevarsratplldliktaltphppqkqaygvtlptsvlfiaghdtDlanlggalelnwtlpgqpdntppggelvferwrrlsdnsqwiqvslvfqtlqqmrdktplsIntppgevkltlagceernaqgmcslagftqivnearipacsl
The ph optimum of this Sumizyme PHY APPA-M is 4.5, when pH 2.0, still has 80% relative phytase activity, and pH 3.0 and 4.0 o'clock all have about 95% relative reactivity.And the APPA Sumizyme PHY of improvement does not only have 35% relative phytase activity when pH 2.0, and pH 3.0 and 4.0 o'clock are also only less than 60% relative phytase activity.The Sumizyme PHY APPA-M that improvement of the present invention is described has high hydrolysis of phytic acid activity in whole acidic conditions environment, reached the better requirement of hydrolysis phytic acid under the pH condition in gastric environment.
The present invention also provides the gene order of the Sumizyme PHY APPA-M of above-mentioned optimization improvement, its above-mentioned Sumizyme PHY APPA-M that encodes, and particularly, its base sequence can be shown in SEQ ID NO.2:
cagagtgagcctgagttgaaactggaatccgttgtcatcgtctctagacatggtgttagagcaccaaccaaggccacccaacttat
gcaagatgtcaccccagacgcttggccaacctggccagtcaagctgggttggttgacacctagaggtggtgagctcattgcttact
tgggtcactaccaaagacagcgtcttgttgccgacggattgttggccaagaagggttgtccacaatctggtcaagtagctattattg
ctgacgtcgacgaaagaacccgtaagacaggtgaagccttcgccgccggtcttgctcctgactgtgccattaccgttcacaccca
agctgacacttcttctccagatccattgttcaaccctttgaagactggtgtttgccaattggacaacgctaacgttactgacgctatctt
gtccagagctggaggatccattgctgacttcaccggtcacagacagactgccttcagagagttggaaagagttcttaacttcccac
aatccaacttgtgccttaagcgtgagaagcaagacgaatcctgttccttgactcaagcattaccatctgagttgaaggtctccgccg
acaacgtctctttgaccggtgctgtcagcttggcttcctgtttgactgaaatctttcttctgcaacaagctcaaggtatgcctgagcca
ggttggggtagaatcaccgactctcaccaatggaacaccttgttgtccttgcacaacgctcaattctacttgctgcagagaactcca
gaggttgctagatccagagccaccccattgttggacttgatcaagactgctttgactcctcacccacctcaaaagcaagcctacggt
gttaccttgcccacttctgtcttgttcattgccggtcacgatactgatttggcaaatctcggcggtgctttggagttgaactggactcttc
ctggtcaacctgataacactccaccaggtggtgagctcgttttcgaaagatggcgtagactatctgataactctcaatggattcaggt
ttcgttggtcttccaaactttgcagcagatgagagacaagactccactgtctttgaacacgcctccaggagaagtcaaattgaccttg
gctggatgtgaagagagaaatgctcagggtatgtgttccttggctggtttcactcaaatcgttaacgaagctagaatcccagcttgtt
ccttgtaa
The present invention also provides a kind of method of optimizing Sumizyme PHY APPA enzymic activity under acidic conditions of improvement intestinal bacteria Escherichia coli; Particularly; At first pass through three-dimensional structural analysis; Confirm the key amino acid in active site, they are respectively the 16th R, the 20th R, the 90th D, the 92nd R, the 126th N, the 129th K, the 204th N, the 215th S, the 216th M, the 219th E, the 220th I, the 223rd L, the 250th H, the 253rd Q, the 254th F, the 257th L, the 258th Q, the 267th R, the 303rd H, the 304th D, the 305th T, the 306th N.Secondly these amino acid are carried out saturation mutation, and make up the expression library of the saturation mutation of these points respectively, respectively 300 clones of random choose in each sudden change library are carried out the analysis of phytase activity.At last gain mutant is optimized combination.Finally acquire the Sumizyme PHY APPA-M that under whole acidic conditions, all has 80% above relative reactivity, its preserving number is: CGMCC 4434.
The present invention also provides the recombinant vectors of the phytase gene appa-m that comprises above-mentioned optimization improvement; The phytase gene appa-m of optimization of the present invention improvement is inserted between the suitable restriction enzyme site of expression vector, makes that its nucleotide sequence is exercisable to be connected with expression regulation sequence.As a most preferred embodiment of the present invention; Be preferably phytase gene appa-m of the present invention is inserted between the EcoR I and Not I restriction enzyme site on the plasmid pPIC9; Make this nucleotide sequence be positioned at the downstream of AOX1 promotor and regulated and control by it, obtain expression of recombinant yeast plasmid pPIC9-appa-m.
The present invention also provides the recombinant bacterial strain that comprises above-mentioned phytase gene appa-m, and preferred recombinant bacterial strain is Pichi strain GS115.
The present invention also provides above-mentioned Sumizyme PHY APPA-M application in fodder additives; Relate generally to the purposes of said Sumizyme PHY in the preparation fodder additives; And corresponding fodder additives, the effective constituent of said fodder additives can be the host cell of said Sumizyme PHY polypeptide, Expressing Recombinant Phytase polypeptide.
The present invention utilizes genetic engineering means improveing intestinal bacteria Sumizyme PHY APPA; To solve intestinal bacteria Sumizyme PHY APPA better deficiency of hydrolysis phytic acid in the gastric juice sour environment; Can not give full play to the effect of hydrolysis phytic acid in animal body; The activity of Sumizyme PHY APPA-M under acidic conditions through optimizing improvement is greatly improved, and when pH 2.0, still has 80% relative phytase activity.And through high flux screening, obtain the bacterial strain of high expression level amount, further satisfy the requirement that suitability for industrialized production reduces cost, therefore, the Sumizyme PHY of optimization improvement of the present invention can demonstrate huge application potential in fodder industry.
Description of drawings
Fig. 1 Sumizyme PHY APPA and the relative enzyme of APPA-M under different pH values graphic representation alive.
Fig. 2 Sumizyme PHY APPA and the stability of APPA-M under different pH values.
Fig. 3 Sumizyme PHY APPA and the APPA-M relative enzyme under differing temps graphic representation alive.
Fig. 4 Sumizyme PHY APPA and APPA-M are 70 ℃ thermostability.
The present invention the ETEC appa-m (Escherichiacoli) that carries improvement Sumizyme PHY APPA-M be stored in (No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, China Committee for Culture Collection of Microorganisms common micro-organisms center on December 7th, 2010; Institute of Microorganism, Academia Sinica; 100101), its preserving number is: CGMCCNo.4434.
Embodiment
Experiment material and reagent:
1, bacterial strain and carrier
Coli strain Topl0, Escherichia coli BL21 (DE3), expression vector pET-22b (+) (available from Novagen company).Pichia spp GS115, carrier pPIC9 (available from Invitrogen company).
2, enzyme and other biochemical reagents
Fast pfu is available from full formula King Company, and restriction endonuclease is available from TaKaRa company, and ligase enzyme is available from Invitrogen company.Substrates such as X-Gal, IPTG, sodium phytate are available from Sigma company, and other all is a domestic reagent.
3, substratum
The intestinal bacteria substratum be LB (1% peptone, 0.5% yeast extract, 1%NaCl, pH7.0).The yeast culture base is YPD (1% yeast extract, 2% peptone, 2% glucose).The yeast screening assay substratum is MD (glucose 20g/L, agar powder 20g/L, a vitamin H 4 * 10
-4G/L, YNB 13.4g/L).
Yeast inducing culture BMGY (1% yeast extract, 2% peptone, 1.34%YNB, 0.00004%Biotin, 1% glycerine (V/V)) and BMMY (replace glycerine divided by 1% methyl alcohol, all the other compositions are identical with BMGY).
The genetic recombination of using learns a skill and is the routine techniques in this area among the present invention.The technology that in following examples, does not describe in detail is all carried out according to related Sections in following laboratory manual or the document or part, comprising: people such as Sambrook, Molecular Cloning, A Laboratory Manual (the 3rd edition .2001); Kriegler, GeneTransfer and Expression:A Laboratory Manual (1990); With James M.Cregg, PichiaProtocols (first version, 1998).
To remove N-end signal peptide sequence from the phytase gene appa (Dassa, 1990) of Escherichia coli bacterial strain,, under the prerequisite that does not change its aminoacid sequence, carry out the sequence transformation according to the preferences (Zhao Xiang, 2000) of pichia spp codon.Avoid GT in the transformation ... The site of this form of AG, the appearance of as far as possible avoiding being rich in AT (e.g.ATTTA, AATAA, AATTAA etc.) sequence, those sequences are relevant with the stability of mRNA.It is synthetic that the gene order that improvement and design is good send Nanjing Jin Ruisi company to carry out full gene.
The structure of embodiment 2, recombinant expression vector pET-22b (+)-appa-m
Sequences Design PCR primer 5 ' end according to synthetic gene contains Nco I restriction enzyme site, and 3 ' end contains EcoR I restriction enzyme site, and primer sequence is following:
5 ' end primer pET-appa-F:GCAC
CCATGGGACAGAGTGAGCCTGAGTTGAAACTG
3 ' end primer pET-appa-R:GCAC
GAATTCTTACAAGGAACAAGCTGGGATTCTAG
With the synthetic gene is template, carries out pcr amplification with above-mentioned primer, and the fragment cloning that amplification is obtained obtains recombinant vectors pET-22b (+)-appa-m to carrier pET-22b (+).
(1), the key amino acid of avtive spot confirms
From protein three-dimensional structure DB (http://www.rcsb.org/pdb/home/home.do), download the three-dimensional structure file of intestinal bacteria Sumizyme PHY APPA, it is numbered 1DKQ in the PDB DB.This PDB structure is the mixture of enzyme in substrate, contains substrate IP6 in its structure.Use PDBVi ewer that this structure is analyzed, APPA has 410 amino-acid residues, and its 15-23 amino-acid residue is the common conserved sequence of acid Sumizyme PHY: RAGVRAPT.152 the amino-acid residue that 134 amino-acid residues that this albumen has two structural domain: N end and C hold is formed the alpha structural domain jointly; 124 amino-acid residues are formed the alpha-beta structural domain in the middle of all the other, and conserved sequence and active site are between two structural domains.Through analyzing; Confirmed that finally 22 combine relevant key amino acid with substrate, they are respectively the 16th R, the 20th R, the 90th D, the 92nd R, the 126th N, the 129th K, the 204th N, the 215th S, the 216th M, the 219th E, the 220th I, the 223rd L, the 250th H, the 253rd Q, the 254th F, the 257th L, the 258th Q, the 267th R, the 303rd H, the 304th D, the 305th T, the 306th N.
(2), the saturation mutation of gene locus
The goal gene mutational site is unified to be NNK, and wherein, N represents A, T, and C, four kinds of bases such as G, K represents G, two kinds of bases such as T.Forward primer is got 18 bases left side and is got 6 base on the NNK right side, reverse primer is got 18 base right sides and got 6 base and constitute in the NNK left side, and reverse primer and forward primer have the Tumor-necrosis factor glycoproteins of 15bp.PCR product Transformed E .coli BL21 (DE3) after the DpnI enzyme is cut processing clicks competent cell, and clone's number in each mutational site is basically all more than 2000.Warp is 20 cloning and sequencings of picking at random, verify its positive rate, and the result shows the positive clone of the chief's clone 95%, and the mensuration of carrying out phytase activity for next step picking clone lays the foundation.
The primary dcreening operation of embodiment 4 high phytase activity bacterial strains
Through probability analysis, 300 clones of each saturated site picking can comprise 20 amino acid of saturation mutation.Therefore for faster from the sudden change storehouse of all 22 points, screen the positive colony that enzymic activity improves, the present invention adopts flat board to induce screening method that the target clone is carried out preliminary screening.Specific as follows: each mutational site is 300 clones of picking at random, and be inoculated into pH and be 5.5 and comprise the IPTG of 0.5mM, 0.1% phytinic acid, in the LB flat board of 1.5% agarose, 37 ℃ of incubated overnight.And will be by 1M CaCl
2The damping fluid of forming with 0.2M sodium acetate (pH is 5.5) is poured on the flat board.Flat board was placed 1 hour in room temperature, and according to the having or not and height of settling section preliminary evaluation phytase activity, and therefrom the picking settling section screens 509 clones altogether obviously greater than the clone of the contrast of former intestinal bacteria Sumizyme PHY APPA.
The multiple sieve of the high bacterial strain of phytase activity under the embodiment 5 whole acidic conditionss
Basically confirm 509 clones through first round primary dcreening operation; Further cultivate screening through 96 hole depth hole culture plates, every hole contains 500 μ L LB substratum, and 37 ℃ of shaking table 200rpm cultivated after 24 hours; 0 μ L plateau of transferase 45,, the bacterium liquid of growing was dull and stereotyped to 96 new holes; 450 μ L LB substratum are added in dull and stereotyped every hole, and containing final concentration is 0.5mM IPTG, 37 ℃ of shaking table 200rpm abduction delivering Sumizyme PHYs that spend the night.Get the bacterium liquid supernatant that 5 μ L contain the incubated overnight abduction delivering; Add substrate 1.5mmol/L sodium phytate 65 μ L (with the pH 7.0Tris-HCl damping fluid preparation of 0.1mol/L); 37 ℃ of reaction 20min add 50 μ L colour developing liquid (10g Ammonium Molybdate Tetrahydrate+32mL sulfuric acid+73.2g ferrous sulfate adds water and is settled to 1L); And under 700nm, survey its OD value, calculate enzyme and live.Respectively at pH 2.0,3.0,4.0,5.0 analyze the different phytase activities under the whole acidic conditions that are cloned in according to above method.Above 4 all high clones of pH condition enzymic activity, and the more close clone of phytase activity under these pH conditions is picked as positive colony, obtained 5 positive colonies from 96 orifice plates, gets the positive colony DNA and carries out gene sequencing.
Sequencing result is confirmed the positive mutational site of amino acid, and clone 1,2 and 4 mutational site all is that the 216th M replaces with C; Clone 3 and 5 mutational site all is that the 306th N replaces with D.Therefore the positive that finally obtains is that two its mutational sites are divided into M216C and N306D; At last these two sites are suddenlyd change on former intestinal bacteria Sumizyme PHY APPA simultaneously; And in intestinal bacteria, express; And in the condition of different pH enzyme assay, compare with two clones of M216C and N306D, newly obtain phytase gene enzyme when pH2.0 and pH 5.0 and live higher.Sudden change enzymic activity under whole acidic conditions that two points are combined is higher, and the therefore final mutator gene that obtains is the gene that M216C and N306D suddenly change, and called after appa-m.
(1) structure of expression vector reaches and expresses at zymic
Intestinal bacteria phytase gene appa-m with suddenlyd change M216C and N306D is a template; Primer pIC9-appa-F and the pIC9-appa-R that has EcoR I and Not I restriction enzyme site synthesized in design, increased in the coding region of the maturation protein of appa-m.And utilize EcoR I and Not I enzyme to cut the PCR product; Connect and get into expression vector pPIC9 (Invitrogen; San Diego), makes phytase gene appa-m be inserted into the downstream of the signal peptide sequence of above-mentioned expression vector, form correct reading frame with signal peptide; Be built into Yeast expression carrier pPIC9-appa-m, transformed into escherichia coli competent cell JM109.Positive transformant carries out dna sequencing, and order-checking shows that the correct transformant of sequence is used for preparing in a large number recombinant plasmid.About 8 micrograms carry out linearizing expression plasmid carrier DNA with restriction enzyme BglII; Electric shock transformed yeast GS115 competent cell; The RDB that coats histidine defect property is dull and stereotyped, cultivates 2-3 days for 30 ℃, and the transformant that picking is grown on the RDB flat board is further expressed experiment.
The yeast expression primer sequence is following:
pIC9-appa-F：GCAC
GAATTCCAGAGTGAGCCTGAGTTGAAACTG
pIC9-appa-R：GCAC
GCGGCCGCTTACAAGGAACAAGCTGGGATTCTAG
(2) abduction delivering of transformant Sumizyme PHY and purifying
Transformant is transferred among the BMMY through methanol induction two days later after after BMGY cultivates 2 days, and transformant is carried out phytase activity mensuration.There are 47 transformants to be detected phytase activity in 96 transformants, live the unit scope between 35-380U/mL at the enzyme of supernatant of culture medium.Select the highest active transformant and in shaking bottle, cultivate in a large number, induce and detect phytase activity two days later.Induced supernatant process 12000g centrifugal 10 minutes, and removed the yeast thalline, collect and contain the proteic supernatant of Sumizyme PHY.Supernatant to handling carries out ammonium sulfate precipitation, and the ammonium sulfate powder is added in the supernatant, arrives 80% saturation ratio, stirred overnight.Centrifugal collecting precipitation, deposition is used 0.1M, the Tris-HCl damping fluid dissolving of pH 8.0.Filter membrane with 0.22 μ m vacuumizes processing to supernatant, removes other impurity.The solution that is obtained is packed in the dialysis band, and the dialysis band is suspended in 0.1M, and in the Tris-HCl damping fluid of pH8.0, dialyzed overnight is handled, and removes a large amount of salt ions.Dialyzed sample is through after PEG 8000 concentration, and (AmershamPharmacia Biotech Sweden) is further purified the liquid concentrator of 2mL through anionresin HiTrap Q Sepharose XL FPLC column.0.1M the Tris-HCl of pH8.0 contains 1M NaCl and carries out gradient elution, collects the elutriant 5mL of purpose peak point.To collect the liquid electrophoresis detection, show that target protein is by purifying.
(1) ph optimum of Sumizyme PHY APPA and APPA-M and pH stability
Under condition of different pH, Sumizyme PHY APPA-M after the improvement of the intestinal bacteria Sumizyme PHY APPA of previous purifying and purifying of the present invention is carried out the mensuration of ph optimum respectively, measuring method is measured by ordinary method, and the result is as shown in Figure 1.Visible from Fig. 1, former intestinal bacteria Sumizyme PHY APPA has evident difference with the pH response curve of improvement back Sumizyme PHY APPA-M, and the relative phytase activity of APPA-M under whole acidic conditions is all apparently higher than former intestinal bacteria Sumizyme PHY APPA generally.For example: when pH 2.0, the relative reactivity of APPA is 35%, and APPA-M is 80%; PH 3.0 is that the relative reactivity of APPA is 40%, and APPA-M is 95%; PH 3.0 is that the relative reactivity of APPA is 50%, and APPA-M is 98%.
Former Sumizyme PHY APPA that purifying is good and improvement Sumizyme PHY APPA-M are diluted to finite concentration, in the damping fluid of pH 1-10, place 1h for 37 ℃.And then under 37 ℃, the condition of pH 4.5, measure enzyme and live, come the stability of comparison recombinase through calculating relative enzyme work to pH.The result is as shown in Figure 2, and is visible from Fig. 2, and the pH beta stability line of these two Sumizyme PHY APPA and APPA-M not have change basically, is APPA-M stable point more when pH 1.0.
(2) optimum temperuture of Sumizyme PHY APPA and APPA-M and thermostability
Concentration shown in the former Sumizyme PHY APPA that purifying is good is diluted to improvement Sumizyme PHY APPA-M is got 50 μ L and under 30,40,45,50,55,60,70,80 ℃ of temperature, is measured enzyme respectively and live, and calculates relative enzyme and lives, to confirm the optimum temperuture of this enzyme.Its result is as shown in Figure 3, and the optimum temperuture curve of these two Sumizyme PHY APPA and APPA-M does not have to change basically, and optimum temperuture is all between 50-60 ℃.
Concentration shown in the former Sumizyme PHY APPA that purifying is good is diluted to improvement Sumizyme PHY APPA-M is got 2mL 60 ℃ of insulations.Then respectively 2,4,6,8,10min take out 100 μ L enzyme liquid be diluted to shown in concentration.At 37 ℃, measure enzyme under the condition of pH 4.5 and live, as 100% contrast, it is as shown in Figure 4 that it measures the result with untreated original enzyme liquid.The thermostability curve of these two Sumizyme PHY APPA and APPA-M does not have to change basically,
(3) specific activity of Sumizyme PHY APPA and APPA-M
In order to confirm that former Sumizyme PHY APPA is alive with the ratio of improvement Sumizyme PHY APPA-M, at first confirmed the protein concentration of the Sumizyme PHY of purifying through the Lowry method.And, through the ferrous sulfate molybdenum blue method, respectively these two enzymes are carried out the mensuration of enzyme activity under the condition of pH 4.5 at 37 ℃.Through calculating, this former Sumizyme PHY APPA is respectively 3135 ± 83U/mg and 3406 ± 92U/mg with the ratio work of improvement Sumizyme PHY APPA-M.
Claims (9)
1. an intestinal bacteria Sumizyme PHY APPA-M who optimizes improvement is characterized in that, its 216th amino acids is that halfcystine, the 306th amino acids are aspartic acid.
2. an intestinal bacteria Sumizyme PHY APPA-M who optimizes improvement is characterized in that it has the aminoacid sequence shown in SEQ ID NO.1.
3. an intestinal bacteria phytase gene appa-m who optimizes improvement is characterized in that, the described intestinal bacteria of its coding claim 1 come Sumizyme PHY APPA-M.
4. an intestinal bacteria phytase gene appa-m who optimizes improvement is characterized in that it has the nucleotide sequence shown in SEQ IDNO.2.
5. the recombinant vectors that comprises the intestinal bacteria phytase gene appa-m of the described optimization improvement of claim 3.
6. recombinant vectors according to claim 5 is characterized in that, said recombinant vectors is reorganization expression plasmid of yeast pPIC9-appa-m.
7. be expressed in the intestinal bacteria of the intestinal bacteria Sumizyme PHY of the optimization improvement that catalytic activity improves in the acid range, it is characterized in that preserving number is: CGMCC 4434.
8. the recombinant bacterial strain that comprises the intestinal bacteria phytase gene appa-m of the described optimization improvement of claim 3.
9. recombinant bacterial strain according to claim 8 is characterized in that, said bacterial strain is Pichi strain GS115.
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|---|---|---|---|---|
| CN104342418A (en) * | 2013-07-24 | 2015-02-11 | 东莞泛亚太生物科技有限公司 | Phytase with improved enzyme activity |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104342418A (en) * | 2013-07-24 | 2015-02-11 | 东莞泛亚太生物科技有限公司 | Phytase with improved enzyme activity |
| CN106399273A (en) * | 2013-07-24 | 2017-02-15 | 东莞泛亚太生物科技有限公司 | Phytase with enzymatic activity elevating function |
| CN104342418B (en) * | 2013-07-24 | 2017-05-31 | 东莞泛亚太生物科技有限公司 | The phytase of the enzymatic activity with lifting |
| CN106399273B (en) * | 2013-07-24 | 2019-09-20 | 东莞泛亚太生物科技有限公司 | The phytase of enzymatic activity with promotion |
| WO2016078168A1 (en) * | 2014-11-21 | 2016-05-26 | 青岛蔚蓝生物集团有限公司 | Phytase mutants |
| US11104908B2 (en) | 2014-11-21 | 2021-08-31 | Qingdao Vland Biotech Group Co., Ltd. | Phytase mutants |
| US11739336B2 (en) | 2014-11-21 | 2023-08-29 | Qingdao Vland Biotech Group Co., Ltd. | Phytase mutants |
| CN105969750A (en) * | 2016-06-24 | 2016-09-28 | 北京昕大洋科技发展有限公司 | Phytase mutant and application thereof |
| CN105969750B (en) * | 2016-06-24 | 2019-04-26 | 北京昕大洋科技发展有限公司 | A kind of phytic acid enzyme mutant and its application |
| CN110117583A (en) * | 2018-02-05 | 2019-08-13 | 广东溢多利生物科技股份有限公司 | Thermostabilization and the ratio phytase ECAPPA mutant living improved and its gene and application |
| CN110117583B (en) * | 2018-02-05 | 2023-08-04 | 广东溢多利生物科技股份有限公司 | Phytase ECAPPA mutant with heat stability and specific activity improvement and gene and application thereof |
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