CN103849607B - High temperature resistant phytase and uses thereof - Google Patents
High temperature resistant phytase and uses thereof Download PDFInfo
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- CN103849607B CN103849607B CN201410105675.2A CN201410105675A CN103849607B CN 103849607 B CN103849607 B CN 103849607B CN 201410105675 A CN201410105675 A CN 201410105675A CN 103849607 B CN103849607 B CN 103849607B
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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)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/189—Enzymes
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K50/00—Feeding-stuffs specially adapted for particular animals
- A23K50/70—Feeding-stuffs specially adapted for particular animals for birds
- A23K50/75—Feeding-stuffs specially adapted for particular animals for birds for poultry
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y301/00—Hydrolases acting on ester bonds (3.1)
- C12Y301/03—Phosphoric monoester hydrolases (3.1.3)
- C12Y301/03026—4-Phytase (3.1.3.26), i.e. 6-phytase
Abstract
The present invention relates to high temperature resistant phytase and uses thereof, belong to biological technical field.Technical problem to be solved by this invention is to provide a kind of high temperature resistant phytase.Is the aminoacid sequence of the present invention is high temperature resistant phytase as seq? ID? shown in NO1.The present invention is high temperature resistant, and phytase activity power reaches 5062FTU/g, 90 DEG C of insulation 30min enzyme activities still have 4708FTU/g, enzyme high temperature resistance is good, moisture content is only 1.62% simultaneously, heavy metal aluminium and arsenic are respectively 0.63mg/kg and 0.47mg/kg, and above index all meets or exceeds current international advanced product standard.The present invention is high temperature resistant, and phytase is on probation through poulty house, effect stability, well.The present invention is that field of fodder provides a kind of new organic feed additive, has broad application prospects.
Description
Technical field
The present invention relates to high temperature resistant phytase and uses thereof, belong to biological technical field.
Background technology
Phosphorus is the required a kind of macro minerals element of animal, and the interpolation of phosphorus has material impact to feed production cost of compound feed and quality product.As the corn, soybean cake, cavings, cotton cake dregs etc. of pig, fowl feed main source, the phosphorus contained by them is about 40-70% to be existed with the form of phytate phosphorus.And the monogastric animal such as pig, fowl, owing to lacking the microorganism decomposing digestion phytate phosphorus in digestive tube, only can utilize the 25-35% of phosphorus in the 10-20% of maize seed phosphorus, soya-bean cake, in typical swine rations, the utilization ratio of phosphorus only has 15%, and all the other phosphorus of about 85% are discharged from ight soil.Therefore, on the one hand for meeting the needs of pig fowl growth to phosphorus, enough inorganic phosphates must be added in feed, which adds feed cost; On the other hand, a large amount of phosphorus of discharging in ight soil causes again the pollution to soil and water source.Phosphorus is present in soil just face, can not only permeate in soil and spread, and also can form insoluble mixture with elements such as copper, aluminium, calcium and be retained in soil.If Soil Phosphorus gathers in a large number and overflows, the erosion along with soil is flowed, causes the pollution of nature water body.Some countries have proposed to intensive stock-farms the requirement that phosphorus is arranged in restriction from the angle of environmental protection.Holland have passed the new mineral element checkout system of MINAS(mono-kind in 1998) legislation, wherein strictly limit phosphatic quantity discharged in animal excrement.In addition, phytate also can reduce the utilization ratio of animal to main trace element such as zinc, manganese, iron, calcium, potassium by sequestering action, reduce the digestibility of animal to protein by becoming phytic acid complex body with protein bound, thus the utilization ratio of whole feed is reduced.
Along with people are to the increase of green food consumers demand and the attention to ecological environmental protection in Animal husbandry production and process, the features such as fodder enzyme preparation is efficient with it, safety, low cost, have become the focus of world's novel fodder additive investigation and application.Phytase (Phytase) is i.e. phytinic acid lytic enzyme (Myo-inositolhexaphosphatephosphohydrotase), is the general name that catalysis phytic acid (i.e. phytinic acid) and phytate hydrolysis become the class of enzymes of inositol and phosphoric acid (or phosphoric acid salt).Large quantity research shows, in pig, fowl daily ration, add phytase, and the utilization ratio of Dietary phosphorus can be made to improve 40-60%, and in ight soil, the output of phosphorus reduces about 30-50%.The current whole world is generally acknowledged, improve the utilization ratio of phytate phosphorus in feed, and reduce pig, phytate phosphorus is to the pollution of environment in poultry manure, reduce the impact of phytate phosphorus on other trace element and protein utilization, application phytase is a kind of effective means.The phytase PhyA wherein deriving from ficoin (A.ficuum) is considered to the most effective feed phytase at present because it possesses superperformance.
As the zymin for fodder industry, due to the requirement of feedstuff pelletization technology and animal physiological biochemistry, the phytase being really promoted utilization must be have good thermal stability, must have higher enzymic activity again under animal normal body temperature simultaneously.How to solve and make its resistance to of short duration granulation high temperature, this conflict of enzymatic activity high can be had again under animal normal body temperature to be the problem that all fodder enzyme preparations comprising phytase at present all need to solve.In addition, phytic acid enzymatic reaction pH also must adapt with animal digestive tract.The pH value of different animal digestive tracts is different, even a kind of animal is also different in gastral different sites pH value, general pH value stomach is 1.5-3.5, and small intestine is 5-7, large intestine is about 7, therefore requires that feeding enzyme has larger subject range to pH value.
Summary of the invention
Technical problem to be solved by this invention is to provide a kind of high temperature resistant phytase.
The aminoacid sequence of the present invention is high temperature resistant phytase is as shown in seqIDNO1.
Wherein, present invention also offers the purposes that described high temperature resistant phytase is used as fodder additives.
In addition, present invention also offers the feed containing above-mentioned phytase.
Phytase of the present invention is prepared by following method:
1, (the self-editing software of Biosist and Columbia University combines to adopt 3D to build in conjunction with software
http:// www.sbg.bio.ic.ac.uk/servers/3dpssm/) prediction, select the amino acid of rite-directed mutagenesis.Adopt software predicting technology, predict that non-conservative district selects amino acid whose sudden change to cause the change of the Tm value of enzyme own, rite-directed mutagenesis is carried out in the amino acid change selecting Tm value to increase.By to the non-conservative avtive spot (1-33 of Aspergillus ficuum (Aspergillusficuum) phytase (EC3.1.3.26); 361-441) carry out serial rite-directed mutagenesis, find that 27 amino acids are undergone mutation by software rebuild (software of Biosist and Columbia University) and Tm value had the greatest impact, but change not obvious to active centre space structure.Utilize software to carry out 3D and rebuild phytase unfolding temperature, after result shows that 27 amino acids sport Gly by Gln, its Tm value is increased to 83.3 DEG C by 72.5 DEG C, and enzyme activity is substantially constant.
2, design a pair Oligonucleolide primers containing the complete complementary of predetermined sudden change, mutating alkali yl is positioned at primer central authorities, and both sides are 10 ~ 15 correct bases; Primer length 25 ~ 45 bases, GC content is greater than 40%, and 3 ˊ ends should be bases G or C, Tm value is not less than 78 DEG C.Stratagene company gives mentioned above principle and have developed online mutant primer design software, with QuikChange
tMsiteDirectedMutagenesisKit with the use of.
The mutant plasmid that Catastrophic selection is correct, the electroporated plate screening carrying out recombination yeast.Electric shock cultivates the transformant on MD flat board, can be observed the oyster white bacterium colony that diameter is about 2mm after 3d.Cultivate after 5d, by all single bacterium colony difference dibblings to MM flat board and MD flat board, and establish positive control GS115Albumin (His
+mut
s) and negative control GS115 β-gal (His
+mut
+), cultivate 2d or longer time, to filter out on MD growth normal for 30 DEG C, on MM, poor growth or the bacterium colony that do not grow, be positive bacterium colony.By the positive transformant YPD liquid nutrient medium shake-flask culture 16-20h of plate screening, get 200 μ l cell suspensions, collecting cell, after washing 2 times with sterile purified water equal-volume, resuspended with 200 μ l deionized waters, getting 1 μ l cell suspension is in the PCR reaction system of 50 μ l in cumulative volume, and compares with the PCR primer of corresponding expression fragment.The be positive transformant of result of PCR carries out expression study.
Recombination yeast after detecting through plate screening and PCR is inoculated in the BMGY of 10ml, cultivates 16h (OD600=10 ~ 20), be inoculated in the BMGY of 200ml by the inoculum size of 5% for 36 DEG C.After cultivating 20h, room temperature leaves standstill 4h, and incline nutrient solution, adds the BMMY(2% methanol induction of 200ml).Intracellular expression recombination yeast 30 DEG C continues inducing culture 4d, harvested cell.The recombination yeast 30 DEG C of secreting, expressing continues inducing culture 48h, results nutrient solution supernatant.By enzyme activity determination, enzyme is lived and is up to 243FTU/ml, and the recombination yeast choosing the highest enzyme alive is cultivated, and collects, is separated, obtain the thick enzyme of phytase.
3, the thick enzyme purification of phytase, purifying adopts concentrated, spraying dry, and spray condition is: inlet temperature is 90 DEG C, and air outlet temperature is 60 DEG C, and blow pressure is 0.2Mpa, collects and obtains phytase powder.
Phytase powder mensuration enzyme adds granulation protective material according to the proportioning of 5100FTU/g, enters fluidized bed prilling, finally obtain phytase particle after living.
When the present invention is high temperature resistant phytase is below temperature 50 C, phytase activity is more stable; The enzyme of phytase sample of the present invention before and after granulation processing rate of loss < 5% alive.Enzyme stability after processing increases, 90 DEG C, and after 30 minutes, the enzyme rate of recovery alive also can reach more than 95%.Enzyme sample after processing is preserved at normal temperatures, and its loss of activity is lower than undressed sample by more than 90%.Granule preservation period at room temperature can reach 1 year.This enzyme pH stability boundary broadens (1.5-7.0) simultaneously.
The present invention is high temperature resistant, and phytase detects through Institute of Analysis of academy of agricultural sciences of Sichuan Province, enzyme activity reaches 5062FTU/g, 90 DEG C of insulation 30min enzyme activities still have 4708FTU/g, enzyme high temperature resistance is good, moisture content is only 1.62% simultaneously, heavy metal aluminium and arsenic are respectively 0.63mg/kg and 0.47mg/kg, and above index all meets or exceeds current international advanced product standard.The present invention is high temperature resistant, and phytase is on probation through poulty house, effect stability, well.The present invention is that field of fodder provides a kind of new organic feed additive, has broad application prospects.
Accompanying drawing explanation
Fig. 1: purifying and after-processing technology schema;
Fig. 2: phytase SDS-PAGE electrophorogram;
Fig. 3: temperature-enzyme graphic representation alive;
Fig. 4: temperature-enzyme graphic representation alive relatively;
Fig. 5: pH-enzymic activity graphic representation;
Fig. 6: ph stability graphic representation;
Fig. 7: the influence curve figure that pH value is lived to thalli growth and enzyme;
Fig. 8: the influence curve figure that temperature is lived to thalli growth and enzyme;
Fig. 9: the influence curve figure that aeration condition is lived to Fungal biodiversity and enzyme;
Figure 10: inoculum size is to the influence curve figure producing enzyme;
Figure 11: produce enzyme graphic representation;
Figure 12: mixture homogeneity experimental curve diagram.
Embodiment
The aminoacid sequence of the present invention is high temperature resistant phytase is as shown in seqIDNO1.
Wherein, present invention also offers the purposes that described high temperature resistant phytase is used as fodder additives.
In addition, present invention also offers the feed containing above-mentioned phytase.
Phytase of the present invention is prepared by following method:
1, (the self-editing software of Biosist and Columbia University combines to adopt 3D to build in conjunction with software
http:// www.sbg.bio.ic.ac.uk/servers/3dpssm/) prediction, select the amino acid of rite-directed mutagenesis.Adopt software predicting technology, predict that non-conservative district selects amino acid whose sudden change to cause the change of the Tm value of enzyme own, rite-directed mutagenesis is carried out in the amino acid change selecting Tm value to increase.By to the non-conservative avtive spot (1-33 of Aspergillus ficuum (Aspergillusficuum) phytase (EC3.1.3.26); 361-441) carry out serial rite-directed mutagenesis, find that 27 amino acids are undergone mutation by software rebuild (software of Biosist and Columbia University) and Tm value had the greatest impact, but change not obvious to active centre space structure.Utilize software to carry out 3D and rebuild phytase unfolding temperature, after result shows that 27 amino acids sport Gly by Gln, its Tm value is increased to 83.3 DEG C by 72.5 DEG C, and enzyme activity is substantially constant.
2, design a pair Oligonucleolide primers containing the complete complementary of predetermined sudden change, mutating alkali yl is positioned at primer central authorities, and both sides are 10 ~ 15 correct bases; Primer length 25 ~ 45 bases, GC content is greater than 40%, and 3 ˊ ends should be bases G or C, Tm value is not less than 78 DEG C.Stratagene company gives mentioned above principle and have developed online mutant primer design software, with QuikChange
tMsiteDirectedMutagenesisKit with the use of.
The mutant plasmid that Catastrophic selection is correct, the electroporated plate screening carrying out recombination yeast.Electric shock cultivates the transformant on MD flat board, can be observed the oyster white bacterium colony that diameter is about 2mm after 3d.Cultivate after 5d, by all single bacterium colony difference dibblings to MM flat board and MD flat board, and establish positive control GS115Albumin (His
+mut
s) and negative control GS115 β-gal (His
+mut
+), cultivate 2d or longer time, to filter out on MD growth normal for 30 DEG C, on MM, poor growth or the bacterium colony that do not grow, be positive bacterium colony.By the positive transformant YPD liquid nutrient medium shake-flask culture 16-20h of plate screening, get 200 μ l cell suspensions, collecting cell, after washing 2 times with sterile purified water equal-volume, resuspended with 200 μ l deionized waters, getting 1 μ l cell suspension is in the PCR reaction system of 50 μ l in cumulative volume, and compares with the PCR primer of corresponding expression fragment.The be positive transformant of result of PCR carries out expression study.
Recombination yeast after detecting through plate screening and PCR is inoculated in the BMGY of 10ml, cultivates 16h (OD600=10 ~ 20), be inoculated in the BMGY of 200ml by the inoculum size of 5% for 36 DEG C.After cultivating 20h, room temperature leaves standstill 4h, and incline nutrient solution, adds the BMMY(2% methanol induction of 200ml).Intracellular expression recombination yeast 30 DEG C continues inducing culture 4d, harvested cell.The recombination yeast 30 DEG C of secreting, expressing continues inducing culture 48h, results nutrient solution supernatant.By enzyme activity determination, enzyme is lived and is up to 243FTU/ml, and the recombination yeast choosing the highest enzyme alive is cultivated, and collects, is separated, obtain the thick enzyme of phytase.
3, the thick enzyme purification of phytase, purifying adopts concentrated, spraying dry, and spray condition is: inlet temperature is 90 DEG C, and air outlet temperature is 60 DEG C, and blow pressure is 0.2Mpa, collects and obtains phytase powder.
Phytase powder mensuration enzyme adds granulation protective material according to the proportioning of 5100FTU/g, enters fluidized bed prilling, finally obtain phytase particle after living.
When the present invention is high temperature resistant phytase is below temperature 50 C, phytase activity is more stable; The enzyme of phytase sample of the present invention before and after granulation processing rate of loss < 5% alive.Enzyme stability after processing increases, 90 DEG C, and after 30 minutes, the enzyme rate of recovery alive also can reach more than 95%.Enzyme sample after processing is preserved at normal temperatures, and its loss of activity is lower than undressed sample by more than 90%.Granule preservation period at room temperature can reach 1 year.This enzyme pH stability boundary broadens (1.5-7.0) simultaneously.
Below in conjunction with embodiment, the specific embodiment of the present invention is further described, does not therefore limit the present invention among described scope of embodiments.
Embodiment 1
1, materials and methods
1.1 plasmids used and bacterial strain as shown in table 1.
Table 1
1.2 main agents:
ThequickchangeTMkit (Invitrogen); Restriction enzyme (EcoRV, EcoRI, BamHI, SnaBI, NotI, BglII)/T4DNA ligase enzyme/dNTP is purchased from precious biotechnology (Dalian) company limited, TaqplusDNA polysaccharase is purchased from Shanghai biotechnology company limited, DNA purification kit is purchased from Hua Shun biotech firm, and penbritin/kalamycin is import packing; IPTG/X-gal matches hundred biotechnology company limiteds purchased from Beijing; Sodium phytate purchased from American Sigma company; DEAE-SepharoseFF, SephacraylS-200 are GE product; All the other reagent are domestic analytical pure.
1.3 substratum:
LB substratum: Tryptones 1%, yeast extract 0.5%, NaCl1%, pH7.0.
YPD substratum: 1% yeast extract, 2% peptone, 2% glucose.
MD is dull and stereotyped: 1.34%YNB, 4 × 10
-5% vitamin H, 2% glucose, 1.5% agar powder.
MM substratum: 1.34%YNB, 4 × 10
-5% vitamin H, 0.5% methyl alcohol, 1.5% agar powder.
BMGY:1% yeast extract, 2% peptone, 1.34%YNB, 4 × 10
-5% vitamin H, 1% glycerine.
BMMY: replace the glycerine in BMGY with 2% methyl alcohol.
Wort semisynthetic medium: Fructus Hordei Germinatus leaching extracting solution adds peptone and damping fluid etc.
The measuring method of 1.4 phytase activities
According to MikioShimizu(1992) method measure phytase activity.Test group 250 μ l enzyme liquid adds 1ml substrate solution, after 37 DEG C of water-bath 30min, adds 1.25ml stop buffer and stops enzyme reaction; Control group 250 μ l enzyme liquid adds 1.25ml stop buffer and stops enzyme reaction, after 37 DEG C of water-bath 30min, adds 1ml substrate solution.Test group and control group all add 2.5ml nitrite ion, and the phosphomolybdate of generation measures OD value at 700 nm.After the colour developing of different concns phosphate solution, production standard curve.A phytase activity unit is: under this condition determination, and per minute discharges the enzyme amount required for 1nmol inorganic phosphorus from sodium phytate solution.
1.5 phytase activities measure solution
Substrate solution: 2mmol/L sodium phytate, 100mmol/LTris-HClpH7.0,2mmol/LCaCl
2
Stop buffer: 5% trichoroacetic acid(TCA) (TCA)
Nitrite ion: 1.5% molybdenum acid ammonia/5.5% sulfuric acid and 2.7% ferrous sulfate mix in 4:1 ratio
Phosphate standard solution: by 0.6214gKH
2pO
4be dissolved in redistilled water, constant volume, in 500ml volumetric flask, is the phosphoric acid salt mother liquor of 9mmol/l.During production standard curve, with redistilled water by the phosphate solution of mother liquor by pipe dilution to be concentration be 22.5nmol/l, 45nmol/l, 90nmol/l, 180nmol/l.
2, the amino acid of sudden change is selected
By to Aspergillus ficuum (Aspergillusficuum) phytase (EC3.1.3.26) (Aspergillusficuum aminoacid sequence is as shown in seqIDNO2) non-conservative avtive spot (1-33; 361-441) carry out serial rite-directed mutagenesis, find that 27 amino acids are undergone mutation by software rebuild (software of Biosist and Columbia University) and Tm value had the greatest impact, but change not obvious to active centre space structure.
3, software carries out rite-directed mutagenesis screening
Utilize software to carry out 3D and rebuild phytase unfolding temperature, change as shown in table 2:
Table 2
| Mutational site | Tm before sudden change | Tm after sudden change | Enzyme activity |
| Gln-27-Gly | 72.5℃ | 83.3℃ | Substantially constant |
| Gln-27-Thr | 72.5℃ | 65.5℃ | Substantially constant |
| Gln-27-Leu | 72.5℃ | 73.5℃ | Substantially constant |
| Gln-27-Ile | 72.5℃ | 74℃ | Substantially constant |
| Gln-27-Phe | 72.5℃ | 74.5℃ | Substantially constant |
| Gln-27-Asn | 72.5℃ | 68.5℃ | Substantially constant |
| Gln-27-Val | 72.5℃ | 75.5℃ | Substantially constant |
| Gln-27-Ala | 72.5℃ | 76.5℃ | Substantially constant |
| Gln-27-Asp | 72.5℃ | 70.5℃ | Substantially constant |
| Gln-27-Glu | 72.5℃ | 66.5℃ | Substantially constant |
| Gln-27-Arg | 72.5℃ | 56.5℃ | Substantially constant |
Therefore choose Gln-27-Gly and carry out carrier construction expression checking.
4, carrier construction expresses checking Gln-27-Gly sudden change.
Design a pair Oligonucleolide primers containing the complete complementary of predetermined sudden change, mutating alkali yl is positioned at primer central authorities, and both sides are 10 ~ 15 correct bases; Primer length 25 ~ 45 bases, GC content is greater than 40%, and 3 ˊ ends should be bases G or C, Tm value is not less than 78 DEG C.Stratagene company gives mentioned above principle and have developed online mutant primer design software, with QuikChange
tMsiteDirectedMutagenesisKit with the use of.
The mutant plasmid that Catastrophic selection is correct, the electroporated plate screening carrying out recombination yeast.Electric shock cultivates the transformant on MD flat board, can be observed the oyster white bacterium colony that diameter is about 2mm after 3d.Cultivate after 5d, by all single bacterium colony difference dibblings to MM flat board and MD flat board, and establish positive control GS115Albumin (His
+mut
s) and negative control GS115 β-gal (His
+mut
+), cultivate 2d or longer time, to filter out on MD growth normal for 30 DEG C, on MM, poor growth or the bacterium colony that do not grow, be positive bacterium colony.By the positive transformant YPD liquid nutrient medium shake-flask culture 16-20h of plate screening, get 200 μ l cell suspensions, collecting cell, after washing 2 times with sterile purified water equal-volume, resuspended with 200 μ l deionized waters, getting 1 μ l cell suspension is in the PCR reaction system of 50 μ l in cumulative volume, and compares with the PCR primer of corresponding expression fragment.The be positive transformant of result of PCR carries out expression study.
Recombination yeast after detecting through plate screening and PCR is inoculated in the BMGY of 10ml, cultivates 16h (OD600=10 ~ 20), be inoculated in the BMGY of 200ml by the inoculum size of 5% for 36 DEG C.After cultivating 20h, room temperature leaves standstill 4h, and incline nutrient solution, adds the BMMY(2% methanol induction of 200ml).Intracellular expression recombination yeast 30 DEG C continues inducing culture 4d, harvested cell.The recombination yeast 30 DEG C of secreting, expressing continues inducing culture 48h, results nutrient solution supernatant.By enzyme activity determination, the recombination yeast choosing the highest enzyme alive is for further study.
Finally show: secretion wants that expressing enzyme activity is 243FTU/ml.
5, Gln-27-Gly expressing protein carries out Purification research and formulation
Purifying and after-processing technology flow process be as shown in Figure 1:
6, serial index verification and investigation
1) protein molecular weight detects
Find after the phytase electrophoresis detection of purified rear (ammonium sulfate precipitation, ion-exchange, molecular sieve purification).
(the phytase SDS-PAGE electrophoresis result of secreting, expressing is as shown in Figure 2) protein band is the protein band that a molecular weight is about 41.5KD, with starting strain to produce the performance molecular weight 42.04KD of native phytase suitable.
2) enzyme activity determination
By the fermented liquid after mutation expression with survey phytase activity (parallel survey is averaged for three times) under non-mutant strain equal conditions, compare:
Result: enzyme activity: 246U/ml, non-mutant enzyme vigor 232U/ml after sudden change.
Phytase after purifying is measured enzyme activity and shows that enzyme activity is 10650U/g, slightly high compared with bibliographical information.
Result illustrates: after the non-conservative district of enzyme carries out rite-directed mutagenesis, enzyme activity change is not obvious, consistent with zymetology basic principle.
3) optimum temperuture and thermostability
Optimum temperuture: phytase at different temperatures measures vigor according to enzyme activity determination condition, does temperature-enzyme curve alive.According to this curve, known temperature is on the impact of enzyme activity.This enzyme optimum temperuture is 45 DEG C.As shown in Figure 3.
Thermostability: certain phytase reaction solution is placed on respectively (30 DEG C-90 DEG C) insulation 30min in the water-bath of differing temps, then press enzyme activity determination method mensuration enzyme and live, result shows, enzyme is stable in this temperature range.As shown in Figure 4
Result shows: the thermostability that phytase of the present invention compares existing phytase improves greatly, still keeps most of enzyme activity in 90 degree of situations, can ignore the high-temperature factor impact in feed granulation process.
4) optimum pH and pH stability
Optimum pH: (pH1.5-8.5) measures enzyme activity by enzyme activity determination condition at various ph values, and make pH-enzymic activity curve (as shown in Figure 5) according to the changing conditions of enzyme work with pH, the optimal pH of this phytase activity known is 2.5 and 7.0.
Ph stability: certain enzyme liquid is placed on (pH1.5-8.0) in different pH buffer respectively, 37 DEG C are incubated 1 hour, then press enzyme activity determination method mensuration enzyme and live.Result shows, enzyme is stable in the pH value range of 1.5-7.5.As shown in Figure 6
Result shows: strengthen through improved enzyme enzyme pH stability alive.And there are two optimal pHs, be animal digestive tract (pH of stomach and enteron aisle).
7, zymotechnique test
The selection of 7.1 carbon sources
In basal fermentation medium, add 9 kinds of different sorts carbon sources of 2% respectively, carry out carbon source test, after access kind of daughter bacteria liquid, carry out fermentation culture under proper condition, the supernatant liquor then getting tunning measures enzyme activity and thalli growth amount.The results are shown in Table 3.
Table 3 carbon source is on the impact of thalli growth and phytase activity
As can be seen from Table 3, different carbon sources is very large on enzymic activity impact, and best with wort, sucrose takes second place, and considers from production, adopts wort to be that carbon source is fermented.
7.2 the selection of nitrogenous source
Adopt the nitrogenous source in 9 kinds of nitrogenous sources replacement basal fermentation medium, add 2% respectively, the supernatant liquor then getting tunning measures enzyme activity and thalli growth amount.The results are shown in Table 4.
Table 4 nitrogenous source is lived on the enzyme of thalli growth to be affected
This bacterial strain is poor in inorganic nitrogen-sourced middle growth as can be seen from Table 4, and the enzymatic production activity level of organic nitrogen source is apparently higher than inorganic nitrogen-sourced, and wherein soy peptone is that nitrogenous source enzyme activity is the highest, considers from production cost, selects bean powder.
The selection of 7.3 the suitableeest initial pH
By fermention medium NaOH and the different pH value of HCl solution furnishing, the impact that research pH value is lived on thalli growth and enzyme, the results are shown in Figure 7.All can grow in thalline pH value range in test, pH value is little on biomass impact, and the suitableeest thalli growth pH value is 7.5.
The selection of 7.4 optimum temperutures
Cultivate at being placed in 20 DEG C, 30 DEG C, 35 DEG C, 40 DEG C, 45 DEG C, 50 DEG C, 55 DEG C, 65 DEG C respectively after being inoculated by fermention medium, observe the impact that differing temps is lived on thalli growth and enzyme, the results are shown in Figure 8.40 DEG C is the suitableeest leavening temperature of phytase.
The selection of 7.5 liquid amounts
In the triangular flask of 250ml, fermention medium is dressed up 5 respectively, 10,20,30,40,50, the different loading amount of 60ml, by 5% inoculum size inoculation, the impact that research aeration condition is lived on Fungal biodiversity and enzyme, result (Fig. 9) shows, air flow is larger on biomass impact, air flow is larger, and thalli growth is better.Consider, select the bottled amount of 20ml.
The selection of 7.6 inoculum sizes
Inoculum size is on producing the impact of enzyme as shown in Figure 10.Best with the inoculum size of 5%.
7.7 orthogonal experimental design
Select L
9(3
4) show medium component bean powder, wort, NaCl tri-factor design orthogonal tests.According to orthogonal test table result, the 9th combination product enzyme is best, and enzyme is lived and reached 243U/ml, and the Optimal compositions of fermentation medium of this bacterial strain is defined as bean powder 2%, wort 2%, NaCl0.5%.
Table 5 orthogonal experiments
| Test number | Bean powder | Wort | NaCl | Enzyme work/U/ml |
| 1 | 1 | 0.5 | 0.3 | 95 |
| 2 | 1 | 1.5 | 0.5 | 125 |
| 3 | 1 | 2 | 0.7 | 148 |
| 4 | 1.5 | 0.5 | 0.5 | 98 |
| 5 | 1.5 | 1.5 | 0.7 | 127 |
| 6 | 1.5 | 2 | 0.3 | 168 |
| 7 | 2 | 0.5 | 0.7 | 185 |
| 8 | 2 | 1.5 | 0.3 | 212 |
| 9 | 2 | 2 | 0.5 | 243 |
7.8 fermentation time
Fermentation results shows: the abduction delivering research carrying out phytase in wort semisynthetic medium, it produces enzyme curve as shown in Figure 11.As seen from the figure, methanol induction is cultivated in 60h, and enzyme is lived and raised very soon, and phytase activity reaches 215U/ml.Afterwards in the situation that slowly rises, 102h reaches climax, can gather in the crops phytase at 60h, shortens enzyme harvest time, significant to reduction production cost.Consider, fermentation time adopts 60h.
Finally determine fermentative medium formula and condition: bean powder 2%, wort 2%, NaCl0.5%, inductor addition is 0.4%, and pH value is 7.5, fermentation time 60 hours.
8, purifying process and granulation process research
Purifying adopts to be done to calculate rear spraying dry, and spray condition is: inlet temperature is 90 DEG C, and air outlet temperature is 60 DEG C, and blow pressure is 0.2Mpa, collects and obtains phytase powder.
Phytase powder mensuration enzyme adds granulation protective material according to the proportioning of 5100FTU/g, enters fluidized bed prilling, finally obtain phytase particle after living.
Pack after the assay was approved.
9, zymetology stability experiment
Shown by research, when temperature is below 50 DEG C, phytase activity is more stable; Place for a long time under undressed enzyme specimen chamber temperature, enzyme rate of loss alive is larger; By our technique, the enzyme of phytase sample before and after granulation processing rate of loss < 5% alive.Enzyme stability after processing increases, 90 DEG C, and after 30 minutes, the enzyme rate of recovery alive also can reach more than 95%.Enzyme sample after processing is preserved at normal temperatures, and its loss of activity is lower than undressed sample by more than 90%.Granule preservation period at room temperature can reach 1 year.
10, mixture homogeneity experiment
Interpolation must ensure the good fluidity of additive, and guarantee mixes, and ensures the homogeneity of the finished product.Product after granulation carries out mixture homogeneity test, checking mobility.
Standard content (theoretical content) in setting feed after phytase interpolation, adds the phytase of identical amount in different samples, investigates the relative content of nelatively standard element in each sample, calculates after mapping.As shown in figure 12
The final variation coefficient is 8%, with this parameter of foreign products suitable (8%-10%).
Sum up: the present invention is high temperature resistant phytase by after Fahrenheit yeast expression through being processed as granule type phytase later, there is good workability.
11, being compared as follows shown in table of the present invention's high temperature resistant phytase indicators of overall performance and domestic and international similar technique.
Table 6 phytase of the present invention compares with domestic and international main products
Therefore, compare from the enzyme aspect such as release rate and thermostability, pH stability, mixture homogeneity alive, product of the present invention exceedes domestic and international product comprehensively, and overall product quality occupies international most advanced level.
12, the price advantage of phytase of the present invention
Due to cultured product sluggish market, feeds product price declines by a big margin, feedstuff raw material price is then stabilized in high price always, make Feedstuff Enterprises benefit very low, lowest manufactured feed per ton is 30 yuan of profits (laying hen concentrated fodder) only, the highest also only 200 yuan of profits (concentrated feed for pig), by the price of phytase on former market, add the production cost that phytase improves feed, for reducing production cost, China's Feedstuff Enterprises does not mostly use phytase, and adopts the method for adding Non-phytate phosphorus.
Current fodder additives is along with the reduction of phytase price, and feed adds the cost of phytase increase not too significantly (table 7).
Table 7 uses the cost of phytase and secondary calcium phosphate to drop into and contrasts in egg feedstuff
To ferment with conventional microbiological or compared with feeding phytase preparation that other genetically engineered is produced, phytase price of the present invention is low, and performance high (high temperature resistant), has good cost performance.Simultaneously due to reasons such as this phytase are high temperature resistant, not only adapt to high temperature when fodder industry is granulated, and its interpolation equivalent at feed can be made to reduce, be about the 60-80% of general production by biological phytase.Reach the requirement of fodder industry to feeding phytase preparation.
Test example 1
Select 5 age in days broiler chicken 400, be divided into 4 process at random, each process 4 repetition, in research feed, add the high temperature resistant phytase of the present invention to the impact of chicken growth performance.
Test process is as follows:
Control group: basal diet;
Test 1. group: basal diet+0.01% phytase;
Test 2. group: basal diet+0.02% cellulase preparation; (cellulase preparation is purchased from Dan Niyue bio tech ltd, Shanghai)
Test 3. group: basal diet+0.02% phytase.
Result shows, 3. day weight gain and feed efficiency test are organized best, and be secondly test 1. group, 2. test organizes the 3rd, control group minimum (p > 0.05); In chicken manure urine nitrogen content test 1., 2., 3. group compare control group and have dropped 11.08%, 5.01%, 14.07% respectively, 1., 2., 3. phosphorus content test is organized comparatively control group and be have dropped 1.04%, 30.01% and 35.60% respectively; In house, 1., 2., 3. ammonia content test is organized comparatively control group and be have dropped 20.01%, 10.07% and 29.08% respectively.
Test example 2
Select the hybridization piglet 96 of 30 ages in days, be divided into 4 treatment group at random, each process 4 repetition, carries out two nitrogen balance tests to it, and the high temperature resistant phytase of the present invention adding 400IU/kg in research diet is on the impact of piglet growth performance and gal4 amino acid utilization ratio.
Test process is as follows:
Control group: basal diet;
Test 1. group: basal diet+400IU/kg phytase;
Test 2. group: basal diet-5% Methionin;
Test 3. group: basal diet-5% Methionin+400IU/kg phytase.
Result shows, after reducing Different Dietary Protein Levels (Methionin), compares control group, and 2. test is organized day weight gain, food consumption and efficiency of feed utilization and to be declined respectively 16.0%, 7.2% and 11.0% (P<0.01); Compare with not enzyme-added two groups, the average effect of adding phytase is: day weight gain, food consumption and efficiency of feed utilization improve 12.0%, 7.0% and 3.8% (P<0.05) respectively, and in ight soil, ammonia content declines 36.0%.
Claims (3)
1. high temperature resistant phytase, is characterized in that: the aminoacid sequence of described high temperature resistant phytase is as shown in SEQIDNO1.
2. high temperature resistant phytase according to claim 1 is used as the purposes of fodder additives.
3. the feed containing phytase according to claim 1.
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