Method for producing a phytase variant with improved thermal stability, and a phytase variant and the use thereof

Abstract

The present invention relates to the field of genetic engineering, in particular, the present invention relates to a method for producing a phytase variant with an improved thermal stability, and a phytase variant and the use thereof. The phytase variant contains at least one proline modification, compared to the phytase from Escherichia coli and other mutants thereof. The phytase variants with the modification have preferably improved properties, such as the thermal stability, optimal reaction temperature, pH property, specific activity, protease resistance and performance in animal feeds.

Claims

1. A method of preparing phytase variants having improved thermostability, including the step of introducing prolines at the sites of S80, S151, T161, N176, S187 and A380 of the phytase with the amino acid sequence set forth in SEQ ID NO:2.

2. Phytase variants having improved thermostability with the following characteristics: having prolines introduced at the sites of S80, S151, T161, N176, S187 and A380 of the amino acid sequence set forth in SEQ ID NO:2.

3. A polynucleotide encoding the phytase variants having improved thermostability of claim 2.

4. A nucleotide construct comprising the polynucleotide of claim 3, and optionally a regulating sequence connected to said polynucleotide to guide expression in an expression host.

5. A recombinant expression vector comprising the nucleotide construct of claim 4.

6. A recombinant host cell comprising the recombinant expression vector of claim 5.

7. The recombinant host cell according to claim 6, wherein said host cell is a bacteria cell or a fungal cell.

8. A method of producing a phytase variant having improved thermostability, comprising the steps of: (a) cultivating the recombinant host cell of claim 6 to produce a supernatant containing said phytase variant; and (b) recovering said phytase.

9. A method of preparing an animal feed, comprising adding one of the phytase variants of claim 2 as a feed additive.

10. A method of preparing an animal feed, comprising using recombinant host cell of claim 6 to manufacture a feed additive.

11. An animal feed, comprising a phytase variant of claim 2 as a feed additive.

Description

BRIEF DESCRIPTIONS OF THE DRAWINGS

(1) FIG. 1 shows SDS-PAGE electrophoresis of the different purified deglycosylated phytase variants responding to the two electrophoresis strips.

(2) FIG. 2 shows the thermostability of the phytase variants at the temperature of 80 C.

EMBODIMENT

Example 1, Designing the Introduction of the Proline to Obtain the Phytase Variants, Constructing the Expression Vector of the Phytase Variant Gene and Expressing in Pichia pastoris

(3) (1) Rational Design the Phytase

(4) A three-dimensional model is researched based on the three-dimensional structure of phytase APPA of E. coli recorded in Protein Databank (http://www.rcsb.org/pdb/). B-factor of the protein is an important indicator of the stability of it specific amino acid, which the higher B-factor value of the amino acid, and the lower structure stability of said amino acid or the section containing it. Finally, the sites of the phytase APPA with higher flexibility are determined as S80, S151, T161, N176, S187 and A380 by calculating and ranking the B-Factor value of each amino acid, optimizing based on the research on the amino acid and the section containing it with the higher B-Factor value in connection with the assay results. Then, the prolines are introduced at these sites so as to increase the rigidity of said section and further improve the phytase's thermostability.

(5) (2) Expressing the Phytase Variants:

(6) Site-directed mutagenesis is performed with DpnI digestion and homologous recombination, using only two mutation primers, and constructing the mutant molecules taking 4 hours, which is quicker and more convenient than Overlap PCR. The mutant genes being confirmed by sequencing are correctly inserted into the downstream of the signal peptide of pPIC9 (Invitrogen, San Diego, Calif.) at the EcoRI and NotI sites to construct the correct ORF, and the yeast expression vectore having been constructed is transformed into JM109 cells. 8 g of the extracted plasmid DNA is linerized with BglII, followed by being transformed into Pichia GS115 by electroporation. The transformed cells are plated in Histidine deficient RDB agar medium and cultivated at 30 C. for 2 to 3 days to obtain the transformants for the further expression assay.

(7) (3) Screening the Transformants with High Phytase Activity

(8) The transformants on the RDB plate are picked, numbered and transferred to MM plate and MD plate respectively, placing in the incubated at 30 C. for 1-2 days until growing the colony. The transformants on the MD plate are picked in the numbering sequence, and inoculated into 3 mL of BMGY medium in the centrifuge tube, placing in the shaker with the rate of 260 rpm at 30 C. for 48 hours, followed by spinning down (3000 g, 15 min) to remove the supernatant and being suspended in 1 mL of 0.5% methanol medium (BMMY) to induce the expression of the phytase gene at 30 C. and 260 rpm. After 48 hours, the supernatant is obtained by centrifugal (3000 g, 5 min), for measuring the phytase activity to screen the transformant with the highest activity.

Example 2, Preparing the Phytase Variants and Measuring their Activity

(9) (1) Concentrate

(10) 3 L of supernatant is collected by centrifugal the BMMY medium after induced the expression of the phytase at 4 C. at the rate of 10,000 rpm for 10 mm, concentrated to 250 mL, and further concentrated in a Filtron ultrafiltration unit with 5 kDa cutoff filters to 50 mL;

(11) (2) Ammonium Sulfate Fractionation Precipitation

(12) The concentrated crude phytase is precipitated with the concentration of 40-75% ammonium sulfate, and the very low phytase activity is remained in the supernatant at 75% saturation. The precipitant collected by centrifugal is dissolved in 5 mL of Tris-HCl buffer solution with pH 8.0, and dialyzed, followed by concentrating with PEG 8000.

(13) (3) Anion Exchange Chromatography

(14) The dialyzed phytase is purified with HiTrap Q Sepharose XL, anion exchange chromatography column, equilibrated with 20 mmol/L Tris-HCl solution (pH 8.0), eluted with 20 mmol/L Tris-HCl solution (pH 8.0) containing 1 mol/L NaCl, and loaded with 2 mL of phytase, followed by linear gradient eluting at 0-100% at rate of 4 mL/min, and fractionally collecting the peak to obtain the interest protein with a molecular weight of about 48-53 kDa and a purity more than 95% having been purified by SDS page, as shown in FIG. 1. The protein concentration of the purified phytase is measured with the Bradford method.

(15) (4) Mearing the Phytase Activity

(16) The purified phytase is diluted with 0.25 mol/L NaAc-HAc containing 0.05% BSA, and 0.05% Triton X-100 (pH 4.5), and 50 L of diluted phytase solution is added to 950 L substrate of 1.5 mmol/L sodium hyaluronate (0.25 mol/L sodium phytase (pH 4.5) in 1.5 mmol/L sodium acetate buffer (Sigma Cat. No. P0109)), kept at 37 C. for 15 mm, followed by adding 1 mL of 10% (m/v) TCA to stop the reaction, and 2 mL of color reagent. Then, OD is measure at 700 nm to calculate the phytase activity. 1 unit of phytase activity is determined to be the enzyme amount releasing 1 mol of phosphate for 1 minute. The absolute value of the measured phytase activity may be calculated based on the standard curve of inorganic phosphate in dilution.

Example 3, Optimal Temperature and Thermostability of the Phytase Variants

(17) (1) Temperature Characteristic

(18) The activity of the interest dilution of the phytase variants purified in the Example 2 is measured in 0.25 mol/L sodium acetate buffer (pH 5.5) from 30 C. to 90 C., followed by calculating the specific activity as the average of three measurements repeated at each temperature, so as to determine the optimal temperature of said phytase, as list in the table 1 showing the mean of the specific activity at each temperature.

(19) TABLE-US-00001 TABLE 1 Reacting temperature ( 0-100 meaned in relative activity and Phytase 100 stand for the optimum temperature of the variant) Variants 30 C. 40 C. 50 C. 60 C. 70 C. 75 C. 80 C. 85 C. 90 C. Appa-WT 20 41 68 100 39 18 10 2 0 Appa-A 20.7 34.5 53.1 76.5 100 88.5 59.6 4.9 0 Appa-B 21.8 42.3 63.7 88 95.1 100 66.7 7.6 0.9 Appa-C 14.5 27.4 50 71.1 91.0 98.7 100 80.2 45.3

(20) Wherein, specific activity is remarked as 0 to 100, and the temperature with 100 of the specific activity is the optimal temperature.

(21) (2) Determining the Thermostability

(22) 2 mL of the interest dilution of the phytase variants purified in the Example 2 is kept for 30 mm at 60 C., 65 C., 70 C., 75 C., 80 C., 85 C., respectively, the phytase sample is taken and placed on the ice when 2 min, 4 min, 10 min, 5 min, 20 min, 30 min to measure the phytase activity of the phytase diluted for 10 times at 37 C. and the optimal pH using the untreated crude phytase as control, so as to determine the optimal temperature of said phytase, as list in the table 1 showing the mean of the specific activity at each temperature, wherein the specific activity is the average of three measurements repeated at each temperature. The thermostability data of the phytase variants with the improved thermostability is shown as FIG. 2.

(23) As shown in FIG. 2, the thermostability of several phytase variants is obviously improved, remaining 40% of activity being left at 80 C. for 10 mm, and remaining 25.8% of activity being left at 80 C. for 30 min. In comparison, the specific activity of the wild phytase APPA is high only at 65 C., obviously decreases with the increase of the temperature, almost losing at 80 C. for 2 min. The thermostability of the several phytase variants at 85 C. indicates the resistance to the short high temperature treatment at 80 C. during feed pelleting. The phytase variants show the great potential of industrial application due to their improved thermostability at 80 C. and 85 C.

Example 4, the Optimal pH and pH Stability of the Phytase Variants

(24) The desalted phytase fermentation supernatant is performed the enzymatic reactions in pH 1.0 to 10.0 using 0.1 mol/L of Glycine-HCl buffer (pH1.03.0), acetic acid-sodium acetate buffer (pH3.55.5), acetic acid buffer (pH6.06.5), Tris-Hcl buffer (pH7.08.5) and glycine-sodium hydroxide buffer (pH9.010.0) at 37 C. to determine the optimal pH.

(25) The phytase is treated in the different pH buffers at 37 C. for 1 hour, followed by measuring the specific activity in 0.1 mol/L of sodium acetate buffer (pH4.5) at 37 C. in order to research its pH stability.

(26) The optimal pH and pH stability of the phytase variants don't obviously change, and the optimal pH is 4.5, except that the stability and activity of the only part of the phytase variants have been improved under the acidic condition.

Example 5, Measuring the Specific Activity of the Phytase Variants

(27) The protein concentration of the purified phytase variants is determined with Bradford kit for the order of their specific activity. The unit of the specific activity of the recumbent phytase is determined with the molybdenum blue spectrophotometry method at 37 C. and pH 5.5. 1 unit of phytase activity was determined to be the enzyme amount for releasing 1 mol of inorganic phosphate in 4 mM of sodium phytate (pH5.5) at 37 C. for 1 minute, and the specific activity of the phytase variant may be calculated.

(28) The specific activity of the phytase variants of the present invention ranges from 2500 to 3100 U/mg at 37 C. and pH 5.5, being little different from that of the wild phytase as control, and therefore not being affected with the improvement on their thermostability.

Example 6, Effect of Proteases on the Enzyme Activity of the Phytase Variants

(29) 0.1 mg/mL of the purified phytase variants was incubated with 0.01 mg/ml of pepsin and trypsin in equal volume at 37 C. for the order of determining effect of proteases on the enzyme activity of the phytase variants. And then, sample incubated was collected respectively at 5 min, 10 min, 20 min, 30 min, 60 mm, 90 mm and 120 min to measure the enzyme activity at 37 C. and pH5.5. The specific activity is calculated, as list in the table 2, with untreated phytase as the control of 100%. As list in the table 2, the phytase variants have the greatly improvement on the trypsin resistance, and little change on the pepsin resistance.

(30) TABLE-US-00002 TABLE 2 Phytase Pepsin ( Treatment time (min)) Trypsin ( Treatment time (min)) variants 5 10 30 60 120 5 10 30 60 120 Appa-WT 105.6 111.2 115 116.8 117.3 59.4 45.7 43.2 39.6 35.1 Appa-A 103.7 108.4 113.9 115.7 116.9 80.9 69.6 58.3 52.3 48.4 Appa-B 102.2 107.2 113.3 115.1 116.2 89.3 83.4 69.9 65.3 59.2 Appa-C 101.3 106.9 110.6 113.4 115.6 97.9 90.3 84.4 76.3 69.4

Example 7, Performance in Animal Feeding and the Simulated Assay In Vitro

(31) Further, it is reached the stability and the hydrolytic ability for the phytate of the purified phytase variant in the artificial simulation gastric juices, in order to compare the degradation ability to the phosphorus in the beat meal of the phytase variants with wild phytase as control in the artificial simulation gastric juices.

(32) (1) The Stability of the Phytase Variants in the Artificial Simulation Gastric Juices

(33) The remained phytase activity is measured respectively at the optimal pH by adding the same units of phytase variants to the artificial simulation gastric juices until the concentration of the phytase is 1 U/mL, followed by keeping at 37 C. for 20 mm, showing the control wild phyase having the 30% of relative activity remained, and the phytase variants of the present invention having the remaining relative activity more than 70% after one hour's treatment in the artificial simulation gastric juices. Therefore, the phytase variants with the improve thermostability have the more stable structure so as to be capable of resisting to the strong acid and the hydrolysis of the high concentration of the protease.

(34) (2) The Hydrolytic Ability for the Phytate of the Purified Phytase Variant in the Artificial Simulation Gastric Juices

(35) 1 g of the bean meal is dissolved in 9 mL of the artificial simulation gastric juices, followed by keep shaking for 1 hour at 37, respectively adding 1 mL of phytase variants and control diluted in the artificial simulation gastric juices, under the ice condition and reacting for 1 hour at 37 C. The released inorganic phosphorus is measured to evaluate the hydrolysis of the phytase, and a test of pH gradient cumulative effect is performed in order to further simulate gastro enteric environment, showing that the amount of released the inorganic phosphorus by the several thermal stable phytase variants is increased by 50-300%, being more than that by the control phytase. Therefore, the phytase variants of the present invention with the improved thermostability may more efficiently degrade phytate in the artificial simulation gastric juices, have a prominent application prospect.