Phytase-pullulan hydrolase fusion protein Y-L4-T and application thereof

Abstract

The invention discloses a phytase-pullulan hydrolase fusion protein Y-L4-T and application thereof, and belongs to the technical field of enzyme engineering. The amino acid sequence of the phytase-pullulan hydrolase fusion protein Y-L4-T disclosed by the invention is as shown in SEQ ID NO. 46. The phytase-pullulan hydrolase fusion protein Y-L4-T is constructed, the phytase activity of the fusion protein is basically consistent with that of phytase YiAPPA, and the amylase activity of the fusion protein is remarkably higher than that of pullulan hydrolase TK-PUL. The wheat bread treated by the fusion protein is obviously increased in specific volume, obviously reduced in hardness and obviously improved in elasticity; the increase of the hardness and the reduction of the elasticity of the wheat bread in the storage period can be effectively relieved. The fusion protein Y-L4-T can effectively improve the quality of bread and prolong the shelf life of the bread, and has a good application prospect in the field of baking.

Description

Technical Field

[0001] The present invention relates to the technical field of enzyme engineering, in particular to a phytase-pullulan hydrolase fusion protein Y-L4-T and applications thereof. Background Art

[0002] Phytase, also known as phytase, degrades phytic acid to produce inorganic phosphorus and inositol phosphates. Adding phytase to plant-based food ingredients can help eliminate the anti-nutritional effects of phytic acid and improve the absorption and utilization of phosphorus in foods. Therefore, phytase can be used as a new food additive in food processing. For example, adding phytase to breadmaking can reduce phytic acid content and increase mineral and protein content. Phytase treatment also improves the aroma, mouthfeel, and chewiness of bread. Starch hydrolases such as α-amylase and pullulanase play an important role in breadmaking as natural additives. α-amylase and pullulanase hydrolyze α-1,4-glycosidic bonds and α-1,6-glycosidic bonds in starch, respectively. The synergistic action of these two enzymes can effectively convert starch into a larger amount of smaller reducing sugars. During breadmaking, the increased production of reducing sugars provides more substrate for yeast, affecting the fermentation rate and fermentation products, thereby improving the volume, firmness, physical, and sensory properties of bread. Furthermore, the addition of starch hydrolases to breadmaking can help extend its shelf life. Studies have shown that the synergistic effect of adding phytase and amylolytic enzyme simultaneously during bread making can help optimize the fermentation process of bread and improve the texture and taste of bread.

[0003] Protein fusion technology involves fusing two or more proteins via a linker peptide to create a desired multifunctional fusion protein. Producing fusion proteins through protein fusion technology can significantly reduce the production cost of enzyme preparations and improve the enzymatic properties of the fusion protein. Therefore, in the bread-making process, the use of phytase and amylolytic enzyme fusion proteins as substitutes for phytase and amylolytic enzymes is expected to reduce enzyme production costs, improve the enzymatic properties of phytase or amylolytic enzymes, and increase the efficiency of the enzyme preparation.

[0004] The phytase YiAPPA, derived from Yersinia intermedia, is the most active phytase known to date, with an activity 40 times that of the most widely used phytase, Aspergillus niger PhyA. The thermoacidophilic type III pullulan hydrolase TK-PUL, derived from Thermococcus kodakarensis, combines the functions of both an α-amylase and a pullulanase, effectively hydrolyzing both α-1,4- and α-1,6-glycosidic bonds in starch to form low-molecular-weight reducing sugars. The enzymatic properties of YiAPPA and TK-PUL suggest their potential for application in breadmaking. The construction of a fusion protein of YiAPPA and TK-PUL using protein fusion technology is expected to improve the enzymatic properties of the enzyme preparation, maximize the synergistic effects between the enzymes, increase the efficiency of the enzymes, and enable the application of the fusion protein in breadmaking. Summary of the Invention

[0005] The purpose of the present invention is to provide a phytase-pullulan hydrolase fusion protein Y-L4-T and its application to solve the problems existing in the above-mentioned prior art. The present invention constructs a phytase-pullulan hydrolase fusion protein Y-L4-T, the phytase activity of the fusion protein is basically the same as that of phytase YiAPPA, and the amylase activity is significantly higher than that of pullulan hydrolase TK-PUL. The specific volume of wheat bread treated with the fusion protein is significantly increased, the hardness is significantly reduced, and the elasticity is significantly improved; and it can effectively alleviate the increase in hardness and the decrease in elasticity of wheat bread during storage. The fusion protein Y-L4-T of the present invention can effectively improve the quality of bread and extend the shelf life of bread, and has good application prospects in the field of baking.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a phytase-pullulan hydrolase fusion protein Y-L4-T, the amino acid sequence of which is shown in SEQ ID NO.46.

[0008] The present invention also provides a gene encoding the phytase-pullulan hydrolase fusion protein Y-L4-T, the nucleotide sequence of which is shown in SEQ ID NO.45.

[0009] The present invention also provides a recombinant vector containing the above gene.

[0010] The present invention also provides a recombinant microorganism comprising the recombinant vector.

[0011] The present invention also provides application of the phytase-pullulan hydrolase fusion protein Y-L4-T in degrading phytic acid and starch.

[0012] The present invention also provides application of the phytase-pullulan hydrolase fusion protein Y-L4-T in producing pasta.

[0013] Optionally, the pasta comprises bread.

[0014] The present invention also provides a method for improving the specific volume and / or texture characteristics of bread, comprising the step of adding the phytase-pullulan hydrolase fusion protein Y-L4-T during the bread making process.

[0015] The present invention also provides the use of the above gene, the above recombinant vector or the above recombinant microorganism in producing the phytase-pullulan hydrolase fusion protein Y-L4-T.

[0016] The present invention discloses the following technical effects:

[0017] The present invention uses protein fusion technology to construct a fusion protein of phytase YiAPPA and pullulan hydrolase TK-PUL. Phytase YiAPPA and pullulan hydrolase TK-PUL were connected in different fusion orders via different linker peptides to construct eight fusion proteins. The phytase and amylase activities of each fusion protein were then compared to screen for a fusion protein with enhanced enzymatic activity, Y-L4-T. The phytase activity of this fusion protein, Y-L4-T, was 1802.59 U / mg, essentially the same as that of phytase YiAPPA; and the amylase activity was 60.94 U / mg, a 1.42-fold increase compared to pullulan hydrolase TK-PUL. Compared to a mixture of phytase YiAPPA and pullulan hydrolase TK-PUL, wheat bread treated with the fusion protein Y-L4-T exhibited a significantly increased specific volume, decreased hardness, and improved elasticity. Furthermore, the fusion protein Y-L4-T effectively mitigated the increased hardness and decreased elasticity of wheat bread during storage. The fusion protein Y-L4-T constructed by this invention can effectively improve bread quality and extend its shelf life, and has promising application prospects in the baking industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0019] Figure 1 Schematic diagram of the construction and screening process of phytase-pullulan hydrolase fusion protein;

[0020] Figure 2 This is the SDS-PAGE detection result of phytase YiAPPA, pullulan hydrolase TK-PUL and phytase-pullulan hydrolase fusion protein Y-L4-T. DETAILED DESCRIPTION

[0021] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0022] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0023] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0024] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0025] The materials and reagents involved in the following examples are as follows:

[0026] Escherichia coli HST08, Bacillus subtilis RIK1285, and Bacillus subtilis expression vector pBE-S were purchased from Bio-Rad Biotechnology (Beijing) Co., Ltd.

[0027] KOD-Plus-neo DNA polymerase was purchased from Toyobo, DNA restriction endonucleases were purchased from Fermentase, PCR product purification kits and plasmid extraction kit EZNA were purchased from Omega Bio-tek, and ClonExpress The Ultra One Step Cloning Kit was purchased from Nanjing Novozymes Biotechnology Co., Ltd., Chelating Sepharose™ Fast Flow was purchased from GE Healthcare, USA, the Bradford protein concentration assay kit was purchased from Shanghai Sangon Biotechnology Co., Ltd., and high-activity yeast was purchased from Angel Yeast Co., Ltd. Gene synthesis was performed by Shanghai Boyi Biotechnology Co., Ltd., and polymerase chain reaction primer synthesis and sequencing were performed by Shanghai Sangon Biotechnology Co., Ltd. All other chemical reagents were domestically produced or imported of analytical grade.

[0028] LB medium (g / L): tryptone 10, yeast extract 5, NaCl 10, pH 7.0. The screening medium used LB medium containing 100 μg / mL ampicillin and 10 μg / mL kanamycin.

[0029] The molecular cloning and protein detection techniques used in the present invention are conventional techniques in the art. Techniques not described in detail in the following examples were performed according to the relevant sections of the following experimental manual: Green MR, Sambrook J. Molecular cloning: a laboratory manual [M]. New York: Cold Spring Harbor Laboratory Press, 2012.

[0030] Example 1 Construction of phytase-pullulan hydrolase fusion protein

[0031] 1. Gene synthesis

[0032] The gene sequence of the phytase YiAPPA from Yersinia intermedia is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2; the gene sequence of the pullulan hydrolase TK-PUL from Thermococcus kodakarensis is shown in SEQ ID NO.3, and the amino acid sequence is shown in SEQ ID NO.4; the gene sequence of the connecting peptide L1 is shown in SEQ ID NO.5, and the amino acid sequence is shown in SEQ ID NO.6; the gene sequence of the connecting peptide L2 is shown in SEQ ID NO.7, and the amino acid sequence is shown in SEQ ID NO.8; the gene sequence of the connecting peptide L3 is shown in SEQ ID NO.9, and the amino acid sequence is shown in SEQ ID NO.10; the gene sequence of the connecting peptide L4 is shown in SEQ ID NO.11, and the amino acid sequence is shown in SEQ ID NO.12. The above genes were handed over to Shanghai Boyi Biotechnology Co., Ltd. for full gene synthesis.

[0033] SEQ ID NO.1:

[0034]

[0035] SEQ ID NO.2:

[0036] NSYAISAAPVAIQPTGYTLERVVILSRHGVRSPTKQTQLMNDVTPDTWPQWPVAAGYLTPRGAQLVTLMGGFYGDYFRSQGLLAAGCPTDAVIYAQADVDQRTRLTGQAFLDGIAPGCGLKVHYQADLKKVDPLFHPVDAGVCKLDSTQTHKAVEERLGGPLSELSKRYAKPFAQMGEILNFAASPYCKSLQQQGKTCDFANFAANKITVNKPGTKVSLSGPLALSSTLGEIFLLQNSQAMPDVAWHRLTGEDNWISLLSLHNAQFDLMAKTPYIARHKGTPLLQQIETALVLQRDAQGQTLPLSPQTKILFLGGHDTNIANIAGMLGANWQLPQQPDNTPPGGGLVFELWQNPDNHQRYVAVKMFYQTMGQLRNAEKLDLKNNPAGRVPVAIDGCENSGDDKLCQLDTFQKKVAQAIEPACHI。

[0037] SEQ ID NO.3:

[0038]

[0039] SEQ ID NO.4:

[0040] SGCISESNENQTATASTVPPTSVTPSQSSTPTTSTSTYGPSERTELKLPSVNYTPIYVGIEKGCPSGRVPVKFTYNPGNKTVKSVSLRGSFNNWGEWPMELKNGTWETTVCLRPGRYEYKYFINGQWVKDMSDDGTGRPYDPDADAYAPDGYGGKNAVRVVEGREAFYVEFDPRDPAYLSIADKRTVVRFEAKRDTVESAVLVTDHGNYTMKLQVWWDFGETWRAEMPVEPADYYILVTSSDGGKFAVLNTSESPFFHFDGVEGFPQLEWVSNGITYQIFPDRFNNGNKSNDALALDHDELILNQVNPGQPILSNWSDPITPLHCCHQYFGGDIKGITEKLDYLQSLGVTIIYINPIFLSGSAHGYDTYDYYRLDPKFGTEDELREFLDEAHRRGMRVIFDFVPNHCGIGNPAFLDVWEKGNESPYWDWFFVKKWPFKLGDGSAYVGWWGFGSLPKLNTANQEVREYLIGAALHWIEFGFDGIRVDVPNEVLDPGTFFPELRKAVKEKKPDAYLVGEIWTLSPEWVKGDRFDSLMNYALGRDILLNYAKGLLSGESAMKMMGRYYASYGENVVAMGFNLVDSHDTSRVLTDLGGGKLGDTPSNESIQRLKLLSTLLYALPGTPVTFQGDERGLLGDKGHYDEQRYPIQWDTVNEDVLNHYRALAELRKRVPALRSSAMRFYTAKGGVMAFFRGHHDEVLVVANSWKKPALLELPEGEWKVIWPEDFSPELLRGTVEVPAIGIIILERG。

[0041] SEQ ID NO.5:GAGGGTAAGTCTTCGGGCTCAGGCTCAGAGTCAAAATCCACC。

[0042] SEQ ID NO.6:EGKSSGSGSESKST。

[0043] SEQ ID NO. 7: GGTTCCGCTGGATCAGCTGCTGGATCAGGAGAATTC.

[0044] SEQ ID NO. 8: GSAGSAAGSGEF.

[0045] SEQ ID NO.9:

[0046] GCTGAAGCTGCTGCTAAGGAAGCTGCTGCTAAGGAAGCTGCTGCTAAGGCT.

[0047] SEQ ID NO. 10: AEAAAKEAAAKEAAAKA.

[0048] SEQ ID NO.11:

[0049] GAAGCTGCTGCTAAGGAAGCTGCTGCTAAGGGTGGTGGTGGTTCCGGTGGTGGTGGTTCCGGTGGTGGTGGTTCC.

[0050] SEQ ID NO. 12: EAAAKEAAAKGGGGSGGGGSGGGGS.

[0051] 2. Construction of recombinant expression plasmids for phytase YiAPPA and pullulan hydrolase TK-PUL

[0052] PCR primers YF and YR were designed based on the gene sequence of the phytase YiAPPA (Table 1). PCR amplification was performed using the synthetic YiAPPA gene as a template and primers YF and YR. The PCR amplification system consisted of 5 μL of 10× buffer I, 5 μL of dNTPs, 5 μL of MgSO₄, 2 μL of each upstream and downstream primer, 1 μL of template, 2 μL of KOD-Plus-neo DNA polymerase, and 28 μL of ddH₂O. PCR amplification conditions were: 98°C for 5 min; 30 cycles of 98°C for 20 s, 60°C for 40 s, and 74°C for 2 min; and 74°C for 10 min. The amplified product was double-digested with Nde I and Xba I and ligated into the pBE-S vector, which had been treated with the same enzymes, to construct the recombinant plasmid pBE-S-yiappa.

[0053] PCR primers TF and TR were designed based on the pullulan hydrolase TK-PUL gene sequence (Table 1). PCR amplification was performed using the synthetic TK-PUL gene as a template and primers TF and TR. The PCR amplification system was the same as above. PCR amplification conditions were: 98°C for 5 min; 30 cycles of 98°C for 20 s, 60°C for 40 s, and 74°C for 2 min 10 s; and 74°C for 10 min. The amplified product was double-digested with Nde I and Xba I and ligated into the pBE-S vector, which had been treated with the same double-digestion enzymes, to construct the recombinant plasmid pBE-S-tkpul.

[0054] Table 1 Primers used to construct recombinant plasmids

[0055] Primer name Primer sequences YF 5'-GCACATATGAATAGTTATGCGATTAGTG-3'(SEQ ID NO.13) YR 5'-ATGTCTAGAAATATGGCAAGCAGGTTC-3'(SEQ ID NO.14) TF 5'-GCACATATGAGCGGATGTATCTCGGAGAG-3'(SEQ ID NO.15) TR 5'-ATGTCTAGAACCCCGCTCAAGGATG-3'(SEQ ID NO.16)

[0056] Note: The underlined part is the restriction enzyme cleavage site.

[0057] 3. Construction of phytase-pullulanase fusion protein

[0058] Protein fusion technology was used to construct phytase-pullulanase fusion protein, and the construction diagram is shown in the figure. Figure 1 According to ClonExpress Ultra One Step Cloning Kit instructions and primers required for designing each gene sequence (Table 2).

[0059] The steps for constructing the fusion protein T-L1-Y are as follows:

[0060] (1) Design PCR primers TL1Y-F and TL1Y-R (Table 2) based on the gene sequence of vector pBE-S. Using vector pBE-S as a template and TL1Y-F and TL1Y-R as primers, PCR amplification was performed to prepare a linearized vector. Design PCR primers based on the gene sequence of the fusion unit. Using the synthetic gene of pullulan hydrolase TK-PUL as a template, PCR amplification was performed with TL1Y-F1 and TL1Y-R1 as primers to prepare insert 1. Using the synthetic gene of linker peptide L1 as a template, PCR amplification was performed with TL1Y-F2 and TL1Y-R2 as primers to prepare insert 2. Using the synthetic gene of phytase YiAPPA as a template, PCR amplification was performed with TL1Y-F3 and TL1Y-R3 as primers to prepare insert 3. Purify the PCR amplified products using a PCR product purification kit. The PCR amplification system is the same as above. PCR amplification conditions were as follows: 98°C for 5 min; 30 cycles of 98°C for 20 s, 60°C for 40 s, and 74°C for 4 min; and 74°C for 10 min.

[0061] (2) The purified PCR amplification products were mixed and then The Ultra One Step Cloning Kit instructions describe the recombination reaction system. The recombination reaction system includes: 1 μL of linearized vector; 12 μL of insert; 20.5 μL of insert; 31.5 μL of insert; and 5 μL of 2×CE Mix. The recombination reaction conditions are: 50°C for 30 min. After completion, the recombination reaction system is immediately cooled on ice to obtain a recombinant product consisting of the linearized vector and the three inserts.

[0062] (3) The recombinant product was transformed into E. coli HST08 competent cells, and transformants were screened on ampicillin-resistant plates. The recombinant plasmid was extracted. The recombinant plasmid was sent to Shanghai Sangon Biotechnology Co., Ltd. for sequencing and compared with the corresponding gene sequence to confirm that the recombinant plasmid pBE-S-tl1y was successfully constructed.

[0063] The construction methods of fusion proteins T-L2-Y, T-L3-Y, T-L4-Y, Y-L1-T, Y-L2-T, Y-L3-T, and Y-L4-T were all carried out with reference to the construction method of fusion protein T-L1-Y.

[0064] Table 2 Primers used to construct phytase-fusion protein

[0065]

[0066]

[0067]

[0068] Example 2 Screening of Phytase-Pullulan Hydrolase Fusion Protein

[0069] 1. Expression of phytase YiAPPA, pullulan hydrolase TK-PUL, and phytase-pullulan hydrolase fusion protein

[0070] The recombinant expression plasmids of phytase YiAPPA, pullulan hydrolase TK-PUL and phytase-pullulan hydrolase fusion protein were transformed into Bacillus subtilis RIK1285 competent cells, and pBE-S was transformed as a negative control Contr. to obtain recombinant Bacillus subtilis.

[0071] The seed culture conditions for the recombinant Bacillus subtilis were as follows: 10 mL of LB liquid medium containing 10 μg / mL kanamycin was cultured in a 100 mL Erlenmeyer flask, the culture temperature was 37°C, the rotation speed was 180 rpm, and the culture time was 24 hours. The fermentation culture conditions for the recombinant Bacillus subtilis were as follows: 50 mL of LB liquid medium containing 10 μg / mL kanamycin was cultured in a 500 mL Erlenmeyer flask, the culture volume was 50 mL, the inoculum size was 1%, the culture temperature was 37°C, the rotation speed was 180 rpm, and the culture time was 36 hours.

[0072] 2. Purification of phytase YiAPPA, pullulan hydrolase TK-PUL and phytase-pullulan hydrolase fusion protein

[0073] Using Ni 2+ The target protein in the fermentation supernatant was purified using an affinity chromatography column and eluted with 200 mmol / L imidazole elution buffer to obtain the purified fusion protein. The purity of the fusion protein was determined by SDS-PAGE, and the concentration of the fusion protein was determined by the Bradford method.

[0074] 3. Screening of phytase-pullulan hydrolase fusion protein

[0075] Phytase activity assay: Take 250 μL of enzyme solution in a centrifuge tube, add 750 μL of 0.25 mol / L sodium acetate buffer (pH 4.5), and mix well. Add 2 mL of 1.5 mmol / L sodium phytate solution (0.25 mol / L sodium acetate buffer, pH 4.5) to the experimental group tube, and add 2 mL of color / endpoint mixture (ammonium molybdate / ammonium vanadate / nitric acid) to the control group tube, and shake well. After reacting at 30°C for 30 minutes, immediately add 2 mL of color / endpoint mixture to the experimental group and 2 mL of 1.5 mmol / L sodium phytate solution to the control group, mix well, and measure the light absorption value at 415 nm. The conversion formula for the inorganic phosphorus content in the reaction solution and the absorbance value of the reaction solution at 415 nm is as follows: Inorganic phosphorus (mmol / L) = 26.5510 × OD 415nm +0.3113. One unit of phytase activity (U) is defined as the amount of phytase required to release 1 μmol / L of inorganic phosphorus per minute from a 1.5 mmol / L sodium phytate solution at 37°C and pH 4.5. The phytase activity test results for phytase YiAPPA and the phytase-pullulan hydrolase fusion protein are shown in Table 3.

[0076] Amylase activity assay: 10 μL of enzyme solution was mixed with 490 μL of 50 mmol / L MES buffer (pH 4.5) containing 1% (m / v) wheat starch. The mixture was incubated at 30°C for 30 minutes, then immediately placed in an ice-water bath to terminate the reaction. The amount of reducing sugar in the reaction system was then determined using the 3,5-dinitrosalicylic acid method. The resulting reducing sugar was converted to maltose mass using a maltose standard working curve. One unit of enzyme activity (U) is defined as the amount of enzyme that catalyzes the production of 1 μmol of maltose per minute under defined reaction conditions. The amylase activity assay results for pullulan hydrolase TK-PUL and the phytase-pullulan hydrolase fusion protein are shown in Table 3.

[0077] The results of phytase and amylase activity assays of the phytase-pullulan hydrolase fusion protein showed that the phytase specific activity of the fusion protein Y-L4-T was 1802.59 U / mg, which was basically consistent with the phytase specific activity of phytase YiAPPA; the amylase specific activity of the fusion protein Y-L4-T was 60.94 U / mg, which was 1.42 times higher than that of pullulan hydrolase. The SDS-PAGE analysis of phytase YiAPPA, pullulan hydrolase TK-PUL and fusion protein Y-L4-T is shown in the figure below. Figure 2 shown.

[0078] Table 3 Enzyme activity determination results of phytase-pullulan hydrolase fusion protein (37°C, pH 4.5)

[0079] recombinant protein Phytase specific activity (U / mg) recombinant protein Amylase specific activity (U / mg) YiAPPA 1785.32±109.05 TK-PUL 25.22±2.31 T-L1-Y 1053.33±100.26 T-L1-Y 20.34±2.09 T-L2-Y 1089.53±109.03 T-L2-Y 19.82±1.83 T-L3-Y 1389.51±110.68 T-L3-Y 24.36±1.47 T-L4-Y 1492.16±134.15 T-L4-Y 24.69±1.43 Y-L1-T 1391.23±119.02 Y-L1-T 25.02±1.33 Y-L2-T 1433.15±128.25 Y-L2-T 25.71±1.73 Y-L3-T 1675.26±154.04 Y-L3-T 39.96±2.95 Y-L4-T 1802.59±128.27 Y-L4-T 60.94±2.59

[0080] The nucleotide sequence of the fusion protein Y-L4-T is shown in SEQ ID NO.45:

[0081]

[0082] The amino acid sequence of the fusion protein Y-L4-T is shown in SEQ ID NO.46:

[0083]

[0084] Example 3 Bread baking application of phytase-pullulan hydrolase fusion protein Y-L4-T

[0085] Wheat bread making method:

[0086] (1) Wheat bread recipe: 300 g of Golden Dragon Fish high-gluten wheat flour, 4.5 g of high-activity yeast, 18 g of sugar, 3 g of salt, and 180 g of water were used as raw materials. Fusion protein Y-L4-T (3.25, 6.5, or 13 mg of fusion protein per kg of wheat flour) or a mixed enzyme of phytase YiAPPA and pullulan hydrolase TK-PUL (1.15 mg YiAPPA + 2.1 mg TK-PUL, 2.3 mg YiAPPA + 4.2 mg TK-PUL, or 4.6 mg YiAPPA + 8.4 mg TK-PUL per kg of wheat flour) were added to the above raw materials, and wheat bread without enzyme addition was used as a control.

[0087] (2) Processing flow of wheat bread: Weigh each component according to the above formula system, pour it into the mixing tank and stir until the ingredients are uniform, then pour in an appropriate amount of water, stir at low speed for 3 minutes and then stir at high speed for 5 minutes. Take out the mixed dough and let it stand for 5 minutes, then cut the dough into approximately 90g / piece. Incubate the dough in an incubator at 37℃ and 90% relative humidity for 90 minutes to proof. Bake the proofed dough at 170℃ on the upper fire and 210℃ on the lower fire for 20 minutes. After baking, cool the wheat bread at room temperature for 2 hours and then store it at 4℃ and 46% relative humidity.

[0088] The wheat bread made by adding 3.25 mg of fusion protein per kg of wheat flour was named wheat bread No. ①; the wheat bread made by adding 6.5 mg of fusion protein per kg of wheat flour was named wheat bread No. ②; the wheat bread made by adding 13 mg of fusion protein per kg of wheat flour was named wheat bread No. ③; the wheat bread made by adding 1.15 mg of YiAPPA + 2.1 mg of TK-PUL mixed enzyme per kg of wheat flour was named wheat bread No. ④; the wheat bread made by adding 2.3 mg of YiAPPA + 4.2 mg of TK-PUL mixed enzyme per kg of wheat flour was named wheat bread No. ⑤; the wheat bread made by adding 4.6 mg of YiAPPA + 8.4 mg of TK-PUL mixed enzyme per kg of wheat flour was named wheat bread No. ⑥.

[0089] Determination of Wheat Bread Specific Volume: The specific volume of wheat bread on storage day 0 was determined using the rapeseed displacement method. A wheat bread sample was weighed and placed in a container of a specified volume. Rapeseed was added to the container, completely covering the bread sample and shaking to fill it. The filler was smoothed with a ruler. The bread was removed and the rapeseed was poured into a graduated cylinder to measure its volume. The volume of the bread was calculated by subtracting the volume of the rapeseed from the volume of the bread. The specific volume (SV) of the bread was calculated as: bread volume (V) / bread mass (m). The specific volume determination results for wheat bread are shown in Table 4. The specific volumes of the enzyme-treated wheat breads (wheat breads Nos. 1-6) were all greater than those of the control. When equal amounts of enzyme were added, the specific volumes of the wheat breads treated with the fusion protein (wheat breads Nos. 1-3) were all greater than those of the mixed enzyme-treated wheat breads (wheat breads Nos. 4-6). Wheat bread No. 3 had the highest specific volume, meaning that the wheat bread prepared with 13 mg of fusion protein per kg of wheat flour had the highest specific volume.

[0090] Table 4 Specific volume of wheat bread

[0091]

[0092] Wheat bread texture properties were determined: Bread stored on days 0, 1, 3, 5, and 7 was sliced ​​into 3 cm slices. The hardness and elasticity parameters of the wheat bread at different storage days were measured using a texture analyzer. The textural properties of the wheat bread are shown in Table 5. The addition of the fusion protein and enzyme mix significantly reduced the hardness and increased the elasticity of the wheat bread. After storage at 4°C, the addition of the fusion protein and enzyme mix also significantly improved the hardness and elasticity of the wheat bread. The fusion protein and enzyme mix were highly effective in improving the textural properties of wheat bread, and the improvement was proportional to the enzyme dosage. When the same amount of enzyme was added, the fusion protein significantly outperformed the enzyme mix in improving the textural properties of wheat bread. The textural properties of wheat bread No. 3 showed the most significant improvement, namely, the wheat bread prepared with 13 mg of fusion protein per kg of wheat flour showed the most significant improvement.

[0093] Table 5 Texture characteristics of wheat bread

[0094]

[0095] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A phytase-pullulan hydrolase fusion protein Y-L4-T, characterized in that: Its amino acid sequence is shown in SEQ ID NO.

46.

2. The gene encoding the phytase-pullulan hydrolase fusion protein Y-L4-T according to claim 1, characterized in that Its nucleotide sequence is shown in SEQ ID NO.

45.

3. A recombinant vector, characterized in that The recombinant vector contains the gene according to claim 2.

4. A recombinant microorganism, characterized in that The recombinant microorganism comprises the recombinant vector according to claim 3.

5. Use of the phytase-pullulan hydrolase fusion protein Y-L4-T according to claim 1 in degrading phytic acid and starch.

6. Use of the phytase-pullulan hydrolase fusion protein Y-L4-T according to claim 1 in producing pasta.

7. The use according to claim 6, characterized in that The pasta includes bread.

8. A method for improving the specific volume and / or texture properties of bread, characterized in that: The method comprises the step of adding the phytase-pullulan hydrolase fusion protein Y-L4-T according to claim 1 during the bread making process.

9. Use of the gene according to claim 2, the recombinant vector according to claim 3, or the recombinant microorganism according to claim 4 in producing the phytase-pullulan hydrolase fusion protein Y-L4-T.

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