Phytase-pullulanase fusion protein y-l4-t and application thereof

Abstract

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

Description

Technical Field

[0001] This invention relates to the field of enzyme engineering technology, and in particular to a phytoacidase-pullulan hydrolase fusion protein Y-L4-T and its applications. Background Technology

[0002] Phytase, also known as inositol hexaphosphatase, can degrade phytic acid to produce inorganic phosphorus and phosphoinositol. Adding phytase to plant-based food ingredients helps to counteract the anti-nutritional effects of phytic acid and improves the absorption and utilization of phosphorus in food. Therefore, phytase can be used as a novel food additive in food processing. For example, adding phytase during bread making can reduce phytic acid content and increase mineral and protein content. Furthermore, phytase treatment also improves the aroma, texture, and chewiness of bread. Starch hydrolases such as α-amylase and pullulanase play important roles as natural additives in bread making. α-amylase and pullulanase can hydrolyze α-1,4-glycosidic bonds and α-1,6-glycosidic bonds in starch, respectively. The synergistic effect of these two enzymes can better convert starch into more reducing sugars with smaller molecular weights. During bread making, the increased production of reducing sugars provides more substrate for yeast, affecting the fermentation rate and fermentation products, thereby improving the volume, hardness, physical, and sensory properties of bread. In addition, adding starch hydrolases during bread making also helps to extend its shelf life. Existing studies have shown that adding phytase and starch hydrolase simultaneously during bread making can optimize the fermentation process and improve the texture and taste of bread.

[0003] Protein fusion technology refers to the fusion of two or more proteins through linker peptides to obtain the desired multifunctional fusion protein. Producing fusion proteins using this technology can significantly reduce the production cost of enzyme preparations and improve their enzymatic properties. Therefore, in bread making, attempting to use fusion proteins of phytase and starch hydrolase to replace phytase and starch hydrolase respectively holds promise for reducing enzyme preparation production costs, improving the enzymatic properties of phytase or starch hydrolase, and increasing the efficiency of enzyme preparations.

[0004] YiAPPA, a phytase derived from *Yersinia intermedia*, is currently the most active phytase known, with an activity 40 times greater than that of *Aspergillus niger* PhyA, the most widely used phytase. TK-PUL, a thermophilic acidic type III pullulan hydrolase derived from *Thermococcus kodakarensis*, possesses both α-amylase and pullulanase functions, simultaneously hydrolyzing α-1,4-glycosidic and α-1,6-glycosidic bonds in starch, efficiently converting starch into low-molecular-weight reducing sugars. The enzymatic properties of YiAPPA and TK-PUL make them promising for bread making. Constructing a fusion protein of YiAPPA and TK-PUL using protein fusion technology is expected to improve the enzymatic properties of enzyme preparations, fully leverage the synergistic effects between enzymes, increase enzyme efficiency, and enable the application of fusion proteins in bread making. Summary of the Invention

[0005] The purpose of this invention is to provide a phytase-pullulan hydrolase fusion protein Y-L4-T and its applications to solve the problems existing in the prior art. This invention constructs a phytase-pullulan hydrolase fusion protein Y-L4-T. The phytase activity of this fusion protein is basically consistent with that of phytase YiAPPA, while its amylase activity is significantly higher than that of pullulan hydrolase TK-PUL. Wheat bread treated with this fusion protein shows a significant increase in specific volume, a significant decrease in hardness, and a significant increase in elasticity; it can also effectively alleviate the increase in hardness and the decrease in elasticity of wheat bread during storage. The fusion protein Y-L4-T of this invention can effectively improve bread quality and extend its shelf life, showing good application prospects in the baking field.

[0006] To achieve the above objectives, the present invention provides the following solution:

[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 above-mentioned 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-mentioned gene.

[0010] The present invention also provides a recombinant microorganism, wherein the recombinant microorganism contains the above-mentioned recombinant vector.

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

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

[0013] Optionally, the pasta includes bread.

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

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

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

[0017] This invention utilizes protein fusion technology to construct a fusion protein of phytase YiAPPA and pullulan hydrolase TK-PUL. Phytase YiAPPA and pullulan hydrolase TK-PUL were linked through different linking peptides in different fusion sequences to construct eight fusion proteins. Further, by comparing the phytase and amylase activities of each fusion protein, the fusion protein Y-L4-T with enhanced enzyme activity was selected. The phytase activity of this fusion protein Y-L4-T is 1802.59 U / mg, which is essentially the same as that of phytase YiAPPA; its amylase activity is 60.94 U / mg, which is 1.42 times higher than that of 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 showed a significant increase in specific volume, a significant decrease in hardness, and a significant increase in elasticity. Furthermore, the fusion protein Y-L4-T effectively mitigated the increase in hardness and the decrease in elasticity of wheat bread during storage. The fusion protein Y-L4-T constructed in this invention can effectively improve bread quality and extend its shelf life, showing promising application prospects in the baking industry. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 This is a schematic diagram illustrating the construction and screening process of the phytase-pullulan hydrolase fusion protein.

[0020] Figure 2 The image shows the SDS-PAGE results of phytase YiAPPA, pullulan hydrolase TK-PUL, and phytase-pullulan hydrolase fusion protein Y-L4-T. Detailed Implementation

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

[0022] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0023] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This specification and embodiments are merely exemplary.

[0024] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are 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 all purchased from Baori Biotechnology (Beijing) Co., Ltd.

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

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

[0029] The molecular cloning and protein detection techniques used in this invention are conventional techniques in the field. Techniques not described in detail in the following examples were performed according to the relevant sections of the following laboratory 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 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 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 linker 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 linker 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 linker 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 linker peptide L4 is shown in SEQ ID NO.11, and the amino acid sequence is shown in SEQ ID NO.12. These genes were synthesized in their entirety by Shanghai Boyi Biotechnology Co., Ltd.

[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 phytase YiAPPA recombinant expression plasmid and pullulan hydrolase TK-PUL recombinant expression plasmid

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

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

[0054] Table 1 Primers used to construct recombinant plasmids

[0055] Primer name Primer sequence 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 parts are restriction enzyme cleavage sites.

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

[0058] Phytase-pullulanase fusion protein was constructed using protein fusion technology. A schematic diagram of the construction is shown below. Figure 1 As shown. According to ClonExpress The Ultra One Step Cloning Kit instruction manual and the primers required for designing each gene sequence are listed in Table 2.

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

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

[0061] (2) Mix the purified PCR amplification products and follow the instructions in ClonExpress. The Ultra One Step Cloning Kit instructions describe the preparation of the recombination reaction system. The recombination reaction system includes: 1 μL linearized vector; 12 μL insert fragment; 20.5 μL insert fragment; 31.5 μL insert fragment; and 5 μL 2×CE Mix. The recombination reaction conditions are: 50 °C for 30 min. After the reaction is complete, immediately place the recombination reaction system on ice to cool, obtaining the recombination product composed of the linearized vector and the three insert fragments.

[0062] (3) The recombinant product was transformed into Escherichia coli HST08 competent cells, and transformants were screened using ampicillin-resistant plates. The recombinant plasmid was then extracted. The recombinant plasmid was sent to Shanghai Sangon Biotech 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 based on the construction method of fusion protein T-L1-Y.

[0064] Table 2 Primers used for constructing the 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] Recombinant expression plasmids of phytase YiAPPA, pullulan hydrolase TK-PUL, and phytase-pullulan hydrolase fusion protein were transformed into Bacillus subtilis RIK1285 competent cells, while pBE-S was transformed as a negative control to obtain recombinant Bacillus subtilis.

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

[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 affinity chromatography, eluted with 200 mmol / L imidazole elution buffer, yielding the purified fusion protein. The purity of the fusion protein was assessed using SDS-PAGE, and its concentration was determined using the Bradford method.

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

[0075] Phytase activity assay: 250 μL of enzyme solution was placed in a centrifuge tube, and 750 μL of 0.25 mol / L sodium acetate buffer (pH 4.5) was added and mixed well. 2 mL of 1.5 mmol / L sodium phytate solution (0.25 mol / L sodium acetate buffer, pH 4.5) was added to the experimental group tubes, and 2 mL of color / endpoint mixture (ammonium molybdate / ammonium vanadate / nitric acid) was added to the control group tubes. The mixture was shaken well. After reacting at 30℃ for 30 min, 2 mL of color / endpoint mixture was immediately added to the experimental group tubes, and 2 mL of 1.5 mmol / L sodium phytate solution was added to the control group tubes. The absorbance was measured at 415 nm. The conversion formula between the inorganic phosphorus content in the reaction solution and the absorbance at 415 nm is as follows: Inorganic phosphorus (mmol / L) = 26.5510 × OD 415nm +0.3113. Phytase activity unit (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℃ and pH 4.5. The phytase activity detection results of 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 containing 1% (m / v) wheat starch, pH 4.5. The mixture was reacted at 30℃ for 30 min, and then the reaction was quickly terminated by placing the mixture in an ice-water bath. The amount of reducing sugar in the reaction system was then determined using the 3,5-dinitrosalicylic acid method. The generated reducing sugar was converted to maltose mass using a maltose standard curve. Enzyme activity unit (U) definition: Under certain reaction conditions, the amount of enzyme that catalyzes the production of 1 μmol of maltose per minute is defined as one enzyme activity unit (U). The amylase activity assay results of pullulan hydrolase TK-PUL and 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 specific activity of phytase in the fusion protein Y-L4-T was 1802.59 U / mg, which was basically consistent with that of phytase YiAPPA; the specific activity of amylase in the fusion protein Y-L4-T was 60.94 U / mg, which was 1.42 times higher than that of pullulan hydrolase. SDS-PAGE images of phytase YiAPPA, pullulan hydrolase TK-PUL, and fusion protein Y-L4-T are shown below. Figure 2 As shown.

[0078] Table 3. Enzyme activity assay results of phytase-pullulan hydrolase fusion protein (37℃, 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: Application of phytase-pullulan hydrolase fusion protein Y-L4-T in bread baking

[0085] Wheat bread making method:

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

[0087] (2) Processing procedure for wheat bread: Weigh each component according to the above formula system, pour them into a mixing bowl and mix until the ingredients are uniform. Then add an appropriate amount of water, mix at low speed for 3 minutes and then at high speed for 5 minutes. Take out the mixed dough and let it stand for 5 minutes. Then divide the dough into approximately 90g pieces. Incubate these doughs in an incubator at 37℃ and 90% relative humidity for 90 minutes to proof. Bake the proofed dough at 170℃ (top heat) and 210℃ (bottom heat) 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] Wheat bread made with 3.25 mg of fusion protein per kg of wheat flour is named Wheat Bread No. 1; wheat bread made with 6.5 mg of fusion protein per kg of wheat flour is named Wheat Bread No. 2; wheat bread made with 13 mg of fusion protein per kg of wheat flour is named Wheat Bread No. 3; wheat bread made with 1.15 mg of YiAPPA and 2.1 mg of TK-PUL mixed enzyme per kg of wheat flour is named Wheat Bread No. 4; wheat bread made with 2.3 mg of YiAPPA and 4.2 mg of TK-PUL mixed enzyme per kg of wheat flour is named Wheat Bread No. 5; and wheat bread made with 4.6 mg of YiAPPA and 8.4 mg of TK-PUL mixed enzyme per kg of wheat flour is named Wheat Bread No. 6.

[0089] Specific volume determination of wheat bread: The specific volume of wheat bread stored on day 0 was determined using the rapeseed displacement method. A sample of wheat bread was weighed and placed in a container of a certain volume. Rapeseed was added to the container, completely covering the bread sample and firmly packed. The filler was leveled 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 obtained by subtracting the volume of the rapeseed from the volume of the bread. Specific volume of bread (SV) = bread volume (V) / bread mass (m). The results of the specific volume determination of wheat bread are shown in Table 4. The specific volumes of enzyme-treated wheat bread (wheat breads ① to ⑥) were all greater than those of the control group. With the addition of equal amounts of enzyme, the specific volumes of wheat bread treated with fusion protein (wheat breads ① to ③) were all greater than those of wheat bread treated with mixed enzymes (wheat breads ④ to ⑥). Wheat bread ③ had the largest specific volume, meaning that wheat bread made by adding 13 mg of fusion protein per kg of wheat flour had the largest specific volume.

[0090] Table 4 Specific volume of wheat bread

[0091]

[0092] Texture properties of wheat bread were determined: Bread stored for 0, 1, 3, 5, and 7 days was sliced ​​into 3cm pieces, and the hardness and elasticity parameters of wheat bread stored for different days were measured using a texture analyzer. The results of the texture property determination are shown in Table 5. The addition of fusion protein and mixed enzyme significantly reduced the hardness of wheat bread and improved its elasticity. After storage at 4℃, the addition of fusion protein and mixed enzyme also significantly improved the hardness and elasticity of wheat bread. Fusion protein and mixed enzyme are very effective in improving the texture properties of wheat bread, and the improvement effect is directly proportional to the enzyme dosage. Under the condition of adding equal amounts of enzyme, the effect of fusion protein in improving the texture properties of wheat bread is significantly better than that of mixed enzyme. The results of the texture property determination of wheat bread showed that wheat bread No. 3 had the most significant improvement in texture properties, that is, wheat bread made by adding 13mg of fusion protein per kg of wheat flour showed the most significant improvement in texture properties.

[0093] Table 5 Texture properties of wheat bread

[0094]

[0095] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope 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 as described in 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 described in claim 2.

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

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

6. The application of the phytase-pullulan hydrolase fusion protein Y-L4-T as described in claim 1 in the production of pasta.

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

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

9. The use of the gene of claim 2, the recombinant vector of claim 3, or the recombinant microorganism of claim 4 in the production of phytase-pullulan hydrolase fusion protein Y-L4-T.

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