CN120683075A

CN120683075A - A phytase-pullulan hydrolase fusion protein Y-L4-T and its application

Info

Publication number: CN120683075A
Application number: CN202510814691.7A
Authority: CN
Inventors: 曾静; 罗暄儿; 袁林; 郭建军; 熊大维; 章帅文; 王通
Original assignee: Institute Of Microbiology Jiangxi Academy Of Sciences Jiangxi Institute Of Watershed Ecology
Current assignee: Institute Of Resources And Environment Jiangxi Academy Of Sciences
Priority date: 2025-06-18
Filing date: 2025-06-18
Publication date: 2025-09-23
Anticipated expiration: 2045-06-18
Also published as: CN120683075B

Abstract

The present invention discloses a phytase-pullulan hydrolase fusion protein Y-L4-T and its application, which belongs to the field of enzyme engineering technology. The amino acid sequence of the phytase-pullulan hydrolase fusion protein Y-L4-T of the present invention is shown in SEQ ID NO.46. 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 after treatment with the fusion protein is significantly increased, the hardness is significantly reduced, and the elasticity is significantly improved; and the increase in hardness and the decrease in elasticity of wheat bread during storage can be effectively alleviated. The fusion protein Y-L4-T of the present invention can effectively improve bread quality and extend the shelf life of bread, and has good application prospects in the field of baking.

Description

Phytase-pullulanase fusion protein Y-L4-T and application thereof

Technical Field

The invention relates to the technical field of enzyme engineering, in particular to a phytase-pullulanase fusion protein Y-L4-T and application thereof.

Background

Phytase is also known as phytase and can degrade phytic acid to inorganic phosphorus and phosphoinositides. The phytase is added into the plant food raw material, which is beneficial to relieving the anti-nutrition effect of the phytic acid and improving the absorption and utilization rate of phosphorus in the food. Therefore, the phytase can be used as a novel food additive to be applied to the field of food processing. For example, the addition of phytase during bread making can result in reduced phytic acid content and increased mineral and protein content. In addition, phytase treatment also improves the aroma, mouthfeel and chewiness of the bread. Starch hydrolases such as alpha-amylase and pullulanase play an important role as natural additives in the bread making process. Alpha-amylase and pullulanase are capable of hydrolyzing alpha-1, 4-glycosidic bonds and alpha-1, 6 glycosidic bonds in starch respectively, and the synergistic effect of the two enzymes can better convert starch into more reducing sugar with smaller molecular weight. An increase in the amount of reducing sugars produced during bread making can provide more substrate for the yeast, affecting the rate of fermentation and the fermentation products of the yeast, thereby improving the volume, firmness, physical and organoleptic properties of the bread. In addition, the addition of starch hydrolyzing enzymes in bread making is also beneficial to prolonging the shelf life of the bread. Studies have shown that the simultaneous addition of phytase and starch hydrolase in the bread making process, the synergistic effect of phytase and starch hydrolase is beneficial to optimizing the fermentation process of bread, improving the texture and taste of bread, etc.

The protein fusion technique is to fuse two or more proteins with a linker peptide to obtain a desired multifunctional fusion protein. The production of fusion proteins by protein fusion techniques can greatly reduce the cost of production of enzyme preparations and facilitate the improvement of the enzymatic properties of fusion proteins. Therefore, in the bread making process, the fusion protein of phytase and starch hydrolase is tried to replace phytase and starch hydrolase, so that the production cost of the enzyme preparation is hopeful to be reduced, the enzymatic properties of the phytase or starch hydrolase are improved, and the action efficiency of the enzyme preparation is hopeful to be improved.

Phytase YiAPPA from YERSINIA INTERMEDIA is the currently known phytase with the highest activity, and its enzymatic activity is 40 times that of the currently most widely used phytase Aspergillus NIGER PHYA. The thermophilic acidic pullulanase TK-PUL from Thermococcus kodakarensis has the functions of alpha-amylase and pullulanase, can hydrolyze alpha-1, 4-glycosidic bond and alpha-1, 6-glycosidic bond in starch at the same time, and can hydrolyze the starch into low molecular weight reducing sugar with high efficiency. The enzymatic properties of phytase YiAPPA and pullulanase TK-PUL make these two enzymes show good application potential in bread making. The fusion protein of phytase YiAPPA and pullulanase TK-PUL is constructed by adopting a protein fusion technology, so that the enzymatic characteristics of enzyme preparations are hopeful to be improved, the synergistic effect among the enzyme preparations is fully exerted, the action efficiency of the enzyme preparations is improved, and the application of the fusion protein in bread making is realized.

Disclosure of Invention

The invention aims to provide a phytase-pullulanase fusion protein Y-L4-T and application thereof, so as to solve the problems in the prior art. The invention 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 obviously higher than Yu Pulu blue hydrolase TK-PUL. The specific volume of the wheat bread treated by the fusion protein is obviously increased, the hardness is obviously reduced, 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 relieved. The fusion protein Y-L4-T provided by the invention can effectively improve the bread quality and prolong the shelf life of bread, and has a good application prospect in the baking field.

In order to achieve the above object, the present invention provides the following solutions:

The invention provides a phytase-pullulanase fusion protein Y-L4-T, and the amino acid sequence of the phytase-pullulanase fusion protein is shown as SEQ ID NO. 46.

The invention also provides a gene for encoding the phytase-pullulanase fusion protein Y-L4-T, and the nucleotide sequence of the gene is shown as SEQ ID NO. 45.

The invention also provides a recombinant vector, which contains the gene.

The invention also provides a recombinant microorganism, which comprises the recombinant vector.

The invention also provides application of the phytase-pullulanase fusion protein Y-L4-T in degradation of phytic acid and starch.

The invention also provides application of the phytase-pullulanase fusion protein Y-L4-T in the production of cooked wheaten food.

Optionally, the pasta comprises bread.

The invention also provides a method for improving specific volume and/or texture characteristics of bread, which comprises the step of adding the phytase-pullulanase fusion protein Y-L4-T in the bread making process.

The invention also provides application of the gene, the recombinant vector or the recombinant microorganism in producing phytase-pullulanase fusion protein Y-L4-T.

The invention discloses the following technical effects:

The invention adopts a protein fusion technology to construct the fusion protein of the phytase YiAPPA and the pullulanase TK-PUL. The phytase YiAPPA and the pullulanase TK-PUL are connected through different connecting peptides according to different fusion sequences, 8 fusion proteins are constructed, and the fusion proteins Y-L4-T with improved enzyme activity are obtained through comparing the phytase activity and the amylase activity of each fusion protein. The phytase activity of the fusion protein Y-L4-T is 1802.59U/mg, which is basically consistent with that of phytase YiAPPA, and the amylase activity is 60.94U/mg, which is improved by 1.42 times compared with that of pullulanase TK-PUL. Compared with the mixed enzyme of phytase YiAPPA and pullulan hydrolase TK-PUL, the specific volume of the wheat bread treated by the fusion protein Y-L4-T is obviously increased, the hardness is obviously reduced, 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 relieved by the fusion protein Y-L4-T. The fusion protein Y-L4-T constructed by the invention can effectively improve the bread quality and prolong the shelf life of the bread, and has good application prospect in the baking field.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a schematic diagram of a process for constructing and screening a phytase-pullulanase fusion protein;

FIG. 2 is a diagram showing SDS-PAGE detection result of phytase YiAPPA, pullulanase TK-PUL and phytase-pullulanase fusion protein Y-L4-T.

Detailed Description

Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions of certain aspects, features and embodiments of the invention.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.

It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the invention described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present invention. The specification and examples of the present invention are exemplary only.

As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.

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

Coli ESCHERICHIA COLI HST, bacillus subtilis Bacillus subtilis RIK1285, and bacillus subtilis expression vector pBE-S were all purchased from baori doctor technology (beijing) inc.

KOD-Plus-neo DNA polymerase from Toyobo Co., ltd., DNA restriction endonuclease from FERMENTASE, PCR product purification kit, plasmid extraction kit E.Z.N.A. from Omega Bio-tek Co., clonExpressUltra One Step Cloning Kit kit is purchased from Nanjinouzan Biotechnology Co., ltd, CHELATING SEPHAROSETM FAST FLOW from U.S. GE HEALTHCARE, bradford method protein concentration determination kit is purchased from Shanghai Biotechnology Co., ltd, high activity yeast is purchased from Angel Yeast Co., ltd, gene synthesis is completed by Shanghai Bo Biotechnology Co., ltd, polymerase chain reaction primer synthesis and sequencing is completed by Shanghai Biotechnology Co., ltd, and other chemical reagents are all domestic or imported analytical pure.

LB medium (g/L) tryptone 10, yeast extract 5, naCl 10, pH 7.0. The screening medium used was LB medium containing 100. Mu.g/mL ampicillin and LB medium containing 10. Mu.g/mL kanamycin.

The molecular cloning techniques and protein detection techniques used in the present invention are conventional in the art. The techniques not described in detail in the examples below are all carried out according to the relevant parts of the following laboratory manuals ：Green M R,SambrookJ.Molecular cloning:alaboratory manual[M].NewYork:Cold Spring Harbor Laboratory Press,2012.

EXAMPLE 1 construction of Phytase-pullulanase fusion proteins

1. Synthesis of Gene

The gene sequence of the phytase YiAPPA from YERSINIA INTERMEDIA is shown as SEQ ID NO.1, the amino acid sequence is shown as SEQ ID NO.2, the gene sequence of the pullulanase TK-PUL from Thermococcus kodakarensis is shown as SEQ ID NO.3, the amino acid sequence is shown as SEQ ID NO.4, the gene sequence of the connecting peptide L1 is shown as SEQ ID NO.5, the amino acid sequence is shown as SEQ ID NO.6, the gene sequence of the connecting peptide L2 is shown as SEQ ID NO.7, the amino acid sequence is shown as SEQ ID NO.8, the gene sequence of the connecting peptide L3 is shown as SEQ ID NO.9, the amino acid sequence is shown as SEQ ID NO.10, the gene sequence of the connecting peptide L4 is shown as SEQ ID NO.11, and the amino acid sequence is shown as SEQ ID NO. 12. The genes are delivered to Shanghai Bo Yi Biotechnology Co.Ltd for complete gene synthesis.

SEQ ID NO.1:

AATAGTTATGCGATTAGTGCCGCGCCGGTTGCCATACAACCCACGGGCTATACATTGGAGCGAGTGGTTATTTTGAGCCGCCATGGTGTTCGCTCGCCAACCAAACAAACACAGTTAATGAATGATGTTACCCCTGACACGTGGCCGCAATGGCCGGTCGCCGCAGGATACTTAACCCCCCGAGGTGCACAATTAGTGACATTGATGGGCGGATTCTATGGTGATTACTTCCGTAGCCAAGGGTTACTCGCAGCAGGGTGCCCAACTGACGCGGTTATTTATGCTCAGGCCGATGTTGATCAACGAACGCGTTTAACGGGGCAGGCATTCCTTGATGGAATAGCACCGGGGTGTGGACTGAAAGTACATTATCAGGCTGATTTGAAAAAAGTGGATCCGCTGTTTCATCCCGTCGACGCGGGGGTGTGTAAGTTAGATTCGACACAAACCCATAAGGCTGTTGAGGAGCGACTAGGTGGGCCATTAAGTGAACTGAGCAAACGCTATGCTAAGCCCTTTGCCCAGATGGGTGAGATTCTGAATTTTGCGGCATCTCCTTACTGTAAATCACTGCAACAGCAAGGGAAAACCTGTGATTTTGCCAACTTTGCAGCGAATAAGATCACGGTGAACAAGCCGGGGACAAAAGTCTCGCTCAGCGGACCACTGGCACTGTCATCAACCTTAGGTGAGATCTTTTTGCTACAAAATTCACAAGCGATGCCTGATGTTGCCTGGCATCGGTTAACGGGAGAAGATAATTGGATCTCGTTATTATCGTTGCACAATGCGCAATTTGATTTAATGGCAAAAACACCTTATATCGCTCGTCATAAGGGCACACCGTTGCTGCAACAGATCGAGACTGCCCTCGTCCTTCAGCGTGATGCTCAGGGGCAAACATTGCCATTATCACCTCAAACCAAAATTCTGTTCCTCGGGGGACATGATACAAACATCGCCAATATTGCTGGAATGTTGGGGGCTAACTGGCAATTACCACAGCAGCCCGATAATACCCCACCTGGGGGGGGATTGGTCTTCGAGCTATGGCAAAACCCAGATAATCATCAACGTTATGTCGCGGTGAAAATGTTCTATCAAACAATGGGCCAATTGCGAAATGCTGAGAAACTAGACCTGAAAAACAATCCGGCTGGTAGGGTCCCTGTTGCAATAGACGGTTGTGAAAATAGTGGTGATGACAAACTTTGTCAGCTTGATACCTTCCAAAAGAAAGTAGCTCAGGCGATTGAACCTGCTTGCCATATT.

SEQ ID NO.2:

NSYAISAAPVAIQPTGYTLERVVILSRHGVRSPTKQTQLMNDVTPDTWPQWPVAAGYLTPRGAQLVTLMGGFYGDYFRSQGLLAAGCPTDAVIYAQADVDQRTRLTGQAFLDGIAPGCGLKVHYQADLKKVDPLFHPVDAGVCKLDSTQTHKAVEERLGGPLSELSKRYAKPFAQMGEILNFAASPYCKSLQQQGKTCDFANFAANKITVNKPGTKVSLSGPLALSSTLGEIFLLQNSQAMPDVAWHRLTGEDNWISLLSLHNAQFDLMAKTPYIARHKGTPLLQQIETALVLQRDAQGQTLPLSPQTKILFLGGHDTNIANIAGMLGANWQLPQQPDNTPPGGGLVFELWQNPDNHQRYVAVKMFYQTMGQLRNAEKLDLKNNPAGRVPVAIDGCENSGDDKLCQLDTFQKKVAQAIEPACHI.

SEQ ID NO.3:

AGCGGATGTATCTCGGAGAGCAACGAAAATCAAACTGCAACGGCTTCGACCGTTCCACCGACTTCAGTGACACCCTCACAGTCTTCCACTCCCACAACCTCGACCTCGACGTACGGCCCTTCCGAAAGAACGGAGCTTAAACTTCCTTCGGTTAACTACACTCCCATCTACGTCGGCATAGAGAAAGGCTGTCCCTCCGGAAGAGTCCCGGTGAAGTTCACGTACAACCCCGGAAACAAGACCGTAAAGTCTGTCAGCCTCCGCGGGAGCTTCAACAACTGGGGAGAGTGGCCGATGGAGCTGAAGAACGGCACGTGGGAGACGACCGTCTGTCTCCGCCCTGGAAGGTATGAGTATAAGTACTTCATCAACGGCCAGTGGGTCAAGGACATGTCCGACGACGGGACGGGAAGGCCCTACGACCCCGATGCAGACGCCTATGCCCCCGATGGCTACGGGGGAAAGAACGCCGTGAGGGTAGTTGAGGGCCGCGAAGCGTTCTACGTGGAGTTCGATCCAAGAGACCCAGCCTACCTCAGCATCGCGGACAAAAGAACCGTGGTCAGGTTCGAGGCTAAGAGAGACACCGTCGAGTCTGCGGTTCTCGTTACGGATCACGGGAACTACACGATGAAGCTTCAGGTCTGGTGGGACTTCGGCGAAACCTGGCGCGCCGAGATGCCAGTTGAACCCGCTGATTATTACATTCTCGTAACCTCCTCCGACGGCGGGAAGTTTGCCGTCCTAAACACAAGCGAAAGCCCGTTCTTCCACTTTGATGGCGTTGAGGGGTTCCCCCAGCTGGAGTGGGTGAGCAACGGGATAACCTACCAGATATTCCCCGACAGGTTCAACAACGGCAATAAAAGCAACGATGCCCTAGCTTTGGATCACGACGAGCTAATTTTGAACCAGGTTAATCCAGGGCAGCCAATCCTCTCCAACTGGAGCGACCCGATAACGCCCCTCCACTGCTGCCACCAGTACTTCGGCGGCGACATAAAGGGAATAACGGAGAAGCTCGACTACCTTCAGAGCCTAGGTGTTACTATAATCTACATCAACCCGATTTTCCTCTCGGGAAGCGCCCACGGCTACGACACCTACGACTACTACCGGCTCGACCCCAAGTTCGGGACCGAGGATGAGCTGAGAGAGTTCCTCGATGAGGCCCACAGGAGGGGAATGAGGGTAATCTTCGATTTCGTGCCCAACCACTGCGGCATAGGGAATCCAGCCTTCCTCGACGTCTGGGAGAAGGGCAACGAAAGCCCATACTGGGACTGGTTCTTCGTCAAGAAGTGGCCCTTCAAGCTCGGCGATGGGAGCGCCTACGTCGGCTGGTGGGGCTTTGGGAGCCTTCCGAAGCTCAACACTGCCAACCAGGAGGTCAGGGAGTACCTGATAGGAGCGGCCCTCCACTGGATAGAGTTCGGCTTTGACGGCATTAGGGTGGATGTGCCGAACGAAGTCCTCGACCCGGGGACGTTCTTCCCGGAGCTGAGAAAGGCAGTTAAGGAGAAAAAGCCCGACGCGTACCTCGTCGGCGAGATATGGACGCTCTCCCCGGAGTGGGTGAAGGGAGACCGCTTCGACTCCCTCATGAACTACGCCCTCGGGAGGGACATCCTCCTGAACTACGCTAAGGGCCTGCTCAGCGGAGAAAGTGCAATGAAAATGATGGGACGTTACTACGCTTCCTACGGCGAGAACGTAGTTGCGATGGGCTTCAACCTCGTTGATTCGCACGACACTTCGAGGGTTCTCACTGACCTCGGTGGTGGCAAACTGGGAGACACACCGTCAAACGAGTCAATTCAGAGGCTCAAGCTCCTCTCAACGCTCCTCTATGCCCTGCCCGGAACTCCCGTCACCTTCCAGGGGGACGAGAGGGGACTGCTCGGAGACAAGGGACACTACGATGAGCAACGCTATCCGATACAGTGGGATACTGTGAACGAGGACGTCCTGAACCACTACAGGGCACTGGCGGAGCTCAGAAAAAGAGTTCCCGCATTGAGGAGCAGCGCAATGAGGTTCTACACTGCCAAAGGCGGCGTTATGGCCTTCTTCAGGGGACATCATGACGAGGTTCTCGTCGTTGCCAACAGCTGGAAGAAGCCAGCCCTACTGGAGCTTCCCGAGGGAGAGTGGAAAGTAATCTGGCCTGAGGATTTCAGCCCGGAACTGCTTCGCGGCACAGTTGAAGTGCCAGCCATAGGGATAATCATCCTTGAGCGGGGT.

SEQ ID NO.4:

SGCISESNENQTATASTVPPTSVTPSQSSTPTTSTSTYGPSERTELKLPSVNYTPIYVGIEKGCPSGRVPVKFTYNPGNKTVKSVSLRGSFNNWGEWPMELKNGTWETTVCLRPGRYEYKYFINGQWVKDMSDDGTGRPYDPDADAYAPDGYGGKNAVRVVEGREAFYVEFDPRDPAYLSIADKRTVVRFEAKRDTVESAVLVTDHGNYTMKLQVWWDFGETWRAEMPVEPADYYILVTSSDGGKFAVLNTSESPFFHFDGVEGFPQLEWVSNGITYQIFPDRFNNGNKSNDALALDHDELILNQVNPGQPILSNWSDPITPLHCCHQYFGGDIKGITEKLDYLQSLGVTIIYINPIFLSGSAHGYDTYDYYRLDPKFGTEDELREFLDEAHRRGMRVIFDFVPNHCGIGNPAFLDVWEKGNESPYWDWFFVKKWPFKLGDGSAYVGWWGFGSLPKLNTANQEVREYLIGAALHWIEFGFDGIRVDVPNEVLDPGTFFPELRKAVKEKKPDAYLVGEIWTLSPEWVKGDRFDSLMNYALGRDILLNYAKGLLSGESAMKMMGRYYASYGENVVAMGFNLVDSHDTSRVLTDLGGGKLGDTPSNESIQRLKLLSTLLYALPGTPVTFQGDERGLLGDKGHYDEQRYPIQWDTVNEDVLNHYRALAELRKRVPALRSSAMRFYTAKGGVMAFFRGHHDEVLVVANSWKKPALLELPEGEWKVIWPEDFSPELLRGTVEVPAIGIIILERG.

SEQ ID NO.5:GAGGGTAAGTCTTCGGGCTCAGGCTCAGAGTCAAAATCCACC。

SEQ ID NO.6:EGKSSGSGSESKST。

SEQ ID NO.7:GGTTCCGCTGGATCAGCTGCTGGATCAGGAGAATTC。

SEQ ID NO.8:GSAGSAAGSGEF。

SEQ ID NO.9:

GCTGAAGCTGCTGCTAAGGAAGCTGCTGCTAAGGAAGCTGCTGCTAAGGCT。

SEQ ID NO.10:AEAAAKEAAAKEAAAKA。

SEQ ID NO.11:

GAAGCTGCTGCTAAGGAAGCTGCTGCTAAGGGTGGTGGTGGTTCCGGTGGTGGTGGTTCCGGTG GTGGTGGTTCC。

SEQ ID NO.12:EAAAKEAAAKGGGGSGGGGSGGGGS。

2. Construction of recombinant expression plasmid for Phytase YiAPPA and recombinant expression plasmid for pullulanase TK-PUL

PCR was performed by designing a PCR primer Y-F, Y-R (Table 1) based on the gene sequence of phytase YiAPPA, using the synthetic gene of YiAPPA as a template and Y-F, Y-R as a primer. The PCR amplification system was 10 Xbuffer I5. Mu. L, dNTP. Mu. L, mgSO ₄. Mu.L, 2. Mu.L of each of the upstream and downstream primers, and 2. Mu. L, ddH ₂ O28. Mu.L of the template 1. Mu. L, KOD-Plus-neo DNA polymerase. The PCR amplification conditions were 98℃for 5min, 98℃for 20s,60℃for 40s,74℃for 2min,30 cycles, and 74℃for 10min. The amplified product was digested with NdeI and XbaI, ligated into vector pBE-S digested with the same double digestion, and recombinant plasmid pBE-S-yiappa was constructed.

PCR primers T-F, T-R (Table 1) were designed based on the gene sequence of the pullulanase TK-PUL, and PCR amplification was performed using the synthetic gene of TK-PUL as a template and T-F, T-R as a primer. The PCR amplification system is the same as above. The PCR amplification conditions were 98℃for 5min, 98℃for 20s,60℃for 40s,74℃for 2min for 10 s,30 cycles, and 74℃for 10min. The amplified product was digested with NdeI and XbaI, ligated into vector pBE-S digested with the same double digestion, and recombinant plasmid pBE-S-tkpul was constructed.

TABLE 1 primers for construction of recombinant plasmids

Primer name	Primer sequences
		Y-F	5’-GCACATATGAATAGTTATGCGATTAGTG-3’(SEQ ID NO.13)
Y-R	5’-ATGTCTAGAAATATGGCAAGCAGGTTC-3’(SEQ ID NO.14)
		T-F	5’-GCACATATGAGCGGATGTATCTCGGAGAG-3’(SEQ ID NO.15)
T-R	5’-ATGTCTAGAACCCCGCTCAAGGATG-3’(SEQ ID NO.16)

Note that the underlined parts are restriction enzyme cleavage sites.

3. Construction of Phytase-pullulanase fusion proteins

The phytase-pullulanase fusion protein is constructed by adopting a protein fusion technology, and the construction schematic diagram is shown in figure 1. According to ClonExpressUltra One Step Cloning Kit kit instructions and primers required for designing each gene sequence (Table 2).

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

(1) PCR primers TL1Y-F, TL Y-R (Table 2) were designed based on the gene sequence of vector pBE-S, and PCR amplification was performed using vector pBE-S as template and TL1Y-F, TL Y-R as primer to prepare linearized vector. Designing PCR primers according to the gene sequence of the fusion unit, carrying out PCR amplification by taking the synthetic gene of the pullulan hydrolase TK-PUL as a template, taking TL1Y-F1 and TL1Y-R1 as primers to prepare an insert fragment 1, taking the synthetic gene of the connecting peptide L1 as a template, taking TL1Y-F2 and TL1Y-R2 as primers to prepare an insert fragment 2, carrying out PCR amplification by taking the synthetic gene of the phytase YiAPPA as a template and taking TL1Y-F3 and TL1Y-R3 as primers to prepare an insert fragment 3. And purifying the PCR amplified product by using a PCR product purification kit. The PCR amplification system is the same as above. The PCR amplification conditions were 98℃for 5min, 98℃for 20s,60℃for 40s,74℃for 4min,30 cycles, and 74℃for 10min.

(2) Mixing the purified PCR amplified products according to ClonExpressUltra One Step Cloning Kit the kit instruction book configures a recombination reaction system. The recombination reaction system included 1. Mu.L of linearization vector, 12. Mu.L of insert, 20.5. Mu.L of insert, 31.5. Mu.L of insert, and 5. Mu.L of 2 XCE Mix. The recombination reaction condition is 50 ℃ for 30min. After the reaction is completed, the recombination reaction system is immediately placed on ice for cooling, and a recombination product composed of a linearization vector and 3 inserts is obtained.

(3) The recombinant product is transformed into escherichia coli HST08 competent cells, and ampicillin resistance plates are used for screening transformants, so as to extract recombinant plasmids. The recombinant plasmid was sent to Shanghai Bioengineering Co., ltd for sequencing and aligned with the corresponding gene sequence, confirming the success of construction of the recombinant plasmid pBE-S-tl1 y.

The construction method of the fusion protein T-L2-Y, T-L3-Y, T-L4-Y, Y-L1-T, Y-L2-T, Y-L3-T, Y-L4-T is carried out according to the construction method of the fusion protein T-L1-Y.

TABLE 2 primers for the construction of phytase-fusion proteins

Example 2 screening of Phytase-pullulanase fusion proteins

1. Expression of Phytase YiAPPA, pullulanase TK-PUL and Phytase-pullulanase fusion proteins

Recombinant expression plasmids of phytase YiAPPA, pullulanase TK-PUL and phytase-pullulanase fusion proteins are respectively transformed into bacillus subtilis RIK1285 competent cells, and pBE-S is transformed as negative control Contr to obtain recombinant bacillus subtilis.

The seed culture condition of the recombinant bacillus subtilis is that LB liquid medium containing 10 mug/mL kanamycin is adopted, and a 100mL triangular flask is used for culture, wherein the liquid loading amount of the medium is 10mL, the culture temperature is 37 ℃, the rotating speed is 180rpm, and the culture time is 24 hours. The fermentation culture condition of the recombinant bacillus subtilis is that LB liquid medium containing 10 mug/mL kanamycin is adopted, and a 500mL triangular flask is used for culture, wherein the liquid loading amount of the medium is 50mL, the inoculation amount is 1%, the culture temperature is 37 ℃, the rotating speed is 180rpm, and the culture time is 36h.

2. Purification of Phytase YiAPPA, pullulanase TK-PUL and Phytase-pullulanase fusion proteins

Purifying the target protein in the fermentation supernatant by adopting a Ni ²⁺ affinity chromatographic column, and eluting by using 200mmol/L imidazole elution buffer solution to obtain the purified fusion protein. Purity of the fusion protein was checked by SDS-PAGE and concentration of the fusion protein was determined by the Bradford method.

3. Screening of Phytase-pullulanase fusion proteins

Enzyme activity measurement of phytase, namely, 250 mu L of enzyme solution is taken in a centrifuge tube, 750 mu L of 0.25mol/L sodium acetate buffer solution (pH 4.5) is added, and the mixture is uniformly mixed. 2mL of 1.5mmol/L sodium phytate solution (0.25 mol/L sodium acetate buffer, pH 4.5) was added to the experimental tube, and 2mL of the color/end point mixture (ammonium molybdate/ammonium vanadate/nitric acid) was added to the control, and shaken well. Immediately after 30min of reaction at 30 ℃, 2mL of the color/endpoint mixture was added to the experimental group, 2mL of 1.5mmol/L sodium phytate solution was added to the control group, and the mixture was mixed well, and the light absorption value was measured at 415 nm. The conversion formula of the inorganic phosphorus content in the reaction solution and the absorbance of the reaction solution at 415nm is shown as follows, inorganic phosphorus (mmol/L) = 26.5510 ×OD _415nm +0.3113. The phytase activity unit (U) is defined as the amount of phytase required to release 1. Mu. Mol/L inorganic phosphorus from 1.5mmol/L sodium phytate solution per minute at 37℃ C, pH 4.5, as one enzyme activity unit (U). The results of the phytase activity assay of the phytase YiAPPA and phytase-pullulanase fusion proteins are shown in Table 3.

Measurement of the enzyme Activity of amylase 10. Mu.L of enzyme solution was mixed with 490. Mu.L of 50mmol/L MES containing 1% (m/v) wheat starch, pH 4.5 buffer, reacted at 30℃for 30min, and then rapidly placed in an ice-water bath to terminate the reaction, and then the amount of reducing sugar in the reaction system was measured by the 3, 5-dinitrosalicylic acid method. The resulting reducing sugars were converted to maltose mass representation by means of a maltose standard working curve. The enzyme activity unit (U) is defined as the amount of enzyme catalyzing the production of 1. Mu. Mol of maltose per minute under certain reaction conditions. The results of the amylase activity assay for the pullulanase TK-PUL and the phytase-pullulanase fusion protein are shown in Table 3.

The phytase activity and amylase activity measurement results of the phytase-pullulanase fusion protein show that the phytase specific activity of the fusion protein Y-L4-T is 1802.59U/mg, the phytase specific activity is basically consistent with that of phytase YiAPPA, the amylase specific activity of the fusion protein Y-L4-T is 60.94U/mg, and the phytase specific activity is improved by 1.42 times compared with that of pullulanase. SDS-PAGE detection of phytase YiAPPA, pullulanase TK-PUL and fusion protein Y-L4-T is shown in FIG. 2.

TABLE 3 enzyme Activity measurement of phytase-pullulanase fusion protein (37 ℃ C., pH 4.5)

Recombinant proteins	Specific activity of phytase (U/mg)	Recombinant proteins	Specific activity of amylase (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

The nucleotide sequence of the fusion protein Y-L4-T is shown as SEQ ID NO. 45:

AATAGTTATGCGATTAGTGCCGCGCCGGTTGCCATACAACCCACGGGCTATACATTGGAGCGAGTGGTTATTTTGAGCCGCCATGGTGTTCGCTCGCCAACCAAACAAACACAGTTAATGAATGATGTTACCCCTGACACGTGGCCGCAATGGCCGGTCGCCGCAGGATACTTAACCCCCCGAGGTGCACAATTAGTGACATTGATGGGCGGATTCTATGGTGATTACTTCCGTAGCCAAGGGTTACTCGCAGCAGGGTGCCCAACTGACGCGGTTATTTATGCTCAGGCCGATGTTGATCAACGAACGCGTTTAACGGGGCAGGCATTCCTTGATGGAATAGCACCGGGGTGTGGACTGAAAGTACATTATCAGGCTGATTTGAAAAAAGTGGATCCGCTGTTTCATCCCGTCGACGCGGGGGTGTGTAAGTTAGATTCGACACAAACCCATAAGGCTGTTGAGGAGCGACTAGGTGGGCCATTAAGTGAACTGAGCAAACGCTATGCTAAGCCCTTTGCCCAGATGGGTGAGATTCTGAATTTTGCGGCATCTCCTTACTGTAAATCACTGCAACAGCAAGGGAAAACCTGTGATTTTGCCAACTTTGCAGCGAATAAGATCACGGTGAACAAGCCGGGGACAAAAGTCTCGCTCAGCGGACCACTGGCACTGTCATCAACCTTAGGTGAGATCTTTTTGCTACAAAATTCACAAGCGATGCCTGATGTTGCCTGGCATCGGTTAACGGGAGAAGATAATTGGATCTCGTTATTATCGTTGCACAATGCGCAATTTGATTTAATGGCAAAAACACCTTATATCGCTCGTCATAAGGGCACACCGTTGCTGCAACAGATCGAGACTGCCCTCGTCCTTCAGCGTGATGCTCAGGGGCAAACATTGCCATTATCACCTCAAACCAAAATTCTGTTCCTCGGGGGACATGATACAAACATCGCCAATATTGCTGGAATGTTGGGGGCTAACTGGCAATTACCACAGCAGCCCGATAATACCCCACCTGGGGGGGGATTGGTCTTCGAGCTATGGCAAAACCCAGATAATCATCAACGTTATGTCGCGGTGAAAATGTTCTATCAAACAATGGGCCAATTGCGAAATGCTGAGAAACTAGACCTGAAAAACAATCCGGCTGGTAGGGTCCCTGTTGCAATAGACGGTTGTGAAAATAGTGGTGATGACAAACTTTGTCAGCTTGATACCTTCCAAAAGAAAGTAGCTCAGGCGATTGAACCTGCTTGCCATATTGAAGCTGCTGCTAAGGAAGCTGCTGCTAAGGGTGGTGGTGGTTCCGGTGGTGGTGGTTCCGGTGGTGGTGGTTCCAGCGGATGTATCTCGGAGAGCAACGAAAATCAAACTGCAACGGCTTCGACCGTTCCACCGACTTCAGTGACACCCTCACAGTCTTCCACTCCCACAACCTCGACCTCGACGTACGGCCCTTCCGAAAGAACGGAGCTTAAACTTCCTTCGGTTAACTACACTCCCATCTACGTCGGCATAGAGAAAGGCTGTCCCTCCGGAAGAGTCCCGGTGAAGTTCACGTACAACCCCGGAAACAAGACCGTAAAGTCTGTCAGCCTCCGCGGGAGCTTCAACAACTGGGGAGAGTGGCCGATGGAGCTGAAGAACGGCACGTGGGAGACGACCGTCTGTCTCCGCCCTGGAAGGTATGAGTATAAGTACTTCATCAACGGCCAGTGGGTCAAGGACATGTCCGACGACGGGACGGGAAGGCCCTACGACCCCGATGCAGACGCCTATGCCCCCGATGGCTACGGGGGAAAGAACGCCGTGAGGGTAGTTGAGGGCCGCGAAGCGTTCTACGTGGAGTTCGATCCAAGAGACCCAGCCTACCTCAGCATCGCGGACAAAAGAACCGTGGTCAGGTTCGAGGCTAAGAGAGACACCGTCGAGTCTGCGGTTCTCGTTACGGATCACGGGAACTACACGATGAAGCTTCAGGTCTGGTGGGACTTCGGCGAAACCTGGCGCGCCGAGATGCCAGTTGAACCCGCTGATTATTACATTCTCGTAACCTCCTCCGACGGCGGGAAGTTTGCCGTCCTAAACACAAGCGAAAGCCCGTTCTTCCACTTTGATGGCGTTGAGGGGTTCCCCCAGCTGGAGTGGGTGAGCAACGGGATAACCTACCAGATATTCCCCGACAGGTTCAACAACGGCAATAAAAGCAACGATGCCCTAGCTTTGGATCACGACGAGCTAATTTTGAACCAGGTTAATCCAGGGCAGCCAATCCTCTCCAACTGGAGCGACCCGATAACGCCCCTCCACTGCTGCCACCAGTACTTCGGCGGCGACATAAAGGGAATAACGGAGAAGCTCGACTACCTTCAGAGCCTAGGTGTTACTATAATCTACATCAACCCGATTTTCCTCTCGGGAAGCGCCCACGGCTACGACACCTACGACTACTACCGGCTCGACCCCAAGTTCGGGACCGAGGATGAGCTGAGAGAGTTCCTCGATGAGGCCCACAGGAGGGGAATGAGGGTAATCTTCGATTTCGTGCCCAACCACTGCGGCATAGGGAATCCAGCCTTCCTCGACGTCTGGGAGAAGGGCAACGAAAGCCCATACTGGGACTGGTTCTTCGTCAAGAAGTGGCCCTTCAAGCTCGGCGATGGGAGCGCCTACGTCGGCTGGTGGGGCTTTGGGAGCCTTCCGAAGCTCAACACTGCCAACCAGGAGGTCAGGGAGTACCTGATAGGAGCGGCCCTCCACTGGATAGAGTTCGGCTTTGACGGCATTAGGGTGGATGTGCCGAACGAAGTCCTCGACCCGGGGACGTTCTTCCCGGAGCTGAGAAAGGCAGTTAAGGAGAAAAAGCCCGACGCGTACCTCGTCGGCGAGATATGGACGCTCTCCCCGGAGTGGGTGAAGGGAGACCGCTTCGACTCCCTCATGAACTACGCCCTCGGGAGGGACATCCTCCTGAACTACGCTAAGGGCCTGCTCAGCGGAGAAAGTGCAATGAAAATGATGGGACGTTACTACGCTTCCTACGGCGAGAACGTAGTTGCGATGGGCTTCAACCTCGTTGATTCGCACGACACTTCGAGGGTTCTCACTGACCTCGGTGGTGGCAAACTGGGAGACACACCGTCAAACGAGTCAATTCAGAGGCTCAAGCTCCTCTCAACGCTCCTCTATGCCCTGCCCGGAACTCCCGTCACCTTCCAGGGGGACGAGAGGGGACTGCTCGGAGACAAGGGACACTACGATGAGCAACGCTATCCGATACAGTGGGATACTGTGAACGAGGACGTCCTGAACCACTACAGGGCACTGGCGGAGCTCAGAAAAAGAGTTCCCGCATTGAGGAGCAGCGCAATGAGGTTCTACACTGCCAAAGGCGGCGTTATGGCCTTCTTCAGGGGACATCATGACGAGGTTCTCGTCGTTGCCAACAGCTGGAAGAAGCCAGCCCTACTGGAGCTTCCCGAGGGAGAGTGGAAAGTAATCTGGCCTGAGGATTTCAGCCCGGAACTGCTTCGCGGCACAGTTGAAGTGCCAGCCATAGGGATAATCATCCTTGAGCGGGGT.

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

NSYAISAAPVAIQPTGYTLERVVILSRHGVRSPTKQTQLMNDVTPDTWPQWPVAAGYLTPRGAQLVTLMGGFYGDYFRSQGLLAAGCPTDAVIYAQADVDQRTRLTGQAFLDGIAPGCGLKVHYQADLKKVDPLFHPVDAGVCKLDSTQTHKAVEERLGGPLSELSKRYAKPFAQMGEILNFAASPYCKSLQQQGKTCDFANFAANKITVNKPGTKVSLSGPLALSSTLGEIFLLQNSQAMPDVAWHRLTGEDNWISLLSLHNAQFDLMAKTPYIARHKGTPLLQQIETALVLQRDAQGQTLPLSPQTKILFLGGHDTNIANIAGMLGANWQLPQQPDNTPPGGGLVFELWQNPDNHQRYVAVKMFYQTMGQLRNAEKLDLKNNPAGRVPVAIDGCENSGDDKLCQLDTFQKKVAQAIEPACHIEAAAKEAAAKGGGGSGGGGSGGGGSSGCISESNENQTATASTVPPTSVTPSQSSTPTTSTSTYGPSERTELKLPSVNYTPIYVGIEKGCPSGRVPVKFTYNPGNKTVKSVSLRGSFNNWGEWPMELKNGTWETTVCLRPGRYEYKYFINGQWVKDMSDDGTGRPYDPDADAYAPDGYGGKNAVRVVEGREAFYVEFDPRDPAYLSIADKRTVVRFEAKRDTVESAVLVTDHGNYTMKLQVWWDFGETWRAEMPVEPADYYILVTSSDGGKFAVLNTSESPFFHFDGVEGFPQLEWVSNGITYQIFPDRFNNGNKSNDALALDHDELILNQVNPGQPILSNWSDPITPLHCCHQYFGGDIKGITEKLDYLQSLGVTIIYINPIFLSGSAHGYDTYDYYRLDPKFGTEDELREFLDEAHRRGMRVIFDFVPNHCGIGNPAFLDVWEKGNESPYWDWFFVKKWPFKLGDGSAYVGWWGFGSLPKLNTANQEVREYLIGAALHWIEFGFDGIRVDVPNEVLDPGTFFPELRKAVKEKKPDAYLVGEIWTLSPEWVKGDRFDSLMNYALGRDILLNYAKGLLSGESAMKMMGRYYASYGENVVAMGFNLVDSHDTSRVLTDLGGGKLGDTPSNESIQRLKLLSTLLYALPGTPVTFQGDERGLLGDKGHYDEQRYPIQWDTVNEDVLNHYRALAELRKRVPALRSSAMRFYTAKGGVMAFFRGHHDEVLVVANSWKKPALLELPEGEWKVIWPEDFSPELLRGTVEVPAIGIIILERG.

EXAMPLE 3 bread baking application of phytase-pullulanase fusion protein Y-L4-T

The wheat bread making method comprises the following steps:

(1) The formula of the wheat bread is that 300g of high-gluten goldenseal wheat flour, 4.5g of high-activity yeast, 18g of sugar, 3g of salt and 180g of water are taken as raw materials, fusion protein Y-L4-T (3.25, 6.5 or 13mg of fusion protein is added to each Kg of wheat flour) or phytase YiAPPA and pullulanase TK-PUL mixed enzyme (1.15mg YiAPPA+2.1mg TK-PUL, 2.3mgYiAPPA+4.2mg TK-PUL or 4.6mgYiAPPA+8.4mg TK-PUL are added to each Kg of wheat flour) are respectively added to the raw materials, and the wheat bread without enzyme is taken as a control.

(2) The processing flow of the wheat bread comprises the steps of weighing each component according to the formula system, pouring the components into a stirring tank, stirring until the ingredients are uniform, pouring a proper amount of water, stirring at a low speed for 3min, and stirring at a high speed for 5min. The mixed dough was taken out and left to stand for 5min, after which the dough was roughly cut into 90 g/pieces. These doughs were incubated in an incubator at 37 ℃ with 90% relative humidity for 90min for proofing. The proofed dough was baked for 20min at 170℃on fire and 210℃on fire. After baking, the wheat bread was cooled at room temperature for 2 hours and then stored at 4 ℃ under conditions of 46% relative humidity.

Wheat bread prepared by adding 3.25mg of the fusion protein to each kg of wheat flour is named as wheat bread ①, wheat bread prepared by adding 6.5mg of the fusion protein to each kg of wheat flour is named as wheat bread ②, wheat bread prepared by adding 13mg of the fusion protein to each kg of wheat flour is named as wheat bread ③, wheat bread prepared by adding 1.15mg YiAPPA+2.1mg TK-PUL enzyme to each kg of wheat flour is named as wheat bread ④, wheat bread prepared by adding 2.3mg YiAPPA+4.2mg TK-PUL enzyme to each kg of wheat flour is named as wheat bread ⑤, and wheat bread prepared by adding 4.6mgYiAPPA+8.4mg TK-PUL enzyme to each kg of wheat flour is named as wheat bread ⑥.

Specific volume measurement of wheat bread: the specific volume of wheat bread on day 0 of storage was measured using the rapeseed displacement method. Taking a wheat bread sample to be measured, weighing, putting the wheat bread sample into a container with a certain volume, adding rapeseeds into the container, completely covering the bread sample, shaking up, filling the bread sample, scraping the filling agent with a ruler, taking out the bread, pouring the rapeseeds into a measuring cylinder to measure the volume, and subtracting the volume of the rapeseeds from the volume to obtain the volume of the bread. Specific volume of bread (SV) =volume of bread (V)/mass of bread (m). As shown in Table 4, the specific volume of the wheat bread was larger than that of the control group for the enzyme-treated wheat bread (wheat bread ①～⑥. RTM.) and that of the fusion protein-treated wheat bread (wheat bread ①～③. RTM.) for the enzyme-treated wheat bread (wheat bread ④～⑥. RTM.) for the enzyme-treated wheat bread, respectively, and the specific volume of the wheat bread ③ was the largest, i.e., the specific volume of the wheat bread prepared by adding 13mg of the fusion protein per Kg of wheat flour was the largest.

Table 4 specific volume of wheat bread

Texture characteristics of wheat bread were measured by slicing the breads stored on days 0,1, 3, 5 and 7 into 3cm slices, and measuring the hardness and elasticity parameters of the wheat breads on different days of storage using a texture analyzer. The results of the texture property measurement of the wheat bread are shown in Table 5, and the addition of the fusion protein and the mixed enzyme significantly reduced the hardness of the wheat bread and improved the elasticity of the wheat bread, and after the wheat bread was stored at 4 ℃, the addition of the fusion protein and the mixed enzyme also significantly improved the hardness and the elasticity of the wheat bread. The fusion protein and the mixed enzyme are very effective for improving the texture characteristics of the wheat bread, the improvement effect is in direct proportion to the enzyme dosage, and the effect of the fusion protein on improving the texture characteristics of the wheat bread is obviously better than that of the mixed enzyme under the condition of adding the same amount of enzyme. The texture characteristic measurement result of the wheat bread shows that the texture characteristic improvement degree of wheat bread ③ is the most obvious, namely the texture characteristic improvement of the wheat bread prepared by adding 13mg of fusion protein to every Kg of wheat flour is the most obvious.

Table 5 texture characteristics of wheat bread

The above embodiments are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solutions of the present invention should fall within the protection scope defined by the claims of the present invention without departing from the design spirit of the present invention.

Claims

1. The phytase-pullulanase fusion protein Y-L4-T is characterized in that the amino acid sequence is shown as SEQ ID NO. 46.

2. A gene encoding the phytase-pullulanase fusion protein Y-L4-T according to claim 1, characterized in that the nucleotide sequence thereof is shown in SEQ ID No. 45.

3. A recombinant vector, characterized in that, the recombinant vector comprises the gene of claim 2.

4. A recombinant microorganism characterized in that, the recombinant microorganism comprises the recombinant vector of claim 3.

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

6. Use of the phytase-pullulanase fusion protein Y-L4-T according to claim 1 for the production of pasta.

7. The use according to claim 6, wherein the pasta comprises bread.

8. A method for improving specific volume and/or texture characteristics of bread, comprising the step of adding the phytase-pullulanase fusion protein Y-L4-T according to claim 1 during the bread making process.

9. Use of the gene of claim 2, the recombinant vector of claim 3 or the recombinant microorganism of claim 4 for the production of phytase-pullulanase fusion protein Y-L4-T.