CN1831109A - Recombinant bacteria of coding macrotherm phytase gene, synthesis, cloning and expression of said gene - Google Patents
Recombinant bacteria of coding macrotherm phytase gene, synthesis, cloning and expression of said gene Download PDFInfo
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- CN1831109A CN1831109A CN 200610038936 CN200610038936A CN1831109A CN 1831109 A CN1831109 A CN 1831109A CN 200610038936 CN200610038936 CN 200610038936 CN 200610038936 A CN200610038936 A CN 200610038936A CN 1831109 A CN1831109 A CN 1831109A
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- phytase
- ncphyn
- gene
- oligonucleotides
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- 229910052749 magnesium Inorganic materials 0.000 description 1
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- 238000012986 modification Methods 0.000 description 1
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- 230000000050 nutritive effect Effects 0.000 description 1
- 235000019198 oils Nutrition 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 235000021317 phosphate Nutrition 0.000 description 1
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 description 1
- 230000004962 physiological condition Effects 0.000 description 1
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- 230000003389 potentiating effect Effects 0.000 description 1
- 244000144977 poultry Species 0.000 description 1
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- 239000002243 precursor Substances 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 230000036632 reaction speed Effects 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 239000001632 sodium acetate Substances 0.000 description 1
- 235000017281 sodium acetate Nutrition 0.000 description 1
- 239000007974 sodium acetate buffer Substances 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 229940083982 sodium phytate Drugs 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 229910021655 trace metal ion Inorganic materials 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
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Abstract
The invention relates to a recombination and the gene and its compounding, cloning and expression of a coding high temperature phytase gene that could gain a coded high temperature phytase gene NcpyN, supply nucleotide sequence, recombinant plasmid pUC-NcphyN and yeast expression carrier pNCphyN, and the recombination yeast of pNCphyN to gain recombination by chemical and biology method. The invention could be used to improve the level and quality of phytase and achieve the purpose of improving species quality.
Description
Technical Field
A recombinant bacterium for coding a high-temperature phytase gene, the gene, synthesis, cloning and expression thereof belong to the field of microbial genetic engineering. Specifically, a set of oligonucleotides for coding novel high-temperature phytase is designed by means of genetic engineering, a gene total synthesis method is applied to obtain a gene NcphyN for coding novel high-temperature phytase, the gene NcphyN is cloned into a yeast expression vector, recombinant bacteria are obtained by the principle of homologous recombination, and then efficient expression and preparation of recombinant protein in yeast can be realized.
Technical Field
Phytic acid (phytic acid) is known by the chemical name inositol hexaphosphate, also known as inositol hexaphosphate, and consists of one molecule of inositol and six molecules of phosphoric acid. Molecular formula C6H18O24P6And the molecular weight is 660.08.
Phytic acid is widely found in plants, and is particularly abundant in grains, beans and oil crop seeds. The phosphorus in the plant seeds is mainly stored in the form of phytate phosphorus. Phytic acid is easily assimilated to many divalent metal ions, such as Ca2+、Mg2+、Fe2+、Zn2+、Mn2+、Cu2+And the combination forms phytate (complex salt or single salt), and the concentration of free ions is reduced.
Phytases (myo-inositol hexakisphosphate hydrolases; EC3.1.3.8) are a class of acid phosphatases that hydrolyze phytic acid and catalyze the decomposition of phytic acid to inorganic phosphate and inositol. This property makes phytase an important application in the feed industryIt has the meaning of usage. The plant-derived components account for a large proportion of the feed, and the phosphorus element in the plants is mainly in the form of phytic acid. The phytic acid content in some cereals and oil crops is even as high as 1-3%. Because the digestive tract of the monogastric animals is lack of an enzyme system for decomposing the phytic acid, the phytic acid cannot be decomposed by the monogastric animals to generate inorganic phosphorus, and the phytic acid is discharged out of the body along with the feces and enters the environment. Therefore, a large amount of phosphorus is wasted, people are required to add the phosphorus into the feed additionally, and a large amount of phosphorus is discharged into the environment, so that the environmental pollution is aggravated. In addition, phytic acid is also a potent anti-nutritional factor, and can chelate Ca2+、Fe2+、Zn2+The metal ions form insoluble phytate complex, and can form insoluble complex with positively charged protein and vitamins, which affects the absorption of protein and vitamins and reduces the nutritive value of feed. The feeding effect of phytase as a feed additive for monogastric animals has been well established: the utilization rate of phosphorus in the vegetable feed can be improved by 60 percent, the addition amount of inorganic phosphorus in the feed is reduced, the discharge amount of the inorganic phosphorus in animal manure is reduced by 40 percent, and the phosphorus pollution amount of the environment is reduced; the phytase can also relieve the chelation of phytic acid on metal ions and proteins, improve the absorption of monogastric animals on the metal ions, particularly trace metal ions, and improve the utilization rate of the proteins.
Phytase is mostly a single subunit glycoprotein with a composition and ratio of mannose to galactose to N-acetylglucosamine of 9: 1: 3, a molecular weight of 40-120kDa, an optimum pH range of 4.5-6.0, and an optimum temperature of 45-60 ℃. There are 3 types of Phytase known, phytate-3-phosphohydrolase (3-Phytase, EC3.1.3.8), phytate-6-phosphohydrolase (6-Phytase, EC3.1.3.26) and the non-characteristic orthophosphoric monoester phosphohydrolase (EC 3.1.3.2). The phytase enzyme is commonly referred to as phytate-3-phosphohydrolase and the gene encoding it is called phyA (Wys 1999, appl. environ. Microbiol). PhyA has 5 disulfide bonds in its higher structure. The disulfide bond mainly plays a role in the formation of a space structure in the enzyme activity center, so any denaturant which breaks the disulfide bond will cause the loss of the enzyme activity. In addition, disulfide bonds are also associated with thermotolerance of phytases. The crystal structure of PhyA consists of one large α' domain and one smaller α domain. The α' domain is centered on a β -sheet composed of 6 corresponding amino acid sequences. The alpha domain is composed of 14 alpha helices. The inner surfaces of the two domains have a deep recess in which the essential amino acids for the active center, Arg58 and His59, are the first two amino acids in the conserved sequence of the phytase active site, RHG × R × P. The zymoprotein molecule has 19 Arg. Arg58 in the RHGXRXP sequence is directly involved in its enzymatic activity, and Arg58 is also positioned in its crystal structure in agreement with its function. Arg58 in the substrate binding site RHGXRXP is considered as a residue necessary for maintaining enzyme activity, i.e., the substrate binding site forms an ES complex with a specific conformation by electrostatic adsorption using a positively charged residue thereof and a negatively charged substrate, and hydrolyzes the substrate by virtue of the function of the catalytic site. The loss of enzymatic activity is due to modification of the arginine R group of RHGXRXP in the substrate binding site. The crystal structure of PhyA in the microorganism has high similarity with the crystal structure of the murine low molecular acid phosphatase, indicating that it belongs to the histidine family of acid phosphatases. PhyB in the microorganism belongs to histidine acid phosphatase family and also has an active site conserved sequence RHG x R x P, but the optimal substrate of the PhyB is not phytate, so that the PhyB can only be regarded as the acid phosphatase with phytase activity. The bacterial phytase coding gene phyC is short, the gene has the full length of 1152, 383 amino acids are coded, the first 26 amino acids at the N end are signal peptides, the molecular weight of the coded enzyme protein is small, the nucleotide sequence and the coded amino acid sequence of the bacterial phytase coding gene have no homology with the phosphatase genes reported by phyA and phyB, and also have active site RHGXRXP conserved sequences of the amino acid sequences coded by phyA and phyB, which indicates that the bacterial phytase coding gene is not in a histidine acid phosphatase family. The phyA gene contains 1 intron and 2 optimal pH values; the phyB gene contains 3 introns and 1 optimal pH.
The phytase acts on the phytic acid, the phosphate groups on the phytic acid molecules are cut off one by one to form an intermediate product IP5、IP4、IP3、IP2The final products are inositol and phosphoric acid. Phytases can be divided into two classes, depending on the site of initiation of action: 3-phytase (EC 3.1.3.8) and 6-phytase (EC 3.1.3.26). The 3-phytase firstly hydrolyzes ester bonds from the 3 rd carbon site of phytic acid to release inorganic phosphorus, and then hydrolyzes ester bonds of other sites in sequence, and the enzyme needs divalent magnesium ions as prosthetic groups and is mainly present in microorganisms. The 6-phytase first hydrolyzes ester bonds starting from the 6 th carbon site of phytic acid, and such enzymes are mainly present in plants. Phytase does not completely decompose inositol phosphates, which requires the aid of acid phosphatase. The phytase is the best to be studied, namely the pseudomonas phytase, and the action mechanisms of the wheat bran phytase and the aspergillus ficuum phytase are also preliminarily understood. It is theorized that 281.6mg of inorganic phosphorus could be released by complete decomposition of 1g of phytic acid.
Phytase is widely found in nature and is found in animals, plants and microorganisms. The phytase protein sequences registered at present are reported to be close to 200, most of which are found in nature, and also comprise a plurality of artificially modified phytase proteins, and new natural enzymes are continuously discovered. However, the natural phytases which can be used in the feed industry are not very numerous. This is mainly limited by the following two factors: firstly, the optimum pH value of the phytase fed to monogastric animals is preferably consistent with the pH environment of the digestive system organs of the animals, such as stomach, intestine and the like, so that the ingested phytase can be favorably exerted. It has been reported (Hanzhengkang, 1991) that chyme pH in pig stomach is between 1.8 and 3.6 and that duodenal chyme pH is between 3.9 and 7.0. The pH of chicken stomach paste is 4.5-4.8, and the pH of duodenum is 5.7-6.0. Therefore, when the optimum pH range of the phytase is wider, the phytase has stronger environmental adaptability to the pH of the digestive tract of animals such as pigs, chickens and the like. Secondly, the heat treatment during the feed processing, such as the processes of granulating, expanding and the like, has a direct influence on the activity of the phytase, and many phytases cannot withstand relatively high processing temperatures (usually 75 ℃ to 93 ℃), thereby limiting the application thereof (Wanghongning 2000, proceedings of Sichuan agriculture university).
The emphasis in the study of phytases is currently on phytases of microbial origin, in particular of fungal origin, as has been reported for example in Aspergillus ficuum, A. fumigatus, A. niger, A. terrus, A. oryzae, Emericella nidulans, Mycleophthora thermophila, Neurosporacracrassa, Thermomyces lanuginose (Berka 1998, Appl. environ. Microbiol.; Mitchell 1997, Microbiology; Zhou, et al 2006, FEMS Microbiol Lett) and the like. Compared with phytase from plant sources, microbial phytase has a wide pH range, some pH values are between 2.5 and 5.5, and the pH value is close to the gastrointestinal physiological conditions of monogastric animals. The phytase from plant has pH value of 5.0-7.5, poor heat resistance, and can not tolerate granulation temperature of 80 deg.C during feed processing (Simons 1990, Br J Nutr). The microorganisms currently used for the industrial production of phytases are mainly aspergillus niger, aspergillus ficuum, aspergillus oryzae, etc., which are capable of secreting extracellular enzymes with higher activity and have an action pH in the acidic range. These enzymes have good enzymatic activity at 37 ℃ but are also not well able to withstand the high temperatures of granulation. The high-temperature phytase separated from mesophilic microorganisms M, thermophila, A, terreus and the like has the optimum temperature of 70-80 ℃, has good temperature resistance, but has extremely low enzyme activity at 37 ℃ (the action temperature of the phytase in animal bodies), and has no use value. Therefore, how to make the enzyme capable of resisting high temperature for a short time and having high enzyme activity at normal body temperature of animals is a problem which needs to be solved urgently by the feeding enzyme preparation including phytase at present. In addition, the phytase fed to monogastric livestock and poultry belongs to an acidic phytase, which is not suitable for freshwater-cultured carps, because the carps have no stomach and only have intestinal tracts with neutral pH, and the neutral phytase with the optimal pH being neutral is required to be used for the phytase added to fish feed to play a role. The development of neutral phytases is also a focus of research. Recently reported bacillus phytase has nearly neutral optimum pH value, high enzyme activity and good heat stability, and is possibly widely applied to fish feed (Choi 2001, J protein chem).
Phytase has been studied for nearly 40 years. In the mid-70's of the 20 th century, limiting phosphorus excretion from the aquaculture became one of the core contents of the environmental protection council guide implemented by the members of the European Union. Europe has begun to strive to find solutions to the overall problem of inorganic phosphorus contamination. Until 1980, university of Wageningen, the Netherlands found phytase gene, and the function of phytase was confirmed by Gist-brocades company (the precursor of DSM), and the development of phytase became possible. Pasf in Germany (BASF) and Gist-brocades have led to the first successful commercial production of phytase in A.niger in 1989 using gene transfer technology. Full approval by the european feed supplement authority was obtained in 1990, and the enzyme tamehos (Natuphos) is marketed under the trade name. From now on, a great amount of manpower and material resources are invested in development and research in various countries mainly including universities, research institutes and various commercial research institutions. At present, at least 10 varieties are produced by several companies in international markets, and at least 3-5 enterprises in China have developed phytase products. There are also more enterprises and research institutions engaged in the development of phytases.
There are roughly two approaches to the development of phytase: firstly, improving the existing strain by a traditional genetic method to obtain a strain with better performance; secondly, cloning phytase genes with excellent performance by means of genetic engineering and introducing the phytase genes into a proper host to obtain excellent recombinant bacteria. Both of the above approaches have been reported as successful examples. For example, Marisa et al, 1994, UV-induced mutagenesis of Aspergillus ficuum NRLL3115, selected a mutant strain with 3.3 times enzyme yield as that of the wild strain. In 1997, Chenhongge et al, perform ultraviolet and nitrosoguanidine mutagenesis on Aspergillus niger MAO21 to obtain a phytase high-activity strain, and the enzyme activity of the phytase high-activity strain is 3.6 times of that of the original strain. In 1991 Van Gorcom et al cloned phytase gene phyA of Aspergillus ficuum into Aspergillus niger CBS513.88 under the control of amyloglucosidase promoter for expression, and the enzyme yield was increased 1400 times. In 1998, the screened phytase gene of the aspergillus niger with high phytase yield is cloned into pichia pastoris, and the expression quantity is 3000 times higher than that of the original strain. And simultaneously develops a process for producing the feed additive phytase with a bioreactor in a large scale and at low cost. However, this phytase has a not negligible drawback: cannot tolerate higher temperatures and the unit of enzyme activity is largely lost during processing (Kim 1998, Enz microbiological Tech). The phytase isolated from A.fumiga is able to withstand high temperatures of 90-100 ℃ and to degrade phytic acid over a wide pH range, and therefore has great market potential (Vall Loon 1998, Appl Environ Microbiol). The phytase gene sequence of A.fumigatus is used for efficiently expressing the phytase in yeast by using the technologies of chemical synthesis, in vitro gene rearrangement and the like, and the enzyme activity is 130,000 u/ml. The expression level is 13 times of that of the wild type gene. After high-density fermentation, the protein expression amount is 5.6g per liter. However, the enzyme has the following disadvantages: the specific enzyme activity of the recombinant phytase after gene rearrangement is only 23,000u/mg (Chinese patent 02136531.8), which is only one fourth of the wild type A.niger phytase.
Gene Ncphy (GenBank AY536581) encoding phytase was previously cloned from Neurospora crassa (N.crassa) CICICIM F00021 in this laboratory and preliminary expression and analysis of enzymatic properties were performed in Pichia pastoris (Zhou et al 2006, FEMS Microbiol Lett; Shenmi et al 2006, microbiological report). The characteristics of the enzyme are suitable for the application of the feed industry, such as obvious high temperature resistance, 80 ℃ and 20min, 58% of enzyme activity is reserved, and under the same condition, the Aspergillus niger phytase only saves 28% of enzyme activity; has wide pH validity and stability, has phytase activity between pH2.0-8.0, and has better pH stability between pH3.5-9.5; has better resistance to environmental factors, Mg2+、Ca2+、Al3+、Fe2+、Co2+、Zn2+And EDTA and the like have little influence on the enzyme activity.
Disclosure of Invention
The invention aims to provide a recombinant bacterium for coding a high-temperature phytase gene, a gene, synthesis, cloning and expression thereof, wherein a high-temperature phytase gene efficiently expressed in yeast is searched by adopting chemical synthesis and molecular in vitro combination technologies, and the recombinant bacterium is obtained by the principle of homologous recombination, so that efficient expression and preparation of recombinant protein in the yeast can be realized.
The technical scheme of the invention is as follows: provides a recombinant bacterium for coding a high-temperature phytase gene, which is named as Pichia pastoris CICICIM MMY0018, is preserved in China center for type culture Collection with the preservation number of CCTCC M206008. The preparation method of the recombinant strain CCTCC M206008 of the phytase gene is that the constructed recombinant plasmid pNCphyN is transferred into Pichia pastoris ACCC2124 by an electric transformation method according to the homologous recombination principle, and the obtained recombinant strain CCTCC M206008 finally realizes the expression of the high-temperature phytase gene NcphyN in the recombinant strain. And analyzing genomes of the saccharomyces cerevisiae and the pichia pastoris by tRNA analysis software to obtain tRNA coding gene characteristics and codon usage frequency parameters. Based on the nucleotide sequence of Neurospora crassa (N.crassa) CICICIM F00021 phytase encoding gene (GenBank AY536581), DNMAN software is used to design a group of 80 oligonucleotides for new gene synthesis, and a DNA synthesizer is used to obtain the oligonucleotide group through chemical synthesis, wherein the oligonucleotides in the group are adjacent on the same chain, the complementary chain has several overlapped basic groups, and the melting temperature of the overlapped region is controlled at 45-50 ℃. The full-length gene NcphyN is obtained by the synthesis of the whole gene by using a two-step in vitro splicing and amplification technology (see example 2 for details). The gene NcphyN was cloned into the EcoRI site of pUC19 to obtain a recombinant plasmid pUC-NcphyN. The recombinant plasmid pUC-NcphyN was used for sequencing NcphyN and for providing NcphyN for subsequent experiments. EcoRI enzyme cuts recombinant plasmid pUC-NcphyN, electrophoresis separation, tapping recovery obtain full length NcphyN, the upstream of the 5' end is connected with a methanol inducible promoter and an MF alpha signal peptide sequence which are obtained by synthesis, the combined gene fragment is cloned into the multiple cloning sites of a yeast integrative vector pRS303K (Christof taxi and Michael Knop. biotechnique 40: 73-78), and the yeast expression vector pNCphyN of the high temperature NcphyN is obtained; the recombinant plasmid pNCphyN is transferred into pichia pastoris ACCC2124 by an electrotransformation method according to a homologous recombination principle, recombinant bacteria pichia pastoris CCTCC M206008 containing gene Ncphy N is constructed, and recombinase is prepared.
The nucleotide sequence of gene NcphyN is as follows:
gaattcatgt taagggttct atctccaaat ccagctagtt gcgactctcc agaattgggg 60
tatcagtgca attctgaaac tacacatact tggggccaat acagcccttt tttttctgtc 120
ccgagtgaaa tcagcccaag cgtgccagag ggttgcagac tgactttcgc gcaagtactg 180
agtagacatg gcgccagatt ccccactcca ggcaaagctg ccgccatttc cgctgtttta 240
accaagatta agacgtctgc tacttggtat gcgcccgatt ttgaatttat caaggattat 300
aactatgtat tgggtgtaga tcacctaaca gctttcggtg agcaggaaat ggttaactct 360
ggtattaagt tctaccagcg ttatgctagt ctgattagag attatacaga tcctgaatct 420
ctgcctttta tcagagcatc aggtcaggaa agggtaattg cgtcggcaga aaatttcact 480
accggatttt actctgcgtt gcttgccgat aagaaccctc ctccaagctc tctgccactt 540
ccacgtcaag aaatggtcat tatatcggaa tcccctacgg caaacaatac tatgcatcat 600
ggtttgtgta gagcgttcga ggattctaca acaggtgatg ctgcccaagc taccttcatc 660
gctgcaaact ttccacctat cactgctaga ctgaacgcac aaggttttaa aggagtgacg 720
ttatctgata ccgatgtcct ttcattgatg gacctaaggc ccttcgatac tgtcgcctac 780
ccaccttcat cgagcttaac aacctcaagc tcaccatccg ggggcagtaa actatctccc 840
ttctgtagtc tttttacggc gcaggatttt accgtttatg actatttaca atccctaggc 900
aaattttacg gctacgggcc aggtaactct ttggcagcta ctcagggtgt cggctacgtc 960
aacgagttac tagcgagatt gacagtctca ccggtcgtag ataacactac tacaaattct 1020
acactggatg gaaatgaaga tactttccca ttatcgagaa atcgtactgt ttttgcagat 1080
tttagtcacg acaacgatat ggtcggtata ttgactgccc taaggatctt cgaaggtgtc 1140
gatgccgaga agatgatgga caatacaact atcccaagag agtatggtga gacgggcgac 1200
gatcctgcta acttgaagga aagggagggg ctatttaagg taggttgggt tgtcccattt 1260
gcagctagag tttattttga gaaaatgata tgcgatggtg atggaagtgg tgagatggtt 1320
caatctgagg aggaacagga caaagagtta gtacgtatac tagtcaacga tcgtgtggtt 1380
aagttgaatg gatgtgaagc tgacgagctg gggagatgca aacttgataa gttcgtagaa 1440
tctatggaat ttgcaaggag aggtggtgat tgggacaaat gttttgct 
attc 1498 wherein at least one of,
is a stop codon.
The invention has the beneficial effects that:
1. the phytase of the invention has obvious high specific enzyme activity, and the specific enzyme activity is 125,000 u/mg. Is 1.2 times of Aspergillus niger wild phytase and 5.1 times of A. Fumigatus recombinant phytase.
2. The recombinant bacterium of the invention expresses phytase and is greatly improved, and the enzyme production level is improved by 3050 times compared with that of a wild strain. The loss amount of the phytase in the preparation process is effectively improved.
Drawings
FIG. 1 shows the constructed bacterial cloning phytase vector.
FIG. 2 Yeast expression Phytase vectors.
FIG. 3 shows the protein electrophoresis pattern of recombinant yeast expressing phytase. Lane 1: protein molecular weight markers comprising a ligand phosphatase b (97,400), bone serum albumin (66,200), a ligand action (43,000), a bone carbonic anhydride (31,000), a trypsin inhibitor (20,100), a hen eg white lysozyme (14, 400); lane 2: purifying phytotase; lane 3: unpurified phytase.
Biological material sample preservation
The recombinant strain Pichia pastoris CICIM MMY0018 containing the gene coding the high-temperature phytase has been preserved in China center for type culture collection (CCTCCM206008) with the preservation date of 2006, 1 month and 14 days.
Detailed Description
Example 1: design of oligonucleotide sequences for the total synthesis of high temperature phytase genes and chemical synthesis of oligonucleotides.
Based on the nucleotide sequence of the gene (GenBank AY536581) encoding phytase of Neurospora crassa (N.crassa) CICICIM F00021(AS 3.1604), a group of 80 oligonucleotides is designed by using DNAMAN software, and the group of oligonucleotides is obtained by chemical synthesis by using a DNA synthesizer, wherein the nucleotide sequences of the 80 oligonucleotides are AS follows:
F0 cat gaattca tgttaagggt tctatctcca aatccagcta gttgc
R0 ccccaattct ggagagtcgc aactagctgg atttgg
F1 gactctccag aattggggta tcagtgcaat tctgaaac
R1 ggccccaagt atgtgtagtt tcagaattgc actgata
F2 tacacatact tggggccaat acagcccttt tttttctg
R2 gctgatttca ctcgggacag aaaaaaaagg gctgtatt
F3 tcccgagtga aatcagccca agcgtgccag agg
R3 cgaaagtcag tctgcaaccc tctggcacgc ttgg
F4 gttgcagact gactttcgcg caagtactga gtagac
R4 gggaatctgg cgccatgtct actcagtact tgcg
F5 atggcgccag attccccact ccaggcaaag ctg
R5 acagcggaaa tggcggcagc tttgcctgga gtg
F6 ccgccatttc cgctgtttta accaagatta agacgtc
R6 gcgcatacca agtagcagac gtcttaatct tggttaaa
F7 tgctacttgg tatgcgcccg attttgaatt tatcaagga
R7 tctacaccca atacatagtt ataatccttg ataaattcaa aatcgg
F8 ttataactat gtattgggtg tagatcacct aacagctttc g
R8 taaccatttc ctgctcaccg aaagctgtta ggtga
F9 gtgagcagga aatggttaac tctggtatta agttctacc
R9 tcagactagc ataacgctgg tagaacttaa taccagagt
F10 agcgttatgc tagtctgatt agagattata cagatcctga a
R10 ctctgataaa aggcagagat tcaggatctg tataatctct aa
F11 tctctgcctt ttatcagagc atcaggtcag gaaagg
R11 ctgccgacgc aattaccctt tcctgacctg atg
F12 gtaattgcgt cggcagaaaa tttcactacc ggatttta
R12 ggcaagcaac gcagagtaaa atccggtagt gaaatttt
F13 ctctgcgttg cttgccgata agaaccctcc tcca
R13 gaagtggcag agagcttgga ggagggttct tatc
F14 agctctctgc cacttccacg tcaagaaatg gtca
R14 cgtaggggat tccgatataa tgaccatttc ttgacgtg
F15 ttatatcgga atcccctacg gcaaacaata ctatgcatca
R15 acgctctaca caaaccatga tgcatagtat tgtttgc
F16 tggtttgtgt agagcgttcg aggattctac aacagg
R16 tagcttgggc agcatcacct gttgtagaat cctcga
F17 tgatgctgcc caagctacct tcatcgctgc aaa
R17 gcagtgatag gtggaaagtt tgcagcgatg aagg
F18 ctttccacct atcactgcta gactgaacgc acaag
R18 ataacgtcac tcctttaaaa ccttgtgcgt tcagtcta
F19 gttttaaagg agtgacgtta tctgataccg atgtcctttc
R19 ggccttaggt ccatcaatga aaggacatcg gtatcag
F20 attgatggac ctaaggccct tcgatactgt cgcc
R20 ctcgatgaag gtgggtaggc gacagtatcg aag
F21 tacccacctt catcgagctt aacaacctca agctca
R21 tactgccccc ggatggtgag cttgaggttg ttaag
F22 ccatccgggg gcagtaaact atctcccttc tgtagt
R22 tcctgcgccg taaaaagact acagaaggga gatagtt
F23 ctttttacgg cgcaggattt taccgtttat gactatttac a
R23 cgtaaaattt gcctagggat tgtaaatagt cataaacggt aaaa
F24 atccctaggc aaattttacg gctacgggcc aggtaac
R24 ctgagtagct gccaaagagt tacctggccc gtagc
F25 tctttggcag ctactcaggg tgtcggctac gtcaa
R25 caatctcgct agtaactcgt tgacgtagcc gacacc
F26 cgagttacta gcgagattga cagtctcacc ggtcgta
R26 tgtagaattt gtagtagtgt tatctacgac cggtgagact gt
F27 gataacacta ctacaaattc tacactggat ggaaatgaag atact
R27 cgatttctcg ataatgggaa agtatcttca tttccatcca g
F28 ttcccattat cgagaaatcg tactgttttt gcagatttta gt
R28 accatatcgt tgtcgtgact aaaatctgca aaaacagta
F29 cacgacaacg atatggtcgg tatattgact gccc
R29 gacaccttcg aagatcctta gggcagtcaa tataccg
F30 taaggatctt cgaaggtgtc gatgccgaga agatgatg
R30 tcttgggata gttgtattgt ccatcatctt ctcggcatc
F31 gacaatacaa ctatcccaag agagtatggt gagacgggc
R31 caagttagca ggatcgtcgc ccgtctcacc atactc
F32 gacgatcctg ctaacttgaa ggaaagggag gggc
R32 caacccaacc taccttaaat agcccctccc tttcctt
F33 tatttaaggt aggttgggtt gtcccatttg cagctagag
R33 cgcatatcat tttctcaaaa taaactctag ctgcaaatgg ga
F34 tttattttga gaaaatgata tgcgatggtg atggaagtgg t
R34 cctcagattg aaccatctca ccacttccat caccat
F35 gagatggttc aatctgagga ggaacaggac aaagagt
R35 tcgttgacta gtatacgtac taactctttg tcctgttcct
F36 tagtacgtat actagtcaac gatcgtgtgg ttaagttgaa
R36 cgtcagcttc acatccattc aacttaacca cacga
F37 tggatgtgaa gctgacgagc tggggagatg caa
R37 tagattctac gaacttatca agtttgcatc tccccagct
F38 acttgataag ttcgtagaat ctatggaatt tgcaaggaga g
R38 tttgtcccaa tcaccacctc tccttgcaaa ttcca
F39 gtggtgattg ggacaaatgt tttgcttaat gaattcgc
R39 ctgccaagtt caagactagc gaattcatta agcaaaaca
example 2 Total Synthesis and cloning of the hyperthermostase Gene NcphyN
The total synthesis of the high-temperature phytase gene NcphyN is carried out in two steps by adopting oligonucleotide in-vitro splicing and amplification technology. In the first step, 5. mu.l of 10 XPCR buffer, 4. mu.l of 2.5mmol/L dNTPs, 1-20. mu. mol/L of all 80 oligonucleotides and 0.5U of Pfu DNA polymerase plus double distilled water are added to 50. mu.l of the reaction system. Oligonucleotide splicing PCR amplification conditions were 1 × (95 ℃ for 2 min); 35 × (94 ℃ for 30s, 45-52 ℃ for 30s, 70 ℃ for 2min for 30 s); 1 × (70 ℃ C. for 10 min). In the second step, 2. mu.l of the oligonucleotide ligation product, 5. mu.l of 10 XPCR buffer, 4. mu.l of 2.5mmol/L dNTPs, 4. mu.l of 1mmol/L primer F01. mu.l, 1mmol/L primer R391. mu.l and 0.5U of Pfu DNA polymerase were added to a 50. mu.l reaction system and made up to 50. mu.l with double distilled water. PCR amplification conditions were 1 × (95 ℃ C. for 2 min); 35 × (95 ℃ 30s, 52 ℃ 30s, 70 ℃ 2min30 s); 1 × (70 ℃ C. for 10 min). The underlined part of the sequence of primer F0 is an artificially introduced EcoRI cleavage site, and the underlined part of the sequence of primer R39 is an artificially introduced EcoRI cleavage site.
The oligonucleotides designed by NcphyN were synthesized, the codons were yeast-preferred codons, the length of the oligonucleotides varied from 28 to 41 bases, and both ends of adjacent oligonucleotides contained complementary sequences of 15 to 25 bases.
Cloning of NcphyN: the synthesized gene NcphyN was artificially introduced with EcoRI cleavage sites at both ends of the gene by primer F0 and primer R39. The obtained synthetic gene was purified, digested with EcoRI and cloned into the EcoRI site of pUC19 to obtain a cloning vector pUC-NcphyN, and sequencing confirmed that the gene synthesis was successful, as shown in FIG. 1. The homology of the gene NcphyN obtained by synthesis with the gene encoding Neurospora crassa (N. crassa) CICICIMF 00021 phytase (GenBank AY536581) is 73.7%.
Example 3: construction of Yeast expression Phytase vector
Cutting pUC-NcphyN with EcoRI, electrophoretically separating, cutting to recover NcphyN, connecting the methanol inducible promoter obtained by synthesis and MF alpha signal peptide sequence (gccatccgacatccacaggtccattct) at 5' end upstreamcacacataagtgccaaacgcaacaggaggggatacactagcagcagaccgttgcaaacgcaggacctccactcctcttctcctcaacacccacttttgccatcgaaaaaccagcccagttattgggcttgattggagctcgctcattccaattccttctattaggctactaacaccatgactttattagcctgtctatcctggcccccctggcgaggttcatgtttgtttatttccgaatgcaacaagctccgcattacacccgaacatcactccagatgagggctttctgagtgtggggtcaaatagtttcatgttccccaaatggcccaaaactgacagtttaaacgctgtcttggaacctaatatgacaaaagcgtgatctcatccaagatgaactaagtttggttcgttgaaatgctaacggccagttggtcaaaaagaaacttccaaaagtcgccataccgtttgtcttgtttggtattgattgacgaatgctcaaaaataatctcattaatgcttagcgcagtctctctatcgcttctgaaccccggtgcacctgtgccgaaacgcaaatggggaaacacccgctttttggatgattatgcattgtctccacattgtatgcttccaagattctggtgggaatactgctgatagcctaacgttcatgatcaaaatttaactgttctaacccctacttgacagcaatatataaacagaaggaagctgccctgtcttaaacctttttttttatcatcattattagcttactttcataattgcgactggttccaattgacaagcttttgattttaacgacttttaacgacaacttgagaagatcaaaaaacaactaattattcgaaggatccaaacgatgagatttccttcaatttttactgcagttttattcgcagcatcctccgcattagctgctccagtcaacactacaacagaagatgaaacggcacaaattccggctgaagctgtcatcggttactcagatttagaaggggatttcgatgttgctgttttgccattttccaacagcacaaataacgggttattgtttataaatactactattgccagcattgctgctaaagaagaaggggtatctctcgagaaaagagaggctgaagcttacgta gaattc) Cloning the combined gene fragment into a multiple cloning site of a yeast integrative vector pRS303K to obtain a yeast expression vector pNCphyN of the high-temperature NcphyN; as shown in fig. 2.
Example 4: construction of recombinant Yeast expressing Phytase
The activated Pichia pastoris ACCC2124 strain was cultured in 500ml YPD (1% yeast extract, 2% peptone, 2% glucose) at 30 ℃ for 18h to OD600The cells were collected by centrifugation at 5000r/min, washed with 500ml of pre-cooled sterile water, 250ml of pre-cooled sterile water, centrifuged to remove the supernatant, and suspended with 20ml of pre-cooled 1mol/L sorbitol. After centrifugation, the thalli are suspended by 0.5ml of precooled sorbitol and used as competent cells for electric shock transformation.
A large amount of yeast expression vector pNCphyN was extracted, 0.1. mu.g of purified pNCphyN was added to 50. mu.l of P.pastoris ACCC2124 competent cells, and the cells were shocked with a Bio-Rad GenePulser shock apparatus for 5min in ice bath with parameters of 2.5Kv and 25 uF. After the electric shock is finished, 1.0ml of precooled 1mol/L sorbitol is immediately added, 200ul of the mixture is taken and coated on YPD-G418 (2 mg/ml of antibiotic G418 is supplemented in a YPD culture medium and purchased from Sigma company), a transformant is identified by a yeast colony PCR method for a well-grown colony, and the positive recombinant yeast Pichia pastoris CICICICIM MMY0001-MMY0081 is used for a phytase production fermentation test.
High-density fermentation of recombinant yeast strains: the obtained 5 recombinant Pichia pastoris strains were inoculated in 20mL YPD liquid medium, and shake-cultured at 30 ℃ until stationary phase. The cells were collected by centrifugation and transferred to 100mL of MGY medium (1.34% YNB, 1% glycerol, 4X 10)-5% biotin) at 30 deg.C under vigorous shaking at 200r/min for 48h, centrifuging to collect thallus, transferring into 30mL MM medium (1.34% YNB, 0.5% methanol, 4X 10-5% biotin) was cultured in a liquid medium at 30 ℃ for 7 days with vigorous shaking, and phytase activity was measured every 12 hours. The enzyme activity of the recombinant bacterium fermentation liquor is different from 10,000 to 56,000 u/mL. Wherein, the highest phytase production level of the recombinant yeast CICICIM MMY0018(CCTCC M206008) is 56,800u/mL, which is 3050 times higher than the enzyme production level of the wild strain Neurospora crassa (N.crassa) CICIM F00021.
Example 5: preparation and purification of Phytase
Freezing and centrifuging at 8,000r/min × 10min to collect fermentation liquid of recombinant yeast CICIM MMY0018(CCTCCM206008), placing into a dialysis belt, dialyzing and desalting, suspending the dialysis belt in physiological saline, maintaining the temperature at 4 deg.C during dialysis, and dialyzing for 2d until the fermentation liquid is substantially transparent and colorless. 15mL of the dialyzed fermentation broth was centrifuged at 4500g for 25min using Amicon Ultrafiltration-15 (Biomax 10K; Millipore) to obtain a final volume of about 500. mu.L of liquid with a concentration factor of about 30. Then, 500. mu.L of the resulting concentrated solution was separated by Superdex 200column (pharmacia Biotech) using 0.25M, pH5.5 sodium acetate solution as an eluent. The eluates were collected in fractions, 1mL of liquid was collected per tube. And (4) according to the test tube number corresponding to the detected protein peak, determining the phytase activity. Mixing the eluates with enzyme activity, loading onto anion exchange chromatography column DEAE-52, and gradient eluting with 0-1.0mol/L NaCl, 0.25mol/L NaAc, pH6.0. The collection tubes from which significant enzyme activity was detected were concentrated by amicon Ultrafiltration-15 (Biomax 10K; Millipore) and subjected to SDS-PAGE. Protein concentration was determined by the Bradford method using bovine serum albumin as a standard. Compared with the empty vector control, the recombinant bacterium fermentation solution has an obviously more protein band. The electrophoresis pattern result is shown in figure 3, the recombinase is separated and purified to have uniform electrophoresis, the molecular weight is close to 60kDa, and the recombinase is a single subunit protein. The specific enzyme activity was 125,000 u/mg.
Example 6: phytase enzymological Property analysis
Optimum pH of recombinant enzyme
pH buffer solution: 0.2mol/L glycine-hydrochloric acid buffer (pH 2.0, 2.5, 3.0, 3.5), 0.2mol/L sodium acetate buffer (pH 4.0, 4.5, 5.0, 5.5), 0.2mol/L imidazole-hydrochloric acid buffer (pH 6.0, 6.5), 0.2mol/L Tris buffer (pH 7.0, 8.0). 120. mu.L of recombinase pure enzyme (30. mu.g/mL) was diluted to 480. mu.L, and 40. mu.L of the enzyme activity was measured at pH2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, and 8.0, respectively. Calculating relative enzyme activity to determine the optimum pH. Under the determination condition, the optimum pH value of the recombinase is 3.5-5.5, and the enzyme activity reaches the maximum value when the pH value is 5.5. Has activity at pH of 2.0-8.0.
Determination of optimum reaction temperature for recombinant enzyme
120 mu L of recombinase pure enzyme (30 mu g/mL) is diluted to 480 mu L, 40 mu L of recombinase pure enzyme is taken to measure the enzyme activity at the temperature of 25, 37, 45, 50, 55, 60, 65, 70 and 80 ℃, and the relative enzyme activity is calculated to determine the optimal temperature. Under the measurement conditions, the optimum temperature of the recombinant enzyme was 60 ℃. Maintaining good activity at 25-70 deg.C.
Determination of the stability of the recombinant enzyme to temperature
70 mu L of recombinase pure enzyme (30 mu g/mL) is diluted to 280 mu L, 15 mu L of recombinase pure enzyme is taken out and is respectively bathed in temperature of 30, 40, 50, 60, 70, 80 and 90 ℃ for 10, 20 and 60min, the recombinase pure enzyme is immediately taken out, and the enzyme activity is measured at 37 ℃ after ice bath for 30 min. And calculating relative enzyme activity and comparing the stability of the recombinase to the temperature. The study on the temperature stability of the recombinase shows that 39-58% of relative enzyme activity still remains after the recombinase is heated at 70-90 ℃ for 20min, and the recombinase is higher than the aspergillus niger phytase expressed by pichia pastoris reported in documents.
Comparison of recombinase and commercial Phytase thermostability
Heating the recombinant enzyme and the commercial enzyme at 80 ℃ for 5min, 10min, 15 min and 20min respectively, immediately taking out, carrying out ice bath for 30min, measuring the enzyme activity at 37 ℃, and calculating the relative enzyme activity. The recombinant enzyme shows heat activation phenomenon after being heated at 80 ℃. The recombinase and the commercial enzyme are compared in thermal stability at 80 ℃, and the decrease of the recombinase is smaller than that of the commercial enzyme when the recombinase is heated for 15 min and 20 min.
Determination of the pH stability of the recombinant enzymes
70. mu.L of recombinase-purified enzyme (30. mu.g/mL) was diluted to 280. mu.L, and 15. mu.L of the enzyme was left at room temperature for 24 hours at pH2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, and 9.5. Then, the enzyme activity is measured under the conditions of 37 ℃ and pH5.5, and the relative enzyme activity is calculated to compare the stability of the recombinase to the pH value. The enzyme has a pH value of 3.5-9.5, and has good stability.
Recombinase kinetic parameter KmAnd VmaxDetermination of value
70 mu L of recombinase pure enzyme (30 mu g/mL) is diluted to 280 mu L, 20 mu L of recombinase pure enzyme is placed in sodium phytate with different concentrations (5, 2.5, 1.25, 1, 0.5 and 0.25mM) of substrate for enzyme activity determination, the reaction speed is expressed by the amount of inorganic phosphorus generated per min (mu mol), and K is calculated by adopting a Lineweaver-Burk double reciprocal plot methodmAnd VmaxThe value is obtained. KmAnd VmaxThe values were 228M and 128 mol/mg. min, respectively.
Effect of different Metal ions on the Activity of recombinant enzymes
mu.L of recombinase-pure enzyme (30. mu.g/mL) was added to 280. mu.L of 1mM Zn, respectively2+、Mn2+、Mg2+、Ca2+、Al3+Of equal metalStanding in saline solution at room temperature for 30 min. Then 200 mul NaAc is added until the volume is 500 mul, then the enzyme activity is measured, the enzyme activity measuring method is the same as the previous method, and the relative enzyme activity is calculated. Mg (magnesium)2+、Ca2+、Al3+、Fe2+、Co2+、Zn2+And EDTA and the like have little influence on the enzyme activity.
Claims (5)
1. A recombinant bacterium for coding a high-temperature phytase gene is named as Pichia pastoris CICICIMMMY 0018, which is preserved in China center for type culture Collection with the preservation number of CCTCC M206008.
2. The preparation method of the recombinant strain CCTCC M206008 of the phytase gene as claimed in claim 1, which is characterized in that the constructed recombinant plasmid pNCphyN is transferred into Pichia pastoris ACCC2124 by an electric transformation method according to the homologous recombination principle, and the obtained recombinant strain CCTCC M206008 finally realizes the expression of the high-temperature phytase gene Ncphy N in the recombinant strain.
3. An artificially synthesized high-temperature phytase gene NcphyN which realizes the expression in the recombinant strain CCTCC M206008 of claim 1 and has the nucleotide sequence as follows:
gaattcatgt taagggttct atctccaaat ccagctagtt gcgactctcc agaattgggg 60
tatcagtgca attctgaaac tacacatact tggggccaat acagcccttt tttttctgtc 120
ccgagtgaaa tcagcccaag cgtgccagag ggttgcagac tgactttcgc gcaagtactg 180
agtagacatg gcgccagatt ccccactcca ggcaaagctg ccgccatttc cgctgtttta 240
accaagatta agacgtctgc tacttggtat gcgcccgatt ttgaatttat caaggattat 300
aactatgtat tgggtgtaga tcacctaaca gctttcggtg agcaggaaat ggttaactct 360
ggtattaagt tctaccagcg ttatgctagt ctgattagag attatacaga tcctgaatct 420
ctgcctttta tcagagcatc aggtcaggaa agggtaattg cgtcggcaga aaatttcact 480
accggatttt actctgcgtt gcttgccgat aagaaccctc ctccaagctc tctgccactt 540
ccacgtcaag aaatggtcat tatatcggaa tcccctacgg caaacaatac tatgcatcat 600
ggtttgtgta gagcgttcga ggattctaca acaggtgatg ctgcccaagc taccttcatc 660
gctgcaaact ttccacctat cactgctaga ctgaacgcac aaggttttaa aggagtgacg 720
ttatctgata ccgatgtcct ttcattgatg gacctaaggc ccttcgatac tgtcgcctac 780
ccaccttcat cgagcttaac aacctcaagc tcaccatccg ggggcagtaa actatctccc 840
ttctgtagtc tttttacggc gcaggatttt accgtttatg actatttaca atccctaggc 900
aaattttacg gctacgggcc aggtaactct ttggcagcta ctcagggtgt cggctacgtc 960
aacgagttac tagcgagatt gacagtctca ccggtcgtag ataacactac tacaaattct 1020
acactggatg gaaatgaaga tactttccca ttatcgagaa atcgtactgt ttttgcagat 1080
tttagtcacg acaacgatat ggtcggtata ttgactgccc taaggatctt cgaaggtgtc 1140
gatgccgaga agatgatgga caatacaact atcccaagag agtatggtga gacgggcgac 1200
gatcctgcta acttgaagga aagggagggg ctatttaagg taggttgggt tgtcccattt 1260
gcagctagag tttattttga gaaaatgata tgcgatggtg atggaagtgg tgagatggtt 1320
caatctgagg aggaacagga caaagagtta gtacgtatac tagtcaacga tcgtgtggtt 1380
aagttgaatg gatgtgaagc tgacgagctg gggagatgca aacttgataa gttcgtagaa 1440
tctatggaat ttgcaaggag aggtggtgat tgggacaaat gttttgct 
attc 1498
wherein,
is a stop codon.
4. The method for synthesizing, cloning and expressing the hyperthermostase gene NcphyN as claimed in claim 3, wherein the method comprises
(A) Synthesis of NcphyN: based on the nucleotide sequence (GenBank AY536581) of the cloned gene encoding the phytase of Neurospora crassa (N.crassa) CICICIM F00021(AS 3.1604), a group of 80 oligonucleotides is designed by using DNAMAN software, the group of oligonucleotides is obtained by chemical synthesis by using a DNA synthesizer, and the nucleotide sequences of the 80 oligonucleotides are AS follows:
F0 cat gaattca tgttaagggt tctatctcca aatccagcta gttgc
R0 ccccaattct ggagagtcgc aactagctgg atttgg
F1 gactctccag aattggggta tcagtgcaat tctgaaac
R1 ggccccaagt atgtgtagtt tcagaattgc actgata
F2 tacacatact tggggccaat acagcccttt tttttctg
R2 gctgatttca ctcgggacag aaaaaaaagg gctgtatt
F3 tcccgagtga aatcagccca agcgtgccag agg
R3 cgaaagtcag tctgcaaccc tctggcacgc ttgg
F4 gttgcagact gactttcgcg caagtactga gtagac
R4 gggaatctgg cgccatgtct actcagtact tgcg
F5 atggcgccag attccccact ccaggcaaag ctg
R5 acagcggaaa tggcggcagc tttgcctgga gtg
F6 ccgccatttc cgctgtttta accaagatta agacgtc
R6 gcgcatacca agtagcagac gtcttaatct tggttaaa
F7 tgctacttgg tatgcgcccg attttgaatt tatcaagga
R7 tctacaccca atacatagtt ataatccttg ataaattcaa aatcgg
F8 ttataactat gtattgggtg tagatcacct aacagctttc g
R8 taaccatttc ctgctcaccg aaagctgtta ggtga
F9 gtgagcagga aatggttaac tctggtatta agttctacc
R9 tcagactagc ataacgctgg tagaacttaa taccagagt
F10 agcgttatgc tagtctgatt agagattata cagatcctga a
R10 ctctgataaa aggcagagat tcaggatctg tataatctct aa
F11 tctctgcctt ttatcagagc atcaggtcag gaaagg
R11 ctgccgacgc aattaccctt tcctgacctg atg
F12 gtaattgcgt cggcagaaaa tttcactacc ggatttta
R12 ggcaagcaac gcagagtaaa atccggtagt gaaatttt
F13 ctctgcgttg cttgccgata agaaccctcc tcca
R13 gaagtggcag agagcttgga ggagggttct tatc
F14 agctctctgc cacttccacg tcaagaaatg gtca
R14 cgtaggggat tccgatataa tgaccatttc ttgacgtg
F15 ttatatcgga atcccctacg gcaaacaata ctatgcatca
R15 acgctctaca caaaccatga tgcatagtat tgtttgc
F16 tggtttgtgt agagcgttcg aggattctac aacagg
R16 tagcttgggc agcatcacct gttgtagaat cctcga
F17 tgatgctgcc caagctacct tcatcgctgc aaa
R17 gcagtgatag gtggaaagtt tgcagcgatg aagg
F18 ctttccacct atcactgcta gactgaacgc acaag
R18 ataacgtcac tcctttaaaa ccttgtgcgt tcagtcta
F19 gttttaaagg agtgacgtta tctgataccg atgtcctttc
R19 ggccttaggt ccatcaatga aaggacatcg gtatcag
F20 attgatggac ctaaggccct tcgatactgt cgcc
R20 ctcgatgaag gtgggtaggc gacagtatcg aag
F21 tacccacctt catcgagctt aacaacctca agctca
R21 tactgccccc ggatggtgag cttgaggttg ttaag
F22 ccatccgggg gcagtaaact atctcccttc tgtagt
R22 tcctgcgccg taaaaagact acagaaggga gatagtt
F23 ctttttacgg cgcaggattt taccgtttat gactatttac a
R23 cgtaaaattt gcctagggat tgtaaatagt cataaacggt aaaa
F24 atccctaggc aaattttacg gctacgggcc aggtaac
R24 ctgagtagct gccaaagagt tacctggccc gtagc
F25 tctttggcag ctactcaggg tgtcggctac gtcaa
R25 caatctcgct agtaactcgt tgacgtagcc gacacc
F26 cgagttacta gcgagattga cagtctcacc ggtcgta
R26 tgtagaattt gtagtagtgt tatctacgac cggtgagact gt
F27 gataacacta ctacaaattc tacactggat ggaaatgaag atact
R27 cgatttctcg ataatgggaa agtatcttca tttccatcca g
F28 ttcccattat cgagaaatcg tactgttttt gcagatttta gt
R28 accatatcgt tgtcgtgact aaaatctgca aaaacagta
F29 cacgacaacg atatggtcgg tatattgact gccc
R29 gacaccttcg aagatcctta gggcagtcaa tataccg
F30 taaggatctt cgaaggtgtc gatgccgaga agatgatg
R30 tcttgggata gttgtattgt ccatcatctt ctcggcatc
F31 gacaatacaa ctatcccaag agagtatggt gagacgggc
R31 caagttagca ggatcgtcgc ccgtctcacc atactc
F32 gacgatcctg ctaacttgaa ggaaagggag gggc
R32 caacccaacc taccttaaat agcccctccc tttcctt
F33 tatttaaggt aggttgggtt gtcccatttg cagctagag
R33 cgcatatcat tttctcaaaa taaactctag ctgcaaatgg ga
F34 tttattttga gaaaatgata tgcgatggtg atggaagtgg t
R34 cctcagattg aaccatctca ccacttccat caccat
F35 gagatggttc aatctgagga ggaacaggac aaagagt
R35 tcgttgacta gtatacgtac taactctttg tcctgttcct
F36 tagtacgtat actagtcaac gatcgtgtgg ttaagttgaa
R36 cgtcagcttc acatccattc aacttaacca cacga
F37 tggatgtgaa gctgacgagc tggggagatg caa
R37 tagattctac gaacttatca agtttgcatc tccccagct
F38 acttgataag ttcgtagaat ctatggaatt tgcaaggaga g
R38 tttgtcccaa tcaccacctc tccttgcaaa ttcca
F39 gtggtgattg ggacaaatgt tttgcttaat gaattcgc
R39 ctgccaagtt caagactagc gaattcatta agcaaaaca
the total synthesis of NcphyN was performed in two steps using oligonucleotide in vitro splicing and amplification techniques: firstly, adding 5 ul of 10 XPCR buffer, 4 ul of 2.5mmol/L dNTPs, 1-20 ul mol/L of all 80 oligonucleotides and 0.5U of Pfu DNA polymerase into a 50 ul reaction system, and adding double distilled water to make up for 50 ul; PCR amplification conditions were 95 ℃ for 2 min; (94 ℃ 30s, 45-52 ℃ 30s, 70 ℃ 2min30s) for 35 cycles; 10min at 70 ℃; secondly, adding 2. mu.l of the oligonucleotide splicing product, 5. mu.l of 10 XPCR buffer, 4. mu.l of 2.5mmol/L dNTPs, 4. mu.l of 1mmol/L primer F01. mu.l, 1mmol/L primer R391. mu.l and 0.5U of PfuDNA polymerase into a 50. mu.l reaction system, and adding double distilled water to make up for 50. mu.l; PCR amplification conditions were 95 ℃ for 2 min; (95 ℃ for 30s, 52 ℃ for 30s, 70 ℃ for 2min for 30s) for 35 cycles; 10min at 70 ℃; the underlined part of the sequence of the primer F0 is an artificially introduced EcoRI restriction site, and the underlined part of the sequence of the primer R39 is an artificially introduced EcoRI restriction site;
(B) cloning of NcphyN: EcoRI restriction sites are artificially introduced into both ends of the synthesized NcphyN, and the NcphyN is cloned into the EcoRI site of pUC19 through EcoRI restriction, so as to obtain a cloning vector pUC-NcphyN;
(C) expression of NcphyN: EcoRI cuts the carrier pUC-NcphyN, electrophoresis separation, tapping recovery to obtain full length NcphyN, the upstream of 5' end is connected with methanol inducible promoter and MF alpha signal peptide sequence, the combined gene fragment is cloned into the multiple cloning site of yeast integration carrier pRS303K to obtain high temperature NcphyN yeast expression carrier pNCphyN.
5. The method according to claim 4, wherein oligonucleotides are designed so that the codons are yeast-preferred codons and the length of the oligonucleotides varies from 28 to 41 bases, and the two ends of adjacent oligonucleotides have complementary sequences of 15 to 25 bases.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101368175B (en) * | 2007-08-16 | 2010-12-29 | 中国农业科学院饲料研究所 | Novel phytase, encoding gene, cell and feedstuff additive including the enzyme |
| CN103467850A (en) * | 2013-09-04 | 2013-12-25 | 深圳市兴盛迪新材料有限公司 | Halogen-free flame-retardant polypropylene composition and preparation method thereof |
| CN114015672A (en) * | 2021-12-06 | 2022-02-08 | 江南大学 | Pfu DNA polymerase |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101935617A (en) * | 2010-07-13 | 2011-01-05 | 湖北大学 | Heat-resisting phytase Pichia pastoris engineering bacterial strain and production method of heat-resisting phytase |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2000050864A (en) * | 1998-08-06 | 2000-02-22 | Ajinomoto Co Inc | Production of phytase |
| KR100381374B1 (en) * | 2000-06-22 | 2003-04-23 | 주식회사 중앙바이오텍 | Expression vector(pGP1) for a novel phytase and Pichia pastoris-pGP1 transformant producing recombinant phytase |
| CN100354421C (en) * | 2002-08-16 | 2007-12-12 | 广东肇庆星湖生物科技股份有限公司 | High expressed high temperature resistant phytase gene in methanol yeast |
| CN1302112C (en) * | 2003-09-17 | 2007-02-28 | 广东肇庆星湖生物科技股份有限公司 | Production for phytase with high living rate high temp. resisting by pichia |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101368175B (en) * | 2007-08-16 | 2010-12-29 | 中国农业科学院饲料研究所 | Novel phytase, encoding gene, cell and feedstuff additive including the enzyme |
| CN103467850A (en) * | 2013-09-04 | 2013-12-25 | 深圳市兴盛迪新材料有限公司 | Halogen-free flame-retardant polypropylene composition and preparation method thereof |
| CN114015672A (en) * | 2021-12-06 | 2022-02-08 | 江南大学 | Pfu DNA polymerase |
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