CN1616659A - A phytase gene with high temperature resistance and high specific activity and its cloning and expression - Google Patents

Abstract

The present invention relates to high temperature resistant high specific activity phytase gene and its cloning and expression, and belongs to the field of microbial gene engineering. The present invention provides N.crassa originated AS3.1604 encoded high temperature resistant high specific activity phytase gene Ncphy, the nucleotide sequence of Ncphy and corresponding protein amino acid sequence, Ncphy containing cloning carrier pUC-Ncphy and yeast expression carrier pP-Ncphy, the construction of recombinant yeast with the yeast expression carrier and the enzymological property of the recombinant enzyme. The present invention may be used in the construction of gene engineering bacteria for industrial phytase production and the molecule reformation of available phytase gene to raise the level and quality of phytase produced through microbial fermentation, and may be also used in the gene transformation of animal and plant to improve variety quality.

Description

A phytase gene with high temperature resistance and high specific activity and its cloning and expression

technical field

A high-temperature-resistant, high-specific activity phytase gene and its cloning and expression, the invention belongs to the field of microbial genetic engineering, more specifically clones a phytase gene with high-temperature resistance, high specific activity and the ability to withstand high temperatures through genetic engineering methods A phytase gene that remains active for a long time.

technical background

The chemical name of phytic acid is cyclohexyl hexaphosphate, also known as inositol hexaphosphate, which is composed of one molecule of inositol and six molecules of phosphoric acid. The molecular formula is C ₆ H ₁₈ O ₂₄ P ₆ , and the molecular weight is 660.08.

Phytic acid is widely found in plants, especially in the seeds of cereals, beans and oil crops. Phosphorus in plant seeds is mainly stored in the form of phytate phosphorus. Phytic acid is easy to combine with many divalent metal ions, such as Ca ²⁺ , Mg ²⁺ , Fe ²⁺ , Zn ²⁺ , Mn ²⁺ , Cu ²⁺ , etc. to form phytate (complex salt or single salt), reducing concentration of free ions.

Phytase (myo-inositol hexakisphosphate phosphohydrolase; EC 3.1.3.8) is a kind of acid phosphatase that can hydrolyze phytic acid, and can catalyze the decomposition of phytic acid to generate inorganic phosphate and inositol. This property makes phytase have very important application significance in feed industry. Plant-derived components account for a large proportion in feed, and phosphorus in plants is mainly in the form of phytic acid. The phytic acid content in some grains and oil crops is even as high as 1% to 3%. Since the digestive tract of monogastric animals lacks an enzyme system for decomposing phytic acid, phytic acid cannot be decomposed by monogastric animals to produce inorganic phosphorus, but is excreted with feces and enters the environment. This has not only caused a large amount of waste of phosphorus, but also requires people to add additional phosphorus to the feed, and a large amount of phosphorus is discharged into the environment, which aggravates environmental pollution. In addition, phytic acid is also a strong anti-nutritional factor, which can chelate Ca ²⁺ , Fe ²⁺ , Zn ²⁺ and other metal ions to form insoluble phytate complexes, and can form insoluble phytate complexes with positively charged proteins and vitamins. Compounds affect the absorption of protein and vitamins and reduce the nutritional value of feed. As a feed additive for monogastric animals, the feeding effect of phytase has been fully confirmed: it can increase the utilization rate of phosphorus in plant feed by 60%, reduce the amount of inorganic phosphorus added in feed, and reduce the amount of inorganic phosphorus in animal manure. Phosphorus discharge is reduced by 40%, which reduces the amount of phosphorus pollution in the environment; phytase can also relieve the chelation effect of phytic acid on metal ions and proteins, improve the absorption of metal ions, especially trace metal ions, and increase protein production in monogastric animals. utilization rate.

Phytases are mostly single-subunit glycoproteins, and their glycosyl composition and ratio are mannose:galactose:N-acetylglucosamine=9:1:3, and their molecular weight is generally 40-120kDa. Their optimum pH range is 4.5～6.0, the optimum temperature is between 45～60℃. Now known phytase has 3 types, phytase-3-phosphate hydrolase (3-Phytase, EC3.1.3.8), phytase-6-phosphate hydrolase (6-Phytase, EC 3.1.3.26) and uncharacterized orthophosphate monoester phosphohydrolase (EC 3.1.3.2). The so-called phytase refers to phytase-3-phosphate hydrolase, and the gene encoding it is called phyA (Wyss 1999, Appl.Environ.Microbiol). There are 5 pairs of disulfide bonds in the advanced structure of PhyA. The role of disulfide bonds is mainly to participate in the formation of the spatial structure of the enzyme active center, so any denaturant that destroys disulfide bonds will lead to the loss of enzyme activity. In addition, disulfide bonds are also related to the heat resistance of phytase. The crystal structure of PhyA consists of a large α' domain and a smaller α domain. The center of the α' domain is a β-sheet composed of 6 corresponding amino acid sequences. There are 14 alpha helices in the alpha domain. There is a deep recess on the inner surface of the two domains. In the recess, there are essential amino acids of the enzyme active center-Arg58 and His59, which are the first two in the conserved sequence RHG×R×P of the phytase active site. amino acids. Enzyme protein molecules have a total of 19 Args. Arg58 in the RHG×R×P sequence is directly involved in its enzymatic activity, and the position of Arg58 in its crystal structure also coincides with its function. Arg58 in the substrate binding site RHG×R×P is considered to be a residue necessary to maintain enzyme activity, that is, the substrate binding site uses its positively charged residues to form by electrostatic adsorption with negatively charged substrates The ES complex with a specific conformation hydrolyzes the substrate with the help of the catalytic site function. The loss of enzyme activity is due to the modification of the arginine R group of RHG×R×P in the substrate binding site. The crystal structure of PhyA in microorganisms is very similar to the crystal structure of low-molecular-weight acid phosphatase in mice, indicating that it belongs to the histidine family of acid phosphatases. Although PhyB in microorganisms belongs to the family of histidine acid phosphatases and has the conserved sequence RHG×R×P at the active site, its optimal substrate is not phytate, so it can only be regarded as an acidic substance with phytase activity. Phosphatase. The bacterial phytase coding gene phyC is relatively short, the full length of the gene is 1152, and it encodes 383 amino acids. The first 26 amino acids at the N-terminal are signal peptides, and the molecular weight of the encoded enzyme protein is also relatively small. The sequence has no homology with phyA and phyB and the reported phosphatase genes, nor does it have the RHG×R×P conservative sequence of the amino acid sequence encoded by phyA and phyB, indicating that it does not belong to the histidine acid phosphatase family. phyA gene contains 1 intron, 2 optimal pH; phyB gene contains 3 introns, 1 optimal pH.

When phytase acts on phytic acid, the phosphate groups on the phytic acid molecule are cut off one by one to form intermediate products IP ₅ , IP ₄ , IP ₃ , IP ₂ , and the final products are inositol and phosphate. According to different starting sites, phytases can be divided into two categories: 3-phytase (EC 3.1.3.8) and 6-phytase (EC 3.1.3.26). 3-Phytase first hydrolyzes the ester bond from the 3rd carbon of phytic acid to release inorganic phosphorus, and then hydrolyzes the ester bond at other positions in turn. This type of enzyme needs divalent magnesium ions as prosthetic groups and mainly exists in microorganisms . 6-phytase first hydrolyzes the ester bond from the 6th carbon of phytic acid, and this type of enzyme mainly exists in plants. Phytase can not completely decompose inositol phosphate, to completely decompose inositol phosphate, you need the help of acid phosphatase. The most thorough research on the mechanism of phytase is Pseudomonas phytase. In addition, the mechanism of action of wheat bran phytase and Aspergillus fig phytase has also been preliminarily understood. Theoretically, 1g of phytic acid can be completely decomposed to release 281.6mg of inorganic phosphorus.

Phytase widely exists in nature and is found in animals, plants, and microorganisms. It is reported that there are currently nearly 200 registered phytase protein sequences, most of which are found in nature, including many artificially modified ones, and new natural enzymes are constantly being discovered. However, there are not many natural phytases that can be used in the feed industry. This is mainly because the following two factors have played a limiting role: 1. The optimum pH value of phytase fed to monogastric animals should preferably be consistent with the pH environment of the animal's digestive system such as the stomach, intestines and other organs, so that it is conducive to being fed Ingested phytase works better. According to reports (Han Zhengkang, 1991), the pH value of chyme in pig stomach is between 1.8 and 3.6, and the pH value of chyme in duodenum is between 3.9 and 7.0. The pH of chicken stomach is between 4.5 and 4.8, and the pH of duodenum is between 5.7 and 6.0. Therefore, the wider the optimum pH range of phytase, the stronger the adaptability to the pH environment of the digestive tract of pigs, chickens and other animals. 2. Heat treatment in the process of feed processing, such as granulation, puffing and other processes, has a direct impact on the activity of phytase. Many phytases cannot withstand relatively high processing temperatures (usually 75 ° C ~ 93 ° C), so it is also limited Its application (Wang Hongning 2000, Journal of Aochuan Agricultural University).

At present, the focus of phytase research is microbial-derived phytase, especially fungal-derived phytase, which has been reported in many ways, such as Aspergillus ficuum, A.fumigatus, A.niger, A.terrus, A.oryzae, Emericella nidulans, Mycleiophthora thermophila, Thermomyces lanuginose (Berka 1998, Appl. Environ. Microbiol; Mitchell 1997, Microbiology), etc. Compared with plant-derived phytase, microbial phytase has a larger pH range, and some pH values are between 2.5 and 5.5, which are close to the gastrointestinal physiological conditions of monogastric animals. The pH value of plant-derived phytase is between 5.0 and 7.5, and its heat resistance is not good. It cannot tolerate the pelleting temperature of 80°C during feed processing (Simons 1990, Br JNutr). The microorganisms currently used for industrial production of phytase are mainly Aspergillus niger, Aspergillus fig, Aspergillus oryzae, etc. They can secrete extracellular enzymes with high activity, and their pH is in the acidic range. These enzymes have good enzymatic activity at 37°C, but they are not able to withstand the high temperature during granulation. The optimum temperature of the high-temperature phytase isolated from mesophilic microorganisms M.thermophila, A.terreus, etc. is 70-80°C. The activity temperature of the enzyme is extremely low when it is used, and it has no use value. Therefore, how to make the enzyme capable of short-term high temperature resistance and high enzyme activity at the normal body temperature of animals is a problem urgently needed to be solved for feed enzyme preparations including phytase. In addition, the phytase fed to monogastric livestock and poultry is an acidic phytase, which is not suitable for freshwater cultured carps, because carps have no stomach and only have a neutral pH intestinal tract. Phytase in fish feed must use neutral phytase whose optimum pH is neutral. At present, the development of neutral phytase has also become a research hotspot. The Bacillus phytase reported in recent years has a near-neutral optimum pH value, high enzyme activity, and good thermal stability, and may be widely used in fish feed (Choi 2001, J Protein Chem).

Phytase research has a history of nearly 40 years. In the mid-1970s, limiting the amount of phosphorus excretion from the farming industry became one of the core contents of the guidelines of the Environmental Protection Council implemented by the member states of the European Community. Europe began to work on finding a comprehensive solution to inorganic phosphorus pollution. Until 1980, Wageningen University in the Netherlands found the phytase gene, Gist-brocades (the predecessor of DSM) confirmed the function of phytase, and the development of phytase became possible. Germany's BASF (BASF) and Gist-brocades took the lead in using gene transfer technology and successfully used A. niger to produce commercial phytase on an industrial scale for the first time in 1989. In 1990, it was fully approved by the European feed additive management agency, and Natuphos was listed as a trade name. Since then, various countries, mainly universities, research institutes and various commercial research institutions, have invested a lot of human and material resources in development and research. Now, there are nearly 10 varieties produced by at least several companies in the international market, and at least 3 to 5 companies in China have developed phytase products. There are more companies and research institutions are engaged in the development of phytase.

There are roughly two approaches to the research and development of phytase: 1. Improve the existing strains through traditional genetic methods to obtain strains with better performance; 2. Clone phytase with excellent performance by means of genetic engineering Gene and introduce it into a suitable host to obtain excellent recombinant bacteria. Both approaches have been reported with successful examples. For example, in 1994, Marisa et al. carried out ultraviolet mutagenesis on Aspergillus figum NRLL3115, and selected a mutant strain whose enzyme production was 3.3 times that of the wild-type strain. In 1997, Chen Hongge and others carried out ultraviolet and nitrosoguanidine mutagenesis on Aspergillus niger MAO21 to obtain a strain with high phytase activity, whose enzyme activity was 3.6 times that of the original strain. In 1991, Van Gorcom et al cloned the phytase gene phyA of Aspergillus fig into Aspergillus niger CBS513.88 under the control of the promoter of amyloglucosidase for expression, and the enzyme production was increased by 1400 times. In 1998, Yao Bin etc. cloned the phytase gene of the high-yield phytase Aspergillus niger into Pichia pastoris, and the expression level was 3000 times higher than that of the original strain (Zhu Jinghuan 2002 Microbiology Journal). At the same time, a large-scale and low-cost production process of feed additive phytase was developed using bioreactors. However, this phytase has a defect that cannot be ignored: it cannot tolerate higher temperatures, and a large number of enzyme activity units are lost during processing (Kim 1998, Enz Microbial Tech). The phytase isolated from A. fumiga can withstand high temperature of 90-100 ℃, and can degrade phytic acid in a wide pH range, so it has great market potential (Vall Loon1998, Appl Environ Microbiol). According to the phytase gene sequence of A. fumigatus, the enzyme was highly expressed in yeast by chemical synthesis and gene rearrangement in vitro, and the enzyme activity was 130,000u/ml. The expression level is 13 times that of the wild-type gene. After high-density fermentation, the protein expression is 5.6g per liter. But the disadvantage of this enzyme is that after gene rearrangement, the specific enzyme activity of the recombinant phytase is only 23,000u/mg (Chinese invention patent 02136531.8), which is only a quarter of the wild type A.niger phytase one.

Contents of the invention

The purpose of the present invention is to find a kind of phytase with high temperature resistance and high specific activity and its encoding gene, and utilize the method for expressing and preparing the phytase in yeast.

The invention provides a heat-resistant and high specific activity phytase gene sequence and its corresponding amino acid sequence derived from Neurospora crassa AS 3.1604. The cloning vector pUC-Ncphy containing N.crassa AS3.1604 phytase gene and the expression vector pP-Ncphy expressed in yeast are provided. Utilize achievement of the present invention, can be used for the construction of the genetically engineered bacterium of industrialized production of phytase and the molecular transformation of existing phytase gene, improve the level and the quality of microbial fermentation method to produce phytase, can also be used for the production of animals and plants Gene transformation to achieve the purpose of improving the quality of species.

Technical scheme of the present invention: based on the highly conserved region sequence of the phytase gene derived from all Aspergillus species, through homologous comparison of the whole genome sequence of N. crassa OR74A, a section with 40% homology to Aspergillus niger phyA is obtained Sexual regions of unknown gene function. The phytase mature peptide gene was amplified by PCR technology using N. crassa AS 3.1604 chromosomal DNA as a template and NC-phy801, 5'-ACCGGAATTCATGCTACGAGTACTATCCCCAAATCC-3' and NC-phy901, 5'-ACCGGAATTCccgcttcggtAATTCGCAGC-3' as primers And remove the intron at the 5' end. Introduce EcoR I restriction sites at both ends of the amplified fragment, recover a 1.5kb phytase gene fragment, digest it with EcoR I, insert it into pUC19, and obtain pUC-Ncphy; The yeast expression vector pP-Ncphy of live phytase gene. pUC-Ncphy was used for sequence determination, and pP-Ncphy was used for the transformation of yeast and the construction of recombinant yeast containing phytase gene Ncphy.

The nucleotide sequence of gene Ncphy is as follows:

atgctacgag tactatcccc aaatccagca tcatgcgaca gcccagagct tggttaccaa 60

tgtaactcag agacaaccca cacatggggt caatactcgc ccttcttctc cgtcccgtcg 120

gaaatctccc cctccgttcc cgagggttgc cgcctcacct ttgcccaagt tctttctcgc 180

cacggcgctc gcttcccaac tcccggaaaa gccgccgcca tctccgccgt tctcaccaag 240

atcaaaacct ccgccacctg gtacgccccc gacttcgagt tcatcaaaga ctacaactat 300

gtcctcggcg tcgaccacct cactgccttt ggcgagcaag agatggtcaa ctcgggcatc 360

aaattctacc aacgctacgc ttccctcatc cgggaactaca ctgacccaga atccctcccc 420

tttatccgtg cctcggggca ggagcgcgtc attgcctcag ctgagaactt caccactggg 480

ttctactctg ccctccttgc cgacaagaac ccaccccctt cctccctccc gcttccccgc 540

caggagatgg tcatcatctc cgaatcgccc acggccaaca accacatgca ccacggtctc 600

tgccgcgcct ttgaggactc caccaccggc gatgcggccc aggcgacctt tatagctgcc 660

aatttcccgc ccatcaccgc gcggttgaat gcgcagggtt tcaaaggcgt cactctttcc 720

gacacggacg tgctctcgct catggatctc cgcccctttg aacccgtcgc ttacccgccc 780

tcctcctctc tcaccacctc gtcctctccc tcggggggaa gcaagctctc ccccttttgc 840

tcccttttta ccgctcaaga ctttaccgtg tacgactacc tccagtccct cggcaagttc 900

tacggctacg gcccgggtaa ttctctggct gccacgcagg gggtggggta cgtgaacgag 960

cttttggctc gcctcacggt ttccccggtg gtggataaca cgaccaccaa ttccacgctg 1020

gacgggaacg aggacacgtt tccgctgagt aggaacagga cggtgtttgc ggatttcagt 1080

catgataatg atatggtggg gatcttgact gctttgagaa tctttgaggg ggtggatgcg 1140

gagaagatga tggataatac gaccataccg agagagtacg gggagactgg cgatgatccg 1200

gcaaatttga aagagaggga gggcttgttc aaggttggtt gggtggtgcc atttgcggcg 1260

agggtgtatt ttgaaaagat gatttgtgat ggggatggga gtggagagat ggttcagagc 1320

gaggaggaac aggacaagga gttggtgagg atcttggtta acgatagagt ggttaaacta 1380

aatggatgtg aggccgatga gttggggagg tgtaagttgg ataaatttgt agagagtatg 1440

gagtttgcta ggaggggtgg agattgggac aagtgttttg cttag 1485

为终止密码子。in, is the artificially added transcription initiation codon,

for the stop codon.

According to the above nucleotide sequence, the amino acid sequence of the phytase of N.crassa AS 3.1604 is as follows:

Met Leu Arg Val Leu Ser Pro Asn Pro Ala Ser Cys Asp Ser Pro

1 5 10 15

Glu Leu Gly Tyr Gln Cys Asn Ser Glu Thr Thr His Thr Trp Gly

20 25 30

Gln Tyr Ser Pro Phe Phe Ser Val Pro Ser Glu Ile Ser Pro Ser

35 40 45

Val Pro Glu Gly Cys Arg Leu Thr Phe Ala Gln Val Leu Ser

50 55 60

Thr Pro Gly Lys Ala Ala Ala Ile Ser

Thr Pro Gly Lys Ala Ala Ala Ile Ser

65 70 75

Ala Val Leu Thr Lys Ile Lys Thr Ser Ala Thr Trp Tyr Ala Pro

80 85 90

Asp Phe Glu Phe Ile Lys Asp Tyr Asn Tyr Val Leu Gly Val Asp

95 100 105

His Leu Thr Ala Phe Gly Glu Gln Glu Met Val Asn Ser Gly Ile

110 115 120

Lys Phe Tyr Gln Arg Tyr Ala Ser Leu Ile Arg Asp Tyr Thr Asp

125 130 135

Pro Glu Ser Leu Pro Phe Ile Arg Ala Ser Gly Gln Glu Arg Val

140 145 150

Phe Thr Thr Gly Phe Tyr Ser Ala LeuIle Ala Ser Ala Glu

Phe Thr Thr Gly Phe Tyr Ser Ala Leu

155 160 165

Leu Ala Asp Lys Asn Pro Pro Pro Ser Ser Leu Pro Leu Pro Arg

170 175 180

Gln Glu Met Val Ile Ile Ser Glu Ser Pro Thr Ala Asn Thr

185 190 195

Met His His Gly Leu Cys Arg Ala Phe Glu Asp Ser Thr Thr Gly

200 205 210

Asp Ala Ala Gln Ala Thr Phe Ile Ala Ala Asn Phe Pro Pro Ile

215 220 225

Thr Ala Arg Leu Asn Ala Gln Gly Phe Lys Gly Val Thr Leu Ser

230 235 240

Asp Thr Asp Val Leu Ser Leu Met Asp Leu Arg Pro Phe Asp Thr

245 250 255

Val Ala Tyr Pro Pro Ser Ser Ser Ser Leu Thr Thr Ser Ser Ser Ser Pro

260 265 270

Ser Gly Gly Ser Lys Leu Ser Pro Phe Cys Ser Leu Phe Thr Ala

275 280 285

Gln Asp Phe Thr Val Tyr Asp Tyr Leu Gln Ser Leu Gly Lys Phe

290 295 300

Tyr Gly Tyr Gly Pro Gly Asn Ser Leu Ala Ala Thr Gln Gly Val

305 310 315

Gly Tyr Val Asn Glu Leu Leu Ala Arg Leu Thr Val Ser Pro Val

320 325 330

Thr Thr Thr

Ser Thr Leu Asp Gly Asn Glu AspVal Asp

Thr Thr Thr

Ser Thr Leu Asp Gly Asn Glu Asp

335 340 345

Thr Phe Pro Leu Ser Arg Arg Thr Val Phe Ala Asp Phe Ser

350 355 360

His Asp Asn Asp Met Val Gly Ile Leu Thr Ala Leu Arg Ile Phe

365 370 375

Glu Gly Val Asp Ala Glu Lys Met Met Asp Thr Thr Ile Pro

380 385 390

Arg Glu Tyr Gly Glu Thr Gly Asp Asp Pro Ala Asn Leu Lys Glu

395 400 405

Arg Glu Gly Leu Phe Lys Val Gly Trp Val Val Pro Phe Ala Ala

410 415 420

Arg Val Tyr Phe Glu Lys Met Ile Cys Asp Gly Asp Gly Ser Gly

425 430 435

Glu Met Val Gln Ser Glu Glu Glu Gln Asp Lys Glu Leu Val Arg

440 445 450

Ile Leu Val Asn Asp Arg Val Val Lys Leu Asn Gly Cys Glu Ala

455 460 465

Asp Glu Leu Gly Arg Cys Lys Leu Asp Lys Phe Val Glu Ser Met

470 475 480

Glu Phe Ala Arg Arg Gly Gly Asp Trp Asp Lys Cys Phe Ala

485 490 494

和一个近氨基端活性中心保守序列 The phytase consists of 494 amino acid residues, and contains 6 glycosylation sites from the analysis of the primary structure of the protein

and a conserved sequence near the amino-terminal active center

Beneficial effects of the present invention:

1. The phytase of the present invention has obvious high specific enzyme activity, which is 125,000u/mg. It is 1.2 times of the wild-type phytase of Aspergillus niger and 5.1 times of the recombinant phytase of A. fumigatus.

2. The phytase of the present invention has obvious high temperature resistance, and retains 58% of the enzyme activity at 80°C and 20 min. Under the same conditions, Aspergillus niger phytase only retained 28% of the enzyme activity.

3. The phytase of the present invention has wide pH availability and stability, has phytase activity between pH 2.0-8.0, and has good pH stability between pH 3.5-9.5.

4. The phytase of the present invention has good resistance to environmental factors, and Mg ²⁺ , Ca ²⁺ , Al ³⁺ , Fe ²⁺ , Co ²⁺ , Zn ²⁺ and EDTA have a great influence on its enzyme activity. Small.

Description of drawings:

Figure 1. Construction of bacterial cloning phytase vectors.

Figure 2 Yeast expression phytase vector.

Figure 3 The protein electrophoresis profile of recombinant yeast expressing phytase. lane 1: protein molecular weightmarkers comprising babbit phosphorylase b(97,400), bovine serum albumin(66,200), rabbit actin(43,000), bovine carbonic anhydrase(31,000), trypsin inhibitor(20,100), henegglanwhite( 4; purified phytase; lane 3: unpurified phytase; lane 4: culture broth of the vector control

Fig. 4 Relative activity of phytase under different pH conditions.

Figure 5 Optimum reaction temperature of recombinase.

Figure 6 Comparison of thermal stability of recombinant enzymes and commercial enzymes.

Figure 7 Determination of pH stability of recombinant enzymes.

Detailed ways

Example 1: Preliminary screening of N. crassa producing phytase

A total of 11 strains of N.crassa preserved in our laboratory or from the China Culture Collection Management Committee were used as the starting strains. Enzyme production medium composition: 1.5% glucose, 0.3% peptone, 0.2% (NH ₄ ) ₂ SO ₄ , 0.05% MgSO ₄ 7H ₂ O, 0.05% KCl, 0.003% FeSO ₄ 7H ₂ O, 0.003% MnSO ₄ · 4H ₂ O, pH 5.7. Culture conditions: inoculate an appropriate amount of N. crassa spores into 50 mL of enzyme-producing medium, shake the flask at 28° C. and 200 r/min for 4 days. The culture solution was filtered to remove mycelium, and then centrifuged at 8000r/min for 10min. Aspirate the supernatant to measure the phytase activity. The method for measuring phytase activity is carried out according to the national standard GB/T 18634-2002. The unit of phytase activity (u) is defined as: the enzyme required to decompose phytate and release 1 nmol of inorganic phosphate per minute at 37°C The amount is 1u. The N..crassa AS3.1604 of the China Culture Collection Management Committee screened out (see "Bacteria Catalog Database" for details). The phytase activity in its fermentation broth is the highest, with an enzyme activity of 18.5u/mL.

Embodiment 2: Cloning of N.crassa AS 3.1604 phytase gene

By comparing with the highly conserved region sequence of microbial phytase genes such as Aspergillus niger and Aspergillus terreus, it was found that there is a nucleotide sequence encoding a polypeptide with unknown function in the N. crassa genome, which is likely to be the gene encoding phytase. Further, the Signal P V2.0 program deduced the signal peptide sequence of N. crassa phytase, and deduced that the 88th amino acid (the third methionine) was the start codon. Among them, the 68 amino acid residues at the amino terminal are pseudogene coding sequences. Therefore, the following oligonucleotides were used as primers:

NC-phy801, 5'-ACCGGAATTCATGCTACGAGTACTATCCCCAAATCC-3'

NC-phy901, 5'-ACCGGAATTCCCGCTTCGGTAATTCGCAGC-3'

By PCR technology, the phytase mature peptide gene was amplified and the intron at the 5' end was amplified using N. crassa AS 3.1604 chromosomal DNA as a template.

N.crassa AS 3.1604 chromosomal DNA was extracted as follows. The hyphae were collected by filtration, and washed 1 or 2 times with normal saline. Place in a 5mL centrifuge tube and centrifuge at maximum speed to remove residual saline. Place the collected thalline in a 5mL centrifuge tube, add 1mL lysis buffer (50mmol/L Tris-HCl pH8.0, 180mmol/L EDTA pH8.0, 1% SDS (Huamei Company) for every 0.5g thalline, Add 3/10 of the total volume of quartz sand at the same time, cover the centrifuge tube cap tightly, shake it on the vortex shaker for 5min-8min, shake the centrifuge tube vigorously up and down for 30s every 1min, to mix the contents evenly. Place at 65°C for 10 minutes, add 600 μL of 7.5 mol/L ammonium acetate solution, and ice-bath for 8 minutes. Centrifuge at the maximum speed for 5 minutes, transfer the supernatant to another sterile 5mL centrifuge tube, add 0.1 times the volume of 3mol/L NaAc and 0.6 times the volume of isopropanol, mix by inverting, and ice-bath for 8 minutes. Collect the precipitate by centrifugation at the maximum speed at room temperature, discard the supernatant, dissolve the precipitate with 200 μL TE, add an appropriate amount of RNase, and incubate at 65 °C for 10 min. Take it out, add 200 μL of chloroform:isoamyl alcohol (24:1) to extract once, transfer the supernatant to another sterile 1.5mL centrifuge tube, add 0.1 times the volume of 3mol/L NaAc and 2.5 times the volume of Water and ethanol, centrifuge at the maximum speed for 8 minutes to collect chromosomal DNA, wash once with 70% ethanol, and dissolve the precipitate with an appropriate amount of TE solution after the ethanol is completely evaporated.

Using PCR technology, N. crassa AS 3.1604 chromosomal DNA was used as a template, and primers NC-phy801, 5'-ACCGGAATTCATGCTACGAGTACTATCCCCAAATCC-3' and NC-phy901, 5'-ACCGGAATTCCCGCTTCGGTAATTCGCAGC-3' were used for PCR amplification. The PCR amplification conditions were: denaturation at 94°C for 10 min, adding 1 unit of Pyrobest ^TM DNA polymerase (Dalian Bao Company), mixed well and adding about 100 l of mineral oil, then denaturation at 94°C for 1 min, annealing at 56°C for 1.5 min, 72 Extend at ℃ for 3 min, and after 30 cycles, extend at 72°C for 7 min.

Both ends of the PCR amplified product were introduced with EcoR I restriction sites, and a 1.5kb phytase gene fragment was recovered, digested with EcoR I, inserted into pUC19, and obtained pUC-Ncphy, as shown in Figure 1. pUC-Ncphy was used for sequence determination, and the nucleotide sequence of the inserted Ncphy was determined by Sanger chain termination method.

Example 3: Yeast expression phytase vector construction

Introduce EcoRI digestion sites at both ends of the amplified fragment, recover the 1.5kb phytase gene fragment, digest with EcoRI, insert into pUC19, obtain pUC-Ncphy, digest pUC-Ncphy with EcoRI, cut the gel to recover the Ncphy fragment, It was forward inserted into the EcoR I site of the pPIC9K vector (from Invitrogen Company) to construct the yeast expression vector pP-Ncphy, as shown in Figure 2.

Example 4: Construction of recombinant yeast expressing phytase

The activated Pichia pastoris strain Pichia pastoris KM71 (from Invitrogen) was cultured in 500ml YPD (1% yeast extract, 2% peptone, 2% glucose) at 30°C for 18h until OD ₆₀₀ =1.7, and collected by centrifugation at 5000r/min For the thallus, wash the thalline successively with 500ml and 250ml of pre-cooled sterile water, centrifuge to remove the supernatant, and suspend the thalline with 20ml of pre-cooled 1mol/L sorbitol. After centrifugation, the bacteria were suspended with 0.5ml pre-cooled sorbitol, and used as competent cells for electroshock transformation.

A large amount of yeast expression vector pP-Ncphy was extracted, pP-Ncphy was digested with Nco I to linearize pP-Ncphy, the fragments were added to 50uL competent cells, ice-bathed for 5min, and electroshocked with Bio-Rad GenePulser electric shocker with parameters of 2.5Kv and 25uF. Immediately after the electric shock, 1.0 ml of pre-cooled 1 mol/L sorbitol was added, 200 ul was spread on YNBG medium (1.34% YNB medium, 2% glucose), and cultured at 30° C. until transformants appeared. Use a toothpick to plant the transformant into YNBG-G418 (2 mg/ml antibiotic G418 (purchased from Sigma) is added to the YNBG medium), and the well-growing colonies are identified by yeast colony PCR. The positive recombinant yeast is used for Fermentation test for phytase production.

High-density fermentation of recombinant yeast strains: Inoculate the obtained 5 strains of recombinant Pichia yeast into 20 mL YPD liquid medium respectively, and cultivate them with shaking at 30°C until the stationary phase. The cells were collected by centrifugation, transferred to 100 mL MGY medium (1.34% YNB, 1% glycerol, 4× ^10-5 % biotin) at 30°C, 200 r/min for 48 hours with vigorous shaking, collected cells by centrifugation, and transferred to 30 mL MM Culture medium (1.34% YNB, 0.5% methanol, 4×10 ^-5 % biotin) was cultured in a liquid medium with vigorous shaking at 30°C for up to 7 days, and the phytase activity was measured every 12 hours. The enzyme activity of the fermented broth of the recombinant bacteria ranged from 1,000 to 13,000u/mL. Among them, the highest level of phytase produced by recombinant yeast Y79a was 13,200u/mL. Compared with the wild strain, the enzyme production level was increased by 710 times.

Embodiment 5: the preparation and purification of phytase

8,000r/min×10min refrigerated and centrifuged to collect the fermentation broth of recombinant yeast Y79a, put it into a dialysis belt for dialysis and desalination, the dialysis belt was suspended in normal saline, keep the temperature at 4°C during dialysis, and dialyze for 2 days until the fermentation broth is basically transparent and colorless . 15 mL of the fermented broth after dialysis was centrifuged with Amico Ultrafitration-15 (Biomax 10K; Millipore) at 4500 g for 25 min to obtain a liquid with a final volume of about 500 μL, and the concentration factor was about 30 times. Then, 0.25 M, pH5.5 sodium acetate solution was used as the eluent, and the obtained 500 μL concentrated solution was separated by Superdex 200 column (Pharmacia Biotech). The eluate was collected step by step, and 1 mL of liquid was collected in each tube. According to the test tube number corresponding to the detected protein peak, the determination of phytase activity was carried out. Combine the eluent with enzymatic activity, apply it to anion exchange chromatography column DEAE-52, and use 0-1.0mol/L NaCl, 0.25mol/L NaAc, pH6.0 gradient elution. The collection tubes with obvious enzyme activity were detected, concentrated with Amico Ultrafitration-15 (Biomax 10K; Millipore) and then subjected to SDS-PAGE electrophoresis. The protein concentration was determined by the Bradford method with bovine serum albumin as the standard. There was one more protein band in the fermentation broth of the recombinant bacteria than in the empty vector control. The results of the electrophoretic spectrum are shown in Figure 3. After separation and purification, the recombinant enzyme was homogeneous in electrophoresis, with a molecular weight close to 60kDa, and it was a single subunit protein. The specific enzyme activity is 125,000u/mg.

Embodiment 6: Phytase enzymatic property analysis

Optimum pH for recombinant enzymes

pH buffer: 0.2mol/L glycine-hydrochloric acid buffer (pH2.0, 2.5, 3.0, 3.5), 0.2mol/L sodium acetate buffer (pH4.0, 4.5, 5.0, 5.5), 0.2mol/L imidazole - hydrochloric acid buffer (pH6.0, 6.5), 0.2mol/L Tris buffer (pH7.0, 8.0). Dilute 120 μL of recombinant enzyme pure enzyme (28.3 μg/mL) to 480 μL, and take 40 μL to measure the enzyme activity at pH 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, and 8.0. Calculate the relative enzyme activity to determine the optimum pH. Under the determination conditions, the optimum pH of the recombinant enzyme is 3.5-5.5, and the enzyme activity reaches the maximum value at pH 5.5. It is active between pH 2.0 and 8.0 (Figure 4).

Determination of Optimum Reaction Temperature of Recombinase

Dilute 120 μL of recombinant enzyme pure enzyme (28.3 μg/mL) to 480 μL, take 40 μL to measure the enzyme activity at 25, 37, 45, 50, 55, 60, 65, 70, and 80 °C, and calculate the relative enzyme activity to determine the maximum moderate temperature. Under the assay conditions, the optimum temperature of the recombinant enzyme is 60°C. It maintains good activity between 25-70°C (Figure 5).

Determination of Stability of Recombinases to Temperature

Dilute 70 μL of recombinant enzyme pure enzyme (28.3 μg/mL) to 280 □ L, take 15 □ L at 30, 40, 50, 60, 70, 80, 90 ° C for 10, 20, 60 min, take it out immediately, and put it in an ice bath After 30 min, the enzyme activity was measured at 37°C. Calculate the relative enzyme activity and compare the stability of the recombinase to temperature. The study on the stability of the recombinant enzyme to temperature shows that after the recombinant enzyme is heated at 70-90°C for 20 minutes, the relative enzyme activity still remains 39-58%, which is higher than that of the Aspergillus niger phytase expressed by Pichia pastoris reported in the literature some.

Comparison of Thermostability of Recombinant Enzyme and Commercial Enzyme

At 80°C, the recombinant enzyme and the commercial enzyme were heated for 5, 10, 15, and 20 minutes, respectively, and immediately taken out. After 30 minutes in ice bath, the enzyme activity was measured at 37°C, and the relative enzyme activity was calculated. The recombinase was thermally activated after heating at 80°C. Comparing the thermostability of the recombinant enzyme with the commercial enzyme at 80°C, it was found that when heated to 15 and 20 minutes, the enzyme activity of the recombinant enzyme decreased less than that of the commercial enzyme (Figure 6).

Determination of pH Stability of Recombinase

Dilute 70 μL of recombinant enzyme pure enzyme (28.3 μg/mL) to 280 μL, take 15 μL at pH 2.0, 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 , 9.5 room temperature for 24h. Then the enzyme activity was measured at 37°C and pH 5.5, and the relative enzyme activity was calculated to compare the stability of the recombinant enzyme to pH. The enzyme has good stability between pH 3.5 and 9.5 (Figure 7).

Determination of K _m and V _max Values of Kinetic Parameters of Recombinase

Dilute 70 μL of recombinant enzyme pure enzyme (28.3 μg/mL) to 280 μL, take 20 μL and place it in different concentrations (5, 2.5, 1.25, 1, 0.5, 0.25 mM) of the substrate sodium phytate to measure the enzyme activity, and the reaction speed is determined by The amount of inorganic phosphorus produced per minute (μmol..) was expressed, and the K _m and V _max values were calculated by the Lineweaver-Burk double reciprocal plotting method. The K _m and V _max values were 228 μM and 128 μmol/mg pro.min ^-1 , respectively.

Effects of Different Metal Ions on the Activity of Recombinase

Add 20 μL of recombinant enzyme pure enzyme (28.3 μg/mL) to 280 μL of 1 mM metal salt solutions such as Zn ²⁺ , Mn ²⁺ , Mg ²⁺ , Ca ²⁺ , Al ³⁺ , and place at room temperature for 30 min. Add another 200 μL of NaAc to a volume of 500 μL and measure the enzyme activity again. The enzyme activity determination method is the same as before, and the relative enzyme activity is calculated. Mg ²⁺ , Ca ²⁺ , Al ³⁺ , Fe ²⁺ , Co ²⁺ , Zn ²⁺ and EDTA had little effect on its enzyme activity.

Claims (4)

1, a kind of high temperature resistance, high specific activity phytase gene Ncphy derived from Neurospora crassa (Neurospora crassa) AS 3.1604, its nucleotide sequence is: atgctacgag tactatcccc aaatccagca tcatgcgaca gcccagagct tggttaccaa 60 tgtaactcag agacaaccca cacatggggt caatactcgc ccttcttctc cgtcccgtcg 120 gaaatctccc cctccgttcc cgagggttgc cgcctcacct ttgcccaagt tctttctcgc 180 cacggcgctc gcttcccaac tcccggaaaa gccgccgcca tctccgccgt tctcaccaag 240 atcaaaacct ccgccacctg gtacgccccc gacttcgagt tcatcaaaga ctacaactat 300 gtcctcggcg tcgaccacct cactgccttt ggcgagcaag agatggtcaa ctcgggcatc 360 aaattctacc aacgctacgc ttccctcatc cgggaactaca ctgacccaga atccctcccc 420 tttatccgtg cctcggggca ggagcgcgtc attgcctcag ctgagaactt caccactggg 480 ttctactctg ccctccttgc cgacaagaac ccacccccctt cctccctccc gcttccccgc 540 caggagatgg tcatcatctc cgaatcgccc acggccaaca accacatgca ccacggtctc 600 tgccgcgcct ttgaggactc caccaccggc gatgcggccc aggcgacctt tatagctgcc 660 aatttcccgc ccatcaccgc gcggttgaat gcgcagggtt tcaaaggcgt cactctttcc 720 gacacggacg tgctctcgct catggatctc cgcccctttg aacccgtcgc ttacccgccc 780 tcctcctctc tcaccacctc gtcctctccc tcggggggaa gcaagctctc ccccttttgc 840 tcccttttta ccgctcaaga ctttaccgtg tacgactacc tccagtccct cggcaagttc 900 tacggctacg gcccgggtaa ttctctggct gccacgcagg gggtggggta cgtgaacgag 960 cttttggctc gcctcacggt ttccccggtg gtggataaca cgaccaccaa ttccacgctg 1020 gacgggaacg aggacacgtt tccgctgagt aggaacagga cggtgtttgc ggatttcagt 1080 catgataatg atatggtggg gatcttgact gctttgagaa tctttgaggg ggtggatgcg 1140 gagaagatga tggataatac gaccataccg agagagtacg gggagactgg cgatgatccg 1200 gcaaatttga aagagaggga gggcttgttc aaggttggtt gggtggtgcc atttgcggcg 1260 agggtgtatt ttgaaaagat gatttgtgat ggggatggga gtggagagat ggttcagagc 1320 gaggaggaac aggacaagga gttggtgagg atcttggtta acgatagagt ggttaaacta 1380 aatggatgtg aggccgatga gttggggagg tgtaagttgg ataaatttgt agagagtatg 1440 gagtttgcta ggaggggtgg agattgggac aagtgttttg cttag 1485 2. The gene Ncphy according to claim 1, whose amino acid composition is: Met Leu Arg Val Leu Ser Pro Asn Pro Ala Ser Cys Asp Ser Pro 1 5 10 15 Glu Leu Gly Tyr Gln Cys Asn Ser Glu Thr Thr His Thr Trp Gly 20 25 30 Gln Tyr Ser Pro Phe Phe Ser Val Pro Ser Glu Ile Ser Pro Ser 35 40 45 Val Pro Glu Gly Cys Arg Leu Thr Phe Ala Gln Val Leu Ser

50 55 60

Thr Pro Gly Lys Ala Ala Ala Ile Ser 65 70 75 Ala Val Leu Thr Lys Ile Lys Thr Ser Ala Thr Trp Tyr Ala Pro 80 85 90 Asp Phe Glu Phe Ile Lys Asp Tyr Asn Tyr Val Leu Gly Val Asp 95 100 105 His Leu Thr Ala Phe Gly Glu Gln Glu Met Val Asn Ser Gly Ile 110 115 120 Lys Phe Tyr Gln Arg Tyr Ala Ser Leu Ile Arg Asp Tyr Thr Asp 125 130 135 Pro Glu Ser Leu Pro Phe Ile Arg Ala Ser Gly Gln Glu Arg Val 140 145 150 Ile Ala Ser Ala Glu

Phe Thr Thr Gly Phe Tyr Ser Ala Leu 155 160 165 Leu Ala Asp Lys Asn Pro Pro Pro Ser Ser Leu Pro Leu Pro Arg 170 175 180 Gln Glu Met Val Ile Ile Ser Glu Ser Pro Thr Ala Asn Thr 185 190 195 Met His His Gly Leu Cys Arg Ala Phe Glu Asp Ser Thr Thr Gly 200 205 210 Asp Ala Ala Gln Ala Thr Phe Ile Ala Ala Asn Phe Pro Pro Ile 215 220 225 Thr Ala Arg Leu Asn Ala Gln Gly Phe Lys Gly Val Thr Leu Ser 230 235 240 Asp Thr Asp Val Leu Ser Leu Met Asp Leu Arg Pro Phe Asp Thr 245 250 255 Val Ala Tyr Pro Pro Ser Ser Ser Ser Leu Thr Thr Ser Ser Ser Ser Pro 260 265 270 Ser Gly Gly Ser Lys Leu Ser Pro Phe Cys Ser Leu Phe Thr Ala 275 280 285 Gln Asp Phe Thr Val Tyr Asp Tyr Leu Gln Ser Leu Gly Lys Phe 290 295 300 Tyr Gly Tyr Gly Pro Gly Asn Ser Leu Ala Ala Thr Gln Gly Val 305 310 315 Gly Tyr Val Asn Glu Leu Leu Ala Arg Leu Thr Val Ser Pro Val 320 325 330 Val Asp

Thr Thr Thr

Ser Thr Leu Asp Gly Asn Glu Asp 335 340 345 Thr Phe Pro Leu Ser Arg Arg Thr Val Phe Ala Asp Phe Ser 350 355 360 His Asp Asn Asp Met Val Gly Ile Leu Thr Ala Leu Arg Ile Phe 365 370 375 Glu Gly Val Asp Ala Glu Lys Met Met Asp

Thr Thr Ile Pro 380 385 390 Arg Glu Tyr Gly Glu Thr Gly Asp Asp Pro Ala Asn Leu Lys Glu 395 400 405 Arg Glu Gly Leu Phe Lys Val Gly Trp Val Val Pro Phe Ala Ala 410 415 420 Arg Val Tyr Phe Glu Lys Met Ile Cys Asp Gly Asp Gly Ser Gly 425 430 435 Glu Met Val Gln Ser Glu Glu Glu Gln Asp Lys Glu Leu Val Arg 440 445 450 Ile Leu Val Asn Asp Arg Val Val Lys Leu Asn Gly Cys Glu Ala 455 460 465 Asp Glu Leu Gly Arg Cys Lys Leu Asp Lys Phe Val Glu Ser Met 470 475 480 Glu Phe Ala Arg Arg Gly Gly Asp Trp Asp Lys Cys Phe Ala 485 490 494 Contains 6 glycosylation sites

an amino-terminal active center 3, as claimed in claim 1, the cloning of high temperature resistance, high specific activity phytase gene Ncphy is characterized in that using PCR technology, with Neurospora crassa (Neurospora crassa) AS 3.1604 chromosomal DNA as template, with NC-phy801, 5'-ACCGGAATTCATGCTACGAGTACTATCCCCAAATCC-3' and NC-phy901, 5'-ACCGGAATTCCCGCTTCGGTAATTCGCAGC-3' are used as primers to amplify the phytase mature peptide gene and remove the intron at the 5'end; PCR amplification conditions are: denaturation at 94°C for 10 minutes , add 1 unit of Pyrobest ^TM DNA polymerase, mix well and add mineral oil, then denature at 94°C for 1min, anneal at 56°C for 1.5min, extend at 72°C for 3min, after 30 cycles, extend at 72°C for 7min. 4, as claimed in claim 1, the expression of high temperature resistance, high specific activity phytase gene Ncphy is characterized in that EcoRI enzyme cutting sites are introduced at the two ends of the amplified fragment, and the phytase gene fragment of 1.5kb is reclaimed, EcoRI enzyme Cut and insert into pUC19 to obtain pUC-Ncphy; forwardly insert into pPIC9K vector to construct a yeast expression vector pP-Ncphy with high temperature resistance and high specific activity phytase gene.

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