阅读说明：本技术 一种d-阿洛酮糖-3-差向异构酶突变体及其编码基因、重组载体、重组菌株和应用 (D-psicose-3-epimerase mutant and encoding gene, recombinant vector, recombinant strain and application thereof ) 是由 徐虹 黄月园 徐铮 李莎 于 2019-11-12 设计创作，主要内容包括：本发明公开了一种D-阿洛酮糖-3-差向异构酶突变体,将D-阿洛酮糖-3-差向异构酶AtDPEase的氨基酸发生如下任意一种的突变：H39N、H39N/I33L、H39N/S213C和H39N/I33L/S213C。本发明还公开了D-阿洛酮糖-3-差向异构酶突变体编码基因、重组载体、重组菌株及其在生产D-阿洛酮糖中的应用。突变体H39N、H39N/I33L、H39N/S213C和H39N/I33L/S213C在55℃的半衰期t<Sub>1/2</Sub>分别是96min、259min、113min、316min；T<Sub>50</Sub><Sup>20</Sup>分别比AtDPEase提高了1.5℃、2.1℃、1.7℃和2.6℃。(The invention discloses a D-psicose-3-epimerase mutant, wherein the amino acid of D-psicose-3-epimerase AtDPEase is mutated as follows: H39N, H39N/I33L, H39N/S213C and H39N/I33L/S213C. The invention also discloses a D-psicose-3-epimerase mutantA coding gene, a recombinant vector, a recombinant strain and application thereof in producing D-psicose. Mutants H39N, H39N/I33L, H39N/S213C and H39N/I33L/S213C have half-lives t at 55 ℃ 1/2 Respectively 96min, 259min, 113min and 316 min; t is 50 20 Respectively increased by 1.5 ℃, 2.1 ℃, 1.7 ℃ and 2.6 ℃ compared with AtDPEase.)

A D-psicose-3-epimerase mutant characterized by being obtained by mutating D-psicose-3-epimerase AtDPEase having an amino acid sequence shown in SEQ ID No. 1 in any one of the following cases:

(1) mutating the 39 th histidine in the amino acid sequence of AtDPEase into asparagine to obtain a D-psicose-3-epimerase mutant H39N;

(2) in the amino acid sequence of AtDPEase, the 39 th histidine is mutated into asparagine, and the 33 th isoleucine is mutated into leucine, so as to obtain a D-psicose-3-epimerase mutant H39N/I33L;

(3) in the amino acid sequence of AtDPEase, the 39 th histidine is mutated into asparagine, and the 213 th serine is mutated into cysteine, so as to obtain a D-psicose-3-epimerase mutant H39N/S213C;

(4) in the amino acid sequence of AtDPEase, the 39 th histidine is mutated into asparagine, the 33 th isoleucine is mutated into leucine, and the 213 th serine is mutated into cysteine, so that a D-psicose-3-epimerase mutant H39N/I33L/S213C is obtained.

2. The D-psicose-3-epimerase mutant according to claim 1, wherein the amino acid sequence of AtDPEase has a mutation of histidine 39 to asparagine, isoleucine 33 to leucine, and serine 213 to cysteine, resulting in D-psicose-3-epimerase mutant H39N/I33L/S213C.

3. An AtDPEase mutant gene encoding the D-psicose-3-epimerase mutant of claim 1.

4. The AtDPEase mutant gene according to claim 3, wherein (1) when the mutant gene is a mutant gene encoding D-psicose-3-epimerase mutant H39N, its nucleotide sequence is shown in SEQ ID No. 6; (2) when the mutant gene is a mutant gene for coding the D-psicose-3-epimerase mutant H39N/I33L, the nucleotide sequence is shown as SEQ ID No. 8; (3) when the mutant gene is a mutant gene for coding the D-psicose-3-epimerase mutant H39N/S213C, the nucleotide sequence is shown as SEQ ID No. 10; (4) when the mutant gene is a mutant gene for coding the D-psicose-3-epimerase mutant H39N/I33L/S213C, the nucleotide sequence is shown as SEQ ID No. 12.

5. A recombinant vector comprising the AtDPEase mutant gene of claim 3.

6. The recombinant vector according to claim 5, wherein the starting vector is pET-28a (+) or ptrc99 a.

7. A recombinant strain comprising the recombinant vector of claim 5.

8. The recombinant strain of claim 7, wherein the original strain is E.coli BL21(DE3) or E.coli MG 1655.

9. Use of the D-psicose-3-epimerase mutant of any one of claims 1 to 2, the AtDPEase mutant gene of any one of claims 3 to 4, the recombinant vector of any one of claims 5 to 6, the recombinant strain of any one of claims 7 to 8 for producing D-psicose.

Technical Field

The invention relates to a site-directed mutagenesis technology of enzyme, in particular to a D-psicose-3-epimerase mutant and a coding gene, a recombinant vector, a recombinant strain and application thereof.

Background

Diabetes is one of the most serious health problems in the world at present, according to data of 2017 of the international diabetes alliance, the number of people suffering from adult diabetes in the world reaches 4.25 hundred million, the number of people possibly suffering from diabetes in 2045 years is estimated to reach 6.29 hundred million, and 1.14 million of China becomes the country with the largest absolute number of patients suffering from diabetes. However, the current medical research on the treatment scheme of diabetes brings inconvenience and pain to the life of patients to a certain extent, and in recent years, the research on the rare sugar becomes a hot point of research in the diabetes diet treatment direction. D-psicose (D-allose/D-psicose) is an aldoketose isomer of D-allose, a rare sugar, and is also an epimer at C-3 position of fructose. As a functional rare sugar, the D-psicose has negligible heat (0.007kcal/g), and has health-care functions of reducing fat, protecting islet beta-cells, stabilizing blood sugar, improving glucose tolerance and the like, which are beneficial to human bodies. In the future, the research on the diabetes treatment strategy needs to adopt a plurality of disciplinary methods, and clinical treatment, drug research and food science are integrated to reduce the risk of diabetes to the maximum extent and provide guarantee for human health.

Biological synthesis of D-psicose D-fructose is generally catalyzed by D-psicose3-epimerase (DPEase) or D-tagatose 3-epimerase (DTEase) to convert D-fructose into D-psicose. The catalytic conversion efficiency obtained after expression of the DPEase gene extracted from Agrobacterium tumefaciens (Agrobacterium tumefaciens) in Escherichia coli is the highest (-33%) of all strains at present. However, a. tumefaciens dpease (atdpease) has a short half-life at 50 ℃ (63min) and poor stability. Therefore, the improvement of the thermal stability and the enzyme activity of the AtDPEase is important for promoting the subsequent development of the D-psicose.

In order to enable the industrial production of D-psicose, researchers use molecular biology technology to carry out genetic engineering transformation on D-psicose3-epimerase, so that the D-psicose3-epimerase has the potential of industrial application. Kim et al further explored the catalytic mechanism of AtDPEase by analyzing its protein crystal structure in 2006 using X-ray diffraction techniques. AtDPEase was found to be an isomerase with a tetrameric structure by means of molecular modeling. The enzyme is strictly metal ion dependent, Glu150, Asp183, His209 and Glu244 can be combined with metal ions to form the active center of the enzyme, and Trp112, Glu156 and Arg215 are key sites for combination of enzyme substrates.

Random mutagenesis techniques, such as error-prone PCR and DNA shuffling, are common strategies for increasing the thermostability of enzymes. In Choi et al 2011, random mutation is carried out on AtDPEase by an error-prone PCR method, two mutant strains with high stability, namely S213C and I33L are screened, site-directed mutation is carried out on S213 and I33 sites respectively, and serine at the 213 th site and leucine at the 33 th site are found to be amino acid mutation sites which are most suitable for enhancing the thermal stability of the enzyme. On the basis, the activity half-life period of the constructed double-site variant strain I33L/S213C at 55 ℃ is 26 times, 9 times and 4 times of that of wild type, S213C and I33L respectively. In subsequent studies, I33L/S213C catalyzed 700g/L fructose in whole cells to 230g/L D-psicose under optimal reaction conditions.

The existing research on the improvement of the thermal stability of the AtDPEase has been developed to a certain extent, but the research on rational design is not many, the theoretical basis of universality is lacked, the operation is complicated, the cost is high, and great improvement spaces exist in the aspects.

Disclosure of Invention

The purpose of the invention is as follows: in order to solve the problems of low thermal stability and low catalytic efficiency of wild AtDPEase, the invention provides a D-psicose-3-epimerase mutant in a first aspect, provides a coding gene, a recombinant vector and a recombinant strain of the D-psicose-3-epimerase mutant in a second aspect, and provides application of the enzyme mutant, the coding gene, the recombinant vector and the recombinant strain in production of D-psicose in a third aspect.

The design principle of the AtDPEase mutant in the invention is as follows: the AtDPEase is transformed by computer aided design and in vitro site-directed saturation mutation technology. The amino acid site with site-directed saturation mutation is an amino acid site with a high B-factor value obtained by predicting and calculating the amino acid of wild AtDPEase through YARASA software, and a site closer to an active center is excluded through Pymol analysis; the B-factor (also called Debye-Waller factor) is used for describing ray attenuation or scattering phenomenon caused by atom thermal motion when the protein crystal structure is subjected to X-ray diffraction, the value (B value) embodied by the B-factor can be used for identifying the mobility and flexibility of atoms, amino acid side chains and loop regions in the protein structure, and the higher the B-factor is, the more unstable or flexible the conformation of the corresponding part is. The in vitro single-site saturation mutation technology is an important technology in protein engineering, and mutants in which target site amino acids are respectively replaced by any other 19 amino acids are obtained in a short time by modifying a coding gene of a target protein; the technology is not only a powerful tool for protein directed modification, but also an important means for researching the structure-function relationship of the protein.

The technical scheme of the invention is as follows: the D-psicose-3-epimerase mutant is obtained by mutating D-psicose-3-epimerase AtDPEase with an amino acid sequence shown as SEQ ID No. 1 under any one of the following conditions:

(1) mutating the 39 th histidine in the amino acid sequence of AtDPEase into asparagine to obtain a D-psicose-3-epimerase mutant H39N;

(2) in the amino acid sequence of AtDPEase, the 39 th histidine is mutated into asparagine, and the 33 th isoleucine is mutated into leucine, so as to obtain a D-psicose-3-epimerase mutant H39N/I33L;

(3) in the amino acid sequence of AtDPEase, the 39 th histidine is mutated into asparagine, and the 213 th serine is mutated into cysteine, so as to obtain a D-psicose-3-epimerase mutant H39N/S213C;

(4) in the amino acid sequence of AtDPEase, the 39 th histidine is mutated into asparagine, the 33 th isoleucine is mutated into leucine, and the 213 th serine is mutated into cysteine, so that a D-psicose-3-epimerase mutant H39N/I33L/S213C is obtained.

The AtDPEase mutant is obtained by starting from D-psicose3-epimerase (AtDPEase) from agrobacterium tumefaciens by site-directed saturation mutagenesis and site-directed mutagenesis, wherein the AtDPEase consists of 289 amino acids, and the amino acid sequence is shown as SEQ ID No. 1; the nucleotide sequence is shown as SEQ ID No. 2; the AtDPEase is obtained by codon optimization of D-psicose3-epimerase (WT-AtDPEase) of wild type Agrobacterium tumefaciens by Nanjing Kingsry Biotech Co.

Wherein, WT-AtDPEase, Genebank accession number ANH56792.1, is composed of 289 amino acids, the amino acid sequence is shown as SEQ ID No. 3, and the nucleotide sequence is shown as SEQ ID No. 4.

Preferably, the amino acid sequence of AtDPEase has the mutation of histidine 39 to asparagine, isoleucine 33 to leucine and serine 213 to cysteine, resulting in D-psicose-3-epimerase mutant H39N/I33L/S213C.

The invention further provides an AtDPEase mutant gene encoding the D-psicose-3-epimerase mutant H39N, H39N/I33L, H39N/S213C or H39N/I33L/S213C.

Preferably, (1) when the mutant gene is a mutant gene encoding D-psicose-3-epimerase mutant H39N, the amino acid sequence of mutant H39N is shown as SEQ ID No. 5, and the nucleotide sequence of the mutant gene is shown as SEQ ID No. 6;

(2) when the mutant gene is a mutant gene for coding the D-psicose-3-epimerase mutant H39N/I33L, the amino acid sequence of the mutant H39N/I33L is shown as SEQ ID No. 7, and the nucleotide sequence of the mutant gene is shown as SEQ ID No. 8;

(3) when the mutant gene is a mutant gene for coding the D-psicose-3-epimerase mutant H39N/S213C, the amino acid sequence of the mutant H39N/S213C is shown as SEQ ID No. 9, and the nucleotide sequence of the mutant gene is shown as SEQ ID No. 10;

(4) when the mutant gene is a mutant gene for coding the D-psicose-3-epimerase mutant H39N/I33L/S213C, the amino acid sequence of the mutant H39N/I33L/S213C is shown as SEQ ID No. 11, and the nucleotide sequence of the mutant gene is shown as SEQ ID No. 12.

The invention further provides a recombinant vector containing the AtDPEase mutant gene.

Preferably, the starting vector is pET-28a (+) or ptrc99 a.

The present invention further provides a recombinant strain comprising the above recombinant vector.

Preferably, the original strain is e.coli BL21(DE3) or e.coli MG 1655.

The recombinant strain mutant H39N is constructed by the following steps:

1) cloning the AtDPEase fragment to a position between NdeI and BamHI enzyme cutting sites of an initial vector to obtain a recombinant vector;

2) taking the recombinant vector constructed in the step 1) as a template, designing a degenerate S/A primer, and carrying out PCR amplification to obtain a site-specific saturation mutation recombinant vector, namely mutating the 39 th histidine of the D-psicose-3-epimerase into asparagine; the nucleotide sequence of the degenerate primer is as follows:

His39-S：GAAGTTGCGGCGCACNNNATCAACGAATACTCT

His39-A：AGAGTATTCGTTGATNNNGTGCGCCGCAACTTC

3) transforming the site-directed mutagenesis recombinant vector obtained in the step 2) into an original strain to obtain a recombinant strain mutant H39N.

The recombinant strain mutant H39N/I33L is constructed by the following steps:

1) cloning the AtDPEase fragment to a position between NdeI and BamHI enzyme cutting sites of an initial vector to obtain a recombinant vector;

2) taking the recombinant vector constructed in the step 1) as a template, designing a degenerate S/A primer, and carrying out PCR amplification to obtain a site-specific saturation mutation recombinant vector H39N, namely mutating the 39 th histidine of the D-psicose-3-epimerase into asparagine; the nucleotide sequence of the degenerate primer is as follows:

His39-S：GAAGTTGCGGCGCACNNNATCAACGAATACTCT

His39-A：AGAGTATTCGTTGATNNNGTGCGCCGCAACTTC

3) transforming the site-directed mutagenesis recombinant vector obtained in the step 2) into an original strain to obtain a recombinant strain mutant H39N;

4) designing a site-directed mutagenesis primer by taking the gene of the mutant H39N obtained in the step 3) as a template, and carrying out PCR amplification to obtain a site-directed mutagenesis recombinant vector H39N/I33L, namely mutating the 33 rd isoleucine of the mutant H39N into leucine;

the nucleotide sequence of the site-directed mutagenesis primer is as follows:

I33L-S：5’-AAATTAGGTTTCGATATCTTAGAAGTTGCGGCG-3’

I33L-A：5’-CGCCGCAACTTCGATATCTAAGAAACCTAATTT-3’

wherein, the amino acid sequence of the mutant H39N is shown as SEQ ID No. 5;

5) transforming the recombinant vector of the site-directed mutagenesis obtained in the step 4) into an original strain to obtain a recombinant strain mutant H39N/I33L, wherein the amino acid sequence of the recombinant strain mutant is shown as SEQ ID No. 7.

Wherein, the recombinant strain mutant H39N/S213C is constructed by the following steps:

1) cloning the AtDPEase fragment to a position between NdeI and BamHI enzyme cutting sites of an initial vector to obtain a recombinant vector;

2) taking the recombinant vector constructed in the step 1) as a template, designing a degenerate S/A primer, and carrying out PCR amplification to obtain a site-specific saturation mutation recombinant vector H39N, namely mutating the 39 th histidine of the D-psicose-3-epimerase into asparagine; the nucleotide sequence of the degenerate primer is as follows:

His39-S：GAAGTTGCGGCGCACNNNATCAACGAATACTCT

His39-A：AGAGTATTCGTTGATNNNGTGCGCCGCAACTTC

3) transforming the site-directed mutagenesis recombinant vector obtained in the step 2) into an original strain to obtain a recombinant strain mutant H39N;

4) designing a site-directed mutagenesis primer by taking the gene of the mutant H39N obtained in the step 3) as a template, and carrying out PCR amplification to obtain a site-directed mutagenesis recombinant vector H39N/S213C, namely mutating the 213 th serine of the mutant H39N into cysteine;

the nucleotide sequence of the degenerate primer is as follows:

S213C-S：5’-TTCCACACCGGTGAATGTAACCGTCGCGTTCCG-3’

S213C-A：5’-CGGAACGCGACGGTTACATTCACCGGTGTGGAA-3’

wherein, the H39N amino acid sequence is shown as SEQ ID No. 5;

5) transforming the site-directed mutagenesis recombinant vector obtained in the step 4) into an original strain to obtain a recombinant strain mutant H39N/S213C, wherein the amino acid sequence of the recombinant strain mutant is shown as SEQ ID No. 9.

The recombinant strain mutant H39N/I33L/S213C is constructed by the following steps:

1) cloning the AtDPEase fragment to a position between NdeI and BamHI enzyme cutting sites of an initial vector to obtain a recombinant vector;

2) taking the recombinant vector constructed in the step 1) as a template, designing a degenerate S/A primer, and carrying out PCR amplification to obtain a site-specific saturation mutation recombinant vector H39N, namely mutating the 39 th histidine of the D-psicose-3-epimerase into asparagine; the nucleotide sequence of the degenerate primer is as follows:

His39-S：GAAGTTGCGGCGCACNNNATCAACGAATACTCT

His39-A：AGAGTATTCGTTGATNNNGTGCGCCGCAACTTC

3) transforming the site-directed mutagenesis recombinant vector obtained in the step 2) into an original strain to obtain a recombinant strain mutant H39N;

4) designing a site-directed mutagenesis primer by taking the gene of the mutant H39N obtained in the step 3) as a template, and carrying out PCR amplification to obtain a site-directed mutagenesis recombinant vector H39N/I33L, namely mutating the 33 rd isoleucine of the mutant H39N into leucine;

the nucleotide sequence of the degenerate primer is as follows:

I33L-S：5’-AAATTAGGTTTCGATATCTTAGAAGTTGCGGCG-3’

I33L-A：5’-CGCCGCAACTTCGATATCTAAGAAACCTAATTT-3’

wherein, the H39N amino acid sequence is shown as SEQ ID No. 5;

5) transforming the recombinant vector of the site-directed mutagenesis obtained in the step 4) into an original strain to obtain a recombinant strain mutant H39N/I33L, wherein the amino acid sequence of the recombinant strain mutant is shown as SEQ ID No. 7;

6) designing a site-directed mutagenesis primer by taking the gene of the mutant H39N/I33L obtained in the step 5) as a template, and carrying out PCR amplification to obtain a site-directed mutagenesis recombinant vector, namely mutating the 213 th serine of the mutant H39N/I33L into cysteine:

the nucleotide sequence of the degenerate primer is as follows:

S213C-S：5’-TTCCACACCGGTGAATGTAACCGTCGCGTTCCG-3’

S213C-A：5’-CGGAACGCGACGGTTACATTCACCGGTGTGGAA-3’

wherein the H39N/I33L amino acid sequence is shown as SEQ ID No. 7;

7) transforming the recombinant vector of the site-directed mutagenesis obtained in the step 6) into an original strain to obtain a mutant H39N/I33L/S213C, wherein the amino acid sequence of the mutant is shown as SEQ ID No. 11.

The invention further provides the D-psicose-3-epimerase mutant, the AtDPEase gene, the recombinant vector and application of the recombinant strain in production of D-psicose.

Preferably, the D-psicose-3-epimerase mutant is used as an immobilized enzyme for producing D-psicose.

Has the advantages that: the invention is based on the rational analysis of the flexibility and the motility of the amino acid structure of the protein and calculates and predicts the mutation sites by the aid of computer-aided design software, and compared with the traditional mutation method, the invention has high targeting property and obviously avoids the blindness of screening. The invention combines the DPEase mutant H39N (His39Asn) obtained by screening with the mutant I33L/S213C (Ile33Leu/Ser213Cys) with the best AtDPEase stability obtained by screening, designs multi-site mutation, combines the site-specific saturation mutation with the site-specific mutation to obtain the AtDPEase mutant with greatly improved thermal stability, and finally obtains 4 mutants with high thermal stability. Mutant H39N/I33L/S213C is the most stable strain with half-life t at 55 ℃ compared to AtDPEase and mutant I33L/S213C_1/2Increases by 306min and 51min respectively. In the research on the effect of the immobilized enzyme, the immobilized enzyme prepared by using the Alg (Ti) PDA material has good use stability. The batch use experiment shows that Alg (Ti) PDA-H39N/I33L/S213C is still used for 10 times63% of the original enzyme activity was retained, while Alg (Ti) PDA-AtDPEase had only 21% of residual enzyme activity. And when the concentration of fructose is 500g/L, the yield of D-psicose obtained by catalyzing H39N/I33L/S213C can reach 164g/L, the catalytic conversion efficiency is not influenced, and the method has a high market application prospect. In industrial application, immobilized enzyme is usually used for catalytic production, the mutant H39N/I33L/S213C has improved thermal stability and good tolerance to environment, and the immobilized enzyme can be stored for a long time or used in batches, so that the cost of industrial production is greatly reduced.

Drawings

FIG. 1 is a calculation analysis of B-factor values for amino acids of AtDPEase in example 2;

FIG. 2 is a simulated analysis of the protein structure of AtDPEase in example 2;

FIG. 3 is the relative residual enzyme activities of AtDPEase and mutants E42K, E42N, H39N, Q188G and H38Q in example 3 after incubation at 55 ℃ for 15 min;

FIG. 4 is the relative residual enzyme activities of AtDPEase and mutants H39N, H39N/S213C, H39N/I33L, H39N/S213C/I33L in example 4 after incubation at 55 ℃ for 15 min;

FIG. 5 is an SDS-PAGE analysis of the expression and purification of AtDPEase and mutant H39N in example 5;

FIG. 6 shows the T of AtDPEase and its mutants H39N, H39N/S213C, H39N/I33L, H39N/S213C/I33L in example 7₅₀ ²⁰；

FIG. 7 shows the analysis of the storage stability at room temperature of AtDPEase and its mutants H39N, H39N/S213C, H39N/I33L, H39N/S213C/I33L in example 7;

FIG. 8 shows the activity half-lives t at 55 ℃ of AtDPEase and mutants H39N, H39N/S213C, H39N/I33L, H39N/S213C/I33L in example 7_1/2Measuring results;

FIG. 9 shows 3 different immobilized enzymes;

FIG. 10 is the stability of AtDPEase immobilized enzyme batch use;

FIG. 11 shows the stability of mutant H39N/S213C/I33L immobilized enzyme batch.

Detailed Description