CN108239632B

CN108239632B - Mutant of D-psicose-3-epimerase with improved thermal stability and application thereof

Info

Publication number: CN108239632B
Application number: CN201611217801.9A
Authority: CN
Inventors: 余允东; 祝俊
Original assignee: Livingzone Shanghai Bio Chem Technology Co ltd
Current assignee: Livingzone Shanghai Bio Chem Technology Co ltd
Priority date: 2016-12-26
Filing date: 2016-12-26
Publication date: 2023-05-05
Anticipated expiration: 2036-12-26
Also published as: CN108239632A

Abstract

The present invention provides a mutant of D-psicose-3-epimerase having improved thermostability and uses thereof, and in particular, the present invention provides a mutant D-psicose-3-epimerase having very excellent thermostability, which has very high catalytic efficiency (up to 29%) in wild-type D-psicose-3-epimerase corresponding to lysine (K) at position 122 and glycine (G) of SEQ ID No.: 1.

Description

Mutant of D-psicose-3-epimerase with improved thermal stability and application thereof

Technical Field

The invention relates to the field of biotechnology, in particular to a mutant of D-psicose-3-epimerase with improved thermal stability and application thereof.

Background

D-psicose is novel functional rare sugar with special health care function, the sweetness of the novel functional rare sugar is 70% of that of fructose, the energy of the novel functional rare sugar is only 0.3% of that of sucrose, and the novel functional rare sugar has the physiological functions of low energy, improving intestinal flora, reducing blood sugar, resisting decayed tooth, preventing obesity and the like. The U.S. FDA approved D-psicose in 2011 as a food additive. The development of D-psicose has been rapidly advanced.

In nature, D-psicose is extremely small in content and extremely difficult to obtain. DPE enzyme derived from wild strain and not modified is poor in thermal stability, and has certain limitation in industrial application, so that the use time of the enzyme is short, the application cost is high, and the large-scale industrial production of D-psicose is limited. Therefore, the DPE enzyme with good heat stability has less resources, and is not beneficial to industrialized application and popularization of D-psicose.

Therefore, there is an urgent need in the art to develop a D-psicose-3-epimerase having good thermostability and high catalytic efficiency.

Disclosure of Invention

The invention aims to provide the D-psicose-3-epimerase which has good heat stability and high catalytic efficiency.

In a first aspect the invention provides a mutated D-psicose-3-epimerase, which mutated D-psicose-3-epimerase has a mutation in lysine (K) at position 122, glycine (G) corresponding to SEQ ID NO: 1 of a wild-type D-psicose-3-epimerase.

In another preferred embodiment, the D-psicose-3-epimerase is derived from Paenibacillus (Paenibacillus senegalensis).

In another preferred embodiment, lysine (K) at position 122 is mutated to alanine (A); and/or

Glycine (G) at position 195 was mutated to proline (P).

In another preferred embodiment, the amino acid sequence of the mutant D-psicose-3-epimerase is shown in SEQ ID NO. 2.

In another preferred embodiment, the mutant D-psicose-3-epimerase has one or more characteristics selected from the group consisting of:

(a) The catalytic efficiency is more than or equal to 25%, preferably 26-40%, more preferably 27-30%;

(b) At 50-70 ℃ (preferably 60 ℃), the half-life of the mutant D-psicose-3-epimerase is 130% -350%, preferably 150% -280% of the half-life of the wild-type D-psicose-3-epimerase;

(c) The half-life of the mutant D-psicose-3-epimerase at 50-70 ℃ (preferably 60 ℃) is more than or equal to 200min, preferably 220-500min, more preferably 230-400min;

(d) The specific enzyme activity of the mutant D-psicose-3-epimerase is reduced by 40-80%, preferably 50-70%, of the specific enzyme activity of the wild-type D-psicose-3-epimerase after treatment at 50-70 ℃ (preferably 60 ℃).

In another preferred embodiment, the mutant D-psicose-3-epimerase has the same or substantially the same sequence as shown in SEQ ID NO. 1, except for amino acids 122 and 195.

In another preferred embodiment, the substantial identity is up to 50 (preferably 1-20, more preferably 1-10) amino acids different, wherein the differences include amino acid substitutions, deletions or additions and the muteins still have the activity of catalyzing synthesis of psicose.

In another preferred embodiment, the sequence shown in SEQ ID No. 1 has a homology of at least 80%, preferably at least 85% to 90%, more preferably at least 95%, most preferably at least 98%.

In another preferred embodiment, the mutant D-psicose-3-epimerase is a wild-type D-psicose-3-epimerase shown in SEQ ID NO. 1.

In a second aspect, the invention provides an isolated polynucleotide selected from the group consisting of:

(a) A polynucleotide encoding a polypeptide as set forth in SEQ ID No. 2;

(b) A polynucleotide having a sequence as set forth in SEQ ID NO. 3;

(c) A polynucleotide which has a nucleotide sequence which has a homology of 95% (preferably 98%) to the sequence shown in SEQ ID NO. 3 and which encodes a polypeptide shown in SEQ ID NO. 1 or 2;

(d) A polynucleotide complementary to the polynucleotide of any one of (a) - (c).

In another preferred embodiment, the polynucleotide additionally comprises an auxiliary element selected from the group consisting of: a signal peptide, a secretory peptide, a tag sequence (e.g., 6 His), or a combination thereof.

In another preferred embodiment, the polynucleotide is selected from the group consisting of: a DNA sequence, an RNA sequence, or a combination thereof.

In a third aspect, the invention provides a vector comprising a polynucleotide according to the second aspect of the invention.

In another preferred embodiment, the carrier is selected from the group consisting of: pET, pCW, pUC, pPIC9k, pMA5, or combinations thereof.

In a fourth aspect, the invention provides a host cell comprising a vector according to the third aspect of the invention or having integrated in its genome a polynucleotide according to the second aspect of the invention.

In another preferred embodiment, the host cell is selected from the group consisting of: prokaryotic cells, eukaryotic cells, or a combination thereof.

In another preferred embodiment, the host cell is selected from the group consisting of: coli, bacillus subtilis, pichia pastoris, streptomyces, or combinations thereof.

In another preferred embodiment, the prokaryotic cells comprise E.coli, B.subtilis, and/or C.glutamicum, preferably E.coli.

In another preferred embodiment, the eukaryotic cell comprises pichia pastoris, aspergillus niger, and/or streptomyces, preferably pichia pastoris.

In a fifth aspect, the present invention provides a method of producing a mutant D-psicose-3-epimerase according to the first aspect of the present invention, comprising the steps of:

culturing the host cell of the fourth aspect of the invention under conditions suitable for expression, thereby expressing the mutant D-psicose-3-epimerase; and

isolating the mutated D-psicose-3-epimerase.

In a sixth aspect the invention provides an enzyme preparation comprising a mutated D-psicose-3-epimerase according to the first aspect of the invention.

In another preferred embodiment, the enzyme preparation further comprises one or more components selected from the group consisting of: cobalt chloride (preferably cobalt chloride hexahydrate), sodium dihydrogen phosphate, disodium hydrogen phosphate, or a combination thereof.

In another preferred embodiment, the enzyme preparation comprises an injectable preparation, and/or a lyophilized preparation.

In a seventh aspect, the present invention provides a method for preparing psicose, comprising the steps of:

(i) Contacting the mutant D-psicose-3-epimerase according to the first aspect of the present invention with a reaction substrate to perform a catalytic reaction, thereby obtaining the psicose; and

(ii) Optionally, isolating and purifying the psicose.

In another preferred embodiment, the psicose is D-psicose.

In another preferred embodiment, in step (i), the reaction substrate comprises fructose.

In another preferred embodiment, the reaction substrate is selected from the group consisting of: crystalline fructose, fructose solution, high fructose syrup, or a combination thereof.

In another preferred embodiment, in step (i), the catalytic reaction is carried out for a period of time ranging from 0.5 to 10 hours, preferably from 0.8 to 8 hours, more preferably from 1 to 5 hours.

In another preferred embodiment, in step (i), the temperature of the catalytic reaction is from 20 to 80 ℃, preferably from 30 to 75 ℃, more preferably from 40 to 70 ℃.

In an eighth aspect the invention provides the use of a mutant D-psicose-3-epimerase according to the first aspect of the invention or a host cell according to the fourth aspect of the invention for the production of psicose.

In another preferred embodiment, the psicose is D-psicose.

It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.

Drawings

FIG. 1 shows the retention time of D-fructose standards on HPLC.

Figure 2 shows the retention time of D-psicose on HPLC.

FIG. 3 shows the stability of wild-type DPE enzyme (diamond-solid) and K122A/G195P mutant (■) at 60 ℃.

FIG. 4 shows a sample HPLC plot of wild-type DPE enzyme reaction recovered by ultrafiltration for 3 h.

FIG. 5 shows a sample HPLC plot of ultrafiltration recovered K122A/G195P mutant enzyme reaction for 3 h.

Detailed Description

The present inventors have unexpectedly obtained a D-psicose-3-epimerase derived from a paenibacillus (Paenibacillus senegalensis) mutation, and as a result of experiments, the half-life of the mutated D-psicose-3-epimerase is 130% to 350% (e.g., 163%) of that of the wild-type D-psicose-3-epimerase, and the decrease in specific enzyme activity of the mutated D-psicose-3-epimerase is significantly lower than that of the wild-type D-psicose-3-epimerase at 60 ℃, and thus, the mutated D-psicose-3-epimerase of the present invention has very excellent thermal stability, and furthermore, the D-psicose-3-epimerase of the present invention has very high catalytic efficiency (up to 29%). The invention also provides the use of the mutant D-psicose-3-epimerase.

As used herein, the terms "DPE enzyme", "PsDPE", "D-psicose-3-epimerase", "wild-type D-psicose-3-epimerase" are used interchangeably.

Wild-type D-psicose-3-epimerase

As used herein, "wild-type D-psicose-3-epimerase" refers to a naturally occurring, non-engineered D-psicose-3-epimerase, the nucleotides of which can be obtained by genetic engineering techniques, such as genomic sequencing, polymerase Chain Reaction (PCR), etc., the amino acid sequence of which can be deduced from the nucleotide sequence. The amino acid sequence of the wild-type D-psicose-3-epimerase is shown in SEQ ID NO. 1, and the source of the wild-type D-psicose-3-epimerase is not particularly limited, and a preferred source is Paenibacillus (Paenibacillus senegalensis).

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Mutant D-psicose-3-epimerase and coding sequence thereof

As used herein, the terms "mutein", "mutein of the invention", "mutated D-psicose-3-epimerase of the invention", "DPE enzyme mutant", "mutant enzyme", "mutant DPE enzyme", "mutant of D-psicose-3-epimerase" are used interchangeably and refer to a mutated D-psicose-3-epimerase which does not occur naturally and which is an artificially engineered protein of the protein shown in SEQ ID No. 1.

It will be appreciated that when the amino acid numbering in the mutant D-psicose-3-epimerase of the present invention is based on SEQ ID NO: 1, and when the homology of a particular mutein to the sequence shown in SEQ ID NO: 1 is 80% or more, the amino acid numbering of the mutein may be shifted from the amino acid numbering of SEQ ID NO: 1, such as by 1-5 positions to the N-terminal or C-terminal end of the amino acid, while such a shift is generally understood to be within a reasonable range by those skilled in the art using conventional sequence alignment techniques, and a mutein having the same or similar glycosyltransferase activity should not be within the range of the mutein of the present invention due to the amino acid numbering shift.

The muteins of the present invention are synthetic or recombinant proteins, i.e., can be the product of chemical synthesis, or can be produced from a prokaryotic or eukaryotic host (e.g., bacteria, yeast, plants) using recombinant techniques. Depending on the host used in the recombinant production protocol, the muteins of the present invention may be glycosylated or may be non-glycosylated. The muteins of the present invention may or may not also include an initial methionine residue.

The invention also includes fragments, derivatives and analogues of the muteins. As used herein, the terms "fragment," "derivative," and "analog" refer to a protein that retains substantially the same biological function or activity of the mutein.

The mutein fragment, derivative or analogue of the present invention may be (i) a mutein having one or more conserved or non-conserved amino acid residues, preferably conserved amino acid residues, substituted, which may or may not be encoded by the genetic code, or (ii) a mutein having a substituent in one or more amino acid residues, or (iii) a mutein formed by fusion of a mature mutein with another compound, such as a compound that extends the half-life of the mutein, e.g. polyethylene glycol, or (iv) a mutein formed by fusion of an additional amino acid sequence to the mutein sequence, such as a leader or secretory sequence or a sequence used to purify the mutein or a pro-protein sequence, or a fusion protein formed with an antigen IgG fragment. Such fragments, derivatives and analogs are within the purview of one skilled in the art and would be well known in light of the teachings herein. In the present invention, conservatively substituted amino acids are preferably generated by amino acid substitution according to Table I.

TABLE I

Initial residues	Representative substitution	Preferred substitution
			Ala(A)	Val；Leu；Ile	Val
Arg(R)	Lys；Gln；Asn	Lys
			Asn(N)	Gln；His；Lys；Arg	Gln
Asp(D)	Glu	Glu
			Cys(C)	Ser	Ser
Gln(Q)	Asn	Asn
			Glu(E)	Asp	Asp
Gly(G)	Pro；Ala	Ala
			His(H)	Asn；Gln；Lys；Arg	Arg
Ile(I)	Leu；Val；Met；Ala；Phe	Leu
			Leu(L)	Ile；Val；Met；Ala；Phe	Ile
Lys(K)	Arg；Gln；Asn	Arg
			Met(M)	Leu；Phe；Ile	Leu
Phe(F)	Leu；Val；Ile；Ala；Tyr	Leu
			Pro(P)	Ala	Ala
Ser(S)	Thr	Thr
			Thr(T)	Ser	Ser
Trp(W)	Tyr；Phe	Tyr
			Tyr(Y)	Trp；Phe；Thr；Ser	Phe
Val(V)	Ile；Leu；Met；Phe；Ala	Leu

The active mutant protein of the present invention has an activity of catalyzing the production of psicose.

Preferably, the mutein is shown as SEQ ID NO. 2.

It will be appreciated that the muteins of the invention generally have a higher homology (identity) than the sequence shown in SEQ ID No. 2, preferably the muteins have a homology of at least 80%, preferably at least 85% -90%, more preferably at least 95%, most preferably at least 98% to the sequence shown in SEQ ID No. 2.

In addition, the mutant proteins of the present invention may be modified. Modified (typically without altering the primary structure) forms include: chemically derivatized forms of muteins such as acetylated or carboxylated in vivo or in vitro. Modifications also include glycosylation, such as those resulting from glycosylation modifications during synthesis and processing of the mutein or during further processing steps. Such modification may be accomplished by exposing the mutein to an enzyme that performs glycosylation (e.g., mammalian glycosylase or deglycosylase). Modified forms also include sequences having phosphorylated amino acid residues (e.g., phosphotyrosine, phosphoserine, phosphothreonine). Also included are muteins modified to enhance their proteolytic resistance or to optimize their solubility properties.

The term "polynucleotide encoding a mutein" may be a polynucleotide comprising a mutein of the invention or may also comprise additional coding and/or non-coding sequences, preferably in the present invention the sequence of the mutein-encoding polynucleotide is shown in SEQ ID NO. 3.

The invention also relates to variants of the above polynucleotides which encode fragments, analogs and derivatives of the polypeptides or muteins having the same amino acid sequence as the invention. Such nucleotide variants include substitution variants, deletion variants and insertion variants. As known in the art, an allelic variant is a substitution of a polynucleotide, which may be a substitution, deletion, or insertion of one or more nucleotides, without substantially altering the function of the mutein encoded thereby.

The invention also relates to polynucleotides which hybridize to the sequences described above and which have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences. The invention relates in particular to polynucleotides which hybridize under stringent conditions (or stringent conditions) to the polynucleotides of the invention. In the present invention, "stringent conditions" means: (1) Hybridization and elution at lower ionic strength and higher temperature, e.g., 0.2 XSSC, 0.1% SDS,60 ℃; or (2) adding denaturing agents such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll,42℃and the like during hybridization; or (3) hybridization only occurs when the identity between the two sequences is at least 90% or more, more preferably 95% or more.

The muteins and polynucleotides of the invention are preferably provided in isolated form, and more preferably purified to homogeneity.

The full-length polynucleotide sequence of the present invention can be obtained by PCR amplification, recombinant methods or artificial synthesis. For the PCR amplification method, primers can be designed according to the nucleotide sequences disclosed in the present invention, particularly the open reading frame sequences, and amplified to obtain the relevant sequences using a commercially available cDNA library or a cDNA library prepared according to a conventional method known to those skilled in the art as a template. When the sequence is longer, it is often necessary to perform two or more PCR amplifications, and then splice the amplified fragments together in the correct order.

Once the relevant sequences are obtained, recombinant methods can be used to obtain the relevant sequences in large quantities. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods.

Furthermore, the sequences concerned, in particular fragments of short length, can also be synthesized by artificial synthesis. In general, fragments of very long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them.

At present, it is already possible to obtain the DNA sequences encoding the proteins of the invention (or fragments or derivatives thereof) entirely by chemical synthesis. The DNA sequence can then be introduced into a variety of existing DNA molecules (or vectors, for example) and cells known in the art. In addition, mutations can be introduced into the protein sequences of the invention by chemical synthesis.

Methods of amplifying DNA/RNA using PCR techniques are preferred for obtaining polynucleotides of the invention. In particular, when it is difficult to obtain full-length cDNA from a library, it is preferable to use RACE method (RACE-cDNA end rapid amplification method), and primers for PCR can be appropriately selected according to the sequence information of the present invention disclosed herein and synthesized by a conventional method. The amplified DNA/RNA fragments can be isolated and purified by conventional methods, such as by gel electrophoresis.

Vectors and host cells

The invention also provides a vector comprising the mutated D-psicose-3-epimerase gene of the invention, and a host cell comprising the vector.

In a preferred embodiment of the invention, the vector has the ability to be expressed in E.coli, more preferably in E.coli BL21 (DE 3) strain.

The mutated D-psicose-3-epimerase gene sequences of the present invention can be obtained by conventional methods, such as total artificial synthesis or PCR synthesis, which can be used by those skilled in the art. One preferred synthesis method is an asymmetric PCR method. The asymmetric PCR method is to amplify a large amount of single-stranded DNA (SSDNA) by PCR using a pair of primers in unequal amounts. The pair of primers is referred to as non-limiting primer and limiting primer, respectively, in a ratio of typically 50-100:1. During the first 10-15 cycles of the PCR reaction, the amplified product is mainly double stranded DNA, but when the restriction primer (low concentration primer) is consumed, the non-restriction primer (high concentration primer) directed PCR will produce a large amount of single stranded DNA. Primers for PCR can be appropriately selected according to the sequence information of the present invention disclosed herein, and can be synthesized by a conventional method. The amplified DNA/RNA fragments can be isolated and purified by conventional methods, such as by gel electrophoresis.

The polynucleotide sequences of the present invention may be used to express or produce a protein of interest by conventional recombinant DNA techniques comprising the steps of:

(1) Transforming or transducing a suitable host cell, preferably an E.coli cell, with a polynucleotide (or variant) encoding a protein of the invention, or with a recombinant expression vector comprising the polynucleotide;

(2) Culturing the host cell in a suitable medium;

(3) Separating and purifying the protein from the culture medium or the cells.

Methods well known to those skilled in the art can be used to construct expression vectors comprising the coding DNA sequences of the proteins of the invention and appropriate transcriptional/translational control signals, preferably commercially available vectors: bacterial plasmids, phage, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses or other vectors. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like. The DNA sequence may be operably linked to an appropriate promoter in an expression vector to direct mRNA synthesis. Representative examples of these promoters are: the lac or trp promoter of E.coli; PL promoter of lambda phage: eukaryotic promoters include CMV early promoter, HSV thymidine kinase promoter, early and late SV40 promoter, LTRs from retroviruses, and other known promoters that control the expression of genes in prokaryotic or eukaryotic cells or viruses thereof. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator, and insertion of an enhancer sequence into the vector will result in enhanced transcription in higher eukaryotic cells. Enhancers are cis-acting elements of DNA, usually about 10-300bp, that act on a promoter to increase the transcription of a gene. Such as adenovirus enhancers. In addition, the expression vector preferably comprises one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells.

The present invention also provides a recombinant vector comprising the DNA sequence of the mutated D-psicose-3-epimerase gene of the present invention. In a preferred embodiment, the promoter downstream of the recombinant vector comprises a multiple cloning site or at least one cleavage site. When it is desired to express the gene of interest, the gene of interest is ligated into a suitable multiple cloning site or cleavage site, thereby operably linking the gene of interest to a promoter.

In another preferred embodiment, the recombinant vector comprises in the 5 'to 3' direction: a promoter, a gene of interest and a terminator. If desired, the recombinant vector may further comprise the following elements: a protein purification tag; a 3' polynucleotide acidification signal; an untranslated nucleic acid sequence; transport and targeting nucleic acid sequences; selection markers (antibiotic resistance genes, fluorescent proteins, etc.); an enhancer; or an operator.

Methods for preparing recombinant vectors are well known to those of ordinary skill in the art. The expression vector may be a bacterial plasmid, phage, yeast plasmid, plant cell virus, mammalian cell virus or other vector, and in a preferred embodiment, the expression vector for the DPE enzyme and its mutants may be pET, pCW, pUC, pPIC k, pMA5 or other vector. In general, any plasmid or vector may be used as long as it is capable of replication and stability in a host.

The person skilled in the art can construct vectors containing the promoter and/or the gene sequence of interest of the present invention by means of well known methods. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like.

The expression vectors of the invention may be used to transform an appropriate host cell to allow the host to transcribe the RNA of interest or to express the protein of interest. The host cell may be a prokaryotic cell such as E.coli, corynebacterium glutamicum, brevibacterium flavum, streptomyces, agrobacterium: or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells such as plant cells, preferably rape, tobacco, soybean; insect cells such as Drosophila S2 or Sf9; animal cells such as CHO, COS or Bowes melanoma cells, etc. In a preferred embodiment, the expression host may be E.coli, B.subtilis, pichia pastoris, streptomyces or other host cells. It will be clear to one of ordinary skill in the art how to select appropriate vectors and host cells. Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryote (e.g., E.coli), caCl may be used ₂ The treatment can also be carried out by electroporation. When the host is eukaryotic, the following DNA transfection methods may be used: calcium phosphate coprecipitation methodConventional mechanical methods (e.g., microinjection, electroporation, liposome encapsulation, etc.). The method is carried out by growing or culturing the host cell according to methods known to those skilled in the art. For example, the microbial cells are usually at a temperature of 0-100℃and preferably 10-60℃with oxygen. The culture medium contains a carbon source such as glucose; nitrogen sources, typically in the form of organic nitrogen, such as yeast extract, amino acids; salts, such as ammonium sulfate, trace elements, such as iron, magnesium salts; vitamins if desired. The pH of the medium may be maintained at a fixed value during this period, that is, with or without control during the cultivation. The cultivation may be carried out in batch, semi-discontinuous or continuous culture. After culturing, the cells are collected, disrupted or used directly. D-fructose can be converted to D-psicose by culturing D-fructose with the mutant enzyme of the present invention or a cell containing the mutant enzyme of the present invention. The transformed plant may also be transformed by Agrobacterium or gene gun, such as leaf disc method, embryo transformation method, flower bud soaking method, etc. Plants can be regenerated from the transformed plant cells, tissues or organs by conventional methods to obtain transgenic plants.

The transformant obtained can be cultured by a conventional method to express the polypeptide encoded by the gene of the present invention. The medium used in the culture may be selected from various conventional media depending on the host cell used. The culture is carried out under conditions suitable for the growth of the host cell. After the host cells have grown to the appropriate cell density, the selected promoters are induced by suitable means (e.g., temperature switching or chemical induction) and the cells are cultured for an additional period of time.

The recombinant polypeptide in the above method may be expressed in a cell, or on a cell membrane, or secreted outside the cell. If desired, the recombinant proteins can be isolated and purified by various separation methods using their physical, chemical and other properties. Such methods are well known to those skilled in the art. Examples of such methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (salting-out method), centrifugation, osmotic sterilization, super-treatment, super-centrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high Performance Liquid Chromatography (HPLC), and other various liquid chromatography techniques and combinations of these methods.

The term "operably linked" refers to the attachment of a gene of interest to be expressed by transcription to its control sequences in a manner conventional in the art.

Preparation of enzyme preparation compositions

The invention also provides an enzyme preparation composition comprising the mutated D-psicose-3-epimerase of the invention.

The enzyme preparation composition of the present invention may further comprise: cobalt chloride (preferably cobalt chloride hexahydrate), disodium hydrogen phosphate, sodium dihydrogen phosphate, or a combination thereof.

Preparation method of psicose

The invention also provides a preparation method of psicose, which comprises the following steps:

(1) Contacting the mutant D-psicose-3-epimerase of the present invention with a reaction substrate to perform a catalytic reaction, thereby producing the psicose;

(2) Optionally, isolating and purifying the psicose.

The invention also relates to the application of the D-psicose prepared by the method in the production of human foods, animal feeds, cosmetics or medicines.

The main advantages of the invention include:

(1) The invention provides a DPE enzyme mutant with obviously improved thermal stability, which has a half-life period at 60 ℃ which is improved from a wild type 151min to 245.8min and is 1.63 times of that of the wild type DPE enzyme, so that the service period of the enzyme in the process of synthesizing D-psicose is greatly prolonged, and the DPE enzyme mutant has important industrial application value.

(2) The DPE enzyme mutant of the invention has about 25% of unit enzyme activity reduced by 60U/mg after heat treatment at 60 ℃ for 2 hours, and the reduced degree is far lower than the reduced degree of the enzyme activity (about 40% reduced and 45U/mg) after treatment under the same conditions of the wild DPE enzyme.

(3) The polynucleotide sequence for coding the mutant DPE enzyme has high expression quantity in escherichia coli, stable expression and capability of obviously reducing the cost for preparing the DPE enzyme.

(4) The preparation method of the mutant DPE enzyme has the advantages of short period and low cost, and is suitable for industrial production.

(5) The mutant DPE enzyme has obviously higher heat stability activity than that of wild DPE enzyme (PsDPE), and can greatly prolong the service period of the enzyme in industrial production, indirectly reduce the use cost of the enzyme and further reduce the production cost of D-psicose.

The present invention will be described in further detail with reference to the following examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedure, in which the detailed conditions are not noted in the following examples, is generally followed by routine conditions such as Sambrook et al, molecular cloning: conditions described in the laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989) or as recommended by the manufacturer. Percentages and parts are by weight unless otherwise indicated.

The experimental materials and reagents used in the following examples were obtained from commercial sources unless otherwise specified.

EXAMPLE 1 construction of a library of mutants of D-psicose-3-epimerase

BLAST alignment was performed based on the reported D-Psicose-3-epimerase (DPE) sequences, and it was found that the amino acid sequence derived from an unknown protein in Paenibacillus (Paenibacillus senegalensis) has a large similarity to several other DPE genes, and it was presumed that the genes have the ability to convert D-fructose into D-Psicose, and are potential DPE genes. The DNA sequence was optimized for total gene synthesis by Changzhou-based biosciences, inc., and recombined onto pET29a (+) vector (purchased from Novagen); the expression experiment and the functional experiment prove that the protein can convert D-fructose into D-psicose, has the function of D-psicose-3-epimerase, belongs to a member of the family, has an amino acid sequence shown as SEQ ID NO. 1, and then carries out directed evolution research on the basis of the wild type protein.

The PsDPE-pET29a (+) plasmid (synthesized by Changzhou-radix Yuan biotechnology Co., ltd.) constructed by total gene synthesis is used as a template, and the primers F1 and R1 are respectively used as forward and reverse primers, error-prone PCR is performed, and a mutant library is constructed.

The primer sequences were as follows: f1:5'GGAATTCCATATGAAATTCGGCACC 3' (SEQ ID NO.: 4); r1:5'CGCGGATCCTTACGGGGTGCTTTT 3' (SEQ ID NO: 5) having NdeI and BamHI restriction sites at each end

The error-prone PCR reaction system is as follows:

error-prone PCR was performed on a Bio-Rad T100 thermal cycler, and the PCR procedure was as follows:

the PCR reaction solution is subjected to 1% agarose gel electrophoresis, the fragment with the size of 880bp is recovered by cutting gel (specific operation is shown in the operating procedure of agarose gel recovery kit of Tiangen Biochemical technology (Beijing)), and after restriction enzyme digestion by restriction enzymes NdeI and BamHI, the fragment is connected with pET29a (+) (purchased from Novagen) carrier subjected to double enzyme digestion, BL21 (DE 3) (purchased from Tiangen Biochemical technology (Beijing)) is converted, and after inversion and overnight culture at 37 ℃, DPE mutant library is obtained, and the long monoclonal is used for activity screening

Example 2 activation and Induction of expression of mutants

From the above overnight-cultured plates, the single clone was picked up into 96-well plates containing 1ml of LB liquid medium (formulation: tryptone 10g/L, yeast Extract 5g/L, sodium chloride 10g/L, wherein Tryptone and Yeast Extract were purchased from Oxoid, sodium chloride was purchased from Guog Chemicals Co., ltd.) containing kanamycin sulfate at a final concentration of 50. Mu.g/ml, and cultured overnight at 37℃with shaking at 220 rpm. The following day, 100. Mu.l of the culture broth was aspirated from the above overnight-cultured 96 empty plates, added to a fresh 96-well plate of 1ml of LB liquid medium containing kanamycin sulfate at a final concentration of 50. Mu.g/ml, cultured for 4 hours at 37℃with shaking at 220rpm, and then induced by adding IPTG at a final concentration of 1mM, followed by further culturing at 30℃for 20 hours. A total of 400 were picked for activity screening.

The induced bacterial liquid is centrifuged at 4000rpm at 4 ℃ for 15min, and the supernatant is discarded. The cells were suspended in 100. Mu.l of 50mM sodium phosphate buffer (pH 7.0), and lysozyme was added at a final concentration of 0.5g/l, treated at 37℃for 1 hour, centrifuged at 4000rpm for 30 minutes, and the supernatant was transferred to a new 96-well plate and subjected to heat treatment at 60℃for 2 hours. Subsequently, 50. Mu.l of the supernatant was transferred to a new 96-well plate for use as a template for activity screening by centrifugation at 4000rpm at 4℃for 30 min.

Example 3 establishment of high Performance liquid chromatography HPLC high throughput screening method

High performance liquid chromatography HPLC was performed as follows:

shimadzu LC-20A HPLC with RID detector or equivalent;

analytical column: waters Sugar-Pak I, 6.5X100 mm column;

mobile phase: water;

flow rate: 0.6ml/min;

column temperature: 80 ℃;

a detector: the identity of the RID,

the temperature of the detector is 60 DEG C

The pure products of D-fructose and D-psicose produced by Sigma were used as standard products, and the loading amount was 20. Mu.l. The chromatographic analysis results are shown in fig. 1 and 2. The results showed that D-fructose retention time was 9.389min (FIG. 1) and D-psicose retention time was 12.233min (FIG. 2), both of which were large in separation, and were used as a method for screening enzyme mutants.

EXAMPLE 4 Activity screening of mutants

In the above 96-well plate containing 50. Mu.l of mutant enzyme supernatant, 400. Mu.l of 25% fructose aqueous solution (final concentration: 200 g/l), 50. Mu.l of 5mM CoCl were sequentially added ₂ The solution (final concentration 0.5 mM) was reacted at 60℃for 10min, then the enzyme was inactivated by treatment at 100℃for 10min, centrifuged at 4000rpm for 10min, and the supernatant was diluted 50-fold and subjected to HPLC detection, whereby mutants were screened for the amount of D-psicose produced by wild-type DPE as a control.

HPLC analysis revealed that the production of D-psicose was significantly higher for 1 mutant than for the wild-type control, and the numbers of the mutants were clone No. 200, respectively. This clone was then subjected to extensive culture to verify whether its thermostability was significantly improved.

Example 5 extensive cultivation and Activity verification of enzyme mutants

Wild-type DPE, clone 200 was inoculated into 5ml tubes containing LB liquid medium at a final concentration of 50. Mu.g/ml kanamycin sulfate, and cultured overnight at 37℃with shaking at 220 rpm. The following day, 1% of the culture medium was transferred to 100ml of LB liquid medium containing kanamycin sulfate at a final concentration of 50. Mu.g/ml, cultured at 37℃with shaking at 220rpm for 3 to 4 hours, IPTG at a final concentration of 1mM was added, and then induced overnight at 30℃at 200 rpm.

The induced bacterial solution was centrifuged at 8000rpm at 4℃for 10min, and the bacterial cells were washed twice with 20ml of pH 7.0.50 mM sodium phosphate buffer, and then suspended by adding 10ml of pH 7.0.50 mM sodium phosphate buffer. Followed by ultrasonication under ice bath conditions (ultrasonic power 200W, ultrasonic 3S/intermittent 5S, ultrasonic 10 min). Centrifuging the ultrasonic sample at 4deg.C and 12000rpm for 20min, subjecting the supernatant to heat treatment at 60deg.C for 2 hr, centrifuging at 4deg.C and 12000rpm for 20min, and lyophilizing the centrifuged supernatant to obtain lyophilized powder for activity detection.

The reaction conditions and HPLC detection conditions were the same as in example 4, and the lyophilized powder was dissolved in a buffer solution to a concentration of 1mg/ml, and the reaction system was 100ml. The result shows that the unit enzyme activity of the wild DPE is 75U/mg, and the unit enzyme activity is reduced to about 45U/mg by about 40% after heat treatment for 2 hours at 60 ℃; the unit enzyme activity of clone 200 was 80U/mg, and after heat treatment at 60℃for 2 hours, the unit enzyme activity was reduced by about 25% to 60U/mg.1U corresponds to the amount of enzyme required to produce 1. Mu. Mol of D-psicose per unit time (1 min) at 60℃and pH 7.0.

The thermostability of this clone was significantly higher than that of the wild-type DPE enzyme. The clone was then sequenced and aligned with the wild type DPE amino acid sequence, and clone 200 was found to carry two mutations, K122A and G195P, designated K122A/G195P, with a protein sequence shown as SEQ ID NO. 2 and a nucleotide sequence shown as SEQ ID NO. 3.

Example 6 thermostability of DPE enzyme at 60℃

The wild-type DPE and K122A/G195P mutants were incubated in a water bath at 60℃and their residual enzyme activities were periodically determined, and half-lives of the DPE enzyme and the mutants at this temperature were obtained according to the curve relationship between the residual enzyme activities and time (FIG. 3). The half-life of the wild-type DPE at 60℃was 151min and the half-life of the K122A/G195P mutant was 245.8min.

EXAMPLE 7 Synthesis of D-psicose Using mutants

100ml of 50% fructose solution and 11.9mg of cobalt chloride hexahydrate solid powder are respectively added into two 250ml Erlenmeyer flasks, after stirring for 10min under the water bath condition of 60 ℃, 0.1G of wild PsDPE enzyme lyophilized powder and K122A/G195P mutant enzyme lyophilized powder are respectively added, the reaction is started, and the reaction is completed after about 29% of the time is reached. Then the reaction liquid is ultrafiltered at 4 deg.c to obtain ultrafiltrate film with 10kD pore size, and the recovered enzyme liquid is reacted continuously. The reaction conditions were the same as above, and the conversion rate of the wild-type PsDPE reached only 8.6% at 3h (FIG. 4), while the conversion rate of the K122A/G195P mutant enzyme still reached about 28% (FIG. 5).

The result shows that the heat stability activity of the mutant enzyme is obviously higher than that of the wild PsDPE, the service cycle of the enzyme can be greatly prolonged in industrial production, and the use cost of the enzyme is indirectly reduced, so that the production cost of D-psicose is reduced.

All documents mentioned in this application are incorporated by reference as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the claims appended hereto.

Sequence listing

<110> Shanghai stand Biotech Co., ltd

<120> mutant of D-psicose-3-epimerase having improved thermostability and use thereof

<130> P2016-1229

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Met Lys Phe Gly Thr Tyr Phe Ala Tyr Trp Glu Gln Ser Trp Asp Thr

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Asp Tyr Leu Lys Tyr Val Lys Lys Val Ala Asp Leu Gly Phe Asp Val

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Leu Glu Val Gly Ala Ala Gly Ile Val Asn Met Ser Asp Asp Ala Leu

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Ser Ala Leu Lys Ser Glu Ala Glu Asn Tyr Ala Ile Thr Leu Thr Ala

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Gly Ile Gly Leu Pro Lys Gln Phe Asp Val Ser Ser Glu Asn Glu Ser

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Val Arg Gln Asp Gly Ile Ala Phe Met Lys Lys Ile Leu Asp Ala Leu

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Asn Asp Ala Lys Glu Ala Val Asp Tyr Val Lys Ala Val Asn Lys Pro

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Asn Val Lys Val Met Leu Asp Ser Phe His Met Asn Ile Glu Glu Asp

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Ile Lys Val Trp Arg Asp Leu Ser Asp Asn Ala Asp Glu Ala Lys Leu

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Met Lys Phe Gly Thr Tyr Phe Ala Tyr Trp Glu Gln Ser Trp Asp Thr

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Asp Tyr Leu Lys Tyr Val Lys Lys Val Ala Asp Leu Gly Phe Asp Val

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Leu Glu Val Gly Ala Ala Gly Ile Val Asn Met Ser Asp Asp Ala Leu

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Ser Ala Leu Lys Ser Glu Ala Glu Asn Tyr Ala Ile Thr Leu Thr Ala

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Gly Ile Gly Leu Pro Lys Gln Phe Asp Val Ser Ser Glu Asn Glu Ser

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Val Arg Gln Asp Gly Ile Ala Phe Met Lys Lys Ile Leu Asp Ala Leu

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aacgcaccgg cagttcgtaa acagagcatc aaaagcatgc aagaactggc agattatgca 420

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Claims

1. A mutant D-psicose-3-epimerase, characterized in that the mutant D-psicose-3-epimerase is mutated at lysine (K) 122, glycine (G) 195 of the wild-type D-psicose-3-epimerase corresponding to SEQ ID No. 1, the lysine (K) 122 is mutated to alanine (a), and glycine (G) 195 is mutated to proline (P).

2. The mutant D-psicose-3-epimerase of claim 1, wherein the D-psicose-3-epimerase is derived from paenibacillusPaenibacillus senegalensis。

3. The mutant D-psicose-3-epimerase of claim 1, wherein the amino acid sequence of the mutant D-psicose-3-epimerase is shown in SEQ ID No.: 2.

4. The mutant D-psicose-3-epimerase of claim 1, wherein the mutant D-psicose-3-epimerase has one or more characteristics selected from the group consisting of:

(a) The catalytic efficiency is more than or equal to 25%;

(b) The half-life of the mutant D-psicose-3-epimerase is 130% -350% of the half-life of the wild-type D-psicose-3-epimerase at 50-70 ℃;

(c) The half-life of the mutant D-psicose-3-epimerase at 50-70 ℃ is more than or equal to 200min;

(d) The specific enzyme activity of the mutant D-psicose-3-epimerase is reduced by 40-80% of the specific enzyme activity of the wild-type D-psicose-3-epimerase after treatment at 50-70 ℃.

5. An isolated polynucleotide, wherein the polynucleotide is selected from the group consisting of:

(a) A polynucleotide encoding a polypeptide as set forth in SEQ ID No. 2;

(b) A polynucleotide having a sequence as set forth in SEQ ID NO. 3;

(c) A polynucleotide which has a nucleotide sequence which has a homology of 95% or more with the sequence shown in SEQ ID NO. 3 and which encodes a polypeptide shown in SEQ ID NO. 2;

6. A vector comprising the polynucleotide of claim 5.

7. A host cell comprising the vector of claim 6 or having integrated into its genome the polynucleotide of claim 5.

8. A method of producing the mutant D-psicose-3-epimerase of claim 1, comprising the steps of:

culturing the host cell of claim 7 under conditions suitable for expression, thereby expressing the mutant D-psicose-3-epimerase; and

isolating the mutated D-psicose-3-epimerase.

9. An enzyme preparation comprising the mutated D-psicose-3-epimerase of claim 1.

10. The enzyme preparation according to claim 9, further comprising one or more components selected from the group consisting of: cobalt chloride, sodium dihydrogen phosphate, disodium hydrogen phosphate, or a combination thereof.

11. A process for preparing psicose, comprising the steps of:

(i) Contacting the mutant D-psicose-3-epimerase of claim 1 with a reaction substrate to perform a catalytic reaction, thereby obtaining the psicose; and

(ii) Optionally, isolating and purifying the psicose.

12. The method of claim 11, wherein said psicose is D-psicose.

13. The method of claim 11, wherein in step (i), the reaction substrate comprises fructose.

14. Use of a mutant D-psicose-3-epimerase according to claim 1 or a host cell according to claim 7 for the production of psicose.

15. The use according to claim 14, wherein said psicose is D-psicose.