CN107446900A

CN107446900A - A kind of trehalose synthase and its preparation method and application

Info

Publication number: CN107446900A
Application number: CN201710776849.1A
Authority: CN
Inventors: 吴敬; 宿玲恰; 张悦
Original assignee: Hunan Huisheng Biological Science & Technology Co Ltd
Current assignee: Hunan Jindai Technology Development Co.,Ltd.
Priority date: 2015-04-28
Filing date: 2015-04-28
Publication date: 2017-12-08
Anticipated expiration: 2035-04-28
Also published as: CN104789539A; CN104789539B; CN107446900B; CN107384903A; CN107384902A; CN107384903B; CN107384902B

Abstract

The invention discloses a kind of trehalose synthase and its preparation method and application, belongs to genetic engineering and enzyme engineering field.The glutamic acid mutation of the 289th near Thermobifida fusca YX trehalose synthase activated centre is obtained mutant E289G by the present invention into glycine；By the Histidine mutagenesis of the 295th into asparagine mutant H295N；The methionine of the 344th is mutated into lysine mutation body M344K；The methionine of the 367th is mutated into leucine mutant M367L, and carries out double mutation on the basis of H295N and obtains mutant H295N/E289G, H295N/M344K, H295N/M367L, H295N/M344K/M367L.Even if mutant is realized containing certain glucose in substrate, the transformation efficiency that trehalose synthase prepares trehalose will not be still greatly affected, and have higher industrial value.

Description

A kind of trehalose synthase and its preparation method and application

The application is Application No.：201510210023.X the applying date is：On April 28th, 2015, application are entitled：One The divisional application of the mutant of kind trehalose synthase and its preparation method and application.

Technical field

The present invention relates to a kind of trehalose synthase and its preparation method and application, belongs to genetic engineering and enzyme engineering field.

Background technology

Trehalose is a kind of safe and non-toxic non-reducing disaccharide formed with 1,1- glycosidic bonds, have three kinds of isomers i.e. (α, α), isotrehalose (β, β), neotrehalose (α, β), exist typically in the form of two hydrates.With protein or amino acid Common heating, will not produce Maillard reaction, and can keep certain stabilization in acidity, alkalescence, high temperature, ultra-low temperature surroundings Property.Its unique bioactivity so that trehalose is widely used.Substantial amounts of research shows that trehalose is unicellular life The protective agent of thing, animal tissue and organ, protein, biomembrane, pharmaceutical preparation etc., can suppress lipid acidifying, age of starch, Protein denaturation, there is flavoring to rectify smelly function, do the properties such as glass transition temperature, agent of low hygroscopicity, low sugariness, can be applied To pharmaceutical sector, agricultural, biochemical product industry, cosmetic industry, food processing industry.

Commercialized trehalose extracts from yeast in early days.About 700 dollars/kg of nineteen ninety price, recovery rate is too low, Cost is too high.Nineteen ninety-five, Japan using two enzymes method realized industrialized production so that trehalose price is by 20,000 yen original/kg Significantly drop to the 280 yen/kg of 1997.The Chinese industrialization for realizing trehalose with two enzymes method first in 2002,79 yuan of price/ kg.Two enzymes method is using starch as raw material, in the effect of malt oligosaccharide based mycose hydrolase and malt oligosaccharide based mycose synthetase Lower generation trehalose, this method complex manufacturing, it is difficult to promote, the whole world only has several companies to produce at present.And trehalose For synthase using maltose as substrate, a step is converted into trehalose, is the production method of relatively economical, but still has many problem needs Research and solve, wherein trehalose synthase is crucial.Therefore, the trehalose synthase for being adapted to produce trehalose is excavated for promoting marine alga The heavy industrialization of sugar, reduction industry cost are significant.

Maltose can be obtained by hydrolysis starch, and certain glucose, industrialized production sea can be produced in production process Algae sugar is too high using pure maltose cost.Trehalose synthase also has faint hydrolysis in addition to turning glycosides effect, thus gives birth to Into accessory substance glucose.If the Thermobifidafusca YX trehalose synthases used in the present invention that derive from are with technical grade Maltose (glucose containing 1%-10%) is substrate, and trehalose synthase enzymatic conversion rate can be influenceed by certain.The present invention Trehalose synthase is transformed by rite-directed mutagenesis, weakens inhibitory action of the glucose to enzymatic conversion.

The content of the invention

A technical problem to be solved by this invention is to provide a kind of mutant of trehalose synthase, its activated centre position Point nearby undergo mutation by amino acid, obtains being influenceed the trehalose synthase of decrease by grape Glyco inhabiting.The mutant includes containing One, two or three relative to Thermobifidafusca YX trehalose synthase reactive amino acid residues substitution.

Thermobifidafusca YX in the amino acid sequence and ncbi database of the parent of the trehalose synthase Trehalose synthase is consistent (accession number WP_011291031.1).

The mutant is that the glutamic acid (Glu) of the 289th of parent's trehalose synthase is sported into glycine (Gly), It is named as E289G；Or the histidine (His) of the 295th is mutated into asparagine (Asn), gained mutant is named as H295N；Or the methionine (Met) of the 344th is mutated into lysine (Lys), gained mutant is named as M344K；Or will The methionine (Met) of the 367th is mutated into leucine (Leu), and gained mutant is named as M367L.

The mutant can also be sports glycine by the glutamic acid (Glu) of the 289th in single-mutant enzyme H295N (Gly), gained mutant is named as H295N/E289G；Or by the methionine (Met) of the 344th in single-mutant enzyme H295N Lysine (Lys) is mutated into, gained mutant is named as H295N/M344K；Or by the 367th in single-mutant enzyme H295N genes The methionine (Met) of position is mutated into leucine (Leu), and gained mutant is named as H295N/M367L.

The mutant can also be the methionine (Met) of the 367th in double-mutant enzyme H295N/M344K genes Leucine (Leu) is mutated into, gained mutant is named as H295N/M344K/M367L.

Another technical problem to be solved by this invention is to provide a kind of trehalose for being influenceed to weaken by grape Glyco inhabiting The preparation method of synthase mutant, comprises the following steps：

(1) mutational site is determined on the basis of Thermobifidafusca YX trehalose synthase amino acid sequences；If The mutant primer of rite-directed mutagenesis is counted, rite-directed mutagenesis is carried out as template using the carrier for carrying trehalose synthase gene；Structure is containing mutation The plasmid vector of body；

(2) mutant plasmid is transformed into host cell；

(3) select positive colony and carry out fermented and cultured, and purify and obtain trehalose synthase mutant.

In one embodiment of the invention, the plasmid vector is pUC series, and pET is serial, or any in pGEX It is a kind of.

In one embodiment of the invention, the host cell is bacterium or fungal cell.

In one embodiment of the invention, described bacterium is Gram-negative bacteria or gram-positive bacteria.

The present invention is weakened with technical grade maltose (containing glucose 10%) when being substrate, and glucose is given birth to trehalose synthase The inhibitory action of trehalose is produced, with technical grade maltose (containing glucose 10%) for substrate, wild enzyme produces trehalose conversion ratio For 62.2%, and mutant E289G, H295N, M344K, M367L, H295N/E289G, H295N/M344K, H295N/M367L, H295N/M344K/M367L, the conversion ratio for producing trehalose respectively reaches 69.7%, 70.5%, 70.3%, 69.6%, 70.4%th, 70.9%, 72.3%, 73.7%, reach wild enzyme using pure maltose be substrate when production trehalose conversion ratio (70.7%).

Embodiment

Embodiment 1：Recombinant bacterium is built

Laboratories Accession has the plasmid TreS/pMD 18T of the gene containing encoding trehalose synthase built early stage.For The plasmid for building Escherichia coli is pET24a (+), with T7 promoters.By pET24a (+) plasmid and matter containing TreS genes Grain carries out Nde I and the double digestions of Hind III respectively, and digestion products are tapped rubber after recovery, then are connected with T4 ligases, connection product conversion E.coli JM109 competent cells, 8h is cultivated through 37 DEG C of cultures, chooses transformant in the LB containing 30mg/L kanamycins liquid Concussion and cultivate in culture medium, extracts plasmid, and digestion verification obtains expression plasmid TreS/pET24a (+).

By plasmid TreS/pET24a (+) Transformed E .coli BL21 (DE3) Host Strains, (30mg/L) containing kanamycins is coated with LB flat boards, 37 DEG C culture 8h, be named as TreS/pET24a (+)/E.coli BL21 (DE3).Single bacterium is chosen to fall on containing 30mg/ In L kanamycins LB liquid mediums, 37 DEG C of overnight incubations, glycerol tube is preserved.

Embodiment 2：The preparation of mutant

(1) single mutation

From mutant enzyme E289G, H295N of Thermobifidafusca YX trehalose synthase, M344K, M367L。

According to the gene order of Thermobifidafusca YX trehalose synthase, separately design and synthesize introducing The primer of E289G, H295N, M344K, M367L mutation, rite-directed mutagenesis is carried out to trehalose synthase gene, determines DNA encoding sequence Row, identify the Glu codons of the 289th and become Gly codons respectively, and the His codons of the 295th become Asn codons, 344th Met codon becomes Lys codons, and the 367th Met codon becomes Leu codons.Mutant gene is placed in Appropriate expression vector and importing in Escherichia coli is expressed, and obtains single mutation trehalose synthase.Single mutation E289G, H295N, M344K, M367L rite-directed mutagenesis：Using fast PCR technology, with expression vector TreS/pET24a (+) for template,

Introducing the rite-directed mutagenesis primer that E289G is mutated is：

Forward primer：5’-GAATCCGGCGGCGACGGTTGCCACATGAACT-3 ' (underscore is mutating alkali yl)

Reverse primer：5’-AGTTCATGTGGCAACCGTCGCCGCCGGATTCG-3 ' (underscore is mutating alkali yl)

Introducing the rite-directed mutagenesis primer that H295N is mutated is：

Forward primer：5’-ATGCCACATGAACTTCAACTTCCCGCTGATGCC-3 ' (underscore is mutating alkali yl)

Reverse primer：5’-GGCATCAGCGGGAAGTTGAAGTTCATGTGGCA-3 ' (underscore is mutating alkali yl)

Introducing the rite-directed mutagenesis primer that M344K is mutated is：

Forward primer：5’-CGAGCTGACCTTGGAGAAAGTCAGCGATGAAG-3 ' (underscore is mutating alkali yl)

Reverse primer：5’-TCTTCATCGCTGACTTTCTCCAAGGTCAGCTCG-3 ' (underscore is mutating alkali yl)

Introducing the rite-directed mutagenesis primer that M367L is mutated is：

Forward primer：5’-GCGGATGCGCGCCAACTTAGGGATCCGCCGCCGGC-3 ' (underscore is mutating alkali yl)

Reverse primer：5’-GCCGGCGGCGGATCCCTAAGTTGGCGCGCATCCGC-3 ' (underscore is mutating alkali yl)

PCR reaction systems are：5 × PS buffer 10 μ L, dNTPs Mix (2.5mM) 4 μ L, forward primer (10 μM) 1 μ L, the μ L of reverse primer (10 μM) 1, template DNA 1 μ L, PrimerStar HS (5U/ μ L) 0.5 μ L, distilled water are added to 50 μ L.

PCR amplification conditions are：94 DEG C of pre-degeneration 4min；Subsequent 30 circulations (98 DEG C of 10s, 55 DEG C of 5s, 72 DEG C of 8min)；72 DEG C continue to extend 10min.

PCR primer digests through Dpn I, converts e. coli jm109 competence, and competent cell (contains in LB solid mediums 30 μ g/mL kanamycins) after overnight incubation, choose be cloned in cultivated in LB fluid nutrient mediums (containing 30 μ g/mL kanamycins) after carry Plasmid is taken, mutant plasmid translation table is reached into host e. coli BL21 (DE3) competent cell, all mutant plasmids are sequenced just Really.

Enzymatic production

(2) double mutation

Double-mutant enzyme H295N/E289G, H295N/M344K of Thermobifidafusca YX trehalose synthase, H295N/M367L：The glutamic acid (Glu) of the 289th in single-mutant enzyme H295N genes is mutated into glycine (Gly), or The methionine (Met) of the 344th is mutated into lysine (Lys), or the methionine (Met) of the 367th is mutated into bright ammonia Sour (Leu), is respectively designated as H295N/E289G, H295N/M344K, H295N/M367L.The preparation method of double-mutant enzyme, with Single mutant H295N encoding genes are template, separately design and synthesize the primer for introducing E289G, M344K, M367L mutation, right Single mutant H295N encoding genes carry out rite-directed mutagenesis, determine sequence, identify the Glu of the 289th and become Gly codons, the The Met codons of 344 become Lys codons, or the Met of the 367th becomes Leu codons, and mutant gene is placed in Appropriate expression vector and importing in Escherichia coli is expressed, and obtains double mutation trehalose synthase mutant.

Double mutation H295N/E289G, H295N/M344K, H295N/M367L rite-directed mutagenesis：Using fast PCR technology, With expression vector H295N/pET24a (+) for template,

Introducing the rite-directed mutagenesis primer that E289G is mutated is：

Introducing the rite-directed mutagenesis primer that M344K is mutated is：

Introducing the rite-directed mutagenesis primer that M367L is mutated is：

Forward primer：5’-CGGATGCGCGCCAACTTAGGGATCCGCCGCCG-3 ' (underscore is mutating alkali yl)

Reverse primer：5’-CCGGCGGCGGATCCCTAAGTTGGCGCGCATCC-3 ' (underscore is mutating alkali yl)

Method of the sequence measurement of PCR reaction systems, reaction condition and mutator with single mutant.

(3) three mutation

The double-mutant enzyme H295N/M344K/M367L of Thermobifidafusca YX trehalose synthase：Will be double prominent The methionine (Met) of the 367th is mutated into leucine (Leu) in variant enzyme H295N/M344K genes, is named as H295N/ M344K/M367L.The preparation method of Trimutant enzyme, using double-mutant H295N/M344K encoding genes as template, design and close Into the primer for introducing M367L mutation, rite-directed mutagenesis is carried out to double-mutant H295N/M344K encoding genes, determines sequence, is differentiated Go out the Met of the 367th and become Leu codons, mutant gene is placed in appropriate expression vector and imports in Escherichia coli Row expression, obtains three mutation trehalose synthase mutant.

Introducing the rite-directed mutagenesis primer that M367L is mutated is：

(4) fermentation and purifying of mutant enzyme

The positive colony that picking is transferred to expressive host e. coli bl21 (DE3) (contains 30 μ g/mL cards in LB fluid nutrient mediums That mycin) 8~10h of growth, seed fermentation liquid is connected in TB culture mediums (containing 30 μ g/mL kanamycins) by 5% inoculum concentration, After cultivating 48h in 37 DEG C of shaking tables, zymotic fluid is removed into thalline in 4 DEG C, 8000rpm centrifugations 10min, collects centrifuged supernatant.

Embodiment 3：HPLC detects the yield of trehalose

In the reactor add maltose 300g/L (containing glucose 10%), add in a certain amount of example 2 obtain it is wild The concentration enzyme liquid of enzyme and mutant, pH is adjusted to 8.0 with 20% sodium hydrate aqueous solution, in 30 DEG C, 150rpm water-bath 30-50 hour timing samplings are reacted in shaking table, boils and sample 12000rpm is centrifuged into 10min after 10min terminating reactions, take supernatant Liquid carries out HPLC analyses moderately after dilution with 0.45 μm of ultrafiltration membrance filter.Chromatographic condition is as follows：Differential refraction detector, NH2 Post (APS-2HYPERSIL, Thermo Scientific), mobile phase (water:Acetonitrile=1:4), flow velocity：0.8mL·min^-1, post Temperature：40℃.

Table 1 produces the conversion ratio of trehalose using technical grade maltose as substrate

Enzyme	Conversion ratio (%)
		Wild enzyme	62.2%
E289G	69.7%
		H295N	70.5%
M344K	70.3%
		M367L	69.6%
H295N/E289G	70.4%
		H295N/M344K	70.9%
H295N/M367L	71.3%
		H295N/M344K/M367L	73.7%

1 is the results are shown in Table, the mutant enzyme that mutant expression obtains is compared with wild enzyme, it is found that mutant realizes sea Algae sugar synthase prepares the raising of trehalose transformation efficiency.Wild enzyme production trehalose conversion ratio is 62.2%, and mutant E289G、H295N、M344K、M367L、H295N/E289G、H295N/M344K、H295N/M367L、H295N/M344K/ M367L, the conversion ratio for producing trehalose respectively reaches 69.7%, 70.5%, 70.3%, 69.6%, 70.4%, 70.9%, 72.3%th, 73.7%, reach wild enzyme using pure maltose be substrate when production trehalose conversion ratio (70.7%).

<110>Hunan Huisheng Bio-Technology Co., Ltd.

<120>A kind of trehalose synthase and its preparation method and application

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Claims

1. a kind of trehalose synthase mutant, it is characterised in that relative to trehalose synthase parent, by the first sulphur ammonia of the 344th Acid mutation is into lysine；The amino acid sequence of the trehalose synthase parent is as shown in SEQ ID NO.1.

2. prepare the method for mutant described in claim 1, it is characterised in that comprise the following steps：

(1) mutational site is determined on the basis of parent's trehalose synthase amino acid sequence；The mutant primer of rite-directed mutagenesis is designed, Carrier to carry trehalose synthase gene carries out rite-directed mutagenesis as template and builds the plasmid vector containing mutant；

(2) plasmid for the gene for carrying encoding mutant body is transformed into host cell；

3. according to the method for claim 2, it is characterised in that the plasmid vector is pUC series, pET series, or pGEX In any one.

4. application of the mutant described in claim 1 in trehalose is prepared.

5. encode the gene of mutant described in claim 1.

6. carry the cell of gene described in claim 5.

7. application of the cell described in claim 6 in trehalose is prepared.