CN107384902B - A kind of trehalose synthase and its preparation method and application that maltose conversion ratio improves - Google Patents
A kind of trehalose synthase and its preparation method and application that maltose conversion ratio improves Download PDFInfo
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- CN107384902B CN107384902B CN201710776826.0A CN201710776826A CN107384902B CN 107384902 B CN107384902 B CN 107384902B CN 201710776826 A CN201710776826 A CN 201710776826A CN 107384902 B CN107384902 B CN 107384902B
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
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- C12N9/1048—Glycosyltransferases (2.4)
- C12N9/1051—Hexosyltransferases (2.4.1)
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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- C12Y204/01—Hexosyltransferases (2.4.1)
- C12Y204/01245—Alpha,alpha-trehalose synthase (2.4.1.245)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses the trehalose synthases and its preparation method and application that a kind of maltose conversion ratio improves, and belong to genetic engineering and enzyme engineering field.The 289th near the trehalose synthase activated centre of Thermobifida fusca YX glutamic acid mutation is obtained mutant E289G at glycine by the present invention；By the 295th Histidine mutagenesis at asparagine mutant H295N；344th methionine is mutated into lysine mutation body M344K；367th methionine 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, industrial value with higher.
Description
The application is that application No. is 201510210023.X, the applyings date are as follows: applies for title are as follows: one on April 28th, 2015
The divisional application of the mutant of kind trehalose synthase and its preparation method and application.
Technical field
The present invention relates to the trehalose synthases and its preparation method and application that a kind of maltose conversion ratio improves, and belong to gene
Engineering and enzyme engineering field.
Background technique
Trehalose is a kind of safe and non-toxic non-reducing disaccharide constituted with 1,1- glycosidic bond, be there are three types of isomers (α,
α), isotrehalose (β, β), neotrehalose (α, β) exist generally in the form of two hydrates.With protein or amino acid
Common heating, will not generate Maillard reaction, and can keep certain stabilization in acidity, alkalinity, high temperature, ultra-low temperature surroundings
Property.Its unique bioactivity, so that trehalose is widely used.Largely studies have shown that trehalose is unicellular life
The protective agent of object, animal tissue and organ, protein, biomembrane, pharmaceutical preparation etc., be able to suppress lipid acidification, age of starch,
There is flavoring to rectify smelly function, do the properties such as glass transition temperature, agent of low hygroscopicity, low sugariness, can be applied for protein denaturation
To pharmaceutical sector, agricultural, biochemical product industry, cosmetic industry, food processing industry.
Commercialized trehalose is extracted from yeast in early days.About 700 dollars/kg of nineteen ninety price, recovery rate is too low,
Cost is excessively high.Nineteen ninety-five Japan realizes industrialized production using two enzymes method, so that trehalose price is by 20,000 yen original/kg
Substantially drop to 280 yen/kg in 1997.China 2002 for the first time with two enzymes method realize trehalose industrialization, 79 yuan of price/
kg.Two enzymes method is made from starch, in the effect of malt oligosaccharide based mycose hydrolase and malt oligosaccharide based mycose synthetase
Lower generation trehalose, this method production technology are complicated, it is difficult to promote, the only several companies in the whole world can 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
It researchs and solves, wherein trehalose synthase is crucial.Therefore, it excavates and is suitble to the trehalose synthase of production trehalose for pushing seaweed
The heavy industrialization of sugar, reduction industry cost are of great significance.
Maltose can be obtained by hydrolyzing starch, and certain glucose, industrialized production sea can be generated in production process
Algae sugar is excessively high using pure maltose cost.There are also faint hydrolysis other than turning glycosides effect for trehalose synthase, thus give birth to
At by-product glucose.If the Thermobifidafusca YX trehalose synthase used in the present invention that derives from is with technical grade
Maltose (glucose containing 1%-10%) is substrate, and trehalose synthase enzymatic conversion rate will receive certain influence.The present invention
Trehalose synthase is transformed by rite-directed mutagenesis, weakens glucose to the inhibiting effect of enzymatic conversion.
Summary of the invention
A technical problem to be solved by this invention is to provide a kind of mutant of trehalose synthase, activated centre position
Nearby amino acid mutates point, obtains the trehalose synthase for being influenced to weaken by grape Glyco inhabiting.The mutant includes containing
Substitution of the one, two or three relative to Thermobifidafusca YX trehalose synthase reactive amino acid residues.
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 289th glutamic acid (Glu) of parent's trehalose synthase is sported glycine (Gly),
It is named as E289G；Or the 295th histidine (His) is mutated into asparagine (Asn), gained mutant is named as
H295N；Or the 344th methionine (Met) is mutated into lysine (Lys), gained mutant is named as M344K；Or it will
367th methionine (Met) is mutated into leucine (Leu), and gained mutant is named as M367L.
The mutant can also be that the glutamic acid (Glu) by the 289th in single-mutant enzyme H295N sports glycine
(Gly), gained mutant is named as H295N/E289G；Or by the 344th in single-mutant enzyme H295N methionine (Met)
It is mutated into lysine (Lys), gained mutant is named as H295N/M344K；Or it will be the 367th in single-mutant enzyme H295N gene
The methionine (Met) of position is mutated into leucine (Leu), and gained mutant is named as H295N/M367L.
The mutant can also be the 367th in double-mutant enzyme H295N/M344K gene methionine (Met)
It is mutated into leucine (Leu), 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 influenced to weaken by grape Glyco inhabiting
The preparation method of synthase mutant, includes the following steps:
(1) mutational site is determined on the basis of Thermobifidafusca YX trehalose synthase amino acid sequence；If
The mutant primer for counting rite-directed mutagenesis, the carrier to carry trehalose synthase gene carry out rite-directed mutagenesis as template；Building is containing mutation
The plasmid vector of body；
(2) mutant plasmid is transformed into host cell；
(3) it selects positive colony and carries out fermented and cultured, and purify and obtain trehalose synthase mutant.
In one embodiment of the invention, the plasmid vector be pUC series, pET series or pGEX in it is any
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, the bacterium is Gram-negative bacteria or gram-positive bacteria.
It is raw to trehalose synthase that the present invention weakens with technical grade maltose (containing glucose 10%) glucose when for substrate
The inhibiting effect of trehalose is produced, with technical grade maltose (containing glucose 10%) for substrate, wild enzyme produces trehalose conversion ratio
Be 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%, 73.7%, reach conversion ratio of the wild enzyme using pure maltose to produce trehalose when substrate
(70.7%).
Specific embodiment
Embodiment 1: recombinant bacterium building
The plasmid TreS/pMD 18T for the gene containing encoding trehalose synthase that laboratory preservation has construct early period.For
The plasmid for constructing Escherichia coli is pET24a (+), has T7 promoter.By pET24a (+) plasmid and matter containing TreS gene
Grain carries out Nde I and III double digestion of Hind respectively, after digestion products are tapped and recovered, then is connected with T4 ligase, connection product converts
E.coli JM109 competent cell cultivates 8h through 37 DEG C of cultures, chooses transformant in the LB containing 30mg/L kanamycins liquid
Shake culture in culture medium, extracts plasmid, and digestion verification obtains expression plasmid TreS/pET24a (+).
By plasmid TreS/pET24a (+) Transformed E .coli BL21 (DE3) host strain, it is coated with (30mg/L) containing kanamycins
LB plate, 37 DEG C of culture 8h are named as TreS/pET24a (+)/E.coli BL21 (DE3).Single bacterium is chosen to fall on containing 30mg/
In L kanamycins LB liquid medium, 37 DEG C of overnight incubations save glycerol tube.
Embodiment 2: the preparation of mutant
(1) single mutation
From mutant enzyme E289G, H295N of the trehalose synthase of Thermobifidafusca YX, M344K,
M367L。
According to the gene order of the trehalose synthase of Thermobifidafusca YX, separately designs and synthesize introducing
The primer of E289G, H295N, M344K, M367L mutation carries out rite-directed mutagenesis to trehalose synthase gene, measures DNA encoding sequence
Column, identify the 289th Glu codon respectively and become Gly codon, and the 295th His codon becomes Asn codon,
344th Met codon becomes Lys codon, and the 367th Met codon becomes Leu codon.Mutant gene is placed in
Expression vector appropriate and importing in Escherichia coli is expressed, and single mutation trehalose synthase is obtained.Single mutation E289G,
The rite-directed mutagenesis of H295N, M344K, M367L: utilizing fast PCR technology, with expression vector TreS/pET24a (+) for template,
Introduce the rite-directed mutagenesis primer of E289G mutation are as follows:
Forward primer: 5 '-GAATCCGGCGGCGACGGTTGCCACATGAACT-3 ' (underscore is mutating alkali yl)
Reverse primer: 5 '-AGTTCATGTGGCAACCGTCGCCGCCGGATTCG-3 ' (underscore is mutating alkali yl)
Introduce the rite-directed mutagenesis primer of H295N mutation are as follows:
Forward primer: 5 '-ATGCCACATGAACTTCAACTTCCCGCTGATGCC-3 ' (underscore is mutating alkali yl)
Reverse primer: 5 '-GGCATCAGCGGGAAGTTGAAGTTCATGTGGCA-3 ' (underscore is mutating alkali yl)
Introduce the rite-directed mutagenesis primer of M344K mutation are as follows:
Forward primer: 5 '-CGAGCTGACCTTGGAGAAAGTCAGCGATGAAG-3 ' (underscore is mutating alkali yl)
Reverse primer: 5 '-TCTTCATCGCTGACTTTCTCCAAGGTCAGCTCG-3 ' (underscore is mutating alkali yl)
Introduce the rite-directed mutagenesis primer of M367L mutation are as follows:
Forward primer: 5 '-GCGGATGCGCGCCAACTTAGGGATCCGCCGCCGGC-3 ' (underscore is mutating alkali yl)
Reverse primer: 5 '-GCCGGCGGCGGATCCCTAAGTTGGCGCGCATCCGC-3 ' (underscore is mutating alkali yl)
PCR reaction system is equal are as follows: 5 × PS buffer 10 μ L, dNTPs Mix (2.5mM) 4 μ L, forward primer (10 μM) 1
Distilled water is added to 50 μ L in μ L, 1 μ L of reverse primer (10 μM), template DNA 1 μ L, PrimerStar HS (5U/ μ L) 0.5 μ L.
PCR amplification condition are as follows: 94 DEG C of initial denaturation 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 product is digested through Dpn I, converts e. coli jm109 competence, and competent cell (contains in LB solid medium
30 μ g/mL kanamycins) after overnight incubation, chooses to be cloned in LB liquid medium (containing 30 μ g/mL kanamycins) and mentioned after culture
Plasmid is taken, mutant plasmid is converted into expression 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 the trehalose synthase of Thermobifidafusca YX,
H295N/M367L: the 289th in single-mutant enzyme H295N gene glutamic acid (Glu) is mutated into glycine (Gly), or
344th methionine (Met) is mutated into lysine (Lys) or the 367th methionine (Met) 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 gene is template, separately designs and synthesize the primer for introducing E289G, M344K, M367L mutation, right
Single mutant H295N encoding gene carries out rite-directed mutagenesis, measures sequence, identifies the 289th Glu and become Gly codon, the
344 Met codons become Lys codon or the 367th Met becomes Leu codon, and mutant gene is placed in
Expression vector appropriate and importing in Escherichia coli is expressed, and double mutation trehalose synthase mutant are obtained.
The rite-directed mutagenesis of double mutation H295N/E289G, H295N/M344K, H295N/M367L: utilizing fast PCR technology,
With expression vector H295N/pET24a (+) for template,
Introduce the rite-directed mutagenesis primer of E289G mutation are as follows:
Forward primer: 5 '-GAATCCGGCGGCGACGGTTGCCACATGAACT-3 ' (underscore is mutating alkali yl)
Reverse primer: 5 '-AGTTCATGTGGCAACCGTCGCCGCCGGATTCG-3 ' (underscore is mutating alkali yl)
Introduce the rite-directed mutagenesis primer of M344K mutation are as follows:
Forward primer: 5 '-CGAGCTGACCTTGGAGAAAGTCAGCGATGAAG-3 ' (underscore is mutating alkali yl)
Reverse primer: 5 '-TCTTCATCGCTGACTTTCTCCAAGGTCAGCTCG-3 ' (underscore is mutating alkali yl)
Introduce the rite-directed mutagenesis primer of M367L mutation are as follows:
Forward primer: 5 '-CGGATGCGCGCCAACTTAGGGATCCGCCGCCG-3 ' (underscore is mutating alkali yl)
Reverse primer: 5 '-CCGGCGGCGGATCCCTAAGTTGGCGCGCATCC-3 ' (underscore is mutating alkali yl)
Method of the sequencing approach of PCR reaction system, reaction condition and mutated gene with single mutant.
(3) three mutation
The double-mutant enzyme H295N/M344K/M367L of the trehalose synthase of Thermobifidafusca YX: will be double prominent
The 367th methionine (Met) is mutated into leucine (Leu) in variant enzyme H295N/M344K gene, is named as H295N/
M344K/M367L.The preparation method of Trimutant enzyme is designed and is closed using double-mutant H295N/M344K encoding gene as template
At the primer for introducing M367L mutation, rite-directed mutagenesis is carried out to double-mutant H295N/M344K encoding gene, measures sequence, is identified
The 367th Met becomes Leu codon out, mutant gene is placed in expression vector appropriate and import in Escherichia coli into
Row expression, obtains three mutation trehalose synthase mutant.
Introduce the rite-directed mutagenesis primer of M367L mutation are as follows:
Forward primer: 5 '-CGGATGCGCGCCAACTTAGGGATCCGCCGCCG-3 ' (underscore is mutating alkali yl)
Reverse primer: 5 '-CCGGCGGCGGATCCCTAAGTTGGCGCGCATCC-3 ' (underscore is mutating alkali yl)
Method of the sequencing approach of PCR reaction system, reaction condition and mutated gene with single mutant.
(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 liquid medium
That mycin) 8~10h of growth, seed fermentation liquid is connected in TB culture medium (containing 30 μ g/mL kanamycins) by 5% inoculum concentration,
After cultivating 48h in 37 DEG C of shaking tables, fermentation liquid is removed into thallus in 4 DEG C, 8000rpm centrifugation 10min, collects centrifuged supernatant.
The yield of embodiment 3:HPLC detection trehalose
Be added in the reactor maltose 300g/L (contain glucose 10%), be added in a certain amount of example 2 obtain it is wild
The concentration enzyme solution of enzyme and mutant is adjusted pH to 8.0, in 30 DEG C, the water-bath of 150rpm with 20% sodium hydrate aqueous solution
30-50 hours timing samplings are reacted in shaking table, boil and sample 12000rpm is centrifuged 10min after 10min termination is reacted, take supernatant
Liquid carries out HPLC analysis moderately after dilution with 0.45 μm of ultrafiltration membrance filter.Chromatographic condition is as follows: differential refraction detector, NH2
Column (APS-2HYPERSIL, Thermo Scientific), mobile phase (water: acetonitrile=1:4), flow velocity: 0.8mLmin-1, column
Temperature: 40 DEG C.
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% |
It the results are shown in Table 1, 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%, 73.7%, reach conversion ratio (70.7%) of the wild enzyme using pure maltose to produce trehalose when substrate.
<110>Hunan Huisheng Bio-Technology Co., Ltd.
<120>trehalose synthase and its preparation method and application that a kind of maltose conversion ratio improves
<160> 17
<170> PatentIn version 3.3
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Claims (7)
1. a kind of trehalose synthase mutant, which is characterized in that relative to trehalose synthase parent, by the 295th histidine
It is mutated into asparagine；Or by the 295th Histidine mutagenesis at asparagine, while the 344th methionine being mutated
At lysine；Or the 295th Histidine mutagenesis is mutated into lysine at asparagine, the 344th methionine, simultaneously
367th methionine is mutated into leucine；The amino acid sequence of the trehalose synthase parent such as SEQ ID NO.1 institute
Show.
2. the method for preparing mutant described in claim 1, which comprises the steps of:
(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 constructs the plasmid vector containing mutant；
(2) plasmid for carrying the gene of encoding mutant body is transformed into host cell；
(3) it selects positive colony and carries out fermented and cultured, and purify and obtain trehalose synthase mutant.
3. according to the method described in claim 2, it is characterized in that, the plasmid vector is pUC series, pET series or pGEX
In any one.
4. mutant described in claim 1 is preparing the application in trehalose.
5. encoding the gene of mutant described in claim 1.
6. carrying the cell of gene described in claim 5.
7. cell described in claim 6 is preparing the application in trehalose.
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| CN110592060B (en) * | 2017-09-13 | 2021-03-02 | 江南大学 | Maltooligosyl trehalose synthetase mutant with improved enzyme activity |
| CN108753747B (en) * | 2018-06-05 | 2021-02-26 | 江南大学 | MTSase mutant with improved thermal stability and trehalose yield |
| CN108753746B (en) * | 2018-06-05 | 2021-03-30 | 江南大学 | Maltooligosyl trehalose synthase mutant with improved thermal stability |
| CN111500566B (en) * | 2019-01-28 | 2022-02-15 | 江南大学 | Trehalose synthetase mutant and preparation method and application thereof |
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2015
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- 2015-04-28 CN CN201710776847.2A patent/CN107384903B/en active Active
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| CN101629166A (en) * | 2009-08-20 | 2010-01-20 | 南宁中诺生物工程有限责任公司 | Mutant of trehalose synthetase from corynebacterium glutamicum and application thereof |
| CN103421761A (en) * | 2013-08-23 | 2013-12-04 | 南宁中诺生物工程有限责任公司 | Heatproof trehalose synthase fusion enzyme mutant gene and application thereof |
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| Publication number | Publication date |
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| CN107446900A (en) | 2017-12-08 |
| CN104789539A (en) | 2015-07-22 |
| CN104789539B (en) | 2017-10-31 |
| CN107384902A (en) | 2017-11-24 |
| CN107384903B (en) | 2019-05-17 |
| CN107384903A (en) | 2017-11-24 |
| CN107446900B (en) | 2019-05-17 |
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