CN109576240A

CN109576240A - A kind of amylosucrase mutant and the preparation method and application thereof

Info

Publication number: CN109576240A
Application number: CN201811547297.8A
Authority: CN
Inventors: 吴敬; 吴丹; 宿玲恰; 赵雅琪
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2018-12-18
Filing date: 2018-12-18
Publication date: 2019-04-05
Anticipated expiration: 2038-12-18
Also published as: CN109576240B

Abstract

本发明公开了一种淀粉蔗糖酶突变体及其制备方法与应用，属于基因工程和酶工程领域。本发明将来源于Deinococcus Geothermalis的蔗糖淀粉酶第399位的甘氨酸突变为丝氨酸，所得淀粉蔗糖酶突变体应用于松二糖的生产。该淀粉蔗糖酶突变体在温度为35℃，初始pH为7.0的条件下得到了松二糖的产率达到32％，是野生酶生产松二糖转化率的2倍。因此，本发明的淀粉蔗糖酶突变体能够应用于松二糖的制备中，使松二糖的转化率得到进一步的提高。The invention discloses an amylosucrase mutant and a preparation method and application thereof, belonging to the fields of genetic engineering and enzyme engineering. In the present invention, the glycine at position 399 of the sucrose amylase derived from Deinococcus Geothermalis is mutated to serine, and the obtained amylosucrase mutant is applied to the production of turanose. The amylosucrase mutant obtained 32% of turanose at the temperature of 35°C and the initial pH of 7.0, which was twice the conversion rate of turanose produced by the wild enzyme. Therefore, the amylosucrase mutant of the present invention can be applied to the preparation of turanose, so that the conversion rate of turanose can be further improved.

Description

A kind of amylosucrase mutant and the preparation method and application thereof

Technical field

The present invention relates to a kind of amylosucrase mutant and the preparation method and application thereof, belong to genetic engineering and enzyme engineering Field.

Background technique

Turanose is to be formed by a molecule glucose and a molecule fructose with α -1,3 glucosides key connections, is used as sucrose A kind of isomer, sugariness is only the half of sucrose.It has be easy crystallization, high soluble, hydrolysis rate is slow, be easy to and Other substances reconcile, not by cariogenic microbial fermentation the features such as, be suitable for suffer from obesity, hyperlipidemia, hypertension, diabetes et al. Group is edible.Therefore, turanose has the potentiality for becoming a new generation's functional sweetener low in calories in field of food.In addition, Turanose is also widely used in field of medicaments, can be used as the inhibitor of acid alpha-D-glucosidase, the medical treatment for Pang Beishi disease Diagnosis；Also there is antiinflammation, the inflammatory reaction etc. that lipopolysaccharides and glucose can be inhibited to mediate is a kind of with high added value Product.

At present enzyme process prepare the report of turanose only there are two types of.A kind of method is to be with the mixture of alpha-cyclodextrin and fructose Substrate synthesizes turanose using cyclodextrin glycosyltransferase, and the reachable maximum output of gained turanose is 45%.But the method The higher cost of the substrate utilized and the processing that need to separately be saccharified.Another method is to utilize amylosucrase using sucrose as substrate Sucrose isomerase is directly turned into turanose, cost is relatively low, technique is more succinct for the method substrate.

Amylosucrase belongs to alpha-amylase family (GH13), using sucrose as natural substrate, major catalytic polymerization and isomery This two big glycosides that turns reacts, and this two generation for turning glycosides reaction greatly depends on initial sucrose concentration.The starch when sucrose concentration is higher Based on invertase can be reacted with catalytic isomerization, primary product is turanose；When sucrose concentration is lower, amylosucrase then can be with Based on catalytic polymerization, generate largely with insoluble α-Isosorbide-5-Nitrae glycosidic linkage and with soluble malto-oligosaccharide.Cause This, in the preparation process of turanose, although polymerization reaction can be inhibited to a certain extent by the sucrose of high concentration, reaction When reducing to sucrose concentration, the generation of polymerization reaction still will affect the yield of final product.Therefore, a kind of polymerization activity is obtained The amylosucrase mutant of decrease has important commercial application potentiality for the conversion ratio for improving turanose.

Summary of the invention

A technical problem to be solved by this invention is to provide a kind of starch sugarcane that isoversion respond is promoted The mutant of carbohydrase.

The present invention pass through will set out amino acid sequence be SEQ ID NO.1 the 399th amino acids of amylosucrase by sweet Histidine mutations are serine, obtained amylosucrase mutant, have higher turanose transformation efficiency compared with parent.

In one embodiment of the invention, the source of the amylosucrase is moderate Thermophilic Bacteria (Deinococcus Geothermalis)。

In one embodiment of the invention, the amino acid sequence of the amylosucrase mutant such as SEQ ID Shown in NO.2.

Another technical problem to be solved by this invention is to provide a kind of method for preparing amylosucrase mutant, packet Include following steps:

(1) mutational site is determined on the basis of amylosucrase amino acid sequence；The mutant primer of rite-directed mutagenesis is designed, Carrier to carry amylosucrase gene carries out rite-directed mutagenesis as template；Construct the plasmid vector containing mutant；

(2) mutant plasmid is transformed into host cell；

(3) it selects positive colony and carries out fermented and cultured, prepare enzyme solution.

In one embodiment of the invention, the amino acid sequence such as SEQ ID NO.1 that sets out of the amylosucrase It is described.

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.

Another technical problem to be solved by this invention is to provide a kind of method for preparing turanose, the method be with Sucrose is substrate, is converted using sucrose starches enzyme mutant as catalyst；The sucrose enzyme mutant is by the ammonia that will set out The 399th amino acids of amylosucrase that base acid sequence is SEQ ID NO.1 are what serine obtained by glycine mutation.

In one embodiment of the invention, the conversion is 45~50h of progress under conditions of 30~40 DEG C.

Application of the amylosucrase mutant in terms of preparing the product containing turanose.

The utility model has the advantages that

The present invention constructs a kind of amylosucrase mutant and the preparation method and application thereof, amylosucrase mutant It is silk that G399S, which is by that will derive from the 399th glycine mutation of the amylosucrase of Deinococcus Geothermalis, What propylhomoserin obtained.Amylosucrase mutant G399S is applied to the production of turanose, at 35 DEG C, pine two under conditions of pH7.0 The conversion ratio of sugar is 2 times of wild enzymatic conversion rate up to 32.2%.Therefore, amylosucrase mutant of the invention can answer For making the conversion ratio of turanose be further improved in the preparation of turanose.

Detailed description of the invention

Fig. 1: the HPLC of turanose detects figure in amylosucrase mutant G399S transformation system.

Specific embodiment

LB solid medium: tryptone 10gL^-1, yeast powder 5gL^-1, sodium chloride 10gL^-1, agar powder 20g L^-1。

LB liquid medium: tryptone 10gL^-1, yeast powder 5gL^-1, sodium chloride 10gL^-1。

TB culture medium: tryptone 12gL^-1, yeast powder 24gL^-1, glycerol 5gL^-1, KH₂PO₄2.31g·L^-1, K₂HPO₄·3H₂O 16.43g·L^-1。

PBS buffer solution: sodium chloride 8.18gL^-1, potassium chloride 0.2gL^-1, disodium hydrogen phosphate 1.42gL^-1, di(2-ethylhexyl)phosphate Hydrogen potassium 0.25gL^-1, pH is adjusted to 7.4 with 2mol/L hydrochloric acid.

The calculation formula of turanose conversion ratio: (quality of turanose/initial sucrose quality) × 100%

The enzyme activity determination method of amylosucrase: using the side of 3,5- dinitrosalicylic acid system (DNS) and reduced sugar colour developing Method measures enzyme activity.With 50mM pH 7.0Tris-HCl buffer 0.3M sucrose solution, 1.9mL is added in tool plug test tube Substrate is added 0.1mL enzyme solution after 35 DEG C of water-baths preheat 10min, shakes and mix, 3mL DNS is added after reaction 30min and terminates instead It answers, then boil 7min and is cooled down with ice water rapidly, 10mL distilled water is finally added into above-mentioned reaction system, and in 540nm Lower measurement light absorption value.

The enzyme activity of amylosucrase defines: enzyme amount needed for catalysis generation per minute is equivalent to 1 μm of ol fructose is a work Unit of force.

Embodiment 1: recombinant bacterium building

According to the amino acid sequence (PDB ID:3UER) of amylosucrase dgas on NCBI, synthesized using chemical synthesis The dgas gene of amylosucrase.Plasmid for constructing Escherichia coli is pET24a (+), has T7 promoter.By pET24a (+) plasmid and dgas gene use Nde I and Hind III double digestion respectively, after digestion products are tapped and recovered, then with T4 ligase Connection, connection product Transformed E .coli JM109 competent cell obtain recombinant cell.Recombinant cell is cultivated through 37 DEG C of cultures 8h chooses transformant shake culture in LB liquid medium (card containing 30mg/L receive mycin), extracts plasmid, obtain after digestion verification Expression plasmid dgas/pET24a (+).

By plasmid dgas/pET24a (+) Transformed E .coli BL21 (DE3) host strain, it is coated with LB plate (card containing 30mg/L Receive mycin), 37 DEG C of culture 8h, obtained recombinant bacterium is named as E.coli J BL21 (DE3)/dgas/pET24a (+).Choose single bacterium It falls in LB liquid medium (card containing 30mg/L receive mycin), 37 DEG C of overnight incubations are stored in glycerol tube.

Embodiment 2: the preparation of single mutant

According to the dgas gene order for the amylosucrase that chemical synthesis in embodiment 1 synthesizes, introducing is designed and synthesized The primer of G399S mutation carries out rite-directed mutagenesis to amylosucrase dgas gene, measures DNA encoding sequence, identify the 399th The Gly codon of position becomes Ser codon.To carry the vector introduction bacillus subtilis of mutant gene, Escherichia coli or It is expressed in bacillus pumilus, obtains single mutation amylosucrase.

The rite-directed mutagenesis of G399S: utilizing fast PCR technology, is carried with carrying the expression of gene of wild type starch invertase Body dgas/pET24a (+) is template.

Introduce the rite-directed mutagenesis primer of G399S mutation are as follows:

Nucleotides sequence is classified as the forward primer of SEQ ID NO.3:

5’-GTCATGATGATATTAGCTGGGCAATTAGCG-3 ' (underscore is mutating alkali yl)

Nucleotides sequence is classified as the reverse primer of SEQ ID NO.4:

5’-CGCTAATTGCCCAGCTAATATCATCATGAC-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 20 circulations (98 DEG C of 10s, 55 DEG C of 30s, 72 DEG C of 8min)； 72 DEG C are continued 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 cards receive mycin) after overnight incubation, chooses to be cloned in LB liquid medium (receiving mycin containing 30 μ g/mL cards) and mentioned after culture Plasmid is taken, all mutant plasmids are sequenced correctly, and obtained recombinant bacterium is named as E.coli JM109/dgas/pET24a (+) (G399S)。

Correct mutant is sequenced, is seeded to LB culture medium from glycerol tube, is incubated overnight, extracts plasmid, plasmid is converted Expressive host e. coli bl21 (DE3) competent cell, mutant plasmid are sequenced correctly, and obtained recombinant bacterium is named as E.coli J BL21(DE3)/dgas/pET24a(+)(G399S)。

Embodiment 3: the fermentation of amylosucrase mutant

Picking recombinant bacterium E.coli J BL21 (DE3)/dgas/pET24a (+) (G399S) (contains 30 in LB liquid medium μ g/mL card receives mycin) 8~10h of growth, seed fermentation liquid is connected to TB culture medium by 5% inoculum concentration and (is received containing 30 μ g/mL cards mould Element) in, 37 DEG C of shaking table culture OD₆₀₀To after 0.2,0.4mM isopropyl ss-D-1- Thiogalactopyranoside (IPTG) is added and carries out Induction after fermenting for 24 hours at 25 DEG C, fermentation liquid is discarded supernatant in 4 DEG C, 8000rpm centrifugation 10min, collection thallus to 30OD, And redissolve laggard horizontal high voltage with the PBS buffer solution of pH7.4 and be homogenized broken wall, supernatant, which is collected, after 8000rpm centrifugation 10min is dashed forward Variant crude enzyme liquid.

Using same method, with recombinant bacterium E.coli J BL21 (DE3)/dgas/pET24a (+) fermentation in embodiment 1 Obtained broken wall enzyme solution is wild enzyme crude enzyme liquid.

Gained mutant crude enzyme liquid and wild enzyme crude enzyme liquid are subjected to enzyme activity determination respectively.The result shows that the enzyme of mutant Living is 1.5U/mL, and the enzyme activity of wild enzyme is 2.2U/mL.

The yield of embodiment 4:HPLC detection turanose

Mutant crude enzyme liquid or open country in the reactor of 10mL, after the broken wall obtained in 2g sucrose and 2mL example 3 is added Raw enzyme crude enzyme liquid is reacted 48 hours in 150rpm shaking bath, after reaction, is boiled under conditions of 35 DEG C, initial pH7.0 Boiling 10min inactivates enzyme, and 12000rpm is centrifuged 10min, collects supernatant, is diluted to 4 times with 50% (v/v) acetonitrile solution, finally It is filtered with 0.22 μm of filter.Gained filtrate measures the content of turanose, product absorption peak as sample by HPLC chromatogram As shown in Figure 1.

The condition of HPLC chromatogram analysis detection are as follows: Agilent 1200HPLC chromatograph, Agilent Composition distribution, color 5 μm of Syncronis Amino Column of column 4.6mm × 250mm are composed, mobile phase is the acetonitrile solution of 80% (v/v), flow velocity 0.8mL/min, 35 DEG C of column temperature.

It the results are shown in Table 1, it is the 2 of wild enzymatic conversion rate that the conversion ratio that single mutant G399S produces turanose, which is 32.2%, Times.

The conversion ratio of the wild enzyme of table 1 and mutant production turanose

Enzyme	Produce the conversion ratio % of turanose
		Wild enzyme	15.8
G399S	32.2

Although the present invention has been described by way of example and in terms of the preferred embodiments, it is not intended to limit the invention, any to be familiar with this skill The people of art can do various change and modification, therefore protection model of the invention without departing from the spirit and scope of the present invention Enclosing subject to the definition of the claims.

SEQUENCE LISTING

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Claims

What is claimed is: 1. An amylosucrase mutant, characterized in that the enzyme mutant is obtained by mutating the amino acid at position 399 of the amylosucrase whose starting amino acid sequence is SEQ ID NO. 1.

2. The amylosucrase mutant according to claim 1, wherein the enzyme mutant is obtained by mutating the amino acid at position 399 of the amylosucrase whose starting amino acid sequence is SEQ ID NO.1 from glycine to serine Obtained, named G399S.

3. The amylosucrase mutant according to claim 1 or 2, wherein the source of the amylosucrase is Deinococcus Geothermalis.

4. A gene encoding an amylosucrase mutant according to any one of claims 1-3.

5. a method for preparing the arbitrary described amylosucrase mutant of claim 1-3, is characterized in that, comprises the steps:

(1) Determine the mutation site on the basis of the amino acid sequence of amylosucrase; design mutation primers for site-directed mutation, and carry out site-directed mutation with the vector carrying the amylosucrase gene as a template; construct a plasmid vector containing the mutant;

(2) transforming the mutant plasmid into the host cell;

(3) Select positive clones for fermentation and culture, prepare enzyme solution, and obtain amylosucrase mutant.

6. preparation method according to claim 5 is characterized in that, described plasmid carrier is any one in pUC series, pET series, or pGEX; Described host cell is bacterium or fungal cell; Described bacterium is Gram-negative or Gram-positive bacteria.

7. A method for producing turanose, characterized in that the method takes sucrose as a substrate, and uses the amylosucrase mutant described in any one of claims 1-3 as a catalyst to convert sucrose into Turanose.

The method according to claim 7, wherein the concentration of the sucrose is 180-220 g/L, and the concentration of the amylosucrase mutant is 0.2-0.5 U/mL.

9 . The method according to claim 7 , wherein the transformation is carried out at 30 to 40° C. for 45 to 50 h. 10 .

10. The application of the amylosucrase mutant described in any one of claims 1-3 in the preparation of turanose-containing products.