CN109576240B - Amylosucrase mutant and preparation method and application thereof - Google Patents

Amylosucrase mutant and preparation method and application thereof Download PDF

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CN109576240B
CN109576240B CN201811547297.8A CN201811547297A CN109576240B CN 109576240 B CN109576240 B CN 109576240B CN 201811547297 A CN201811547297 A CN 201811547297A CN 109576240 B CN109576240 B CN 109576240B
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amylosucrase
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吴敬
吴丹
宿玲恰
赵雅琪
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Jiangnan University
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    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
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Abstract

The invention discloses an amylosucrase mutant and a preparation method and application thereof, belonging to the field of genetic engineering and enzyme engineering. The invention mutates the 399 th glycine of the sucrose amylase from Deinococcus Geothermalis into serine, and the obtained amylosucrase mutant is applied to the production of turanose. The yield of the turanose obtained by the amylosucrase mutant reaches 32 percent under the conditions that the temperature is 35 ℃ and the initial pH is 7.0, and the yield is 2 times of the conversion rate of the turanose produced by the wild enzyme. Therefore, the amylosucrase mutant of the invention can be applied to the preparation of turanose, so that the conversion rate of turanose is further improved.

Description

Amylosucrase mutant and preparation method and application thereof
Technical Field
The invention relates to an amylosucrase mutant and a preparation method and application thereof, belonging to the field of genetic engineering and enzyme engineering.
Background
The turanose is formed by connecting α -1,3 glycosidic bonds of one molecule of glucose and one molecule of fructose, is an isomer of sucrose, has the sweetness only half of that of the sucrose, has the characteristics of easy crystallization, high solubility, slow hydrolysis rate, easy regulation with other substances, no fermentation by cariogenic microorganisms and the like, is suitable for people suffering from obesity, hyperlipidemia, hypertension, diabetes and the like, and therefore, has the potential of becoming a new generation of low-calorie functional sweetener in the food field.
The other method is that sucrose is used as a substrate, and the amylosucrase is used for directly isomerizing the sucrose into the turanose, so that the cost of the substrate is lower and the process is simpler.
The amylosucrase belongs to α -amylase family (GH13), which takes sucrose as a natural substrate and mainly catalyzes two major transglycosylation reactions, namely polymerization and isomerization, wherein the two major transglycosylation reactions occur depending on initial sucrose concentration, the amylosucrase mainly catalyzes the isomerization reaction and mainly produces a large amount of insoluble α -1,4 glucoside chains and soluble malto-oligosaccharide when the sucrose concentration is higher, and the amylosucrase mainly catalyzes the polymerization reaction and produces a large amount of insoluble α -1,4 glucoside chains and soluble malto-oligosaccharide when the sucrose concentration is lower.
Disclosure of Invention
The invention aims to solve the technical problem of providing an amylosucrase mutant with improved catalytic isomerization reaction capability.
The invention mutates the 399 th amino acid of the amylosucrase with the starting amino acid sequence of SEQ ID NO.1 from glycine to serine to obtain the amylosucrase mutant which has higher turanose conversion efficiency compared with the parent.
In one embodiment of the invention, the source of the amylosucrase is moderately thermophilic (deinococcus geothermalis).
In one embodiment of the invention, the amino acid sequence of the amylosucrase mutant is shown in SEQ ID No. 2.
Another technical problem to be solved by the present invention is to provide a method for preparing an amylosucrase mutant, comprising the steps of:
(1) determining a mutation site on the basis of an amino acid sequence of the amylosucrase; designing a mutation primer of site-directed mutagenesis, and carrying out site-directed mutagenesis by taking a vector carrying an amylosucrase gene as a template; constructing a plasmid vector containing the mutant;
(2) transforming the mutant plasmid into a host cell;
(3) and selecting positive clones, performing fermentation culture, and preparing an enzyme solution.
In one embodiment of the invention, the starting amino acid sequence of the amylosucrase is depicted in SEQ ID NO. 1.
In one embodiment of the invention, the source of the amylosucrase is moderately thermophilic (deinococcus geothermalis).
In one embodiment of the invention, the amino acid sequence of the amylosucrase mutant is shown in SEQ ID No. 2.
In one embodiment of the present invention, the plasmid vector is any one of pUC series, pET series, or pGEX.
In one embodiment of the invention, the host cell is a bacterial or fungal cell.
In one embodiment of the invention, the bacterium is a gram-negative bacterium or a gram-positive bacterium.
Another technical problem to be solved by the invention is to provide a method for preparing turanose, which uses sucrose as a substrate and a glucoamylase mutant as a catalyst for conversion; the sucrase mutant is obtained by mutating the 399 th amino acid of amylosucrase with the starting amino acid sequence of SEQ ID NO.1 from glycine to serine.
In one embodiment of the invention, the conversion is carried out at 30-40 ℃ for 45-50 h.
The amylosucrase mutant is applied to the preparation of products containing turanose.
Has the advantages that:
the invention constructs an amylosucrase mutant and a preparation method and application thereof, wherein the amylosucrase mutant G399S is obtained by mutating 399 th glycine of amylosucrase derived from Deinococcus Geothermalis into serine. The amylosucrase mutant G399S is applied to the production of turanose, and the conversion rate of turanose can reach 32.2 percent under the conditions of 35 ℃ and pH7.0, and is 2 times of the conversion rate of wild enzyme. Therefore, the amylosucrase mutant of the invention can be applied to the preparation of turanose, so that the conversion rate of turanose is further improved.
Drawings
FIG. 1-HP L C assay of turanose in the amylosucrase mutant G399S transformation system.
Detailed Description
L B solid culture medium tryptone 10 g. L-1Yeast powder 5 g. L-1Sodium chloride 10 g. L-120 g. L of agar powder-1。
L B liquid culture medium tryptone 10 g. L-1Yeast powder 5 g. L-1Sodium chloride 10 g. L-1。
TB culture medium tryptone 12 g-L-1Yeast powder 24 g. L-1Glycerol 5 g-L-1，KH2PO42.31g·L-1，K2HPO4·3H2O 16.43g·L-1。
PBS buffer 8.18 g-L g of sodium chloride-10.2 g-L g of potassium chloride-1Disodium hydrogen phosphate 1.42 g. L-1Potassium dihydrogen phosphate 0.25 g. L-1The pH was adjusted to 7.4 with 2 mol/L hydrochloric acid.
Calculation formula of turanose conversion (mass of turanose/mass of initial sucrose) × 100%
The enzyme activity determination method of amylosucrase comprises the steps of determining the enzyme activity by adopting a 3, 5-dinitrosalicylic acid method (DNS) and a reducing sugar color development method, preparing 0.3M sucrose solution by using 50mM Tris-HCl buffer solution with the pH value of 7.0, adding 1.9M L substrate into a test tube with a plug, preheating in a water bath at 35 ℃ for 10min, adding 0.1M L enzyme solution, shaking and uniformly mixing, reacting for 30min, adding 3M L DNS to terminate the reaction, boiling for 7min, rapidly cooling with ice water, finally adding 10M L distilled water into the reaction system, and determining the light absorption value at 540 nm.
Definition of the enzymatic activity of amylosucrases: the amount of enzyme required to catalyze the production of fructose corresponding to 1. mu. mol per minute is one unit of activity.
Example 1: construction of recombinant bacterium
According to the amino acid sequence (PDB ID: 3UER) of amylosucrase dgas on NCBI, the chemical synthesis method is adopted to synthesize the dgas gene of amylosucrase, the plasmid for constructing escherichia coli is pET24a (+) and is provided with a T7 promoter, pET24a (+) plasmid and the dgas gene are subjected to double digestion by Nde I and Hind III respectively, after the digestion product is cut and recovered, the digestion product is connected by T4 ligase, the connection product is converted into E.coli JM109 competent cells to obtain recombinant cells, the recombinant cells are cultured and cultured for 8 hours at 37 ℃, transformants are picked and subjected to shake culture in L B liquid culture medium (containing 30 mg/L kanamycin), the plasmids are extracted, and the expression plasmid dgas/pET24a (+) is obtained after enzyme digestion verification.
E.coli B L21 (DE3) host bacteria are transformed by plasmid dgas/pET24a (+), coated with L B plates (containing 30 mg/L kanamycin) and cultured for 8 hours at 37 ℃, the obtained recombinant bacteria are named as E.coli J B L21 (DE3)/dgas/pET24a (+). the recombinant bacteria are selected to fall into L B liquid culture medium (containing 30 mg/L kanamycin), cultured overnight at 37 ℃ and stored in a glycerol tube.
Example 2: preparation of Single mutants
According to the sequence of the gene dgas of the amylosucrase synthesized by the chemical synthesis method in example 1, primers for introducing the G399S mutation were designed and synthesized, the amylosucrase dgas gene was subjected to site-directed mutagenesis, the DNA coding sequence was determined, and the Gly codon at position 399 was identified as the Ser codon. And (3) introducing the vector carrying the mutant gene into bacillus subtilis, escherichia coli or bacillus pumilus for expression to obtain the single-mutation amylosucrase.
Site-directed mutagenesis of G399S: using rapid PCR technology, expression vector dgas/pET24a (+) carrying wild type amylosucrase gene was used as template.
The site-directed mutagenesis primers for introducing the G399S mutation were:
a forward primer with a nucleotide sequence of SEQ ID NO. 3:
5’-GTCATGATGATATTAGCTGGGCAATTAGCG-3' (the mutated base is underlined)
Reverse primer with nucleotide sequence of SEQ ID NO. 4:
5’-CGCTAATTGCCCAGCTAATATCATCATGAC-3' (the mutated base is underlined)
The PCR reaction system was 5 × PS buffer 10. mu. L Mix (2.5mM) 4. mu. L, forward primer (10. mu.M) 1. mu. L, reverse primer (10. mu.M) 1. mu. L, template DNA 1. mu. L HS (5U/. mu. L) 0.5. mu. L, and double distilled water was added to 50. mu. L.
The PCR amplification conditions were: pre-denaturation at 94 ℃ for 4 min; followed by 20 cycles (98 ℃ for 10s, 55 ℃ for 30s, 72 ℃ for 8 min); extension was continued for 10min at 72 ℃.
The PCR product was digested with Dpn I, transformed into E.coli JM109 competent cells, cultured overnight in L B solid medium (containing 30. mu.g/m L kanamycin), selected and cloned in L B liquid medium (containing 30. mu.g/m L kanamycin), and plasmids were extracted, all mutated plasmids were sequenced correctly, and the resulting recombinant strain was named E.coli JM109/dgas/pET24a (+) (G399S).
The mutant with correct sequencing is inoculated to L B culture medium from a glycerol pipe, the overnight culture is carried out, plasmids are extracted, the plasmids are transformed to express competent cells of a host Escherichia coli B L21 (DE3), all the mutant plasmids have correct sequencing, and the obtained recombinant bacterium is named as E.coli J B L21 (DE3)/dgas/pET24a (+) (G399S).
Example 3: fermentation of amylosucrase mutants
Selecting recombinant bacteria E.coli J B L21 (DE3)/dgas/pET24a (+) (G399S) to grow in L B liquid culture medium (containing 30 mu G/m L kanamycin) for 8-10 h, inoculating the seed fermentation liquid to TB culture medium (containing 30 mu G/m L kanamycin) according to the inoculation amount of 5 percent, and performing shake culture at 37 ℃ on OD600And after the temperature reaches 0.2, adding 0.4mM isopropyl β -D-1-thiogalactopyranoside (IPTG) for induction, fermenting at 25 ℃ for 24 hours, centrifuging the fermentation liquor at 4 ℃ and 8000rpm for 10min, discarding the supernatant, collecting the thallus to 30OD, re-dissolving the thallus by PBS buffer solution with pH7.4, performing high-pressure homogenization and wall breaking, centrifuging at 8000rpm for 10min, and collecting the supernatant to obtain the crude mutant enzyme solution.
In the same way, the recombinant bacterium E.coli J B L21 (DE3)/dgas/pET24a (+) in example 1 was fermented to obtain a crude enzyme solution of the wild enzyme.
And respectively carrying out enzyme activity determination on the obtained crude enzyme solution of the mutant and the crude enzyme solution of the wild enzyme, wherein the result shows that the enzyme activity of the mutant is 1.5U/m L, and the enzyme activity of the wild enzyme is 2.2U/m L.
Example 4 HP L C determination of the yield of turanose
2g of sucrose and 2m of the crude enzyme solution of the mutant after wall breaking or the crude enzyme solution of the wild enzyme obtained in example 3 were added to a reactor of 10m L and reacted at 35 ℃ and an initial pH of 7.0 for 48 hours in a water bath shaker of 150rpm, after the reaction was completed, the enzyme was inactivated by boiling for 10 minutes, centrifuged at 12000rpm for 10 minutes, the supernatant was collected, diluted to 4-fold with a 50% (v/v) acetonitrile solution, and finally filtered through a 0.22 μm filter head, and the resulting filtrate was used as a sample to determine the content of turanose by HP L C chromatography, and the absorption peak of the product is shown in FIG. 1.
The HP L C chromatographic analysis detection conditions comprise an Agilent 1200HP L C chromatograph, an Agilent differential detector, a chromatographic Column of 4.6mm × 250mm 5 μm Syncronis Amino Column, a mobile phase of 80% (v/v) acetonitrile solution, a flow rate of 0.8m L/min and a Column temperature of 35 ℃.
The results are shown in Table 1, and the conversion rate of the single mutant G399S to produce turanose is 32.2%, which is 2 times the conversion rate of the wild enzyme.
TABLE 1 conversion of wild enzymes and mutants to turanose
Enzyme Conversion to turanose%
Wild enzyme 15.8
G399S 32.2
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
SEQUENCE LISTING
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Claims (8)

1. An amylosucrase mutant is characterized in that the amino acid sequence of the amylosucrase mutant is shown as SEQ ID No. 2.
2. A gene encoding an amylosucrase mutant according to claim 1.
3. A method for preparing the amylosucrase mutant of claim 1, comprising the steps of:
(1) determining a mutation site on the basis of an amino acid sequence of the amylosucrase; designing a mutation primer of site-directed mutagenesis, and carrying out site-directed mutagenesis by taking a vector carrying an amylosucrase gene as a template; constructing a plasmid vector containing the mutant;
(2) transforming the mutant plasmid into a host cell;
(3) and selecting positive clones, performing fermentation culture, and preparing enzyme solution to obtain the amylosucrase mutant.
4. The method according to claim 3, wherein the plasmid vector is any one of pUC series, pET series, or pGEX; the host cell is a bacterial or fungal cell; the bacteria are gram-negative bacteria or gram-positive bacteria.
5. A method for producing turanose, said method comprising converting sucrose to turanose using sucrose as a substrate and an amylosucrase mutant of claim 1 as a catalyst.
6. The method of claim 5, wherein the concentration of sucrose is 180-220 g/L, and the concentration of the amylosucrase mutant is 0.2-0.5U/m L.
7. The method according to claim 5, wherein the conversion is carried out at 30-40 ℃ for 45-50 h.
8. Use of an amylosucrase mutant according to claim 1 for the preparation of a product containing turanose.
CN201811547297.8A 2018-12-18 2018-12-18 Amylosucrase mutant and preparation method and application thereof Active CN109576240B (en)

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CN114908072B (en) * 2022-03-10 2023-08-15 江苏省奥谷生物科技有限公司 Beta-amylase mutant and application thereof in maltose preparation

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CN110747245B (en) * 2019-11-29 2021-07-27 江南大学 Method for preparing malt oligosaccharide syrup by using complex enzyme

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CN114908072B (en) * 2022-03-10 2023-08-15 江苏省奥谷生物科技有限公司 Beta-amylase mutant and application thereof in maltose preparation

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