CN113564092B - Fusion enzyme for directionally synthesizing dextran, construction method and application thereof - Google Patents

Fusion enzyme for directionally synthesizing dextran, construction method and application thereof Download PDF

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CN113564092B
CN113564092B CN202110938594.0A CN202110938594A CN113564092B CN 113564092 B CN113564092 B CN 113564092B CN 202110938594 A CN202110938594 A CN 202110938594A CN 113564092 B CN113564092 B CN 113564092B
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dextran
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escherichia coli
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CN113564092A (en
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张洪斌
张宇馨
杨静文
胡雪芹
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Hefei University of Technology
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Abstract

The invention discloses a construction method of fusion enzyme escherichia coli engineering bacteria capable of directionally synthesizing low molecular weight dextran and application thereof, which takes P473S/P856S double-mutant thermostable dextrorotase gene and streptococcus-derived dextrorotase gene as templates, and takes (EAAAK) n (n=0, 1, 2) is connecting peptide, two enzyme genes are subjected to homologous recombination according to a catalytic sequence and are transformed into BL21 (DE 3) escherichia coli, and the obtained fusion enzyme escherichia coli BL21 (DE 3)/dex-YG-nG-a 1dex (n=0, 1, 2) genetic engineering bacteria capable of directionally synthesizing different low molecular weight dextrans is obtained. The molecular weight of the dextran synthesized by the dex-YG-nG-a1dex (n=0, 1, 2) genetic engineering bacteria constructed by the invention is concentrated, and most of sucrose can be directly converted into dextran with different low molecular weights by the dex-YG-nG-a1dex (n=0, 1, 2) genetic engineering bacteria constructed by the invention through controlling conditions and connecting peptide, wherein the weight average molecular weight of the dextran can be directly 1000-2000Da,5000Da and 10000Da.

Description

Fusion enzyme for directionally synthesizing dextran, construction method and application thereof
Technical Field
The invention relates to the technical field of biology, in particular to a method for constructing fusion enzyme for directionally synthesizing low-molecular-weight dextran and application thereof.
Background
Dextran and its derivatives have been widely used in food systems such as thickeners, stabilizers, prebiotics, and improving the quality of wheat bread. In addition, dextran and its derivatives are also used in the pharmaceutical industry (as carriers, blood volume expanders, antithrombotics, anticoagulants), as well as in biotechnology fields (molecular sieves). Since human alpha-amylase only hydrolyzes the alpha (1, 6) branch of glycogen slowly, and can hydrolyze the alpha (1, 4) branch of starch and glycogen rapidly. The allergic reaction (DIAR) caused by dextran appears to be related to chemical structure. The non-alpha (1, 6) linkage ratio in high molecular weight dextran and/or dextran is associated with a high incidence of allergic reactions. Therefore, it is important that dextran produced by Leuconostoc (Leuconostoc mesenteroides 0326) has low antigenicity and a high percentage (95%) of alpha (1, 6) branches. Furthermore, the α (1, 3) -bond has higher water solubility than the α (1, 3) -bond and the β -bond. In order to obtain clinical grade dextran, acid hydrolysis or fermentation of high molecular weight dextran is generally employed. The dextran obtained by the methods has wide and uneven molecular weight distribution, and the dextran can be obtained by fractionation. In summary, these methods are environmentally unfriendly, expensive and time consuming. The production of the enzyme is green and the enzyme may be modified. The result of some complex chemical processes is achieved by directed modification of enzymes. For example, the synthesis of oligomeric dextran with truncated dextran sucrase. The oligosaccharide is obtained by taking the transglycosylation function of dextran sucrase and taking sucrose as a donor and taking the participation of a sugar acceptor such as isomaltose or maltose as a catalyst. The molecular weight of the product is shifted to low molecular weight by fine-tuning the enzyme active center through site-directed mutagenesis based on the characteristics of residues around the catalytic site and the binding pattern to the product. Dextran sucrase and dextran enzyme co-catalyze the production of low molecular weight dextran. However, there is no report of one-step production of dextran with more concentrated molecular weight.
Dextran sucrase (EC 2.4.1.5) is a glucosyltransferase produced by leuconostoc mesenteroides (Leuconstoc mesenteriodes) and streptococcus stomatae (Oral Strepotococcus), belongs to family 70 of glycoside hydrolase, is composed of 1250 to 1600 different amino acids, has a molecular weight of about 170KDa, and has important applications in a plurality of fields such as polysaccharide drug synthesis, pharmaceutical excipients, health foods and preparation of degradable biological materials. The enzyme is catalyzed by taking sucrose as a substrate, releasing fructose after enzyme digestion, and carrying out catalytic polymerization on the rest D-glucosyl group in an alpha (1, 6) bond connection mode to generate the dextran with different molecular weights.
Dextranase (Dextranase; E.C.3.2.1.11) is a hydrolase capable of specifically degrading alpha-1, 6 glycosidic linkages in dextran molecules. The enzyme is generally classified into endo-dextranase (endodextranase EC.2.1.11, α -1, 6-glucopyranose-6-glucohydrolase; also referred to as dextranase) and exo-dextranase (exoglucanase EC 3.2.1.70, glucoan-1, 6- α -glucolase; also referred to as dextran glucosidase) according to the hydrolysis mode of dextran. Exo-dextranase mainly degrades alpha- (1, 6) bond from the non-reducing end of dextran chain, releasing glucose. Endo-dextranase randomly hydrolyzes alpha- (1, 6) bonds in the dextran chain to generate low molecular weight polysaccharides in the series. From the reported research results, the exo-dextranase is mainly produced by enterobacteria, while the endo-dextranase has a relatively large number of producing bacteria, and the common bacteria are penicillium, streptococcus, bacillus and arthrobacter.
The double enzyme synergistic catalysis of dextran sucrase and dextran enzyme is the biochemical reaction process of synthesizing and degrading biological polysaccharide in the solution system of substrate sucrose to produce medium and low molecular weight polysaccharide. Currently, studies on the co-catalysis of dextran sucrase and dextranase double enzymes have focused mainly on the preparation of functional oligosaccharides. A.K Goulas et al, which use dextran sucrase and dextran enzyme in combination to prepare low molecular weight dextran, solve the problem of product binding with proteins, but the molecular weight distribution of the product is wider, and the polymerization degree is from 10DP to 60DP; previously, the subject group carries out research on preparing low molecular weight dextran by combining double enzymes under the condition that dextran sucrase and dextranase are effectively expressed, the double enzyme synergistic catalysis flow is shown in a figure 3, and experimental results also obtain the dextran with different molecular weights, so that expected results are achieved.
However, in the process of double-enzyme co-catalysis, enzymes are in a free state, and the distance between enzyme molecules is difficult to control, so that the catalysis efficiency is restricted; the oligosaccharides prepared under the existing conditions are all mixed sugar with the polymerization degree of 3-10, and the oligosaccharides with single polymerization degree or high concentration are very difficult to prepare at present. The synergistic effect of the double enzymes makes it difficult to obtain large amounts of dextran of uniform molecular weight, especially below 5000 Da.
Disclosure of Invention
The invention aims to provide a method for constructing fusion enzyme for directionally synthesizing low-molecular-weight dextran and application thereof, which can solve the technical problems in the prior art. Compared with natural enzymes (single enzyme or a mixture of multiple enzymes such as double enzyme synergistic catalysis), the construction of the fusion enzyme can bring numerous advantages in function and application, so that the fusion enzyme is suitable for industrial production in order to obtain products with concentrated molecular weight, the cost is reduced, the practical application value is expanded, two enzymes participating in sequential catalytic reaction are fused and connected through protein engineering, and dextran with different low molecular weights, uniform molecular weights and concentration can be directly obtained through one-step catalysis of the fusion enzyme.
In order to achieve the above purpose, the present invention provides the following technical solutions:
a method for constructing fusion enzyme escherichia coli engineering bacteria capable of directionally synthesizing dextran uses P473S/P856S double-mutant thermostable type dextran sucrase gene and streptococcus-derived dextran enzyme gene as templates, and uses (EAAAK) n (n=0, 1, 2) as connecting peptide, carrying out homologous recombination on two enzyme genes in sequence and converting the two enzyme genes into escherichia coli BL21 (DE 3) to obtain fusion enzyme escherichia coli engineering bacteria BL21 (DE 3)/dex-YG-nG-a 1dex (n=0, 1, 2) capable of directionally synthesizing different low molecular weight dextrans. Wherein dex-YG is a dextran sucrase, nG is a connecting peptide, a1dex is a dextran enzyme, and dex-YG-nG-a1dex (n=0, 1, 2) refers to a fusion enzyme. The escherichia coli engineering bacteria BL21 (DE 3)/dex-YG-G-a 1dex are preserved in the common microorganism center of China Committee for culture Collection of microorganisms, and are classified and named as escherichia coli BL21 (DE 3) which is a low molecular weight dextran fusion enzyme directionally synthesized, the preservation date is 2021, 07, 08, and the preservation number is CGMCC No.22849.
The construction method specifically comprises the following steps: molecular simulation, primer design, recombinant plasmid construction and host cell transformation were performed using (EAAAK) n (n=0, 1, 2) as a connecting peptide. The design and use of the fusion enzyme was studied using the connecting peptide (EAAAK) n (n=0, 1, 2), reference being made to Huang Z, zhang C, wu X, et al, return progress in fusion enzyme design and applications, journal of bioengineering, 2012,28 (4): 393-409.
In the above construction method, preferably, the specific steps of molecular simulation using (EAAAK) n (n=0, 1, 2) as the connecting peptide are as follows: the P473S/P856S double mutant thermostable type dextrorotase protein and streptococcus source dextrorotase are subjected to sequence similarity comparison by Discovery Studio2019 software to search templates, the templates are subjected to structure comparison and overlapping, a fusion enzyme model is generated by using MODELLER for target sequence and template sequence comparison, and then feasibility analysis is performed.
In the above construction method, preferably, the primer design is a thermostable type dextrorotatory sucrase gene and vector pET-28a- (+) according to P473S/P856S double mutation and a streptococcus-derived dextrorotatory enzyme sequence, and the mutation primer is designed by means of snapge software as follows:
n=0:
pET-28a- (+) -P473S/P856S template:
an upstream primer:
5’-----GGTTACTTTGCTCCATTGCTGACACAGCATTTCCATTATTATC-----3’
a downstream primer:
5’-----AGAACGACCTCGAGCACCACCACCACCACTGA-----3’。
a1dex template:
an upstream primer:
5’-----GTGTCAGCAATGGAGCAAAGTAACCGCCAG-----3’
a downstream primer:
5’-----GCTCGAGGTCGTTCTTACGGCCTTTGATCAG-----3’。
when n=1
pET-28a- (+) -P473S/P856S template:
an upstream primer:
5’-----TTTCGCGGCGGCTTCTGCTGACACAGCATTTCCATTATTATCAAAT-----3’
a downstream primer:
5’-----TAAGAACGACCTCGAGCACCACCACCACCACCACTG-----3’。
a1dex template:
an upstream primer:
5’-----CAGCAGAAGCGGCCGCGAAAATGGAGCAAAGTAACCGCCAG-----3’
a downstream primer:
5’-----GCTCGAGGTCGTTCTTACGGCCTTTGATCAG-----3’。
when n=2
pET-28a- (+) -P473S/P856S template:
an upstream primer:
5’-----GGCCGCTTCTTTCGCGGCCGCTTCTGCTGACACAGCATTTCCATTATTATCAAAT-----3’
a downstream primer:
5’-----AGAACGACCTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAAC-----3’
a1dex template:
an upstream primer:
5’-----GCCGCGAAAGAAGCGGCCGCGAAAATGGAGCAAAGTAACCGCCAG-----3’
a downstream primer:
5’-----GCTCGAGGTCGTTCTTACGGCCTTTGATCAG-----3’。
in the above construction method, preferably, the recombinant plasmid construction is based on P473S/P856S double mutant thermostable dextrorotatory sucrase gene and vector pET-28a (+) and streptococcus-derived dextrorotatory enzyme gene sequence template, and the recombinant expression plasmid pET-28a- (+) -dex-YG-nG-a1dex (n=0, 1, 2) is obtained by digesting the original template with DMT by PCR amplification technique and seamless cloning technique using the designed primers. The vector pET-28a (+) is a universal plasmid vector and can be obtained from a professional supplier or reference or homemade.
In the above construction method, preferably, the host bacterium transformation is to transform a recombinant expression plasmid pET-28a- (+) -dex-YG-nG-a1dex (n=0, 1, 2) into an escherichia coli competent cell BL21 (DE 3), and obtain a fusion enzyme escherichia coli engineering bacterium BL21 (DE 3)/dex-YG-nG-a 1dex (n=0, 1, 2) for directionally synthesizing the dextran after kanamycin resistance screening, enzyme digestion, bacterial liquid PCR and DNA sequencing verification.
The invention also provides application of the fusion enzyme escherichia coli engineering bacteria capable of directionally synthesizing the dextran in preparing the low-molecular-weight dextran, which is characterized in that escherichia coli engineering bacteria BL21 (DE 3)/dex-YG-nG-a 1dex (n=0, 1, 2) is fermented to express the fusion enzyme, and then sucrose is taken as a substrate to directly obtain the dextran with different low molecular weights through double-function catalysis of the fusion enzyme. Preferably, dextran of 1000-15000Da in molecular weight concentration is obtained; more preferably, dextran can be obtained directly with a weight average molecular weight of 1000-2000Da (n=0), 5000Da (n=1, 2) and 10000Da (n=1, 2). The sucrose conversion rate is more than 90%.
In the above application, preferably, the fermentation expresses a fusion enzyme by a method comprising the steps of: inoculating genetically engineered bacterium BL21 (DE 3)/dex-YG-nG-a 1dex (n=0, 1, 2) into LB culture medium containing 40-60 mug/ml kanamycin according to the inoculum size of 0.5% of volume fraction, culturing at 37 ℃ for 16 hours at the rotating speed of 250 r/min; 2mL of the culture solution is sucked up and added into 200mL of A culture medium, and the culture medium is placed at 37 ℃ for shake culture, and when the enriched culture bacterial liquid is diluted by 10 times by distilled water, the OD is obtained 600 Adding 500 mu L of IPTG (inducer, isopropyl thiogalactoside) to start to induce enzyme production when the temperature is 0.20-0.24, keeping the temperature of 25 ℃ to induce fermentation for 3.5-4 hours, centrifuging the bacterial suspension after the induced fermentation at the temperature of 0 ℃ for 15min at 8000r/min, adding distilled water to shake and clean one separating tube corresponding to one bottle of bacterial suspension, and centrifuging again; adding 15-20mL of acetic acid-calcium acetate buffer solution with pH value of 5.4 into each of the separation tubes, shaking uniformly, adding ice water bath, carrying out ultrasonic crushing for 15min, and carrying out centrifugal separation, wherein the supernatant is fusion enzyme for directionally synthesizing dextran, and the enzyme activity is 80-100U/mL.
Preferably, each liter of the A medium contains 5g of glycerol, 5g of glucose, 10g of peptone, 10g of potassium nitrate and 17.105g of Na 2 HPO 4 ·12H 2 O、3g KH 2 PO 4 、1g NH 4 Cl、0.1mM MgSO 4 ·7H 2 O。
Compared with the prior art, the invention has the beneficial effects that:
1) The glycoside synthesized by the dex-YG-nG-a1dex (n=0, 1, 2) genetic engineering bacteria constructed by the invention has concentrated molecular weight, higher conversion rate and simple separation and purification, and under the same condition, the yield calculation finds that the dex-YG-nG-a1dex (n=0, 1, 2) genetic engineering bacteria constructed by the invention can directly convert most of sucrose into dextran with molecular weight of 1000-15000Da by controlling conditions and connecting peptide, wherein the weight average molecular weight of the dextran can be directly obtained to 1000-2000Da,5000Da and 10000Da. .
2) Fusion enzyme for directed Synthesis of dextran Using thermostable dextran sucrase (Genbank No. DQ 345760) and Streptococcus-derived dextranase (Genbank No. HQ 711852.1) genes with double mutation of P473S/P856S as templates, was prepared using (EAAAK) n (n=0, 1, 2) is connecting peptide, two enzyme genes are subjected to homologous recombination in sequence and are transformed into BL21 (DE 3)/dex-YG-nG-a 1dex (n=0, 1, 2) gene engineering bacteria obtained in BL21 (DE 3) escherichia coli, sucrose is taken as a substrate, and 1000-15000Da dextran with concentrated molecular weight can be directly obtained through double-function catalysis. Taking the product as an example for reacting the dextran 10, the reaction is carried out for the same time under the same condition, the fusion enzyme expressed by engineering bacteria BL21 (DE 3)/dex-YG-G-a 1dex has concentrated molecular weight compared with the double-enzyme free enzyme synergistic catalysis product (the double-enzyme free enzyme synergistic catalysis product system contains heterogeneous dextran with molecular weight of 20000Da,10000Da and 5000 Da), and the yield of the dextran 10 can reach 78% in the reaction time of 6 h. Taking the weight average molecular weight 1000-2000Da of dextran as an example, the fusion enzyme expressed by engineering bacteria BL21 (DE 3)/dexYG-a 1dex reacts for the same time under the same condition, and the molecular weight is smaller and more concentrated than that of the double-enzyme free enzyme synergistic catalysis product (the double-enzyme free enzyme synergistic catalysis product system contains non-uniform dextran with 20000, 10000 and 5000 molecular weight).
3) The invention can specifically produce dextran 1000-15000Da, simplify the separation and purification steps, reduce the production cost and provide a certain foundation for better application in production.
Drawings
FIG. 1 is a diagram of construction of a dextran sucrase and dextranase fusion plasmid;
FIG. 2 is a diagram of a simulation of the homologous modeling of the dextran sucrase and the dextranase fusion enzyme by Discovery Studio2019 software;
FIG. 3 is a diagram of a two-step catalytic mechanism of the one-step process of the present invention versus the background art;
FIG. 4 is a graph showing the molecular weight characterization of dextran 10, a product of the present invention; the high performance liquid chromatogram is characterized in that the ordinate represents peak height, namely content, and the abscissa represents peak time and molecular weight, and the molecular weight is marked.
FIG. 5 is a graph showing the characterization of the weight average molecular weight of dextran, a product of example 5, according to the present invention, from 1000 to 2000.
FIG. 6 is a graph showing the characterization of the weight average molecular weight of dextran, a product of example 6, according to the present invention.
FIG. 7 is a one-dimensional hydrogen spectrum of dextran 10, a product of the present invention.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and fully with reference to the accompanying drawings, in which it is evident that the embodiments described are only some, but not all embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
A method for constructing fusion enzyme escherichia coli capable of directionally synthesizing dextran uses P473S/P856S double-mutant thermostable type dextran sucrase gene and streptococcus-derived dextran enzyme gene as templates, and uses (EAAAK) 1 And (3) carrying out homologous recombination on the two enzyme genes according to a catalytic sequence for connecting peptides, and transforming the two enzyme genes into fusion enzyme escherichia coli BL21 (DE 3)/dex-YG-G-a 1dex gene engineering bacteria which can directionally synthesize the dextran, wherein the fusion enzyme escherichia coli BL21 (DE 3) is obtained. The other two fusion enzyme genetic engineering bacteria and the construction method are consistent with BL21 (DE 3)/dex-YG-G-a 1dex genetic engineering bacteria. The BL21 (DE 3)/dex-YG-G-a 1dex genetically engineered bacteria are preserved inThe China general microbiological culture Collection center (North Xili No. 1, 3) of the Korean area of Beijing, which is classified and named as directed synthesis of low molecular weight dextran fusion enzyme, such as Escherichia coli BL21 (DE 3), has a preservation date of 2021, 07, 08 and a preservation number of CGMCC No.22849.
The construction method of the invention specifically comprises the following steps: in order (EAAAK) 1 For connecting peptides, molecular simulation, primer design, recombinant plasmid construction and host bacteria transformation are performed.
More specifically, in order to (EAAAK) 1 For linking peptides, specific steps of molecular modeling were as follows, sequence similarity alignment of P473S/P856S double mutated thermostable dextran sucrase protein and Streptococcus derived dextran enzyme by Discovery Studio2019 software to search templates (homology modeling), structural alignment of templates to overlap, alignment of target sequences with template sequences to generate fusion enzyme models using MODELLER (a module within Discovery Studio2019 software) and feasibility analysis.
Wherein, the primer design is based on P473S/P856S double mutant thermostable type dextran sucrase gene and carrier pET-28a- (+) and streptococcus source dextran enzyme sequence, the mutation primer is designed by Snap Gene software as follows: pET-28a- (+) -P473S/P856S template:
an upstream primer:
5’-----TTTCGCGGCGGCTTCTGCTGACACAGCATTTCCATTATTATCAAAT-----3’
a downstream primer:
5’-----TAAGAACGACCTCGAGCACCACCACCACCACCACTG-----3’。
a1dex template:
an upstream primer:
5’-----CAGCAGAAGCGGCCGCGAAAATGGAGCAAAGTAACCGCCAG-----3’
a downstream primer:
5’-----GCTCGAGGTCGTTCTTACGGCCTTTGATCAG-----3’。
the recombinant plasmid construction is to digest the original template by a DMT enzyme (full gold company) through a PCR amplification technology and a seamless cloning technology by using designed primers according to a P473S/P856S double-mutation thermostable dextrorotatory sucrase gene, a vector pET-28a- (+) and a streptococcus-derived dextrorotatory enzyme sequence template to obtain a recombinant expression plasmid pET-28a- (+) -dex-YG-G-a1dex.
The host bacteria transformation is to transform a recombinant expression plasmid pET-28a- (+) -dex-YG-G-a1dex into an escherichia coli competent cell BL21 (DE 3), and obtain a fusion enzyme escherichia coli engineering bacterium BL21 (DE 3)/dex-YG-G-a 1dex gene engineering bacterium suitable for directionally synthesizing the dextran after kanamycin resistance screening, enzyme digestion, bacterial liquid PCR and DNA sequencing verification. Specifically, the host bacterium transformation includes the following steps:
(1) Taking 100 μl of competent cell (purchased from Beijing Quan Jinshengmo Co., ltd.) suspension of Escherichia coli BL21 (DE 3) from-80deg.C refrigerator, and thawing on ice;
(2) Adding the prepared recombinant expression plasmid solution, adding about 5ul plasmid DNA per 50ul competent cells, shaking gently, and standing on ice for 30 min;
(3) Heat shock in a 42 ℃ water bath for 45 seconds, and rapidly cooling on ice for 2 minutes after the heat shock;
(4) Adding 500ml of sterilized LB liquid medium (without antibiotics) into the tube, uniformly mixing, culturing at 37 ℃ for 1 hour to enable bacteria to recover to a normal growth state, and expressing a resistance gene coded by a plasmid;
(5) Shaking the bacterial liquid uniformly, then taking 100 mu L of the bacterial liquid to be coated on a screening plate containing antibiotics, standing the bacterial liquid for half an hour in the front side, inverting the culture medium after the bacterial liquid is completely absorbed by the culture medium, and culturing the bacterial liquid at 37 ℃ for 16-18 hours; and selecting positive colonies, and performing PCR (polymerase chain reaction) verification on bacterial liquid to obtain fusion enzyme escherichia coli engineering bacteria BL21 (DE 3)/dex-YG-G-a 1dex gene engineering bacteria capable of directionally synthesizing dextran.
Example 2
Expression of fusion enzyme escherichia coli engineering bacterium BL21 (DE 3)/dex-YG-G-a 1dex gene engineering bacterium capable of directionally synthesizing dextran
Inoculating the genetically engineered bacterium BL21 (DE 3)/dex-YG-G-a 1dex obtained in the example 1 into LB culture medium containing 40-60 mug/ml kanamycin according to the inoculation amount of 0.5%, and culturing for 16 hours at 37 ℃ at the rotating speed of 250 r/min; sucking the culture solution2mL of the culture medium is added into 200mL of A culture medium, shaking culture is carried out at 37 ℃, and OD after 10 times dilution of enrichment culture bacterial liquid with distilled water is carried out 600 And adding 500 mu L of IPTG to start to induce enzyme production at 0.20-0.24, carrying out crushing and centrifugation after inducing fermentation at 25 ℃ for 3.5-4 hours, and carrying out high performance liquid chromatography analysis on the sampled product, wherein the molecular weight of the expressed fusion enzyme protein is about 265kDa and is consistent with a predicted value. Wherein each liter of the A culture medium contains 5g of glycerol, 5g of glucose, 10g of peptone, 10g of potassium nitrate and 17.105g of Na 2 HPO 4 ·12H 2 O、3g KH 2 PO 4 、1g NH 4 Cl、0.1mM MgSO 4 ·7H 2 O。
Example 3
The fermentation and enzyme production of the fusion enzyme escherichia coli engineering bacterium BL21 (DE 3)/dex-YG-G-a 1dex gene engineering bacterium capable of directionally synthesizing the dextran specifically comprises the following steps:
inoculating genetically engineered bacterium BL21 (DE 3)/dex-YG-G-a 1dex into LB culture medium containing 40-60 mug/ml kanamycin according to the inoculum size of 0.5% by volume fraction, culturing at the rotating speed of 250r/min for 16 hours at the temperature of 37 ℃; 2mL of the culture solution is sucked up and added into 200mL of A culture medium, and the culture medium is placed at 37 ℃ for shake culture, and when the enriched culture bacterial liquid is diluted by 10 times by distilled water, the OD is obtained 600 Adding 500 mu L of IPTG to start inducing enzyme production when the temperature is 0.20-0.24, keeping the temperature of 25 ℃ to induce fermentation for 3.5-4 hours, centrifuging the bacterial suspension after the induced fermentation at the temperature of 4 ℃ for 15min at 8000r/min, enabling a separation tube to correspond to one bottle of bacterial suspension, adding distilled water to shake and clean, and centrifuging again; adding 15-20mL of acetic acid-calcium acetate buffer solution with pH value of 5.4 into each of the separation tubes, shaking uniformly, adding ice water bath, carrying out ultrasonic crushing for 15min, and carrying out centrifugal separation, wherein the supernatant is fusion enzyme suitable for directional synthesis of dextran, and the enzyme activity is 80-100U/mL. Wherein each liter of the A culture medium contains 5g of glycerol, 5g of glucose, 10g of peptone, 10g of potassium nitrate and 17.105g of Na 2 HPO 4 ·12H 2 O、3g KH 2 PO 4 、1g NH 4 Cl、0.1mM MgSO 4 ·7H 2 O。
Example 4
The use of a fusion enzyme that can be directed to the synthesis of dextran 10 is as follows:
the crude enzyme solution of the fusion enzyme dexYG-G-a1dex obtained in the method of example 3 was used for reacting sucrose, an enzyme reaction system was prepared into 150mM sucrose, acetic acid-calcium acetate buffer (pH=5.4), 10U/ml enzyme activity reaction system was reacted at 30℃for 6-8 hours on a shaking table of 120-150r/min, the reaction solution was taken out, 3 times of absolute ethanol was added, dextran 10 was precipitated with ethanol, and drying was performed. Detecting the dried product by using a differential liquid phase under the following detection conditions: GPC column, differential detector, mobile phase pure water, flow rate 0.6ml/min, high performance liquid chromatography detection as shown in figure 4, and one-dimensional hydrogen spectrum analysis for characterization of dextran as shown in figure 7.
1 H NMR spectra
A proton peak around 4.93ppm demonstrates the presence of a-1,6 glycosidic linkages on the dextran backbone
The proton peak at 5.20ppm indicates that the branched glycosidic bond of dextran is an a-1,3 dextran bond
Reference is made to: li MQ, zhang HB, li Y, hu XQ, yang JW The thermoduric effects of site-directed mutagenesis of proline and lysine on dextransucrase from Leuconostoc mesenteroides 0326.International Journal of Biological Macromolecules 2018,107 (Pt B): 1641-1649.
Example 5
The application of the fusion enzyme with the weight average molecular weight of 1000-2000Da for directionally synthesizing the dextran is as follows:
using a method similar to that of example 1-3 above, obtaining a crude enzyme solution of fusion enzyme dex-YG-a1dex, reacting sucrose, preparing an enzyme reaction system of 150mM sucrose, acetic acid-calcium acetate buffer (ph=5.4), reacting for 6-8 hours at 35 ℃ in a shaker of 120-150r/min, taking out the reaction solution, adding 3 times absolute ethanol, precipitating with ethanol to obtain dextran of more than 3000Da, dialyzing the supernatant to remove small molecule byproducts (fructose and a small portion of disaccharide), drying, and detecting the dried products by using differential liquid phase under the following detection conditions: GPC column, differential detector, mobile phase pure water, flow rate 0.6ml/min, high performance liquid chromatography detection result as shown in figure 5. The fusion enzyme used was different to link the peptides due to different catalytic temperatures, so that the molecular weight of the resulting dextran was also different from example 4.
Example 6
The application of the fusion enzyme with weight average molecular weight of 5000Da for directionally synthesizing dextran is as follows:
a crude enzyme solution of the fusion enzyme dex-YG-2G-a1dex was obtained by a method similar to that of examples 1 to 3, and sucrose was reacted with each other to prepare an enzyme reaction system comprising 150mM sucrose, acetic acid-calcium acetate buffer (pH=5.4) and 100mM NaCl 2 Adding a reaction system with 5U/ml enzyme activity, reacting for 6-8 hours at 30 ℃ in a shaking table with the speed of 120-150r/min, taking out the reaction solution, adding 3 times of absolute ethyl alcohol, precipitating the dextran with the alcohol of more than 5000Da, drying, and detecting the dried product by using a differential liquid phase under the following detection conditions: GPC column, differential detector, mobile phase pure water, flow rate 0.6ml/min, high performance liquid chromatography detection result shown in FIG. 6. The molecular weight of the resulting dextran was also different from examples 4 and 5 due to the increased calcium ion concentration.
The foregoing is merely illustrative and explanatory of the invention, as it is well within the scope of the invention, as it is intended to provide those skilled in the art with various modifications, additions and substitutions to the specific embodiments disclosed and those skilled in the art without departing from the scope of the invention as disclosed in the accompanying claims.

Claims (9)

1. The application of a fusion enzyme escherichia coli engineering bacterium capable of directionally synthesizing the dextran in preparing the low-molecular-weight dextran is characterized in that escherichia coli engineering bacterium BL21 (DE 3)/dex-YG-nG-a 1dex (n=1, 2) is fermented to express the fusion enzyme, and then sucrose is taken as a substrate to directly obtain the dextran with different low molecular weights through the difunctional catalysis of the fusion enzyme;
the construction method of the fusion enzyme escherichia coli engineering bacteria capable of directionally synthesizing the dextran takes a gene of P473S/P856S double mutation of a dextrorotase gene of Genbank No. DQ345760 and a dextrorotase gene of streptococcus source of Genbank No. HQ711852.1 as templates, and takes (EAAAK) n (n=1, 2) two enzyme genes are linked to the peptideHomologous recombination is carried out in sequence and the recombinant strain is transformed into escherichia coli BL21 (DE 3) to obtain fusion enzyme escherichia coli engineering bacteria BL21 (DE 3)/dex-YG-nG-a 1dex (n=1, 2) capable of directionally synthesizing different low molecular weight dextrans.
2. The use according to claim 1, wherein: the escherichia coli engineering bacteria BL21 (DE 3)/dex-YG-G-a 1dex are preserved in the common microorganism center of China Committee for culture Collection of microorganisms, and are classified and named as escherichia coli BL21 (DE 3) which is a low molecular weight dextran fusion enzyme directionally synthesized, the preservation date is 2021, 07, 08, and the preservation number is CGMCC No.22849.
3. The use according to claim 1, wherein the construction method of the fusion enzyme escherichia coli engineering bacteria capable of directionally synthesizing the dextran comprises the following steps: in order (EAAAK) n (n=1, 2) is a connecting peptide, and molecular simulation, primer design, recombinant plasmid construction and host bacteria transformation are performed.
4. The use according to claim 3, characterized in that (EAAAK) n (n=1, 2) is a connecting peptide, the specific steps of molecular simulation are as follows, the P473S/P856S double mutant thermostable dextrorotatory sucrase protein and streptococcus-derived dextrorotatory enzyme are subjected to sequence similarity comparison by Discovery Studio2019 software to search templates, the templates are subjected to structure comparison and superposition, and the target sequence is compared with the template sequence to generate a fusion enzyme model.
5. The use according to claim 3, wherein the primer design is based on the P473S/P856S double mutated thermostable dextrorotatory sucrase gene and the vector pET-28a- (+) and the streptococcus-derived dextrorotatase sequence, and the mutated primer design is based on the snap gene software as follows:
when n=1, the number of the groups,
pET-28a- (+) -P473S/P856S template:
an upstream primer:
5’----- TTTCGCGGCGGCTTCTGCTGACACAGCATTTCCATTATTATCAAAT-----3’
a downstream primer:
5’----- TAAGAACGACCTCGAGCACCACCACCACCACCACTG-----3’；
a1dex template:
an upstream primer:
5’----- CAGCAGAAGCGGCCGCGAAAATGGAGCAAAGTAACCGCCAG-----3’
a downstream primer:
5’ ----- GCTCGAGGTCGTTCTTACGGCCTTTGATCAG-----3’；
when n=2, the number of the groups,
pET-28a- (+) -P473S/P856S template:
an upstream primer:
5’-----GGCCGCTTCTTTCGCGGCCGCTTCTGCTGACACAGCATTTCCATTATTATCAAAT-----3’
a downstream primer:
5’-----AGAACGACCTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAAC-----3’；
a1dex template:
an upstream primer:
5’----- GCCGCGAAAGAAGCGGCCGCGAAAATGGAGCAAAGTAACCGCCAG-----3’
a downstream primer:
5’ ----- GCTCGAGGTCGTTCTTACGGCCTTTGATCAG-----3’。
6. the use according to claim 3, wherein the recombinant plasmid construction is carried out by using the gene of the dextran sucrase gene of Genbank No. DQ345760 for double mutation of P473S/P856S and the vector pET-28a- (+) and the dextran enzyme gene of Streptococcus source of Genbank No. HQ711852.1 as templates, and by using PCR amplification technique and seamless cloning technique and digesting the original templates by DMT with the designed primers, the recombinant expression plasmid pET-28a- (+) -dex-YG-nG-a1dex (n=1, 2) is obtained.
7. The use according to claim 3, wherein the host bacterium transformation is to transform a recombinant expression plasmid pET-28a- (+) -dex-YG-nG-a1dex (n=1, 2) into an escherichia coli competent cell BL21 (DE 3), and obtain a fusion enzyme escherichia coli engineering bacterium BL21 (DE 3)/dex-YG-nG-a 1dex (n=1, 2) suitable for directionally synthesizing the dextran after kanamycin resistance screening, enzyme digestion, bacterial liquid PCR and DNA sequencing verification.
8. The use according to claim 1, wherein the fermentation expresses a fusion enzyme by a method comprising the steps of:
inoculating the genetically engineered bacterium BL21 (DE 3)/dex-YG-nG-a 1dex (n=1, 2) into LB culture medium containing 40-60 mug/ml kanamycin according to the inoculum size of 0.5% of volume fraction, culturing at the rotating speed of 250r/min for 16 hours at the temperature of 37 ℃; 2mL of the culture solution is sucked up and added into 200mL of A culture medium, and the culture medium is placed at 37 ℃ for shake culture, and when the enriched culture bacterial liquid is diluted by 10 times by distilled water, the OD is obtained 600 Adding 500 mu L of IPTG to start inducing enzyme production when the temperature is 0.20-0.24, keeping the temperature of 25 ℃ to induce fermentation for 3.5-4 hours, centrifuging the bacterial suspension after the induced fermentation at the temperature of 4 ℃ for 15min at 8000r/min, enabling a separation tube to correspond to one bottle of bacterial suspension, adding distilled water to shake and clean, and centrifuging again; adding 15-20mL of acetic acid-calcium acetate buffer solution with pH value of 5.4 into each of the separation tubes, shaking uniformly, adding ice water bath, carrying out ultrasonic crushing for 15min, and carrying out centrifugal separation, wherein the supernatant is the fusion enzyme capable of directionally synthesizing the dextran, and the enzyme activity is 80-100U/mL.
9. The use according to claim 8, wherein each liter of said A medium contains 5g glycerol, 5g glucose, 10g peptone, 10g potassium nitrate, 17.105g Na 2 HPO 4 ·12H 2 O、3g KH 2 PO 4 、1g NH 4 Cl、0.1mM MgSO 4 ·7H 2 O。
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