CN115948433A - Encoding gene of alkali-resistant levan synthetase for high-yield levan polysaccharide - Google Patents

Abstract

The invention relates to the field of enzyme engineering, in particular to a coding gene of alkali-resistant levan synthetase for high-yield levan. The existing levan synthetase has the problem of low activity, and the catalytic efficiency is particularly poor under special conditions such as strong alkali, so that the application range of the enzyme is influenced. The levan synthetase gene capable of being efficiently expressed in escherichia coli is high in activity and substrate conversion rate, high in tolerance to strong basicity and good in production and application prospects.

Description

Encoding gene of alkali-resistant levan synthetase for high-yield levan polysaccharide

Technical Field

The invention relates to the field of enzyme engineering, in particular to a coding gene of alkali-resistant levan synthetase for high-yield levan glycan, which catalyzes sucrose to generate levan glycan with high yield and particularly has high relative activity under a strong alkaline condition.

Background

Fructose polymers are polysaccharide polymerases formed by the linkage of multiple fructose groups that are widely found in nature. Fructose groups in the fructose polymer have various different connection modes, inulin is connected by a beta-2,1 glycosidic bond, and the inulin is an important energy storage substance in a plurality of plant cells; connected by beta-2,6 glycosidic linkages are levan glycans. Studies have shown that levan-type glycans are predominantly formed in microorganisms. In addition, there is a new class of fructose polymers, which are linked by a number of different glycosidic bonds (Zhang W.et al. Appl Microbiol Biotechnol.2019;103 (19): 7891-7902), and which are mainly present in the cells of plants of the Liliaceae family. Compared with inulin and novel fructose polymers, the levan glycan is still not deeply researched, but the related research results have been remarkably advanced, and the glycan has wide application prospect (the research results show)

ET et al.Biotechnol Adv.2016；34(5):827-844)。

Researches prove that the Levan polysaccharide has various unique physiological and biochemical functions and has wide application prospects in the fields of food, pharmacy and the like. The average molecular weight of the Levan glycans is generally 2X 10 ⁶ Da to 100X 10 ⁶ Da, has relatively low viscosity. The Levan polysaccharide is mixed with glycerol and heated together to form a film having both some tackiness and flexibility. Additionally, levan polysaccharides can also be used as thickeners in food processing (Bae IY et al 2008.42 (1): 10-13). Levan polysaccharide is difficult to be digested and absorbed by human intestinal tracts, and is a prebiotic polysaccharide. Has effects in inhibiting obesity, reducing cholesterol, resisting oxidation, reducing blood lipid, regulating immunity, and resisting tumor (Zhang W.et al.appl Microbiol)Biotechnol.2019;103 (19):7891-7902.). In microorganisms, levan glycans are produced from sucrose as a substrate under the catalytic action of levan synthase. Currently, enzymatic synthesis based on levan synthetase is the most rational method for the production of levan. The enzyme catalysis synthesis method has the advantages of high catalysis efficiency, simple reaction process, low requirement on a reactor and the like, so the enzyme catalysis synthesis method has better application prospect. levan synthase (EC2.4.1.10) is a glycosyltransferase that specifically catalyzes the fructosyl transfer reaction. The fructose group donor of the catalytic reaction of the levan synthetase is sucrose, and the catalytic reaction can be divided into three types of reactions according to the difference of fructose group acceptors, wherein the first type of reaction is a polymerization reaction: when only sucrose substrate exists in the reaction system, levan synthetase can transfer fructose groups to sucrose molecules by using a beta-2,6 connecting bond to form fructo-oligosaccharide, and then continuously prolongs glycan chains in the same transfer mode to form levan glycan with high polymerization degree; the second type of reaction is a fructosyl-conversion reaction: besides sucrose substrates, when monosaccharide, alcohol, disaccharide or oligosaccharide and the like exist in the reaction system, levan synthetase can transfer sucrose-derived fructosyl to the molecules to form some novel polysaccharide molecules or saccharide derivatives; the third type of reaction is a hydrolysis reaction: levan synthase can also act as an acceptor for the fructose group in sucrose by using water, thereby catalyzing sucrose to generate glucose and fructose monomers. Although levan synthase is known to be contained in various microorganisms (Srikanth R.et al. Carbohydr Polymer.2015; 120-14), the levan synthase known so far still needs to be further improved in catalytic activity and levan glycan conversion rate and the like (Zhang W.et al. Appl Microbiol Biotechnol.2019;103 (19): 7891-7902). More importantly, the optimum pH of the currently known levan synthetase is generally in a neutral range (pH = 7.0), and the activity of the currently known levan synthetase is very low under special catalytic reaction conditions such as acidity or alkalinity, so that the wide application of the levan synthetase is limited.

The levan synthetase related by the invention has the highest levan polysaccharide production efficiency of catalyzing sucrose to reach about 80%, is superior to the currently reported levan synthetase, and shows good application prospect; more particularly, levan synthetase is most suitable at pH =6.0, but can maintain up to 90% of catalytic activity in an alkaline reaction system at pH = 9.0. This feature makes the enzyme more advantageous than the existing levan synthetase in catalyzing reactions under special circumstances. Meanwhile, the alkali resistance characteristic also makes the enzyme have special value in the aspect of the research of a catalytic mechanism.

Disclosure of Invention

The invention aims to provide a coding gene of an alkali-resistant levan synthetase for high-yield levan glycan.

Taking soil from forest of forest park of chessboard mountain in Shenyang city, carrying out microorganism enrichment culture, extracting metagenome DNA from the soil, carrying out PCR amplification on a DNA fragment with a target size through degenerate primers, and identifying and obtaining a coding gene of the alkali-resistant levan synthetase for high-yield levan polysaccharide through the steps of cloning, expressing, functional verification, DNA sequencing and the like (shown in figure 1). The specific research scheme is as follows:

1) And (3) extracting metagenome DNA. Taking soil from forest in the forest park of the chessboard mountain in Shenyang city, adding sucrose according to 1% (w/w), spraying water to wet, and culturing in an incubator at 37 ℃ for 7d. And (3) extracting the DNA by adopting a metagenome DNA extraction kit.

2) Obtaining of Mg-sucB Gene: the existing degenerate primers Mg-sucB-For and Mg-sucB-Rev in the laboratory are used For carrying out PCR amplification by taking metagenome DNA as a template, and the reaction system is as follows: mu.l of metagenomic DNA, 0.5. Mu.l each of 40mmol/L of the primer Mg-sucB-For and the primer Mg-sucB-Rev, 0.5. Mu.l of Pfu DNA polymerase, 0.8. Mu.l of 100mmol/L dNTP,1 XPfu Buffer, and water were added to 40. Mu.l. The reaction conditions are as follows: preheating at 94 ℃ for 3min,30 cycles of heating denaturation at 94 ℃ for 30s, annealing at 60 ℃ for 40s, extension reaction at 72 ℃ for 3min, and finally reaction at 72 ℃ for 10min.

3) Construction of pETM11-Mg-sucB recombinant plasmid: the PCR product was subjected to agarose gel electrophoresis, a band of a desired size was gel-recovered using a gel recovery kit, and the recovered DNA product was digested with BamHI and EcoRI, respectively, and then ligated to a pETM11 vector, which was also digested with BamHI and EcoRI. The specific enzyme digestion reaction system is as follows: bamHI and EcoRI 1.0. Mu.l each, 16. Mu.l each of the pETM11 linear vector or gel purified PCR product was recovered, 1 Xrestriction enzyme reaction Buffer, added to 100. Mu.l of water and reacted at 37 ℃ for 12 hours. PCR products recovered and purified from the digested pETM11 linear vector or gel were mixed at a molar ratio of 3:1, ligated with T4 DNA ligase, and transformed into Escherichia coli JM109 strain. Colonies grown on the transformed plates were inoculated into an LB medium containing Kana and liquid-cultured.

4) Activity screening and DNA sequencing. Liquid culture was performed in LB medium to OD600=0.4, and IPTG was added to induce expression for 10h. Then, the cells were collected by centrifugation, 50mmol of phosphate buffer (pH = 7.0) containing 10% sucrose was added to the cells, the cells were disrupted by sonication, reacted at 37 ℃ for 4 hours, the reaction product was centrifuged, and the supernatant was collected and tested for sucrose degradability by the DNS method. Plasmids are extracted from the bacteria with the strongest vitality and DNA sequencing is carried out, and the reading frame is shown as SEQ ID No. 1. Through identification, the gene coding protein is levan synthetase, and has the characteristics of high conversion rate and alkali resistance. The specific analysis method is shown in the examples.

Compared with the currently known levan synthetase, the levan synthetase encoded by the gene has the following outstanding advantages:

1) The levan synthetase coded by the gene obtained by screening can realize 80 percent of levan glycan conversion rate under the optimized reaction condition, and a reaction system does not need special reaction conditions. Meanwhile, compared with the substrate sucrose, the added value of the reaction product levan is high. Therefore, the gene obtained by screening and the levan synthetase coded by the gene have good application prospect.

2) The optimum pH of the levan synthetase encoded by the gene obtained by screening of the invention is =6.0, but the catalytic activity can be kept up to 90% in an alkaline reaction system with pH = 9.0. This feature makes the enzyme more advantageous than the existing levan synthetase in catalyzing reactions under special circumstances. Meanwhile, the alkali resistance characteristic also enables the enzyme to have special value in the aspect of researching a catalytic mechanism.

Drawings

FIG. 1 is a physical map of plasmid pETM11-Mg-sucB constructed in the present invention. Mg-sucB is a gene encoding an alkali-resistant levan synthase.

FIG. 2 is a diagram of a real object of the Mg-sucB gene encoded protein obtained by the present invention catalyzing production of milky levan glycan with sucrose as a substrate. 1,2 is a reaction product catalyzed 2d,4d at 4 ℃;3 is the reaction product of 2d catalyzed by the crude extract of Escherichia coli transformed by pETM11 original plasmid.

FIG. 3 is a diagram showing the optimum temperature detection of the Mg-sucB gene-encoded protein obtained by the present invention. The average of three replicates was taken for each reaction and the percent ratio of sample activity to maximum activity was the relative activity.

FIG. 4 is a thermal stability test of the Mg-sucB gene-encoded protein obtained by the present invention. The relative activity was determined after 30 minutes of treatment at each temperature by averaging three replicates for each reaction and the relative activity was determined as the percentage ratio of the activity of the sample to the maximum activity.

FIG. 5 is an optimum pH test of the Mg-sucB gene-encoded protein obtained by the present invention. The relative activity was determined at each pH by averaging three replicates per reaction and the percent activity of the sample relative to the maximum activity.

FIG. 6 is an HPLC detection of a catalytic product of the Mg-sucB gene encoded protein obtained by the present invention with sucrose as a substrate. In the figure, G represents glucose, GF represents sucrose, GF2 represents kestose, GF3 represents nystose, and GF4 represents nystose.

FIG. 7Mg-sucB catalyzed sucrose production of opalescent precipitate Polymer after purification ¹³ C NMR was carried out for composition analysis. a is the polymer of the ethanol precipitate of the catalytic product ¹³ C NMR chart; b. method for catalyzing product ethanol precipitate polymer ¹³ Chemical shifts of the C NMR peaks are compared to chemical shifts of levan standards.

Detailed Description

The gene capable of coding levan synthetase is obtained from the soil metagenome DNA, the protein coded by the gene can realize 80 percent of levan glycan conversion rate under the optimized reaction condition, and the reaction system does not need special reaction conditions. Meanwhile, compared with the substrate sucrose, the added value of the reaction product levan is high. The levan synthetase encoded by the gene has the optimum pH =6.0, but can maintain the catalytic activity of up to 90% in an alkaline reaction system with the pH = 9.0. This feature makes the enzyme more advantageous than the existing levan synthetase in catalyzing reactions under special circumstances.

Example 1: the expression and the heat stability of the Mg-sucB gene coding protein Mg-sucB obtained by the invention and the determination of the optimal temperature are as follows:

the pETM11-Mg-sucB recombinant plasmid or pETM11 original plasmid is transformed into an escherichia coli BL21 (DE 3) strain, inoculated into a TB culture solution and cultured to a logarithmic growth phase, and induced and expressed for 12 hours by using 0.1mmol/L IPTG. Then, the cells were collected by centrifugation, resuspended in 50mmol/L phosphate buffer (pH = 7.0), and disrupted by sonication. The supernatant was collected by centrifugation and purified by Ni-NTA purification column. The method for catalyzing the reaction by using pure enzyme liquid and sucrose as a substrate comprises the following steps: the reaction was carried out in a phosphate buffer solution (pH = 7.0) having a concentration of 50mmol/L, and 3ml of a phosphate buffer solution (pH = 7.0) containing 10% sucrose in the reaction system was mixed with 1ml of the enzyme solution, and reacted at 4 ℃ for 4d. The product was analyzed by HPLC using an amino column with 65% acetonitrile as the mobile phase, 10. Mu.L loading, 1mL/min flow rate, using Shodex detector. The results show that the crude enzyme solution extracted from the strain expressing the original plasmid pETM11 can not convert sucrose to produce milky levan glycan, so that the Escherichia coli does not contain levan synthetase per se; the protein expressed by the strain expressing pETM11-Mg-sucB can effectively catalyze sucrose to generate milky levan glycan (shown in figure 2) after being purified, and the reaction time is prolonged under the optimal reaction condition, so that the maximum levan yield of 80 percent can be obtained. To determine the optimum reaction temperature for Mg-sucB, reactions were measured every 10 ℃ in the temperature range of 20 ℃ to 90 ℃, and 3ml of 50mmol/L phosphate buffer containing 10% sucrose (pH = 7.0) was mixed with 1ml of enzyme solution for 10min. After the reaction was completed, the product was analyzed by HPLC under the conditions as described above. The relative activity is the percentage of the activity of the sample in relation to the maximum activity, averaged over three replicates for each assay. The results show that the optimal temperature for Mg-sucB is 40 ℃ while 70 ℃ retains 40% of the relative activity (as shown in FIG. 3). To determine the thermal stability of Mg-sucB, the reaction was measured every 10 ℃ in the temperature range of 20 ℃ to 90 ℃ and incubated for 30min at the corresponding temperature. After the end of the warm bath, the residual enzyme activity was measured at 40 ℃ by the above-mentioned measuring method. The average of three replicates was taken for each assay and the percent ratio of sample activity to maximum activity was the relative activity. As shown in fig. 4, the residual enzyme activity decreased significantly with increasing temperature, and 50% of the enzyme activity was lost by incubation at 50 ℃ for 30 min; while the enzyme activity is lost by 30min of warm bath at 60 ℃. Therefore, the optimum use temperature of Mg-sucB should be 40 ℃.

Example 2: the activity analysis of levan glycan produced by Mg-sucB gene coding protein Mg-sucB obtained by the invention under different pH conditions:

measuring a reaction at intervals of 1pH within the range of pH 3.0 to pH 10.0, setting the reaction time to 10min, and after the reaction is finished, carrying out product detection analysis by HPLC (high performance liquid chromatography) under the detection conditions shown above, thereby determining the activity of the enzyme for producing levan under different pH conditions. The relative activity is the percentage of the activity of the sample in relation to the maximum activity, averaged over three replicates for each assay. As shown in fig. 5, mg-sucB had an optimum pH =6.0, but was able to retain 90% of the relative activity in the strongly alkaline reaction solution having a pH of 9.0. The levan synthetase which has been reported so far is mostly used at neutral pH, and the Mg-sucB obtained by the present invention is the enzyme which has the strongest alkali resistance among known levan synthetases.

Example 3: under the optimal reaction condition, the HPLC analysis of the Mg-sucB gene coding protein Mg-sucB catalytic product obtained by the invention comprises the following steps:

Mg-sucB pure enzyme was prepared, catalyzed on sucrose substrate at optimum temperature and pH and the supernatant of the product was analyzed by HPLC, see example 2 for specific steps. The results showed that the supernatant of the reaction product was mainly glucose, whereas fructooligosaccharides such as kestose were hardly detectable (as shown in FIG. 6). This indicates that the main catalytic product of Mg-sucB is levan glycan, and the specificity of the product is high, which is beneficial to the subsequent separation and purification of levan glycan. Precipitating the catalytic product with 60% final alcohol on ice, centrifuging at high speed, discarding supernatant, washing with 75% alcohol for 3 times, lyophilizing, and making into oral liquid ¹³ C NMR analysis. ¹³ C NMR results showed that Mg-sucB is predominantThe bond type of the milky white precipitated polymer of the catalytic product is beta-2,6-fructoside bond, so that the catalytic product is levan, and finally the Mg-sucB is determined to be levan synthetase.

Claims (3)

1. A coding gene of an alkali-resistant levan synthetase for high-yield levan, which is characterized in that: can be efficiently expressed in escherichia coli, and the expressed protein can catalyze sucrose to generate levan glycan.

2. The gene encoding an alkali-resistant levan synthase for highly yielding levan glycans of claim 1, wherein: the yield of levan glycan generated by catalyzing sucrose by protein coded by the gene reaches nearly 80%; pH optimum =6.0. At the same time, it also has a relative activity of up to 90% under alkaline conditions of pH = 9.0.

3. The gene encoding an alkali-resistant levan synthase for highly yielding levan glycans of claim 1, wherein: has the DNA sequence shown in SEQ ID No. 1.

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