CN115725558A - High-stability ethanol-resistant mannose isomerase and coding gene thereof - Google Patents
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
- Y02E50/10—Biofuels, e.g. bio-diesel
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Abstract
The invention relates to the field of enzyme engineering, in particular to high-stability ethanol-resistant mannose isomerase and a coding gene thereof. The mannose isomerase which has been characterized in the prior art has limited quantity and low activity, and the catalytic efficiency is particularly poor under the special conditions of high temperature, strong alkalinity, organic solvent ethanol and the like, thereby affecting the application range of the mannose isomerase. The mannose isomerase gene provided by the invention can be efficiently expressed in escherichia coli, has high activity, has high tolerance to high temperature, strong alkalinity and ethanol, and shows good production and application prospects.
Description
Technical Field
The invention relates to the field of enzyme engineering, in particular to high-stability ethanol-resistant mannose isomerase and a coding gene thereof.
Background
Mannose is a monosaccharide commonly found in nature, and has a molecular weight of 180, is an isomer of fructose and glucose, is slightly soluble in ethanol, and is readily soluble in water. The crystals of sugar were white powder with a sweetness of 70% of sucrose and a slightly bitter aftertaste. Mannose is widely applied in various fields, wherein the mannose can be used as a health food for regulating sweet taste and is suitable for the daily diet of the vast population suffering from diabetes and obesity. The mannose can also be applied to animal feed, and has a certain inhibiting effect on the proliferation of pathogenic microorganisms in poultry intestinal tracts. Meanwhile, mannose has functions of immune regulation, anti-inflammation and the like, and has important effects on immune regulation and glycoprotein synthesis (Hu X et al. Comprehensive Reviews in Food Science and Food Safety,2016,15 (4): 773-785). At present, the preparation method of mannose is divided into two major categories, namely a chemical method and a biological enzyme method. The chemical method requires high temperature reaction, and has many byproducts, and the requirement of various chemical raw materials causes high cost. The biological enzyme method takes fructose as a substrate and generates D-mannose through the catalysis of D-mannose isomerase. The reaction condition of the biological enzyme method is mild, the cost of the raw material fructose is low, the by-product is less, and the product is easy to separate and purify. Therefore, the biological enzyme method is a better choice for preparing mannose.
D-mannose isomerase is a class of aldone isomerase, and can reversibly catalyze fructose to produce mannose. D-mannose isomerase was first found in Pseudomonas saccharophila in 1956, and its presence was later found in Xanthomonas. It has been reported that approximately 25% of fructose is converted to D-galactose when the D-mannose isomerase derived from Agrobacterium actinobacillus M-1 increases the fructose concentration from 5% to 40% (Hirose J.et al bioscience, biotechnology, and biochemistry,2001,65 (3): 658-661). In 2015, jiangfeng et al designed a production process for producing D-mannose by efficiently converting D-fructose by pseudomonas D-mannose isomerase (Jiangfeng et al, a strain producing D-mannose isomerase and a method for producing D-mannose by using the strain, wherein in China, CN201510195854.4[ P ]. 2015-07-15.). It has been proved that related enzyme genes can be separated from pseudomonas, streptomyces, escherichia coli and other bacteria, and escherichia coli is used as a carrier to synthesize engineering bacteria (Wu H.et al. Appl Microbiol Biotechnol.2019;103 (21-22): 8753-8761.). Mannose isomerase has been reported to be mainly derived from bacteria such as Pseudomonas saccharophila, xanthomonas hydrophila, streptomyces chromophilus, agrobacterium radiobacter, escherichia coli, pseudomonas cepacia, etc. Most of D-mannose isomerases have the highest enzymatic activity within a range of pH 7.0 to 8.0, except that the optimum pH of D-mannose isomerase of Pseudomonas cepacia is weakly acidic (Allenza P et al applied Biochemistry and Biotechnology,1990,24/25 (1): 171-182). Until now, the mannose isomerase which has been characterized has limited quantity, low activity, low catalytic efficiency under special conditions of strong alkalinity and organic solvent (such as ethanol) and the like, and the application range of the mannose isomerase is influenced. The mannose isomerase gene provided by the invention can be efficiently expressed in escherichia coli, has high activity and high tolerance on strong basicity and ethanol, and shows good production and application prospects.
Disclosure of Invention
The invention aims to provide alkali-resistant and ethanol-resistant mannose isomerase and a coding gene thereof.
Soil is taken from a forest of a botanical garden of Shenyang university for microorganism enrichment culture, metagenome DNA is extracted from the soil, a DNA fragment with a target size is amplified through degenerate primer PCR, and an alkali-resistant and ethanol-resistant mannose isomerase coding gene capable of efficiently converting fructose to generate mannose is identified and obtained through steps of molecular cloning, heterologous expression, catalytic function verification, DNA sequencing and the like. The specific research scheme is as follows:
1) And (3) extracting metagenome DNA. Taking soil from the forest of a vegetable garden of Shenyang agricultural university, adding fructose according to the proportion of 1% (w/w), adding water for wetting, and culturing in an incubator at 37 ℃ for 10 days to extract high-quality metagenomic DNA.
2) Obtaining Mg-yihS gene: the existing degenerate primers Mg-yihS-For and Mg-yihS-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 metagenomic DNA, 0.5. Mu.l Taq DNA polymerase, 1 XTaq Buffer, 0.5. Mu.l each of the 40mmol/L Mg-yihS-For and Mg-yihS-Rev primers, 0.8. Mu.l 100mmol/L dNTP, and water 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 52 ℃ for 40s, extension reaction at 72 ℃ for 3min, and reaction at 72 ℃ for 10min after the circulation is finished.
3) Construction of pANY2-Mg-yihS recombinant plasmid: the PCR product was subjected to agarose gel electrophoresis, and the band of the desired size was recovered with a gel recovery kit. And (3) performing secondary PCR by using the recovered DNA as a template and degenerate primers Mg-yihS-For and Mg-yihS-Rev, wherein the PCR reaction conditions are the same as the above steps. The PCR product was subjected to agarose gel electrophoresis, and the band of the desired size was gel recovered using a gel recovery kit. The recovered DNA product was digested with NdeI and BamHI, respectively, and ligated to pANY2 vector which was also digested in two. The specific enzyme digestion reaction system is as follows: ndeI and BamHI 1.0. Mu.l each, 1 Xrestriction enzyme reaction Buffer, 16. Mu.l pANY2 linear vector or gel purified PCR product was recovered, water was added to 100. Mu.l, and reaction was carried out at 16 ℃ for 5 hours. And (3) mixing PCR products recovered and purified from the gel after enzyme digestion or pANY2 linear vectors according to the molar ratio of 1. Colonies grown on the transformed plate were picked, inoculated into LB medium containing kanamycin, and subjected to liquid culture.
4) Activity primary screening and DNA sequencing analysis. The picked colonies are cultured in LB culture medium to the logarithmic growth phase, and 0.1mmol/L IPTG is added for inducing expression for 10h. The cells were collected by centrifugation, resuspended in 50mmol/L phosphate buffer (pH 7.0), sonicated, and the supernatant was collected by centrifugation as a crude enzyme solution. 30. Mu.L of the crude enzyme solution was added with 30. Mu.L of 10% fructose and 10. Mu.L of 1mol/L magnesium sulfate dissolved in 50mmol/L phosphate buffer (pH 7.0), mixed well, and reacted at 37 ℃ for 2 hours. Centrifuging the reaction product, taking the supernatant, and measuring the mannose production by a cysteine-carbazole method. Selecting one of the bacteria with activity and strongest activity, liquid culturing in a culture medium containing kanamycin, extracting plasmid, and performing DNA sequencing, wherein the reading frame is shown as SEQ ID No:1, and the physical map of the plasmid is shown as figure 1. Through identification, the gene coding protein is mannose isomerase, and has the characteristics of high activity, alkali resistance, ethanol resistance and the like. The specific analysis method is shown in the examples.
Compared with the mannose isomerase coded by the currently known mannose isomerase, the mannose isomerase coded by the gene disclosed by the invention has the following outstanding advantages:
1) The mannose isomerase encoded by the gene obtained by screening in the invention has the optimum pH of 7.0, but still maintains the relative activity of up to 60% under the strong alkaline condition of pH = 10.0. Therefore, the enzyme activity is high, and the catalytic reaction can be carried out under neutral conditions or strong alkaline conditions, so that the application range is very wide.
2) The mannose isomerase coded by the gene obtained by screening in the invention is most suitable for being used at 40 ℃, but can still maintain 20% of activity after being subjected to warm bath at 70 ℃ for 30min. Therefore, the enzyme can be used under the condition of normal temperature, and can catalyze the reaction under the condition of 60-70 ℃. This feature therefore also increases the range of applications of the enzyme.
3) The mannose isomerase coded by the gene obtained by screening has unique ethanol tolerance. Most known enzymes are denatured, either completely or partially, in the presence of organic solvents such as ethanol. The enzyme activity in 10% (v/v) ethanol solution is not reduced, but increased by 10%. This indicates that the enzyme is more suitable for catalyzing the reaction under the condition of containing ethanol than the general enzyme. In addition, the characteristic also prompts a user to add a certain amount of ethanol into the catalytic reaction system, so that the catalytic efficiency of the mannose isomerase is promoted, and simultaneously, the action of other enzymes or microorganisms is inhibited, thereby achieving better catalytic effect.
Drawings
FIG. 1 is a physical map of the plasmid pANY2-Mg-yihS constructed according to the present invention. Mg-yihS is a coding gene of high-stability ethanol-resistant mannose isomerase.
FIG. 2 is the optimum temperature detection of the mannose isomerase encoded by the Mg-yihS gene 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. 3 is a graph showing the thermal stability test of the mannose isomerase encoded by the Mg-yihS gene obtained by the present invention, and the relative activity of the mannose isomerase is measured after each temperature treatment for 30min. The specific method is to take the average value of three times of repetition of each reaction, and the percentage of the ratio of the activity of the sample to the highest activity is the relative activity.
FIG. 4 detection of the optimum pH of the Mg-yihS gene encoding mannose isomerase 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. 5 detection of pH stability of the Mg-yihS gene encoding mannose isomerase obtained by the present invention, the relative activity of which was measured after 30min of each pH treatment. The specific method is to take the average value of three times of repetition of each reaction, and the percentage of the ratio of the activity of the sample to the highest activity is the relative activity.
FIG. 6 shows the relative activities of the Mg-yihS gene encoding mannose isomerase obtained in the present invention in the presence of organic solvents at different concentrations.
Detailed Description
The invention screens and obtains a gene which can code mannose isomerase from the soil metagenome DNA, the optimum pH of the enzyme is 7.0, but the relative activity of the enzyme still keeps up to 60 percent under the strong alkaline condition that the pH = 10.0; meanwhile, the optimum temperature of the enzyme is 40 ℃, but 20% of activity can still be maintained after the enzyme is subjected to warm bath at 70 ℃ for 30min. In addition, the enzyme has unique ethanol tolerance. The enzyme activity in 10% (v/v) ethanol solution is not only not reduced, but also increased by 10%. These characteristics make the enzyme have more advantages than the existing mannose isomerase in catalyzing the reaction under special environment.
Example 1: the expression and the determination of the optimal temperature and the temperature stability of the Mg-yihS gene coding protein obtained by the invention are as follows:
the pANY2-Mg-yihS recombinant 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 5h by using 0.2mmol/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. mu.L of purified enzyme was taken, and 30. Mu.L of 10% fructose and 10. Mu.L of 1mol/L magnesium sulfate dissolved in 50mmol/L phosphate buffer (pH 7.0) were added thereto, and the mixture was mixed and reacted at 37 ℃ for 2 hours. The reaction product was centrifuged, and the supernatant was collected and analyzed by HPLC. The Shodex NH 2P-50E chromatographic column, the RID-10A differential detector and the LC-10AT liquid pump are adopted, the mobile phase is 75% acetonitrile (v/v), the flow rate is 1ml per min, and the column temperature is 40 ℃. To determine the optimum reaction temperature for Mg-yihS. 0.5ml of the enzyme solution was mixed with 3ml of 50mmol/L phosphate buffer (pH = 7.0) containing 10% fructose, and the reaction time was 10min. The reaction was measured every 10 ℃ over the temperature range of 20 ℃ to 90 ℃ and after the reaction was complete the product was detected by HPLC, the detection conditions being as indicated above. The average of three replicates was taken for each assay and the percent ratio of sample activity to maximum activity was the relative activity. The results show that the optimum temperature for Mg-yihS is 40 ℃ and that 40% of the relative activity is retained at 70 ℃ (as shown in FIG. 2). To determine the thermal stability of Mg-yihS, the reaction was measured every 10 ℃ in the temperature range of 20 ℃ to 90 ℃ and incubated at the corresponding temperature for 30min. After the completion of the warm bath, the residual enzyme activity was measured at the optimum temperature of 40 ℃ 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. As shown in fig. 3, the residual enzyme activity decreased significantly with increasing temperature, and 50% of the enzyme activity remained after 30min of warm bath at 50 ℃; and carrying out warm bath at 60 ℃ for 30min, wherein 30% of activity of enzyme remains; and 20% of activity of the enzyme still remains after 30min of warm bath at 70 ℃. Therefore, the optimum use temperature of Mg-yihS should be 40 ℃, but the enzyme has good thermal stability.
Example 2: the optimal pH and pH stability of the Mg-yihS gene coding protein obtained by the invention are determined as follows:
the protein expression, purification and activity determination methods of Mg-yihS are the same as above. In order to examine the optimum pH of the enzyme, the reaction was measured every 1 pH in the range of pH 3.0 to pH10.0, and the reaction time was set to 10min. After the reaction is finished, the product is detected and analyzed by HPLC, and the detection conditions are the same as 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. As shown in fig. 4, mg-yihS has an optimum pH =7.0, but can retain 40% of the relative activity in a strongly alkaline reaction solution having a pH of 10.0. The currently known mannose isomerases mostly have neutral pH range, while the Mg-yihS obtained in the present invention shows very outstanding alkali tolerance. To determine the pH stability of Mg-yihS, the reaction was measured every 1 pH in the range of pH 3.0 to pH10.0, treated at the corresponding pH for 30min, and then the residual enzyme activity was measured under the conditions of optimum temperature and optimum pH using the 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 residual enzyme activity was significantly reduced at both pH > 7.0 and pH < 7.0, but the enzyme still retained 60% relative activity when treated for 3min in a strongly alkaline reaction solution at pH10.0 compared to the control (as shown in FIG. 5), further demonstrating the outstanding stability characteristics of the enzyme under alkaline conditions.
Example 3: the relative activity of the Mg-yihS gene coding protein obtained by the invention in the presence of different organic solvents is as follows:
in order to study the relative activity of the enzyme in the presence of an organic solvent, the enzyme is mixed with 10-50% (v/v) methanol, ethanol or acetone to carry out a catalytic reaction, and the specific catalytic reaction conditions are the same as above. The average of three replicates was taken for each reaction and the percent ratio of sample activity to maximum activity was the relative activity. The results show that 10% ethanol has a promoting effect on the relative activity of the enzyme. In general, the presence of organic solvents causes complete or partial loss of enzymatic activity, and the mechanism of the enzymatic activity-promoting effect of low-concentration ethanol is worth intensive study. Methanol and acetone at different concentrations, and ethanol at high concentration all had stronger inhibitory effects on enzyme activity, of which the inhibitory effect of methanol was most significant (as shown in fig. 6). The characteristic of ethanol tolerance prompts a user to add a certain amount of ethanol into a catalytic reaction system, so that the catalytic efficiency of mannose isomerase is promoted, and the action of other enzymes or microorganisms is inhibited, thereby achieving a better catalytic effect.
Claims (4)
1. A high-stability ethanol-resistant mannose isomerase and a coding gene thereof are characterized in that: can be efficiently expressed in Escherichia coli, and the expressed protein can convert fructose to produce mannose.
2. The high-stability ethanol-resistant mannose isomerase and the coding gene thereof as claimed in claim 1, wherein the high-stability ethanol-resistant mannose isomerase is characterized in that: the enzyme has the optimum pH =7.0, shows extremely strong stability, still keeps up to 60% of relative activity under the strong alkaline condition of pH =10.0, and still can keep 20% of activity after being bathed for 30min at 70 ℃.
3. The high-stability ethanol-resistant mannose isomerase and the coding gene thereof as claimed in claim 1, wherein the high-stability ethanol-resistant mannose isomerase is characterized in that: the enzyme has unique ethanol tolerance, and the enzyme activity is not reduced but increased by 10% in a 10% (v/v) ethanol solution.
4. The high-stability ethanol-resistant mannose isomerase and the coding gene thereof as claimed in claim 1, wherein the high-stability ethanol-resistant mannose isomerase is characterized in that: has the DNA sequence shown in SEQ ID No. 1.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1347455A (en) * | 1999-04-22 | 2002-05-01 | 协和发酵工业株式会社 | Novel meannose isomerase and DNA encoding enzyme |
| CN111944796A (en) * | 2020-08-13 | 2020-11-17 | 浙江农林大学 | D-mannose isomerase and application thereof |
| CN113512544A (en) * | 2021-07-14 | 2021-10-19 | 江南大学 | Mannose isomerase mutant with improved heat stability |
-
2022
- 2022-09-01 CN CN202211066863.XA patent/CN115725558A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1347455A (en) * | 1999-04-22 | 2002-05-01 | 协和发酵工业株式会社 | Novel meannose isomerase and DNA encoding enzyme |
| CN111944796A (en) * | 2020-08-13 | 2020-11-17 | 浙江农林大学 | D-mannose isomerase and application thereof |
| CN113512544A (en) * | 2021-07-14 | 2021-10-19 | 江南大学 | Mannose isomerase mutant with improved heat stability |
Non-Patent Citations (1)
| Title |
|---|
| ""Aldose-ketose isomerase YihS [Salmonella enterica subsp. enterica serovar Inverness str. R8-3668]，GenBank: EHC49369.1", 《GENBANK》, 21 October 2011 (2011-10-21) * |
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