Sucrose phosphorylase with enhanced catalytic activity and application

Abstract

The invention relates to the technical field of enzyme catalysis, in particular to sucrose phosphorylase with enhanced catalytic activity and application, the amino acid sequence of the sucrose phosphorylase is shown as SEQ ID NO: 3, and compared with wild type sucrose phosphorylase, the sucrose phosphorylase has higher catalytic activity. According to the sucrose phosphorylase with enhanced catalytic activity, the reaction efficiency can be improved, the activation energy of chemical reaction is remarkably reduced, and the reaction process is accelerated; the energy consumption and environmental influence are reduced, the production cost is reduced, the adaptability is enhanced, the production flexibility is improved, and the development of the green chemical industry can be promoted.

Description

Technical Field

[0001] The invention relates to the technical field of enzyme catalysis, and specifically relates to a sucrose phosphorylase with enhanced catalytic activity and its application. Background Art

[0002] Arbutin can be divided into α-arbutin, β-arbutin and deoxyarbutin, of which α-arbutin and β-arbutin are both water-soluble. It is a skin depigmenting component that originated from the leaf cells of the perennial evergreen shrub of the genus Ursula in the Ericaceae family. It also exists in other plants such as cranberries, blueberries, and wheat. It is reported that α-arbutin is more effective in inhibiting human tyrosinase than its isomer β-arbutin, and the whitening effect of α-arbutin is more than 10 times higher than that of β-arbutin.

[0003] Currently, α-arbutin can be produced by chemical synthesis and bioconversion. The biological method has several advantages: simple cell post-processing, high synthesis yield and simple product separation and purification. In addition, the catalyst can be recovered by simple centrifugation, thereby reducing the cost of industrial production.

[0004] CN112300977A integrates the optimized gene expression frame P43-gtfA into the B.subtilis WB600 genome to obtain a genetically engineered strain. When the substrate concentration is 50 g / L, the sucrose concentration is 310.9 g / L, the bacterial concentration OD600 is 20, and the catalytic system is catalyzed in a shake flask at 30°C and 220 rpm for 20 hours, the α-arbutin yield is 61.1 g / L, and the molar conversion rate of the substrate hydroquinone (HQ) is 49.4%. The overall conversion rate is low, which will lead to high production costs.

[0005] Deng Li et al. have achieved a yield of 61.7 g / L of α-arbutin and a conversion rate of 94.5% by adding hydroquinone. However, this process requires the use of macroporous adsorption resin to immobilize hydroquinone, which has a complicated operation process and brings difficulties to industrial production.

[0006] CN112646762A uses medium-copy plasmid to overexpress endogenous genes such as shikimate kinase (AroL), pyruvate kinase (ppsA), transketolase (tktA), 3-deoxy-7-phosphoheptanoate synthase (aroGfbr) and isochorismate synthase (EntC), and low-copy plasmid to overexpress exogenous gene malEFGK2, and uses carbon sources such as maltose and / or glycerol to produce α-arbutin, thus achieving the de novo synthesis of α-arbutin. However, the final α-arbutin yield is only 685±17 mg / L, which does not yet have the basis for industrialization.

[0007] Another report used recombinant starch sucrase from Cellulomonas carboniz T26 (accession number: KGM11272.1), and the conversion reaction conditions were: substrate concentration range of 1% to 30%, enzyme dosage of 1 to 3 U / mg hydroquinone, pH 5.5 to 7.0, conversion reaction temperature of 35 to 45°C, conversion reaction time of 3 to 4 hours, to obtain a reaction solution containing α-arbutin with a mass concentration of >60%.

[0008] CN107236696B uses a recombinant Bacillus subtilis expressing sucrose phosphorylase from L. mesenteroides, and the reaction system includes: adding sucrose and HQ in a molar ratio of 4:1 to the acceptor and 7500U / g of hydroquinone SPase in 20mM sodium phosphate buffer (pH 7.0), and reacting it at 30°C for 24h. Then, it is treated with saccharifying enzyme at 40°C for 3h and further heated to inactivate. When the reaction is completed, the conversion rate can reach 83.64%.

[0009] At present, there are still some challenges in the biological method, including improving the tolerance to HQ and enhancing the catalytic activity of the enzyme to reduce the amount of bacteria used, etc. Therefore, the present invention aims to screen and verify a sucrose phosphorylase mutant with enhanced activity and establish its application in a simple and efficient α-arbutin preparation process. Summary of the invention

[0010] The purpose of the present invention is to provide a sucrose phosphorylase with enhanced catalytic activity and application thereof.

[0011] To achieve the above object, the present invention provides the following technical solutions:

[0012] A sucrose phosphorylase with enhanced catalytic activity, the amino acid sequence of which is shown in SEQ ID NO:3.

[0013] The sucrose phosphorylase of the present invention has stronger catalytic activity than the wild-type sucrose phosphorylase, and the amino acid sequence of the wild-type sucrose phosphorylase is shown in SEQ ID NO: 1 or 2.

[0014] The present invention also provides a polynucleotide which can encode the above-mentioned sucrose phosphorylase.

[0015] Furthermore, the polynucleotide sequence is shown in SEQ ID NO:4.

[0016] The sucrose phosphorylase with enhanced catalytic activity of the present invention can be used for catalytic preparation of α-arbutin. The method of using the sucrose phosphorylase is as follows: sucrose, hydroquinone and VC are added, crude enzyme solution of sucrose phosphorylase is added, and the mixture is reacted at pH 7.0 and 35°C.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] (1) Improving reaction efficiency: The sucrose phosphorylase of the present invention, as a highly efficient α-arbutin biocatalyst, can significantly reduce the activation energy of the chemical reaction and accelerate the reaction process.

[0019] (2) Reduction of post-processing and purification steps: The increased activity of the sucrose phosphorylase of the present invention can reduce the amount of enzyme input, reduce post-processing and purification steps, and thus reduce production costs.

[0020] (3) Improving the efficiency of raw material and energy utilization: The increased activity of the sucrose phosphorylase of the present invention can reduce the fermentation volume and energy consumption, and has the characteristics of green manufacturing and sustainable development.

[0021] (4) Enhanced structural rigidity: The improved activity of the sucrose phosphorylase of the present invention can enhance its structural rigidity, thereby maintaining stability during multiple uses of the immobilized enzyme and extending its service life.

[0022] (5) Improving the flexibility of industrial production: The increased activity of enzymes can enable them to play a catalytic role under wider process parameters, thereby improving production intensity and operational flexibility.

[0023] (6) The enhanced activity of sucrose phosphorylase of the present invention has many benefits in industrial biocatalysis, including improving reaction efficiency, reducing energy consumption and environmental impact, reducing production costs, enhancing adaptability, improving production flexibility, and promoting the development of the green chemical industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is the spectrum of WT1 in Example 5 after reaction at 35°C for 3 hours.

[0025] Figure 2 This is the spectrum of WT1 in Example 5 after reaction at 35°C for 18 hours.

[0026] Figure 3 This is the spectrum of WT2 in Example 5 after reaction at 35°C for 3 hours.

[0027] Figure 4 This is the spectrum of WT2 in Example 5 after reaction at 35°C for 18 hours.

[0028] Figure 5 This is the spectrum of MUT11 in Example 5 after reaction at 35°C for 3 hours.

[0029] Figure 6 This is the spectrum of MUT11 in Example 5 after reaction at 35°C for 18 hours.

[0030] Figure 7This is the spectrum of MUT11 in Example 6 after reaction at 35°C for 22 hours.

[0031] Figure 8 This is the standard HPLC spectrum of hydroquinone, with a peak time of 23 minutes.

[0032] Fig. 9 This is the HPLC spectrum of the standard product of α-arbutin, with a peak time of 17 minutes.

[0033] Fig.10 This is the protein gel detection result of MUT11. The three lanes are whole cells, supernatant, and precipitate respectively. DETAILED DESCRIPTION

[0034] The technical scheme in the embodiment of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention and the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of the present invention. The instruments and reagents used in the present embodiment are all commercially available products unless otherwise specified.

[0035] The HPLC detection conditions for the following experiments are as follows:

[0036] Chromatographic column: Shim-pack GIS 4.6x250mm 5um C18 (or equivalent C18 column).

[0037] Detection wavelength: 280nm.

[0038] Flow rate: 0.3mL / min.

[0039] Column temperature: 45℃.

[0040] Injection volume: 10 μL.

[0041] Running time: 28min.

[0042] Mobile phase: 0.1% formic acid aqueous solution: methanol = 90:10.

[0043] Elution mode: isocratic elution.

[0044] Example 1 Obtaining the wild-type sucrose phosphorylase gene sequence

[0045] According to the whole genome information of Leuconostoc mesenteroides, the sucrose phosphorylase gene from Leuconostoc mesenteroides was synthesized by whole gene synthesis. PrimerPremier (http: / / primer3.ut.ee / ) and OPTIMIZER (http: / / genomes.urv.es / OPTIMIZER / ) were used for design, and the difference in annealing temperature (Tm) was controlled within 3°C, the primer length was controlled within 60 bases, and the obtained primers were dissolved in double distilled water and added to the following reaction system, so that the final concentration of each primer was 30nM, and the final concentration of the first and last primers was 0.6μM.

[0046] 2mM dNTPmix (2mM each dNTP) 5μL 10×Pfubuffer 5μL PfuDNApolymerase (10U / μL) 0.5μL <![CDATA[ddH2O]]> Make the total volume of the reaction system to 50 μL

[0047] Place the prepared PCR reaction system in the Biori XP cycler gene amplification instrument and perform amplification according to the following program: 98°C for 30s, 55°C for 45s, 72°C for 120s, 35x.

[0048] The DNA fragment obtained by PCR was purified by gel excision and cloned into the NdeI / XhoI site of pET30a by homologous recombination. A single clone was selected for sequencing. The DNA sequence successfully sequenced was SEQ ID NO: 1, named WT1.

[0049] Example 2 Obtaining the wild-type sucrose phosphorylase gene sequence

[0050] According to the whole genome information of Streptococcus mutans UA159, the sucrose phosphorylase gene from Streptococcus mutans UA159 was synthesized by whole gene synthesis. PrimerPremier (http: / / primer3.ut.ee / ) and OPTIMIZER (http: / / genomes.urv.es / OPTIMIZER / ) were used for design, and the difference in annealing temperature (Tm) was controlled within 3°C, the primer length was controlled within 60 bases, and the obtained primers were dissolved in double distilled water and added to the following reaction system, so that the final concentration of each primer was 30nM, and the final concentration of the first and last primers was 0.6μM.

[0051] 2mM dNTPmix (2mM each dNTP) 5μL 10×Pfubuffer 5μL PfuDNApolymerase (10U / μL) 0.5μL <![CDATA[ddH2O]]> Make the total volume of the reaction system to 50 μL

[0052] Place the prepared PCR reaction system in the Biori XP cycler gene amplification instrument and perform amplification according to the following program: 98°C for 30s, 55°C for 45s, 72°C for 120s, 35x.

[0053] The DNA fragment obtained by PCR was purified by gel excision and cloned into the NdeI / XhoI site of pET30a by homologous recombination. A single clone was selected for sequencing. The DNA sequence successfully sequenced was SEQ ID NO: 2, named WT2.

[0054] Example 3 Mutation of wild-type sucrose phosphorylase

[0055] Sucrose phosphorylase mutants are derived from the wild-type sucrose phosphorylase of Example 2, and the goal is to obtain mutants with enhanced catalytic activity, which show stronger catalytic activity than wild-type sucrose phosphorylase. Sucrose phosphorylase mutants and polynucleotides encoding such mutants can be prepared using methods commonly used by those skilled in the art. Mutants can be obtained by in vitro recombination, polynucleotide mutagenesis, DNA shuffling, error-prone PCR, and directed evolution methods, etc., encoding the enzyme.

[0056] Through the method of total gene synthesis, the secondary structure and codon preference of the gene were adjusted to achieve high expression in E. coli. Primer Premier (http: / / primer3.ut.ee / ) and OPTIMIZER (http: / / genomes.urv.es / OPTIMIZER / ) were used for design, and the difference in annealing temperature (Tm) was controlled within 3°C, the primer length was controlled within 60 bases, and the obtained primers were dissolved in double distilled water and added to the following reaction system, so that the final concentration of each primer was 30nM, and the final concentration of the first and last primers was 0.6μM.

[0057] 2mM dNTPmix (2mM each dNTP) 5μL 10×Pfubuffer 5μL PfuDNApolymerase (10U / μL) 0.5μL <![CDATA[ddH2O]]> Make the total volume of the reaction system to 50 μL

[0058] Place the prepared PCR reaction system in the Biori XP cycler gene amplification instrument and perform amplification according to the following program: 98°C for 30s, 55°C for 45s, 72°C for 120s, 35x.

[0059] The DNA fragment obtained by PCR was gel-cleaved and purified, and cloned into the NdeI / XhoI site of pET30a by homologous recombination. A single clone was selected for sequencing. The mutant sequence successfully sequenced was named MUT11, the DNA sequence was SEQ ID NO: 4, and the corresponding amino acid sequence was SEQ ID NO: 3, which contained C72R and E436D double mutations.

[0060] Example 4 Preparation of enzyme solution in shake flasks and fermentation tanks

[0061] A single colony of E. coli containing the expression vector was picked and inoculated into 10 mL of autoclaved medium: 10 g / L tryptone, 5 g / L yeast extract, 3.55 g / L disodium hydrogen phosphate, 3.4 g / L potassium dihydrogen phosphate, 2.68 g / L ammonium chloride, 0.71 g / L sodium sulfate, 0.493 g / L magnesium sulfate heptahydrate, 0.027 g / L ferric chloride hexahydrate, 5 g / L glycerol, 0.8 g / L glucose, and kanamycin was added to 50 mg / L. Incubate overnight at 30°C, 250 rpm. The next day, take a 1L Erlenmeyer flask and inoculate it into 100mL of autoclaved culture medium at an inoculation ratio of 1:100: 10g / L tryptone, 5g / L yeast extract, 3.55g / L disodium hydrogen phosphate, 3.4g / L potassium dihydrogen phosphate, 2.68g / L ammonium chloride, 0.71g / L sodium sulfate, 0.493g / L magnesium sulfate heptahydrate, 0.027g / L ferric chloride hexahydrate, 5g / L glycerol, 0.3g / L glucose, and add kanamycin to 50mg / L. Cultivate at 30℃ until the bacterial OD is 5-6, and immediately place the Erlenmeyer flask in a 25℃ shaker at 250rpm for 1 hour. Add IPTG to a final concentration of 0.1mM and continue to culture at 25℃ and 250rpm for 16 hours. After the culture is completed, the culture solution is centrifuged at 4℃ and 12000g for 20 minutes to collect the wet bacteria. Then the bacterial precipitate was washed twice with distilled water, and the bacterial cells were collected and stored at -70°C. At the same time, a small amount of bacterial cells was taken for SDS-PAGE detection.

[0062] Batch fed fermentation was carried out in a computer-controlled 7L pressure-resistant stainless steel bioreactor (Jiangsu B. Braun) with a working volume of 4L. The culture medium used was a modified TB culture medium, with specific components of 24g / L yeast extract, 12g / L peptone, 0.4% glucose, 2.31g / L dihydrogen phosphate and 12.54g / L dipotassium hydrogen phosphate, pH 7.0. About 200mL of the primary seed culture of the shake flask was connected to the fermenter when OD2.0 was reached. The temperature was maintained at 37°C during the initial fermentation process. The dissolved oxygen concentration was automatically controlled at 30% by the stirring rate (rpm) and the ventilation supply cascade control during the fermentation process, and the pH value of the culture medium was maintained at 6.7-7.1 by 50% (v / v) orthophosphoric acid and 30% (v / v) ammonia water. When a substantial rise in dissolved oxygen occurred, feeding began. The feed solution contained 9% w / v peptone, 9% w / v yeast extract, and 14% w / v glycerol. When OD600 was about 15.0, the temperature was lowered to 25°C, and IPTG was added to a final concentration of 0.2 mM to induce expression.

[0063] Example 5 Comparison of catalytic efficiency with wild-type protein

[0064] Sucrose phosphorylase from Streptococcus mutans UA159 and Leuconostoc mesenteroides is the most mature sucrose phosphorylase in industrial application, and is also the two most widely studied sucrose phosphorylases in the α-arbutin biocatalytic process, so it is used as a control protein for synchronous reaction and named WT1 and WT2 respectively. Prepare 100mL reaction system: 25mM PB7.0, final concentration 30g / L hydroquinone, 300g / L sucrose, 5.3g / L VC, 60g / L WT1 crude enzyme solution, adjust pH to 7.0, dilute to 100mL, start reaction and timing, control the water bath temperature to 35℃ and operate in dark throughout the process.

[0065] Similarly, the reaction systems of WT2 and MUT11 crude enzyme solutions were prepared and reacted at 35° C. Samples were taken at 3 hours, 18 hours, and 24 hours to detect the amount of α-arbutin produced by HPLC.

[0066] The results showed that the catalytic efficiency of MUT11 at 3 hours and 18 hours was significantly higher than that of the two wild proteins, and reached the highest level at 18 hours. The product decreased significantly at 24 hours, and it was speculated that the reason was that α-arbutin was oxidized and consumed over time after reaching the peak. The two wild proteins WT1 and WT2 continued to catalyze for 24 hours and still did not reach the peak, indicating that their catalytic efficiency was significantly lower than that of MUT11. Therefore, the use of MUT11 for production can significantly shorten the reaction time, or reduce the amount of enzyme solution used, and has higher economic efficiency.

[0067]

[0068]

[0069] Example 6 Catalytic reaction of the amplified system

[0070] Prepare 1000mL reaction system: 25mM PB7.0, final concentration 30g / L hydroquinone, 300g / L sucrose, 3g / L VC, 50g / L MUT11 crude enzyme solution, adjust pH to 7.0, make the volume to 1000mL, start the reaction and start timing, control the water bath temperature at 35℃, react overnight and take a 22-hour reaction sample for HPLC detection.

[0071] The results showed that the substrate of MUT11 was almost completely reacted after 22 hours of reaction, with a conversion rate of up to 96% and a final product concentration of 71 g / L.

[0072] Example 7 Effect of different buffer systems on the reaction

[0073] Prepare 100 mL PB buffer system: 25 mM PB pH 7.0, final concentration 350 g / L sucrose, final concentration 40 g / L hydroquinone, 5.4 g / L VC, 55 g / L MUT11 crude enzyme solution, adjust pH to 7.0, make the volume to 100 mL, start the reaction and start timing, control the water bath temperature at 35 °C to react overnight and take a 48-hour reaction sample for HPLC detection.

[0074] Prepare 100 mL of MES buffer system: 25 mM MES pH 7.0, final concentration 350 g / L sucrose, 40 g / L hydroquinone, 5.4 g / LVC, 55 g / L MUT11 crude enzyme solution, adjust pH to 7.0, make the volume to 100 mL, start the reaction and start timing, control the water bath temperature at 35 °C to react overnight and take a 48-hour reaction sample for HPLC detection.

[0075] The results of 48 hours of reaction showed that the concentration of α-arbutin in the PB system was 92.17 g / L, and the concentration of α-arbutin in the MES system was 91.69 g / L. Different buffer systems had little effect on the catalytic results after extended time.

[0076] Example 8 Effect of adding saccharifying enzyme on the reaction

[0077] Reaction system: 25mM PB7.0, final concentration 15g / L hydroquinone, 300g / L sucrose, 5.4g / L VC, 30g / L MUT2 crude enzyme solution, adjust pH 7.0, dilute to 100mL, shake at 35℃ and avoid light. After 19 hours of reaction, add 50g / L saccharifying enzyme, continue the reaction for 3 hours, and take samples for HPLC detection. The results showed that the concentration of α-arbutin dropped from 37.4g / L to 24.77g / L, indicating that the product dropped significantly after the saccharifying enzyme treatment was used after the reaction. The results show that it is not necessary to add saccharifying enzyme to the α-arbutin process of sucrose process, which is inconsistent with some previous literature reports.

[0078] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A sucrose phosphorylase with enhanced catalytic activity, characterized in that: The amino acid sequence is shown in SEQ ID NO: 3, which contains double mutations of G48V and S268G.

2. A polynucleotide encoding the sucrose phosphorylase according to claim 1.

3. The polynucleotide according to claim 2, characterized in that: The sequence is shown in SEQ ID NO:

4.

4. Use of the sucrose phosphorylase with enhanced catalytic activity according to claim 1 in catalytic preparation of α-arbutin.

5. Use of the sucrose phosphorylase with enhanced catalytic activity according to claim 4 in catalytic preparation of α-arbutin, characterized in that: Take sucrose, hydroquinone and VC, add crude enzyme solution of sucrose phosphorylase, and react at pH 7.0 and 35°C.

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