Sucrose phosphorylase with enhanced catalytic activity and its application

Abstract

The present invention relates to the field of enzyme catalysis technology, and in particular to a sucrose phosphorylase with enhanced catalytic activity and its application. The amino acid sequence of the sucrose phosphorylase is shown in SEQ ID NO: 3, and the sucrose phosphorylase has stronger catalytic activity than the wild-type sucrose phosphorylase. The sucrose phosphorylase with enhanced catalytic activity of the present invention can improve reaction efficiency, significantly reduce the activation energy of the chemical reaction, and accelerate the reaction process; it also reduces energy consumption and environmental impact, reduces production costs, enhances adaptability, improves production flexibility, and can promote the development of the green chemical industry.

Description

Technical Field

[0001] The present 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. α-arbutin and β-arbutin are both water-soluble. Originally derived from the leaf cells of bearberry, a perennial evergreen shrub of the Ericaceae family, it is a skin-depigmenting compound also found in other plants, such as cranberries, blueberries, and wheat. α-arbutin is reportedly more effective at inhibiting human tyrosinase than its isomer β-arbutin, and its whitening effect is over 10 times greater than that of β-arbutin.

[0003] Currently, α-arbutin can be produced through both chemical synthesis and bioconversion. The bioconversion method offers several advantages: simple cell post-processing, high synthesis yields, and simple product separation and purification. Furthermore, the catalyst can be recovered by simple centrifugation, thus reducing industrial production costs.

[0004] CN112300977A integrates the optimized gene expression cassette P43-gtfA into the genome of B. subtilis WB600 to generate a genetically engineered strain. When the substrate concentration is 50 g / L, the sucrose concentration is 310.9 g / L, and the bacterial cell concentration is OD600 = 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 of the substrate hydroquinone (HQ) is 49.4%. This overall low conversion rate will lead to high production costs.

[0005] Deng Li and colleagues achieved a yield of 61.7 g / L of α-arbutin with a conversion rate of 94.5% by feeding hydroquinone. However, this process requires immobilizing the hydroquinone with a macroporous adsorption resin, resulting in a complex process and difficulties in industrial production.

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

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

[0008] CN107236696B utilizes a recombinant Bacillus subtilis expressing sucrose phosphorylase derived from L. mesenteroides. The reaction system involves adding sucrose and HQ at a donor-to-acceptor molar ratio of 4:1, along with 7500 IU / g of hydroquinone, to 20 mM sodium phosphate buffer (pH 7.0). The reaction is then carried out at 30°C for 24 hours. The enzyme is then treated with saccharifying enzyme at 40°C for 3 hours and heat-inactivated. Upon completion of the reaction, the conversion rate reaches 83.64%.

[0009] Currently, biological methods still face several challenges, including improving tolerance to HQ and enhancing the catalytic activity of the enzyme, thereby reducing the amount of bacterial cells used. 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 process for preparing α-arbutin. Summary of the Invention

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

[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 nucleotide sequence of the wild-type sucrose phosphorylase is shown in SEQ ID NO: 1 or 2.

[0014] The present invention also provides a polynucleotide that 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 to catalyze the preparation of α-arbutin. The method of using the sucrose phosphorylase is as follows: sucrose, hydroquinone and VC are added into the crude enzyme solution of sucrose phosphorylase, 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 increased 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) Improve 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 HPLC spectrum of the standard hydroquinone, with a peak time of 23 minutes.

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

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

[0034] Below in conjunction with the embodiment of the present invention and accompanying drawing, the technical scheme in the embodiment of the present invention is clearly and completely described. Obviously, the embodiment described is only a part of embodiment of the present invention, rather than all embodiments. Based on the embodiment in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work premise belong to the scope of protection of the present invention. The instruments and reagents used in the present embodiment are 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: 0.3 mL / min.

[0039] Column temperature: 45℃.

[0040] Injection volume: 10 μL.

[0041] Running time: 28 minutes.

[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] Based on the complete genome of Leuconostoc mesenteroides, the sucrose phosphorylase gene from Leuconostoc mesenteroides was synthesized using whole-genome synthesis. Primer design was performed using PrimerPremier (http: / / primer3.ut.ee / ) and OPTIMIZER (http: / / genomes.urv.es / OPTIMIZER / ), ensuring that the annealing temperature (Tm) difference was within 3°C and the primer length was within 60 bases. The resulting primers were dissolved in double-distilled water and added to the following reaction system, achieving a final concentration of 30 nM for each primer and 0.6 μM for the first and last primers.

[0046] 2mM dNTP mix (2mM each dNTP) 5μL 10×Pfu buffer 5μL Pfu DNA polymerase (10U / μL) 0.5μL <![CDATA[ddH2O]]> Make the total volume of the reaction system to 50 μL

[0047] The prepared PCR reaction system was placed in a Biori XP cycler gene amplification instrument and amplified 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 gel-purified and cloned into the NdeI / XhoI sites of pET30a using homologous recombination. A single clone was selected for sequencing. The successfully sequenced DNA sequence is SEQ ID NO: 1 and is designated WT1.

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

[0050] Based on the complete genome of Streptococcus mutans UA159, the sucrose phosphorylase gene from Streptococcus mutans UA159 was synthesized using whole-genome synthesis. Primer Premier (http: / / primer3.ut.ee / ) and Optimizer (http: / / genomes.urv.es / Optimizer / ) were used for design, ensuring that the annealing temperature (Tm) difference was within 3°C and the primer length was within 60 bases. The resulting primers were dissolved in double-distilled water and added to the following reaction system, achieving a final concentration of 30 nM for each primer and 0.6 μM for the first and last primers.

[0051] 2mM dNTP mix (2mM each dNTP) 5μL 10×Pfu buffer 5μL Pfu DNA polymerase (10U / μL) 0.5μL <![CDATA[ddH2O]]> Make the total volume of the reaction system to 50 μL

[0052] The prepared PCR reaction system was placed in a Biori XP cycler gene amplification instrument and amplified 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 gel-purified and cloned into the NdeI / XhoI sites of pET30a using homologous recombination. A single clone was selected for sequencing. The successfully sequenced DNA sequence is SEQ ID NO: 2 and is designated 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. The goal is to obtain mutants with enhanced catalytic activity, exhibiting greater catalytic activity than the 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 of enzymes encoding the enzyme, mutagenesis of polynucleotides, DNA shuffling, error-prone PCR, and directed evolution.

[0056] Through whole-genome 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, ensuring that the annealing temperature (Tm) difference was within 3°C and the primer length was within 60 bases. The resulting primers were dissolved in double-distilled water and added to the following reaction system, achieving a final concentration of 30 nM for each primer and 0.6 μM for the first and last primers.

[0057] 2mM dNTP mix (2mM each dNTP) 5μL 10×Pfu buffer 5μL Pfu DNA polymerase (10U / μL) 0.5μL <![CDATA[ddH2O]]> Make the total volume of the reaction system to 50 μL

[0058] The prepared PCR reaction system was placed in a Biori XP cycler gene amplification instrument and amplified 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-purified and cloned into the NdeI / XhoI sites of pET30a using homologous recombination. A single clone was selected for sequencing. The mutant sequence that was successfully sequenced was named MUT11, with the DNA sequence as SEQ ID NO: 4 and the corresponding amino acid sequence as SEQ ID NO: 3, which contains the double mutations C72R and E436D.

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

[0061] A single colony of E. coli harboring the expression vector was inoculated into 10 mL of autoclaved culture medium containing 10 g / L tryptone, 5 g / L yeast extract, 3.55 g / L sodium hydrogen phosphate (NaHPO), 3.4 g / L potassium dihydrogen phosphate (KHPO), 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, and 0.8 g / L glucose. Kanamycin was added to the culture medium to a concentration of 50 mg / L. The culture was incubated overnight at 30°C, 250 rpm. The next day, a 1L Erlenmeyer flask was inoculated with 100mL of autoclaved culture medium at a 1:100 ratio: 10g / L tryptone, 5g / L yeast extract, 3.55g / L sodium hydrogen phosphate (DAP), 3.4g / L potassium dihydrogen phosphate (KDP), 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 kanamycin supplemented to 50mg / L. Culture the cells at 30°C until the OD value reached 5-6. Immediately, the flask was placed in a shaker at 25°C, 250 rpm, and incubated for 1 hour. IPTG was added to a final concentration of 0.1mM and incubated for an additional 16 hours at 25°C, 250 rpm. After completion of the incubation period, the culture was centrifuged at 12,000g for 20 minutes at 4°C to collect the wet cells. The bacterial pellet was then washed twice with distilled water, and the cells were collected and stored at -70°C. A small amount of cells was taken for SDS-PAGE analysis.

[0062] Fed-batch fermentation was conducted in a computer-controlled 7-L pressure-resistant stainless steel bioreactor (Jiangsu B. Braun) with a working volume of 4 L. The culture medium used was a modified TB medium composed of 24 g / L yeast extract, 12 g / L peptone, 0.4% glucose, 2.31 g / L dihydrogen phosphate, and 12.54 g / L dipotassium phosphate, pH 7.0. Approximately 200 mL of primary seed culture in a shake flask was transferred to the fermentor when the OD reached 2.0. During the initial fermentation, the temperature was maintained at 37°C. Dissolved oxygen concentration was automatically controlled at 30% by agitation speed (rpm) and aeration cascade control, while the pH of the culture medium was maintained at 6.7-7.1 using 50% (v / v) orthophosphoric acid and 30% (v / v) ammonia. Feeding was initiated when a significant increase in dissolved oxygen occurred. The feed solution contained 9% w / v peptone, 9% w / v yeast extract, and 14% w / v glycerol. When OD600 reached approximately 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 phosphorylases from Streptococcus mutans UA159 and Leuconostoc mesenteroides are the most mature sucrose phosphorylases in industrial applications and the two most extensively studied sucrose phosphorylases in α-arbutin biocatalysis. Therefore, they were used as control proteins in the simultaneous reactions and designated WT1 and WT2, respectively. Prepare a 100 mL reaction system: 25 mM PB7.0, a final concentration of 30 g / L hydroquinone, 300 g / L sucrose, 5.3 g / L VC, and 60 g / L crude WT1 enzyme solution. Adjust the pH to 7.0 and bring the volume to 100 mL. Start the reaction and start the timer. Maintain a water bath temperature of 35°C and protect from light throughout the reaction.

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

[0066] The results showed that MUT11's catalytic efficiency was significantly higher than that of the two wild-type proteins at both 3 and 18 hours, reaching its peak at 18 hours. Its production decreased significantly after 24 hours, presumably due to oxidative consumption of α-arbutin after peak production. However, the wild-type proteins WT1 and WT2 continued to catalyze after 24 hours and still did not reach their peak, indicating that their catalytic efficiency was significantly lower than that of MUT11. Therefore, using MUT11 for production can significantly shorten reaction times or reduce enzyme usage, resulting in higher economic efficiency.

[0067]

[0068]

[0069] Example 6 Catalytic reaction of the amplified system

[0070] Prepare a 1000 mL reaction system: 25 mM PB7.0, final concentration 30 g / L hydroquinone, 300 g / L sucrose, 3 g / L VC, 50 g / L MUT11 crude enzyme solution, adjust the pH to 7.0, and adjust the volume to 1000 mL. Start the reaction and start the timer. Control the water bath temperature to 35°C and react overnight. Take a 22-hour reaction sample for HPLC analysis.

[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 of 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 the pH to 7.0, and adjust the volume to 100 mL. Start the reaction and start the timer. Control the water bath temperature to 35°C and react overnight. Take a 48-hour reaction sample for HPLC analysis.

[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 / L VC, 55 g / L MUT11 crude enzyme solution, adjust the pH to 7.0, and adjust the volume to 100 mL. Start the reaction and start the timer. Control the water bath temperature to 35°C, react overnight, and take a 48-hour reaction sample for HPLC analysis.

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

[0076] Example 8 Effect of Adding Glucoamylase 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 to 7.0, dilute to 100mL, and react at 35℃ on a shaker in the dark. After 19 hours of reaction, 50g / L saccharifying enzyme was added, and the reaction continued for 3 hours. Samples were taken for HPLC analysis. The results showed that the concentration of α-arbutin decreased from 37.4g / L to 24.77g / L, indicating that the product decreased significantly after saccharifying enzyme treatment after the reaction. The results show that the addition of saccharifying enzyme to the α-arbutin process of the sucrose process is not necessary, which is inconsistent with some previous literature reports.

[0078] While 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 these embodiments without departing from the principles and spirit of the invention, and that the scope of the 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 C72R and E436D. A polynucleotide encoding the sucrose phosphorylase according to claim 1 .

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

4.

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

5. Use of the sucrose phosphorylase with enhanced catalytic activity according to claim 4 in the 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.

Images