A sucrose phosphorylase with enhanced heat resistance and its application in the preparation of α-arbutin by high-temperature catalysis

Abstract

The present invention relates to the field of enzyme catalysis technology, in particular to a sucrose phosphorylase with enhanced heat resistance and its application in the preparation of α-arbutin by high-temperature catalysis. Its amino acid sequence is shown in SEQ ID NO: 3, and it has stronger heat resistance compared with the wild-type sucrose phosphorylase. The sucrose phosphorylase with enhanced heat resistance of the present invention realizes industrial biocatalysis at high temperature, can improve the catalytic efficiency, reduce the risk of microbial contamination, and at the same time helps to reduce the substrate viscosity and increase the transfer rate, which is of great significance for shortening the reaction time and improving the production efficiency.

Description

Technical Field

[0001] The present invention relates to the technical field of enzyme catalysis, and specifically relates to a sucrose phosphorylase with enhanced heat resistance and its application in the preparation of α-arbutin by high-temperature catalysis. Background Art

[0002] α-Arbutin is an α-glucoside composed of hydroquinone (HQ) and glucose. α-Arbutin is widely recognized for its significant whitening properties. By effectively inhibiting the tyrosinase activity of melanocytes, it blocks the synthesis of melanin. α-Arbutin is a recognized new type of highly efficient freckle-removing and whitening agent with no irritation, no allergy, and strong compatibility. In addition to its whitening effect, α-arbutin also has high antioxidant, anti-microbial, and anti-inflammatory activities and is widely used in the pharmaceutical and cosmetic industries. The global demand for α-arbutin has been increasing year by year, but whether α-arbutin can compete with β-arbutin depends entirely on the cost of its commercial production.

[0003] Currently, α-arbutin can be produced by two methods: chemical synthesis and biotransformation. Chemical synthesis requires protection and deprotection of hydroxyl groups, with harsh reaction conditions and a complex process. Biotransformation is considered a potential candidate method because it has a simpler synthesis process, mild reaction conditions, and less pollution, including enzyme conversion, microbial fermentation, and whole-cell biocatalysis. Using hydroquinone as the acceptor substrate, its phenolic hydroxyl group can form an α-glycosidic bond with the glycosyl moiety of different donor substrates.

[0004] Liu et al. expressed a heterologous sucrose phosphorylase isolated from Streptococcus mutans in Bacillus subtilis, knocked out four genes (sacB, sacC, levB, and sacA), dynamically regulated the expression of PfkA, and strengthened the pgi and pgcA genes, thereby increasing the sucrose conversion rate and the synthesis rate of α-arbutin. Finally, a product concentration of 120 g / L and a sucrose conversion rate of 60.4% (mol / mol) were achieved.

[0005] Deng et al. used Xanthomonas campestris BT-112 for biotransformation reactions (α-arbutin synthesis), using sucrose as the glycosyl donor and HQ as the acceptor. To reduce the impact of HQ on X. maltophilia BT-112 to achieve a high concentration of α-arbutin, the effects of different continuous feeding strategies on biomass and α-arbutin content were studied, including continuous feeding at a constant feeding rate (CRFB), continuous feeding at a constant HQ concentration (CHFB), exponential continuous feeding (EFB), and DO-controlled pulsed continuous feeding (DPFB).

[0006] A sucrose amylase DpAMY bacterial cell was obtained from the Deinococcus puniceus strain. Sucrose was 30%, hydroquinone was 3% (3 g / 100 mL), the wet bacterial cells of sucrose amylase were 20 g / L, and ammonia water was used to adjust the pH of the reaction system to 7.3. The reaction was carried out at 35 °C for 8 h. The content of α-arbutin obtained was 73.2 g / L.

[0007] CN106148256A introduced the amylosucrase gene derived from Xanthomonas campestris CGMCC 1.3408 into Escherichia coli. When VC and Triton X-100 were present, 180 mM of hydroquinone could be converted to obtain 45.36 g / L of α-arbutin, and the production intensity was 7.56 g / L / h. It did not use the relatively expensive maltose as a donor and did not require enzyme purification. However, the exact dosage of the catalyst was not given, and the raw material cost could not be effectively evaluated.

[0008] Currently, there are still some challenges in the biological method, including low substrate concentration, low conversion rate, and excessive dosage of bacterial cells (enzyme solution), resulting in a relatively high production cost of α-arbutin. Therefore, the present invention aims to find a sucrose phosphorylase mutant with significantly enhanced stability and activity at high temperatures and establish a method for producing α-arbutin with a high conversion rate and rapid reaction. Summary of the Invention

[0009] The purpose of the present invention is to provide a sucrose phosphorylase with enhanced heat resistance and its application in the catalytic preparation of α-arbutin at high temperatures.

[0010] To achieve the above purpose, the present invention provides the following technical solutions:

[0011] A sucrose phosphorylase with enhanced heat resistance, whose amino acid sequence is as shown in SEQ ID NO: 3.

[0012] The sucrose phosphorylase described in the present invention has stronger heat resistance compared with the wild-type sucrose phosphorylase, and the amino acid sequence of the wild-type sucrose phosphorylase is as shown in SEQ ID NO: 1 or 2.

[0013] The present invention also provides a polynucleotide that can encode the above sucrose phosphorylase.

[0014] Furthermore, the polynucleotide sequence is as shown in SEQ ID NO: 4.

[0015] The sucrose phosphorylase with enhanced heat resistance in the present invention can be used for the catalytic preparation of α-arbutin at high temperatures. Its usage method is as follows: Take sucrose, hydroquinone, and VC, add them to the crude enzyme solution of sucrose phosphorylase, and react at pH 7.0 and 35 °C - 50 °C.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0017] (1) Improving the catalytic rate and reducing the reaction time: The reaction of thermostable sucrose phosphorylase at high temperatures can achieve significantly higher catalytic rates, which is of great significance for shortening the reaction time and improving production efficiency.

[0018] (2) Reducing the risk of microbial contamination: The use of thermostable sucrose phosphorylase allows industrial processes to be carried out at higher temperatures, thus minimizing the risk of microbial contamination, especially in reactions where sucrose is present, which is more likely to breed microorganisms.

[0019] (3) Prolonging the catalyst life: Sucrose phosphorylase with improved heat resistance has better stability at high temperatures, meaning they can be used for a longer time in industrial catalysis, thereby reducing the frequency of catalyst replacement.

[0020] (4) Improving the product purity: The stability and activity of thermostable sucrose phosphorylase at high temperatures can reduce the generation of by-products and improve the purity of the final product.

[0021] (5) Adapting to harsh environments: Thermostable sucrose phosphorylase can maintain its activity under extreme conditions (such as high temperature, high salt, high acidity or alkalinity, etc.) and is suitable for various industrial applications.

[0022] (6) The heat resistance of the sucrose phosphorylase of the present invention is enhanced, enabling industrial biocatalysis at high temperatures, which can improve the catalytic efficiency, reduce the risk of microbial contamination, and at the same time contribute to reducing the substrate viscosity and increasing the transfer rate, bringing multiple benefits such as increasing solubility, reducing costs and promoting green manufacturing in the reaction operation. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 It is the HPLC chromatogram of the hydroquinone standard, and the peak emergence time is 23 min.

[0024] Figure 2 It is the HPLC chromatogram of the α - arbutin standard, and the peak emergence time is 17 min.

[0025] Figure 3 It is the chromatogram of WT2 in Example 5 after reacting at 35 °C for 3 hours.

[0026] Figure 4 It is the chromatogram of WT2 in Example 5 after reacting at 50 °C for 3 hours, with a large amount of substrate remaining.

[0027] Figure 5 It is the chromatogram of MUT2 in Example 5 after reacting at 35 °C for 3 hours.

[0028] Figure 6The spectrum of MUT2 after reacting at 50 °C for 3 hours in Example 5.

[0029] Figure 7 The spectrum of MUT2 after reacting at 50 °C for 18 hours in Example 6, and the substrate has been nearly completely converted. Detailed implementation manners

[0030] The technical solutions in the embodiments 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. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention. The instruments and reagents used in this embodiment are all commercially available products unless otherwise specified.

[0031] The HPLC detection conditions for the following experiments are as follows:

[0032] Chromatographic column: Shim-pack GIS 4.6x250mm 5um C18 (or equivalent C18 column).

[0033] Detection wavelength: 280 nm.

[0034] Flow rate: 0.3 mL / min.

[0035] Column temperature: 45 °C.

[0036] Injection volume: 10 μL.

[0037] Running time: 28 min.

[0038] Mobile phase: 0.1% formic acid aqueous solution: methanol = 90:10.

[0039] Elution mode: isocratic elution.

[0040] Obtaining the wild-type sucrose phosphorylase gene sequence in Example 1

[0041] According to the whole genome information of Leuconostoc mesenteroides, the whole gene synthesis method was used to synthesize the whole gene of the sucrose phosphorylase gene derived from Leuconostoc mesenteroides. Primer Premier (http: / / primer3.ut.ee / ) and OPTIMIZER (http: / / genomes.urv.es / OPTIMIZER / ) were used for design, and it was ensured that the difference in annealing temperature (Tm) was controlled within 3 °C, the primer length was controlled within 60 bases. After dissolving the obtained primers in double-distilled water, they were added to the following reaction system so that the final concentration of each primer was 30 nM, and the final concentration of the head and tail primers was 0.6 μM.

[0042] 2 mM dNTP mix (2 mM each dNTP) 5 μL 10×Pfu buffer 5 μL Pfu DNA polymerase (10 U / μL) 0.5 μL <![CDATA[ddH2O]]> Make the total volume of the reaction system up to 50 μL

[0043] The prepared PCR reaction system was placed in a Biorad XP cycler gene amplifier and amplified according to the following program: 98 °C for 30 s, 55 °C for 45 s, 72 °C for 120 s, 35 cycles.

[0044] The DNA fragment obtained by PCR was subjected to gel purification and cloned into the NdeI / XhoI site of pET30a by homologous recombination. Single colonies were picked for sequencing. The successfully sequenced DNA sequence was SEQ ID NO: 1, named WT1.

[0045] Example 2 Obtaining the wild-type sucrose phosphorylase gene sequence

[0046] According to the whole genome information of Streptococcus mutans UA159, the whole gene synthesis method was used to synthesize the whole gene of the sucrose phosphorylase gene derived from Streptococcus mutans UA159. Primer Premier (http: / / primer3.ut.ee / ) and OPTIMIZER (http: / / genomes.urv.es / OPTIMIZER / ) were used for design, and it was ensured that the difference in annealing temperature (Tm) was controlled within 3 °C, the primer length was controlled within 60 bases. After dissolving the obtained primers in double-distilled water, they were added to the following reaction system so that the final concentration of each primer was 30 nM, and the final concentration of the head and tail primers was 0.6 μM.

[0047] 2 mM dNTP mix (2 mM each dNTP) 5 μL 10×Pfu buffer 5 μL Pfu DNA polymerase (10 U / μL) 0.5 μL <![CDATA[ddH2O]]> Make the total volume of the reaction system up to 50 μL

[0048] The prepared PCR reaction system was placed in a Biorad XP cycler gene amplifier and amplified according to the following program: 98 °C for 30 s, 55 °C for 45 s, 72 °C for 120 s, 35 cycles.

[0049] The DNA fragment obtained by PCR was subjected to gel cutting and purification, and cloned into the NdeI / XhoI site of pET30a by homologous recombination. Single colonies were picked for sequencing. The successfully sequenced DNA sequence was SEQ ID NO: 2, named WT2.

[0050] Example 3 Mutagenesis of wild-type sucrose phosphorylase

[0051] Sucrose phosphorylase mutants, which are derived from the wild-type sucrose phosphorylase of Example 2, aim to obtain mutants with enhanced heat resistance and exhibit stronger heat resistance compared to 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 the polynucleotide encoding the enzyme, polynucleotide mutagenesis, DNA shuffling, error-prone PCR, and directed evolution methods, etc.

[0052] By means of gene synthesis, the secondary structure of the gene and codon preference were adjusted to achieve high expression in Escherichia coli. Designed using Primer Premier (http: / / primer3.ut.ee / ) and OPTIMIZER (http: / / genomes.urv.es / OPTIMIZER / ), and ensuring that the difference in annealing temperature (Tm) was controlled within 3°C and the primer length was controlled within 60 bases. After dissolving the obtained primers in double-distilled water, they were added to the following reaction system so that the final concentration of each primer was 30 nM and the final concentration of the head and tail primers was 0.6 μM.

[0053] 2 mM dNTP mix (2 mM each dNTP) 5 μL 10×Pfu buffer 5 μL Pfu DNA polymerase (10 U / μL) 0.5 μL <![CDATA[ddH2O]]> Make the total volume of the reaction system up to 50 μL

[0054] The prepared PCR reaction system was placed in a Bori XP cycler gene amplifier and amplified according to the following program: 98°C for 30 s, 55°C for 45 s, 72°C for 120 s, 35 cycles.

[0055] The DNA fragment obtained by PCR was subjected to gel cutting and purification, and cloned into the NdeI / XhoI site of pET30a by homologous recombination. Single colonies were picked for sequencing. The two successfully sequenced mutant sequences were named MUT1 and MUT2 respectively. The DNA sequence of MUT2 was SEQ ID NO: 4, and its corresponding amino acid sequence was SEQ ID NO: 3, which contained the double mutations G48V and S268G.

[0056] Example 4 Shake flask expression test and fed-batch fermentation

[0057] Pick a single colony of Escherichia coli containing the expression vector and inoculate it 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 add kanamycin to 50 mg / L. Incubate overnight at 30 °C with shaking at 250 rpm. The next day, take a 1 L Erlenmeyer flask and inoculate it into 100 mL of autoclaved medium at an inoculation ratio of 1:100: 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, 0.3 g / L glucose, and add kanamycin to 50 mg / L. Incubate at 30 °C until the cell OD reaches 5 - 6, then immediately place the Erlenmeyer flask in a shaker at 25 °C and shake at 250 rpm for 1 hour. Add IPTG to a final concentration of 0.1 mM and continue to incubate at 25 °C with shaking at 250 rpm for 16 hours. After the incubation, centrifuge the culture broth at 4 °C and 12,000 g for 20 minutes to collect the wet cells. Then wash the cell pellet twice with distilled water, collect the cells, and store them at -70 °C. At the same time, take a small amount of cells for SDS-PAGE detection.

[0058] Fed-batch fermentation was carried out in a computer-controlled 7 L pressure-resistant stainless steel bioreactor (Jiangsu Bailang), with a working volume of 4 L. The medium used was a modified TB medium, and the specific components were 24 g / L yeast extract, 12 g / L peptone, 0.4% glucose, 2.31 g / L phosphodiesterase, and 12.54 g / L dipotassium hydrogen phosphate, pH 7.0. Approximately 200 mL of shake flask primary seed culture was inoculated into the fermenter when the OD reached 2.0. During the initial fermentation process, the temperature was maintained at 37 °C. During the fermentation process, the dissolved oxygen concentration was automatically controlled at 30% by the cascade control of the stirring rate (rpm) and the ventilation supply, and the pH value of the medium was maintained at 6.7 - 7.1 by 50% (v / v) orthophosphoric acid and 30% (v / v) ammonia water. When a significant increase in dissolved oxygen occurred, feeding began. The feeding solution contained 9% w / v peptone, 9% w / v yeast extract, and 14% w / v glycerol. When the OD600 was approximately 15.0, the temperature was lowered to 25 °C, and IPTG was added at a final concentration of 0.2 mM to induce expression.

[0059] Example 5 Comparison of catalytic efficiency at different temperatures

[0060] Prepare a 100 mL reaction system: 25 mM PB 7.0, hydroquinone with a final concentration of 30 g / L, sucrose 300 g / L, VC 5.3 g / L, 60 g / L of crude WT1 enzyme solution, adjust the pH to 7.0, make up the volume to 100 mL, start the reaction and record the time, control the water bath temperature at 35 °C and operate in the dark throughout the process. The same reaction system is carried out at 50 °C.

[0061] Similarly, prepare reaction systems for crude WT2, MUT1, and MUT2 enzyme solutions, and carry out reactions at 35 °C and 50 °C respectively. Take samples for HPLC detection of the production amount of α-arbutin after 3 hours.

[0062] As Figures 3 - 6 shown, the results show that only MUT2 can efficiently catalyze the formation of α-arbutin from hydroquinone at high temperature. A large amount of products can be formed after 3 hours of reaction, and the reaction rate at this time exceeds that at 35 °C; after the MUT1 mutation, the activity loss is large, and the enzyme activities at 35 °C and 50 °C are both lower than before the mutation, which is a non-functional mutation. The wild-type WT1 and WT2 have a fast reaction rate at 35 °C, but are strongly inhibited at 50 °C, probably due to the rapid inactivation of the protein caused by intolerance to high temperature. Overall, WT2 is slightly better than WT1 at low temperature. From the results, it can be seen that the catalytic rate of MUT2 at 50 °C far exceeds that of other proteins, and also exceeds the reaction rate of MUT2 at 35 °C.

[0063] 35℃ 50℃ WT1 24 g / L 16 g / L WT2 30 g / L 12 g / L MUT1 17 g / L 19 g / L MUT2 41 g / L 51 g / L

[0064] Example 6 High-temperature catalytic reaction of the enlarged system

[0065] Prepare a 1000 mL reaction system: 25 mM PB 7.0, hydroquinone with a final concentration of 30 g / L, sucrose 300 g / L, VC 3 g / L, 50 g / L of crude MUT2 enzyme solution, adjust the pH to 7.0, make up the volume to 1000 mL, start the reaction and record the time, control the water bath temperature at 50 °C, react overnight and take a reaction sample after 18 hours for HPLC detection.

[0066] Use the wild-type protein WT2 as a control, react the same reaction system at 50 °C overnight and take a reaction sample after 18 hours to detect the formation of α-arbutin.

[0067] As Figure 7 , the results show that for MUT2, the substrate is almost completely reacted after 18 hours of reaction, and the conversion rate is as high as 94.5%. The conversion rate of the control reaction WT2 is only 18.8%.

[0068] 50℃ MUT2 70 g / L WT2 14 g / L

[0069] Example 7 High-temperature catalytic reaction of the enlarged system

[0070] Prepare a 1000 mL reaction system: 25 mM PB 7.0, hydroquinone with a final concentration of 25 g / L, sucrose 250 g / L, VC 3 g / L, 40 g / L of crude MUT2 enzyme solution. Adjust the pH to 7.0, make up the volume to 1000 mL, start the reaction and record the time. Control the water bath temperature at 50 °C for overnight reaction and take a reaction sample at 22 hours for HPLC detection. The results show that the concentration of α-arbutin is 60 g / L, the conversion rate is 97%, and there is almost no residual hydroquinone after extending the reaction time.

[0071] Example 8 Effect of reducing sucrose concentration on the reaction

[0072] The reaction system is the same as that in Example 7. The concentrations of sucrose are reduced to 100 g / L, 150 g / L, and 200 g / L respectively. Control the water bath temperature at 50 °C for overnight reaction and take a reaction sample at 22 hours for HPLC detection. The results show that the concentration of α-arbutin is below 45 g / L in all cases, and a large amount of substrate hydroquinone remains, indicating that sucrose must be added in excess to effectively reduce substrate residue.

[0073] Example 9 Effect of increasing sucrose concentration on the reaction

[0074] The reaction system is the same as that in Example 7. The concentrations of sucrose are increased to 400 g / L and 500 g / L respectively, with other conditions unchanged. Control the water bath temperature at 50 °C for overnight reaction and take a reaction sample at 22 hours for HPLC detection. The results show that the concentration of α-arbutin is about 60 g / L in both cases, and the small amount of remaining hydroquinone no longer decreases, indicating that increasing the sucrose concentration further cannot improve the conversion rate.

[0075] Although the embodiments of the present invention have been shown and described, those of ordinary skill in the art can understand that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A sucrose phosphorylase with enhanced heat resistance, characterized in that: The amino acid sequence is shown in SEQ ID NO: 3, which contains double mutations of G48V and S268G. 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 heat-resistant sucrose phosphorylase according to claim 1 in the high-temperature catalytic preparation of α-arbutin.

5. Use of the heat-resistant sucrose phosphorylase according to claim 4 in the high-temperature 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℃~50℃.

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