Sucrose phosphorylase with enhanced heat resistance and application of sucrose phosphorylase in high-temperature catalytic preparation of alpha-arbutin

Abstract

The invention relates to the technical field of enzyme catalysis, in particular to sucrose phosphorylase with enhanced heat resistance and application of the sucrose phosphorylase in high-temperature catalytic preparation of alpha-arbutin, 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 heat resistance. According to the sucrose phosphorylase with enhanced heat resistance, industrial biological catalysis at high temperature is realized, the catalytic efficiency can be improved, the risk of microbial pollution is reduced, meanwhile, the viscosity of a substrate is reduced, the transfer speed is increased, and the sucrose phosphorylase has important significance in shortening the reaction time and improving the production efficiency.

Description

Technical Field

[0001] The invention relates to the technical field of enzyme catalysis, and specifically relates to a sucrose phosphorylase with enhanced heat resistance and application thereof in the preparation of alpha-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 remarkable whitening properties, which effectively inhibits the tyrosinase activity of melanocytes and blocks the synthesis of melanin. α-Arbutin is recognized as a new type of non-irritating, non-allergic, highly compatible and highly effective freckle whitening agent. In addition to its whitening effect, α-Arbutin also has high antioxidant, antimicrobial and anti-inflammatory activities and is widely used in the pharmaceutical and cosmetic industries. The global demand for α-Arbutin is 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 chemical synthesis and biotransformation. Chemical synthesis requires the protection and deprotection of hydroxyl groups, and the reaction conditions are harsh and the process is complicated. Biotransformation is considered a potential candidate method because it has a simpler synthetic process, mild reaction conditions and less pollution, including enzymatic transformation, microbial fermentation and whole-cell biocatalysis. Using hydroquinone as an acceptor substrate, its phenolic hydroxyl group can form an α-glycosidic bond with the glycosyl part of different donor substrates.

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

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

[0006] CN107828752A obtains a sucrose amylase DpAMY bacterial cell from Deinococcus puniceus strain, 30% sucrose, 3% hydroquinone (3g / 100mL), 20g / L sucrose amylase wet bacterial cell, and ammonia water to adjust the pH of the reaction system to 7.3, and the reaction is carried out at 35°C for 8h. The obtained α-arbutin content is 73.2g / L.

[0007] CN106148256A introduces the amylosucrase gene from Xanthomonas campestris CGMCC 1.3408 into Escherichia coli. In the presence of VC and TritonX-100, 180mM hydroquinone can be converted into 45.36g / L α-arbutin, with a production intensity of 7.56g / L / h. It does not use relatively expensive maltose as a donor, and does not require enzyme purification. However, the exact amount of catalyst used is not given, so it is impossible to effectively evaluate the cost of raw materials.

[0008] At present, biological methods still have some challenges, including low substrate concentration, low conversion rate, and excessive use of bacteria (enzyme solution), resulting in high production costs of α-arbutin. Therefore, the present invention aims to find a sucrose phosphorylase mutant with significantly enhanced stability and activity at high temperatures, and to establish a method for producing α-arbutin with high conversion rate and rapid reaction. Summary of the invention

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

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

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

[0012] The sucrose phosphorylase of the present invention has stronger heat resistance 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.

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

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

[0015] The heat-resistant sucrose phosphorylase of the present invention can be used for high-temperature catalytic preparation of α-arbutin. The method of using the sucrose phosphorylase is as follows: sucrose, hydroquinone and VC are added, and crude enzyme solution of the sucrose phosphorylase is added, and the reaction is carried out at pH 7.0 and 35°C to 50°C.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

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

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

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

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

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

[0022] (6) The heat resistance of the sucrose phosphorylase of the present invention is enhanced, and industrial biocatalysis at high temperature is realized, which can improve the catalytic efficiency and reduce the risk of microbial contamination. At the same time, it helps to reduce the viscosity of the substrate and increase the transfer rate, thereby increasing the solubility in the reaction operation, reducing the cost and promoting green manufacturing. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is the standard HPLC spectrum of hydroquinone, with a peak time of 23 minutes.

[0024] Figure 2 This is the HPLC spectrum of the standard product of α-arbutin, with a peak time of 17 minutes.

[0025] Figure 3 This is the spectrum of WT2 in Example 5 after reaction at 35°C for 3 hours.

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

[0027] Figure 5 This is the spectrum of MUT2 in Example 5 after reaction at 35°C for 3 hours.

[0028] Figure 6This is the spectrum of MUT2 in Example 5 after reaction at 50°C for 3 hours.

[0029] Figure 7 This is the spectrum of MUT2 in Example 6 after reaction at 50°C for 18 hours, and the substrate has been almost completely converted. DETAILED DESCRIPTION

[0030] 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.

[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: 280nm.

[0034] Flow rate: 0.3mL / min.

[0035] Column temperature: 45℃.

[0036] Injection volume: 10 μL.

[0037] Running time: 28min.

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

[0039] Elution mode: isocratic elution.

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

[0041] 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.

[0042] 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

[0043] 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.

[0044] 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.

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

[0046] 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.

[0047] 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

[0048] 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.

[0049] 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.

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

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

[0052] 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.

[0053] 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

[0054] 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.

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

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

[0057] 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.

[0058] 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.

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

[0060] 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 water bath temperature at 35℃ and keep away from light throughout the process. The same reaction system is carried out at 50℃.

[0061] Similarly, reaction systems of crude enzyme solutions of WT2, MUT1 and MUT2 were prepared and reacted at 35° C. and 50° C., respectively. Samples were taken at 3 hours for HPLC detection of the amount of α-arbutin produced.

[0062] like Figure 3-6 As shown, the results show that only MUT2 can efficiently catalyze hydroquinone to produce α-arbutin at high temperature, and a large amount of product can be generated after 3 hours of reaction, and the reaction rate exceeds 35°C at this time; MUT1 has a large loss of activity after mutation, and the enzyme activity at 35°C and 50°C is not as good as before the mutation, which is an invalid mutation. The wild-type WT1 and WT2 react very quickly at 35°C, but are strongly inhibited at 50°C, which may be due to the rapid inactivation of the protein caused by intolerance to high temperature. Overall, WT2 is slightly better than WT1 at low temperatures. From the results, it can be seen that the catalytic rate of MUT2 at 50°C is much higher than that of several other proteins, and also exceeds the reaction rate of MUT2 at 35°C.

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

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

[0065] Prepare 1000mL reaction system: 25mM PB7.0, final concentration 30g / L hydroquinone, 300g / L sucrose, 3g / L VC, 50g / L MUT2 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 50℃, react overnight and take 18-hour reaction sample for HPLC detection.

[0066] The wild-type protein WT2 was used as a control, and the same reaction system was reacted at 50°C overnight and the 18-hour reaction sample was taken to detect the production of α-arbutin.

[0067] like Figure 7 The results showed that the substrate of MUT2 was almost completely reacted after 18 hours of reaction, with a conversion rate of 94.5%, while the conversion rate of the control reaction WT2 was only 18.8%.

[0068] 50℃ MUT2 70g / L WT2 14g / L

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

[0070] Prepare 1000mL reaction system: 25mM PB7.0, final concentration 25g / L hydroquinone, 250g / L sucrose, 3g / L VC, 40g / L MUT2 crude enzyme solution, adjust pH to 7.0, dilute to 1000mL, start reaction and timing, control water bath temperature to 50℃, react overnight and take 22 hours reaction sample for HPLC detection. The results showed that the concentration of α-arbutin was 60g / L, the conversion rate was 97%, and there was almost no hydroquinone residue after extending the reaction time.

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

[0072] The reaction system was the same as in Example 7, except that the concentration of sucrose was reduced to 100 g / L, 150 g / L, and 200 g / L, respectively. The water bath temperature was controlled to 50°C for overnight reaction and the 22-hour reaction sample was taken for HPLC detection. The results showed that the concentration of α-arbutin was below 45 g / L, and a large amount of substrate hydroquinone remained, indicating that sucrose must be added in excess to effectively reduce the substrate residue.

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

[0074] The reaction system was the same as in Example 7, except that the concentration of sucrose was increased to 400 g / L and 500 g / L respectively, and other conditions remained unchanged. The water bath temperature was controlled to 50°C for overnight reaction and a 22-hour reaction sample was taken for HPLC detection. The results showed that the concentration of α-arbutin was about 60 g / L, and the small amount of residual hydroquinone was no longer reduced, indicating that increasing the sucrose concentration could not further improve the conversion rate.

[0075] 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 heat resistance, 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 heat resistance according to claim 1 in the preparation of α-arbutin by high temperature catalysis.

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°C to 50°C.

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