Application of beta-glucosidase in preparation of salicylic acid by degrading glucoside salicylate

Abstract

The invention discloses application of beta-glucosidase to preparation of salicylic acid by degrading glucoside salicylate, and relates to the technical field of biology. The amino acid sequence of the beta-glucosidase is as shown in SEQ ID NO. 2. Research finds that the beta-glucosidase can rapidly and efficiently degrade glucoside salicylate and produce salicylic acid, and can meet industrial production requirements. In addition, salicylic acid is efficiently prepared through a biological conversion method, the production efficiency is greatly improved, the environmental pollution risk in the chemical synthesis process is reduced, meanwhile, the production cost is further reduced through high conversion rate and stability, and remarkable economic value and social benefits are shown. The beta-glucosidase provided by the invention has outstanding technical advantages in the aspects of improving the production efficiency of salicylic acid, reducing the production cost and reducing environmental pollution, and has wide application prospects and market potential.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to application of beta-glucosidase in degrading salicylic acid glucoside to prepare salicylic acid. Background Art

[0002] β-glucosidase (EC3.2.1.21), also known as amygdalase, gentiobiose, or cellobiase, hydrolyzes β-O-glycosidic bonds on the α-carbon atom of the glucose moiety at the non-reducing end of glycoside molecules or carbohydrates. This enzyme is widely distributed in microorganisms, plants, mammals, and humans, and has important applications in food, medicine, agriculture, and the environment. Compared to β-glucosidases from other species, various β-glucosidases from microorganisms (such as bacteria, fungi, and yeast) exhibit a wider range of biochemical properties, substrate specificity, and stability.

[0003] Based on their amino acid sequence similarity and conserved structural features, β-glucosidases are classified into glycoside hydrolase (GH) families 1, 3, 5, 9, 30, and 116, with the GH1 family being the most numerous. β-glucosidases catalyze the cleavage of β-glucosidic bonds between aromatic or aliphatic groups and sugar moieties. Common natural substrates for hydrolysis include salicin, geniposide, esculin, 4-methylumbelliferyl-β-D-glucopyranoside (4-MUG), and cellobiose. Synthetic substrates include p-nitrophenyl-β-D-glucopyranoside (pNPG) and flavonol-β-D-glucoside.

[0004] Salicylic acid (SA), as an important plant hormone, plays a key role in plant immune response, growth and development, and environmental stress response. However, the content of SA in plants is usually low, and it mainly exists in the form of derivatives, among which salicylate 2-O-β-D-glucoside (SAG) is an important storage and transport form of SA. Currently, there are few known β-glucosidases that can hydrolyze SAG, mainly including Os4BGlu12 and Os4BGlu13 from rice, and FtSAGH1 from buckwheat. However, the catalytic activity of these enzymes is generally low, which makes it difficult to meet the demand for efficient SAG hydrolysis in practical applications. In addition, due to the limited resources of β-glucosidases with SAG hydrolysis function in nature, the development of new and efficient β-glucosidases has become a technical problem that needs to be solved urgently. Summary of the Invention

[0005] The present invention aims to provide a method for preparing salicylic acid by degrading salicylic acid glucoside using a β-glucosidase, thereby solving the problems of the prior art. The present invention finds that the β-glucosidase can improve the hydrolysis efficiency of salicylic acid glucoside, thereby significantly improving the production efficiency of salicylic acid.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides an application of a beta-glucosidase in degrading salicylic acid glucoside to prepare salicylic acid. The amino acid sequence of the beta-glucosidase is shown in SEQ ID NO.2.

[0008] Furthermore, the β-glucosidase is prepared using an Escherichia coli recombinant expression system.

[0009] Furthermore, the nucleotide sequence of the gene encoding the β-glucosidase is shown in SEQ ID NO.6.

[0010] The present invention also provides a method for preparing salicylic acid, comprising the steps of utilizing β-glucosidase to catalyze the degradation of salicylic acid glucoside to generate salicylic acid;

[0011] The amino acid sequence of the β-glucosidase is shown in SEQ ID NO.2.

[0012] Furthermore, the β-glucosidase is prepared using an Escherichia coli recombinant expression system; and / or

[0013] The nucleotide sequence of the gene encoding the β-glucosidase is shown in SEQ ID NO.6.

[0014] Furthermore, the pH range of the catalytic degradation of salicylic acid glucoside by the β-glucosidase is 3.5-5.5, and the temperature range is 30-70°C.

[0015] Preferably, the optimum pH for catalytic degradation of salicylic acid glucoside by the β-glucosidase is 4.0, and the optimum temperature is 60°C.

[0016] The present invention also provides an application of an enzyme preparation product in degrading salicylic acid glucoside to prepare salicylic acid, wherein the active ingredient of the enzyme preparation product includes β-glucosidase;

[0017] The amino acid sequence of the β-glucosidase is shown in SEQ ID NO.2.

[0018] Furthermore, the enzyme preparation product also includes auxiliary materials for enzyme preparation.

[0019] The present invention also provides a use of a biomaterial in preparing an enzyme preparation product for degrading salicylic acid glucoside to prepare salicylic acid, wherein the biomaterial is any one of the following (1) to (3):

[0020] (1) The gene encoding β-glucosidase;

[0021] (2) a recombinant expression vector comprising the encoding gene;

[0022] (3) a recombinant strain comprising the recombinant expression vector;

[0023] The amino acid sequence of the β-glucosidase is shown in SEQ ID NO.2.

[0024] Furthermore, the nucleotide sequence of the encoding gene is shown as SEQ ID NO.6.

[0025] The present invention discloses the following technical effects:

[0026] The present invention discloses the use of a β-glucosidase derived from Globitermes brachycerastes in improving the hydrolysis efficiency of salicylate 2-O-β-D-glucoside (SAG). The present invention uses Escherichia coli as a recombinant expression system and performs codon optimization on the target gene to ensure efficient expression of the target protein in the host cell. Experimental results show that under the reaction conditions of sodium acetate buffer at pH 4.0 and 60°C, the enzyme exhibits extremely high conversion efficiency when using salicylate 2-O-β-D-glucoside as a substrate. When the substrate concentration is 20.9 g / L, the yield of salicylic acid can reach 9.04 g / L, and the molar conversion rate is as high as 93.88%. This result shows that the enzyme can significantly improve the production efficiency of salicylic acid and meet the needs of industrial production. In addition, the present invention efficiently prepares salicylic acid through a biotransformation method, greatly improving production efficiency and reducing the risk of environmental pollution during chemical synthesis. At the same time, the high conversion rate and stability further reduce production costs, showing significant economic value and social benefits.

[0027] The β-glucosidase provided by the present invention has outstanding technical advantages in improving salicylic acid production efficiency, reducing production costs and reducing environmental pollution, and has broad application prospects and market potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0029] Figure 1 The figure is the SDS-PAGE gel electrophoresis of the supernatant and precipitate after the E. coli recombinant bacteria expressing four different β-glucosidases were disrupted;

[0030] Figure 2 The results are from HPLC analysis of salicylic acid glucoside and salicylic acid standards;

[0031] Figure 3 The results are the test results of salicylic acid glucoside and salicylic acid content after salicylic acid glucoside was degraded by different β-glucosidases;

[0032] Figure 4 The results show the detection results of salicylic acid glucoside and salicylic acid contents after the degradation of salicylic acid glucoside by GbBgl17 crude enzyme solution under different pH conditions.

[0033] Figure 5The results show the detection results of salicylic acid glucoside and salicylic acid content after the degradation of salicylic acid glucoside by GbBgl17 crude enzyme solution at different temperatures.

[0034] Figure 6 This is the yield diagram of salicylic acid produced by GbBgl17 crude enzyme solution under the optimal reaction conditions. DETAILED DESCRIPTION

[0035] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0036] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0037] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0038] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0039] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0040] The sequence information involved in the present invention is as follows:

[0041] SEQ ID NO.1:

[0042] MSKITFPKDFIWGSATAAYQIEGAYNEDGKGESIWDRFSHTPGNIADGHTGDVACDHYHRYEEDIKIMKEIGIKSYRFSISWPRIFPEGTGKLNQKGLDFYKRLTNLLLENGIMPAITLYHWDLPQKLQDKGGWKNRDTTDYFTEYSEVIFKNLGDIVPIWFTHNEPGVVSLLGHFLGIHAPGIKDLRTSLEVSHNLLLSHGKAVKLFREMNIDAQIGIALNLSYHYPASEKAEDIEAAELSFSLAGRWYLDPVLKGRYPENALKLYKKKGIELSFPEDDLKLISQPIDFIAFNNYSSEFIKYDPSSESGFSPANSILEKFEKTDMGWIIYPEGLYDLLMLLDRDYGKPNIVISENGAAFKDEIGSNGKIEDTKRIQYLKDYLTQAHRAIQDGVNLKAYYLWSLLDNFEWAYGYNKRFGIVHVNFDTLERKIKDSGYWYKEVIKNNGF.

[0043] SEQ ID NO.2:

[0044] MEKLVFPKDFVWGTATASYQVEGAAREGGRGECIWDVFARKPGAVNAGENGDVACDQYHRYEEDVALMAELGFNSYRFSIAWPRIIPTGSGKVNPEGVAYYRKLCDELHKHNIKACATLYHWDLPQVLEEKGGWADRGVLDAFEEYVKVCYETLGDVIDTWITINEPFCVAYLGYLWGVHAPGQRDLNKALAAVHHINMAHGIAVREYRKTKLKAPIGITYNPATPRPATSSAADARAADISRAFNTEVFMFPALGKGYPELVTKDLNLSFPVQSGDMQLIAQPIDFIGVNYYTEHAVAADEKAQFKTTNKPSWETTTAMGWPIVPGGLERQLLWINEVSNGIPIYITENGCAYDDVVAPDGRVHDKERIKYLHQHLAVCADVIKKGVPLKGYFVWSLMDNFEWAFGYGRRFGIIHIDFKTQKRTVKDSAYFLRDTIAGYGEL.

[0045] SEQ ID NO.3:

[0046] MANFPKGFLFGTATSSYQIEGAVNEDGRTPSIWDTFSKTSGMTYNGDTGDIACDHYHRYKEDVVILKEIGVKAYRFSIAWPRIFPEKGNFNPKGIDFYKRLVEELLKNDIIPVATIYHWDLPQWAGDLGGWLNRDLIYWYSEYSQKLFKEIGNVVPMWITHNEPWCASILSYGIGEHAPGHKDYREALIAAHHILLSHGEAVKIFRDMNIKESQIGITLNLTPAYPASERDVDRLAAQYADGFSNRWFLDPIFKGNYPEDMIELYKEEIGKFDFIKSEDLGIISQPIDFLGINFYSRSIVKYSEKSMLKWIGVEGPGAKTDMGWEIRPESLYDLLKRLDKEYTRIPIYITENGAAFKDIITEDGKVHDQERIEYIKEHLKYANKFIKEGGNLKGYFLWSFLDNFEWAFGYSKRFGIVYVDYKTQKRILKDSALWYKEVINRASIVF.

[0047] SEQ ID NO.4:

[0048] MAKIIFPEDFIWGAATSSYQIEGAFNEDGKGESIWDRFSHTPGKIENGDTGDIACDHYHLYREDIELMKEIGIRSYRFSTSWPRILPEGKGRVNQKGLDFYKRLVDNLLKANIRPMITLYHWDLPQALQDKGGWTNRDTAKYFAEYARLMFEEFNGLVDLWVTHNEPWVVAFEGHAFGNHAPGTKDFKTALQVAHHLLLSHGMAVDIFREEDLPGEIGITLNLTPAYPAGDSEKDVKAASLLDDYINAWFLSPVFKGSYPEELHHIYEQNLGAFTTQPGDMDIISRDIDFLGINYYSRMVVRHKPGDNLFNAEVVKMEDRPSTEMGWEIYPQGLYDILVRVNKEYTDKPLYITENGAAFDDKLTEEGKIHDEKRINYLGDHFKQAYKALKDGVPLRGYYVWSLMDNFEWAYGYSKRFGLIYVDYENGNRRFLKDSALWYREVIEKGQVEAN.

[0049] SEQ ID NO.5:

[0050]

[0051] SEQ ID NO.6:

[0052]

[0053] SEQ ID NO.7:

[0054]

[0055] SEQ ID NO.8:

[0056]

[0057] The recombinant Escherichia coli BL21 (DE3) expressing β-glucosidase used in the present invention was derived from the Coli Genetic Stock Center (CGSC, https: / / cgsc.biology.yale.edu / ).

[0058] The expression plasmid vector pET28a involved in the present invention was purchased from BioVector NTCC (http: / / www.biovector.net / ).

[0059] The salicylic acid glucoside standard used in the present invention was purchased from Shanghai Meryer Biochemical Technology Co., Ltd. (https: / / www.meryer.com / ab_intro.html), and the salicylic acid standard was purchased from Tianjin Alta Technology Co., Ltd. (https: / / www.altascientific.cn / ).

[0060] The primers and heterologous genes used in the present invention were synthesized by Beijing Qingke Biotechnology Co., Ltd. (https: / / www.tsingke.com.cn / ).

[0061] Other biochemical reagents used in the present invention were purchased from Sangon Biotechnology (Shanghai) Co., Ltd. (http: / / www.sangon.com / ).

[0062] Example 1 Construction, culture, induced expression and extraction of recombinant Escherichia coli expressing β-glucosidase

[0063] (1) The amino acid sequences of β-glucosidase from Clostridium thermocellum, Globitermes brachycerastes, Thermoanaerobacterium aotearoense, and Halothermothrix orenii were obtained from the NCBI database. The GenBank accession numbers were X60268.1, JN903693.1, KP772230.1, and KX808501, respectively. The amino acid sequences are shown in SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, and SEQ ID NO. 4. The genes encoding the amino acid sequences shown in SEQ ID NO. 1-4 were optimized according to the codon preference of Escherichia coli. The nucleotide sequences of the optimized encoding genes are shown in SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, and SEQ ID NO. 8, respectively. The optimized coding genes were synthesized by Beijing Qingke Biotechnology Co., Ltd. and cloned into the pET28a expression vector to obtain recombinant plasmids pET28a-CtBglA, pET28a-GbBgl17, pET28a-TaBGL and pET28a-HoB8CYA8 expressing β-glucosidase from different sources.

[0064] (2) The above recombinant plasmids were transformed into Escherichia coli BL21 (DE3) competent cells to obtain recombinant Escherichia coli bacteria heterologously expressing β-glucosidase from different sources.

[0065] (3) Pick a single colony of the recombinant bacteria and inoculate it into a 5 mL LB tube containing 50 μg / mL kanamycin resistance and culture it overnight for 12 h. Then transfer it to 50 mL LB medium at a 2% inoculum volume and culture it at 37°C until the OD 600 The concentration of IPTG was 0.6, and the inducer IPTG was added to a final concentration of 0.2 mM. The culture was continued at 18 °C for 16 h, and the cells were centrifuged at 8000 rpm and 4 °C for 10 min to collect the bacteria for later use.

[0066] (4) The cells collected in step (3) were resuspended in a pH 5.0, 50 mM sodium acetate buffer, centrifuged at 8000 rpm and 4°C for 10 min, the supernatant discarded, and the cells were resuspended in a pH 5.0, 50 mM sodium acetate buffer and ultrasonically disrupted to obtain a crude enzyme solution.

[0067] The SDS-PAGE gel electrophoresis of the supernatant and precipitate of the recombinant E. coli expressing four different β-glucosidases is shown in the figure. Figure 1 shown.

[0068] Example 2 Crude enzyme catalytic screening of β-glucosidase with the best hydrolysis activity

[0069] The same concentration of salicylic acid glucoside was added to the crude enzyme solution prepared in Example 1, and the reaction was catalytically reacted at 37°C for 30 min. The reaction was then terminated by boiling in water for 2 min. The solution was centrifuged at 12000 rpm for 2 min, and the supernatant was filtered through a 0.45 μm filter membrane. The contents of salicylic acid glucoside and salicylic acid were detected by high performance liquid chromatography (HPLC).

[0070] The HPLC detection method of salicylic acid glucoside and salicylic acid is as follows:

[0071] High performance liquid chromatography system: Agilent 1220;

[0072] Chromatographic column: Diamonsil Plus 5 μm C18A, 250 × 4.6 mm, DiKMA;

[0073] Gradient elution: 0 min, A:B = 98:2 (volume ratio); 2 min, A:B = 98:2; 5 min, A:B = 90:10; 12 min, A:B = 60:40; 15 min, A:B = 50:50; 20 min, A:B = 98:2. Solvent A was 0.02 mol / L ammonium formate aqueous solution (pH = 3.0), and solvent B was 100% acetonitrile.

[0074] Flow rate: 1 mL / min; column temperature: 40°C; detection wavelength: 300 nm.

[0075] Among them, the test results of standard salicylic acid glucoside and salicylic acid are as follows Figure 2 As shown, the peak time of salicylic acid glucoside is 10.7 min, and the peak time of salicylic acid is 14.0 min.

[0076] After using different crude enzyme solutions to catalyze the degradation of salicylic acid glucoside, the concentrations of salicylic acid glucoside and salicylic acid were calculated based on the peak area values ​​and the standard curve. The results are as follows: Figure 3 The results showed that when other conditions were the same, the hydrolysis activity of β-glucosidase GbBgl17 from Globitermes brachycerastes was the best.

[0077] Example 3 Optimization of catalytic conditions of crude β-glucosidase GbBgl17

[0078] (1) Effect of reaction pH on the activity of β-glucosidase GbBgl17

[0079] The same concentration of substrate salicylic acid glucoside was added to the crude GbBgl17 enzyme solution prepared in Example 1, and the reaction pH was set to pH = 3.5, pH = 4.0, pH = 4.5, pH = 5.0, and pH = 5.5, respectively. The reaction was catalyzed at 37°C for 30 min, and then boiled in boiling water for 2 min to terminate the reaction. The mixture was centrifuged at 12000 rpm for 2 min, and the supernatant was filtered with a 0.45 μm filter membrane. The content of salicylic acid glucoside and salicylic acid was detected by HPLC.

[0080] The concentrations of salicylic acid glucoside and salicylic acid were calculated based on the peak area values ​​and the standard curve. The test results are as follows: Figure 4 The results showed that the optimal reaction pH for β-glucosidase GbBgl17 was 4.0.

[0081] (2) Effect of reaction temperature on the activity of β-glucosidase GbBgl17

[0082] The same concentration of substrate salicylic acid glucoside was added to the crude GbBgl17 enzyme solution prepared in Example 1, and the reaction temperatures were set to 30°C, 40°C, 50°C, 60°C, and 70°C, respectively. The reaction was catalyzed at pH 5.0 for 30 min, and then boiled in boiling water for 2 min to terminate the reaction. The solution was centrifuged at 12,000 rpm for 2 min, and the supernatant was filtered through a 0.45 μm filter membrane. The contents of salicylic acid glucoside and salicylic acid were detected by HPLC.

[0083] The concentrations of salicylic acid glucoside and salicylic acid were calculated based on the peak area values ​​and the standard curve. The test results are as follows: Figure 5 The results showed that the optimal reaction temperature of β-glucosidase GbBgl17 was 60℃.

[0084] Example 4 β-glucosidase GbBgl17 crude enzyme catalyzes salicylic acid glucoside to produce salicylic acid

[0085] The recombinant E. coli expressing β-glucosidase GbBgl17 collected from fermentation in Example 1 was resuspended in a pH 4.0, 50 mM sodium acetate buffer solution so that the final OD 600 The mixture was ultrasonically crushed to a final concentration of 20.9 g / L of substrate salicylic acid glucoside. The reaction was catalyzed at 60°C for 30 min, then boiled in water for 2 min to terminate the reaction. The mixture was centrifuged at 12000 rpm for 2 min, and the supernatant was filtered with a 0.45 μm filter membrane. HPLC analysis showed a salicylic acid yield of 9.04 g / L and a molar conversion of 93.88%. Figure 6 shown.

[0086] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A use of β-glucosidase in the degradation of salicylic acid glucoside to prepare salicylic acid, characterized in that: The amino acid sequence of the β-glucosidase is shown in SEQ ID NO.

2.

2. The use according to claim 1, characterized in that The beta-glucosidase is prepared by utilizing an Escherichia coli recombinant expression system.

3. The use according to claim 2, characterized in that The nucleotide sequence of the gene encoding the β-glucosidase is shown in SEQ ID NO.

6.

4. A method for preparing salicylic acid, characterized in that: The method comprises the steps of utilizing β-glucosidase to catalyze the degradation of salicylic acid glucoside to generate salicylic acid; The amino acid sequence of the β-glucosidase is shown in SEQ ID NO.

2.

5. The preparation method according to claim 4, characterized in that The β-glucosidase is prepared using an Escherichia coli recombinant expression system; and / or The nucleotide sequence of the gene encoding the β-glucosidase is shown in SEQ ID NO.

6.

6. The preparation method according to claim 5, characterized in that The pH range of the beta-glucosidase catalyzing the degradation of salicylic acid glucoside is 3.5-5.5, and the temperature range is 30-70°C.

7. An application of an enzyme preparation product in the degradation of salicylic acid glucoside to prepare salicylic acid, characterized in that: The active ingredient of the enzyme preparation product includes β-glucosidase; The amino acid sequence of the β-glucosidase is shown in SEQ ID NO.

2.

8. The use according to claim 7, characterized in that The enzyme preparation product also includes enzyme preparation auxiliary materials.

9. Use of a biomaterial in the preparation of an enzyme preparation product for degrading salicylic acid glucoside to prepare salicylic acid, characterized in that: The biomaterial is any one of the following (1)-(3): (1) The gene encoding β-glucosidase; (2) a recombinant expression vector comprising the encoding gene; (3) a recombinant strain comprising the recombinant expression vector; The amino acid sequence of the β-glucosidase is shown in SEQ ID NO.

2.

10. The use according to claim 9, characterized in that The nucleotide sequence of the coding gene is shown in SEQ ID NO.6.

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