Use of beta-glucosidase Lf18920 in the specific hydrolysis of crocin-1 into crocetin monosaccharide derivatives

Abstract

The application discloses application of beta-glucosidase Lf18920 in specifically hydrolyzing crocin-1 into crocin-4 and crocin-5 which are safflor acid monosaccharide derivatives, and the beta-glucosidase Lf18920 is encoded by IT072_18920 gene of leifsonia sp. ZF2019 and is abbreviated as Lf18920 gene. The hydrolysis reaction catalyzed by the enzyme to obtain the safflor acid monosaccharide derivatives crocin-4 and crocin-5 can directly use crocin-1 as a substrate, crocin-1 has good water solubility and is a main crocin compound in plants, and the raw material is abundant. The reaction operation is simple and easy to realize industrial production. The application has important industrial application potential in the preparation of crocin-4 and crocin-5.

Description

Technical Field

[0001] This invention relates to the field of enzyme genetic engineering technology, specifically to the application of β-glucosidase Lf18920 in the specific hydrolysis of crocin-1 to obtain crocin-4 and crocin-5, which are crocin monosaccharide derivatives. Background Technology

[0002] Crocin (also known as saffron glycoside) is the main functional component of saffron, a precious medicinal herb, and is mainly found in plants such as saffron and gardenia fruit. Crocin has many effects, including treating cardiovascular diseases and preventing atherosclerosis, treating central nervous system diseases, anti-tumor, anti-inflammatory, anti-diabetic, hepatoprotective and choleretic effects. Crocin also plays a certain role in the prevention and treatment of Alzheimer's disease, Parkinson's disease, cardiovascular diseases, and cancer (Han Yu, Xie Guoyong, Li Ran, Huang Litao, Qin Minjian. Research progress on the pharmacological activity of crocin [J]. Modern Drugs and Clinical, 2017, 32(09):1806-1814.).

[0003] Crocin is a series of ester compounds formed by the combination of crocinic acid and glucose or gentiobiose. Based on the number and type of glycosidic bonds, crocin can be classified into: crocin-1, crocin-2, crocin-3, crocin-4, crocin-5, and crocinic acid without glycosidic bonds. In saffron and gardenia, crocin mainly exists in the form of crocin-1. However, drug metabolism studies have shown that the presence of gentiobioses at both ends affects the absorption of crocin-1 in vivo. After oral administration of crocin-1 to mice, crocetin compounds in the blood are mainly present as monosaccharide crocin-4 and crocin-5 or saffron acid, which is aglycone. It is speculated that these compounds are the main components that exert its biological functions in vivo (Akira A. et al. Orally Administered Crocetin and Crocins Are Absorbed into Blood Plasma as Crocetin and Its Glucuronide Conjugates in Mice J. Agric. Food Chem. 2005, 53, 18, 7302–7306). Recent studies have shown that crocin acid monosaccharide derivatives have better neuroprotective, antioxidant and anticancer activities (Zhu AL et al. Characterization of Crocetin-Monoglucuronide as a Neuro-Protective Metabolite of Crocin-1.[J]. Molecular nutrition & foodresearch, 2019.).

[0004] Crocin exists primarily in plants as crocin-1, while the monosaccharide glycosides crocin-4 and crocin-5 are difficult to extract from plants through purification. Their scarcity, high cost, and the large amount of organic chemical reagents required for chemical extraction, coupled with the time-consuming and labor-intensive process of separation and purification, severely limit the drug development and clinical application of crocin. Currently, research on the selective synthesis of crocin is limited. Fangyu Ding's team has pioneered the use of a microbial glycosyltransferase to convert crocinic acid into crocin-4 and crocin-5 (Ding Fangyu, Liu Feng, Shao Wenming, Chu Jianlin, Wu Bin, He Bingfang. Efficient synthesis of crocins from crocetin by a microbial glycosyltransferase from Bacillus subtilis 168.[J].Journal of agricultural and food chemistry,2018,66(44):11701–11708). He Bingfang et al. disclosed a microbial glucosyltransferase that can directionally synthesize crocin monoglucose ester (crocin-5) (He Bingfang, Ding Fangyu, Liu Feng, Shao Wenming, Wang Guangji, Ajiye, Tan Chaoyi, Chu Jianlin, Wu Bin. A glucosyltransferase and its application in the synthesis of crocinic acid glucose ester [P]. China: CN106906192B,2020-10-30.). However, the synthesis of crocinic acid monosaccharide derivatives using glucosyltransferase requires the hydrolysis of crocin-1 into crocinic acid, with uridine diphosphate glucose as the glycosylation donor. If the reaction is to proceed continuously, it needs to be regenerated and coupled with the uridine diphosphate glucose system. At the same time, crocinic acid has poor water solubility, which increases the difficulty of large-scale industrial production using this enzyme.

[0005] β-glucosidase (EC 3.2.1.21) can be used to hydrolyze β-glucosidic bonds at non-reducing ends, generating β-glucosides and corresponding ligands. β-glucosidase can directly hydrolyze water-soluble crocin-1, but most known β-glucosidases directly hydrolyze crocin-1 to crocinic acid, making it difficult to obtain crocinic acid monosaccharide derivatives by controlling the enzymatic hydrolysis conditions. Summary of the Invention

[0006] The purpose of this invention is to provide the application of β-glucosidase Lf18920 in the specific hydrolysis of crocin-1 to obtain crocin monosaccharide derivatives crocin-4 and crocin-5, in order to overcome the shortcomings of the prior art.

[0007] The present invention adopts the following technical solution:

[0008] The application of β-glucosidase Lf18920 in the specific hydrolysis of crocin-1 to obtain crocin-4 and crocin-5 monosaccharide derivatives, wherein the β-glucosidase Lf18920 is derived from... leifsonia sp. ZF2019 IT072_ 18920 Gene abbreviation Lf18920 Encoded by genes.

[0009] Furthermore, the aforementioned Lf18920 The gene nucleotide sequence is shown in SEQ ID NO:1, with a full length of 2700 bp; the amino acid sequence of the β-glucosidase Lf18920 it encodes is shown in SEQ ID NO:2.

[0010] Furthermore, the aforementioned Lf18920 Genes are obtained using the following methods: leifsonia sp. Using the whole genome of ZF2019 as a template, and with 18920F and 18920R as primers, the genome was amplified by PCR to obtain the desired genome. Lf18920 Gene; wherein the nucleotide sequence of 18920F is shown in SEQ ID NO:3 and the nucleotide sequence of 18920R is shown in SEQ ID NO:4.

[0011] Furthermore, the recombinant expression of the β-glucosidase Lf18920 was performed via PCR amplification. Lf18920 The gene was used to construct an expression plasmid using pET-28a-HMT as a vector, and expression was induced in Escherichia coli BL21(DE3). The expressed protein was then purified using a Ni-NTA column.

[0012] Furthermore, β-glucosidase Lf18920 specifically hydrolyzes crocin-1 to obtain crocin-4 and crocin-5, which includes the following steps: hydrolyzing crocin-1 and β-glucosidase Lf18920 in a citrate-disodium hydrogen phosphate buffer solution with a pH of 5.5-6.5 at a reaction temperature of 25-40℃ for a reaction time of 120-150 min, and terminating the reaction in a hot water bath after the hydrolysis is completed.

[0013] Furthermore, the addition ratio of crocin-1 to β-glucosidase Lf18920 was 1-2 mg / U.

[0014] Furthermore, the pH of the citrate-disodium hydrogen phosphate buffer solution is 6.0.

[0015] Furthermore, the reaction temperature is 40°C.

[0016] Furthermore, the reaction time is 120 minutes.

[0017] Furthermore, the reaction is terminated in a hot water bath at 80-100°C after the hydrolysis reaction is completed.

[0018] The beneficial effects of this invention are:

[0019] Crocin is an important functional component of the medicinal herbs saffron and gardenia, mainly existing in the form of crocin-1. However, it can only be absorbed by organisms and exert its biological functions after being converted into crocinic acid or crocinic acid monosaccharide derivatives (crocin-4 and crocin-5). The low water solubility of crocinic acid affects its absorption, while crocin-4 and crocin-5 are readily absorbed by organisms. Recent studies have shown that crocinic acid monosaccharide derivatives have better neuroprotective, antioxidant, and anticancer activities. However, currently, β-glucosidase can generally only directly hydrolyze crocin-1 to crocinic acid. Directly converting crocin-1 into crocinic acid monosaccharide derivatives via β-glucosidase remains a technical challenge.

[0020] This invention discloses a β-glucosidase Lf18920 that can specifically hydrolyze crocin-1 to obtain crocin-4 and crocin-5 as the main products. The hydrolysis reaction catalyzed by this enzyme to obtain the crocin-4 and crocin-5 monosaccharide derivatives can directly use crocin-1 as a substrate. Crocin-1 has good water solubility and is the main crocin compound in plants, making it an abundant raw material. The reaction operation is simple and easy to implement for industrial production. This invention has significant industrial application potential in the preparation of crocin-4 and crocin-5.

[0021] The β-glucosidase Lf18920 of this invention, under the conditions of pH 5.5-6.5 and reaction time of 120 min, yields a product with a total content of over 70% of crocin-4 and crocin-5. Under the conditions of pH 6.0 and reaction time of 120 min, the product with a total content of crocin-4 and crocin-5 can reach 89.7%. This product can be directly used as a crocin product, is easily absorbed by the body, and better exerts its biological functions. Attached Figure Description

[0022] Figure 1 Comparison of HPLC chromatograms of the products of hydrolysis of crocin-1 by β-glucosidase Bgl19165, β-glucosidase Bgl1973, and β-glucosidase Lf18920.

[0023] Figure 2 The gene encoding β-glucosidase Lf18920 Lf18920 The PCR amplification agarose gel electrophoresis image is shown. Where: M represents the DNA marker; 1 represents the target gene.

[0024] Figure 3 This is a polyacrylamide gel electrophoresis (SDS-PAGE) image of β-glucosidase Lf18920. Where: M is the protein marker; 1 is the target protein.

[0025] Figure 4 To study the optimal temperature and pH for β-glucosidase Lf18920.

[0026] Figure 5 The HPLC chromatograms of five crocin standards are compared with the HPLC chromatograms of the hydrolysate of crocin-1 by β-glucosidase Lf18920. In the comparison, A represents the standards: crocin-1, crocin-2, crocin-3, crocin-4, and crocin-5; B represents the hydrolysate of crocin-1 by β-glucosidase Lf18920.

[0027] Figure 6 Here are schematic diagrams of the structures of five crocin and crocinic acid.

[0028] Figure 7 The yield of crocin-4 varies over time under different pH conditions.

[0029] Figure 8 The yield of crocin-5 varies over time under different pH conditions. Detailed Implementation

[0030] The present invention will be further explained below with reference to embodiments and accompanying drawings. The following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0031] Example 1: Screening of β-glucosidase Lf18920

[0032] leifsonia sp. ZF2019 was isolated from the intestine of the Gardenia blue butterfly larvae in the fruit of Gardenia jasminoides by our research group. It has been preserved at the China Industrial Microbial Culture Collection Center, with the culture accession number CICC 25134. leifsonia sp The complete genome sequence of ZF2019 (NCBI: GCA_019924635.1) IT072_19730 , IT072_18920 , IT072_ 19165The encoded enzymes (abbreviated as β-glucosidase Bgl19730, β-glucosidase Lf18920, and β-glucosidase Bgl19165, respectively) are annotated as GH3 family β-glucosidases. The hydrolytic activities of the three enzymes on crocin-1 were preliminarily determined according to the method in Example 4. It was found that β-glucosidase Bgl1973 directly hydrolyzed crocin-1 to crocin acid, β-glucosidase Bgl19165 could not hydrolyze crocin, while β-glucosidase Lf18920 could only partially hydrolyze crocin-1 to obtain crocin-4 and crocin-5 (…). Figure 1 Therefore, β-glucosidase Lf18920 was used to continue subsequent experiments.

[0033] Example 2: Recombinant expression and purification of β-glucosidase Lf18920

[0034] extract leifsonia sp. ZF2019 whole genome, with leifsonia sp. Using the ZF2019 whole genome as a template, primers 18920F and 18920R were designed.

[0035] 18920F (SEQ ID N0: 3): 5'-TTTTTT CATATG ACGTCGACCCTTCCCGA-3' (underlined part) Nde I) Enzyme cleavage site

[0036] 18920R (SEQ ID N0: 4): 5'-TTTT GAATTC AGTGCACCTCGAACGG-3' (underlined part) Eco RⅠ restriction site)

[0037] PCR amplification Lf18920 Genes, results as Figure 2 As shown. The correctly sized bands were purified and recovered using a DNA gel purification and recovery kit. The recovered product was then treated with restriction endonucleases. Nde I and Eco RI double digestion. The target gene fragment was ligated with the double-digested expression vector pET-28a-HMT at 16℃ for 12-16 h to obtain the recombinant expression vector pET-28a-HMT-Lf18920.

[0038] The constructed recombinant expression vector pET-28a-HMT-Lf18920 was introduced into BL21(DE3) strain, and expression was induced using isopropyl-β-D-thiogalactoside (0.4 mmol / L). Cells were collected, and lysates were obtained by sonication. The lysates were purified using a pre-equilibrated nickel affinity chromatography (Ni-NTA) column to obtain the target protein tagged with HMT. The HMT tag was removed by TEV enzyme digestion at 4℃ for 12 h. The protein was then dialyzed overnight with buffer (20 mM Tris-HCl, 0.5 mM EDTA, 1 mM DTT, 500 mM NaCl) and loaded onto a Ni-NTA column. The TEV enzyme, HMT tag, and undigested protein, all carrying a 6×His tag, bound to the Ni-NTA column. The target protein, having had its HMT tag removed by enzyme digestion, did not bind to the Ni-NTA column. The fraction that permeated through the Ni-NTA column was the target protein, β-glucosidase Lf18920. The results of its polyacrylamide gel electrophoresis (SDS-PAGE) are shown below. Figure 3 As shown. The obtained target protein can be directly used for subsequent experiments, or it can be stored at -80℃ with 10-20 v / v% glycerol for later use, or freeze-dried to prepare a dry powder and stored at -20℃ for later use.

[0039] Example 3: Study on the optimal temperature and pH of β-glucosidase Lf18920

[0040] Temperature and pH have a significant impact on enzyme activity. This example studies the optimal temperature and pH for β-glucosidase Lf18920, laying the foundation for subsequent experimental research on its hydrolysis of crocin-1.

[0041] 3.1 Test Methods

[0042] The optimal temperature and pH of β-glucosidase Lf18920 were determined using p-nitrophenyl-β-D-galactopyranoside (pNPG) as a substrate.

[0043] Take 130 μL of citrate-disodium hydrogen phosphate buffer solution at different pH (4.0~8.0), add 10 μL of enzyme solution (containing 0.13 U β-glucosidase; the activity unit U is the activity unit measured with pNPG as the substrate, and the enzyme solution is prepared with citrate-disodium hydrogen phosphate buffer solution of the corresponding pH), and 20 μL of 20 mol / L pNPG (pNPG is prepared with citrate-disodium hydrogen phosphate buffer solution of the corresponding pH). Incubate at 40℃ for 10 min, and then add 50 μL of 2 mol / L Na2CO3 solution to terminate the enzyme reaction. Add 200 μL of the reaction solution to an ELISA plate, and measure the absorbance at 405 nm. Determine the optimal pH by taking the point of highest enzyme activity as 100%.

[0044] Under optimal pH conditions, the enzyme activity of β-glucosidase Lf18920 was measured by placing the above enzyme reaction system at 25–50 °C (5 °C intervals). The optimal temperature was determined by taking the highest enzyme activity as 100%.

[0045] 3.2 Results and Analysis

[0046] like Figure 4 As shown, the optimal temperature for β-glucosidase Lf18920 is 40 °C, and the optimal pH is 5.0.

[0047] Example 4: Qualitative analysis of the hydrolysis products of crocin-1 by β-glucosidase Lf18920

[0048] 4.1 Test Methods

[0049] A 20 μg / mL solution of crocin-1 was prepared using a citrate-disodium hydrogen phosphate buffer solution at pH 5.0. 0.13 U of β-glucosidase Lf18920 was hydrolyzed with 10 mL of the 20 μg / mL crocin-1 solution at 40 °C. The reaction was terminated by a water bath at 80 °C after 1 h. Qualitative analysis of the products was performed using high performance liquid chromatography (HPLC). Column temperature: 35℃; injection volume: 10 μL; chromatographic conditions: UV 440 nm; mobile phase: (A) 0.01 mol / L acetate-ammonium acetate buffer (pH 4.0), (B) acetonitrile; flow rate: 1 mL / min; elution program: 0~1 min 20% B, 1~8 min 20%~60% B, 8~8.1 min 60%~70% B, 8.1~13 min 70%~98% B, 13~15 min 98%~20% B, 15~20 min 20% B.

[0050] 4.2 Results and Analysis

[0051] According to HPLC detection ( Figure 5 The comparison with the standard showed that the hydrolysis products of β-glucosidase Lf18920 on crocin-1 were mainly crocin-4 and crocin-5. The structures of the five crocins and crocin acid are as follows: Figure 6 As shown.

[0052] Example 5: Optimization of conditions for hydrolyzing crocin-1 with β-glucosidase Lf18920 to obtain crocin-4 and crocin-5

[0053] 5.1 Test Methods

[0054] Prepare 20 μg / mL solutions of crocin-1 using 10 mL of citrate-disodium hydrogen phosphate buffer solutions at pH 5.0, 5.5, 6.0, 6.5, and 7.0, respectively. Add 0.13 U of β-glucosidase Lf18920 to each solution and react in a 40℃ water bath. Take 1 mL of the reaction solution at 30 min, 60 min, 120 min, and 150 min, respectively, and immediately terminate the reaction by placing it in a 100℃ water bath. Calculate the yields of crocin-4 and crocin-5 based on the increase in characteristic peak area of ​​crocin-4 and crocin-5 using HPLC, and calculate the conversion rate of crocin-1 based on the decrease in characteristic peak area of ​​crocin-1 using HPLC.

[0055] 5.2 Results and Analysis

[0056] At pH 7.0, crocin-4 and crocin-5 were not detected, indicating that β-glucosidase Lf18920 mainly hydrolyzes crocin-1 to yield crocin-4 and crocin-5 under weakly acidic conditions. Figure 7 , Figure 8 (No Crocin-5 was observed to be produced at pH 5.0 and pH 5.5). As shown in Tables 1 and 2, the total yield of crocin-4 and crocin-5 obtained by hydrolyzing crocin-1 with β-glucosidase Lf18920 at pH 6.0 was the highest, and the individual yields of crocin-4 and crocin-5 were also the highest. The optimal reaction time was 120 min.

[0057] Table 1. Conversion rate of crocin-1 under different pH conditions for 120 min

[0058]

[0059] Table 2. Crocin-4 and Crocin-5 under different pH conditions for 120 min

[0060]

Claims

1. The application of β-glucosidase Lf18920 in the specific hydrolysis of crocin-1 to obtain crocin-4 and crocin-5, characterized in that, The β-glucosidase Lf18920 is composed of Lf18920 Encoded by the gene; the Lf18920 The full-length nucleotide sequence of the gene is shown in SEQ ID NO:1, with a length of 2700 bp; the amino acid sequence of the β-glucosidase Lf18920 it encodes is shown in SEQ ID NO:

2.

2. The application according to claim 1, characterized in that, The Lf18920 The coding sequence of a gene is obtained using the following method: Lf18920 Using the gene nucleotide sequence as a template, and with 18920F and 18920R as primers, the gene was amplified by PCR to obtain the desired result. Lf18920 The coding sequence of the gene; wherein, the 18920F nucleotide sequence is shown in SEQ ID NO:3, and the 18920R nucleotide sequence is shown in SEQ ID NO:

4.

3. The application according to claim 1, characterized in that, The recombinant expression of β-glucosidase Lf18920 was performed via PCR amplification. Lf18920 The gene was used to construct an expression plasmid using pET-28a-HMT as a vector, and expression was induced in Escherichia coli BL21(DE3). The expressed protein was then purified using a Ni-NTA column.

4. The application according to claim 1, characterized in that, The specific hydrolysis of crocin-1 by β-glucosidase Lf18920 yields crocin-4 and crocin-5, which includes the following steps: crocin-1 and β-glucosidase Lf18920 are hydrolyzed in a citrate-disodium hydrogen phosphate buffer solution with a pH of 5.5-6.5 at a temperature of 25-40℃ for 120-150 min. The reaction is terminated in a hot water bath after the hydrolysis is completed.

5. The application according to claim 4, characterized in that, The addition ratio of crocin-1 to β-glucosidase Lf18920 was 1-2 mg / U.

6. The application according to claim 4, characterized in that, The pH of the citric acid-disodium hydrogen phosphate buffer solution is 6.

0.

7. The application according to claim 4, characterized in that, The reaction temperature is 40℃.

8. The application according to claim 4, characterized in that, The reaction time is 120 min.

9. The application according to claim 4, characterized in that, The reaction was terminated in a hot water bath at 80-100℃ after the hydrolysis reaction was completed.

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