Oxalate decarboxylase for reducing oxalic acid content of tea drink and application of oxalate decarboxylase

Abstract

The invention relates to the technical field of biological enzymes, and discloses oxalate decarboxylase for reducing the oxalic acid content of tea drinks and application of the oxalate decarboxylase. The oxalate decarboxylase comprises an amino acid sequence as shown in SEQ ID NO. 1. The oxalate decarboxylase provided by the invention can keep higher catalytic activity in tea drinks, the oxalate decarboxylase contained in the tea drinks can be removed to a greater extent, and the oxalate decarboxylase can adapt to wider pH and temperature ranges of the tea drinks.

Description

Technical Field

[0001] The present invention relates to the technical field of biological enzymes, and in particular to an oxalate decarboxylase for reducing the oxalic acid content of tea drinks and an application thereof. Background Art

[0002] Oxalic acid, also known as oxalic acid, is an organic compound commonly found in plants. In particular, plant beverages such as tea have a high content of oxalic acid. Since the human body lacks an enzyme that can effectively decompose oxalic acid, excessive intake of oxalic acid may lead to the formation of calcium oxalate crystals, affecting mineral absorption, leading to calcium loss, affecting bone health and increasing the risk of kidney stones. Oxalic acid is also associated with symptoms such as oral discomfort, joint pain, arrhythmia and skin ulcers, and may be associated with other diseases such as hyperoxaluria, cardiac conduction disorders, Crohn's disease, etc. By removing oxalic acid from tea beverages or other plant beverages, the incidence of kidney stones can be reduced, calcium absorption can be improved, and it helps maintain the overall health of the human body. Therefore, researching and developing effective methods for removing oxalic acid has vital practical significance for the production and consumption of plant beverages such as tea beverages in terms of improving their safety and nutritional value.

[0003] In vivo, the degradation of oxalate mainly depends on several specific enzymes, including oxalate decarboxylase (OxdC), oxalate oxidase and oxalyl-CoA decarboxylase. Among them, OxdC is an enzyme containing manganese ions that can catalyze the decomposition of oxalate into carbon dioxide and formate. It is widely present in plants, bacteria and fungi, such as Aspergillus niger, Bacillus subtilis, lactic acid bacteria, etc. Using OxdC to hydrolyze oxalic acid is expected to reduce the oxalic acid content in tea. However, catechins, flavonoids, and polyphenols such as tannic acid contained in tea have strong antioxidant and metal chelating abilities, and may interact with metal cofactors (such as manganese and iron) of oxalate decarboxylase, thereby inhibiting its enzyme activity. At the same time, caffeine, as a major alkaloid in tea, may also inhibit the activity of oxalate decarboxylase by competitively binding to the active site of the enzyme or changing the conformation of the enzyme. In addition, tea usually contains more metal ions, such as iron and calcium, which may reduce the availability of oxalic acid by binding to oxalic acid, thereby indirectly inhibiting the activity of oxalate decarboxylase. The above reasons can easily lead to a significant reduction in the activity of oxalate decarboxylase in tea, and fail to achieve the ideal effect of reducing the oxalic acid content in tea. Summary of the invention

[0004] In order to solve the above technical problem, that is, the activity of oxalate decarboxylase in tea is greatly reduced, and the effect of reducing the oxalic acid content of tea is not good, the present invention provides an oxalate decarboxylase for reducing the oxalic acid content of tea and its application. The oxalate decarboxylase provided by the present invention can maintain a high catalytic activity in tea, remove the oxalate decarboxylase contained in the tea to a large extent, and the oxalate decarboxylase can adapt to a wider pH and temperature range of tea.

[0005] The specific technical scheme of the present invention is: In a first aspect, the present invention provides an oxalate decarboxylase for reducing the oxalic acid content of tea beverages, wherein the oxalate decarboxylase comprises an amino acid sequence as shown in SEQ ID NO.1.

[0006] The polyphenols (such as catechins, flavonoids, tannic acid), caffeine and metal ions present in tea drinks can easily have an adverse effect on the activity of oxalate decarboxylase, resulting in poor effect of oxalate decarboxylase when used to reduce the oxalic acid content of tea drinks. However, the present invention team found that when using this specific oxalate decarboxylase with an amino acid sequence as shown in SEQ ID NO.1, it has good resistance to components in tea drinks that inhibit enzyme activity (such as polyphenols, caffeine and metal ions), can maintain a high catalytic activity in tea drinks, hydrolyze oxalic acid into carbon dioxide and formate, and more thoroughly remove the oxalic acid contained in tea drinks.

[0007] In addition, the activity of the oxalate decarboxylase used in the present invention is less restricted by the pH and temperature of tea drinks. It can effectively degrade oxalic acid in tea drinks within a wider pH and temperature range, which helps to adapt to tea drinks with different pH values ​​and temperatures at different stages in the tea production process. It has good application prospects in reducing the oxalic acid content in tea drinks, and is expected to reduce the harm of oxalic acid in tea drinks to the human body, which will promote the improvement of the health attributes of the food industry.

[0008] In a second aspect, the present invention provides an application of oxalate decarboxylase in reducing the oxalic acid content of tea beverages, wherein the oxalate decarboxylase comprises an amino acid sequence as shown in SEQ ID NO.1.

[0009] In a third aspect, the present invention provides a method for reducing the oxalic acid content of tea, comprising the following steps: adding oxalate decarboxylase comprising an amino acid sequence as shown in SEQ ID NO.1 to the tea, and performing an enzymatic reaction.

[0010] Preferably, the mass volume ratio of the oxalate decarboxylase to the tea is 0.05-1 mg:1 mL.

[0011] Preferably, the conditions of the enzymatic hydrolysis reaction are as follows: temperature of 45-65° C., stirring speed of 650-750 rpm, and time of 0.5-1 h.

[0012] Preferably, the pH value of the tea beverage is 4.0-6.0.

[0013] Preferably, the oxalate decarboxylase is obtained by chassis cell recombinant expression technology.

[0014] Preferably, the preparation step of the oxalate decarboxylase comprises: S1: integrating the nucleic acid sequence encoding the oxalate decarboxylase into a plasmid vector by homologous recombination to obtain a recombinant expression plasmid; S2: transferring the recombinant expression plasmid into chassis cells to obtain oxalate decarboxylase expressing cells; S3: After culturing the oxalate decarboxylase-expressing cells, the cells are disrupted and the product is purified to obtain the oxalate decarboxylase.

[0015] Preferably, the chassis cells are Escherichia coli.

[0016] Preferably, in step S1, the plasmid vector is pET28a.

[0017] Preferably, in step S3, the culturing process comprises: inoculating the oxalate decarboxylase expressing cells into an LB liquid culture medium containing kanamycin, culturing at 35-37°C for 12-13h, and then inoculating into an LB liquid culture medium containing kanamycin and α-lactose at an inoculum size of 1-3%, and culturing at 30-35°C for 20-24h.

[0018] Furthermore, the content of α-lactose in the LB liquid culture medium is 50-55 μg / mL.

[0019] Preferably, in step S1, a 6×his-tag is connected to the end of the nucleic acid sequence; in step S3, the process of purifying the product comprises: after centrifuging the cell lysate, subjecting the supernatant to nickel column affinity chromatography, washing impurities with a Tris-HCl buffer containing 150-160 mM NaCl and 40-50 mM imidazole, eluting the target protein with a Tris-HCl buffer containing 150-160 mM NaCl and 250-260 mM imidazole, collecting the eluate, and desalting and concentrating.

[0020] Compared with the prior art, the present invention has the following advantages: (1) The present invention uses a specific oxalate decarboxylase, which can maintain a high catalytic activity in tea and can reduce the oxalate decarboxylase content in tea to a large extent. According to tests, the oxalate decarboxylase can reduce the oxalic acid content in tea by more than 99%.

[0021] (2) The oxalate decarboxylase used in the present invention is less restricted by the pH and temperature of tea drinks, and can adapt to a wider pH and temperature range when used to reduce the oxalic acid content of tea drinks. According to tests, under the conditions of pH 4.0-6.0 and temperature 45-65°C, the oxalate decarboxylase can catalyze the degradation of oxalic acid in tea drinks and effectively reduce the oxalic acid content. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the HPLC chromatogram of the tea soup after OxdC1 enzymatic hydrolysis at pH 4.0 and temperature 55°C in Application Example 2.

[0023] Figure 2 It is the HPLC chromatogram of sodium oxalate standard (concentration of 105 mg / L).

[0024] Figure 3 This is the HPLC chromatogram of the blank control sample of tea soup in Application Example 2.

[0025] Figure 4 This is the SDS-PAGE picture of purified OxdC1. DETAILED DESCRIPTION

[0026] The present invention will be further described below in conjunction with the embodiments.

[0027] In the first aspect, the present invention relates to an oxalate decarboxylase for reducing the oxalic acid content of tea drinks, wherein the oxalate decarboxylase comprises an amino acid sequence as shown in SEQ ID NO.1. The oxalate decarboxylase can catalyze the decomposition of oxalate into carbon dioxide and formate. The oxalate decarboxylase used in the present invention can maintain a high catalytic activity in tea drinks, remove the oxalic acid contained in the tea drinks to a large extent, and its activity is less restricted by the pH and temperature of the tea drinks, and can catalyze the degradation of oxalic acid in the tea drinks within a wide pH and temperature range. The above characteristics enable the oxalate decarboxylase of the present invention to play a better role in reducing the oxalic acid content of tea drinks.

[0028] In a second aspect, the present invention relates to the use of oxalate decarboxylase in reducing the oxalic acid content of tea beverages, wherein the oxalate decarboxylase comprises an amino acid sequence as shown in SEQ ID NO.1.

[0029] In the above application, specifically: In some specific embodiments, the use comprises the following steps: adding the oxalate decarboxylase to tea to perform an enzymatic hydrolysis reaction, thereby reducing the oxalic acid content in the tea.

[0030] In the above specific embodiments, optionally or preferably, the mass volume ratio of the oxalate decarboxylase to the tea is 0.05-1 mg:1 mL; the pH value of the tea is 4.0-6.0; the conditions of the enzymatic hydrolysis reaction are as follows: the temperature is 45-65°C, the stirring speed is 650-750 rpm, and the time is 0.5-1h.

[0031] In some specific embodiments, the preparation step of oxalate decarboxylase comprises: S1: integrating the nucleic acid sequence encoding the oxalate decarboxylase into a plasmid vector by homologous recombination to obtain a recombinant expression plasmid; S2: transferring the recombinant expression plasmid into chassis cells to obtain oxalate decarboxylase expressing cells; S3: After culturing the oxalate decarboxylase-expressing cells, the cells are disrupted and the product is purified to obtain the oxalate decarboxylase.

[0032] In the above specific implementation scheme: Optionally or preferably, in step S1, the plasmid vector is pET28a; Optionally or preferably, in step S1, a 6×his-tag label is connected to the end of the nucleic acid sequence; Optionally or preferably, in step S2, the chassis cells are Escherichia coli; Optionally or preferably, in step S2, after the recombinant expression plasmid is transferred into the chassis cells, sequencing is performed to verify whether the recombinant expression plasmid is successfully transferred into the chassis cells; Optionally or preferably, in step S3, the culturing process comprises: inoculating the oxalate decarboxylase expressing cells into an LB liquid culture medium containing kanamycin, culturing at 35-37° C. for 12-13 hours, and then inoculating into an LB liquid culture medium containing kanamycin and 50-55 μg / mL α-lactose at an inoculum size of 1-3%, and culturing at 30-35° C. for 20-24 hours; Optionally or preferably, in step S3, the cell disruption method is ultrasonic cell disruption until clarified.

[0033] Optionally or preferably, in step S3, the process of purifying the product includes: after centrifuging the cell lysate, performing nickel column affinity chromatography on the supernatant, washing impurities with Tris-HCl buffer containing 150-160mM NaCl and 40-50mM imidazole, eluting the target protein with Tris-HCl buffer containing 150-160mM NaCl and 250-260mM imidazole, collecting the eluate, and desalting and concentrating.

[0034] In a third aspect, the present invention relates to a method for reducing the oxalic acid content in tea, comprising the following steps: adding oxalate decarboxylase comprising an amino acid sequence as shown in SEQ ID NO.1 to the tea, performing an enzymatic reaction, thereby reducing the oxalic acid content in the tea.

[0035] In the above method, specifically: In some specific embodiments, the mass volume ratio of the oxalate decarboxylase to the tea beverage is 0.05-1 mg:1 mL.

[0036] In some specific embodiments, the conditions of the enzymatic hydrolysis reaction are as follows: temperature of 45-65°C, stirring speed of 650-750 rpm, and time of 0.5-1 h. Within the above temperature range, the oxalate decarboxylase used in the present invention has a good catalytic effect on the degradation of oxalic acid in tea beverages, and can effectively reduce the oxalic acid content.

[0037] In some specific embodiments, the pH value of the tea drink is 4.0 to 6.0. Within the above pH range, the oxalate decarboxylase used in the present invention has a good catalytic effect on the degradation of oxalic acid in the tea drink and can effectively reduce the oxalic acid content.

[0038] In some specific embodiments, the preparation step of oxalate decarboxylase comprises: S1: integrating the nucleic acid sequence encoding the oxalate decarboxylase into a plasmid vector by homologous recombination to obtain a recombinant expression plasmid; S2: transferring the recombinant expression plasmid into chassis cells to obtain oxalate decarboxylase expressing cells; S3: After culturing the oxalate decarboxylase-expressing cells, the cells are disrupted and the product is purified to obtain the oxalate decarboxylase.

[0039] In the above specific implementation scheme: Optionally or preferably, in step S1, the plasmid vector is pET28a; Optionally or preferably, in step S1, a 6×his-tag label is connected to the end of the nucleic acid sequence; Optionally or preferably, in step S2, the chassis cells are Escherichia coli; Optionally or preferably, in step S2, after the recombinant expression plasmid is transferred into the chassis cells, sequencing is performed to verify whether the recombinant expression plasmid is successfully transferred into the chassis cells; Optionally or preferably, in step S3, the culturing process comprises: inoculating the oxalate decarboxylase expressing cells into an LB liquid culture medium containing kanamycin, culturing at 35-37° C. for 12-13 hours, and then inoculating into an LB liquid culture medium containing kanamycin and 50-55 μg / mL α-lactose at an inoculum size of 1-3%, and culturing at 30-35° C. for 20-24 hours; Optionally or preferably, in step S3, the cell disruption method is ultrasonic cell disruption until clarified.

[0040] Optionally or preferably, in step S3, the process of purifying the product includes: after centrifuging the cell lysate, performing nickel column affinity chromatography on the supernatant, washing impurities with Tris-HCl buffer containing 150-160mM NaCl and 40-50mM imidazole, eluting the target protein with Tris-HCl buffer containing 150-160mM NaCl and 250-260mM imidazole, collecting the eluate, and desalting and concentrating.

[0041] The present invention is described below by specific examples. It should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of the present invention. Without departing from the spirit and scope of the inventive concept, changes and advantages that can be thought of by those skilled in the art are included in the present invention, and the attached claims and any equivalents thereof are the protection scope of the present invention.

[0042] In the following specific examples, the oxalate decarboxylase with an amino acid sequence as shown in SEQ ID NO.1 is recorded as "OxdC1", the oxalate decarboxylase with an amino acid sequence as shown in SEQ ID NO.2 is recorded as "OxdC2", and the oxalate decarboxylase with an amino acid sequence as shown in SEQ ID NO.3 is recorded as "OxdC3".

[0043] In the following specific examples, the E. coli BL21 (DE3) competent cells, 2×TranStartFastPfu Fly PCR SuperMix and 2×Assembly Mix recombinase were purchased from Beijing Quanshijin Biotechnology Co., Ltd., SDS-PAGE was purchased from GenScript Biotech Co., Ltd., Tris and hydrochloric acid were purchased from Sinopharm Chemical Reagent Co., Ltd., and α-lactose was purchased from Shanghai MacLean Biochemical Technology Co., Ltd. However, the protection scope of the present invention is not limited thereto.

[0044] Preparation Example 1: Preparation of OxdC1 The DNA sequence encoding OxdC1 was integrated into the pET28a vector using homologous recombination technology, and a 6×his-tag tag was added to the end of the nucleic acid sequence to obtain the recombinant expression plasmid pET28b-OxdC1. The recombinant plasmid was transformed into E.coli BL21 (DE3) competent cells using the heat shock method, and the OxdC1 expression strain was obtained after sequencing verification.

[0045] A single colony of the OxdC1 expression strain that was verified by sequencing was picked and placed in 5 mL of LB liquid medium (containing 50 μg / mL kanamycin) and cultured with shaking at 37°C for 12 h. A 2% inoculum was transferred to 50 mL of LB liquid medium containing 50 μg / mL kanamycin and 1% α-lactose and cultured with shaking at 30°C and 180 rpm for 20 h.

[0046] After the culture is completed, the obtained bacterial solution is centrifuged at 4000×g for 15 minutes at 4°C, the supernatant is discarded, and the bacteria are collected. After washing the bacteria with 100mM Tris-HCl buffer with a pH value of 8.0, it is resuspended in Tris-HCl buffer and placed in an ice water bath for ultrasonic cell lysis until clarified. The obtained cell lysis solution is centrifuged at 8000×g for 20 minutes at 4°C, the supernatant is taken, and affinity chromatography is performed on a nickel column. The impurities are washed with a washing buffer (100mM Tris-HCl buffer, containing 150mM NaCl and 50mM imidazole, pH value 8.0), and the target protein is eluted with an elution buffer (100mM Tris-HCl buffer, containing 150mM NaCl and 250mM imidazole, pH value 8.0). The separated target protein is placed in an ultrafiltration centrifuge tube for sufficient desalting and concentration to obtain a pure target protein, namely OxdC1. The SDS-PAGE detection results are as follows: Figure 4 As described above, the molecular weight of the obtained target protein is about 44.4 kDa. The purified OxdC1 was rapidly quantified by the A280 of the microplate reader Multiskan SkyHigh.

[0047] The amino acid sequence of OxdC1 is shown in SEQ ID NO.1, with a total length of 385 amino acids. The specific sequence is as follows: MKKQNDIPQP IRGDKGATVK IPRNIERDRQ NPDMLVPPET DHGTVSNMKF SFSDTHNRLE 60 KGGYAREVTV RELPISENLA SVNMRLKPGA IRELHWHKEA EWAYMIYGSA RVTIVDEKGR 120 SFIDDVGEGD LWYFPSGLPH SIQALEEGAE FLLVFDDGSF SENSTFQLTD WLAHTPKEVI 180 AANFGVTKEE ISNLPGKEKY IFENQLPGSL KDDIVEGPNG EVPYPFTYRL LEQEPIESEG 240 GKVYIADSTN FKVSKTIASA LVTVEPGAMR ELHWHPNTHE WQYYISGKAR MTVFASDGHA 300 RTFNYQAGDV GYVPFAMGHY VENIGDEPLV FLEIFKDDHY ADVSLNQWLA MLPETFVQAH 360 LDLGKDFTDV LSKEKHPVVKKKCSK 385.

[0048] Preparation Example 2: Preparation of OxdC2 The DNA sequence encoding OxdC2 was integrated into the pET28a vector using homologous recombination technology, and a 6×his-tag label was added to the end of the nucleic acid sequence to obtain the recombinant expression plasmid pET28b-OxdC2. The recombinant plasmid was transformed into E.coli BL21 (DE3) competent cells using the heat shock method, and the OxdC2 expression strain was obtained after sequencing verification.

[0049] A single colony of the OxdC2 expression strain that was verified by sequencing was picked and placed in 5 mL of LB liquid medium (containing 50 μg / mL kanamycin) and cultured with shaking at 37°C for 12 h. A 2% inoculum was transferred to 50 mL of LB liquid medium containing 50 μg / mL kanamycin and 1% α-lactose and cultured with shaking at 30°C and 180 rpm for 20 h.

[0050] After the culture is completed, the obtained bacterial solution is centrifuged at 4000×g for 15 minutes at 4°C, the supernatant is discarded, and the bacterial cells are collected. After washing the bacterial cells with 100mM Tris-HCl buffer with a pH value of 8.0, they are resuspended in Tris-HCl buffer and placed in an ice water bath for ultrasonic cell lysis until clarified. The obtained cell lysis solution is centrifuged at 8000×g for 20 minutes at 4°C, the supernatant is taken, and affinity chromatography is performed on a nickel column. The impurities are washed with a washing buffer (100mM Tris-HCl buffer, containing 150mM NaCl and 50mM imidazole, pH value 8.0), and the target protein is eluted with an elution buffer (100mM Tris-HCl buffer, containing 150mM NaCl and 250mM imidazole, pH value 8.0). The separated target protein is placed in an ultrafiltration centrifuge tube for sufficient desalting and concentration to obtain a pure target protein, namely OxdC2. The purified OxdC2 was quickly quantified using the A280 microplate reader Multiskan Sky High.

[0051] The amino acid sequence of OxdC2 is shown in SEQ ID NO.2, with a total length of 404 amino acids. The specific sequence is as follows: MNVFSRRGLI AAAGGVALSA GPARSAEGPS PLRGSKGADI LGPRNAPREA EDPFTMAPPK 60 TDHGTMPNLK WSFADSHMRL EEGGWARQTT TRELPISTAM AGVNMRLKAN VVREMHWHKE 120 AEWAYMIKGR ARITAIDGDG RTFADDVGEG DLWYFPSGIP HSIQGLEGPE DGCEFLLVFD 180 DGGFSEDSTF LITDWLAHTP RDVLAKNFGL PESAFDAIPE KELYIFPGPK PGALADDQLG 240 GTGPVPKRFS HRMLAQEPIR TKGGSVRITD SSIFPASATI AAALVELEPG AMRELHWHPN 300 GDEWQYYLSG RGRMTVFGSE SKARSFDYQA GDVGYVPFAM GHYIENTGDT PLTFLEMFRS 360 PRYADISLRQ WMALTPHALV QAHTKLDRAAIDALPQTKSP VVPG 404.

[0052] Preparation Example 3: Preparation of OxdC3 The DNA sequence encoding OxdC3 was integrated into the pET28a vector using homologous recombination technology, and a 6×his-tag label was added to the end of the nucleic acid sequence to obtain the recombinant expression plasmid pET28b-OxdC3. The recombinant plasmid was transformed into E.coli BL21 (DE3) competent cells using the heat shock method, and the OxdC3 expression strain was obtained after sequencing verification.

[0053] A single colony of the OxdC3 expression strain that was verified by sequencing was picked and placed in 5 mL of LB liquid medium (containing 50 μg / mL kanamycin) and cultured with shaking at 37°C for 12 h. A 2% inoculum was transferred to 50 mL of LB liquid medium containing 50 μg / mL kanamycin and 1% α-lactose and cultured with shaking at 30°C and 180 rpm for 20 h.

[0054] After the culture is completed, the obtained bacterial solution is centrifuged at 4000×g for 15 minutes at 4°C, the supernatant is discarded, and the bacterial cells are collected. After washing the bacterial cells with 100mM Tris-HCl buffer with a pH value of 8.0, they are resuspended in Tris-HCl buffer and placed in an ice water bath for ultrasonic cell lysis until clarified. The obtained cell lysis solution is centrifuged at 8000×g for 20 minutes at 4°C, the supernatant is taken, and affinity chromatography is performed on a nickel column. The impurities are washed with a washing buffer (100mM Tris-HCl buffer, containing 150mM NaCl and 50mM imidazole, pH value 8.0), and the target protein is eluted with an elution buffer (100mM Tris-HCl buffer, containing 150mM NaCl and 250mM imidazole, pH value 8.0). The separated target protein is placed in an ultrafiltration centrifuge tube for sufficient desalting and concentration to obtain a pure target protein, namely OxdC3. The purified OxdC3 was quickly quantified using the A280 microplate reader Multiskan Sky High.

[0055] The amino acid sequence of OxdC3 is shown in SEQ ID NO.3, with a total length of 392 amino acids. The specific sequence is as follows: MATFGNPDEP PQGAVNAKSP GSLSDPGPQN PELAKQFPSAQTPPATDVGG LPMDWASFNN 60 APRRIQNGGW ARQVTVEDFAISKEISGVNM RLSAGGIREL HWHQAAEWAI MTYGSCRITV 120 LDTQGRPYVG DLKAGDLWYF PPGAPHSLQG LGPDGCEFVI CFDDGHANEF NTLLVTDWLA180 HTPPEVLAKN FGVPADAFAK IPLHNRWIFQ GTVPGDLADD RAAVSRHAEAPPYPFIHSLG 240 SSPPVKESAA GSIRVADSSN FKVATTVAAA LVTMPPGAVR EMHWHPNADE WQYYIKGKAR 300 MTVFDTGPNA LTTDFSAGDI GYVRRNLGHY VENVGDTELQ FVGVFRAPRY EEVSLSNWLT 360 HTPPKLVAQH FNIDEKLIAQ WPDNGPGVMP KS 392.

[0056] Application Example 1: Effects of different oxalate decarboxylases on reducing oxalic acid content in tea drinks In order to explore the ability of OxdC1 to reduce the oxalic acid content in tea drinks and compare it with other oxalate decarboxylases, this application example tested the ability of OxdC1, OxdC2 and OxdC3 (prepared by Preparation Examples 1 to 3) to degrade oxalic acid in tea drinks.

[0057] The pH value of the concentrated tea sample used in this application example was measured to be 5.8, and the oxalic acid concentration was 200 mg / L. Take 1 mL of the concentrated tea sample, add 0.05 mg of OxdC1, OxdC2 and OxdC3 respectively, and then react for 0.5 h at 55°C and 700 rpm. After the reaction is completed, pass through a 0.22 μm filter membrane and perform high performance liquid chromatography (HPCL) to detect the oxalic acid content. The conditions for HPCL detection are as follows: 1) High performance liquid chromatograph: Waters ARC01 chromatography system; 2) Chromatographic column: Amiex HPX-87H Column 300×78mnm, 9μm; 3) Detector: Waters 2998 PDA detector; 4) Mobile phase: 0.008 mol / L H2SO4; 5) Mobile phase speed: 0.6 mL / min; 6) Column temperature: 30°C; 7) Injection volume: 10 μL; 8) Detection wavelength: 210nm; 9) The sodium oxalate standard was tested by HPLC according to the above parameters, and the retention time was 6.414 min.

[0058] According to the oxalic acid content in the concentrated tea soup before and after enzymatic hydrolysis, the oxalic acid degradation rate was calculated, and the results were as follows: the oxalic acid degradation rates of OxdC1, OxdC2 and OxdC3 were 20.12%, 7.54% and 10.58%, respectively. The above results show that compared with OxdC2 and OxdC3, OxdC1 of the present invention can maintain a higher catalytic activity in tea soup.

[0059] Application Example 2: Effect of OxdC1 on Reducing Oxalic Acid Content in Tea under Different Conditions In order to explore the adaptability of OxdC1 to pH and temperature of tea, this application example tested the ability of OxdC1 (prepared in Preparation Example 1) to degrade oxalic acid in tea under a series of pH and temperature gradients.

[0060] The clear and concentrated Pu'er tea soup obtained after extraction with a solid-liquid ratio of 1:50 was adjusted to pH 4.0, 4.5, 5.0, 5.5, and 6.0 by hydrochloric acid, and 1 mL of samples with different pH values ​​were taken respectively, and 0.1 mg of OxdC1 was added thereto, and then reacted for 1 h at 45°C, 55°C, 65°C, and 700 rpm. After the reaction was completed, the oxalic acid content was detected by HPCL through a 0.22 μm filter membrane. The conditions for HPCL detection were as follows: 1) High performance liquid chromatograph: Waters ARC01 chromatography system; 2) Chromatographic column: Amiex HPX-87H Column 300×78mnm, 9μm; 3) Detector: Waters 2998 PDA detector; 4) Mobile phase: 0.008 mol / L H2SO4; 5) Mobile phase speed: 0.6 mL / min; 6) Column temperature: 30°C; 7) Injection volume: 10 μL; 8) Detection wavelength: 210nm; 9) The sodium oxalate standard was tested by HPLC according to the above parameters, and the retention time was 6.414 min.

[0061] According to the oxalic acid content in the concentrated Pu'er tea soup before and after enzymatic hydrolysis, the oxalic acid degradation rate (removal rate) was calculated, and the results are shown in Table 1. The HPLC chromatogram of the sodium oxalate standard (105 mg / L concentration) is shown in Figure 2 The HPLC chromatogram of the blank control sample of tea soup (i.e., no oxalate decarboxylase was used for enzymatic hydrolysis; oxalic acid concentration was 151 mg / L) is shown in FIG. Figure 3 The HPLC chromatogram of the tea soup after enzymatic hydrolysis at pH 4.0 and temperature 55°C is shown in Figure 1 shown.

[0062] Table 1 Removal rate of oxalic acid in tea soup by OxdC1 under different conditions pH 4.0 pH 4.5 pH 5.0 pH 5.5 pH 6.0 45℃ 99.15％ 80.90％ 60.16％ 29.49％ 41.44％ 55℃ 99.96％ 91.94％ 79.42％ 67.27％ 53.61％ 65℃ 91.12％ 74.11％ 55.64％ 44.09％ 28.62％ It can be seen from Table 1 that the OxdC1 of the present invention can reduce the oxalic acid content in tea soup by more than 99%, and can well enzymatically hydrolyze the oxalic acid in tea soup within a wider pH and temperature range. Therefore, when the oxalate decarboxylase reduces the oxalic acid content in tea, it is less restricted by the pH and temperature of the tea. This characteristic helps it adapt to teas with different pH values ​​and temperatures at different stages in the tea production process.

[0063] Unless otherwise defined, all technical terms and scientific terms used in the present invention have the same meanings as those commonly understood by those skilled in the art to which the present disclosure belongs. In addition, the raw materials and equipment used in the present invention, unless otherwise specified, are conventional raw materials and equipment in the art and can be obtained from conventional commercial channels; the methods used in the present invention, unless otherwise specified, are conventional methods in the art; the cell culture, molecular genetics, and nucleic acid chemistry operation steps used in the present invention, unless otherwise specified, are conventional steps widely used in the corresponding fields.

[0064] The present invention is described in detail above, but the present invention is not limited to the above-mentioned specific implementation schemes and application fields. The above-mentioned specific implementation schemes are only preferred embodiments of the present invention and do not impose any limitations on the present invention. Any simple modifications, changes and equivalent transformations made to the above embodiments based on the technical essence of the present invention still fall within the protection scope of the technical solution of the present invention.

Claims

1. An oxalate decarboxylase for reducing the oxalic acid content of tea, characterized in that: The oxalate decarboxylase comprises the amino acid sequence shown in SEQ ID NO.

1.

2. An application of oxalate decarboxylase in reducing the oxalic acid content of tea, characterized in that: The oxalate decarboxylase comprises the amino acid sequence shown in SEQ ID NO.

1.

3. A method for reducing the oxalic acid content of tea, characterized in that: The following steps are involved: Oxalate decarboxylase containing the amino acid sequence shown in SEQ ID NO.1 is added to the tea to carry out enzymatic hydrolysis reaction.

4. The method according to claim 3, characterized in that The mass volume ratio of the oxalate decarboxylase to the tea beverage is 0.05-1 mg:1 mL.

5. The method according to claim 3, characterized in that: The conditions of the enzymatic hydrolysis reaction are as follows: temperature is 45-65°C, stirring speed is 650-750 rpm, and time is 0.5-1 h.

6. The method according to claim 3, characterized in that The pH value of the tea drink is 4.0-6.

0.

7. The method according to claim 3, characterized in that The oxalate decarboxylase is obtained by adopting chassis cell recombinant expression technology.

8. The method according to claim 7, characterized in that The preparation steps of the oxalate decarboxylase include: S1: integrating the nucleic acid sequence encoding the oxalate decarboxylase into a plasmid vector by homologous recombination to obtain a recombinant expression plasmid; S2: Transform the recombinant expression plasmid into chassis cells to obtain oxalate decarboxylase expressing cells; S3: After culturing the oxalate decarboxylase-expressing cells, the cells are disrupted and the product is purified to obtain the oxalate decarboxylase.

9. The method according to claim 7 or 8, characterized in that: The chassis cells are Escherichia coli.

10. The method according to claim 8, characterized in that In step S1, the plasmid vector is pET28a.

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