Patent Application
20200080070
The entire content of the priority of the application of a Chinese patent (the Patent Application No.: 201610848095.1, titled as “Dry Powder and Fungus Powder Containing Agrocybe aegerita Oxalate Decarboxylase and Preparation Method and Application Thereof” and submitted to the State Intellectual Property Office of the P.R.C. on Sep. 26, 2016), is hereby incorporated by reference to this present application.
The invention relates to glycosylated oxalate decarboxylase and preparation and application thereof.
Kidney stones, 80% of which are calcium oxalate stones, cause pain to tens of millions of people around the world every year and billions of dollars of economic losses. In the United States, about 5 million persons enter the first aid rooms each year because of kidney stones, and it is also one of the seven major hospitalized diseases which cause an annual economic loss of about $5 billion. Each year, the kidney stone patient number is increased at a rate of 7-11%, the situation in China is similar to that in the United States. In addition, kidney stones are also the complication of many modern diseases (e.g., diabetes, obesity, etc.).
Oxalic acid is formed by the coupling of dicarboxylic acid connected via C—C bond. Under the condition of physiological pH value, sodium oxalate and potassium oxalate are soluble in water, but the solubility of calcium oxalate in water is less than 0.06 mM. The concentration of oxalic acid in the blood is 1-6 μM, far below the saturation of calcium oxalate. However, owing to the reabsorption of kidney on water, the concentration of oxalate in the kidney's concentrated crude urine is increased by more than a hundredfold. The concentration of calcium ions in urine is generally about 5 times that of oxalate, and thus, the concentration of calcium oxalate in the kidney is up to 0.1-0.6 mM, which is in the supersaturated state, and calcium oxalate in a highly supersaturated state can be precipitated into a solid, namely, growth calculus. Although the growth calculus is affected by the physiological state of the kidney, crude urine and other components in urine, the degree of supersaturation of calcium oxalate is the decisive factor for the formation and growth of the calculus.
The degree of supersaturation of calcium oxalate in urine is directly related to the source of oxalate in human blood. There are two paths for the sources of oxalate in the blood of the human body: the physiological metabolism of the human body and absorption from food. Except for the patients with primary hyperoxaluria, almost all people metabolize a certain amount of oxalate, but the amount of oxalate absorbed from food varies from person to person. The amount of oxalate absorbed by patients with urolithiasis is more than 50% higher than that of the normal persons (R. Holmes et al., Urol. Res. 32:311-316, 2004), so that calcium oxalate in the pelvis and urethra is significantly higher than its saturation. In the urinalysis of these patients, the amount of urinary oxalate is higher than that of normal people, known as hyperoxaluria. Most of patients with recurrent calcium oxalate stones are those with hyperoxaluria.
Almost all food contains oxalate, and a lot of food, especially some vegetables and cereals, contain high oxalate. Doctors often advise patients with urolithiasis to reduce the intake of oxalate by choosing the food with low oxalate, but there are a few varieties of food with low oxalate content, and the content of oxalic acid in such food is affected by the components of its growing soil, water content and climate, resulting in unreliable selection methods of edible food with low oxalate content and malnutrition. Therefore, people shall not choose it if there is another choice.
There is not a really effective medicine or good treatment method for kidney stones. Only some small stones (diameter or maximum size <0.6 cm) which are found early can be excreted through drinking a large amount of water and doing sports, however, and other treatment methods such as extracorporeal shock wave lithotripsy, percutaneous nephrolithotomy, or open surgical therapy to removal of the stones not only bring trauma to the body and kidney, but also pose a high recurrence rate and high operating expenses. Repeated use of these methods for recurrent patients will eventually lead to renal failure. Therefore, it is urgent to treat and prevent the recurrence of calculus through medicine.
The citrate medicament is the only drug for preventing calculus growth for 20 to 90 percent of patients with urolithiasis under the verification of the clinical trial, but it is mainly used for calcium urate stones. However, the patients need to take 6-10 grains of citrate daily for a long period of time. Citrate changes the composition of salts and acid-base balance in blood and urine. Because of large dosage and high side effects, many patients can't bear the side effects and have to stop treatment.
Degrading the oxalate in stomach with enzyme or microbes to reduce its absorption and further reduce the concentration of oxalate in urine has attracted people's attention. Except of enzyme or microbes can remove materials (oxalate) for growing calcium oxalate calculus, and prevent the formation, growth and recurrence of calcium oxalate stones, the enzyme or microbe method also has the advantages of safety, no trauma and the like.
The microbe method comprises the oral administration of microbes, such as a mixture product (CN00808182.4) of multiple Bacillus and Bifidobacterium, and an intestinally parasitic live bacterial formulation (U.S. Pat. No. 6,699,469) taking oxalate as energy substance. The former microbes are able to consume oxalic acid, however, because of the rich carbohydrates in the intestines, such microbes do not consume oxalic acid preferentially, and the clinical trials are proved to have no effect (D. Goldfarb, et. al., Clin. J. Am. Soc. Nephrol., 2:745-749, 2007); and the latter microbes only consume oxalic acid, but American Oxthera's Pharmaceutical Company completed two clinical trials and did not observe its ability to reduce urinary oxalic acid, and it is likely that oral live bacteria have not been able to colonize and survive in the intestinal tract.
After entering the stomach, the oxalate in the food is dissolved and released under the action of gastric acid with low pH value, and then, it is absorbed by the human body. Therefore, reduction in the absorption of diet oxalate should start from the stomach. The use of oral oxalate degrading enzymes to degrade oxalate in the stomach shall be more effective. The work in this area is mainly done by Altus and Oxthera. Altus Pharmaceutical made cross-linked enzyme crystals with an oxalate oxidase (PCT/US2006/023115) or an oxalate decarboxylase (U.S. patent application U.S. Ser. No. 11/833,082) (Altus-237, Altus), and these crystals are then made into oral preparations for the degradation of oxalate in the stomach. Altus Pharmaceutical's patent technology is inherited by Allena Pharmaceutical, which mainly focus on the oxalate decarboxylase cross-linked enzyme crystal. Clinical trials have shown that Allena Pharmaceutical found that the oral administration of high-dose enzyme preparations can only reduce urinary oxalate by 13% in patients with severe hyperoxaluria. Oxthera has prepared a preparation of another form, oxalate decarboxylase (OxDc) is mixed with an acid-insoluble polymer to prepare into microparticles by spray drying (Oxazyme, Oxthera). Clinical trials have shown that Oxazyme (Oxthera) does not reduce the effect of urinary oxalic acid.
Chinese patent application (No. 201610217032.6) announced a kind of oxalate decarboxylase produced by recombinant expression. There's an oxalate degrading enzyme activity at the pH value of 1.5-7.5, but because of the expression of the prokaryotic system, there is no glycosylated modification in the expressed oxalate degrading enzyme, the activity is not stable in the low pH value and high pH value, and the resistance to pepsin is weaker than that of the glycosylated oxalate decarboxylase (OxDc) in the present patent. Its activity is lower than the glycosylated oxalate decarboxylase of this patent.
For the prior art, the oxalate decarboxylase preparation is inactive or unstable at a low pH value and the technical defect leads to that the urinary oxalate cannot be truly reduced. The invention provides a glycosylated oxalate decarboxylase, which can effectively reduce the content of urinary oxalate.
The technical scheme of the invention is as follows:
The present invention relates to a glycosylated oxalate decarboxylase. It is derived from an edible basidiomycetes and has enzyme activity when the pH value is 1.5-7.5; when the pH value is 1.5-2.0, the activity exceeds 10% of the optimal activity, and the specific activity is more than 2 U/mg. Preferably, when the pH value is 2.0-5.5; the activity exceeds 30% of the optimal activity, and the specific activity is more than 6 U/mg. Further preferably, the optimal pH environment for the enzyme activity of oxalate decarboxylase is pH2.5-3.0. Furthermore, when the oxalate decarboxylase is in the optimal pH range, the enzyme activity is 20-200 U/mg and the Kin value is 0.06 mM.
In one embodiment, the glycosylated oxalate decarboxylase involved in the invention is from edible Agrocybe fungus. Specifically, it can be selected from Agrocybe aegerita, Agrocybe cylindracea, Agrocybe praecox (Pers) Fayod, Agrocybe salicacola or Agrocybe pediades (Fr) Fayod.
In one embodiment, the oxalate decarboxylase is produced by liquid fermentation technology. In one specific embodiment, the induced production of the glycosylated oxalate decarboxylase via the liquid fermentation technology comprises the following steps: inoculating and culturing for 2 to 5 days, adjusting the pH value of fermentation broth to pH2.0-4.0 by adding acid to induce fungi to produce enzyme, and continuing to culture it for 7-10 days.
The invention also relates to a preparation method of the oxalate decarboxylase. According to the method, oxalate decarboxylase is produced with the use of biological fermentation by acid induction under the condition of 0.001-50 mM manganese ions. Strains for producing oxalate decarboxylase are from Agrocybe edible fungus.
In one specific embodiment, according to the preparation method of oxalate decarboxylase, when the enzyme is produced by induction, the pH value of the fermentation broth is adjusted to pH3.2˜4.0, pH3.0˜3.6, pH2.8˜3.2, pH2.6˜3.0 or pH2.0˜2.8, preferably pH2.6˜3.0 or pH2.8˜3.2, and more preferably pH2.8˜3.2; the acid used to adjust the pH value of the fermentation broth is organic acid or inorganic acid, preferably phosphoric acid, hydrochloric acid, citric acid and/or oxalic acid, more preferably phosphate and/or hydrochloric acid.
In one specific embodiment, the materials containing manganese ions added in the enzyme induction production of the preparation method of oxalate decarboxylase include MnCl2, MnSO4, MnCO3, Mn(NO3)2 and/or Mn(CH3COO)2; the concentration of the added manganese ions is 0.001˜0.1 mM, 0.1˜1 mM, 1˜2 mM, 2˜4 mM, 4˜6 mM, 6˜10 mM or 10˜50 mM, preferably 1˜2 mM, 2˜4 mM, 4˜6 mM or 6˜10 mM, and more preferably 2˜4 mM or 4˜6 mM.
In one specific embodiment, according to the preparation method of the oxalate decarboxylase, the feeding culture medium added in the induced enzyme production phase contained glutamic acid, arginine, aspartic acid or glutathione.
In one specific embodiment, according to the preparation method of the oxalate decarboxylase, the fermenter used in production is a mechanical stirring fermenter or an air lifting fermenter, preferably a mechanical stirring fermenter.
In one specific embodiment, according to the preparation method of enzyme powder containing the oxalate decarboxylase, the fermentation liquid obtained by fermentation is concentrated and purified, small molecular substances are removed, then the purified concentrate is dried into powder, i.e., the enzyme powder containing the oxalate decarboxylase.
In one specific embodiment, according to the preparation method of edible fungus powder containing oxalate decarboxylase, mycelia obtained by fermentation are filtered, washed, dried and crushed into fine powder, i.e., the edible fungus powder containing oxalate decarboxylase.
In one specific embodiment, the drying method in the preparation process of the enzyme powder containing the oxalate decarboxylase and the edible fungus powder containing the oxalate decarboxylase is a spray drying method, a vacuum freeze drying method or a vacuum drying method.
The invention also relates to an enzyme preparation containing the glycosylated oxalate decarboxylase. The enzyme preparation is for oral administration.
Specifically, the enzyme preparation is edible fungus powder, a feed additive, a feed, a food additive, food, a health care product, special medical food or a drug containing the glycosylated oxalate decarboxylase.
The invention further relates to the application of glycosylated oxalate decarboxylase and the oxalate decarboxylase in prevention and/or treatment of hyperoxaluria.
The invention further relates to the application of glycosylated oxalate decarboxylase and the oxalate decarboxylase in prevention and/or treatment of urinary calculi containing calcium oxalate.
The oxalate decarboxylase provided by the embodiments of the invention has stability, high activity and very high degradation efficiency on low-concentration oxalic acid in gastrointestinal environment, can be directly orally taken, is safe, and does not have any toxic or side effect.
The glycosylated oxalate decarboxylase and the related enzyme preparations provided in embodiments of the invention are stable and have high activity when the pH value is 1.5-7.5, have the Kin value of 0.06 mM at the optimal pH value (pH2.5-3.0), are significantly superior to the existing oxalate decarboxylase from Bacillus subtilis, and have the stability superior to oxalate decarboxylase obtained by recombinant expression of Escherichia coli.
The glycosylated oxalate decarboxylase and the related enzyme preparations thereof provided by the present invention are from Agrocybe edible fungi, which have a long history as human food, and are proved to be free of any toxic and side effects, so it is safe and can be taken for a long time.
In the prior art, the application of oxalate decarboxylase does not achieve the useful clinical effect, which seems to show that it is not feasible to use oxalate decarboxylase to reduce the content of urinary oxalic acid. Upon a lot of experiments, the inventor is surprised to find that the main reason for the unsatisfactory pharmacodynamics of oxalate decarboxylase in the prior art is that the selected oxalate decarboxylase comes from the same kind of Bacillus subtilis, and has two major disadvantages (acid intolerance and high Kin value), thus resulting in incapability of survival and inefficient degradation of low-concentration oxalic acid in the stomach. The inventor supposed that the oxalate decarboxylase which could adapt to the acid gastrointestinal environment can achieve the effect to control the urinary oxalic acid content. The inventor screened thousands of types of edible materials and their different parts, involving plants, microbes, edible fungi, etc., and found that the oxalate decarboxylase of Agrocybe had high activity at a low pH value and could degrade oxalic acid in the acid environment of the stomach. However, because the content of oxalate decarboxylase is very low in the natural Agrocybe fungi, and it does not have the possibility of industrialization, so it is known that it creates a barrier to degrade oxalic acid in the acid environment of the stomach. The inventor of this application makes a lot of attempts to increase the yield of oxalate decarboxylase and realize its industrialization. The patent application (NO.: 201610217032.6) discloses a method for producing oxalate decarboxylase by using the expression of a prokaryotic expression system, in which the expression yield is greatly improved. However, the inventor finds a liquid culture and induction method enabling the agrocybe to naturally express oxalate decarboxylase through a lot of experiments, and surprisingly finds that the naturally expressed oxalate decarboxylase is of the same sequence as the oxalate decarboxylase expressed by the prokaryotic system, but it has significant advantages. By analyzing, the results show that the oxalate decarboxylase expressed by induction is glycosylated, so that it has significant advantages for improving the resistance to the low pH value in the stomach, the tolerance to pepsin and the enzyme activity. That is to say, the glycosylated oxalate decarboxylase has obvious advantages in treatment of hyperoxaluria over non-glycosylated oxalate decarboxylase.
The gene and the protein sequence of Agrocybe aegirita oxalate decarboxylase in the patent application are all the same as those of the Agrocybe aegirita described in the patent application (No. 201610217032.6). The application is introduced herein and will not be recorded repeatedly.
Since the pH in the stomach is very low, has non-uniform distribution, and is always changed, the pH value generally varies between pH1.5 and pH5.5, and the pH depends on the amount of food eaten, the type of food, the mixture of food and gastric acid, and the residence time in the stomach. In general, the pH value is low in an empty stomach, and the pH value is high when food with a great amount of meat is eaten. This requires that the enzyme products or drugs must be stable and have extremely high activity at the pH value of pH1.5 to pH5.5. In addition, the oxalic acid in the diet is generally present in the form of low soluble calcium oxalate and magnesium oxalate, resulting in low concentration of the soluble oxalic acid in the diet, generally between 0.05 and 0.5 mM. This requires high affinity of oxalate decarboxylase to oxalate, that is to say, the Kin value should be small. The oxalate decarboxylase of Bacillus subtilis is very unstable when the pH value is less than pH3.0, and is going to be denatured in a few seconds and loses the activity permanently. The pH value in the stomach is often lower than pH3.0. In addition, due to the constant secretion of gastric acid, the pH value in the whole stomach is not uniform, even if the average pH value in the stomach is above 3.0, there is still a pH value below pH3.0 locally. This is the main reason for the poor effect of the Bacillus subtilis OxDc enzyme. In addition, the enzyme has poor affinity to oxalate, and even under a condition of an ideal pH value, the Kin value is 4 mM. Moreover, prevention of hyperoxaliduria is a long-term process, so the patient needs long-term and even life-long administration. The Bacillus subtilis in the prior art has no historical habit of long-term administration by people. There is a potential risk of long-term administration of the oxalate decarboxylase from the Bacillus subtilis.
In order to screen a material containing an oxalate degrading enzyme that is stable and has a high activity in the gastrointestinal environment, that has a very high degradation effect on oxalic acid with a low concentration, and that is proved to be non-toxic and harmless for long-term administration by people, the inventor screens thousands of types of edible materials and their different parts, involving plants, microbes, edible fungi, etc., and finds that the enzyme activity and pH range of oxalate decarboxylase from basidiomycetes are suitable for the human gastrointestinal environment. The enzyme activity of the preferred Agrocybe edible fungi is stable and has a high activity at the pH value of pH1.5-7.5. Through a large number of experiments, the inventor finally finds a method for the cultivation of basidiomycetes in a liquid culture to produce enough oxalate decarboxylase, which can be used for experimental analysis and practical application. The oxalate decarboxylase produced by basidiomycetes has been glycosylated compared with the oxalate decarboxylase expressed by Escherichia coli. The stability of the glycosylated oxalate decarboxylase is significantly higher than that of the non-glycosylated oxalate decarboxylase at low pH values. The specific activity of the enzyme is increased significantly, and the resistance to pepsin and trypsin is better.
In this embodiment, Agrocybe aegerita is taken as an example to elaborate the fermentation process of glycosylated oxalate decarboxylase. In the experiments, the conclusions of Agrocybe aegerita are basically the same as those of Agrocybe cylindracea, Agrocybe praecox (pens) fayod, Agrocybe salicacola and Agrocybe pediades (Fr.) fayod, and except part of the comparison results, this application does not set forth them.
Agrocybe aegerita is selected as the strain to cultivate and produce oxalate decarboxylase by shake flask. The culture medium formula is as follows: yeast extract 4 g/L, soy peptone 3 g/L, KH2PO4 2 g/L, MgSO4.7H2O 0.2 g/L, CaCl2 1 g/L, glucose 20 g/L, corn starch 10 g/L and vitamin B1 10 mg/L. The pH value is 5.0-6.0. The shake flask loading volume is about 20-30%, and is sterilized at 121° C. for 30 minutes (vitamin B1 was sterilized by filtration, and added into the culture medium before the inoculation). Pure Agrocybe aegerita mycelium cultivated on a PDA plate was inoculated to a sterilized fluid medium to produce oxalate decarboxylase as follows: culturing the mycelium (100 rpm-350 rpm) at 23-28° C. for 2-5 days, adding phosphoric acid into the broth to adjust its pH value to pH2.8-3.2 to induce OxDc production, and continuing to culture it for 7 days; collecting the fresh mycelium through filtration, washing it with citrate buffer solution (pH 3.0), placing the washed mycelium in a clean ceramic mortar, where liquid nitrogen is added to grind the mycelium into powder, and then adding citrate buffer solution (pH 3.0) into the powder and continuing to grind it into paste, which is then centrifuged (15,000 g) for 10 minutes; collecting the supernatant and the precipitate from the container, respectively, and placing them into new containers, wherein the mycelium precipitate becomes fungus powder containing oxalate decarboxylase after freeze-drying, while the supernatant becomes enzyme powder containing oxalate decarboxylase after freeze-drying; and taking the supernatant to purify and identify oxalate decarboxylase in Embodiment 2.
The supernatant containing oxalate decarboxylase obtained in Embodiment 1 is purified with Q sepharose chromatography to get purified oxalate decarboxylase. The operation process is as follows: adjusting the pH value of the supernatant containing oxalate decarboxylase to pH 6.0 with 0.5M NaOH, removing the precipitated impurities through high-speed centrifugation (15,000 g), and concentrating the supernatant with a 50 KDa ultrafiltration membrane; equilibrating the Q sepharose column by 5 column volumes (CV) by using 25 mM NaH2PO4 buffer solution (pH 6.0), loading the concentrated oxalate decarboxylase sample on the equilibrated Q sepharose column, and washing off the impure protein not bonding to the column with 2 CV equilibration buffer solution (25 mM NaH2PO4, pH 6.0), then gradiently eluting 5 CV with an elution buffer solution (elution buffer solution A: 25 mM NaH2PO4, pH 6.0; and elution buffer solution B: 25 mM NaH2PO4, 1M NaCl, pH 6.0), and collecting the components of the oxalate decarboxylase sample. The molecular weight of the purified glycosylated oxalate decarboxylase is 60-80 KDa by electrophoresis identification with denatured polyacrylamide gel (SDS-PAGE). The oxalate decarboxylase expressed in Escherichia coli is found to have a molecular weight of 45-50 KDa by electrophoresis identification with denatured polyacrylamide gel (SDS-PAGE). The molecular weight identification results of the two oxalate decarboxylases are as shown in
In this invention, high performance liquid chromatography (HPLC) is used to determine the activity of oxalate decarboxylase. The operation process is as follows: preheating 1.0 mL 5 mM (mmol/L) oxalate solution (25 mM citrate buffer solution contained, pH 3.0) at 37° C. for 10 min and adding 0.01-0.1 ml (added volume is adjusted according to enzyme concentration) solution containing oxalate decarboxylase or fungus powder suspension into the oxalate solution for reaction; adding 50 μL 2.5M (molar concentration) H2SO4 to inactivate the enzyme after reacting for 30 minutes; quickly centrifuging the reaction solution and collecting supernatant to determine the concentration of residual oxalate by using HPLC. One enzyme activity unit (U) is defined as the required enzyme amount to degrade 1 micromole oxalate per minute on this condition.
The enzyme activity of the purified glycosylated oxalate decarboxylase obtained in Embodiment 2 was determined at pH 3.0, and the result is shown in Table 1. The oxalate decarboxylases of Agrocybe aegerita and Agrocybe cylindracea are taken as an example to determine the relative enzyme activity at different pH values from pH 1.5 to pH 7.5, and the results (
Agrocybe Aegerita
Agrocybe Cylindracea
Agrocybe Praecox (Pers.) Fayod
Agrocybe Pediades Fayod
Agrocybe Pediades (Fr.) Fayod
The purified glycosylated oxalate decarboxylase of Agrocybe aegerita in Embodiment 2 was digested with TPCK treated trypsin and analyzed by MALDI-TOF-MS. The result is shown in
In this embodiment, oxalate decarboxylase of Agrocybe aegerita is taken as an example to compare the enzymatic property of glycosylated oxalate decarboxylase with that of non-glycosylated oxalate decarboxylase. Oxalate decarboxylase genes of Agrocybe aegerita were recombined expressed according to the operation method of invention patent application (201610217032.6), and purified to obtain non-glycosylated oxalate decarboxylase. The enzymatic property and stability of the oxalate decarboxylase were compared with those of the glycosylated oxalate decarboxylase of Agrocybe aegerita obtained in Embodiment 2 by purification.
(1) Specific Activity Comparison:
The specific activity comparison is as follows: determining the protein concentrations of non-glycosylated oxalate decarboxylase expressed in Escherichia coil and the glycosylated oxalate decarboxylase expressed in Agrocybe aegerita with a Coomassie brilliant blue method, adjusting them to the same protein concentration (0.5 mg/ml), and determining the specific activities of the two oxalate decarboxylases in buffer solutions of different pH values with the method described in Embodiment 3; defining the specific activity of glycosylated oxalate decarboxylase at the optimum pH value as 100%, and comparing its specific activity at different pH values as well as the specific activity of non-glycosylated oxalate decarboxylase at different pH values with it. According to the result shown in
(2) Stability Comparison:
The stability comparison is as follows: adjusting the non-glycosylated oxalate decarboxylase sample expressed in Escherichia coli and the glycosylated oxalate decarboxylase sample expressed in Agrocybe aegerita to the same enzyme activity concentration, adding them to buffer solutions with pH 1.5, pH 2.0, pH 2.5, pH 3.0, pH 3.5, pH 4.0, pH 4.5, pH 5.0, pH 5.5, pH 6.0, pH 6.5, pH 7.0, pH 7.5 and pH 8.0, respectively, incubating them for 30 minutes at 37° C., taking a sample of 25μL from each of them, adding each sample into 10 mL 5 mM oxalate reaction solution (25 mM citrate buffer solution contained, pH value of 3.0) preheated at 37° C., then reacting them for 30 minutes at 37° C., and adding 50 μL 2.5M H2SO4 into the reaction solution to stop reaction; centrifuging the reaction solution immediately and collecting supernatant for activity determination by HPLC; determining the activities of oxalate decarboxylase in the samples by adopting the HPLC method, defining the activity of an original sample as 100%, and calculating the activity of residual oxalate decarboxylase in the sample after being processed by different pH buffer solutions, wherein the results are as shown in
(3) Comparison of Resistance to Proteinase
The comparison is as follows: preparing samples of glycosylated oxalate decarboxylase and non-glycosylated oxalate decarboxylase to into solutions of 10 U/mL, respectively, taking 1 mL OxDc sample respectively and mixing it with 20 mL 50 mM hydrochloric acid buffer solution (pH 2.5) containing 10 mg/mL pepsin, then stirring and incubating it at 37° C. for 5 minutes, 10 minutes, 20 minutes, 40 minutes and 60 minutes, respectively, and sampling it at the time point above to analyze the activity of residual oxalate decarboxylase. The results are as shown in Table 4.
Almost of all kinds of food, beverages, Chinese herbal preparations and feeds made with plants as raw materials contain oxalate, and its content in green vegetables, chocolate, cocoa, peanut and its products, soybean and bean products, tea and tea drinks, coffee and various wheat and grains is very high. Oxalate decarboxylase can be prepared into food additives or drugs to eat together with food, and degrades beverages and food's oxalate in the stomach to prevent excess oxalate from being absorbed by the gastrointestinal tract. Application of enzyme powder and edible bacterial powder containing oxalate decarboxylase that is prepared in the embodiments of this invention will be explained below.
(1) Removal of Oxalate from Tea
1-40 mg oxalate is contained in 1 L tea water, and its content depends on tea type, producing area, concentration and preparation method. The removing process is as follows: taking 20 g green tea, adding it in hot water of 90-95° C. for 5 minutes, filtering out tea leaves, then adjusting its pH value to pH 3.0-4.0 when the water temperature is lowered to less than 50° C., adding 20 U oxalate decarboxylase of Agrocybe aegerita into the tea water, and stirring it for reacting 2 hours, wherein the results of oxalate concentration in green tea determined at different time points are as shown in Table 5. The results show that nearly all the oxalate in tea water is ici removed after 2 hours by the oxalate decarboxylase of Agrocybe aegerita prepared in this invention. This embodiment is fit for making oxalic acid-free bottled tea water, iced tea or tea granules, and it is also suitable for removing oxalate from many Chinese herbal preparations.
(2) Removal of Oxalate from Bean Products
Oxalate content is very high in dry soybean, so various kinds of bean products contain abundant oxalate. As daily foods of Asian people, soybean products are also important sources of protein. Therefore, removing oxalate from bean products is of great significance to those people suffering from enteric hyperoxaluria. The removing process is as follows: adjusting the pH value of soybean milk to pH 3.0-4.0, then adding oxalate decarboxylase of Agrocybe praecox (Pers.) fayod prepared in the embodiments of this invention into the soybean milk, and stirring the soybean milk until oxalate decreased to certain content, wherein the oxalate concentration in soybean milk determined at different time points is as shown in Table 5. The time needed to degrade oxalate depends on its content in soybean milk and the enzyme amount added. If 0.1 g oxalate is contained in 1 L soybean milk, and 1,200-unit oxalate decarboxylase is added in each 10 L soybean milk, 2 hours would be needed to stir it for oxalate removal.
The test process is as follows: feeding 6 male beagles, weighing 7-8 kg, purchased from Hubei Anlu Ruikesen Experimental Animal Co., Ltd., in standard independent dog cages; after their acclimatization for 3-5 days, feeding them with prepared oxalate-free food (Ox-free dog food), collecting 24 h urine, and determining total oxalic acid content; determining the content of oxalic acid in urine for 5 consecutive days when it was kept a stable value, and recording the average as low oxalic acid excretion; then feeding them with commercially available ordinary dog food (ordinary dog food), collecting 24 h urine, determining oxalic acid excretion in urine for 5 consecutive days, and recording the average as normal oxalic acid excretion; then successively feeding them with 1,000 U oxalate decarboxylase (OxDc) samples of different sources (Bacillus subtilis (patent number: 201080029636.9)), glycosylated oxalate decarboxylase (this patent) and non-glycosylated oxalate decarboxylase (patent number: 201610217032.6)) together with ordinary food, collecting 24 h urine for 5 consecutive days, determining oxalic acid excretion in urine, recording the mean value of total oxalic acid amount of the 5 days as oxalic acid excretion in urine, and evaluating the oxalic acid degrading ability of oxalate decarboxylase from different strain sources, wherein the result is as shown in
The test process is as follows: feeding six Beagles, male, weighing 7-8 kg, purchased from Hubei Anlu Ruikesen Experimental Animal Co., Ltd. in a standard independent dog cage, acclimatizing them for 3-5 days, then feeding them the prepared oxalate-free food (Ox-free food), collecting 24 h urine, and determining the total amount of urine oxalic acid; after the oxalic acid in urine is steady, determining it for 5 successive days, recording the average value of oxalic acid excretion as low oxalic acid excretion; while feeding with the marketed ordinary food, feeding them with 500 U, 1000 U, 2000 U and 3000 U enzyme powder containing the glycosylated oxalate decarboxylase in turn, wherein the test period of each sample is 5 days; collecting 24 h urine, and determining urine oxalic acid excretion to evaluate the ability of different doses of glycosylated oxalate decarboxylase to degrade oxalic acid in food. The results are shown in
This embodiment describes the effect of different induction conditions on enzyme production of oxalate decarboxylase of Agrocybe fungi. The specific operation is as follows:
Agrocybe aegirita is selected as the strain to cultivate and produce oxalate decarboxylase by a shake flask. The culture medium formula is as follows: yeast extract 4 g/L, soybean peptone 3 g/L, KH2PO4 2 g/L, MgSO4.7H2O 0.2 g/L, CaCl2 1 g/L, glucose 20 g/L, corn starch 10 g/L, vitamin B1 10 mg/L. The pH value is pH 5.0-6.0. The shake flask loading volume is 20-30%, and the culture medium is sterilized at 121° C. for 30 minutes (Vitamin B1 is sterilized by filtration, and added to the culture medium before inoculation). The mycelia of Agrocybe aegirita cultured on PDA plate were inoculated into 3 groups (6 shake flasks) of the sterilized liquid culture medium while amount of inoculation is kept consistent as follows: culturing the mycelia (100 rpm-350 rpm) at 23-28° C. for 2-5 days; for Group 1 (shake flasks No. 1 and No. 2), adding hydrochloric acid to adjust the pH value to pH 2.8-3.2, and culturing it for 7-10 days to produce enzyme by induction; for Group 2 (shake flasks No. 3 and No. 4), adding 1.0 mM MnCl2 and hydrochloric acid to regulate the pH value to pH 2.8-3.2, and then culturing it for 7-10 days to produce enzyme by induction; for Group 3 (shake flasks No. 5 and No. 6), adding sterilized distilled water of equal volume, and culturing it for 7-10 days to produce enzyme by induction; determining the activity of oxalate decarboxylase by suspension (refer to Embodiment 3 for the determination of enzyme activity) after the fermentation culture is homogenized in a high speed homogenizer. As a result (table 6), the enzyme activity of the acid-induced and manganese-added group (group 2) is significantly higher than that of the acid-induced group (group 1) and the control group (group 3). The enzyme activity of the acid induction group (group 1) is also significantly higher than that of the control group (group 3).
This embodiment provides the effect of mycelium pellet control on the activity of oxalate decarboxylase during the fermentation process by comparing the fermentation of two 7 L fermenters. The specific steps are as follows:
1) Agrocybe aegirita is taken as the strain, and a seed solution is cultivated by adopting a shake flask and divided into two portions. The seed culture medium is as follows: yeast extract, 4 g/L soy peptone, 6 g/L, KH2PO4, 1 g/L, MgSO4.7H2O, 0.5 g/L, CaCl2, 0.1 g/L, glucose, 20 g/L, corn starch, 10 g/L, vitamin B1 10 mg/L. The pH value is pH5.0 to 6.0. The shake flask loading volume is 20˜30%. The strain is cultured at 23-28° C. for 3 days under 100-350 rpm. Mycelium pellets are crushed with a hand-held emulsifier before one portion of the seed solution is inoculated into the fermentation culture medium, and the other portion of the seed solution is directly transferred into the fermentation culture medium as control. The inoculation amount is 10-30%;
2) The fermentation medium comprises the following components: yeast extract 4 g/L, soy peptone 3 g/L, KH2PO4 2 g/L, Na2HPO4 1 g/L, MgSO4.7H2O 0.2 g/L, CaCI2 1 g/L, glucose 10 g/L, sucrose 20 g/L, corn starch 5 g/L, vitamin B1 10 mg/L. The shake flask loading volume is 70%. The pH value of the medium is adjusted to pH 4.5-6.5. The glucose and other components in the medium are sterilized separately. The glucose is sterilized at 115° C. for 15 min and the other components are sterilized together with the glass fermenter at 121° C. for 25 min. After the seed solution is inoculated into 7 L fermenter, the initial culture conditions of the two fermenters are as follows: temperature, 26° C.; stirring speed, 150 rpm; aeration volume, 3 L/min. During the fermentation process, the DO is controlled to greater than 40% by adjusting the stirring speed and the aeration volume. The pH value is adjusted to pH 2.8-3.2 by adding phosphoric acid for induction after fermentation for 3 days (72 h). Meanwhile, MnCl2 is added to final concentration of 5 mM to induce the OxDc production for about 7 days. The size of mycelium pellets at different time points is detected by sampling after inoculation, the enzyme activity change of the fermentation broth is detected at the same time, and the details are as follows (Table 7):
After nearly 10 days of fermentation, the enzyme activity in the fermenter (7L-1) with mycelium pellets uncontrolled reaches 15,000 U/L, but the enzyme activity in the fermenter (7L-2) with mycelium pellets emulsified and controlled is 21,500 U/L. Compared with the uncontrolled mycelium pellets, the final oxalate decarboxylase activity of the controlled the mycelium pellet is improved by 43%.
This embodiment provides a preparation method of enzyme powder and edible fungus powder containing oxalate decarboxylase, comprising the specific steps as follows:
This embodiment provides a preparation method of enzyme powder and edible fungus powder containing oxalate decarboxylase, comprising the specific steps as follows:
This embodiment provides a preparation method of enzyme powder and edible fungus powder containing oxalate decarboxylase, comprising the specific steps as follows:
This embodiment provides a preparation method of enzyme powder and edible fungus powder containing oxalate decarboxylase, comprising the specific steps as follows:
This embodiment provides a preparation method of enzyme powder and edible fungus powder containing oxalate decarboxylase, comprising the specific steps as follows:
This embodiment provides a preparation method of enzyme powder and edible fungus powder containing oxalate decarboxylase, comprising the specific steps as follows:
The activity of oxalate decarboxylase is determined by adopting an HPLC (High Performance Liquid Chromatography) method in the present invention. The specific operation process is as follows: preheating 1.0 mL of 5 mM (millimole concentration) oxalate solution (containing 25 mM citrate buffer, pH3.0) for 10 minutes at 37° C., and then adding 0.01-0.1 ml (the adding volume is determined according to the enzyme concentration) solution or fungus powder suspension containing oxalate decarboxylase to react; adding 50 μL of 2.5M (molar concentration) sulfuric acid to the solution to inactivate the enzyme after reacting 30 minutes; centrifuging the solution immediately and taking supernatant, and determining the concentration of residual oxalate with HPLC. One enzyme activity unit (U) is defined as the enzyme amount required to degrade 1 micromole oxalate per minute on this condition.
The results of activity of oxalate decarboxylase in the enzyme powder and edible fungus powder containing oxalate decarboxylase as prepared in Embodiments 10-15 by adopting HPLC method are shown in table 8 below:
Taking Agrocybe aegerita as an example, Agrocybe aegerita mycelia cultured naturally and Agrocybe aegerita mycelia (before/after induction) samples cultured by liquid fermentation are respectively weighed with the same weight, then the same volume of pure water is added to them, the solutions are homogenized with a high speed homogenizing machine, the enzyme activities of the homogenates are determined, and the results are shown as table 9. The results show that yield of oxalate decarboxylase is greatly improved after induced fermentation of this patent method, and compared with the natural Agrocybe aegerita mycelia or the uninduced liquid fermentation Agrocybe aegerita, the activity is improved by a thousand times.
The fermentation yields of oxalate decarboxylase of different mycelia are studied by comparing the yield of oxalate decarboxylase of fermentation culture in Agrocybe aegerita, Agrocybe cylindracea, Agrocybe praecox (pers.) fayod, Agrocybe pediades (Fr.) Fayod and Agrocybe salicacola in embodiments 10-15. The fermentation yields are shown in table 10, the enzyme yields of Agrocybe aegerita and Agrocybe cylindracea are significantly superior to those of the other three edible fungi. The specific activities of oxalate decarboxylase of Agrocybe pediades (Fr) Fayod and Agrocybe salicacola are higher than those of Agrocybe aegerita and Agrocybe cylindracea, but their growth of mycelia is particularly slow and the product amount of mycelia is very small. Considering the economy of fermentation, Agrocybe aegerita and Agrocybe cylindracea are the most suitable production strains. The inventor would continue to optimize the fermentation conditions to improve the yield of oxalate decarboxylase.
Agrocybe aegerita
Agrocybe Cylindracea
Agrocybe praecox
Agrocybe pediades
Agrocybe salicacola
The present embodiment provides an enlarged production process of enzyme powder and edible fungus powder containing oxalate decarboxylase, comprising the specific steps as follows:
Commercial Agrocybe edible fungi (totally 20 kinds of fresh and dried edible fungi such as Agrocybe aegerita, Agrocybe cylindracea, Agrocybe praecox in different areas) are cleaned with clear water, then shredded with scissors and put into a clean ceramic mortar, the edible fungi are ground into powder while liquid nitrogen is added, and then citrate buffer solution (pH3.0) is added, the edible fungi are continuously ground into paste, the paste is centrifuged at 15000 g for 10 minutes, the supernatant and the sediment are respectively put into new containers, the sediment is freeze-dried into fungus powder containing oxalate decarboxylase, and the supernatant is freeze-dried into enzyme powder containing oxalate decarboxylase. The manganese ion contents of the prepared natural Agrocybe edible fungus powder and enzyme powder as well as the enzyme powder and fungus powder containing oxalate decarboxylase prepared by fermentation in embodiments 10-15 are detected. The results are as follows (Table 11):
It can be seen from table 11, the manganese ion content of the oxalate decarboxylase product prepared in the present invention is over 3 times higher than that of the natural Agrocybe oxalate decarboxylase product, but is still in a safety range.
The activities of oxalate decarboxylase prepared in embodiments 10-15 are consistent with the results in embodiments 5-8. It showed that the expression yield of oxalate decarboxylase prepared in embodiments 10-15 are increased, however, the activities of the prepared oxalate decarboxylase are not affected. So, the industrialization of oxalate decarboxylase can be realized.
| Number | Date | Country | Kind |
|---|---|---|---|
| 201610848095.1 | Sep 2016 | CN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2017/092149 | 7/7/2017 | WO | 00 |
