Glycosylated oxalate decarboxylase and preparation and application thereof
11085033 · 2021-08-10
Assignee
Inventors
- Haifeng Liu (Wuhan, CN)
- Baoping Song (Wuhan, CN)
- Xianqiao Chen (Wuhan, CN)
- Huoqing Chen (Wuhan, CN)
- Pei Tong (Wuhan, CN)
- Wei Wang (Wuhan, CN)
- Xiaofeng Wang (Wuhan, CN)
Cpc classification
A23V2002/00
HUMAN NECESSITIES
International classification
A23L29/00
HUMAN NECESSITIES
Abstract
The present invention discloses a glycosylated oxalate decarboxylase. The oxalate decarboxylase is derived from edible basidiomycetes, the glycosylated oxalate decarboxylase has an enzyme activity at the pH value of 1.5-7.5, the activity at the pH value of 1.5-2.0 is 10% greater than the optimum activity, and a specific activity is more than 2 U/mg. The present invention also discloses preparation and application of the glycosylated oxalate decarboxylase. The glycosylated oxalate decarboxylase disclosed by the present invention can keep the activity under a low pH value, and can effectively prevent and treat kidney stones.
Claims
1. A glycosylated oxalate decarboxylase, wherein: the oxalate decarboxylase is derived from edible fungi of Agrocybe, the glycosylated oxalate decarboxylase has an enzyme activity at the pH value of 1.5-7.5; the optimal pH value of the enzyme activity of the oxalate decarboxylase is pH 2.5-3.0; when the oxalate decarboxylase is at the optimal pH value, the optimal activity is 20-200 U/mg; the activity at the pH value of 1.5-2.0 is greater than 10% of the optimum activity and the specific activity is more than 2 U/mg; the activity at the pH value of 2.0-5.5 is more than 30% of the optimal activity, and the specific activity is more than 6 U/mg; and the oxalate decarboxylase is inductively produced by the following liquid fermentation method: after 2-5 days of culture in a fermentation broth of the edible fungi, an acid is added to adjust the pH value to pH 2.0-4.0 under the condition of 0.1-6 mM manganese ions, and then the enzyme is inductively produced; and continuously cultured 7-10 days.
2. The glycosylated oxalate decarboxylase of claim 1, wherein, when the oxalate decarboxylase is at the optimal pH value, the Km value is 0.06 mM.
3. The glycosylated oxalate decarboxylase of claim 1, wherein the oxalate decarboxylase comes from Agrocybe aegerita, Agrocybe cylindracea, Agrocybe praecox (pers.) fayod, Agrocybe salicacola or Agrocybe pediades (Fr.) Fayod.
4. A method for preparing the oxalate decarboxylase of claim 1, comprising: culturing the edible fungi of Agrocybe in a fermentation broth after inoculation for 2-5 days; adding an acid to adjust the pH value of the fermentation broth to pH 2.0-4.0 and a chemical to adjust manganese ions to 0.1-6 mM, thereby inductively producing the oxalate decarboxylase; and culturing continuously for 7-10 days.
5. The method of claim 4, wherein the pH value of the fermentation broth is adjusted to pH 3.2-4.0, pH 3.0-3.6, pH 2.8-3.2, pH 2.6-3.0 or pH 2.0-2.8 during induced enzyme production; and the acid for adjusting the pH value of the fermentation broth is an organic acid or an inorganic acid.
6. The method of claim 4, wherein the added chemical during induced enzyme production include MnCl.sub.2, MnSO.sub.4, MnCO.sub.3, Mn(NO.sub.3).sub.2 and/or Mn(CH.sub.3COO).sub.2; the final concentration of the added manganese ions is 0.1 to 1 mM, 1 to 2 mM, 2 to 4 mM, or 4 to 6 mM.
7. The method of claim 4, wherein a feeding medium is added when the oxalate decarboxylase is being induced, and the feeding medium contains glutamic acid, arginine, aspartic acid or glutathione.
8. The method of claim 4, wherein the culturing of the edible fungi of Agrocybe is conducted in a fermenter and the fermenter is a mechanical stirring fermenter or an air-lifting fermenter.
9. The method of claim 4, comprising the following steps: when the edible fungi of Agrocybe are cultured to the level that the wet cell mass content reaches 5-20% of the total volume of the fermentation broth, slowly adjusting the pH value of the fermentation broth to pH 2.0-4.0 at the rate of decreasing 0.05-1 pH value unit per hour, at the same time, adding a chemical containing manganese ions, to induce the enzyme for 3-15 days, and after the enzyme production is finished, collecting the fermentation broth and the mycelia respectively.
10. The method of claim 9, further comprising the following steps: 1) culturing Agrocybe fungus strains by liquid fermentation, wherein the seed culture medium consists of yeast extract 2-4 g/L, soy peptone 1-3 g/L, KH.sub.2PO.sub.4 0.5-3 g/L, MgSO.sub.4.7H.sub.2O 0.2-2 g/L, CaCl.sub.2 0.1-1 g/L, glucose 10-30 g/L, corn starch 10-30 g/L and vitamin B.sub.1 5-20 mg/L; adjusting the pH value of the culture medium to pH 5.0-6.0; subculturing the fungi in the form of mycelium in a seed fermenter at 23 to 28° C., 100-350 rpm for 2-5 days; 2) inoculating the fungi in the form of mycelium to a fermenter for fermentation culture, in a liquid fermentation medium that consists of the following components: yeast extract 4-8 g/L, soy peptone 2-6 g/L, KH.sub.2PO.sub.4 0.5-3 g/L, Na.sub.2HPO.sub.4 0.1-1 g/L, MgSO.sub.4.7H.sub.2O 0.2-2 g/L, CaCl.sub.2 0.1-1 g/L, glucose 10-30 g/L, cane sugar 10-30 g/L, corn starch 5-20 g/L and vitamin B.sub.1 5-20 mg/L; adjusting the pH value of the medium to pH 4.5-6.5; culturing the fungi in the form of mycelium at 23 to 28° C. for 3-5 days after inoculation; 3) when the fungi in the form of mycelium are cultured to the level that the wet cell mass content reaches 5-30% of the total volume, slowly adjusting the pH value of the fermentation broth to pH 2.0-4.0 at the rate of decreasing 0.05-1 pH value unit per hour, at the same time, adding a chemical containing manganese ions, to induce the enzyme for 3-15 days, and after the enzyme production is finished, collecting the fermentation broth and the fungi in the form of mycelium mycelia respectively; 4) concentrating the fermentation broth obtained in step 3), purifying the concentrate, removing small molecular substances, and then drying the purified concentrate into powder to obtain an enzyme powder containing oxalate decarboxylase; and filtering, washing, drying and crushing the fungi in the form of mycelium obtained in step 3) into a powder to obtain an edible fungus powder containing oxalate decarboxylase.
11. An enzyme preparation containing the glycosylated oxalate decarboxylase of claim 1.
12. The enzyme preparation of claim 11, wherein the enzyme preparation is an oral preparation.
13. The enzyme preparation of claim 11, wherein the enzyme preparation is edible fungus powder, a feed additive, a feed, a food additive, food, a health-care product, medical food or medicine containing the glycosylated oxalate decarboxylase.
14. A method of treating hyperoxaluria, comprising administering to a subject in need thereof a therapeutically effective amount of a composition, wherein the composition comprises the enzyme preparation of claim 11.
15. A method of treating calcium oxalate urinary calculi, comprising administering to a subject in need thereof a therapeutically effective amount of a composition, wherein the composition comprises the enzyme preparation of claim 11.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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DETAILED DESCRIPTION
(8) 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.
(9) 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.
(10) 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.
(11) 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.
Embodiment 1
(12) 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 (pers.) fayod, Agrocybe salicacola and Agrocybe pediades (Fr.) fayod, and except part of the comparison results, this application does not set forth them.
(13) 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, KH.sub.2PO.sub.4 2 g/L, MgSO.sub.4.7H.sub.2O 0.2 g/L, CaCl.sub.2 1 g/L, glucose 20 g/L, corn starch 10 g/L and vitamin B.sub.1 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 B.sub.1 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.
Embodiment 2: Purification and Identification Oxalate Decarboxylase
(14) 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 NaH.sub.2PO.sub.4 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 NaH.sub.2PO.sub.4, pH 6.0), then gradiently eluting 5 CV with an elution buffer solution (elution buffer solution A: 25 mM NaH.sub.2PO.sub.4, pH 6.0; and elution buffer solution B: 25 mM NaH.sub.2PO.sub.4, 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
Embodiment 3: Determination of Oxalate Decarboxylase Activity and its Enzymatic Property
(15) 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) H.sub.2SO.sub.4 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.
(16) 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 (
(17) TABLE-US-00001 TABLE 1 Specific activity of different oxalate decarboxylases Specific Strain Activity (U/mg) Agrocybe Aegerita 130 Agrocybe Cylindracea 100 Agrocybe Praecox (Pers.) Fayod 20 Agrocybe Pediades Fayod 200 Agrocybe Pediades (Fr.) Fayod 153
(18) TABLE-US-00002 TABLE 2 Km value determination of oxalate decarboxylase at different pH values Range of pH Value Km Value (mM) 2.5-3.0 0.06 ± 0.03 3.0-3.5 0.12 ± 0.04 3.5-4.0 0.20 ± 0.03 4.0-4.5 0.25 ± 0.08 4.5-5.0 0.36 ± 0.13 5.0-6.0 0.7 ± 0.30
Embodiment 4: Peptide Mapping Analysis on Glycosylated Oxalate Decarboxylase
(19) 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
(20) TABLE-US-00003 TABLE 3 Molecular weight list of peptide mapping mass spectrum of glycosylated oxalate decarboxylase Serial Molecular Oxalate decarboxylase Number Weight (Da) Sequence Fragment 1 804.3521 2 842.4955 3 879.6353 VKPIVVGPA 4 888.5458 LQTGGWAR 5 895.5654 6 920.5276 7 1080.6455 TAEWAYVLK 8 1117.6328 WAFSLSHNR 9 1127.6497 10 1177.7084 11 1185.7059 VPPMQLSGGTAK 12 1209.6884 MTIFAAQSNAR 13 1225.6816 14 1269.7098 NFQTDISAFAR 15 1292.677 16 1382.7612 17 1481.9274 AIAAAEVTIEPGAIR 18 1503.8977 19 1549.8677 20 1597.8722 AHLGFDDATMAHLAK 21 1643.9357 GSLGATIIGPTDVDTTK 22 1782.0438 23 1858.9263 24 2063.0854 25 2033.1101 ANPDLLAPPTTDHGSVDNAK 26 2091.1174 EQNIGVMPIATEMASVNMR 27 2103.1228 28 2314.2021 ELHWHPTQDEWSFFIEGR 29 3131.7402 30 3322.8489 31 3643.145 32 3778.1868
Embodiment 5: Comparison of Glycosylated Oxalate Decarboxylase and Non-Glycosylated Oxalate Decarboxylase
(21) 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.
(22) (1) Specific Activity Comparison:
(23) 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
(24) (2) Stability Comparison:
(25) 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 25 μL 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 H.sub.2SO.sub.4 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
(26) (3) Comparison of Resistance to Proteinase
(27) 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.
(28) TABLE-US-00004 TABLE 4 Comparison of resistance to pepsin of glycosylated and non-glycosylated oxalate decarboxylases 5 10 20 40 60 Control minutes minutes minutes minutes minutes Glycosylated 100% 95% 88% 75% 65% 58% oxalate Degrading Enzyme Non- 100% 85% 72% 63% 50% 34% Glycosylated oxalate Degrading Enzyme
Embodiment 6: Removal of Oxalic Acid from Food with Oxalate Decarboxylase
(29) 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.
(30) (1) Removal of Oxalate from Tea
(31) 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.
(32) (2) Removal of Oxalate from Bean Products
(33) 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.
(34) TABLE-US-00005 TABLE 5 0 10 30 60 120 minutes minutes minutes minutes minutes Green Tea 100% 65% 27% 5% 3% Black Tea 100% 59% 32% 6% 2% Soybean 100% 89% 67% 34% 8% Milk
Embodiment 7: Animal Experiment to Reduce Urinary Oxalic Acid Content with Oxalate Decarboxylase
(35) 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
(36) 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
Embodiment 8: Effect of Different Induction Conditions on the Production of Oxalate Decarboxylase
(37) This embodiment describes the effect of different induction conditions on enzyme production of oxalate decarboxylase of Agrocybe fungi. The specific operation is as follows:
(38) 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, KH.sub.2PO.sub.4 2 g/L, MgSO.sub.4.7H.sub.2O 0.2 g/L, CaCl.sub.2 1 g/L, glucose 20 g/L, corn starch 10 g/L, vitamin B.sub.1 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 B.sub.1 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 MnCl.sub.2 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).
(39) TABLE-US-00006 TABLE 6 Acid-induced and Acid-induced group manganese-added Control group (Group 1) group (Group 2) (Group 3) Flask No. 1 2 3 4 5 6 Enzyme 2007 1761 5247 5560 3 5 activity (U/L)
Embodiment 9: Effect of Mycelium Pellet on Enzyme Activity
(40) 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:
(41) 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, KH.sub.2PO.sub.4, 1 g/L, MgSO.sub.4.7H.sub.2O, 0.5 g/L, CaCl.sub.2, 0.1 g/L, glucose, 20 g/L, corn starch, 10 g/L, vitamin B.sub.1 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%;
(42) 2) The fermentation medium comprises the following components: yeast extract 4 g/L, soy peptone 3 g/L, KH.sub.2PO.sub.4 2 g/L, Na.sub.2HPO.sub.4 1 g/L, MgSO.sub.4.7H.sub.2O 0.2 g/L, CaCl.sub.2 1 g/L, glucose 10 g/L, sucrose 20 g/L, corn starch 5 g/L, vitamin B.sub.1 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, MnCl.sub.2 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):
(43) TABLE-US-00007 TABLE 7 Fermentation 7 L-1 (non-emulsified) 7 L -2 (emulsified) time Mycelium morphology Mycelium morphology 40 h The hyphae are elongated. There are The hyphae are dispersed. There many mycelium pellets, and its' are less mycelium pellets, and its' diameters are between 200 μm and diameters are between 50-300 μm. 500 μm. 64 h The mycelium gradually becomes The scattered hyphae are short, and the mycelium pellets decreasing, the tiny mycelium gradually become thick; the mycelium pellets are also gradually bigger, pellets with the diameter of and the diameters of the mycelium 400 μm-800 μm are increased; pellet maintain between 100 μm-300 μm; 72 h The hyphae around the mycelium The hyphae around the mycelium (induced) pellets are getting shorter, and the size pellets are getting shorter, and the of the mycelium pellets basically size of the mycelium pellets is maintain between 600 μm-1,000 μm; almost not changed; 118 h The size of the mycelium pellets is The size of the mycelium pellet is basically kept between 700 μm and basically maintained between 1,200 μm; the hyphae around the 200 μm-500 μm, and the hyphae mycelium pellets showed a decreasing around the mycelium pellets are trend; less than that of the hyphae when being induced. 200 h The change of mycelium pellets is not The change of mycelium pellets is obvious, and there's less and less not obvious, there's less and less hyphae around the mycelium pellets; hyphae around the mycelium pellet, but there are more hyphae around the mycelium pellet; 240 h Total activity 15,000 U/L Total activity 21,500 U/L
(44) 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%.
Embodiment 10
(45) This embodiment provides a preparation method of enzyme powder and edible fungus powder containing oxalate decarboxylase, comprising the specific steps as follows: 1) Agrocybe aegirita is taken as the strain and the seed solution is cultured in a shake flask. The components of seed medium are as follows: yeast extract 4 g/L, soy peptone 3 g/L, KH.sub.2PO.sub.4 2 g/L, MgSO.sub.4.7H.sub.2O 0.2 g/L, CaCl.sub.2 1 g/L, glucose 20 g/L, corn starch 10 g/L, vitamin B.sub.1 10 mg/L, The pH value is pH 5-6. The loading volume in the shake flask is 20-30%. The strain is cultured at 23-28° C. for 2-5 days under 100-350 rpm. The mycelium pellets are crushed by a mechanical crushing method, and then inoculated with 10-30% of inoculation to next-stage seed culture medium; 2) The seed solution mycelium pellets mechanically crushed are inoculated with 10%-30% of inoculation to a 7 L mechanical-stirring fermenter. The liquid fermentation medium consists of the following ingredients: yeast extract 4 g/L, soybean peptone 3 g/L, KH.sub.2PO.sub.4 2 g/L, Na.sub.2HPO.sub.4 1 g/L, MgSO.sub.4.7H.sub.2O 0.2 g/L, CaCl.sub.2 1 g/L, glucose 10 g/L, cane sugar 20 g/L, corn starch 5 g/L, vitamin B.sub.1 10 mg/L. The loading volume is 65-70%. The pH value of the culture medium is adjusted to pH4.5-6.5. The mycelium pellets are cultured at 25-28° C. for 3-5 days after inoculation under the stirring speed of 150-350 rpm. The DO is maintained at over 30% by adjusting the stirring speed. This stage is the growth stage of mycelium, and the size of the mycelium pellets is controlled to be less than 1 mm 3) Induced enzyme production stage: when the wet cell mass content of the mycelium reached 5-30% of total volume, the pH value of the fermentation broth is adjusted slowly to pH 2.6-3.0 at the rate of 1 pH unit per hour at the same time, MnCl.sub.2 is added to the final concentration (5 mM) of manganese ions, and the induction production is carried out for 3-15 days. The size of the mycelium pellets is controlled to be not more than 0.5 mm in the induction process. Feeding medium (soybean peptone: 70-90 g/L, glucose 80-120 g/L, glutamic acid 5 mM) is added from the 3rd day of induction. After the enzyme production is finished, the fermentation broth and mycelium are collected. 4) The solid-liquid separation is carried out via the method of filtering the fermentation broth and mycelium through the frame filter, and then the supernatant of the fermentation broth is concentrated through ultrafiltration. The small molecules in the fermentation broth are removed and the concentrated solution of oxalate decarboxylase is finally obtained. The concentrated solution is dried by spray drying. An enzyme protective material is added while spraying and oxalate decarboxylase powder is collected. Agrocybe aegirita mycelium is obtained through frame filtering, and dried by vacuum freeze drying. The edible fungus powder containing oxalate decarboxylase is finally obtained by superfine grinding the dry mycelium.
Embodiment 11
(46) This embodiment provides a preparation method of enzyme powder and edible fungus powder containing oxalate decarboxylase, comprising the specific steps as follows: 1) Agrocybe aegirita is taken as the strain and a seed solution is cultured in a shake flask. The components of seed medium are as follows: yeast extract 4 g/L, soy peptone 3 g/L, KH.sub.2PO.sub.4 2 g/L, MgSO.sub.4.7H.sub.2O 0.2 g/L, CaCl.sub.2 1 g/L, glucose 20 g/L, corn starch 10 g/L, vitamin B.sub.1 10 mg/L, pH 5-6. The shake flask loading volume is 20-30%. The strain is cultured at 23-28° C., 100-350 rpm for 2-5 days. The mycelium pellets of seed culture are crushed by a mechanical crushing method, and then inoculated with 10-30% of inoculation to next-stage seed culture medium; 2) The seed solution mycelium pellets mechanically crushed are inoculated with 10%-30% inoculation to a 7 L air-lifting fermenter for fermentation culture. The liquid fermentation medium consists of the following ingredients: yeast extract 4 g/L, soybean peptone 3 g/L, KH.sub.2PO.sub.4 2 g/L, Na.sub.2HPO.sub.4 1 g/L, MgSO.sub.4.7H.sub.2O 0.2 g/L, CaCl.sub.2 1 g/L, glucose 10 g/L, sucrose 20 g/L, corn starch 10 g/L, vitamin B.sub.1 10 mg/L. The loading volume is 65-70% and the pH value of the culture medium is adjusted to pH 4.5-6.5. The mycelium pellets are cultured at 23-28° C., 100-350 rpm for 3-5 days after inoculation. The DO is maintained over 30% by adjusting the airflow speed. This stage is the growth stage of mycelium, and the size of the mycelium pellets is controlled to be less than 1 mm 3) Induced enzyme production stage: when the wet cell mass content of the mycelium grows to 5-30% of total volume, the pH value of the fermentation broth is slowly adjusted to pH 2.6-3.0 at the rate of 1 pH unit per hour. At the same time, MnCl.sub.2 is added to the final concentration of manganese ions of 0.5 mM, and induced production is carried out for 3-15 days. During induction, the size of the mycelium pellets is not more than 1 mm, and the feeding medium (soybean peptone 70-90 g/L, glucose 80-120 g/L, citric acid 1 g/L) is added from the 3rd day of induction. After the enzyme production is finished, the fermentation broth and mycelium are collected. 4) The solid-liquid separation is carried out by the method of filtering the fermentation broth and the mycelium through the plate frame filtration, and then the supernatant of the fermentation broth is concentrated by ultrafiltration and concentration. The small molecule substances in the fermentation broth are removed to obtain a concentrated solution. The concentrated solution is dried by spray drying. A protein protectant is added while spraying, and the oxalate decarboxylase powder is collected. Agrocybe aegirita mycelium is obtained through frame filtering, and dried by vacuum freeze drying to obtain dry mycelium, and the edible fungus powder containing oxalate decarboxylase is finally obtained by superfine grinding.
Embodiment 12
(47) This embodiment provides a preparation method of enzyme powder and edible fungus powder containing oxalate decarboxylase, comprising the specific steps as follows: 1) Agrocybe cylindracea is taken as the strain and a seed solution is cultured in a shake flask. The components of seed medium are as follows: yeast extract 2 g/L, soybean peptone 1 g/L, KH.sub.2PO.sub.4 3 g/L, MgSO.sub.4.7H.sub.2O 1 g/L, CaCl.sub.2 0.1 g/L, glucose 30 g/L, corn starch 10 g/L, vitamin B.sub.1 5 mg/L. The pH value is pH 5.0-6.0. The shake flask loading volume is 20-30%. The strain is cultured at 23-28° C., 100-350 rpm for 2-5 days. The mycelium pellets of seed solution are crushed by a mechanical crushing method, and then inoculated with 10-30% of inoculation to next-stage seed culture medium; 2) The seed solution mycelium pellets mechanically crushed are inoculated with 10%-30% inoculation to a 7 L mechanical-stirring fermenter for fermentation culture. The liquid fermentation medium consists of the following ingredients: yeast extract 8 g/L, soybean peptone 3 g/L, KH.sub.2PO.sub.4 1 g/L, Na.sub.2HPO.sub.4 1 g/L, MgSO.sub.4.7H.sub.2O 0.8 g/L, CaCl.sub.2 1 g/L, glucose 10 g/L, cane sugar 30 g/L, corn starch 15 g/L, vitamin B.sub.1 5 mg/L. The loading volume is 60-70% and the pH value of the culture medium is adjusted to pH 4.5-6.5. The mycelium pellets are cultured at 23-28° C., 100-350 rpm for 3-5 days after inoculation. The DO is maintained over 30% by adjusting the stirring speed. This stage is the growth stage of mycelium, and the size of the mycelium pellets is controlled to be less than 1 mm. 3) Induced enzyme production stage: when the wet cell mass content of the mycelium reaches 5-30%, the pH value of the fermentation broth is adjusted slowly to pH 2.0-2.8 at the rate of 0.5 pH unit per hour; at the same time, MnSO.sub.4 is added to the final concentration of manganese ions of 5 mM, and the induction production is carried out for 3-15 days. The size of the mycelium pellets is controlled to be not more than 0.5 mm in the induction process. A feeding medium (soybean peptone: 70-90 g/L, glucose 80-120 g/L, formic acid 2 g/L) is added from the 4th day of induction. After the enzyme production is finished, the fermentation broth and mycelium are collected respectively. 4) The solid-liquid separation is carried out by the method of filtering the fermentation broth and the mycelium through the plate frame filtration, then the supernatant of the fermentation broth is concentrated by ultrafiltration and concentration, the small molecule substances in the fermentation broth are removed, and the final concentrated solution is dried by spray drying while protein protectant is added, to obtain oxalate decarboxylase powder. Agrocybe cylindracea mycelium is obtained through frame filtering, and dried by spray drying after homogenization, wherein the inlet temperature of the spray dryer is 170-190° C., the outlet temperature is 80-95° C., and the other parameters are adjusted according to the size of the spray dryer. Edible fungus powder containing oxalic acid degrading enzyme is finally obtained.
Embodiment 13
(48) This embodiment provides a preparation method of enzyme powder and edible fungus powder containing oxalate decarboxylase, comprising the specific steps as follows: 1) Agrocybe praecox (Pers) Fayod is taken as the strain and a seed solution is cultured in a shake flask. The components of seed medium are as follows: yeast extract 3 g/L, soybean peptone 2 g/L, KH.sub.2PO.sub.4 0.5 g/L, MgSO.sub.4.7H.sub.2O 1 g/L, CaCl.sub.2 0.1 g/L, glucose 20 g/L, corn starch 20 g/L, vitamin B 20 mg/L. The pH value is pH 5.0-6.0. The shake flask loading volume is 20-30%. The strain is cultured at 23-28° C., 100-350 rpm for 2-5 days. The mycelium pellets of seed culture are crushed by a mechanical crushing method, and then inoculated with 10-30% of inoculation to next-stage seed culture medium; 2) The seed solution mycelium pellets mechanically crushed are inoculated with 10%-30% inoculation to a 7 L air-lift fermenter for fermentation culture. The liquid fermentation medium consists of the following ingredients: yeast extract 6 g/L, soybean peptone 5 g/L, KH.sub.2PO.sub.4 0.5 g/L, Na.sub.2HPO.sub.4 0.1 g/L, MgSO.sub.4.7H.sub.2O 2 g/L, CaCl.sub.2 1 g/L, glucose 20 g/L, cane sugar 15 g/L, corn starch 20 g/L, vitamin B.sub.1 20 mg/L. The loading volume is 70% and the pH value of the culture medium is adjusted to pH 4.5-6.5. The mycelium pellets are cultured at 23-28° C., 100-350 rpm for 3-5 days after inoculation. The DO is maintained at over 30% by adjusting the airflow speed. This stage is the growth stage of mycelium, and the size of the mycelium pellets is controlled to be less than 0.5 mm. 3) Induced enzyme production stage: when the wet cell mass content of the mycelium reaches 5-30%, the pH value of fermentation broth is adjusted slowly to 3.2-4.0 at the rate of 0.05-1 pH unit per hour. At the same time, Mn(NO.sub.3).sub.2 is added to the final concentration of manganese ions of 0.001 mM of, and the induction production is carried out for 3-15 days. The size of the mycelium pellets are controlled to be not more than 1 mm in the induction process. A feeding medium (soybean peptone: 70-90 g/L, glucose 80-120 g/L, glutathione 10 mM) is added from the 3rd day of induction. After the enzyme production is finished, the fermentation broth and mycelium are collected respectively. 4) The solid-liquid separation is carried out by the method of filtering the fermentation broth and the mycelium through the plate frame filtration, then the supernatant of the fermentation broth is concentrated by ultrafiltration, the small molecule substances in the fermentation broth are removed, and the final concentrated solution is dried by vacuum freeze to obtain enzyme powder, i.e., oxalate decarboxylase powder. Agrocybe praecox (Pers.) Fayod mycelium is obtained by frame filtering, and dried by spray drying after homogenization. The protectant is added when spraying, and the edible fungus powder containing oxalate decarboxylase is finally obtained.
Embodiment 14
(49) This embodiment provides a preparation method of enzyme powder and edible fungus powder containing oxalate decarboxylase, comprising the specific steps as follows: 1) Agrocybe pediades (Fr.) fayod is taken as the strain and a seed solution is cultured in a shake flask. The components of seed medium are as follows: yeast extract 3 g/L, soybean peptone 3 g/L, KH.sub.2PO.sub.4 1 g/L, MgSO.sub.4.7H.sub.2O 1 g/L, CaCl.sub.2 0.5 g/L, glucose 30 g/L, corn starch 10 g/L, vitamin B.sub.1 8 mg/L. The pH value is pH 5.0-6.0. The shake flask loading volume is 20-30%. The strain is cultured at 23-28° C., 100-350 rpm for 2-5 days. The mycelium pellets of seed culture are crushed by a mechanical crushing method, and then inoculated with 10-30% of inoculation to next-stage seed culture medium. 2) The seed solution mycelium pellets mechanically crushed are inoculated with 10%-30% inoculation to a 7 L mechanical-stirring fermenter for fermentation culture. The liquid fermentation medium consists of the following ingredients: yeast extract 5 g/L, soybean peptone 6 g/L, KH.sub.2PO.sub.4 1 g/L, Na.sub.2HPO.sub.4 0.2 g/L, MgSO.sub.4.7H.sub.2O 2 g/L, CaCl.sub.2 0.1 g/L, glucose 30 g/L, cane sugar 10 g/L, corn starch 15 g/L, vitamin B.sub.1 15 mg/L. The loading volume is 60-70% and the pH value of the culture medium is adjusted to pH 4.5-6.5. The mycelium pellets are cultured at 23-28° C., 100-350 rpm for 3-5 days after inoculation. The DO is maintained at over 30% by adjusting the stirring speed. This stage is the growth stage of mycelium, and the size of the mycelium pellets is controlled to be less than 1 mm. 3) Induced enzyme production stage: when the wet cell mass content of the mycelium reaches 5-30%, the pH value of fermentation broth is adjusted slowly to 2.8-3.2 at the rate of 0.5 pH unit per hour; at the same time, MnCl.sub.2 is added to the final concentration of manganese ions of 2 mM, and the induction production is carried out for 3-15 days. The size of the mycelium pellets is controlled to be not more than 0.5 mm in the induction process. A feeding medium (soybean peptone: 70-90 g/L, glucose 80-120 g/L, arginine 20 mM) is added from the 3rd day of induction. After the enzyme production is finished, the fermentation broth and mycelium are collected respectively. 4) The fermentation broth and the mycelia are separated by frame filtration, then the supernatant of the fermentation broth is concentrated by ultra-filtration concentration, small molecules are removed from the fermentation broth, and the final concentrate is dried by vacuum freeze-drying to obtain the enzyme powder, which is oxalate decarboxylase powder. Fermentation mycelium is obtained through frame filtering and dried by vacuum freeze drying. The mycelium obtained after drying is crushed by superfine grinding to obtain the edible fungus powder containing oxalate decarboxylase.
Embodiment 15
(50) This embodiment provides a preparation method of enzyme powder and edible fungus powder containing oxalate decarboxylase, comprising the specific steps as follows: 1) Agrocybe salicacola is taken as the strain and cultured by liquid fermentation. The components of seed medium are as follows: yeast extract 3 g/L, soy peptone 2 g/L, KH.sub.2PO.sub.4 2 g/L, MgSO.sub.4.7H.sub.2O 2 g/L, CaCl.sub.2 0.5 g/L, glucose 15 g/L, corn starch 25 g/L and vitamin B.sub.1 15 mg/L. The pH value is pH 5.0-6.0. The flask volume loading volume is 20-30%. The strain is cultured at 23-28° C., 100-350 rpm for 2-5 days. The mycelium pellets of seed culture are crushed by a mechanical crushing method, and then inoculated with 10-30% of inoculation to next-stage culture medium. 2) The seed solution mycelium pellets mechanically crushed are inoculated to a 10 L air-lift fermenter with the inoculation amount of 10-30% for fermentation culture. The liquid fermentation medium consists of the following components: yeast extract 5 g/L, soy peptone 6 g/L, KH.sub.2PO.sub.4 1 g/L, Na.sub.2HPO.sub.4 0.2 g/L, MgSO.sub.4.7H.sub.2O 2 g/L, CaCl.sub.2 0.1 g/L, glucose 30 g/L, corn starch 10 g/L and vitamin B.sub.115 mg/L. The loading volume is 60-70% and the pH value of the culture medium is adjusted to pH 4.5-6.5. The mycelium pellets are cultured at 23-28° C., 100-350 rpm for 3-5 days after inoculation. The DO is maintained above 30% by adjusting the stirring speed. This is a mycelium growth stage, and the size of the mycelium pellets is controlled to less than 1 mm; 3) Induced enzyme production stage: when the wet cell mass content of the mycelium reaches 5-30%, the pH value of the fermentation broth is slowly adjusted to pH 3.0-3.6 at the rate of decreasing 1 pH unit per hour, at the same time, induced production is performed 3 to 15 days with Mn(CH.sub.3COO).sub.2 having 50 mM of final concentration of manganese ions, the size of the mycelium pellets is controlled to not more than 0.5 mm in the induction process, a feeding medium (soybean peptone 70-90 g/L, glucose 80-120 g/L, aspartic acid 50 mM) is added from 2nd to 4th day of the induction, and after the enzyme production is finished, the fermentation broth and mycelia are collected respectively; 4) The fermentation broth and the mycelia are separated by plate-frame filtration, then the supernatant of the fermentation broth is concentrated by ultra-filtration concentration, small molecules are removed from the fermentation broth, and the final concentrate is dried by vacuum freeze-drying to obtain enzyme powder, which is oxalate decarboxylase powder. The Agrocybe salicacola mycelia obtained by plate-frame filtration are dried by vacuum drying, and the dried mycelia are crushed by ultrafine crushing to obtain the edible fungus powder containing oxalate decarboxylase.
Embodiment 16: Enzymatic Characteristics of Agrocybe Oxalate Decarboxylase
(51) 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.
(52) 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:
(53) TABLE-US-00008 TABLE 8 Determination of activity of different oxalate decarboxylases Enzymatic activity Activity of of edible enzyme powder fungus powder Strain (U/mg) (U/g) Example 10 50-90 600-2354 (Agrocybe aegerita) Example 11 45-80 500-1890 (Agrocybe aegerita) Example 12 30-72 300-1420 (Agrocybe cylindracea) Example 13 5-8 10-220 (Agrocybe praecox (pers.) fayod) Example 14 80-130 650-2624 (Agrocybe pediades (Fr.) fayod) Example 15 72-127 800-3100 (Agrocybe salicacola)
(54) 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.
(55) TABLE-US-00009 TABLE 9 Comparison of enzyme activities of natural Agrocybe aegerita and fermented Agrocybe aegerita (induced/uninduced) Enzyme activity (U/g) Natural Agrocybe aegerita powder 1-5 Fermented Agrocybe aegerita 1-3 powder (uninduced) Fermented Agrocybe aegerita 600-2354 powder (induced)
Embodiment 17: Comparison of Oxalate Decarboxylase Yield of Different Edible Fungi
(56) 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.
(57) TABLE-US-00010 TABLE 10 Comparison of oxalate decarboxylase yield of different strains Enzyme Mycelia Strain activity (U/L) amount (g/L) Agrocybe aegerita 15000-30000 12-30 Agrocybe Cylindracea 8000-21000 10-22 Agrocybe praecox 200-1500 6-13 (Pers.) Fayod Agrocybe pediades 5500-3200 4.6-8.2 (Fr.) Fayod Agrocybe salicacola 6100-14500 4.4-7.2
Embodiment 18: Pilot Scale Enlargement of Fermentation Conditions
(58) The present embodiment provides an enlarged production process of enzyme powder and edible fungus powder containing oxalate decarboxylase, comprising the specific steps as follows: 1) I-stage seed solution preparation stage: Agrocybe aegerita is taken as a strain and cultured by shake flask fermentation. The seed medium contains yeast extract 2-4 g/L, soy peptone 1-3 g/L, KH.sub.2PO.sub.4 0.5-3 g/L, MgSO.sub.4.7H.sub.2O 0.2-2 g/L, CaCl.sub.2 0.1-1 g/L, glucose 10-30 g/L, corn starch 10-30 g/L and vitamin B.sub.1 5-20 mg/L. The pH value of the culture medium is pH 5.0-6.0. Seeds are cultured in the seed solution at 23-28° C. 100-350 rpm for 2-5 days; 2) II-stage seed solution preparation stage: a 50 L fermenter is adopted for II-stage seed solution culture, and the medium is same as the I-stage seed solution medium. The seed solution is sterilized 30 min with steam of 121° C. and cooled to 23-28° C., the mycelium pellets in the I-stage seed solution of shake flask culture is crushed to less than 1 mm by mechanical crushing, and the crushed mycelium pellets are transferred to the II-stage seed culture medium with the inoculation amount of 10-30% and cultured at 23-28° C., 100-350 rpm for 2-5 days. 2) Fermentation stage: the II-stage seed mycelium pellets crushed mechanically are inoculated to a 500 L mechanical stirring fermenter with the inoculation amount of 10-30% for fermentation culture. The liquid fermentation medium consists of the following components: yeast extract 4-8 g/L, soy peptone 2-6 g/L, KH.sub.2PO.sub.4 0.5-3 g/L, Na.sub.2HPO.sub.4 0.1-1 g/L, MgSO.sub.4.7H.sub.2O 0.2-2 g/L, CaCl.sub.2 0.1-1 g/L, glucose 10-30 g/L, cane sugar 10-30 g/L, corn starch 5-20 g/L and vitamin B.sub.1 5-20 mg/L. The loading volume is 60-70% and the pH value of the culture medium is adjusted to pH 4.5-6.5. The mycelium pellets are cultured at 23-28° C., 100-350 rpm for 3-5 days after inoculation. The DO is maintained over 30% by adjusting the stirring speed. This is a mycelium growth stage, and the size of the mycelium pellets is controlled to less than 1 mm; 3) Induced enzyme production stage: when the wet cell mass content of the mycelium reaches 5-30%, the pH value of the fermentation broth is slowly adjusted to pH 3.0-3.6 at the rate of decreasing 0.5-1 pH unit per hour, at the same time, induced production is performed 3-15 days with MnCl.sub.2 having 5 mM of final concentration of manganese ions, the size of the mycelium pellets is controlled to not more than 1 mm in the induction process, a feeding medium (soybean peptone 70-90 g/L, glucose 80-120 g/L, arginine 20 mM) is added from 2nd to 4th day of the induction, and after the enzyme production is finished, fermentation broth and mycelia are collected respectively; 4) The fermentation broth and the mycelia are separated by plate-frame filtration, then the supernatant of the fermentation broth is concentrated by ultra-filtration concentration, small molecules are removed from the fermentation broth, and the final concentrate is dried by vacuum freeze-drying to obtain enzyme powder. The Agrocybe aegerita mycelia obtained by plate-frame filtration are dried by vacuum drying, and the dried mycelia are crushed by ultrafine crushing to obtain the edible fungus powder containing oxalate decarboxylase.
Embodiment 19: Comparison of Manganese Ion Content of Fermented and Natural Oxalate Decarboxylase Powder
(59) 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 (pH 3.0) 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):
(60) TABLE-US-00011 TABLE 11 Manganese ion content Natural Fermented edible fungi edible fungi Enzyme powder Not detected 1.25-4.65 (mg/g) Edible fungus powder 0.75-1.65 4.56-18.59 (mg/100 g) Enzyme powder 0.38-1.72 (mg/10000 U) Edible fungus powder 0.73-9.58 (mg/10000 U)
(61) 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.
(62) 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.