A liquid glucose oxidase composite heat stabilizer

Abstract

The invention provides a composite heat stabilizer for liquid glucose oxidase, the stabilizer comprising glycerol, lactose, potassium sorbate and water. The liquid glucose oxidase comprising the stabilizer is simple to prepare, convenient to use, low in addition amount, low in price, has good biocompatibility, still has 98% enzyme activity after being treated at 60°C for 2h, can still maintain a high enzyme activity when encountering a high temperature environment during use, and still has more than 90% enzyme activity after being stored at room temperature for 6 months, which plays an important role in the industrialization of liquid glucose oxidase preparations.

Description

Technical Field

[0001] The invention relates to the field of liquid enzyme preparations, and more specifically to a composite heat stabilizer applied to liquid glucose oxidase. Background Art

[0002] Glucose oxidase (GOD) is systematically named β-D-glucose oxidoreductase (EC1.1.3.4), which can efficiently catalyze the reaction of glucose and oxygen to generate gluconic acid and hydrogen peroxide. The catalytic properties of glucose oxidase enable it to have the functions of removing glucose, deoxygenation, and sterilization, and it is safe and has no toxic side effects. Therefore, it is widely used in food processing and preservation, medicine, etc.

[0003] In recent years, glucose oxidase, as a new feed enzyme preparation to replace antibiotics, has multiple functions such as protecting animal intestines, promoting digestion and absorption, and improving animal immunity. In terms of feed application, the annual demand for glucose oxidase can reach 300,000 tons. It is estimated that the annual total output value of glucose oxidase is more than 10 billion yuan, and its demand is also increasing year by year at a rate of 10%-30% per year. However, liquid glucose oxidase often shows weak thermal stability and is easily inactivated and denatured during storage and use, which greatly limits the application and development of glucose oxidase. Therefore, if the thermal stability of liquid glucose oxidase can be effectively improved, low-temperature refrigeration can be avoided to a large extent to reduce resource waste, and the action time of glucose oxidase can be significantly extended. In short, improving the thermal stability of liquid glucose oxidase has important prospects and significance in application and research.

[0004] At present, the main methods to improve the thermal stability of enzymes are: chemical modification, immobilization, protein engineering technology, adding stabilizers, etc. The effects of different methods on improving the thermal stability of glucose oxidase are analyzed and compared based on specific cases:

[0005] Kim et al. used chitosan, acetic acid, sodium tripolyphosphate (TPP), Tween 20, glutaraldehyde (GA), ammonium sulfate, magnetic nanoparticles and other materials to immobilize glucose oxidase. The storage stability of the immobilized glucose oxidase at room temperature was significantly improved (after storage for 16 days, the residual activity can be retained by more than 90%); but on the other hand, this method uses a variety of chemical reagents (including expensive magnetic nanoparticles), the production process is complicated, and it is difficult to prepare on a large scale, and its practical application value is greatly limited {Yeon KM, You J, Adhikari MD, et al. Enzyme-Immobilized Chitosan Nanoparticles as Environmentally Friendly and Highly Effective Antimicrobial Agents[J]. Biomacromolecules, 2019, 20(7).}.

[0006] Qian Junmin et al. used the HEC / SiO2 gel complex embedding method to immobilize glucose oxidase. The optimal reaction temperature of the immobilized glucose oxidase was 4°C higher than that of the free enzyme. However, during the immobilization process, the glucose oxidase activity was nearly 50% lost; at the same time, after the immobilized glucose oxidase was kept at 50°C for 1 hour, its relative activity was only about 65% of the original activity, and its thermal stability was still poor [Qian Junmin, Li Xuxiang. Study on the immobilization of glucose oxidase by embedding HEC / SiO2 gel complex [J]. Applied Chemistry, 2002, 19(002): 153-157.].

[0007] Yao Bin and others from the Feed Research Institute of the Chinese Academy of Agricultural Sciences used protein engineering methods to transform glucose oxidase from Aspergillus niger. After five rounds of mutation, the activity of the mutant glucose oxidase-M5 was 66% higher than that of the parent, and the thermal stability was also improved; however, after being treated at 70°C for 10 minutes, the activity of the mutant was only 55% of the initial value, which is still difficult to meet the needs of actual production applications {Tu Tao, Jiang Xiao, Huang Huoqing, Luo Huiying, Yao Bin. Research on improving the thermal stability and catalytic efficiency of glucose oxidase from Aspergillus niger based on in vitro molecular evolution technology [C]}.

[0008] Du Guocheng and others from Jiangnan University used the method of adding biparental self-assembling peptides (SAPs) to improve the thermal stability of glucose oxidase. After protein engineering and comparative screening, the thermal stability of fusion enzyme 1 was significantly improved compared with the parent enzyme (10.9 times that of the initial enzyme under the same heat treatment conditions); however, after treatment at 60°C for 30 minutes, the activity of the mutant was only 67% of the initial value, which is still far from the actual production application requirements {Ren Chunhui, Zhang Juan, Du Guocheng, Chen Jian. Improving the thermal stability of glucose oxidase based on fusion biparental short peptides [J]. Journal of Biotechnology, 2018, 34(07): 1106-1116.}.

[0009] Although there is no case of using stabilizers to improve the thermal stability of liquid glucose oxidase, this method has a good effect in improving the thermal stability of other enzymes. Yin Peiqiong greatly improved the storage stability of alkaline liquid protease by adding simple and easily available stabilizers such as β-cyclodextrin, calcium chloride, and glycerol to the liquid enzyme. After the liquid enzyme with stabilizers was stored at 27°C for three months, it still retained more than 96% of its activity (the control group only had 0.5% of its activity), showing the great potential of stabilizers to improve the stability of liquid enzymes.

[0010] In summary, among the various methods for improving the thermal stability of enzymes, chemical modification and immobilization methods are complex and costly to operate, and are prone to introduce toxic and harmful chemicals, which also cause certain damage to the catalytic activity of the enzyme; protein engineering technology generally requires a large amount of computer design of the enzyme molecular structure, and the establishment of efficient high-throughput screening methods. The thermal stability and activity of the enzyme must be taken into account during the modification process, which is technically difficult and has limited effect on improving the stability of the enzyme; on the other hand, adding thermal stabilizers to liquid enzyme preparations to improve their stability is the most economical and effective method, which has the advantages of low cost, simple operation, non-toxicity, and significant effect. Summary of the invention

[0011] The present invention aims to solve one of the technical problems in the related art at least to a certain extent. To this end, the inventors of the present application preferably select substances that can promote the stable existence of liquid enzymes, mix them in a certain proportion to form a composite enzyme stabilizer, and add it to liquid glucose oxidase to enhance its thermal stability.

[0012] According to an embodiment of the present invention, the composite thermal stabilizer applied to liquid glucose oxidase has the following technical features:

[0013] According to an embodiment of the present invention, the composite heat stabilizer applied to liquid glucose oxidase comprises two or three selected from glycerol, lactose, potassium sorbate, and water.

[0014] According to an embodiment of the present invention, the composite thermal stabilizer applied to liquid glucose oxidase comprises glycerol, lactose and water, the weight ratio of glycerol to lactose is 2:1, and the weight of water is the sum of the weights of other raw materials.

[0015] According to an embodiment of the present invention, the composite heat stabilizer applied to liquid glucose oxidase comprises glycerol, potassium sorbate and water, the weight ratio of glycerol to potassium sorbate is 5:1, and the weight of water is the sum of the weights of other raw materials.

[0016] According to an embodiment of the present invention, the composite heat stabilizer applied to liquid glucose oxidase comprises lactose, potassium sorbate and water, the weight ratio of lactose to potassium sorbate is 5:1, and the weight of water is the sum of the weights of other raw materials.

[0017] According to an embodiment of the present invention, the composite thermal stabilizer applied to liquid glucose oxidase comprises glycerol, lactose, potassium sorbate and water, the weight ratio of glycerol, lactose and potassium sorbate is (15-25):(5-15):(1-3), and the weight of water is the sum of the weights of other raw materials.

[0018] According to an embodiment of the present invention, the composite thermal stabilizer applied to liquid glucose oxidase comprises glycerol, lactose, potassium sorbate and water, the weight ratio of glycerol, lactose and potassium sorbate is 10:5:1, and the weight of water is the sum of the weights of other raw materials.

[0019] According to an embodiment of the present invention, in the liquid glucose oxidase comprising a composite thermal stabilizer, the mass ratio of the liquid glucose oxidase to the composite thermal stabilizer is 20:(1-5).

[0020] According to an embodiment of the present invention, in the liquid glucose oxidase comprising a composite thermal stabilizer, the mass ratio of the liquid glucose oxidase to the composite thermal stabilizer is 10:1.

[0021] The inventors found that water and one or two other components in the composite heat stabilizer were added to the liquid glucose oxidase at low concentrations, which did not significantly improve its thermal stability or only slightly improved it. However, when the components were combined in a certain proportion and added to the liquid glucose oxidase at low concentrations, its thermal stability was greatly improved, and the liquid glucose oxidase could still retain 98% of its activity after being treated at 60°C for 2 hours, and still retain 91% of its activity after being stored at 30°C for 6 months. The composite heat stabilizer applied to liquid glucose oxidase of the present invention is simple to prepare, easy to use, low in addition amount, low in price, has good biocompatibility, does not cause pollution to the environment, can enable glucose oxidase to still maintain a high enzyme activity when encountering a high temperature environment during use, prolongs its action time, and can significantly reduce the enzyme activity loss when stored at room temperature, which plays an important role in the industrialization of glucose oxidase liquid preparations. DETAILED DESCRIPTION

[0022] In order to clearly illustrate the technical features of this solution, the present invention is described in detail below through specific implementation methods.

[0023] Example 1

[0024] The preparation of the biological enzyme stabilizer is prepared by uniformly mixing the following components: 1 part of glycerol and 3 parts of water.

[0025] Thermal stability test: 0.5 g of the prepared bio-enzyme stabilizer was added to 10 g of liquid glucose oxidase enzyme solution, stored at 60°C for 2 h, and activity was measured.

[0026] Example 2

[0027] The preparation of the biological enzyme stabilizer is prepared by uniformly mixing the following components: glycerol, 1 part; water, 2 parts.

[0028] Thermal stability test: 0.5 g of the prepared bio-enzyme stabilizer was added to 10 g of liquid glucose oxidase enzyme solution, stored at 60°C for 2 h, and activity was measured.

[0029] Example 3

[0030] The preparation of the biological enzyme stabilizer is prepared by uniformly mixing the following components: 1 part of glycerol and 1 part of water.

[0031] Thermal stability test: 0.5 g of the prepared bio-enzyme stabilizer was added to 10 g of liquid glucose oxidase enzyme solution, stored at 60°C for 2 h, and activity was measured.

[0032] Example 4

[0033] The preparation of the biological enzyme stabilizer is prepared by uniformly mixing the following components: lactose, 1 part; water, 10 parts.

[0034] Thermal stability test: 0.5 g of the prepared bio-enzyme stabilizer was added to 10 g of liquid glucose oxidase enzyme solution, stored at 60°C for 2 h, and activity was measured.

[0035] Example 5

[0036] The preparation of the biological enzyme stabilizer is prepared by uniformly mixing the following components: lactose, 1 part; water, 5 parts.

[0037] Thermal stability test: 0.5 g of the prepared bio-enzyme stabilizer was added to 10 g of liquid glucose oxidase enzyme solution, stored at 60°C for 2 h, and activity was measured.

[0038] Example 6

[0039] The preparation of the biological enzyme stabilizer is prepared by uniformly mixing the following components: lactose, 1 part; water, 3 parts.

[0040] Thermal stability test: 0.5 g of the prepared bio-enzyme stabilizer was added to 10 g of liquid glucose oxidase enzyme solution, stored at 60°C for 2 h, and activity was measured.

[0041] Example 7

[0042] The preparation of the biological enzyme stabilizer is prepared by uniformly mixing the following components: 1 part of potassium sorbate and 10 parts of water.

[0043] Thermal stability test: 0.5 g of the prepared bio-enzyme stabilizer was added to 10 g of liquid glucose oxidase enzyme solution, stored at 60°C for 2 h, and activity was measured.

[0044] Example 8

[0045] The preparation of the biological enzyme stabilizer is prepared by uniformly mixing the following components: 1 part of potassium sorbate and 30 parts of water.

[0046] Thermal stability test: 0.5 g of the prepared bio-enzyme stabilizer was added to 10 g of liquid glucose oxidase enzyme solution, stored at 60°C for 2 h, and activity was measured.

[0047] Example 9

[0048] The preparation of the biological enzyme stabilizer is prepared by uniformly mixing the following components: 1 part of potassium sorbate and 50 parts of water.

[0049] Thermal stability test: 0.5 g of the prepared bio-enzyme stabilizer was added to 10 g of liquid glucose oxidase enzyme solution, stored at 60°C for 2 h, and activity was measured.

[0050] Example 10

[0051] The preparation of the biological enzyme stabilizer is prepared by uniformly mixing the following components: 10 parts of glycerol; 5 parts of lactose; and 15 parts of water.

[0052] Thermal stability test: 0.5 g of the prepared bio-enzyme stabilizer was added to 10 g of liquid glucose oxidase enzyme solution, stored at 60°C for 2 h, and activity was measured.

[0053] Embodiment 11

[0054] The preparation of the biological enzyme stabilizer is prepared by uniformly mixing the following components: 10 parts of glycerol; 1 part of potassium sorbate; and 11 parts of water.

[0055] Thermal stability test: 0.5 g of the prepared bio-enzyme stabilizer was added to 10 g of liquid glucose oxidase enzyme solution, stored at 60°C for 2 h, and activity was measured.

[0056] Example 12

[0057] The preparation of the biological enzyme stabilizer is prepared by uniformly mixing the following components: lactose, 10 parts; potassium sorbate, 2 parts; water, 12 parts.

[0058] Thermal stability test: 0.5 g of the prepared bio-enzyme stabilizer was added to 10 g of liquid glucose oxidase enzyme solution, stored at 60°C for 2 h, and activity was measured.

[0059] Embodiment 13

[0060] The preparation of the biological enzyme stabilizer is prepared by uniformly mixing the following components: 15 parts of glycerol; 5 parts of lactose; 1 part of potassium sorbate; and 21 parts of water. Thermal stability test: 0.5 g of the prepared biological enzyme stabilizer is added to 10 g of liquid glucose oxidase enzyme solution, stored at 60° C. for 2 hours, and then the activity is measured.

[0061] Embodiment 14

[0062] The preparation of the biological enzyme stabilizer is prepared by uniformly mixing the following components: glycerol, 20 parts; lactose, 5 parts; potassium sorbate, 1 part; water, 26 parts. Thermal stability test: 0.5 g of the prepared biological enzyme stabilizer is added to 10 g of liquid glucose oxidase enzyme solution, stored at 60°C for 2 hours, and then the activity is measured.

[0063] Embodiment 15

[0064] The preparation of the biological enzyme stabilizer is prepared by uniformly mixing the following components: glycerol, 25 parts; lactose, 5 parts; potassium sorbate, 1 part; water, 31 parts. Thermal stability test: 0.5 g of the prepared biological enzyme stabilizer is added to 10 g of liquid glucose oxidase enzyme solution, stored at 60°C for 2 hours, and then the activity is measured.

[0065] Example 16

[0066] The preparation of the biological enzyme stabilizer is prepared by uniformly mixing the following components: 20 parts of glycerol; 10 parts of lactose; 1 part of potassium sorbate; and 31 parts of water.

[0067] Thermal stability test: 0.5 g of the prepared bio-enzyme stabilizer was added to 10 g of liquid glucose oxidase enzyme solution, stored at 60°C for 2 h, and activity was measured.

[0068] Embodiment 17

[0069] The preparation of the biological enzyme stabilizer is prepared by uniformly mixing the following components: 20 parts of glycerol; 15 parts of lactose; 1 part of potassium sorbate; and 36 parts of water.

[0070] Thermal stability test: 0.5 g of the prepared bio-enzyme stabilizer was added to 10 g of liquid glucose oxidase enzyme solution, stored at 60°C for 2 h, and activity was measured.

[0071] Embodiment 18

[0072] The preparation of the biological enzyme stabilizer is prepared by uniformly mixing the following components: glycerol, 20 parts; lactose, 10 parts; potassium sorbate, 2 parts; water, 32 parts. Thermal stability test: 0.5 g of the prepared biological enzyme stabilizer is added to 10 g of liquid glucose oxidase enzyme solution, stored at 60°C for 2 hours, and then the activity is measured.

[0073] Embodiment 19

[0074] The preparation of the biological enzyme stabilizer is prepared by uniformly mixing the following components: 20 parts of glycerol; 10 parts of lactose; 3 parts of potassium sorbate; and 33 parts of water.

[0075] Thermal stability test: 0.5 g of the prepared bio-enzyme stabilizer was added to 10 g of liquid glucose oxidase enzyme solution, stored at 60°C for 2 h, and activity was measured.

[0076] Embodiment 20

[0077] The preparation of the biological enzyme stabilizer is prepared by uniformly mixing the following components: 20 parts of glycerol; 10 parts of lactose; 2 parts of potassium sorbate; and 32 parts of water.

[0078] Thermal stability test: Take 1 g of the prepared bio-enzyme stabilizer, add it to 10 g of liquid glucose oxidase enzyme solution, store it at 60°C for 2 h, and measure the activity.

[0079] Embodiment 21

[0080] The preparation of the biological enzyme stabilizer is prepared by uniformly mixing the following components: 20 parts of glycerol; 10 parts of lactose; 2 parts of potassium sorbate; and 32 parts of water.

[0081] Thermal stability test: 1.5 g of the prepared bio-enzyme stabilizer was added to 10 g of liquid glucose oxidase enzyme solution, stored at 60°C for 2 h, and activity was measured.

[0082] Embodiment 22

[0083] Blank control: Take 0.5 g of water and add it to 10 g of liquid glucose oxidase enzyme solution, store it at 60°C for 2 h, and then measure the activity.

[0084] Embodiment 23

[0085] The preparation of the biological enzyme stabilizer is prepared by uniformly mixing the following components: 20 parts of glycerol; 10 parts of lactose; 2 parts of potassium sorbate; and 32 parts of water.

[0086] Thermal stability test: Take 1 g of the prepared bio-enzyme stabilizer, add it to 10 g of liquid glucose oxidase enzyme solution, store it at 30°C for 1 month, and measure the activity.

[0087] Embodiment 24

[0088] The preparation of the biological enzyme stabilizer is prepared by uniformly mixing the following components: 20 parts of glycerol; 10 parts of lactose; 2 parts of potassium sorbate; and 32 parts of water.

[0089] Thermal stability test: Take 1 g of the prepared bio-enzyme stabilizer, add it to 10 g of liquid glucose oxidase enzyme solution, store it at 30°C for 2 months, and measure the activity.

[0090] Embodiment 25

[0091] The preparation of the biological enzyme stabilizer is prepared by uniformly mixing the following components: 20 parts of glycerol; 10 parts of lactose; 2 parts of potassium sorbate; and 32 parts of water.

[0092] Thermal stability test: Take 1g of the prepared bio-enzyme stabilizer, add it to 10g of liquid glucose oxidase enzyme solution, store it at 30℃ for 3 months, and measure the activity.

[0093] Embodiment 26

[0094] The preparation of the biological enzyme stabilizer is prepared by uniformly mixing the following components: 20 parts of glycerol; 10 parts of lactose; 2 parts of potassium sorbate; and 32 parts of water.

[0095] Thermal stability test: Take 1g of the prepared bio-enzyme stabilizer, add it to 10g of liquid glucose oxidase enzyme solution, store it at 30℃ for 4 months, and measure the activity.

[0096] Embodiment 27

[0097] The preparation of the biological enzyme stabilizer is prepared by uniformly mixing the following components: 20 parts of glycerol; 10 parts of lactose; 2 parts of potassium sorbate; and 32 parts of water.

[0098] Thermal stability test: 1 g of the prepared bio-enzyme stabilizer was added to 10 g of liquid glucose oxidase enzyme solution, stored at 30°C for 5 months, and then the activity was measured.

[0099] Embodiment 28

[0100] The preparation of the biological enzyme stabilizer is prepared by uniformly mixing the following components: 20 parts of glycerol; 10 parts of lactose; 2 parts of potassium sorbate; and 32 parts of water.

[0101] Thermal stability test: 1 g of the prepared bio-enzyme stabilizer was added to 10 g of liquid glucose oxidase enzyme solution, stored at 30°C for 6 months, and then the activity was measured.

[0102] In the present invention, the glucose oxidase enzyme activity determination method is as follows:

[0103] Add 2.5 ml of o-dianisidine, 0.3 ml of 18% glucose, and 0.1 ml of horseradish peroxidase in sequence, preheat at 37°C for 5 min, then add 0.1 ml of diluted enzyme solution, react at 37°C, accurately time for 3 min, and terminate the reaction with 2 mol / L sulfuric acid.

[0104] The configuration of each solution required in the enzyme activity assay is as follows:

[0105] 1. Phosphate buffer (pH=6.0): Weigh 10.524 g of anhydrous sodium dihydrogen phosphate and 4.4 g of disodium hydrogen phosphate dodecahydrate, adjust the pH to 6.0 with sodium dihydrogen phosphate solution or disodium hydrogen phosphate solution, filter, and store at room temperature. The shelf life is one month.

[0106] 2. 1% o-dianisidine solution: Weigh 0.1 g of o-dianisidine into a 10 mL volumetric flask, add anhydrous methanol and shake to dissolve. After adjusting the volume, place in a brown bottle, seal with sealing film and store in a refrigerator. The shelf life is one month.

[0107] 3. 100U / mL horseradish peroxidase solution: Prepare 100mL of 100U / mL peroxidase solution with water according to the activity of the purchased horseradish peroxidase. Dispense into centrifuge tubes and freeze for storage.

[0108] 4. 2 mol / L sulfuric acid solution: Measure 109 mL of concentrated sulfuric acid in water and dilute to 1000 mL.

[0109] 5. o-Dianisidine buffer: Take 100 μL of 1% o-Dianisidine solution, add it to 12 mL of phosphate buffer, mix well, and store in a dark place. Prepare it just before use.

[0110] The definition of glucose oxidase activity is: at 37°C and pH 6.0, the amount of enzyme required to catalyze the oxidation of glucose to produce 1 μmol of hydrogen peroxide per minute is one enzyme activity unit U.

[0111] The calculation method of glucose oxidase activity is as follows:

[0112] (1) The enzyme activity XD of glucose oxidase, expressed in U / mL, is calculated as follows:

[0113]

[0114] Where:

[0115] XD is the activity of glucose oxidase in the diluted enzyme solution, unit: U / mL;

[0116] A is the absorbance of the enzyme reaction solution;

[0117] K is the slope of the standard curve;

[0118] CO is the intercept of the standard curve;

[0119] t is the enzymatic reaction time, in min;

[0120] 34.01 is the relative molecular weight of hydrogen peroxide.

[0121] The XD value should be between 0.45U / mL and 0.65U / mL. If it is not within this range, the enzyme solution dilution should be reselected before analysis and determination.

[0122] Where:

[0123] X=XD×Df

[0124] X is the activity of glucose oxidase in the sample, in U / ml;

[0125] XD is the activity of glucose oxidase in the diluted enzyme solution, unit: U / mL;

[0126] Df is the total dilution factor of the sample.

[0127] Table 1 Stabilizer screening

[0128]

[0129] Table 2 Effect of stabilizers on the thermal stability of glucose oxidase

[0130]

[0131] It is not difficult to see from Table 1 that after adding the stabilizer, the thermal stability of the liquid glucose oxidase has been significantly improved, among which Example 20 has the best effect. It can be seen from Table 2 that under the condition of adding the optimal stabilizer formula, the liquid glucose oxidase still maintains 91.6% activity after being stored at 30°C for 6 months.

[0132] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A composite heat stabilizer for liquid glucose oxidase, characterized in that: It contains glycerol, lactose, potassium sorbate and water; the weight ratio of glycerol, lactose and potassium sorbate is (15-25): (5-15): (1-3), and the weight of water is the sum of the weights of other raw materials.

2. The composite heat stabilizer for liquid glucose oxidase according to claim 1, characterized in that: It comprises glycerol, lactose, potassium sorbate and water, wherein the weight ratio of glycerol, lactose and potassium sorbate is 10:5:1, and the weight of water is the sum of the weights of other raw materials.

3. A liquid glucose oxidase comprising the composite thermal stabilizer according to any one of claims 1 to 2, characterized in that: The mass ratio of liquid glucose oxidase to composite thermal stabilizer is 20:(1-5).

4. The liquid glucose oxidase comprising the composite thermal stabilizer according to claim 3, characterized in that: The mass ratio of liquid glucose oxidase to the composite thermal stabilizer is 10:1.

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