A granulation process with high retention of beta-glucosidase enzyme activity

Abstract

The present application relates to the field of genetic engineering and genetic engineering, specifically, the present application relates to a kind of granulation method of high retention beta-glucosidase enzyme activity.The method includes the following steps: beta-glucosidase fermentation liquor is sterilized and concentrated to obtain concentrated solution, 5-10% glucose is added to concentrated enzyme solution, and pH is adjusted to 4.5-5.0;Then mixed with adsorption carrier, the mixing ratio of concentrated enzyme solution and adsorption carrier is 1:2.5-3.0, and the adsorption carrier is the mixture of sodium sulfate and starch;Granulation drying after mixing.According to the method of the present application, the enzyme activity retention rate after granulation reaches 76.7%.

Description

Technical Field

[0001] This invention relates to the fields of genetic engineering and genetic engineering, and more specifically, to a granulation method that preserves high levels of β-glucosidase activity. Background Technology

[0002] β-D-Glucosidase (EC3.2.1.21), belonging to the glycoside hydrolase family, hydrolyzes β-D-glucosidic bonds bound to non-reducing ends, releasing β-D-glucose and the corresponding ligand. It is an important component of the cellulolytic enzyme system. β-Glucosidase also has significant applications in the feed industry. Soybean meal, a feed ingredient, is rich in soy isoflavones (1200–4216 mg / kg). Studies have shown that it plays an important role in promoting animal growth, anti-oxidation, improving animal health, increasing feed digestibility, enhancing animal reproductive capacity, reducing carcass fat percentage, and increasing lean meat percentage. In natural soybeans, soy isoflavones mainly exist as bound glycosides. Research indicates that converting glycoside-type soy isoflavones into free aglycones is necessary to exert their pharmacological effects. β-Glucosidase can convert bound soy isoflavones into free active aglycones. However, feed processing requires high temperatures, so improving the heat resistance of β-glucosidase is crucial for its application in animal husbandry. Summary of the Invention

[0003] The purpose of this application is to provide a granulation method that retains high levels of β-glucosidase activity.

[0004] The granulation method according to the present invention, which retains high β-glucosidase activity, comprises the following steps: The fermentation broth is filtered through a plate and frame filter press and then sterilized and concentrated using a hollow fiber membrane. 5-10% glucose is added to the concentrated enzyme solution, and the pH is adjusted to 4.5-5.0. The enzyme solution is mixed with the adsorbent carrier at a ratio of 1:2.5-3.0 (mass ratio). The adsorbent carrier is a mixture of sodium sulfate and starch, with sodium sulfate comprising 70-85 wt%. After mixing, the mixture is passed through a gyratory granulator to form irregular particles, which are then dried by a fluidized bed dryer.

[0005] To improve the retention of β-glucosidase activity, this application involves adding glucose, the catalytic product, before mixing with the carrier, and simultaneously stabilizing the enzyme's conformation by adjusting the pH value, thereby enhancing its stability during drying and storage. Although, theoretically, the catalytic product of an enzyme usually stabilizes its conformation, this is not always the case in practice. This invention combines the effects of both the enzyme product and the pH environment, thus achieving satisfactory enzyme stability.

[0006] The granulation method according to the present invention, which retains high β-glucosidase activity, wherein granulation is performed by a rocking granulator.

[0007] According to the granulation method of the present invention with high retention of β-glucosidase activity, the inlet air temperature of the fluidized bed is controlled at 70-80°C, and the drying ends after the material temperature rises to 50°C.

[0008] According to the method of the present invention, the enzyme activity retention rate after granulation reaches 76.7%. Detailed Implementation

[0009] In the following examples, the β-glucosidase fermentation broth was obtained by fermentation using the Pichia pastoris strain developed by our company, and the preparation method is based on CN 105018444 A. Example 1: Granulation processing of β-glucosidase

[0010] The β-glucosidase fermentation broth was subjected to plate and frame filtration, sterilization and concentration via hollow fiber membrane, and then granulation was carried out using the following method: Option 1: Mix 100 kg of concentrated β-glucosidase solution with an enzyme activity of 3350 U / mL with 100 kg of starch, then granulate using a swing granulator. Dry the enzyme granules in a 120-type fluidized bed, controlling the inlet air temperature at 70-80℃. After the material temperature reaches 50℃, drying is complete, and the material is discharged. The total weight is 112 kg. The theoretical enzyme activity is 2991 U / mL, the actual enzyme activity is 2960 U / mL, and the enzyme activity retention rate is 99%.

[0011] Option 2: Mix 100 kg of concentrated β-glucosidase solution with an enzyme activity of 3350 U / mL with 200 kg of mixed adsorption carrier. The mixed adsorption carrier is a mixture of sodium sulfate and starch pulverized through a 60-mesh sieve, with a sodium sulfate ratio of 80%. Granulate the mixture using a swing granulator. Dry the enzyme granules in a 120-type fluidized bed, with the fluidized bed inlet air temperature controlled at 70-80℃. After the material temperature reaches 50℃, drying is completed, and the material is discharged. The total weight is 210 kg. The theoretical enzyme activity is 1595 U / mL, and the actual enzyme activity is 1520 U / mL, with an enzyme activity retention rate of 99%.

[0012] Option 3: 100 kg of concentrated β-glucosidase solution with an enzyme activity of 3350 U / mL was added to 10 kg of glucose and fully dissolved. The pH was adjusted to 4.5 with sulfuric acid, and then mixed with 200 kg of mixed adsorption carrier. The mixed adsorption carrier was a mixture of sodium sulfate and starch pulverized through a 60-mesh sieve, with a sodium sulfate ratio of 80%. Granulation was then performed using a swing granulator. The enzyme granules were dried in a 120-type fluidized bed with the fluidized bed inlet air temperature controlled at 70-80℃. After the material temperature reached 50℃, drying was completed, and the material was discharged. The total weight was 220 kg. The theoretical enzyme activity was 1523 U / mL, the actual enzyme activity was 1520 U / mL, and the enzyme activity retention rate was 99.5%.

[0013] Based on the above-mentioned addition of different carriers, high processing retention rates were obtained using different carriers. Example 2: Enzyme Particle Stability Test

[0014] The feed ingredients consisted of a mixture of 7 parts crushed corn, 2 parts soybean meal, and 1 part wheat bran. Enzyme granules prepared in Example 1 were added at a ratio of 1 / 100. After mixing, 500g samples were taken from three different locations, mixed, and crushed as initial samples. The remaining mixed feed was fed into a pellet mill to prepare pellet feed. The material temperature was adjusted to 85-88℃ using steam. The extruded pellets were rapidly cooled to room temperature using air cooling. Then, 500g samples were taken from three different locations, mixed, and crushed as post-granulation samples. Enzyme activity was measured at each sample, and the results are shown in Table 1.

[0015] Assay for β-glucosidase activity: The activity of the enzyme hydrolyzed substrate was measured at 405 nm. p The product generated by NPG is p-nitrophenol ( p The amount of NP.

[0016] Reaction steps: Add 100 μL of 10 mM HCl to 800 μL of buffer solution. p NPG substrate and 100 μL of appropriately diluted enzyme solution were reacted at 37 °C for 10 min. The reaction was terminated by adding 1.5 mL of 1 mol / L Na₂CO₃. The OD was measured using a spectrophotometer. 405 Value. Definition of enzyme activity unit: One β-glucosidase activity unit (U) is defined as the amount of substrate that can be broken down per minute. p NPG generates 1 μmol of p-nitrophenol ( p The amount of enzyme required for NP.

[0017] Table 1 Option 1 28.6 U / g 5.3 U / g 18.5% Option 2 14.2 U / g 9.2 U / g 64.8% Option 3 15.4 U / g 11.8 U / g 76.7% .

[0018] From the perspective of actual feed pelleting, although the enzyme activity retention rate is high in different preparation processes, the enzyme preparations prepared using different pelleting processes show huge differences in enzyme activity retention during simulated feed pelleting. The pelleting product using glucose as a stabilizer (Scheme 3) achieved a relatively high retention rate.

[0019] The above embodiments are only used to understand the technical solutions of this application and do not limit the scope of protection of this application.

Claims

1. A granulation method for highly retaining β-glucosidase activity, characterized in that, The method includes the following steps: The β-glucosidase fermentation broth was sterilized and concentrated to obtain a concentrated solution. 5-10 wt% glucose was added to the concentrated enzyme solution and the pH was adjusted to 4.5-5.

0. Then the concentrated enzyme solution is mixed with the adsorption carrier, and the mixing ratio of the concentrated enzyme solution to the adsorption carrier is 1:2.5-3.

0. The adsorption carrier is a mixture of sodium sulfate and starch. After mixing, the mixture is granulated and dried.

2. The granulation method for highly retaining β-glucosidase activity according to claim 1, characterized in that, In the mixture of sodium sulfate and starch, the proportion of sodium sulfate is 70-85 wt%.

3. The granulation method for highly retaining β-glucosidase activity according to claim 1, characterized in that, The enzyme concentrate is mixed with the adsorbent carrier and then passed through a gyratory granulator to form irregular particles.

4. The granulation method for highly retaining β-glucosidase activity according to claim 1, characterized in that, Drying is carried out using a fluidized bed.

5. The granulation method for highly retaining β-glucosidase activity according to claim 4, characterized in that, The inlet air temperature of the fluidized bed is controlled at 70-80℃, and drying ends when the material temperature rises to 50℃.