A method for producing β-glucosidase using Chaetomium sphaerocephalum DX-THS3 and its application

Abstract

The present invention belongs to the technical field of fungal fermentation, and the present invention provides a method and application of producing β-glucosidase using Chaetomium sphaerocephalum DX-THS3. The present invention controls the morphology of Chaetomium sphaerocephalum DX-THS3 by adding different nitrogen sources, and finally obtains that the β-glucosidase yield of Chaetomium sphaerocephalum DX-THS3 is 3.5 times that of the unoptimized nitrogen source. The diameter of the small spheres of Chaetomium sphaerocephalum DX-THS3 under the condition of 7g / L peptone is 1.5±0.65mm, which can hydrolyze isoliquiritigenin in licorice into isoliquiritigenin. In addition, the present invention adopts a 5L bioreactor for β-glucosidase expression, and under the optimal fermentation conditions, the β-glucosidase activity can reach 680U / mL, which provides a strategy for designing the morphology of filamentous fungi using nitrogen sources when industrially producing isoliquiritigenin.

Description

Technical Field

[0001] The invention relates to the technical field of fungal fermentation, and in particular to a method for producing beta-glucosidase by utilizing Chaetomium globosum DX-THS3 and application thereof. Background Art

[0002] Isoliquiritin (ISL) is one of the main bioactive flavonoid components of licorice extract. It has been widely used in the food and pharmaceutical industries because of its physiological activities such as antioxidant, anti-ulcer, treatment of depressive psychosis, AIDS, and adjuvant treatment of diabetes. However, its absorption rate is low and it has multiple adverse effects in animals and humans, which limits its application. Fortunately, isoliquiritin can be hydrolyzed to produce isoliquiritigenin, which has stronger physiological activity, higher bioavailability, and fewer adverse reactions. At present, isoliquiritigenin can be prepared by chemical synthesis or biotransformation. In general, chemical synthesis has poor selectivity, ineffective cost, and causes environmental pollution. The biotransformation of isoliquiritigenin uses β-D-glucosidase (GUS, EC3.2.1.21), which has the advantages of high yield, high selectivity, and environmental friendliness, and has more potential than chemical synthesis. Therefore, it should be strongly considered to study how to efficiently and selectively synthesize isoliquiritigenin to produce isoliquiritigenin.

[0003] β-Glucosidase can be screened from a wide range of microorganisms, plants and animals; most of the isoliquiritigenin produced by the bioconversion of isoliquiritigenin is mainly isolated from microorganisms, especially filamentous fungi, which cannot be directly obtained from the host due to its low content. Therefore, fermentation is the most promising method for producing β-glucosidase using filamentous fungi, but the complex morphological structure of filamentous fungi significantly reduces the mass and oxygen transfer during the fermentation process, hindering its application in production. For example, compared with other fungal morphologies, the granular morphology of filamentous fungi can significantly increase the yield of citric acid or itaconic acid, and there is evidence that the filamentous morphology of fungi has a significant effect on the production of isoliquiritigenin during fermentation. Therefore, designing the morphology of filamentous fungi has become one of the challenges for large-scale production of β-glucosidase or isoliquiritigenin by fungal fermentation.

[0004] In general, the morphology of filamentous fungi is difficult to control, especially during bioreactor fermentation. The morphology of filamentous fungi has an important impact on their metabolism and products during fermentation. In submerged liquid fermentation, filamentous fungi can grow in a variety of morphologies, including free hyphae, hyphae dispersed in the fermentation broth; smooth, fuzzy hyphae masses, with highly entangled hyphae networks visible in the core; loose hyphae masses, large densely aggregated hyphae. The morphology of filamentous fungi during fermentation can be affected by many factors, including pH, medium position, inoculation amount, medium shear, culture temperature, medium viscosity, etc. Previous studies have studied the morphology of filamentous fungi and attempted to engineer them to improve their fermentation performance. However, the morphological control of filamentous fungi is difficult, especially during submerged fermentation. Few scholars have successfully engineered the morphology of filamentous fungi and reported significant improvements in fermentation performance. Hasan B et al. showed that the addition of alumina and talc to Aspergillus fermentation medium significantly increased phytase activity. Zhang et al. found that soybean meal hydrolysate as a nitrogen source had a significant effect on the morphology and fumaric acid production during the subsequent fermentation of Rhizopus oryzae. In our previous study, we screened out a highly efficient and highly substrate-selective GUS (called cg-GUS) from the endophytic fungus Chaetomium globosum DX-THS3. The fermentation process of GAMG and cg-GUS by DX-THS3 was studied in a shake flask. The bacterial morphology of DX-THS3 was suitable for the production of GUS. The particle morphology of DX-THS3 was designed by adding 10 g / L of silica (600 mesh) to the fermentation medium, and 576 U / mL of GUS activity was obtained. However, the process engineering of adding silica faces challenges such as low cost, equipment damage, and product separation difficulties, which brings difficulties to the large-scale production of GUS in bioreactors. Therefore, studying a correct and appropriate production method is of great significance for the large-scale production of β-glucosidase and designing fungal morphology without any additional conditions.

[0005] Nitrogen sources have an important influence on microbial fermentation products. Xing et al. analyzed the effects of five nitrogen sources on fumaric acid production: they found that different nitrogen sources had a great influence on fumaric acid. Li et al. reported that different nitrogen sources had a significant effect on the growth of fermentation cells, glucose utilization and ethanol production of Zymomonas mobile. At present, there are no reports on the effect of nitrogen sources on the fermentation production of β-glucosidase by Chaetomium sphaerocephalum DX-THS3. Summary of the invention

[0006] In order to overcome the above-mentioned defects in the prior art, the present invention controls the morphology of the bulbous algae DX-THS3 by adding different nitrogen sources, and provides a method and application of producing high-activity β-glucosidase using Chaetomium sphaerocephalum DX-THS3.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The present invention provides a method for producing β-glucosidase by using Chaetomium sphaerocephalum DX-THS3, comprising the following steps:

[0009] (1) placing Chaetomium globosum DX-THS3 on a seed culture medium for shaking culture to obtain a Chaetomium globosum DX-THS3 seed suspension;

[0010] (2) placing the above-obtained Chaetomium DX-THS3 seed suspension on a fermentation medium for fermentation culture to obtain a Chaetomium DX-THS3 fermentation product, wherein the nitrogen source of the fermentation medium comprises one of yeast extract, beef extract, peptone, soybean meal and corn syrup powder; and the concentration of the nitrogen source in the fermentation medium is 2 to 7 g / L;

[0011] (3) The fermentation product of Chaetomium sphaerocephalum DX-THS3 obtained above was subjected to amplified fermentation culture to obtain the amplified fermentation culture product of Chaetomium sphaerocephalum DX-THS3, and samples were taken every 11 to 13 hours to determine the dry weight of the bacteria and the enzyme activity.

[0012] Preferably, the inoculation amount of the Chaetomium sphaerocephalum DX-THS3 in step (1) is 1 to 4% of the volume of the seed culture medium, and the seed culture medium uses water as a solvent and comprises the following components in concentrations: 180 to 220 g / L of potato, 18 to 22 g / L of glucose and 0.1 to 0.3 g / L of yeast extract.

[0013] Preferably, the frequency of the shaking culture in step (1) is 120-180 rpm, the temperature of the shaking culture is 25-30° C., and the time of the shaking culture is 2-4 days.

[0014] Preferably, the fermentation medium in step (2) uses water as solvent and includes components with the following concentrations: sucrose 4-6 g / L, KH2PO4 3-4 g / L, nitrogen source 2-7 g / L, NaCl 0.4-0.6 g / L, MgSO4·7H2O 0.4-0.6 g / L, ZnSO4·7H2O 0.002-0.003 g / L, NaMoO4·2H2O 0.0002-0.0003 g / L, MnSO4·H2O 0.001-0.003 g / L, CaCl2 0.01-0.02 g / L and H3BO3 0.00002-0.00004 g / L.

[0015] Preferably, the inoculation amount of the fermentation culture in step (2) is 2-4%;

[0016] The fermentation culture temperature is 25-30°C;

[0017] The fermentation culture time is 6 to 10 days;

[0018] The fermentation culture is a shaking culture, and the shaking frequency is 120-180 rpm.

[0019] Preferably, each 2.5 L culture medium of the scaled-up fermentation culture in step (3) comprises 12-13 g sucrose, 6-10 g KH2PO4, 17-18 g peptone, 1-2 g NaCl, 1-2 g MgSO4·7H2O, 0.007-0.008 g ZnSO4·7H2O, 0.0005-0.0007 g NaMoO4·2H2O, 0.004-0.006 g MnSO4·H2O, 0.03-0.04 g CaCl2, and 0.00007-0.00008 g H3BO3.

[0020] Preferably, the amplified fermentation culture process in step (3) further includes a stirring step, the stirring frequency is 180-220 rpm, and the stirring aeration volume is 0.5-1.5 vvm.

[0021] Preferably, the temperature of the amplified fermentation culture in step (3) is 25-30° C., and the time of the amplified fermentation culture is 150-200 h.

[0022] The present invention also provides a Chaetomium DX-THS3 enlarged fermentation culture product obtained by the above-mentioned method of producing beta-glucosidase by utilizing Chaetomium DX-THS3.

[0023] The present invention also provides the use of the amplified fermentation culture product of Chaetomium sphaerocephalum DX-THS3 in promoting the hydrolysis and conversion of isoliquiritigenin into isoliquiritigenin.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] The present invention studies the influence of nitrogen source on GUS expression and morphological changes of C. globosum DX-THS3 under shake flask culture conditions. The present invention controls the morphology of C. globosum DX-THS3 by adding different nitrogen sources, and finally obtains that the GUS yield of C. globosum DX-THS3 is 3.5 times that of the unoptimized nitrogen source. The diameter of the small spheres of C. globosum DX-THS3 under 7g / L peptone conditions is 1.5±0.65mm. In addition, the present invention uses a 5L bioreactor for GUS expression, and under optimal fermentation conditions, the GUS activity can reach 680U / mL, which provides a strategy for designing the morphology of filamentous fungi using nitrogen sources during the industrial production of GAMG. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Bioconversion of licorice to produce isoliquiritigenin by Chaetomium sphaerocephalum DX-THS3;

[0027] Figure 2 The liquid phase diagram of the biotransformation process of Chaetomium sphaerocephala DX-THS3 in the solid-state fermentation of licorice (S108 gc is the fermentation sample of Chaetomium sphaerocephala DX-THS3 and licorice; gc is the control group of uninoculated licorice samples);

[0028] Figure 3 The isoliquiritigenin was isolated and purified from the solid-state fermentation biotransformation of licorice by Chaetomium sphaerocephalum DX-THS3. 13 C NMR spectra;

[0029] Figure 4 The isoliquiritigenin was isolated and purified from the solid-state fermentation biotransformation of licorice by Chaetomium sphaerocephalum DX-THS3. 13 H NMR spectra. DETAILED DESCRIPTION

[0030] The present invention provides a method for producing β-glucosidase by using Chaetomium sphaerocephalum DX-THS3, comprising the following steps:

[0031] (1) placing Chaetomium globosum DX-THS3 on a seed culture medium for shaking culture to obtain a Chaetomium globosum DX-THS3 seed suspension;

[0032] (2) placing the above-obtained Chaetomium DX-THS3 seed suspension on a fermentation medium for fermentation culture to obtain a Chaetomium DX-THS3 fermentation product, wherein the nitrogen source of the fermentation medium comprises one of yeast extract, beef extract, peptone, soybean meal and corn syrup powder; and the concentration of the nitrogen source in the fermentation medium is 2 to 7 g / L;

[0033] (3) The fermentation product of Chaetomium sphaerocephalum DX-THS3 obtained above was subjected to amplified fermentation culture to obtain the amplified fermentation culture product of Chaetomium sphaerocephalum DX-THS3, and samples were taken every 11 to 13 hours to determine the dry weight of the bacteria and the enzyme activity.

[0034] In the present invention, the inoculation amount of the Chaetomium sphaerocephalum DX-THS3 in step (1) is preferably 1 to 4% of the volume of the seed culture medium, and more preferably 3%; the seed culture medium, with water as a solvent, preferably comprises components with the following concentrations: 180 to 220 g / L of potato, 18 to 22 g / L of glucose and 0.1 to 0.3 g / L of yeast extract, and more preferably comprises components with the following concentrations: 200 g / L of potato, 20 g / L of glucose and 0.2 g / L of yeast extract.

[0035] In the present invention, the frequency of the shaking culture in step (1) is preferably 120-180 rpm, more preferably 140-160 rpm, and further preferably 150 rpm. The temperature of the shaking culture is preferably 25-30°C, and further preferably 28°C. The time of the shaking culture is preferably 2-4 d, and further preferably 3 d.

[0036] In the present invention, the fermentation medium in step (2) uses water as a solvent, and preferably includes the following components in concentrations: sucrose 4-6 g / L, KH2PO4 3-4 g / L, nitrogen source 2-7 g / L, NaCl 0.4-0.6 g / L, MgSO4·7H2O 0.4-0.6 g / L, ZnSO4·7H2O 0.002-0.003 g / L, NaMoO4·2H2O 0.0002-0.0003 g / L, MnSO4·H2O 0.001-0.003 g / L, CaCl2 0.01-0.02 g / L and H3BO3 0.00002-0.00004 g / L, and further preferably includes the following components in concentrations: sucrose 5 g / L, KH2PO4 3.2 g / L, nitrogen source 3 g / L, NaCl 0.5g / L, MgSO4·7H2O 0.5g / L, ZnSO4·7H2O 0.0029g / L, NaMoO4·2H2O 0.00024g / L, MnSO4·H2O 0.002g / L, CaCl20.014g / L and H3BO30.00003g / L.

[0037] In the present invention, the nitrogen source in step (2) comprises one of yeast extract, yeast extract, beef extract, peptone, soybean meal and corn syrup powder, and is more preferably peptone; the concentration of the nitrogen source in the fermentation medium is 2 to 7 g / L, and is more preferably 3 g / L.

[0038] In the present invention, the inoculation amount of the fermentation culture in step (2) is preferably 2-4%, more preferably 3%;

[0039] The fermentation culture temperature is preferably 25-30°C, more preferably 28°C;

[0040] The fermentation culture time is preferably 6 to 10 days, more preferably 8 days;

[0041] The fermentation culture is preferably a shaking culture, and the frequency of the shaking is preferably 120 to 180 rpm, more preferably 140 to 160 rpm, and even more preferably 150 rpm.

[0042] In the present invention, the inoculation amount of the amplified fermentation culture in step (3) is preferably 2-10%, and more preferably 5%.

[0043] In the present invention, each 2.5L culture medium of the enlarged fermentation culture in step (3) preferably comprises 12-13g sucrose, 6-10g KH2PO4, 17-18g peptone, 1-2g NaCl, 1-2g MgSO4·7H2O, 0.007-0.008g ZnSO4·7H2O, 0.0005-0.0007g NaMoO4·2H2O, 0.004-0.006g MnSO4·H2O, 0.03-0.04g CaCl2, and 0.00007-0.00008g H3BO3, and further preferably comprises 12.5g sucrose, 8g KH2PO4, 17.5g peptone, 1.25g NaCl, 1.25g MgSO4·7H2O, and 0.006g ZnSO4·7H2O. 0.00725g, NaMoO4·2H2O 0.0006g, MnSO4·H2O0.005g, CaCl2 0.035g, H3BO3 0.000075g.

[0044] In the present invention, the amplified fermentation culture process in step (3) also preferably includes a stirring step, and the stirring frequency is preferably 180-220 rpm, more preferably 200 rpm, and the aeration volume of the stirring is preferably 0.5-1.5 vvm, more preferably 0.8-1.2 vvm, and even more preferably 1 vvm.

[0045] In the present invention, the temperature of the amplified fermentation culture in step (3) is preferably 25-30°C, more preferably 28°C, and the time of the amplified fermentation culture is preferably 150-200h, more preferably 180h.

[0046] In the present invention, the sampling in step (3) is performed every 11 to 13 hours, and more preferably every 12 hours.

[0047] The present invention also provides a Chaetomium DX-THS3 enlarged fermentation culture product obtained by the above-mentioned method of producing beta-glucosidase by utilizing Chaetomium DX-THS3.

[0048] The present invention also provides the use of the amplified fermentation culture product of Chaetomium sphaerocephalum DX-THS3 in promoting the hydrolysis and conversion of isoliquiritigenin into isoliquiritigenin.

[0049] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0050] The Chaetomium globosum DX-THS3 in the following examples was preserved in China Center for Type Culture Collection (CCTCC, preservation number: CCTCC NO: M 2016005), and was isolated from Dongxiang wild rice. All culture media in the following examples were sterilized at 121° C. for 25 min.

[0051] Example 1

[0052] (1) placing Chaetomium sphaeroceum DX-THS3 on a seed culture medium and shaking culture it at 120 rpm and 25° C. for 2 days to obtain a Chaetomium sphaeroceum DX-THS3 seed suspension, wherein the seed culture medium uses water as a solvent and includes the following components in concentrations: 180 g / L potato, 18 g / L glucose, and 0.1 g / L yeast extract;

[0053] (2) placing the obtained Chaetomium DX-THS3 seed suspension on a fermentation medium and fermenting it at 120 rpm and 25° C. for 6 days to obtain a fermentation product of Chaetomium DX-THS3, wherein the inoculum amount of the fermentation culture is 2%, and the fermentation medium uses water as a solvent and comprises components of the following concentrations: sucrose 4 g / L, KH2PO4 3 g / L, yeast extract 7 g / L, NaCl 0.4 g / L, MgSO4·7H2O 0.4 g / L, ZnSO4·7H2O 0.002 g / L, NaMoO4·2H2O 0.0002 g / L, MnSO4·H2O 0.001 g / L, CaCl2 0.01 g / L and H3BO3 0.00002 g / L;

[0054] (3) The fermentation product of Chaetomium sphaerocephalum DX-THS3 obtained above was placed in a 5L bioreactor for amplified fermentation culture at 25°C, a stirring frequency of 180rpm, and an aeration volume of 0.5vvm for 150h. Each 2.5L culture medium of the amplified fermentation culture includes 12g sucrose, 6g KH2PO4, 7g peptone, 1g NaCl, 1g MgSO4·7H2O, 0.007g ZnSO4·7H2O, 0.0005g NaMoO4·2H2O, 0.004g MnSO4·H2O, 0.03g CaCl2, and 0.00007g H3BO3. Samples were taken every 11h to determine the dry weight of the bacteria and the enzyme activity. All experiments were repeated 3 times, and the values ​​were reported as standard deviations.

[0055] Example 2

[0056] (1) placing Chaetomium sphaeroceum DX-THS3 on a seed culture medium and shaking culture it at 150 rpm and 28° C. for 3 days to obtain a Chaetomium sphaeroceum DX-THS3 seed suspension, wherein the seed culture medium uses water as a solvent and includes the following components in concentrations: 200 g / L potato, 20 g / L glucose, and 0.2 g / L yeast extract;

[0057] (2) placing the obtained Chaetomium DX-THS3 seed suspension on a fermentation medium and fermenting it at 150 rpm and 28° C. for 8 days to obtain a fermentation product of Chaetomium DX-THS3, wherein the inoculum size of the fermentation culture is 3%, and the fermentation medium uses water as a solvent and comprises components of the following concentrations: sucrose 5 g / L, KH2PO4 3.2 g / L, peptone 7 g / L, NaCl 0.5 g / L, MgSO4·7H2O 0.5 g / L, ZnSO4·7H2O 0.0029 g / L, NaMoO4·2H2O 0.00024 g / L, MnSO4·H2O 0.002 g / L, CaCl2 0.014 g / L and H3BO3 0.00003 g / L;

[0058] (3) The fermentation product of Chaetomium sphaerocephalum DX-THS3 obtained above was placed in a 5L bioreactor for amplified fermentation culture at 28°C, a stirring frequency of 150rpm, and an aeration volume of 1vvm for 180h. Each 2.5L culture medium of the amplified fermentation culture includes sucrose 12.5g, KH2PO4 8g, peptone 17.5g, NaCl 1.25g, yeast extract 0.125g, MgSO4·7H2O1.25g, ZnSO4·7H2O 0.00725g, NaMoO4·2H2O 0.0006g, MnSO4·H2O 0.005g, CaCl2 0.035g, and H3BO3 0.000075g. Samples were taken every 12h to determine the dry weight of the bacteria and the enzyme activity. All experiments were repeated 3 times, and the values ​​were reported as standard deviations.

[0059] Example 3

[0060] (1) placing Chaetomium sphaeroceum DX-THS3 on a seed culture medium and shaking culture it at 150 rpm and 28° C. for 3 days to obtain a Chaetomium sphaeroceum DX-THS3 seed suspension, wherein the seed culture medium uses water as a solvent and includes the following components in concentrations: 200 g / L potato, 20 g / L glucose, and 0.2 g / L yeast extract;

[0061] (2) placing the obtained Chaetomium DX-THS3 seed suspension on a fermentation medium and fermenting it at 150 rpm and 28° C. for 8 days to obtain a fermentation product of Chaetomium DX-THS3, wherein the inoculum amount of the fermentation culture is 3%, and the fermentation medium uses water as a solvent and comprises the following components in concentrations: 5 g / L sucrose, 3.2 g / L KH2PO4, 6 g / L beef extract, 0.5 g / L NaCl, 0.5 g / L MgSO4·7H2O, 0.0029 g / L ZnSO4·7H2O, 0.00024 g / L NaMoO4·2H2O, 0.002 g / L MnSO4·H2O, 0.014 g / L CaCl2 and 0.00003 g / L H3BO3;

[0062] (3) The fermentation product of Chaetomium sphaerocephalum DX-THS3 obtained above was placed in a 5L bioreactor for amplified fermentation culture at 28°C, with a stirring frequency of 200rpm and an aeration volume of 1.5vvm for 180h. Each 2.5L culture medium of the amplified fermentation culture includes 12.5g sucrose, 8g KH2PO4, 17.5g peptone, 1.25g NaCl, 0.125g yeast extract, 1.25g MgSO4·7H2O, 0.00725g ZnSO4·7H2O, 0.0006g NaMoO4·2H2O, 0.005g MnSO4·H2O, 0.035g CaCl2, and 0.000075g H3BO3. Samples were taken every 12h to determine the dry weight of the bacteria and the enzyme activity. All experiments were repeated 3 times, and the values ​​were reported as standard deviations.

[0063] Example 4

[0064] (1) placing Chaetomium sphaeroceum DX-THS3 on a seed culture medium and shaking culture it at 140 rpm and 28° C. for 3 days to obtain a Chaetomium sphaeroceum DX-THS3 seed suspension, wherein the seed culture medium uses water as a solvent and includes the following components in concentrations: 200 g / L potato, 20 g / L glucose, and 0.2 g / L yeast extract;

[0065] (2) placing the obtained Chaetomium DX-THS3 seed suspension on a fermentation medium and fermenting it at 140 rpm and 28° C. for 8 days to obtain a fermentation product of Chaetomium DX-THS3, wherein the inoculation amount of the fermentation culture is 3%, and the fermentation medium uses water as a solvent and includes the following components in concentrations: 5 g / L sucrose, 3.2 g / L KH2PO4, 8 g / L soybean meal, 0.5 g / L NaCl, 0.5 g / L MgSO4·7H2O, 0.0029 g / L ZnSO4·7H2O, 0.00024 g / L NaMoO4·2H2O, 0.002 g / L MnSO4·H2O, 0.014 g / L CaCl2 and 0.00003 g / L H3BO3;

[0066] (3) The fermentation product of Chaetomium sphaerocephalum DX-THS3 obtained above was placed in a 5L bioreactor at 28°C, with a stirring frequency of 200rpm and an aeration volume of 1vvm for amplified fermentation culture for 180h. Each 2.5L culture medium of the amplified fermentation culture includes 12.5g sucrose, 8g KH2PO4, 17.5g peptone, 1.25g NaCl, 0.125g yeast extract, 1.25g MgSO4·7H2O, 0.00725g ZnSO4·7H2O, 0.0006g NaMoO4·2H2O, 0.005g MnSO4·H2O, 0.035g CaCl2, and 0.000075g H3BO3. Samples were taken every 12h to determine the dry weight of the bacteria and the enzyme activity. All experiments were repeated 3 times, and the values ​​were reported as standard deviations.

[0067] Example 5

[0068] (1) placing Chaetomium sphaeroceum DX-THS3 on a seed culture medium and shaking culture it at 160 rpm and 28° C. for 3 days to obtain a Chaetomium sphaeroceum DX-THS3 seed suspension, wherein the seed culture medium uses water as a solvent and includes the following components in concentrations: 200 g / L potato, 20 g / L glucose, and 0.2 g / L yeast extract;

[0069] (2) placing the obtained Chaetomium DX-THS3 seed suspension on a fermentation medium and fermenting it at 160 rpm and 28° C. for 8 days to obtain a fermentation product of Chaetomium DX-THS3, wherein the inoculum amount of the fermentation culture is 3%, and the fermentation medium uses water as a solvent and comprises the following components in concentrations: sucrose 5 g / L, KH2PO4 3.2 g / L, corn syrup powder 8 g / L, NaCl 0.5 g / L, MgSO4·7H2O 0.5 g / L, ZnSO4·7H2O 0.0029 g / L, NaMoO4·2H2O 0.00024 g / L, MnSO4·H2O 0.002 g / L, CaCl2 0.014 g / L and H3BO3 0.00003 g / L;

[0070] (3) The fermentation product of Chaetomium sphaerocephalum DX-THS3 obtained above was placed in a 5L bioreactor at 28°C, with a stirring frequency of 200rpm and an aeration volume of 1vvm for amplified fermentation culture for 180h. Each 2.5L culture medium of the amplified fermentation culture includes 12.5g sucrose, 8g KH2PO4, 17.5g peptone, 1.25g NaCl, 0.125g yeast extract, 1.25g MgSO4·7H2O, 0.00725g ZnSO4·7H2O, 0.0006g NaMoO4·2H2O, 0.005g MnSO4·H2O, 0.035g CaCl2, and 0.000075g H3BO3. Samples were taken every 12h to determine the dry weight of the bacteria and the enzyme activity. All experiments were repeated 3 times, and the values ​​were reported as standard deviations.

[0071] Comparative Example 1

[0072] (1) placing Chaetomium sphaeroceum DX-THS3 on a seed culture medium and shaking culture it at 120 rpm and 25° C. for 2 days to obtain a Chaetomium sphaeroceum DX-THS3 seed suspension, wherein the seed culture medium uses water as a solvent and includes the following components in concentrations: 180 g / L potato, 18 g / L glucose, and 0.1 g / L yeast extract;

[0073] (2) placing the obtained Chaetomium DX-THS3 seed suspension on a fermentation medium and fermenting it at 120 rpm and 25° C. for 6 days to obtain a fermentation product of Chaetomium DX-THS3, wherein the inoculum amount of the fermentation culture is 2%, and the fermentation medium uses water as a solvent and comprises the following components in concentrations: sucrose 4 g / L, KH2PO4 3 g / L, (NH4)2SO4 2 g / L, NaCl 0.4 g / L, yeast extract 0.04 g / L, MgSO4·7H2O 0.4 g / L, ZnSO4·7H2O 0.002 g / L, NaMoO4·2H2O 0.0002 g / L, MnSO4·H2O 0.001 g / L, CaCl2 0.01 g / L and H3BO3 0.00002 g / L;

[0074] (3) The fermentation product of Chaetomium sphaerocephalum DX-THS3 obtained above was placed in a 5L bioreactor for amplified fermentation culture at 25°C, a stirring frequency of 180rpm, and an aeration volume of 0.5vvm for 150h. Each 2.5L culture medium of the amplified fermentation culture includes sucrose 12g, KH2PO4 6g, peptone 17g, NaCl 1g, yeast extract 0.1g, MgSO4·7H2O 1g, ZnSO4·7H2O 0.007g, NaMoO4·2H2O 0.0005g, MnSO4·H2O 0.004g, CaCl2 0.03g, and H3BO3 0.00007g. Samples were taken every 11h to determine the dry weight of the bacteria and the enzyme activity. All experiments were repeated 3 times, and the values ​​were reported as standard deviations.

[0075] Comparative Example 2

[0076] (1) placing Chaetomium sphaeroceum DX-THS3 on a seed culture medium and shaking culture it at 150 rpm and 28° C. for 3 days to obtain a Chaetomium sphaeroceum DX-THS3 seed suspension, wherein the seed culture medium uses water as a solvent and includes the following components in concentrations: 200 g / L potato, 20 g / L glucose, and 0.2 g / L yeast extract;

[0077] (2) placing the obtained Chaetomium DX-THS3 seed suspension on a fermentation medium and fermenting it at 150 rpm and 28° C. for 8 days to obtain a fermentation product of Chaetomium DX-THS3, wherein the inoculum amount of the fermentation culture is 3%, and the fermentation medium uses water as a solvent and comprises the following components in concentrations: sucrose 5 g / L, KH2PO4 3.2 g / L, NH4Cl 3 g / L, NaCl 0.5 g / L, yeast extract 0.05 g / L, MgSO4·7H2O 0.5 g / L, ZnSO4·7H2O 0.0029 g / L, NaMoO4·2H2O 0.00024 g / L, MnSO4·H2O 0.002 g / L, CaCl2 0.014 g / L and H3BO3 0.00003 g / L;

[0078] (3) The fermentation product of Chaetomium sphaerocephalum DX-THS3 obtained above was placed in a 5L bioreactor at 28°C, with a stirring frequency of 200rpm and an aeration volume of 1vvm for amplified fermentation culture for 180h. Each 2.5L culture medium of the amplified fermentation culture includes 12.5g sucrose, 8g KH2PO4, 17.5g peptone, 1.25g NaCl, 1.25g MgSO4·7H2O, 0.00725g ZnSO4·7H2O, 0.0006g NaMoO4·2H2O, 0.005g MnSO4·H2O, 0.035g CaCl2, and 0.000075g H3BO3. Samples were taken every 12h to determine the dry weight of the bacteria and the enzyme activity. All experiments were repeated 3 times, and the values ​​were reported as standard deviations.

[0079] Comparative Example 3

[0080] (1) placing Chaetomium sphaeroceum DX-THS3 on a seed culture medium and shaking cultured at 180 rpm and 30° C. for 4 days to obtain a Chaetomium sphaeroceum DX-THS3 seed suspension, wherein the seed culture medium uses water as a solvent and includes the following components in concentrations: 220 g / L potato, 22 g / L glucose, and 0.3 g / L yeast extract;

[0081] (2) placing the obtained Chaetomium DX-THS3 seed suspension on a fermentation medium and fermenting it at 180 rpm and 30° C. for 10 days to obtain a fermentation product of Chaetomium DX-THS3, wherein the inoculum amount of the fermentation culture is 4%, and the fermentation medium uses water as a solvent and comprises the following components: sucrose 6 g / L, KH2PO4 4 g / L, NaNO3 4 g / L, NaCl 0.6 g / L, yeast extract 0.06 g / L, MgSO4·7H2O 0.6 g / L, ZnSO4·7H2O 0.003 g / L, NaMoO4·2H2O 0.0003 g / L, MnSO4·H2O 0.003 g / L, CaCl2 0.02 g / L and H3BO3 0.00004 g / L;

[0082] (3) The fermentation product of Chaetomium sphaerocephalum DX-THS3 obtained above was placed in a 5L bioreactor for amplified fermentation culture at 30°C, with a stirring frequency of 220rpm and an aeration volume of 1.5vvm for 200h. Each 2.5L culture medium of the amplified fermentation culture includes 12.5g sucrose, 8g KH2PO4, 17.5g peptone, 1.25g NaCl, 1.25g MgSO4·7H2O, 0.00725g ZnSO4·7H2O, 0.0006g NaMoO4·2H2O, 0.005g MnSO4·H2O, 0.035g CaCl2, and 0.000075g H3BO3. Samples were taken every 13h to determine the dry weight of the bacteria and the enzyme activity. All experiments were repeated 3 times, and the values ​​were reported as standard deviations.

[0083] Comparative Example 4

[0084] (1) placing Chaetomium sphaeroceum DX-THS3 on a seed culture medium and shaking culture it at 150 rpm and 28° C. for 3 days to obtain a Chaetomium sphaeroceum DX-THS3 seed suspension, wherein the seed culture medium uses water as a solvent and includes the following components in concentrations: 200 g / L potato, 20 g / L glucose, and 0.2 g / L yeast extract;

[0085] (2) placing the obtained Chaetomium DX-THS3 seed suspension on a fermentation medium and fermenting it at 150 rpm and 28° C. for 8 days to obtain a fermentation product of Chaetomium DX-THS3, wherein the inoculum amount of the fermentation culture is 3%, and the fermentation medium uses water as a solvent and comprises the following components in concentrations: sucrose 5 g / L, KH2PO4 3.2 g / L, NH4NO3 3 g / L, NaCl 0.5 g / L, yeast extract 0.05 g / L, MgSO4·7H2O 0.5 g / L, ZnSO4·7H2O 0.0029 g / L, NaMoO4·2H2O 0.00024 g / L, MnSO4·H2O 0.002 g / L, CaCl2 0.014 g / L and H3BO3 0.00003 g / L;

[0086] (3) The fermentation product of Chaetomium sphaerocephalum DX-THS3 obtained above was placed in a 5L bioreactor at 28°C, with a stirring frequency of 200rpm and an aeration volume of 1vvm for amplified fermentation culture for 180h. Each 2.5L culture medium of the amplified fermentation culture includes 12.5g sucrose, 8g KH2PO4, 17.5g peptone, 1.25g NaCl, 1.25g MgSO4·7H2O, 0.00725g ZnSO4·7H2O, 0.0006g NaMoO4·2H2O, 0.005g MnSO4·H2O, 0.035g CaCl2, and 0.000075g H3BO3. Samples were taken every 12h to determine the dry weight of the bacteria and the enzyme activity. All experiments were repeated 3 times, and the values ​​were reported as standard deviations.

[0087] Comparative Example 5

[0088] (1) placing Chaetomium sphaeroceum DX-THS3 on a seed culture medium and shaking culture it at 150 rpm and 28° C. for 3 days to obtain a Chaetomium sphaeroceum DX-THS3 seed suspension, wherein the seed culture medium uses water as a solvent and includes the following components in concentrations: 200 g / L potato, 20 g / L glucose, and 0.2 g / L yeast extract;

[0089] (2) placing the obtained Chaetomium DX-THS3 seed suspension on a fermentation medium and fermenting it at 150 rpm and 28° C. for 8 days to obtain a fermentation product of Chaetomium DX-THS3, wherein the inoculum amount of the fermentation culture is 3%, and the fermentation medium uses water as a solvent and comprises the following components in concentrations: sucrose 5 g / L, KH2PO4 3.2 g / L, urea 3 g / L, NaCl 0.5 g / L, yeast extract 0.05 g / L, MgSO4·7H2O 0.5 g / L, ZnSO4·7H2O 0.0029 g / L, NaMoO4·2H2O 0.00024 g / L, MnSO4·H2O 0.002 g / L, CaCl2 0.014 g / L and H3BO3 0.00003 g / L;

[0090] (3) The fermentation product of Chaetomium sphaerocephalum DX-THS3 obtained above was placed in a 5L bioreactor at 28°C, with a stirring frequency of 200rpm and an aeration volume of 1vvm for amplified fermentation culture for 180h. Each 2.5L culture medium of the amplified fermentation culture includes 12.5g sucrose, 8g KH2PO4, 17.5g peptone, 1.25g NaCl, 0.125g yeast extract, 1.25g MgSO4·7H2O, 0.00725g ZnSO4·7H2O, 0.0006g NaMoO4·2H2O, 0.005g MnSO4·H2O, 0.035g CaCl2, and 0.000075g H3BO3. Samples were taken every 12h to determine the dry weight of the bacteria and the enzyme activity. All experiments were repeated 3 times, and the values ​​were reported as standard deviations.

[0091] Experimental Example 1

[0092] The effects of different nitrogen sources on the morphological properties of Chaetomium DX-THS3 and the GUS activity and biomass produced by Chaetomium DX-THS3 were studied: based on the nitrogen source in the fermentation medium, the morphological properties of Chaetomium DX-THS3 and the GUS activity and biomass produced by Chaetomium DX-THS3 in Examples 1-5 and Comparative Examples 1-5 were compared, and the results are shown in Table 1.

[0093] Table 1 Comparison of morphological properties of Chaetomium sphaerocephalum DX-THS3 under different nitrogen sources

[0094]

[0095] As shown in Table 1, different nitrogen sources have a significant effect on the GUS activity and biomass of Chaetomium sphaerocephalum DX-THS3. Peptone is the best nitrogen source for Chaetomium sphaerocephalum DX-THS3 to produce GUS. When 7g / L peptone is used, the GUS activity reaches 680U / mL. The second is yeast extract (GUS activity is 480U / mL), followed by beef extract, soybean meal, corn syrup powder, NH4NO3, NaNO3, and urea (GUS activities are 395, 405, 252, 128, 172, 128, and 55U / mL, respectively). When (NH4)2SO4 or NH4Cl is used as the nitrogen source for Chaetomium sphaerocephalum DX-THS3 fermentation, no GUS activity is detected.

[0096] It can also be seen from Table 1 that the tested nitrogen sources have a significant effect on the morphology of Chaetomium DX-THS3 during the fermentation process. The morphology of Chaetomium DX-THS3 was observed in the experiment: for yeast extract, black, smooth, dense, small particles with a diameter of 2.2±0.8mm; for peptone, black ellipsoidal particles, smooth and dense particles with a diameter of 1.5±0.65mm; for beef extract, gray, piercing, small block particles with a diameter of 2.2±1.20mm; for (NH4)2SO4 and NH4Cl, white thorny free hyphae; for NaNO3, gray thorny pellets with a particle size of 3.2±1.0mm; for NH4NO3, gray thorny pellets with a diameter of 3.0±1.0mm; for urea, gray thorny pellets with a diameter of 2.9±1.0mm; for corn syrup powder, gray rod-shaped particles with a diameter of 2.6±1.0mm; for soybean meal, gray thorny pellets with a particle size of 2.9±1.22mm. The experimental results of the present invention show that the particles of Chaetomium globosum DX-THS3 are smooth and dense, with a diameter of 1.5 mm (that is, peptone is used as the nitrogen source in the fermentation medium), which is most conducive to the production of GUS in submerged fermentation.

[0097] In this study, Chaetomium DX-THS3 was constructed by adding peptone to the culture medium to form pellets with a diameter of 1.5 mm. This resulted in similar GUS activity. Compared with the addition of silica, the use of peptone to engineer the morphology of Chaetomium DX-THS3 has significant advantages, including low cost and suitability for large-scale industrial production (using bioreactors). In submerged culture, filamentous fungi grow either in a spherical or filamentous form, with fungal pellets being either smooth and coagulated or spiny and non-coagulated. The morphogenesis of filamentous fungi has a crucial influence on the viscosity of the fermentation broth, which in turn significantly affects the material transfer of the culture medium, including carbon source, nitrogen source, and oxygen. Different types of spherical pellets of Chaetomium DX-THS3 were observed in submerged culture in this study and our previous studies. When Chaetomium DX-THS3 was grown with (NH4)2SO4 and NH4Cl as nitrogen sources, spinnable and non-coagulated types were obtained. This type of morphology of Chaetomium DX-THS3 severely hindered the production of GUS in submerged culture, as the viscosity of the broth medium was greatly increased, which significantly hindered the transfer of materials. In addition, the echinococcoid type of the morphology of Chaetomium DX-THS3 increased the viscosity of the broth medium, which severely hindered the transfer of materials. Therefore, GUS activity was low in this morphology of Chaetomium DX-THS. It is worth noting that the size of the smooth pellets of Chaetomium DX-THS3 also had an effect on GUS production. The large-sized pellet-type fungal morphology not only limits the accessibility of nutrients, but also hinders the secretion of products from the inner hyphae. It can be seen that the morphological type of Chaetomium DX-THS3 with smooth pellets of approximately 1.5 mm in diameter is most conducive to the production of GUS (Table 1).

[0098] Experimental Example 2

[0099] Taking Example 2 as an example, the effects of different peptone concentrations (1, 3, 5, 7 and 9 g / L) in the fermentation medium on the fermentation production of GUS by Chaetomium globosum DX-THS3 were studied.

[0100] 7g / L peptone (i.e., the solution in Example 2) is most conducive to the production of GUS. Chaetomium sphaerocephalum DX-THS3 was placed in a 5L bioreactor with 2.5L culture medium for fermentation for the production of GUS. The GUS yield and growth performance of DX-THS3 were evaluated. In the initial period (0-38h), DX-THS3 grew rapidly and accumulated a large amount of biomass. The biomass reached a maximum value of 4.87g / L at 72h. GUS activity was detected during the fermentation process. Compared with the biomass of Chaetomium sphaerocephalum DX-THS3, GUS activity was rarely detected in the early stage of fermentation (0-48h). GUS is rapidly produced by Chaetomium sphaerocephalum DX-THS3. The maximum GUS activity was detected at 680U / mL at 120h, and then the GUS activity decreased rapidly. During the fermentation process, the total nitrogen source concentration continued to drop from 2.15g / L to 0.87g / L. The total carbon source concentration was rapidly utilized by Chaetomium sphaerocephalum DX-THS3 in the initial fermentation period (0-48 h), and then slowly decreased after 48 h. The pH value was also detected during the fermentation process. At the initial stage pH value, the lowest pH value of 4.75 was reached at 36 h. The medium was acidic from 0 to 60 h, and the pH value increased and stabilized at 7.88.

[0101] Cell morphology can significantly affect the operation of bioreactors and the production of GUS in microbial fermentation. The most suitable morphology for the production of GUS by Chaetomium sphaerocephalum DX-THS3 strain is a 1 mm diameter pellet. Previous studies have found that the spherical morphology significantly increased the fumaric acid production of Rhizopus oryzae NRRL 1526. Therefore, pellets with a diameter of less than 1 mm are considered to be the ideal fermentation morphology in bioreactors due to their lower medium viscosity, lower possibility of wrapping around the impeller, and better mass transfer performance. In addition, the fermentation characteristics of Chaetomium sphaerocephalum DX-THS3 were analyzed to provide a reference for the industrial production of GUS. The growth curve showed that the biomass of Chaetomium sphaerocephalum DX-THS3 accumulated rapidly in the early stage of fermentation (0-70 h), and most of the carbon source was utilized. Due to the production of abundant carbon dioxide, the pH of the fermentation broth was significantly reduced. However, the production of GUS began when the strain consumed most of the carbon source. According to the biological activity of GUS in previous studies, it can be seen that Chaetomium sphaerocephalum DX-THS3 first utilizes some carbon sources such as glucose and fructose, and then produces GUS to degrade carbon sources that are difficult to utilize. Therefore, it is recommended to provide moderate availability of carbon sources such as glucose at the early stage of fermentation to allow rapid accumulation of biomass and produce sufficient amount of GUS by fermentation of Chaetomium sphaerocephalum DX-THS3.

[0102] By inoculating the bacterial pellet or bacterial solution obtained in Example 2 into solid licorice, isoliquiritigenin in licorice can be converted into isoliquiritigenin, such as Figure 1 , Figure 2 The transformed product was separated and purified by preparative liquid phase to obtain the transformed product, which was confirmed to be isoliquiritigenin by nuclear magnetic resonance spectroscopy 1HNMR and 13CNMR, as shown in FIG. Figure 3 , Figure 4 shown.

[0103] In summary, the research results of the present invention show that different nitrogen sources have a significant effect on the production of GUS by Chaetomium sphaerocephalum DX-THS3. In particular, peptone has the most significant promoting effect on the production of GUS by the strain. For further analysis, peptone was used to transform the morphology of DX-THS3 to form a small ball with a diameter of 1.5 mm. Under optimal conditions, DX-THS3 was fermented in a bioreactor to obtain a higher GUS activity (680U / mL). The present invention provides a possible strategy for large-scale production of GUS and designing fungal morphology without additional conditions.

[0104] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for producing β-glucosidase using Chaetomium sphaerocephalum DX-THS3, characterized in that: The steps include: (1) Placing Chaetomium globosum DX-THS3 on a seed culture medium for shaking culture to obtain a Chaetomium globosum DX-THS3 seed suspension; (2) placing the obtained Chaetomium DX-THS3 seed suspension on a fermentation medium for fermentation culture to obtain a Chaetomium DX-THS3 fermentation product, wherein the nitrogen source of the fermentation medium comprises one of yeast extract, beef extract, peptone, soybean meal and corn syrup powder; and the concentration of the nitrogen source in the fermentation medium is 2-7 g / L; (3) The fermentation product of Chaetomium sphaerocephalum DX-THS3 obtained above was subjected to amplified fermentation culture to obtain the amplified fermentation culture product of Chaetomium sphaerocephalum DX-THS3, and samples were taken every 11 to 13 hours to determine the dry weight of the fungus and the enzyme activity.

2. The method for producing β-glucosidase using Chaetomium sphaerocephalum DX-THS3 according to claim 1, characterized in that: The inoculation amount of the Chaetomium sphaerocephalum DX-THS3 in step (1) is 1-4% of the volume of the seed culture medium. The seed culture medium uses water as a solvent and comprises the following components in the following concentrations: 180-220 g / L potato, 18-22 g / L glucose and 0.1-0.3 g / L yeast extract.

3. A method for producing β-glucosidase using Chaetomium sphaerocephalum DX-THS3 according to claim 1 or 2, characterized in that: The frequency of the shaking culture in step (1) is 120-180 rpm, the temperature of the shaking culture is 25-30° C., and the time of the shaking culture is 2-4 days.

4. The method for producing β-glucosidase using Chaetomium sphaerocephalum DX-THS3 according to claim 3, characterized in that: The fermentation medium in step (2) uses water as solvent and includes the following components in concentrations: sucrose 4-6 g / L, KH2PO4 3-4 g / L, nitrogen source 2-7 g / L, NaCl 0.4-0.6 g / L, MgSO4·7H2O 0.4-0.6 g / L, ZnSO4·7H2O 0.002-0.003 g / L, NaMoO4·2H2O 0.0002-0.0003 g / L, MnSO4·H2O 0.001-0.003 g / L, CaCl2 0.01-0.02 g / L and H3BO3 0.00002-0.00004 g / L.

5. A method for producing β-glucosidase using Chaetomium sphaerocephalum DX-THS3 according to claim 1 or 4, characterized in that: The inoculation amount of the fermentation culture in step (2) is 2-4%; The fermentation culture temperature is 25-30°C; The fermentation culture time is 6 to 10 days; The fermentation culture is a shaking culture, and the frequency of the shaking is 120-180 rpm.

6. The method for producing β-glucosidase using Chaetomium sphaerocephalum DX-THS3 according to claim 1, characterized in that: Each 2.5 L culture medium of the scaled-up fermentation culture in step (3) comprises 12-13 g sucrose, 6-10 g KH2PO4, 17-18 g peptone, 1-2 g NaCl, 1-2 g MgSO4·7H2O, 0.007-0.008 g ZnSO4·7H2O, 0.0005-0.0007 g NaMoO4·2H2O, 0.004-0.006 g MnSO4·H2O, 0.03-0.04 g CaCl2, and 0.00007-0.00008 g H3BO3.

7. The method for producing β-glucosidase using Chaetomium globosum DX-THS3 according to claim 6, characterized in that: The amplified fermentation culture process in step (3) also includes a stirring step, the stirring frequency is 180-220 rpm, and the stirring aeration volume is 0.5-1.5 vvm.

8. The method for producing β-glucosidase using Chaetomium globosum DX-THS3 according to claim 7, characterized in that: The temperature of the amplified fermentation culture in step (3) is 25-30° C., and the time of the amplified fermentation culture is 150-200 h.

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