Method for improving enzymatic activity of laccase produced by ganoderma lucidum

Abstract

The invention discloses a method for improving enzyme activity of laccase produced by ganoderma lucidum, aims to improve laccase production capacity of ganoderma lucidum, and belongs to the technical field of microbial ferment.The method includes the steps that dominant strains are selected in the mode that strains are screened from all stages for fermentation enzyme production, so that the enzyme activity of laccase used for follow-up production is improved, and the yield of laccase is improved. The method mainly comprises the following steps: (1) screening preservation bacteria; (2) strain recovery and screening; (3) strain amplification culture and screening; and (4) screening strain fermentation enzyme production seed liquid. According to the method, the ganoderma lucidum is screened in different stages, so that the ganoderma lucidum in each stage has the best performance, and the final laccase activity is improved. The enzyme activity of the ganoderma lucidum laccase is improved, the stability of the ganoderma lucidum laccase can be improved, and it is ensured that the enzyme activity of each batch can range from 140000 u / L to 160000 u / L.

Description

Technical Field

[0001] This invention belongs to the field of microbial fermentation technology and relates to a method for improving the activity of laccase produced by Ganoderma lucidum. Background Technology

[0002] In 2019, my country's tobacco leaf production reached approximately 1.6 million tons, generating about 400,000 tons of tobacco stems. Tobacco stems are a byproduct of the tobacco industry. The cellulose in the cell walls of tobacco stems produces a hot and harsh smoke with a burnt paper-like odor when burned, while lignin contributes to a bitter taste. Furthermore, tobacco stems have low levels of endogenous enzymes, resulting in slow degradation of cellulose and lignin. These quality defects lead to the waste of hundreds of thousands of tons of tobacco stems annually, causing significant resource waste and environmental pollution. To improve the quality of tobacco raw materials, the industry has recently sought to use biotechnology to degrade lignin and cellulose, aiming to improve the usability of tobacco raw materials and reduce cigarette costs. Literature indicates that after appropriate fermentation, the woody odor and burning sensation of tobacco stems are reduced, significantly improving the quality and usability of the tobacco raw materials.

[0003] Fermentation of tobacco stems has broad application prospects and has the following advantages compared with commercial enzymes: (1) The enzyme activity of commercial enzymes is unstable and will be greatly lost if not handled properly; (2) Commercial enzymes are highly specific and have limited substrates for single enzyme action, mainly concentrated on a certain component or class of components in tobacco; (3) Commercial enzymes are expensive and will increase costs if used in large quantities.

[0004] Laccase is a polyphenol oxidase with unique catalytic properties, which can be used in the tobacco industry to improve the quality of tobacco stems. However, during the cultivation and enzyme production stages of Ganoderma lucidum, the growth state of the Ganoderma lucidum is unstable, and the enzyme activity of the laccase produced by the fermentation varies greatly, reaching as high as 180,000 enzyme activities and as low as only 80,000 enzyme activities. In order to better apply this bio-fermentation technology to the industrial production of tobacco stem fermentation and reduce the bio-fermentation costs for production enterprises, it is necessary to ensure the stability of enzyme activity in each batch of fermentation broth. Summary of the Invention

[0005] The purpose of this invention is to address the problem of low laccase activity in Ganoderma lucidum fermentation by providing a method and its application for improving laccase activity in Ganoderma lucidum, thereby increasing the stability of laccase production and improving the utilization rate of laccase in tobacco stem fermentation.

[0006] To achieve the above objectives, one embodiment of the present invention adopts the following technical solution:

[0007] A method for increasing the laccase activity of Ganoderma lucidum includes the following steps:

[0008] Screening of preservative strains: Select the highest weight seed liquid from multiple Ganoderma lucidum seed liquids, centrifuge and preserve them;

[0009] Strain resuscitation and screening: The seed culture to be preserved is revived, and colonies with larger and more uniform morphology are selected for rejuvenation;

[0010] Strain expansion and screening: Rejuvenated Ganoderma lucidum strains with more abundant mycelium were transferred and expanded in multiple containers, and the bacterial solution in the heavier container was selected for the next step.

[0011] Seed culture screening for enzyme production by microbial fermentation: The screened microbial culture was cultured in multiple containers for a second expansion, and the microbial culture in the heavier container was selected for enzyme production in the fermenter.

[0012] Optionally, in step (1) of the method for improving the activity of laccase produced by Ganoderma lucidum, seed liquid with higher weight and density is selected, centrifuged at low temperature, and then stored at -70℃; low temperature centrifugation means that the centrifugation temperature is not higher than 0℃.

[0013] Optionally, in step (2) of the method for improving the activity of laccase produced by Ganoderma lucidum, the preserved bacterial solution obtained in step (1) is plate-coated for revival. During the revival process, each colony is numbered, the growth rate and morphology of each colony are recorded, and the mycelial growth is observed under a microscope. Finally, colonies with larger size, uniform morphology and abundant mycelium are selected for rejuvenation.

[0014] Optionally, in step (3) of the method for improving the laccase activity of Ganoderma lucidum, the Ganoderma lucidum strain that has grown rapidly and has abundant mycelium after rejuvenation in step (2) is transferred and expanded. The mycelium near the edge of the plate is selected and inoculated into multiple 250ml conical flasks with known weight and 100ml of fermentation liquid for expansion culture. The conical flasks are numbered, and after five days of culture at 30℃, the bacterial liquid in the conical flask with heavier weight and larger mycelial density and size is selected.

[0015] Optionally, in step (4) of the method for improving the activity of laccase produced by Ganoderma lucidum, the seed liquid selected in step (3) is inoculated into multiple 2000ml conical flasks with known weight of empty bottles and 750ml of fermentation liquid for secondary expansion culture, and the conical flasks are numbered. After five days of culture at 30℃, the conical flasks with heavier weight and larger mycelial density and size are selected for the next step of enzyme production in the fermenter.

[0016] Optionally, the culture medium used in step (2) of the method for improving the activity of laccase produced by Ganoderma lucidum is PDA medium.

[0017] Optionally, the culture medium used in steps (3) and (4) of the method for improving the activity of laccase produced by Ganoderma lucidum is PDW medium.

[0018] Optionally, the method for improving the activity of laccase produced by Ganoderma lucidum, step (4), involves preparing the culture medium used in the fermenter as follows: 0.8 g / L potassium dihydrogen phosphate, 0.8 g / L magnesium sulfate heptahydrate, 1.0 g / L yeast extract, 20 g / L corn steep liquor, 31 g / L wheat bran, 15 g / L tobacco stalk powder, 0.2 g / L vitamin B1, and 0.05% defoamer; pH at rest, sterilized at 121°C for 20 minutes.

[0019] Preferably, the Ganoderma lucidum is preserved at low temperature, with a glycerol-to-liquid ratio of 4:6, and the glycerol is added after the liquid is centrifuged.

[0020] Preferably, the temperature of the Ganoderma lucidum plate culture stage in step (2) is 30℃, and the temperature of the shaker culture stage in steps (3) and (4) is 30℃ and the rotation speed is 150 rpm; the temperature of the Ganoderma lucidum enzyme production culture stage is 30℃ and the rotation speed is 180 rpm.

[0021] Preferably, the inoculum size for the extended culture stage is 5%.

[0022] Preferably, the inoculum size of the enzyme-producing medium is 8%.

[0023] Compared with the prior art, the present invention has at least the following beneficial effects:

[0024] 1. This invention can increase the activity of laccase from the original 80,000 u / L to 160,000 u / L, effectively improving the production efficiency of enzyme production by Ganoderma lucidum fermentation and increasing the utilization rate of laccase solution.

[0025] 2. This invention not only enhances the activity of laccase produced by Ganoderma lucidum, but also improves the stability of laccase produced by Ganoderma lucidum, ensuring that the enzyme activity of each batch is between 140,000 and 160,000 u / L. Detailed Implementation

[0026] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0027] In all specific embodiments of this application, the resuscitation and rejuvenation solid culture medium, the proliferation and expansion liquid culture medium, and the fermentation enzyme production culture medium are prepared according to the following methods:

[0028] (1) Resuscitation and rejuvenation solid culture medium, namely glucose potato agar medium (PDA medium): 26 g / L glucose potato water, 20 g / L agar, natural pH, sterilized at 121℃ for 20 minutes.

[0029] (2) Expanding liquid culture medium, namely glucose potato water medium (PDW medium): 26g / L glucose potato water, natural pH, sterilized at 121℃ for 20 minutes.

[0030] (3) Fermentation enzyme production medium: potassium dihydrogen phosphate 0.8 g / L, magnesium sulfate heptahydrate 0.8 g / L, yeast extract 1.0 g / L, corn steep liquor 20 g / L, wheat bran 31 g / L, tobacco stalk powder 15 g / L, vitamin B1 0.2 g / L, and defoamer 0.05%. The pH was natural, and the medium was sterilized at 121°C for 20 minutes.

[0031] In all specific embodiments of this application, laccase activity was determined according to the following assay method:

[0032] Methods for determining laccase activity

[0033] Plotting the standard curve:

[0034] Take six 5ml centrifuge tubes and add 0.0, 1.0, 2.0, 3.0, 4.0, and 5.0 μL of laccase standard solution (100,000 enzyme activity) to each tube, respectively. Then add 10.0, 9.0, 8.0, 7.0, 6.0, and 5.0 μL of distilled water to each tube, respectively. Shake well. The laccase activities in each tube will be 0, 10,000, 20,000, 30,000, 40,000, and 50,000 U, respectively. Add 2.5 ml of pH 3.0 buffer to each tube, then add 500 μL of ABTS solution. Incubate in a 45℃ water bath with shaking for 5 minutes. Immediately after the time is up, remove the tube and measure the absorbance of the laccase solutions with different enzyme activities at a wavelength of 420 nm.

[0035] After the enzyme was produced by Ganoderma lucidum fermentation, 2.5 μL of the filtered fermentation broth was transferred to a 5 ml centrifuge tube. 2.5 ml of pH 3.0 buffer solution was added to the centrifuge tube, followed by 500 μL of ABTS solution. The tube was then placed in a 45°C water bath with shaking for 5 minutes. Immediately after the time was up, the absorbance was measured at a wavelength of 420 nm, and the laccase activity was calculated based on the regression equation.

[0036] In each embodiment, low-temperature centrifugation refers to centrifugation at a temperature of -4°C.

[0037] In each embodiment, the density of the seed liquid, the density and size of the mycelial pellets were evaluated by visual observation.

[0038] The term "preservation" in the embodiments refers to storage in a laboratory under suitable conditions for the storage of microbial strains.

[0039] The strain used in each embodiment is Ganoderma lucidum.

[0040] Example 1

[0041] (1) Screening of preserved strains: When preserving strains, seed liquids with different weights and densities were selected, centrifuged at low temperature, and then preserved at -70℃ to obtain Ganoderma lucidum seed strains;

[0042] (2) Screening of strain resuscitation: The preserved bacterial solution obtained in step (1) is plate-spread for resuscitation. During the resuscitation process, each colony is numbered, and the growth and morphology of each colony are recorded. The mycelial growth is observed under a microscope. Finally, colonies with larger size, uniform shape and abundant mycelium are selected for rejuvenation.

[0043] (3) Screening of strain expansion culture: The Ganoderma lucidum strains that grew rapidly and had abundant hyphae after rejuvenation in step (2) were transferred and expanded. The strains near the edge of the plate were selected and inoculated into several 250ml Erlenmeyer flasks with known weight and 100ml of fermentation liquid for expansion culture. The Erlenmeyer flasks were numbered and cultured at 150 rpm and 30℃ for five days. The strains with heavier weight and larger density and size of mycelial balls were selected for the next step.

[0044] (4) Screening of seed culture for enzyme production by microbial fermentation: The seed culture selected in step (3) was inoculated into 2000ml conical flasks containing 750ml of fermentation liquid in multiple empty bottles of known weight for secondary expansion culture. The conical flasks were numbered and cultured at 150 rpm and 30℃ for five days. After that, the bacterial culture in the conical flask with heavier weight and larger bacterial density and size was selected and inoculated into the enzyme production medium in the fermenter at 8%. The enzyme production culture was carried out at 180 rpm, 30℃ and 1.0 ventilation ratio for 5 days.

[0045] The fermentation broth was filtered and then subjected to laccase activity assay.

[0046] Experimental results: The effect of the weight of the bacterial culture before preservation on the final laccase activity is shown in Table 1. The greater the weight and density of the preserved Ganoderma lucidum bacterial culture, the higher the enzyme activity of the final fermentation broth can be increased by 30,000-40,000 u / L.

[0047] Table 1:

[0048] Seed liquid weight before preservation Final laccase activity (u / L) 114g 158743 112g 148775 115g 162933 110g 139823 117g 170234

[0049] Example 2

[0050] (1) Screening of preservative strains: When preserving strains, seed liquids with high weight and density are selected, centrifuged at low temperature, and then preserved at -70℃ to obtain high-quality, high-density Ganoderma lucidum seed strains.

[0051] (2) Screening of strain resuscitation: The preserved bacterial solution obtained in step (1) is plate-spread for resuscitation. During the resuscitation process, each colony is numbered, the growth rate and morphology of each colony are recorded, and the mycelial growth is observed under a microscope. Finally, colonies of different sizes and shapes are selected for rejuvenation.

[0052] (3) Screening of strain expansion culture: The Ganoderma lucidum strains that grew rapidly and had abundant mycelium after rejuvenation in step (2) were transferred and expanded. The mycelium near the edge of the plate was selected and inoculated into several 250ml Erlenmeyer flasks with known weight of empty bottles and 100ml of fermentation liquid for expansion culture. The Erlenmeyer flasks were numbered and cultured at 30℃ for five days. The bacterial solution with the largest increase in weight was selected for the next step.

[0053] (4) Screening of seed culture for enzyme production by microbial fermentation: The seed culture selected in step (3) was inoculated into several 2000ml Erlenmeyer flasks containing 750ml of fermentation broth in empty bottles of known weight for secondary expansion culture. The Erlenmeyer flasks were numbered, and after five days of culture at 30℃, the bacterial culture from the heavier Erlenmeyer flasks with larger bacterial density and size was selected for the next step of enzyme production in the fermenter. The fermenter culture conditions were the same as in Example 1.

[0054] The fermentation broth was filtered and then subjected to laccase activity assay.

[0055] Experimental results: The effect of colony morphology on the final laccase activity after the cell recovery and rejuvenation stage is shown in Table 2. The more uniform the cell morphology of Ganoderma lucidum during the cell recovery and rejuvenation stage, the higher the enzyme activity of the final fermentation broth can be by 20,000-30,000 u / L.

[0056] Table 2:

[0057] Colony morphology description Final laccase activity (u / L) Irregular colony morphology 120987 Slow colony growth 74692 The edges of the colonies are serrated. 93231 Low colony mycelial density 108934 Colonies are regularly round. 132398

[0058] Example 3

[0059] (1) Screening of preservative strains: When preserving strains, seed liquids with high weight and density are selected, centrifuged at low temperature, and then preserved at -70℃ to obtain high-quality, high-density Ganoderma lucidum seed strains.

[0060] (2) Screening of strain resuscitation: The preserved bacterial solution obtained in step (1) is plate-spread for resuscitation. During the resuscitation process, each colony is numbered, and the growth and morphology of each colony are recorded. The mycelial growth is observed under a microscope. Finally, colonies with larger size, uniform shape and abundant mycelium are selected for rejuvenation.

[0061] (3) Screening of strain expansion culture: The Ganoderma lucidum strains that grew rapidly and had abundant mycelium after rejuvenation in step (2) were transferred and expanded. The mycelium near the edge of the plate was selected and inoculated into multiple 250ml Erlenmeyer flasks with known weight of empty bottles and 100ml of fermentation liquid for expansion culture. The Erlenmeyer flasks were numbered and cultured at 30℃ for five days. Then, different weights of bacterial liquid were selected for the next step.

[0062] (4) Screening of seed culture for enzyme production by microbial fermentation: The seed culture selected in step (3) was inoculated into several 2000ml Erlenmeyer flasks containing 750ml of fermentation broth in empty bottles of known weight for secondary expansion culture. The Erlenmeyer flasks were numbered, and after five days of culture at 30℃, the bacterial culture from the heavier Erlenmeyer flasks with larger bacterial density and size was selected for the next step of enzyme production in the fermenter. The fermenter culture conditions were the same as in Example 1.

[0063] The fermentation broth was filtered and then subjected to laccase activity assay.

[0064] Experimental results: The effect of the weight of the seed liquid inoculated in the conical flasks during the seed liquid screening stage on the final laccase activity is shown in Table 3. The greater the weight and density of the seeds inoculated during the seed liquid screening stage, the higher the enzyme activity of the final fermentation broth can be increased by 40,000-50,000 u / L.

[0065] Table 3

[0066] Increase the weight of the seed culture liquid Final laccase activity (u / L) 113g 157249 110g 141297 115g 163745 111g 149874 118g 183945

[0067] Example 4

[0068] (1) Screening of preservative strains: When preserving strains, seed liquids with high weight and density are selected, centrifuged at low temperature, and then preserved at -70℃ to obtain high-quality, high-density Ganoderma lucidum seed strains.

[0069] (2) Screening of strain resuscitation: The preserved bacterial solution obtained in step (1) is plate-spread for resuscitation. During the resuscitation process, each colony is numbered, and the growth and morphology of each colony are recorded. The mycelial growth is observed under a microscope. Finally, colonies with larger size, uniform shape and abundant mycelium are selected for rejuvenation.

[0070] (3) Screening of strain expansion culture: The Ganoderma lucidum strains that grew rapidly and had abundant mycelium after rejuvenation in step (2) were transferred and expanded. The mycelium near the edge of the plate was selected and inoculated into several 250ml Erlenmeyer flasks with known weight and 100ml fermentation liquid for expansion culture. The Erlenmeyer flasks were numbered and the bacterial solutions with larger weight and density after five days of culture at 30℃ were used for the next step.

[0071] (4) Screening of seed culture for enzyme production by microbial fermentation: The seed culture selected in step (3) was inoculated into several 2000ml Erlenmeyer flasks containing 750ml of fermentation broth in empty bottles of known weight for secondary expansion culture. The Erlenmeyer flasks were numbered, and after five days of culture at 30℃, the bacterial culture from Erlenmeyer flasks of different weights was selected for the next step of enzyme production in the fermenter. The fermenter culture conditions were the same as in Example 1.

[0072] The fermentation broth was filtered and then subjected to laccase activity assay.

[0073] Experimental results: The effect of the seed liquid weight of the conical flask inoculated during the enzyme production stage of the strain fermentation on the final laccase activity is shown in Table 4. The greater the seed weight and density inoculated during the enzyme production stage of the strain fermentation, the higher the enzyme activity of the final fermentation broth can be increased by 20,000-30,000 u / L.

[0074] Table 4

[0075] Fermentation enzyme-producing seed liquid weight Final laccase activity (u / L) 111g 130345 112g 139855 117g 188944 113g 142934 115g 168334

[0076] Through the above embodiments, it can be concluded that the method of the present invention can effectively improve the activity of laccase produced by Ganoderma lucidum, and the method of the present invention can make the laccase produced by Ganoderma lucidum more stable.

[0077] Although the invention has been described herein with reference to illustrative embodiments, it should be understood that many other modifications and implementations can be devised by those skilled in the art, which will fall within the scope and spirit of the principles disclosed herein. More specifically, various variations and modifications can be made to the components and / or layout of the subject matter combination within the scope of this disclosure. Besides variations and modifications to the components and / or layout, other uses will be apparent to those skilled in the art.

Claims

1. A method for improving the laccase activity of Ganoderma lucidum, characterized in that, Includes the following steps: (1) Screening of preservative strains: Select the seed liquid with the highest weight from multiple Ganoderma lucidum seed liquids, centrifuge and preserve it; (2) Screening of strain resuscitation: Resuscitate the preserved seed liquid and select colonies with larger and more uniform morphology for rejuvenation; (3) Screening of strain expansion culture: The rejuvenated Ganoderma lucidum strain with more abundant mycelium was transferred and expanded in multiple containers, and the bacterial solution in the heavier container was selected for the next step. (4) Screening of seed liquid for enzyme production by fermentation: The screened bacterial liquid is cultured in multiple containers for a second expansion, and the bacterial liquid in the heavier container is selected for enzyme production in the fermenter.

2. The method for improving the laccase activity of Ganoderma lucidum according to claim 1, characterized in that, In step (1), seed liquid with higher weight and density is selected, centrifuged at low temperature, and then stored at -70℃; low temperature centrifugation means that the centrifugation temperature is not higher than 0℃.

3. The method for improving the laccase activity of Ganoderma lucidum according to claim 1, characterized in that, In step (2), the preserved bacterial solution obtained in step (1) is plate-spread for revival. During the revival process, each colony is numbered, and the growth rate and morphology of each colony are recorded. The mycelial growth is observed under a microscope. Finally, colonies with larger size, uniform shape and abundant mycelium are selected for rejuvenation.

4. The method for improving the laccase activity of Ganoderma lucidum according to claim 1, characterized in that, In step (3), the Ganoderma lucidum strain that has grown rapidly and has abundant mycelium after rejuvenation in step (2) is transferred and expanded. The mycelium near the edge of the plate is selected and inoculated into several 250ml Erlenmeyer flasks with known weight and 100ml of fermentation liquid for expansion culture. The Erlenmeyer flasks are numbered. After five days of culture at 30℃, the bacterial liquid in the Erlenmeyer flask with heavier weight and larger mycelial density and size is selected.

5. The method for improving the laccase activity of Ganoderma lucidum according to claim 1, characterized in that, In step (4), the seed liquid selected in step (3) is inoculated into multiple 2000ml conical flasks containing 750ml of fermentation broth and known weight of empty bottles for secondary expansion culture. The conical flasks are numbered, and after five days of culture at 30℃, the conical flasks with heavier weight and larger bacterial density and size are selected for the next step of enzyme production in the fermenter.

6. The method for improving the laccase activity of Ganoderma lucidum according to claim 1, characterized in that, The culture medium used in step (2) is PDA medium.

7. The method for improving the laccase activity of Ganoderma lucidum according to claim 1, characterized in that, The culture medium used in steps (3) and (4) is PDW medium.

8. The method for improving the laccase activity of Ganoderma lucidum according to claim 1, characterized in that, The culture medium used in the fermenter in step (4) is prepared as follows: 0.8 g / L potassium dihydrogen phosphate, 0.8 g / L magnesium sulfate heptahydrate, 1.0 g / L yeast extract, 20 g / L corn steep liquor, 31 g / L wheat bran, 15 g / L tobacco stalk powder, 0.2 g / L vitamin B1, and 0.05% defoamer; pH is natural, and the mixture is sterilized at 121°C for 20 minutes.