CN108285894B - Method for improving laccase yield of Trametes versicolor - Google Patents

Method for improving laccase yield of Trametes versicolor Download PDF

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CN108285894B
CN108285894B CN201810070458.2A CN201810070458A CN108285894B CN 108285894 B CN108285894 B CN 108285894B CN 201810070458 A CN201810070458 A CN 201810070458A CN 108285894 B CN108285894 B CN 108285894B
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trametes versicolor
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王�锋
徐玲
马海乐
孙柯
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    • C12Y110/03—Oxidoreductases acting on diphenols and related substances as donors (1.10) with an oxygen as acceptor (1.10.3)
    • C12Y110/03002—Laccase (1.10.3.2)

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Abstract

The invention discloses a method for improving the yield of Trametes versicolor laccase, which is characterized in that the laccase is subjected to ultrasonic treatment and continuous fermentation in the fermentation process of Trametes versicolor laccase to generate laccase. The thalli in the shake flask are directly subjected to ultrasonic treatment in the fermentation process, so that the operation is simple and the possibility of pollution is reduced; in the hypha growth stage, high-intensity ultrasound is adopted to enable the hypha of the trametes to have hyperbranched hypha forms and corm tops so as to increase the area of secretion sites and improve the permeability of cell membranes so as to accelerate the secretion of laccase, and in the enzyme production stage, low-intensity ultrasound is adopted to induce and generate active oxygen substances so as to stimulate the expression of laccase genes, so that the laccase yield of trametes versicolor is effectively improved. Experiments prove that the laccase yield can be improved by more than 3 times and can be improved by 5-6 times to the maximum extent by adopting the method. The method disclosed by the invention is simple to operate, easy for large-scale production and suitable for large-scale laccase fermentation, so that the production cost of the laccase is reduced, and the requirement of large-scale application is met.

Description

Method for improving laccase yield of Trametes versicolor
Technical Field
The invention relates to the technical field of bioengineering, in particular to a method for improving laccase yield of trametes versicolor.
Background
Laccase (laccase, ec1.10.3.2) is a copper-containing polyphenol oxidase that catalyzes a variety of phenolic compounds by obtaining oxygen to produce the corresponding benzoquinone and water. Trametes versicolor, one of the well-recognized superior strains for producing laccase, is the main "producer" of laccase. In recent years laccases have received increasing attention for their industrial applications, such as waste water treatment, bioremediation, bleaching of pulp, establishment of biosensors and food processing. However, the low expression level of laccase fermentation is one of the reasons for the high production and application costs. Therefore, improving the yield of laccase and reducing the production cost of laccase become one of the researches on laccase in the present stage. At present, most researches on improving the laccase yield are focused on optimizing a strain culture medium and fermentation conditions, but the method has the defects of large workload, low efficiency and limited laccase yield improvement. In addition, co-culture, the use of biosurfactants and inducers, etc. are also common methods for increasing laccase production. However, although the above method has a certain improvement on laccase yield, the amplitude of the method is far from enough for the commercialization of laccase, so that how to greatly improve the laccase yield is still the focus of research.
The ultrasonic is one of effective means for reinforcing the biological cell culture process, the mass transfer of a reaction system can be improved by ultrasonic reinforcement, and the mass transfer and the reaction rate can be improved by ultrasonic in a multiphase or homogeneous reaction system; ultrasound promotes growth and secondary metabolite synthesis, which can be used as a way to increase biomass and secondary metabolite production during biological cell culture; the ultrasonic wave destroys the cells to promote the release of intracellular products, and the high-intensity ultrasonic wave can damage the cells and destroy the cells to release the intracellular products; the ultrasonic reactor can also be used as a bioreactor in cell culture. Researchers have been able to accelerate the growth rate of Anabaena flos-aquae (Anabaena flors-aquae) by means of ultrasonic enhancement and increase biomass by 46% (Franck et al, Effect of low-dose ultrasonic physiological variables in Anabaena flos-aquae and Selenastrum capricanum, 1990,12: 219-. In addition, ultrasound can promote the ion flux of the cell membrane of ginseng (Panax ginseng), and increase the yield of its secondary metabolites (Wu et al, Elichor-like effects of low-energy ultrasounded on-plant (Panax ginseng) cells: Induction of plant decay and second microorganisms production,2002,59: 51-57). However, researchers have been less investigating the biological effects of ultrasound, and the theoretical mechanism of their biological effects is unclear. The inventor group of the invention discovers the physiological mechanism of the ultrasonic wave for regulating the growth and metabolism of the filamentous fungus cells through a great deal of previous researches; on the basis, the invention introduces ultrasonic reinforcement into the laccase fermentation process of trametes versicolor free mycelium, so that the coupling strategy of obtaining hyperbranched mycelium shape through high-frequency ultrasonic in the growth stage to increase protein secretion sites and improve laccase gene expression level through low-frequency ultrasonic in the enzyme production stage can be realized, the enzyme production capacity of the trametes versicolor of filamentous fungi is improved, the expression level of extracellular laccase of the trametes versicolor is greatly improved, an effective way is provided for reducing the laccase production cost, and an efficient yield increase strategy is provided for other fermentation systems for producing extracellular protein by filamentous fungi.
Disclosure of Invention
The invention provides a method for improving the laccase production capacity of trametes versicolor. The method can greatly improve laccase yield of trametes versicolor; and the method is simple to operate and easy to enlarge production.
In order to realize the purpose of the invention, the invention provides the following technical scheme:
a method of increasing Trametes versicolor laccase production, the method comprising: the bacteria are treated by ultrasonic treatment in the fermentation process of trametes versicolor laccase and are continuously fermented to produce laccase. Preferably, the ultrasonic treatment is carried out in a hypha growth stage of 6-48 h and an enzyme production stage of 60-120 h respectively.
The method specifically comprises the following steps:
(1) inoculating the pre-cultured trametes versicolor mycelium into a laccase fermentation culture medium for fermentation culture;
(2) performing ultrasonic treatment on the mycelia for 1-4 times at the frequency of 40-80 kHz and the power of 30-210 w for 6-48 h of fermentation, wherein the total time is 2-20 min, the working interval is 2-30 s;
(3) performing ultrasonic treatment on the mycelia for 1-5 times at the frequency of 10-30 kHz and the power of 30-180 w for 60-120 h of fermentation, wherein the total time is 2-20 min, the working interval is 5-30 s;
(4) and (4) continuing culturing after ultrasonic treatment until the laccase activity in the fermentation liquor reaches the peak value, ending fermentation and collecting the fermentation liquor.
Preferably, the pre-culture method of trametes versicolor mycelium and the composition of the fermentation medium in the step (1) adopt the methods reported in the literature (Wang et al, Ultrasound-induced lactic production from microorganisms versicolor, Ultrasound experiments at the temperature of 2013,20: 118-.
Furthermore, the ultrasonic treatment frequency in the 6 th to 48 th hours of fermentation is 40kHz to 80kHz, the power is 30w to 210w, and the total time is 2min to 20 min. Preferably, the frequency may be 0kHz, 50kHz, 60kHz, 70kHz, 80kHz, preferably 30 to 70kHz, more preferably 45 to 60 kHz. Preferably, the power of ultrasonic treatment is 30w, 60w, 90w, 120w, 150w, 180w, 210w, preferably 60-180 w, more preferably 60-150 w. Preferably, the total time of the ultrasonic treatment is 2min, 3min, 4min, 5min, 6min, 7min, 8min, 9min, 10min, 11min, 12min, 13min, 14min, 15min, 16min, 17min, 18min, 19min, 20min, preferably 3-15 min, more preferably 5-10 min.
Furthermore, the working interval of ultrasonic treatment in 6 to 48 hours of fermentation is 5 to 30 s. Preferably, the working interval is 5s, 10s, 15s, 20s, 25s, 30s, preferably 5-20 s, more preferably 5-15 s.
Furthermore, the number of times of ultrasonic treatment in 6-48 h of fermentation is 1-4, and the time between each ultrasonic treatment is divided evenly. Specifically, the number of the administration may be 1, 2, 3 or 4.
Further, the frequency of ultrasonic treatment in 60-120 h of fermentation is 10-30 kHz, the power is 30-180 w, and the total time of ultrasonic treatment is 2-20 min.
Preferably, the frequency is 10kHz, 15kHz, 20kHz, 28kHz, 30kHz, preferably 15 to 30kHz, more preferably 20 to 30 kHz. Preferably, the power of ultrasonic treatment is 30w, 60w, 90w, 120w, 150w, 180w, preferably 60-150 w, and more preferably 60-120 w. Preferably, the total time of the ultrasonic treatment is 2min, 3min, 4min, 5min, 6min, 7min, 8min, 9min, 10min, 11min, 12min, 13min, 14min, 15min, 16min, 17min, 18min, 19min, 20min, preferably 3-10 min, more preferably 4-6 min.
Furthermore, the working interval of ultrasonic treatment in 60-120 h of fermentation is 5-30 s. For example, 5s, 10s, 15s, 20s, 25s, 30s, preferably 5 to 20s, and more preferably 5 to 15 s.
Furthermore, the ultrasonic treatment times in the 60 th to 120 th hours of fermentation are 1 to 5, and the time between each ultrasonic treatment is divided equally. For example, 1 time, 2 times, 3 times, 4 times, 5 times, preferably 2 to 4 times, more preferably 2 to 3 times, and the time between each ultrasonic treatment is divided equally.
The method for improving the yield of trametes versicolor laccase provided by the invention has the advantages that the thalli in a shake flask are directly subjected to ultrasonic treatment in the fermentation process, the operation is simple, and the possibility of pollution is reduced; in the hypha growth stage, high-intensity ultrasound is adopted to enable the hypha of the trametes to have hyperbranched hypha forms and corm tops so as to increase the area of secretion sites and improve the permeability of cell membranes so as to accelerate the secretion of laccase, and in the enzyme production stage, low-intensity ultrasound is adopted to induce and generate active oxygen substances so as to stimulate the expression of laccase genes, so that the laccase yield of trametes versicolor is effectively improved. Experiments prove that the laccase yield can be improved by more than 3 times and can be improved by 5-6 times to the maximum extent by adopting the method. The method disclosed by the invention is simple to operate, easy for large-scale production and suitable for large-scale laccase fermentation, so that the production cost of the laccase is reduced, and the requirement of large-scale application is met.
Detailed Description
The following description of the preferred embodiments of the present invention is provided for the purpose of illustration and description, and is in no way intended to limit the invention.
Examples
Activation and preculture of trametes versicolor
(1) Activating the mixture
Trametes versicolor (Trametes versicolor) was purchased from China center for Industrial culture Collection of microorganisms, and has the number CICC 14001.
Preparing a culture medium for bevel activation by a common potato culture medium preparation method, wherein 200g/L of potatoes, 20g/L of glucose and 15g/L of agar powder are prepared; trametes versicolor (CICC14001) is inoculated on the culture medium and cultured for 5-7 days at 28 ℃.
(2) And pre-culturing
Preparing a culture medium for pre-culture by adopting a common potato culture medium preparation method, wherein the potato culture medium comprises 200g/L of potato and 20g/L of glucose; inoculating the activated mycelium into the culture medium, and culturing at 30 deg.C and 150r/min for 7 days; homogenizing the culture, inoculating to fresh culture medium according to the inoculation amount of 10% (v/v), and culturing for 5 days to obtain pre-cultured mycelium for laccase fermentation.
Specific examples of trametes versicolor laccase fermentations are described below. The laccase fermentation medium comprises the following components in percentage by weight: KH (Perkin Elmer)2PO40.2g/L，CaCl2·2H2O 0.0755g/L，MgSO4·7H2O 0.05g/L，NH4H2PO40.5g/L，FeSO4·7H2O0.035 g/L, glucose 2g/L, NH4Cl 0.267g/L，CuSO4·5H2O 0.1g/L。
"x h th" or "after x h" in the examples means the time from the time of inoculation and start of fermentation in step (1).
Example 1: trametes versicolor laccase fermentation
(1) Inoculating pre-cultured trametes versicolor mycelium into laccase fermentation culture medium at the initial biomass of 10% (v/v) at 26 deg.C and 150r/min, and performing fermentation culture under the condition;
(2) carrying out ultrasonic treatment on the thalli in the triangular flask in 6-48 h of the fermentation process, wherein the frequency is 45kHz, the ultrasonic power is 120w, the ultrasonic time is 10s for work and 10s for stop, the total time is 5min, and the times are 2; and (3) carrying out ultrasonic treatment on the thalli for 60-120 h in the fermentation process, wherein the frequency is 20kHz, the power is 150w, the times are 3, the other conditions are unchanged, and the thalli are continuously cultured after ultrasonic treatment.
(3) The pH was adjusted to 4.5 from day 1 to day 3 of the fermentation process.
(4) After 7 days of fermentation culture, the fermentation broth was collected and the laccase yield was determined after removal of the mycelia. And (3) testing the culture effect:
the yield of trametes versicolor laccase cultured under the ultrasonic treatment condition is improved by 313 percent compared with that of laccase cultured under the non-ultrasonic condition (see table 1).
Example 2: trametes versicolor laccase fermentation
(1) Inoculating pre-cultured trametes versicolor mycelium into laccase fermentation culture medium at the initial biomass of 10% (v/v) at 26 deg.C and 150r/min, and performing fermentation culture under the condition;
(2) carrying out ultrasonic treatment on the thalli in the triangular flask in 6-48 h of the fermentation process, wherein the frequency is 50kHz, the power is 90w, the time is 10s for operation and 10s for stop, the total time is 10min, and the ultrasonic frequency is 1 time; and (3) carrying out ultrasonic treatment on the thalli for 60-120 h in the fermentation process, wherein the frequency is 28kHz, the power is 120w, the ultrasonic frequency is 2 times, and other conditions are unchanged, and the thalli are continuously cultured after ultrasonic treatment.
(3) The pH was adjusted to 4.5 from day 1 to day 3 of the fermentation process.
(4) After 7 days of fermentation culture, the fermentation broth was collected and the laccase yield was determined after removal of the mycelia.
And (3) testing the culture effect:
the laccase yield of trametes versicolor cultured under the ultrasonic treatment condition is improved by 355 percent compared with the laccase yield under the non-ultrasonic condition (see table 1).
Example 3: trametes versicolor laccase fermentation
(1) Inoculating pre-cultured trametes versicolor mycelium into laccase fermentation culture medium at the initial biomass of 10% (v/v) at 26 deg.C and 150r/min, and performing fermentation culture under the condition;
(2) carrying out ultrasonic treatment on the thalli in the triangular flask in 6-48 h of the fermentation process, wherein the frequency is 60kHz, the power is 60w, the time is 20s for working and 20s for stopping, the total time is 20min, and the ultrasonic frequency is 1 time; and (3) carrying out ultrasonic treatment on the thalli for 60-120 h in the fermentation process, wherein the frequency is 28kHz, the power is 120w, the ultrasonic frequency is 2 times, and other conditions are unchanged, and the thalli are continuously cultured after ultrasonic treatment.
(3) The pH was adjusted to 4.5 from day 1 to day 3 of the fermentation process.
(4) After 7 days of fermentation culture, the fermentation broth was collected and the laccase yield was determined after removal of the mycelia.
And (3) testing the culture effect:
the yield of trametes versicolor laccase cultured under the ultrasonic treatment condition is improved by 459 percent compared with the yield of laccase cultured under the non-ultrasonic condition (see table 1).
Example 4: trametes versicolor laccase fermentation
(1) Inoculating pre-cultured trametes versicolor mycelium into laccase fermentation culture medium at the initial biomass of 10% (v/v) at 26 deg.C and 150r/min, and performing fermentation culture under the condition;
(2) carrying out ultrasonic treatment on the thalli in the triangular flask in 6-48 h of the fermentation process, wherein the frequency is 80kHz, the power is 30w, the time is 10s for work and 10s for stop, the total time is 5min, and the ultrasonic frequency is 2 times; and (3) carrying out ultrasonic treatment on the thalli for 60-120 h in the fermentation process, wherein the frequency is 28kHz, the power is 150w, the ultrasonic frequency is 3 times, and other conditions are unchanged, and continuously culturing after ultrasonic treatment.
(3) The pH was adjusted to 4.5 from day 1 to day 3 of the fermentation process.
(4) After 7 days of fermentation culture, the fermentation broth was collected and the laccase yield was determined after removal of the mycelia.
And (3) testing the culture effect:
the laccase output of trametes versicolor cultured under the ultrasonic treatment condition is improved by 483 percent compared with the laccase output under the non-ultrasonic condition (see table 1).
Example 5: trametes versicolor laccase fermentation
(1) Inoculating pre-cultured trametes versicolor mycelium into laccase fermentation culture medium at the initial biomass of 10% (v/v) at 26 deg.C and 150r/min, and performing fermentation culture under the condition;
(2) carrying out ultrasonic treatment on the thalli in the triangular flask in 6-48 h of the fermentation process, wherein the frequency is 70kHz, the power is 60w, the time is 10s for operation and 10s for stop, the total time is 10min, and the ultrasonic frequency is 1 time; and (3) carrying out ultrasonic treatment on the thalli for 60-120 h in the fermentation process, wherein the frequency is 20kHz, the power is 60w, the ultrasonic frequency is 3 times, and other conditions are unchanged, and continuously culturing after ultrasonic treatment.
(3) The pH was adjusted to 4.5 from day 1 to day 3 of the fermentation process.
(4) After 7 days of fermentation culture, the fermentation broth was collected and the laccase yield was determined after removal of the mycelia.
And (3) testing the culture effect:
the laccase yield of trametes versicolor cultured under the ultrasonic treatment condition is 457 percent higher than that of laccase cultured under the non-ultrasonic condition (see table 1).
Example 6: trametes versicolor laccase fermentation
(1) Inoculating pre-cultured trametes versicolor mycelium into laccase fermentation culture medium at the initial biomass of 10% (v/v) at 26 deg.C and 150r/min, and performing fermentation culture under the condition;
(2) carrying out ultrasonic treatment on the thalli in the triangular flask in 6-48 h of the fermentation process, wherein the frequency is 40kHz, the power is 150w, the time is 10s for operation and 10s for stop, the total time is 10min, and the ultrasonic frequency is 1 time; and (3) carrying out ultrasonic treatment on the thalli within 60-120 h in the fermentation process, combining the thalli with the frequency of 28kHz and the power of 150w for 3 times of ultrasonic treatment, keeping the rest conditions unchanged, and continuously culturing after ultrasonic treatment.
(3) The pH was adjusted to 4.5 from day 1 to day 3 of the fermentation process.
(4) After 7 days of fermentation culture, the fermentation broth was collected and the laccase yield was determined after removal of the mycelia.
And (3) testing the culture effect:
the laccase yield of trametes versicolor cultured under the ultrasonic treatment condition is improved by 603 percent compared with the laccase yield under the non-ultrasonic condition (see table 1).
Example 7: trametes versicolor laccase fermentation
(1) Inoculating pre-cultured trametes versicolor mycelium into laccase fermentation culture medium at the initial biomass of 10% (v/v) at 26 deg.C and 150r/min, and performing fermentation culture under the condition;
(2) carrying out ultrasonic treatment on the thalli in the triangular flask in 6-48 h of the fermentation process, wherein the frequency is 50kHz, the power is 60w, the time is 10s for operation and 10s for stop, the total time is 10min, and the ultrasonic frequency is 1 time; and (3) carrying out ultrasonic treatment on the thalli for 60-120 h in the fermentation process, wherein the frequency is 28kHz, the power is 150w, the ultrasonic frequency is 3 times, the rest conditions are unchanged, and the thalli are continuously cultured after ultrasonic treatment.
(3) The pH was adjusted to 4.5 from day 1 to day 3 of the fermentation process.
(4) After 7 days of fermentation culture, the fermentation broth was collected and the laccase yield was determined after removal of the mycelia.
And (3) testing the culture effect:
the laccase yield of trametes versicolor cultured under the ultrasonic treatment condition is improved by 453 percent compared with the laccase yield under the non-ultrasonic condition (see table 1).
Example 8: trametes versicolor laccase fermentation
(1) Inoculating pre-cultured trametes versicolor mycelium into laccase fermentation culture medium at the initial biomass of 10% (v/v) at 26 deg.C and 150r/min, and performing fermentation culture under the condition;
(2) carrying out ultrasonic treatment on the thalli in the triangular flask in 6-48 h of the fermentation process, wherein the frequency is 40kHz, the power is 120w, the time is 10s for operation and 10s for stop, the total time is 10min, and the ultrasonic frequency is 2 times; and (3) carrying out ultrasonic treatment on the thalli within 60-120 h in the fermentation process, combining the thalli with the frequency of 60kHz and the power of 150w for 3 times of ultrasonic treatment, keeping the rest conditions unchanged, and continuously culturing after ultrasonic treatment.
(3) The pH was adjusted to 4.5 from day 1 to day 3 of the fermentation process.
(4) After 7 days of fermentation culture, the fermentation broth was collected and the laccase yield was determined after removal of the mycelia.
And (3) testing the culture effect:
the yield of trametes versicolor laccase cultured under the ultrasonic treatment condition is 393 percent higher than that of laccase cultured under the non-ultrasonic condition (see table 1).
Determination of laccase enzyme Activity
Catechol was used as substrate, 3mL of 0.1% (w/v) catechol solution (prepared from 0.1mol/L pH 3.0 tartaric acid buffer) was added with a suitable amount of laccase, reacted at 25 ℃ for 5min, absorbance change was measured at 450nm [250], and molar extinction coefficient ∈ 2211L/(mol · cm). Definition of enzyme activity: the amount of laccase required for 1. mu. mol of catechol to be oxidized in 1 minute is defined as one enzyme activity unit (U).
And (3) detecting the laccase activity of the fermentation liquor, and performing activity determination on the fermentation liquor clear liquid obtained by centrifugation. The measurement results are shown in table 1.
TABLE 1 determination of laccase enzyme Activity
Figure BDA0001558005590000091
Note: in the corresponding examples, the other conditions were identical except for the control which was not sonicated.
The applicant states that the present invention is illustrated in detail by the above examples, but the present invention is not limited to the above detailed methods, i.e. the scope of the claims of the present invention is not limited thereto. Therefore, any modifications of the invention, equivalent substitutions of the strains, products and raw materials, and specific operation choices, etc., which are related to the invention, are within the scope of protection and disclosure of the invention.

Claims (2)

1. A method for improving trametes versicolorTrametes versicolor) The laccase production method is characterized by specifically comprising the following steps of:
(1) inoculating the pre-cultured trametes versicolor mycelium into a laccase fermentation culture medium for fermentation culture;
(2) carrying out ultrasonic treatment on the thalli in 6-48 h of fermentation, wherein the ultrasonic frequency is 40kHz, the power is 150w, the time is 10s for operation and 10s for stop, and the ultrasonic treatment is carried out for 1 time in total for 10 min;
(3) carrying out ultrasonic treatment on the thalli in 60-120 h of fermentation, wherein the ultrasonic frequency is 28kHz, the power is 150w, the ultrasonic treatment is carried out for 3 times, and the time of each time is 10s for operation and 10s for stop for 10 min;
(4) adjusting the pH to 4.5 from day 1 to day 3 of the fermentation process;
(5) and (4) continuing culturing after ultrasonic treatment until the laccase activity in the fermentation liquor reaches the peak value, ending fermentation and collecting the fermentation liquor.
2. The method of claim 1, wherein the time between each sonication is averaged.
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Immobilization of Trametes versicolor cultures for improving laccase production in bubble column reactor intensified by sonication;Feng Wang et.al.,;《J Ind Microbiol Biotechnol》;20121128;第40卷;摘要，第142页右栏第2段，第143页左栏第2段 *
Ultrasound-intensified laccase production from Trametes versicolor;Feng Wang et.al.,;《Ultrasonics Sonochemistry》;20120518;第20卷;摘要，第2.2-2.4节 *

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