CN113355298A - Method for producing laccase by using mixed bacteria - Google Patents
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Abstract
The invention discloses a method for producing laccase by using mixed fungi, which utilizes fungi-Cerrena unicolor Han 849, chaetomium betulinus Han 851 and Schizophyllum commune Han 881 to mix and ferment basic culture solution containing lignocellulose materials to produce laccase, the laccase production efficiency is high, the laccase activity is obviously improved, moreover, the laccase activity produced by the method is high, and the mixed fungi have mutual synergistic effect in the mixed fermentation process, thereby synergistically improving the laccase activity, improving the laccase production efficiency and reducing the laccase production cost.
Description
Technical Field
The invention relates to a method for producing laccase by using microorganisms, in particular to a method for producing laccase by using various mixed fungi, belonging to the field of fungal microorganism application.
Background
The rapid development of agriculture and forestry is beneficial to economic development and environmental protection. At the same time, some environmental problems are brought about, and the most important is agricultural and forestry waste. If agricultural and forestry wastes are incinerated, more serious air pollution is caused. Agricultural and forestry waste is an important lignocellulosic biomass. The lignocellulose biomass can be converted into green second-generation energy fuels and valuable industrial chemicals through various biological treatment processes, is a renewable resource with wide application and abundant reserves, and has been widely concerned by people in recent years. The use of various low cost lignocellulosic wastes, such as corncobs, cottonseed hulls, bamboo, coffee hulls, and leaves, to produce enzymes, particularly laccases, has been extensively studied.
Laccases (EC 1.10.3.2, p-benzoquinone: oxydoreductase) are widely distributed in various higher plants, fungi, bacteria and insects. Because laccases have the ability to oxidize a wide variety of phenolic and non-phenolic compounds, laccases can be used in a variety of biotechnological processes, including material science, bioremediation, biofuels, fiber modification, nanotechnology, biosensors, food chemistry, paper and pulp industry, and biodegradation. White rot fungi are one of basidiomycetes, can secrete a plurality of extracellular lignin-decomposing enzymes, and play an important role in degrading lignocellulose. Among them, laccase is the oldest and most important one of lignin-degrading enzymes. Meanwhile, white rot fungi are also regarded as excellent laccase producers, and almost all white rot fungi have certain laccase secretion capacity.
Laccase enzymes have shown a great interest to more and more researchers due to their widespread use in many areas of biotechnology. At the same time, the widespread use of laccases in various bioprocesses requires large quantities of low-cost laccases. In order to improve laccase yield and reduce laccase production cost, optimizing fermentation conditions and fermentation methods and developing new laccase-producing strains are very effective methods. The optimization of the fermentation conditions mainly comprises the following aspects: 1) the type, concentration and proportion of the carbon-nitrogen source; 2) metal ions such as copper ions, ferrous ions, manganese ions, silver ions, zinc ions, magnesium ions, and the like; 3) temperature and pH; 4) secondary metabolites, such as ferulic acid and veratryl alcohol. Different species or strains of fungi have significant effects on laccase activity. Therefore, it is a very interesting work to develop new strains with laccase production capacity.
Studies have shown that Erysipelothrix luteus (Cerrena unicolor) has the ability to secrete laccase. However, no reports have been made on the ability of Fuscoporia obliqua (Lenzites betulinus) and Schizophyllum commune (Schizophyllum commune) to secrete laccase. The co-culture of fungi has been studied in recent years and has a good effect on secreting laccase, and almost all studies have been directed to the production of laccase by the combination of white rot fungi and fungi or yeasts. The research on the joint production of laccase by two or three white rot fungi is less. However, the evaluation of laccase activity by C.chromophilus (Cerrena unicolor), B.betulinus (Lenzites betulinus) and Schizophyllum commune (Schizophyllum commune), and the effect of co-cultivation of these fungi on laccase production, has not been studied to date. Under the condition, the influence of 3 fungi and co-culture thereof on laccase activity is researched. The research result provides a new way for improving the yield of laccase and obtaining the laccase with low cost.
The experiment firstly researches the laccase secretion capacity of the chaetomium cupreum (Lenzites betulinus) and the Schizophyllum commune (Schizophyllum commune), and simultaneously researches the laccase secretion capacity of the chaetomium globosum (Cerrena unicolor), the chaetomium cupreum (Lenzites betulinus) and the Schizophyllum commune (Schizophyllum commune) under the co-culture condition for the first time, and finds that the co-culture of the fungi is beneficial to the improvement of the enzyme production activity, the working efficiency is improved, and a large amount of cost is saved.
Disclosure of Invention
The invention aims to solve the technical problems of low laccase yield, complex fermentation substrate components, high laccase production cost and the like in the existing laccase production process by utilizing fungi, and provides a method for producing laccase by utilizing mixed fungi.
In order to achieve the object of the present invention, in one aspect, the present invention provides a method for producing laccase using mixed bacteria, wherein the laccase is produced by mixed fermentation of basic culture solution containing lignocellulose material using fungi, such as Cerrena unicolor Han 849, Fuscoporia obliqua Han 851, and Schizophyllum commune Han 881.
Wherein, the fungus trichinosis (Cerrena unicolor Han 849), the chaulmoogra (Lenzites betulinus Han 851) and the Schizophyllum commune Han 881 are respectively HFNC852, 853 and 854 in the strain numbers of specimen museums of North China forestry experiment center of China forestry academy of sciences.
Particularly, the strains are fungi trichoderma chromophoris (Cerrena unicolor Han 849), Fuscoporia obliqua (Lenzites betulinus Han 851) and Schizophyllum commune Han 881 which are respectively numbered as HFNC852, 853 and 854 and are preserved in specimen museum of North China forestry experiment center of China forestry academy of sciences; and (4) storage address: the west road No.1 of the sluice in the Ministry ditch area of Beijing.
Wherein the lignocellulose material is phoenix tree wood powder and Chinese pine wood powder, preferably phoenix tree wood powder.
In particular, the particle size of the phoenix tree wood flour is less than 10 meshes, preferably 20-60 meshes.
Wherein the mass of the lignocellulose material contained in each 100ml of the basic culture solution is 1-4g, preferably 2 g.
In particular, the amount of the added phoenix tree wood flour per 100ml of the basic culture medium is 1 to 4g, preferably 2 to 3g, and more preferably 2 g.
Wherein the basic culture solution comprises the following components: 1.0-2.0g of monopotassium phosphate, constant volume to 1L and natural pH.
In particular, the composition of the basal medium is: 1.5g of monopotassium phosphate, constant volume of 1L and natural pH.
Wherein the conditions of the mixed fermentation are as follows: dark culture; the culture temperature is 25-28 deg.C, preferably 26 + -0.5 deg.C.
In particular, the stirring speed in the fermentation culture process is 100-200 r/min.
In particular, the volume ratio of the mixed bacteria to the basic culture solution containing the lignocellulose material is (2-6): 100, preferably 3: 100.
In another aspect, the invention provides a method for producing laccase by using mixed bacteria, which comprises the following steps in sequence:
1) respectively inoculating 3 fungi of Trichium chromocor (Cerrena unicolor Han 849), Fuscoporia obliqua (Lenzites betulinus Han 851) and Schizophyllum commune Han 881 to a solid CYM plate complete culture medium for strain activation treatment;
2) respectively inoculating the activated and cultured strains of 3 strains into a liquid complete culture medium, and respectively performing amplification culture to obtain an Erysipelothrix tropicalis amplification culture mixture, a Inonotus obliquus amplification culture mixture and a Schizophyllum commune amplification culture mixture;
3) respectively homogenizing a trichlorphon culture mixture, a birch-ruffle culture mixture and a schizophyllum culture mixture by a homogenizer under aseptic operation conditions to obtain a trichlorphon culture homogenate, a birch-ruffle culture homogenate and a schizophyllum culture homogenate;
4) mixing and inoculating a trichoderma trichinosum homogeneous body and a birch-ruffle homogeneous body into a basic culture solution containing a lignocellulose material, and performing fermentation culture; or
Mixing and inoculating the trichoderma unicolor homogenate body and the schizophyllum commune homogenate body into a basic culture solution containing a lignocellulose material for fermentation culture; or
Mixing and inoculating the homogeneous body of the birch-pleat fungus and the homogeneous body of the schizophyllum commune into a basic culture solution containing a lignocellulose material for fermentation culture; or
Mixing and inoculating the trichoderma trichinosum homogenate, the birch-ruffle homogenate and the schizophyllum commune homogenate into a basic culture solution containing a lignocellulose material for fermentation culture.
Wherein, the strain activation treatment in the step 1) is dark culture, and the treatment temperature is 25-28 ℃, preferably 26 +/-0.5 ℃; the activation treatment time is 7-8d, preferably 7 d.
In particular, the composition of the solid CYM plate complete medium is: 20g of glucose, 2g of peptone, 2g of yeast extract, 0.5g of magnesium sulfate heptahydrate, 1.0g of dipotassium phosphate trihydrate, 0.46g of potassium dihydrogen phosphate and 15g of agar, wherein the volume is constant to 1L, and the pH is natural.
Wherein, the expanding culture in the step 2) is dark culture; the culture temperature is 25-28 deg.C, preferably 26 + -0.5 deg.C.
In particular, the amplification culture in step 2) is performed on a shaker at a constant temperature, wherein the shaker speed is 100-.
In particular, the time for the expansion culture is 5 to 9 days, preferably 7 days.
In particular, the composition of the liquid complete medium is: 20g of glucose, 2g of peptone, 2g of yeast extract powder, 0.5g of magnesium sulfate heptahydrate, 1.0g of dipotassium phosphate trihydrate and 0.46g of potassium dihydrogen phosphate, wherein the volume is constant to 1L, and the pH is natural.
Wherein, the treatment time of the homogenization treatment in the step 3) is 2-5min, preferably 2 min; the homogenization treatment speed is 3000-8000rpm, preferably 5000 rpm.
Particularly, the trichoderma viride homogenate, the birch-ruffle homogenate and the schizophyllum commune homogenate are mycelium with uniform texture and no large mycelium pellet which are obtained by stirring and expanding culture of a homogenizer.
Wherein the basic culture solution in the step 4) comprises the following components: 1.0-2.0g of monopotassium phosphate, constant volume to 1L and natural pH.
In particular, the composition of the basal medium is: 1.5g of monopotassium phosphate, constant volume of 1L and natural pH.
Wherein the lignocellulose material is phoenix tree wood powder and Chinese pine wood powder, preferably phoenix tree wood powder.
In particular, the particle size of the phoenix tree wood flour is less than 10 meshes, preferably 20-60 meshes.
Wherein the mass of the lignocellulose material contained in each 100ml of the basic culture solution is 1-4g, preferably 2 g.
In particular, the amount of the added phoenix tree wood flour per 100ml of the basic culture medium is 1 to 4g, preferably 2 to 3g, and more preferably 2 g.
Wherein the culture conditions of the fermentation culture are as follows: dark culture; the culture temperature is 25-28 deg.C, preferably 26 + -0.5 deg.C.
In particular, the stirring speed in the fermentation culture process is 100-200 r/min.
Wherein, the odontobutis chromophoris homogeneous body and the birch-ruffle homogeneous body in the step 4) are mixed and inoculated, and the volume ratio of the odontobutis chromophoris homogeneous body to the birch-ruffle homogeneous body in the fermentation culture process is 1: (1-2), preferably 1: 1; the trichoderma viride homogeneous body and the schizophyllum homogeneous body are mixed and inoculated, and the volume ratio of the trichoderma viride homogeneous body to the schizophyllum homogeneous body in the fermentation culture process is 1: (1-2), preferably 1: 1; the homogeneous body of the birch-leaf fungus and the homogeneous body of the schizophyllum commune are mixed and inoculated, and the volume ratio of the homogeneous body of the birch-leaf fungus to the homogeneous body of the schizophyllum commune in the fermentation culture process is 1: (1-2), preferably 1: 1.
Particularly, the fusobacterium chromocor homogeneous body, the fuscoporia obliqua homogeneous body and the schizophyllum commune homogeneous body in the step 4) are mixed and inoculated, and the volume ratio of the fusobacterium chromocor homogeneous body, the fuscophyllum obliqua homogeneous body and the schizophyllum commune homogeneous body in the fermentation culture process is 1:1: 1.
Particularly, the homogeneous bodies of the trichoderma unicolor and the birch-ruffle are mixed and inoculated in the step 4), and the ratio of the sum of the volume of the homogeneous bodies of the trichoderma unicolor and the birch-ruffle to the volume of the basic culture solution containing the lignocellulose material in the fermentation culture process is (2-6): 100, preferably 3: 100; the homogeneous body of the Tricholoma chroococcum and the Schizophyllum commune is mixed and inoculated, and the ratio of the sum of the volume of the homogeneous body of the Tricholoma chroococcum and the Schizophyllum commune to the volume of the basic culture solution containing the lignocellulose material in the fermentation culture process is (2-6): 100, preferably 3: 100; mixing and inoculating the homogeneous body of the schizophyllum commune and the birch-fold fungus, wherein the ratio of the sum of the volumes of the homogeneous body of the schizophyllum commune and the birch-fold fungus to the volume of the basic culture solution containing the lignocellulose material in the fermentation culture process is (2-6): 100, preferably 3: 100; the homogeneous body of the trichoderma trichineum, the birch-ruffle and the schizophyllum is mixed and inoculated, and the ratio of the sum of the volumes of the homogeneous body of the trichoderma trichineum, the birch-ruffle and the schizophyllum to the volume of the basic culture solution containing the lignocellulose material in the fermentation culture process is (2-6): 100, preferably 3: 100.
Compared with the prior art, the invention has the following advantages and benefits:
the invention firstly researches the laccase secretion capacity of the Lenzites betulinus Han 851 and the Schizophyllum commune Han 881, and simultaneously researches the laccase secretion capacity of the Cerrena unicolor Han 849, the Fuscoporia obliqua and the Schizophyllum commune under the co-culture condition, and finds that the co-culture of the fungi is beneficial to the improvement of the enzyme production activity, and the stability and the maximum laccase activity under the co-culture condition are obviously superior to the single strain culture induced laccase production.
The method of the invention obviously improves the activity of laccase production induced by hirsutella chromophoris, birch-ruffle and schizophyllum commune, improves the laccase production efficiency and reduces the laccase production cost.
The method of the invention has rich raw material sources and low cost, can also fully utilize phoenix tree wood cellulose material (phoenix tree wood powder), especially phoenix tree wood processing residues and phoenix tree growth waste, saves resources and is beneficial to environmental protection.
The fermentation medium used by the method has simple components and is easy to obtain; the quality of the fermentation product is stable and reliable; time and labor are saved, the process is easy to repeat, and the process is favorable for wide popularization and use.
The method has the advantages of extremely simple production process, convenient operation, mild and easily controlled operation conditions, high laccase activity, obvious improvement of working efficiency, large cost saving and establishment of a new method for obtaining the high laccase activity.
Drawings
FIG. 1 is a result chart of laccase activity tests of liquid fermentation culture of Tricholoma chrondrifolium Cerrena Han 849, Fuscoporia obliqua Lenzites betulinus Han 851 and Schizophyllum commune Han 881, wherein J1-3 is Tricholoma chrondrifolium, Fuscophyllum obliqua and Schizophyllum commune respectively; t1 is a mixed bacterium of Erysipelothrix chromophoris and Fuscoporia obliqua (the volume ratio of the homogeneous body is 1: 1); t2 is a mixed bacterium of Tricholoma chromophilum and Schizophyllum commune (the volume ratio of the homogeneous body is 1: 1); t3 is a mixture of Fuscoporia obliqua and Schizophyllum commune (the volume ratio of the homogeneous body is 1: 1); t4 is a mixture of Erysipelothrix chromophoris, Fuscoporia Obliqua and Schizophyllum commune (the ratio of the volume of the homogenate is 1:1: 1).
FIG. 2 is a graph showing the results of laccase activity tests on the liquid fermentation culture of Cerrena unicolor Han 849, Lenzites betulinus Han 851 and Schizophyllum commune Han 881, wherein the results are shown in the figure
Is a mixed bacterium of trichoderma viride and birch-ruffle (the volume ratio of the homogeneous body is 1: 2);
is a mixed bacterium of trichoderma chromophoris and schizophyllum commune (the volume ratio of the homogeneous body is 1: 2);
is a mixture of Fuscoporia obliqua and Schizophyllum commune (the volume ratio of the homogeneous body is 1: 2).
Detailed Description
The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. These examples are illustrative only and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1
1. Experimental strains
A chromocor Cerrena unicolor Han 849, a Fuscoporia obliqua Lenzites betulinus Han 851 and a Schizophyllum commune Han 881 are provided by North China forestry science research institute North China forestry experiment center, and strain numbers of a North China forestry science institute North China forestry experiment center specimen house are respectively HFNC852, HFNC853 and HFNC854 in sequence, and a preservation address: the west road No.1 of the sluice in the Ministry ditch area of Beijing. Now in the life science college of the gallery institute of faculty;
2. culture medium
Malt extract agar medium (Malt extract agar, MEA): 10g of glucose, 20g of malt extract powder, 3g of monopotassium phosphate and 20g of agar, wherein the volume of water is fixed to 1L, and the pH value is natural.
Complete Medium (CYM): 20g of glucose, 2g of peptone, 2g of yeast extract, 0.5g of magnesium sulfate heptahydrate, 1.0g of dipotassium phosphate trihydrate, 0.46g of potassium dihydrogen phosphate and 15g of agar, wherein the volume is constant to 1L, and the pH is natural.
Liquid complete medium: 20g of glucose, 2g of peptone, 2g of yeast extract powder, 0.5g of magnesium sulfate heptahydrate, 1.0g of dipotassium phosphate trihydrate and 0.46g of potassium dihydrogen phosphate, wherein the volume is constant to 1L, and the pH is natural.
Basal Medium (BM): 1.5g of monopotassium phosphate, constant volume of 1L and natural pH.
Medium D1: the culture medium comprises 100ml of stock solution, and 1-4g (preferably 2g) of Chinese parasol (Firmiana platanifolia) wood powder with particle size of 20-60 mesh, usually less than 10 mesh.
Culture medium D1-1: 100ml of basic culture solution, and 1g of Chinese parasol tree wood powder with granularity of 20-60 mesh, usually less than 10 mesh.
Culture medium D1-2: 100ml of basic culture solution and 4g of phoenix tree wood powder, wherein the particle size of the phoenix tree wood powder is 20-60 meshes, and is usually smaller than 10 meshes.
Medium D2: 100ml of basic culture solution, 1-4g (preferably 2-3g) of pine wood powder (Pinus tabuliformis), wherein the granularity of the pine wood powder is 20-60 meshes, and is usually less than 10 meshes.
All media were used after sterilization at 121 ℃ for 30 min.
Example 2 preparation of homogeneous bodies of Tricholoma monodentatum, Fuscoporia obliqua and Schizophyllum commune
2-1, activation treatment
Inoculating a chromotrichum C.unicolor Han 849, a birch-ruffle L.betulinus Han 851 and a schizophyllum S.commune Han 881 which are stored on a malt extract powder agar medium MEA and are provided by China forestry science research institute North China forestry experimental center to a solid complete medium (CYM plate complete medium), and respectively culturing at constant temperature for 7-8 days under the condition of 26 +/-0.5 ℃ (usually 25-28 ℃) for strain activation treatment;
2-2, expanded culture
Respectively perforating the activated culture of the strains after 7-8 days of activation culture by using a sterile perforator to respectively obtain 3 kinds of expanded culture inocula with the diameter of 5 mm; then respectively inoculating 3 kinds of expanded culture inocula into a liquid complete culture medium under aseptic conditions, wherein every 100mL of the liquid complete culture medium is inoculated with 5 kinds of the expanded culture inocula; then, carrying out light-resistant liquid amplification culture on a shaking table to respectively obtain an amplification culture mixture of the Erysipelothrix chromophoris, the Inonotus obliquus and the Schizophyllum commune (respectively containing amplification culture to obtain mycelium pellets of the Erysipelothrix chromophoris, the Inonotus obliquus and the Schizophyllum commune); wherein the culture conditions of the light-resistant liquid amplification culture are as follows: the culture temperature is 26 plus or minus 0.5 ℃ (usually 25-28 ℃); the culture time is as follows: 7d (typically 5-9 d); shaking is carried out in the process of light-resistant culture, and the shaking rate is 100-;
2-3, homogenizing
Homogenizing the enlarged culture mixture of the trichoderma viride containing the trichoderma viride mycelium pellets after being cultured for 7d in the dark liquid by using a homogenizer under an aseptic condition, and smashing the trichoderma viride mycelium pellets in the enlarged culture mixture of the trichoderma viride by using the homogenizer to obtain a trichoderma viride homogenate body, wherein the homogenizing time is 2min (usually 2-5min), and the homogenizing speed is 5000rpm (usually 3000 plus 8000 rpm);
homogenizing the enlarged culture mixture of the Inonotus obliquus containing Inonotus obliquus mycelium pellets after being cultured in the dark liquid for 7 days by using a homogenizer under aseptic conditions, and smashing the Inonotus obliquus mycelium pellets in the enlarged culture mixture of the Inonotus obliquus by using the homogenizer to obtain an Inonotus obliquus homogenate body, wherein the homogenizing time is 2min (usually 2-5min), and the homogenizing speed is 5000rpm (usually 3000 plus 8000 rpm);
homogenizing the enlarged culture mixture of the Schizophyllum commune containing Schizophyllum commune mycelium pellets after being cultured in the dark liquid for 7d by using a homogenizer under an aseptic condition, and smashing the Schizophyllum commune mycelium pellets in the enlarged culture mixture of the Schizophyllum commune by using the homogenizer to obtain a Schizophyllum commune homogeneous body, wherein the homogenizing time is 2min (usually 2-5min), and the homogenizing speed is 5000rpm (usually 3000 plus 8000 rpm);
example 3 Induction of laccase production
Respectively adding 3mL (usually 2-5mL) of uniformly stirred homogeneous body of Tricholoma chromophoris (J1), Fuscoporia obliqua (J2) and Schizophyllum commune (J3) into 250mL triangular flasks containing 100mL of culture medium D1, performing constant-temperature dark culture on a shaking table, performing fermentation treatment, and inducing Tricholoma chromophoris, Fuscoporia obliqua and Schizophyllum commune to produce laccase, wherein the ratio of the volume of the inoculated homogeneous body to the volume of the induction treatment culture medium D1 is 3:100 (usually 2-6: 100); the shaking rate in the fermentation treatment process is 150r/min (usually 100-; the fermentation temperature is 26 ℃ (usually 25-28 ℃); each experiment was set up in 3 replicates.
In the present invention, medium D1 contained 2g of phoenix tree wood powder per 100ml of the basic culture solution, and the particle size of the phoenix tree wood powder was 20 to 60 mesh.
The ratio of the volume of the inoculated homogenate to the volume of the induction treatment medium in the embodiment of the invention is illustrated by 3:100, and the other 2-6: a ratio of 100 is also suitable for use in the present invention.
In the fermentation treatment process of induction production of laccase, the fermentation mixture in different fermentation triangular flasks inoculated with homogeneous bodies is filtered by Whatman No.1 filter paper every 24h after inoculation, hypha and Chinese parasol wood powder are removed, the filtrate is centrifuged for 20min at 4 ℃ and 12000rpm, and the supernatant (enzyme solution) obtained after centrifugation is used for laccase activity determination, and the determination method is as follows:
A) according to a 300-microliter reaction system, 2 '-azino-bis (3-ethylbenzothiazole-6-sulfonic Acid) (ABTS), acetic acid-sodium acetate buffer solution and enzyme solution are uniformly mixed, wherein 100 microliter of ABTS (2, 2' -azino-bis (3-ethylbenzoline-6-sulpho) with the concentration of 1mmol/L is contained in the 300-microliter reaction systemnic acid) substrate, 190. mu.l of acetic acid-sodium acetate buffer (pH 4.2) with a concentration of 50mmol/L and 10. mu.L of enzyme solution, mixed and then applied with iMark at 415nmTMThe absorbance change was measured within 5min using a microplate reader (Bio-Rad, Hercules, Calif., USA).
The determination process comprises the following steps: iMarkTMThe 96-well plate used by the microplate reader was added with 100. mu.l ABTS, then 190. mu.l acetic acid-sodium acetate buffer, and finally 10. mu.L enzyme solution and immediately passed through iMarkTMThe absorbance change was measured by a microplate reader.
One unit of laccase activity is defined as: the amount of enzyme required to oxidize 1. mu. mol of 2, 2' -azino-bis (3-ethyllbenzhiazoline-6-sulfonic Acid) (ABTS) per minute at pH 4.2. ABTS molar absorptivity at 415nm of 3.16X 104M-1cm-1。
The enzyme activity of the fermentation broth was measured continuously for 10 days, i.e. during the fermentation process for producing laccase by inducing a single strain of Tricholoma chromophorina, Fuscoporia obliqua and Schizophyllum commune separately for 10 days, and the data of the measured enzyme activity was analyzed by using software Origin 2016(Origin Lab Corporation, Northampton, MA, USA) to generate color pictures, and the measurement results are shown in Table 1 and FIG. 1.
Example 3A Induction of laccase production
Respectively adding 3mL (usually 2-6mL) of uniformly stirred homogeneous body of the trichoderma pigmentosum and the inonotus obliquus (T1), the trichoderma pigmentosum and the schizophyllum (T2), the trichoderma obliquus and the schizophyllum (T3) into 250mL triangular flasks containing 100mL of the culture medium D1, performing constant-temperature dark culture on a shaking table, performing fermentation treatment, and inducing pairwise mixed bacteria of the trichoderma pigmentosum, the trichoderma obliquus and the schizophyllum to produce laccase, wherein the ratio of the volume of the inoculated mixed homogeneous body to the volume of the induction treatment culture medium D1 is 3:100 (usually 2-6: 100); the shaking rate in the fermentation treatment process is 150r/min (usually 100-; the fermentation temperature is 26 ℃ (usually 25-28 ℃);
the volume ratio of the fusobacterium chromotrichum to the fuscoporia obliqua in the mixed bacteria T1 is 1:1, namely 1.5ml of fusobacterium chromotrichum and 1.5ml of fuscoporia obliqua in T1;
the volume ratio of the trichoderma versicolor to the schizophyllum commune in the mixed bacteria T2 is 1:1, namely 1.5ml of trichoderma versicolor and 1.5ml of schizophyllum commune in T1;
the volume ratio of the homogeneous bodies of the birch-leaf fungus and the schizophyllum commune in the mixed fungus T3 is 1:1, namely 1.5ml of the homogeneous bodies of the birch-leaf fungus and 1.5ml of the homogeneous bodies of the schizophyllum commune in T1;
each experiment was set up in 3 replicates.
After inoculation of the mixed bacteria, the enzyme activity after fermentation was measured every 24 hours after inoculation, and the measurement was continued for 10 days in the same manner as in example 3, and the measurement results are shown in table 1 and fig. 1.
Example 3A-1 Induction of laccase production
Respectively adding 3mL (usually 2-5mL) of uniformly stirred homogeneous bodies of the trichoderma pigmentosum and the inonotus obliquus (T1A), the trichoderma pigmentosum and the schizophyllum (T2A), the trichoderma obliquus and the schizophyllum (T3A) into 250mL triangular flasks containing 100mL of the culture medium D1, performing constant-temperature dark culture on a shaking table, performing fermentation treatment, and inducing pairwise mixed bacteria of the trichoderma pigmentosum, the trichoderma obliquus and the schizophyllum to produce laccase, wherein the ratio of the volume of the inoculated mixed homogeneous bodies to the volume of the induction treatment culture medium D1 is 3:100 (usually 2-6: 100); the shaking rate in the fermentation treatment process is 150r/min (usually 100-; the fermentation temperature is 26 ℃ (usually 25-28 ℃);
the volume ratio of the fusobacterium chromotrichum to the fuscoporia obliqua in the mixed bacteria T1A is 1:2, namely 1.0ml of fusobacterium chromotrichum homogenate and 2.0ml of fuscoporia obliqua homogenate in T1;
the volume ratio of the trichoderma versicolor to the schizophyllum commune in the mixed bacteria T2A is 1:2, namely 1.0ml of trichoderma versicolor and 2.0ml of schizophyllum commune in T1;
the volume ratio of the homogeneous bodies of the birch-leaf fungus and the schizophyllum commune in the mixed fungus T3A is 1:2, namely 1.0ml of the homogeneous body of the birch-leaf fungus in T1 and 2.0ml of the homogeneous body of the schizophyllum commune;
each experiment was set up in 3 replicates.
After inoculation of the mixed bacteria, the enzyme activity after fermentation was measured every 24 hours after inoculation, and the measurement was continued for 10 days in the same manner as in example 3, and the measurement results are shown in table 1 and fig. 2.
Example 3B Induction of laccase production
Respectively adding 3mL (usually 2-5mL) of uniformly stirred homogeneous body of the trichoderma palustre, the birch-leaf fungus and the schizophyllum (T4) into 250mL triangular flasks containing 100mL of the culture medium D1, performing constant-temperature dark culture on a shaking table, performing fermentation treatment, and inducing a mixed bacterium of the trichoderma palustre, the birch-leaf fungus and the schizophyllum to produce laccase, wherein the ratio of the volume of the inoculated mixed homogeneous body to the volume of the induction treatment culture medium D1 is 3:100 (usually 2-6: 100); the shaking rate in the fermentation treatment process is 150r/min (usually 100-; the fermentation temperature is 26 ℃ (usually 25-28 ℃);
the volume ratio of the fuscoporia chromophorina, the birch-ruffle and the schizophyllum commune in the mixed bacteria T4 is 1:1:1, namely 1.0ml of the fuscoporia chromophorina homogenate, 1.0ml of the birch-ruffle homogenate and 1.0ml of the schizophyllum commune homogenate in T4.
Each experiment was set up in 3 replicates.
After inoculation of the mixed bacteria, the enzyme activity after fermentation was measured every 24 hours after inoculation, and the measurement was continued for 10 days in the same manner as in example 3, and the measurement results are shown in table 1 and fig. 1.
TABLE 1 time of appearance of maximum laccase activity and maximum laccase activity of Erysipelothrix chromophoris, Fuscoporia Obliqua and Schizophyllum commune on medium D1
Wherein J1-3 is Tricholoma chromophilum, Fuscoporia obliqua, and Schizophyllum commune respectively; t1 is a mixed bacterium of Erysipelothrix chromophoris and Fuscoporia obliqua (the volume ratio of the homogeneous body is 1: 1); t2 is a mixed bacterium of Tricholoma chromophilum and Schizophyllum commune (the volume ratio of the homogeneous body is 1: 1); t3 is a mixture of Fuscoporia obliqua and Schizophyllum commune (the volume ratio of the homogeneous body is 1: 1); T1A is a mixed bacterium of Erysipelothrix chromophoris and Fuscoporia obliqua (the volume ratio of the homogeneous body is 1: 2); T2A is a mixed bacterium of Tricholoma chromophilum and Schizophyllum commune (the volume ratio of the homogeneous body is 1: 2); T3A is a mixture of Fuscoporia obliqua and Schizophyllum commune (the volume ratio of the homogeneous body is 1: 2); t4 is a mixture of Erysipelothrix chromophoris, Fuscoporia Obliqua and Schizophyllum commune (the ratio of the volume of the homogenate is 1:1: 1).
As can be seen from the experimental results of table 1 and fig. 1:
1. fermentation and induction production of laccase by using C.unicolor Han 849, L.betulinus Han 851 and S.commune Han 881 of Trichophyton mentagrophytes in a D1 culture medium respectively, wherein the maximum laccase activity occurrence time is respectively 3 days, 7 days and 7 days of inoculation and fermentation, and the maximum enzyme activity is respectively 552.34 +/-49.14U/L, 309.72 +/-12.53U/L and 5.22 +/-0.35U/L;
2. when the hirsutella sinensis C.unicolor Han 849 and the chaetomium cupreum Han 851 are co-cultured and fermented (D1 culture medium), the maximum laccase activity occurs 10 days after inoculation and fermentation, the maximum laccase activity is 1144.85 +/-34.97U/L, is 2.07 times of the laccase activity produced by single hirsutella sinensis fermentation and is 3.70 times of the laccase activity produced by single chaetomium cupreum fermentation; however, when C.unicolor Han 849 and L.betulina Han 851 were co-cultured and fermented (D1 medium), the added plasmids were 1.5mL and 1.5mL, respectively, and 3mL for fermentation of single C.unicolor or single B.betulina.
3. When the hirsutella sinensis C.unicolor Han 849 and the schizophyllum commune Han 881 are co-cultured and fermented (D1 culture medium), the maximum laccase activity occurs for 6 days of inoculation and fermentation, the maximum laccase activity is 1373.12 +/-55.93U/L, is 2.49 times of the laccase activity produced by single hirsutella sinensis fermentation, and is 263.05 times of the laccase activity produced by single schizophyllum commune fermentation; however, in the case of co-culture fermentation of C.unicolor Han 849 and Schizophyllum S.commune Han 881 (D1 medium), the amount of added homogenate was 1.5mL and 1.5mL, respectively, and the amount of added homogenate was 3mL for fermentation of a single C.unicolor or a single Schizophyllum.
4. When the coprecious fermentation (D1 culture medium) of the betulin L.betulina Han 851 and the schizophyllum S.commune Han 881 is carried out, the maximum laccase activity occurs for 5 days of inoculation fermentation, the maximum laccase activity is 549.83 +/-12.42U/L, and is 1.78 times of the laccase activity produced by single fermentation of the betulin; 105.33 times of the laccase activity produced by single schizophyllum commune fermentation; however, in the case of co-culture fermentation of Fuscoporia obliqua L.betulina Han 851 and Schizophyllum commune Han 881 (D1 medium), the amount of added homogenate was 1.5mL and 1.5mL, respectively, and the amount of added homogenate was 3mL in the case of fermentation of Fuscoporia obliqua or Schizophyllum obliqua, respectively.
5. When the hirsutella sinensis C.unicolor Han 849, the chaetomium cupreum L.beta Han 851 and the schizophyllum S.commune Han 881 are co-cultured and fermented (D1 culture medium), the maximum laccase activity occurs 10 days after inoculation and fermentation, the maximum laccase activity is 774.96 +/-13.79U/L, is 1.40 times of the laccase activity produced by single hirsutella sinensis fermentation, and is 2.50 times of the laccase activity produced by single chaetomium cupreum fermentation; 148.46 times of the laccase activity produced by single schizophyllum commune fermentation; when the trichinosis C.unicolor Han 849, the birch-fold bacterium L.beta-ina Han 851 and the schizophyllum S.commune Han 881 are co-cultured and fermented (D1 culture medium), the added homogentises of the trichinosis C.unicolor Han 849, the added homogentises of the birch-fold bacterium L.beta-ina Han 851 and the added homogentises of the schizophyllum S.commune Han 881 are respectively 1mL, 1mL and 1mL, and the added homogentises of the single trichinosis C.unicolor, the added homogentises of the single birch-fold bacterium or the single schizophyllum fermented are respectively 3mL (figure 1).
6. As is apparent from FIG. 1, the stability of the laccase activity induced and produced by the mixed bacteria is obviously better than that induced and produced by a single strain, and the laccase activity produced by the mixed bacteria is obviously higher than that produced by a single strain.
Example 3C Induction of laccase production
The same procedure as in example 3 was repeated, except that the medium contained in each flask was the medium D1-1. The measurement was continued for 10 days, and the measurement results are shown in Table 2.
Example 3C-1 Induction of laccase production
The same procedure as in example 3A was repeated, except that the medium contained in each flask was the medium D1-1. The measurement was continued for 10 days, and the measurement results are shown in Table 2.
Example 3C-2 Induction of laccase
The same procedure as in example 3A-1 was repeated, except that the medium contained in each flask was the medium D1-1. The measurement was continued for 10 days, and the measurement results are shown in Table 2.
TABLE 2 appearance time of maximum laccase activity and maximum laccase activity of Tricholoma chromophilum, Fuscoporia obliqua and Schizophyllum commune in culture medium D1-1
Example 3D Induction of laccase production
The same procedure as in example 3 was repeated, except that the medium contained in each flask was the medium D1-2. The measurement was continued for 10 days, and the measurement results are shown in Table 3.
Example 3D-1 Induction of laccase production
The same procedure as in example 3A was repeated, except that the medium contained in each flask was the medium D1-2. The measurement was continued for 10 days, and the measurement results are shown in Table 3.
Example 3D-2 Induction of laccase production
The same procedure as in example 3A-1 was repeated, except that the medium contained in each flask was the medium D1-2. The measurement was continued for 10 days, and the measurement results are shown in Table 3.
TABLE 3 time of appearance of maximum laccase activity and maximum laccase activity of Tricholoma chromocor, Fuscoporia Obliqua and Schizophyllum commune on culture medium D1-2
Example 4 Induction of laccase production
The laccase activity assay and the time to maximum laccase activity appearance are shown in Table 4, which is the same as example 3, except that 100mL of medium D2 is contained in a 250mL Erlenmeyer flask.
Example 4A Induction of laccase production
The laccase activity assay and the time to maximum laccase activity appearance are shown in Table 4, which is the same as in example 3A, except that 100mL of medium D2 is contained in a 250mL Erlenmeyer flask.
Example 4B Induction of laccase production
The laccase activity assay and the time to maximum laccase activity appearance are shown in Table 4, which is the same as example 3B, except that 100mL of medium D2 is contained in a 250mL Erlenmeyer flask.
TABLE 4 appearance time of maximum laccase activity and maximum laccase activity of Tricholoma chromocor, Fuscoporia Obliqua and Schizophyllum commune on culture medium D2
The above-described embodiments of the present invention are merely exemplary and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.
Claims (10)
1. A method for producing laccase by using mixed bacteria is characterized in that fungi, namely Cerrena unicolor Han 849, Lenzites betulinus Han 851 and Schizophyllum commune Han 881 are used for mixed fermentation of a basic culture solution containing lignocellulose materials to produce the laccase.
2. The process as claimed in claim 1, wherein the lignocellulosic material is phoenix tree wood flour, Chinese pine wood flour, preferably phoenix tree wood flour.
3. The method according to claim 1 or 2, characterized in that the mass of lignocellulosic material is 1-4g, preferably 2-3g, per 100ml of basic culture medium.
4. The method according to claim 1 or 2, wherein the composition of the basal medium is: 1.0-2.0g of monopotassium phosphate, constant volume to 1L and natural pH.
5. The method according to claim 1 or 2, wherein the conditions of the mixed fermentation are: dark culture; the culture temperature is 25-28 ℃.
6. A method for producing laccase by using mixed bacteria is characterized by comprising the following steps in sequence:
1) respectively inoculating fungi such as Trichium chromophilum (Cerrena unicolor Han 849), Fuscoporia obliqua (Lenzites betulinus Han 851) and Schizophyllum commune Han 881 to a solid CYM plate complete culture medium for strain activation treatment;
2) respectively inoculating the activated and cultured strains of 3 strains into a liquid complete culture medium, and respectively performing amplification culture to obtain an Erysipelothrix tropicalis amplification culture mixture, a Inonotus obliquus amplification culture mixture and a Schizophyllum commune amplification culture mixture;
3) respectively homogenizing a trichlorphon culture mixture, a birch-ruffle culture mixture and a schizophyllum culture mixture by a homogenizer under aseptic operation conditions to obtain a trichlorphon culture homogenate, a birch-ruffle culture homogenate and a schizophyllum culture homogenate;
4) mixing and inoculating a trichoderma trichinosum homogeneous body and a birch-ruffle homogeneous body into a basic culture solution containing a lignocellulose material, and performing fermentation culture; or
Mixing and inoculating the trichoderma unicolor homogenate body and the schizophyllum commune homogenate body into a basic culture solution containing a lignocellulose material for fermentation culture; or
Mixing and inoculating the homogeneous body of the birch-pleat fungus and the homogeneous body of the schizophyllum commune into a basic culture solution containing a lignocellulose material for fermentation culture; or
Mixing and inoculating the trichoderma trichinosum homogenate, the birch-ruffle homogenate and the schizophyllum commune homogenate into a basic culture solution containing a lignocellulose material for fermentation culture.
7. The method as claimed in claim 6, wherein the homogeneous body of Tricholoma mentarius and the homogeneous body of Fuscoporia obliqua in step 4) are inoculated in a mixed manner, and the volume ratio of the homogeneous body of Tricholoma mentarius and the homogeneous body of Fuscoporia obliqua in the fermentation culture process is 1: (1-2), preferably 1: 1; the trichoderma viride homogeneous body and the schizophyllum homogeneous body are mixed and inoculated, and the volume ratio of the trichoderma viride homogeneous body to the schizophyllum homogeneous body in the fermentation culture process is 1: (1-2), preferably 1: 1; the homogeneous body of the birch-leaf fungus and the homogeneous body of the schizophyllum commune are mixed and inoculated, and the volume ratio of the homogeneous body of the birch-leaf fungus to the homogeneous body of the schizophyllum commune in the fermentation culture process is 1: (1-2), preferably 1: 1.
8. The method according to claim 6 or 7, wherein the homogeneous body of Tricholoma mentarius, the homogeneous body of Fuscoporia obliqua and the homogeneous body of Schizophyllum commune in step 4) are inoculated and mixed, and the volume ratio of the homogeneous body of Tricholoma mentarius, the homogeneous body of Fuscoporia obliqua and the homogeneous body of Schizophyllum commune in the fermentation culture process is 1:1: 1.
9. The method as claimed in claim 6 or 7, wherein the homogeneous body of Tricholoma chroothecoides and Fuscoporia obliqua is inoculated in step 4), and the ratio of the sum of the volume of the homogeneous body of Tricholoma chroothecoides and Fuscoporia obliqua to the volume of the basic culture solution containing the lignocellulose material in the fermentation culture process is (2-6): 100, preferably 3: 100; the homogeneous body of the Tricholoma chroococcum and the Schizophyllum commune is mixed and inoculated, and the ratio of the sum of the volume of the homogeneous body of the Tricholoma chroococcum and the Schizophyllum commune to the volume of the basic culture solution containing the lignocellulose material in the fermentation culture process is (2-6): 100, preferably 3: 100; mixing and inoculating the homogeneous body of the schizophyllum commune and the birch-fold fungus, wherein the ratio of the sum of the volumes of the homogeneous body of the schizophyllum commune and the birch-fold fungus to the volume of the basic culture solution containing the lignocellulose material in the fermentation culture process is (2-6): 100, preferably 3: 100; the homogeneous body of the trichoderma trichineum, the birch-ruffle and the schizophyllum is mixed and inoculated, and the ratio of the sum of the volumes of the homogeneous body of the trichoderma trichineum, the birch-ruffle and the schizophyllum to the volume of the basic culture solution containing the lignocellulose material in the fermentation culture process is (2-6): 100, preferably 3: 100.
10. The process as claimed in claim 6 or 7, wherein the lignocellulosic material in step 4) is phoenix tree wood flour, Chinese pine wood flour, preferably phoenix tree wood flour.
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