CN109868224B

CN109868224B - Myrothecium verrucaria with high laccase yield and application thereof

Info

Publication number: CN109868224B
Application number: CN201910105654.3A
Authority: CN
Inventors: 孙旸; 陈�光; 苟泽昌; 苏瑛杰; 王刚; 陈欢; 于潇潇
Original assignee: Jilin Agricultural University
Current assignee: Jilin Agricultural University
Priority date: 2019-02-01
Filing date: 2019-02-01
Publication date: 2021-03-16
Anticipated expiration: 2039-02-01
Also published as: CN109868224A

Abstract

A Myrothecium verrucaria (Myrothecium verrucaria) mutant strain 3H6 with a preservation number of CCTCC NO: M2018800 is provided. The laccase strain is a high-yield laccase strain, the enzyme activity of laccase produced in a basic fermentation culture medium in a shake flask culture mode can reach 3408.69U/L, and the enzyme activity is improved by 55.6% compared with that of the existing Myrothecium verrucaria strain. A culture for high-yield laccase contains corn stalk treated by tannic acid as sole carbon source, and the strain is Myrothecium verrucaria (Myrothecium verrucaria)3H 6. By adopting the laccase-producing culture of the invention, the enzyme activity of the laccase produced by a shake-flask culture mode is obviously improved and can reach 7025.54U/L.

Description

Myrothecium verrucaria with high laccase yield and application thereof

Technical Field

The invention relates to the technical field of applied microorganisms, and in particular relates to a new strain of Myrothecium verrucaria and application thereof.

Background

Laccase (ec1.10.3.2, Lac), abbreviated as β -bisphenol: the oxidoreductase is a copper-containing oxidase, and belongs to the multi-effect oxidase superfamily. Laccases can catalyze the oxidation of a variety of compounds to water by generating unstable free radicals and oxygen four-electron reduction. Laccases have been widely used in the fields of synthetic organic chemistry, food, textile industry, sewage treatment, delignification and bleaching. They can be obtained from bacteria, fungi and plants, and fungi play a dominant role in the production of laccases due to their thermostability and extensive catalytic action.

The potential benefits of laccase in the environmental field need to be further discussed, the activity of the laccase is improved by changing the conditions of a culture medium, ultraviolet mutagenesis, normal-temperature and normal-pressure plasma mutagenesis, directed evolution and other ways, and the production of the laccase is limited by low yield and high cost. How to improve the activity and stabilize the laccase output in a large range is still needed to be further researched. The development of novel microorganisms with high enzyme productivity is an effective approach to solve this problem.

Corn is an agricultural waste that is not well utilized, is a lignocellulose, and mainly consists of cellulose, hemicellulose and lignin. There remains questionable how to increase the utilization and processing levels of corn resources in a cost-effective, eco-friendly and feasible manner. Generally, corn stalks are pretreated by acid-base or steam explosion before being used for biological processing or fiber feed. With respect to acid pretreatment, whatever the acid, the corn stover requires washing after treatment and is usually accompanied by a loss of biomass. In order to realize the comprehensive utilization of the straws, a great deal of work is required. Tannic acid (also called tannic acid) is weakly acidic and is mainly used in the fields of dye mordants, preparation of gallic acid, leather tanning agents and rubber coagulants.

Disclosure of Invention

The invention aims to provide a laccase-producing Myrothecium verrucaria mutant strain 3H 6.

The invention also aims to provide a fermentation production method of the high-yield laccase.

The purpose of the invention is realized by the following technical scheme:

a Myrothecium verrucaria (Myrothecium verrucaria) mutant strain 3H6, comprising:

the Myrothecium verrucaria (Myrothecium verrucaria) mutant strain 3H6 is obtained by separating, screening and mutagenesis from a soil sample collected from Changbai mountain, and is preserved in China Center for Type Culture Collection (CCTCC) with the preservation address: the preservation date of Wuhan university in China is 11 months and 19 days in 2018, and the preservation number is CCTCC NO: M2018800.

The Myrothecium verrucaria strain mutant strain 3H6 is inoculated on a PDA solid culture medium, hyphae of the strain initially appear white flocculent and are divergently grown towards the periphery, the bacterial colony is approximately regular and round, and a black conidium seat appears after the bacterial colony grows on a flat plate for 5 days.

The invention relates to a mutated strain 3H6 of Myrothecium verrucaria, which is a high-yield laccase mutant strain 3H6 obtained by collecting strains of forest soil in natural protected areas of Changbai mountains → separating and screening strains → ultraviolet mutagenesis → normal pressure room temperature plasma mutagenesis (ARTP mutagenesis) → screening. The method comprises the following steps:

1. isolation and screening of strains

Collecting the forest soil in the natural protected area of Changbai mountain, and taking a soil sample to shake for 45min on a constant temperature oscillator at 30 ℃ and 120r/min in sterile water. Standing, collecting supernatant, diluting according to gradient multiple, and diluting with dilution degree of 10 by dilution coating plate method^-6～10^-8The supernatant was transferred to PDA plates, 3 replicates for each dilution, and incubated in an incubator at 29 ℃. After the flora grows out, repeatedly streaking and separating on a PDA plate, and inoculating the pure single flora on a preservation medium after observing and determining the pure single flora under a microscope.

2. Morphological characterization of strains

Inoculating the separated Myrothecium verrucaria strain on a PDA (PDA dextrose agar) solid culture medium, wherein hyphae of the strain are initially white flocculent and are divergently grown towards the periphery, the bacterial colony is approximately regular and round, and a black conidium seat appears after the bacterial colony grows on a flat plate for 5 days.

3. UV and ARTP combined mutagenesis

Diluting Myrothecium verrucaria to 10⁶At a concentration of 75s per ml, UV mutagenesis was carried out at an irradiation power of 100W and a distance of 5 cm. After the UV mutagenesis, ARTP mutagenesis was carried out at a distance of 2mm at 100W for 85s with a pure helium flow rate of 10 standard liters/min.

4. Mutant strain screening

Inoculating the above-mentioned strain after mutagenesis to a shallow well plate containing PD medium (PD medium: potato 200g/L, glucose 20g/L) for culturing, transferring it to a medium containing basic fermentation medium (basic fermentation medium: potato 20g/L, glucose 2.4g/L, peptone 10g/L, yeast powder 5g/L, Tween 801 g/L, KH 801/L)₂PO₄ 1.5g/L,MgSO₄·7H₂O 1.5g/L,CaCl₂·2H₂O0.1 g/L), screening by adopting an HTS (high throughput screening) method, screening 7 positive mutants from 30000 mutants, and finally screening the 3H6 strain with the highest enzyme activity in a basic fermentation culture medium, thereby screening out the high-yield laccase mutant. The screened Myrothecium verrucaria 3H6 strain has enzyme activity of the laccase up to 3408.69U L in the basic fermentation culture medium and in the shake flask culture mode^-1The strain is improved by 55.6 percent compared with the existing strain, and meanwhile, the 3H6 strain cell shows genetic stability in the 8 th generation through genetic stability research.

A culture with high laccase yield is characterized in that: the method comprises the step of using corn straws treated by tannic acid as a unique carbon source, wherein the strain adopts Myrothecium verrucaria (Myrothecium verrucaria)3H 6.

Further, the concentration of the above tannic acid is 1 to 15g/L, and more preferably 1 g/L.

Further, the solid-to-liquid ratio of the corn straws to the tannic acid is 1:8-24, and the unit is corresponding to gram/mL; preferably, the solid-to-liquid ratio of the corn straws to the tannic acid is 1: 20.

Preferably, the temperature for soaking the corn stalks in the tannin is 90-130 deg.C for 50-65min, and most preferably, the soaking temperature is 110 deg.C for 60 min.

Specifically, a culture with high laccase yield is characterized in that: the corn straw treated by tannic acid is used as a carbon source, and potato, peptone, yeast powder, Tween80 and KH₂PO₄、MgSO₄·7H₂O、CaCl₂·2H₂And (C) O.

Most particularly, a culture with high laccase yield is characterized in that: the formula comprises 20g/L of potato, 3.6g/L of corn straw soaked by tannic acid, 10g/L of peptone, 5g/L of yeast powder, 801 g/L of Tween and KH₂PO₄ 1.5g/L,MgSO₄·7H₂O 1.5g/L,CaCl₂·2H₂O is 0.1 g/L; the solid-liquid ratio of the corn straws to the tannic acid is 1:20, the unit is corresponding to gram/mL, the concentration of the tannic acid is 1g/L, the soaking temperature is 110 ℃, and the soaking time is 60 min.

In the development process, various other crop straws are also selected and used as a growth carbon source of the 3H6 bacterial strain after being treated by acid, but the enzyme activity of the laccase obtained is not obviously improved, the enzyme activity of the laccase obtained by finally selecting the corn straws as the carbon source after being treated by tannic acid is greatly improved, and the enzyme activity of the laccase produced by the shake flask culture mode can reach 7025.54U/L. The cheap corn straw is used as a carbon source to completely replace expensive glucose, so that the fermentation production cost is greatly saved, and the method is very beneficial to industrial production.

A production method of laccase is characterized by comprising the following steps:

(1) soaking the crushed corn straws in a tannic acid solution; the concentration of the tannic acid is 1-15g/L, the solid-to-liquid ratio of the corn straw to the tannic acid is 1:8-24, in gram/mL corresponding units, the soaking temperature is 90-130 ℃, and the soaking time is 50-65 min;

(2) after the reaction is finished, carrying out suction filtration on the corn straw powder until the water content is 50-60%, and then drying until the water content is 10-13%;

(3) and (3) preparing a fermentation medium by taking the corn straw powder obtained in the step (2) as a unique carbon source, wherein the formula of the fermentation medium is as follows: the corn straw treated by the tannic acid is used as a carbon source, potato, peptone, yeast powder, Tween80 and KH₂PO₄、MgSO₄·7H₂O、CaCl₂·2H₂O, the strain adopts the Myrothecium verrucaria 3H6 strain;

(4) and shake flask fermentation culture: 160r/min, 30 ℃ and 96 hours;

(5) centrifuging the fermentation liquor to obtain crude enzyme in supernatant, and adding (NH) into the crude enzyme solution₄)₂SO₄The solution precipitates the protein step by step at 4 ℃; enriching, concentrating and desalting laccase with ultrafiltration concentrator with molecular weight cut-off of 50kDa, and storing at 4 deg.C.

The invention has the following beneficial effects:

the Myrothecium verrucaria 3H6 is a high-yield laccase strain, the enzyme activity of laccase produced in a basic fermentation culture medium by a shake flask culture mode can reach 3408.69U/L, and is increased by 55.6% compared with the existing Myrothecium verrucaria strain (see attached figure 3); by adopting the laccase-producing culture of the invention, the enzyme activity of the laccase produced by the shake flask culture mode is obviously improved and can reach 7025.54U/L (see figure 4). The laccase of the invention has simple production process, high purity of the produced laccase and less impure protein, and simultaneously saves water washing process and the like without environmental pollution. The laccase produced by the invention is adopted to decolorize industrial dye methylene blue, and the decolorization rate is up to 70% after incubation for 10h at 30 ℃ and pH4.5; the enzyme has potential application prospect in textile and environmental protection industries. These results provide a new idea for the production of high-activity laccase and a new way for the comprehensive utilization of straws.

Drawings

FIG. 1: a comparison graph of laccase activities produced by basal culture media with washed corn stalks and unwashed corn stalks as unique carbon sources is shown.

FIG. 2: (A) scanning electron microscope of original corn stalk. (B) Scanning electron microscope of corn stalk treated with tannic acid.

FIG. 3: the enzyme activity of the laccase produced by the better forward mutant strain is compared with that produced by the original strain.

FIG. 4: enzyme production history of 3H6 strain in fermentation medium with tannic acid as sole carbon source.

FIG. 5: SDS-PAGE analysis of laccase. M: 97.2kDa protein maker 1: original strain laccase, 2: mutant 3H6 laccase. 3: and purifying the laccase.

FIG. 6: the spectrum property diagram of the laccase produced by the invention.

Detailed Description

The present invention is described in detail below by way of examples, it being necessary to note that the following examples are provided only for illustrating the present invention and are not to be construed as limiting the scope of the present invention, and modifications or substitutions of the method, steps or conditions of the present invention may be made without departing from the spirit and spirit of the present invention.

The conventional media used in the following examples are specifically as follows:

potato medium (PD): potato 200g/L and glucose 20 g/L;

potato agar medium (PDA): PD medium supplemented with 18g/L agar;

basic fermentation medium: 20g/L of potato, 2.4g/L of glucose, 10g/L of peptone, 5g/L of yeast powder, Tween 801 g/L and KH₂PO₄ 1.5g/L,MgSO₄·7H₂O 1.5g/L,CaCl₂·2H₂O 0.1g/L。

Example 1 obtaining of A. verrucosa mutant Strain 3H6

1. Isolation and screening of strains

Collecting the forest soil in the natural protected area of Changbai mountain, and taking 1g of soil sample to put in a 250mL triangular flask containing 99mL sterile water, and oscillating for 45min on a constant temperature oscillator at 30 ℃ and 120 r/min. Standing, collecting supernatant, diluting according to gradient multiple, and diluting with dilution degree of 10 by dilution coating plate method^-6～10^-8The supernatant was transferred to PDA plates, 3 replicates for each dilution, and incubated in an incubator at 29 ℃. After the flora grows out, repeatedly streaking and separating on a PDA plate, and inoculating the pure single flora on a preservation medium after observing and determining the pure single flora under a microscope.

2. Morphological identification

3. UV and ARTP combined mutagenesis

4. Mutant strain screening

Inoculating the mutagenized strain into a 96-shallow pore plate containing 200 mu L PD culture medium, culturing at 30 ℃ for 48H, transferring the strain into a deep pore plate containing 1ml basic fermentation culture medium, continuing to ferment, culturing at 30 ℃ for 96H, screening by adopting an HTS (high throughput screening) method, screening 7 positive mutants from 30000 mutants, and finally screening the 3H6 strain with highest enzyme activity in the basic fermentation culture medium so as to screen out the high-yield laccase mutant.

Example 2 method for producing laccase with high enzyme activity by fermentation

Comprises the following steps:

(1) soaking the crushed corn straws in a tannic acid solution; the concentration of tannic acid is 1-15g/L, preferably 1g/L, the solid-to-liquid ratio of corn stalk and tannic acid is 1:8-24 (preferably 1:20), in gram/mL corresponding unit, the soaking temperature is 90-130 deg.C, and the soaking time is 50-65min (preferably 110 deg.C, and the soaking time is 60 min); the scanning electron microscope image of the corn straw treated with tannic acid is shown in FIG. 2. We have also found that corn stover treated with tannic acid has a significantly increased lignin content in its constituents, which is beneficial in stimulating laccase production by fungi, as shown in Table 1.

TABLE 1 comparison of tanned and acid-treated corn stover compositions in other tables

	Cellulose(％)	Hemicellulose(％)	Lignin(％)
				Original corn strove	35.27±0.28	16.04±0.36	9.75±0.26
Tannic acid	21.63±0.54	13.78±0.30	25.47±0.42
				Sulphuric acid	50.43±0.73	12.32±0.37	12.49±0.53
Hydrochloric acid	55.07±0.44	11.23±0.53	13.22±0.12
				Solid acid	59.47±3.42	5.9±0.41	14.25±0.94

(3) and (3) preparing a fermentation medium by taking the corn straw powder obtained in the step (2) as a unique carbon source, wherein the formula of the fermentation medium is as follows: the formula comprises 20g/L of potato, 3.6g/L of corn straw soaked by tannic acid, 10g/L of peptone, 5g/L of yeast powder, 801 g/L of Tween and KH₂PO₄ 1.5g/L,MgSO₄·7H₂O 1.5g/L,CaCl₂·2H₂O0.1 g/L, wherein the strain adopts the Myrothecium verrucaria 3H6 strain;

(4) and shake flask fermentation culture: 160r/min, 30 ℃ and 96 hours;

(5) centrifuging the fermentation broth at 8500g for 5min to obtain crude enzyme in supernatant, and adding (NH) into 100mL of crude enzyme solution₄)₂SO₄The solution precipitates the protein step by step at 4 ℃ with the final concentration of 10-90% (w/v); enrichment, concentration and desalting of laccase with ultrafiltration concentrator with molecular weight cut-off of 50kDaPrepared and stored at 4 ℃.

The enzyme activity determination method comprises the following steps: determining laccase activity at 30 ℃ by using ABTS as a substrate; enzyme activity is expressed in units of enzyme required to oxidize 1 μ M substrate per minute, all assays being performed in triplicate.

Determination of optimum temperature and thermal stability: under the condition of pH4.5, the temperature of 20-80 ℃ is measured, the optimal temperature and thermal stability of the reaction are determined, and three experiments are carried out.

Determination of optimum pH and stability: the pH-activity curve is determined within a range of 100mm by using a citric acid buffer solution (pH 2.0-3.5), an acetic acid buffer solution (pH 3.5-5.5), a phosphate buffer solution (pH 5.5-7.5) and a Tris-HCl buffer solution (pH 7.5-9.0) respectively at a pH value of 2.0-9.0 and at a temperature of 30 ℃. The pH stability of the purified laccase was determined at pH4.5, 5.0, 5.5, 6.0, 7.0, 8.0. Activity assays were performed according to the standard ABTS method. All experiments were done in triplicate.

Dye decolorization experiment: decolorizing with methylene blue (664nm), preparing 1mg/mL stock solution, diluting to the required concentration for decolorizing test, and adding purified laccase in an amount of 10-50 μ L (7.025U/mL).

Determination of molecular mass and spectral properties: the molecular weight of the laccase was determined by SDS-PAGE and gel filtration chromatography. The absorbance of the enzyme solution at a wavelength of 200-800 nm was recorded with an ultraviolet-visible spectrophotometer (Agilent Technologies, CARY series UV-VIS spectrophotometer), and the spectral properties of the enzyme were studied.

The laccase produced above has a molecular weight of about 65kDa, see FIG. 5; the laccase spectrum characteristics are shown in figure 6, and the ultraviolet-visible spectrum of the purified laccase has a slight shoulder at 340nm, which is a typical type III binuclear copper center, and has no characteristic absorption peak at 620nm, which indicates that the laccase is not blue copper laccase, but is yellow or white laccase. The laccase of the invention has excellent stability in the temperature range of 30-40 ℃ and the pH value of 4.0-5.0, and can still keep more than 80% of the activity of the proenzyme after being incubated for 72 hours under the condition. The laccase produced by the invention is adopted to decolorize industrial dye methylene blue, and the decolorization rate is up to 70% after incubation for 10h at 30 ℃ and pH4.5; the enzyme has potential application prospect in textile and environmental protection industries.

Example 3 Effect of washed and unwashed corn stover after tannic acid treatment on laccase Activity

The corn straws treated by the tannic acid are used as a carbon source to replace the glucose component of a basic fermentation medium, and the influence of the corn straws on laccase enzyme production is examined in the medium respectively. The results show that: the laccase activity of the washed corn stalks has no obvious difference with that of the unwashed corn stalks (figure 1). Typically, acid-treated corn stover needs to be washed to a neutral state before being added to the medium. The results show that the tannin treated corn stalks can be directly supplemented in the culture medium. The method can save water source, reduce the cost of corn straw pretreatment and is favorable for solving the problem of environmental pollution.

Claims

1. A production method of laccase is characterized by comprising the following steps:

(3) and (3) preparing a fermentation medium by taking the corn straw powder obtained in the step (2) as a unique carbon source, wherein the formula of the fermentation medium is as follows: the corn straw treated by the tannic acid is used as a carbon source, potato, peptone, yeast powder, Tween80 and KH₂PO₄、MgSO₄·7H₂O、CaCl₂·2H₂O, using a Myrothecium verrucaria mutant strain 3H6 as a strain;

(4) and shake flask fermentation culture: 160r/min, 30 ℃ and 96 hours;

(5) centrifuging the fermentation liquor obtained in the step (4) to obtain crude enzyme in supernatant, and adding (NH) into the crude enzyme solution₄)₂SO₄The solution precipitates the protein step by step at 4 ℃; enriching, concentrating and desalting laccase with ultrafiltration concentrator with molecular weight cut-off of 50kDa, andstoring at 4 deg.C;

the Myrothecium verrucaria (A), (B), (CMyrothecium verrucaria) The mutant strain 3H6 has the preservation number of CCTCC NO: M2018800.