CN104232599A - Method for increasing yield of ganoderma lucidum laccase by using waste paper crushing materials - Google Patents

Method for increasing yield of ganoderma lucidum laccase by using waste paper crushing materials Download PDF

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CN104232599A
CN104232599A CN201410494078.3A CN201410494078A CN104232599A CN 104232599 A CN104232599 A CN 104232599A CN 201410494078 A CN201410494078 A CN 201410494078A CN 104232599 A CN104232599 A CN 104232599A
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waste paper
crushed material
paper crushed
preparation
laccase
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CN104232599B (en
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王海磊
刘宇峰
杨玉华
李平
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Henan Normal University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0055—Oxidoreductases (1.) acting on diphenols and related substances as donors (1.10)
    • C12N9/0057—Oxidoreductases (1.) acting on diphenols and related substances as donors (1.10) with oxygen as acceptor (1.10.3)
    • C12N9/0061—Laccase (1.10.3.2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y110/00—Oxidoreductases acting on diphenols and related substances as donors (1.10)
    • 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 increasing the yield of ganoderma lucidum laccase by using waste paper crushing materials and belongs to the technical field of enzyme preparations for industrial purposes. According to the technical scheme, the method comprises the following four steps: preparation of the waste paper crushing materials, preparation of a ganoderma lucidum seed solution, liquid fermentation of ganoderma lucidum and preparation of a crude enzyme solution. The waste paper crushing materials are wide in source and low in cost, and the preparation method is simple. No toxic material induction exists in the enzymatic synthesis process, post processing is simple, and environment is not polluted. Enzyme is high in yield and short in cycle, and the method is suitable for industrial production.

Description

A kind of method utilizing waste paper crushed material to improve yield of lucid ganoderma laccase
Technical field
The present invention relates to a kind of preparation method of industrial enzymes, be specifically related to a kind of method utilizing waste paper crushed material to improve yield of lucid ganoderma laccase.
Background technology
Laccase (1accase) is the polyphenoloxidase of a class cupric; this enzyme has Substratspezifitaet widely; multiple phenol and aromatic amine compounds can be oxidized; at paper industry, food-processing, dye decolored [Susana Rodr í guez Couto, the Jos é Luis Toca Herrera. Industrial and biotechnological applications of laccases:A review. Biotechnology Advances. 2006 that to have broad application prospects with fields such as degraded, sewage disposal, biosensor and immunodetection; 24(5): 500-513].Become a study hotspot of biotechnology and environmental science crossing domain in recent years.
Laccase is extensively present in plant, higher fungi, insect and bacterium, and the white-rot fungi wherein in basidiomycetes is the microbe groups that laccase output is higher.Glossy ganoderma ( ganoderma lucidum) be a kind of important medicinal fungi, people produce to have carried out to its laccase and study comparatively widely.But want to obtain higher Laccase from White Rot Fungus output, usual aromatic amine compounds and the metal ion need used induces [Yang Yang, Fuxiang Wei, Rui Zhuo, Fangfang Fan, Huahua Liu, Chen Zhang, Li Ma, Mulan Jiang, Xiaoyu Zhang. Enhancing the laccase production and laccase gene expression in the white-rot fungus trametes velutina5930 with great potential for biotechnological applications by different metal ions and aromatic compounds. PLoS One. 2013; 8 (11): e79307].These inductors are generally toxic, add the post-processing difficulty of fermented liquid after interpolation, and easily cause new secondary pollution.
Waste paper, the various paper products that general reference is discarded through using in productive life, it is low that China's waste paper recycles level, the waste paper reclaimed also is processed into the low-grade product such as cardboard, toilet paper by small business backward in technique in a large number, do not play the resource value of waste paper, also can bring serious secondary pollution.The present invention utilizes waste paper crushed material to improve laccase output, and not only for the recycle of waste paper provides a kind of new way, and fermentation residue can be used as feed, nontoxic pollution-free.
Summary of the invention
The present invention overcomes the deficiencies in the prior art to provide a kind of method utilizing waste paper crushed material to improve yield of lucid ganoderma laccase simply, efficiently.
Technical scheme of the present invention is: a kind of method utilizing waste paper crushed material to improve yield of lucid ganoderma laccase, is characterized in that comprising the following steps:
(1) preparation of waste paper crushed material
The waste paper shredded is soaked 5-8h in tap water, and the water ratio of waste paper reaches 50%-80%, dries to constant weight, breaks into broken powder obtain waste paper crushed material with pulverizer for 100 DEG C;
(2) preparation of glossy ganoderma seed liquor
Be that the ganoderma strain capable of CGMCC 5.765 is inoculated on PDA plate culture medium by deposit number, cultivate 5 days for 30 DEG C, flat board is paved with to mycelia, the bacterium block getting 3-4 block soya bean size with aseptic punch tool is inoculated in seed culture medium, 30 DEG C, 150rpm cultivates 5 days as activated seed liquid, the composition of described PDA plate culture medium is counted with g/L: potato 300, glucose 20, agar 15-20, all the other compositions are water, the composition of described seed culture medium is counted with g/L: Semen Maydis powder 100, wheat bran 100, peanut hull meal 25, KH 2pO 410, MgSO 45, all the other compositions are water;
(3) liquid fermenting of glossy ganoderma
Be inoculated in fermention medium by the activated seed liquid that step (2) obtains according to the inoculum size of volume fraction 10%, 30 DEG C, 150rpm cultivates 7 days, filter removal mycelium and obtain fermented liquid, the composition of described fermention medium is counted with g/L: Semen Maydis powder 100, wheat bran 10-30, peanut hull meal 25, KH 2pO 410, MgSO 45, waste paper crushed material 10-30, all the other compositions are water;
(4) preparation of crude enzyme liquid
Fermented liquid step (3) obtained is centrifugal, gets supernatant liquor and is crude enzyme liquid.
Waste paper crushed material of the present invention is that waste newspaper flour minces, give up kraft paper crushed material or useless book paper crushed material.
Compared with prior art, tool of the present invention has the following advantages: (1) waste paper crushed material wide material sources, with low cost, and preparation method is simple; (2) non-toxic substance induction in enzymic synthesis process, post-treatment process is simple, non-environmental-pollution; (3) production of enzyme is high, and the cycle is short, is applicable to suitability for industrialized production.
Embodiment
By the following examples foregoing of the present invention is described in further details, but this should be interpreted as that the scope of the above-mentioned theme of the present invention is only limitted to following embodiment.All technology realized based on foregoing of the present invention all belong to scope of the present invention.
Embodiment
1, the preparation of waste paper crushed material
The waste paper shredded (old newsprint, book paper and kraft paper) is soaked 5-8 hour in tap water, and waste paper water ratio, at 50%-80%, is dried at 100 DEG C to constant weight, breaks into broken powder obtain waste paper crushed material with pulverizer.
2, PDA plate culture medium
Potato 300g, glucose 20g, agar 18g, distilled water complements to cumulative volume 1L, and sterilizing is for subsequent use.
3, seed culture medium
Semen Maydis powder 100g, wheat bran 100g, peanut hull meal 25g, KH 2pO 410g, MgSO 45g, distilled water complements to cumulative volume 1L, and sterilizing is for subsequent use.
4, fermention medium
Control medium 1: Semen Maydis powder 100g, wheat bran 10g, peanut hull meal 25g, KH 2pO 410g, MgSO 45g, distilled water complements to 1L, and sterilizing is for subsequent use.
Control medium 2: Semen Maydis powder 100g, wheat bran 30g, peanut hull meal 25g, KH 2pO 410g, MgSO 45g, distilled water complements to 1L, and sterilizing is for subsequent use.
Fermention medium 1: Semen Maydis powder 100g, wheat bran 10g, peanut hull meal 25g, KH 2pO 410g, MgSO 45g, waste newspaper flour minces 10g, and distilled water complements to 1L, and sterilizing is for subsequent use.
Fermention medium 2: Semen Maydis powder 100g, wheat bran 10g, peanut hull meal 25g, KH 2pO 410g, MgSO 45g, waste newspaper flour minces 30g, and distilled water complements to 1L, and sterilizing is for subsequent use.
Fermention medium 3: Semen Maydis powder 100g, wheat bran 10g, peanut hull meal 25g, KH 2pO 410g, MgSO 45g, useless kraft paper crushed material 10g, distilled water complements to 1L, and sterilizing is for subsequent use.
Fermention medium 4: Semen Maydis powder 100g, wheat bran 10g, peanut hull meal 25g, KH 2pO 410g, MgSO 45g, useless kraft paper crushed material 30g, distilled water complements to 1L, and sterilizing is for subsequent use.
Fermention medium 5: Semen Maydis powder 100g, wheat bran 10g, peanut hull meal 25g, KH 2pO4 10g, MgSO 45g, useless book paper crushed material 10g, distilled water complements to 1L, and sterilizing is for subsequent use.
Fermention medium 6: Semen Maydis powder 100g, wheat bran 10g, peanut hull meal 25g, KH 2pO 410g, MgSO 45g, useless book paper crushed material 30g, distilled water complements to 1L, and sterilizing is for subsequent use.
Fermention medium 7: Semen Maydis powder 100g, wheat bran 30g, peanut hull meal 25g, KH 2pO 410g, MgSO 45g, waste newspaper flour minces 10g, and distilled water complements to 1L, and sterilizing is for subsequent use.
Fermention medium 8: Semen Maydis powder 100g, wheat bran 30g, peanut hull meal 25g, KH 2pO 410g, MgSO 45g, waste newspaper flour minces 30g, and distilled water complements to 1L, and sterilizing is for subsequent use.
Fermention medium 9: Semen Maydis powder 100g, wheat bran 30g, peanut hull meal 25g, KH 2pO 410g, MgSO 45g, useless kraft paper crushed material 10g, distilled water complements to 1L, and sterilizing is for subsequent use.
Fermention medium 10: Semen Maydis powder 100g, wheat bran 30g, peanut hull meal 25g, KH 2pO 410g, MgSO 45g, useless kraft paper crushed material 30g, distilled water complements to 1L, and sterilizing is for subsequent use.
Fermention medium 11: Semen Maydis powder 100g, wheat bran 30g, peanut hull meal 25g, KH 2pO 410g, MgSO 45g, useless book paper crushed material 10g, distilled water complements to 1L, and sterilizing is for subsequent use.
Fermention medium 12: Semen Maydis powder 100g, wheat bran 30g, peanut hull meal 25g, KH 2pO 410g, MgSO 45g, useless book paper crushed material 30g, distilled water complements to 1L, and sterilizing is for subsequent use.
5, the fermentation of ganoderma lucidum laccase
(1) preparation of glossy ganoderma seed liquor
By ganoderma strain capable (purchased from China General Microbiological culture presevation administrative center (China General Microbiological Culture Collection Center, CGMCC), deposit number is CGMCC 5.765) be inoculated on PDA plate culture medium, cultivate 5 days for 30 DEG C, be paved with flat board to mycelia, the bacterium block getting 3-4 block soya bean size with aseptic punch tool is inoculated in above-mentioned seed culture medium, 30 DEG C, 150rpm cultivates 5 days, as activated seed liquid.
(2) liquid fermenting of ganoderma lucidum laccase
Be inoculated in above-mentioned fermention medium 1-12 by activated seed liquid with the inoculum size of volume fraction 10%, 30 DEG C, 150rpm cultivates 7 days, filters removal mycelium and obtains fermented liquid.
(3) mensuration of Laccase activity
The mensuration of Laccase activity adopts 2,2-azine-bis-(3-ethyl-benzothiazole)-6-sulfonic acid (ABTS) method, and enzyme unit definition (U) of living is oxidized enzyme amount needed for 1 μm of ol ABTS for per minute.
By centrifugal for the fermented liquid of gained, get supernatant liquor and be crude enzyme liquid, spectrophotometric determination enzyme is utilized to live, the results are shown in Table 1, compare with 2 with control medium 1, adding of waste paper crushed material improves Laccase activity significantly, and the laccase activity wherein in fermention medium 7 reaches 80000U/L, is 27-32 times of laccase activity in control medium.
Table 1 different culture media is on the impact of ganoderma lucidum laccase enzyme activity
Embodiment above describes ultimate principle of the present invention, principal character and advantage; the technician of the industry should understand; the present invention is not restricted to the described embodiments; what describe in above-described embodiment and specification sheets just illustrates principle of the present invention; under the scope not departing from the principle of the invention; the present invention also has various changes and modifications, and these changes and improvements all fall in the scope of protection of the invention.

Claims (2)

1. utilize waste paper crushed material to improve a method for yield of lucid ganoderma laccase, it is characterized in that comprising the following steps:
(1) preparation of waste paper crushed material
The waste paper shredded is soaked 5-8h in tap water, and the water ratio of waste paper reaches 50%-80%, dries to constant weight, breaks into broken powder obtain waste paper crushed material with pulverizer for 100 DEG C;
(2) preparation of glossy ganoderma seed liquor
Be that the ganoderma strain capable of CGMCC 5.765 is inoculated on PDA plate culture medium by deposit number, cultivate 5 days for 30 DEG C, flat board is paved with to mycelia, the bacterium block getting 3-4 block soya bean size with aseptic punch tool is inoculated in seed culture medium, 30 DEG C, 150rpm cultivates 5 days as activated seed liquid, the composition of described PDA plate culture medium is counted with g/L: potato 300, glucose 20, agar 15-20, all the other compositions are water, the composition of described seed culture medium is counted with g/L: Semen Maydis powder 100, wheat bran 100, peanut hull meal 25, KH 2pO 410, MgSO 45, all the other compositions are water;
(3) liquid fermenting of glossy ganoderma
Be inoculated in fermention medium by the activated seed liquid that step (2) obtains according to the inoculum size of volume fraction 10%, 30 DEG C, 150rpm cultivates 7 days, filter removal mycelium and obtain fermented liquid, the composition of described fermention medium is counted with g/L: Semen Maydis powder 100, wheat bran 10-30, peanut hull meal 25, KH 2pO 410, MgSO 45, waste paper crushed material 10-30, all the other compositions are water;
(4) preparation of crude enzyme liquid
Fermented liquid step (3) obtained is centrifugal, gets supernatant liquor and is crude enzyme liquid.
2. the method utilizing waste paper crushed material to improve yield of lucid ganoderma laccase according to claim 1, is characterized in that: described waste paper crushed material is that waste newspaper flour minces, give up kraft paper crushed material or useless book paper crushed material.
CN201410494078.3A 2014-09-25 2014-09-25 A kind of method that yield of lucid ganoderma laccase is improved using waste paper crushed material Expired - Fee Related CN104232599B (en)

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Cited By (1)

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Publication number Priority date Publication date Assignee Title
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CN112359026A (en) * 2020-11-18 2021-02-12 山东农业大学 Method for increasing laccase content in auricularia polytricha

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