CN101008005A - Lucid ganoderma laccase preparation and production method thereof - Google Patents
Lucid ganoderma laccase preparation and production method thereof Download PDFInfo
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- CN101008005A CN101008005A CNA2006100380896A CN200610038089A CN101008005A CN 101008005 A CN101008005 A CN 101008005A CN A2006100380896 A CNA2006100380896 A CN A2006100380896A CN 200610038089 A CN200610038089 A CN 200610038089A CN 101008005 A CN101008005 A CN 101008005A
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Abstract
The invention relates to ganoderam lucidum karst laccase preparation, which in detail relates to a blue copperas- containing liquid laccase preparation produced with Ganoderma lucidum through inclined-plane culture and two- stage fermentation, the blue copperas concentration is 0.15- 0.40 mol/l. The culture medium of first- stage comprises amylaceum, maize flour, bran flour and water, and the culture condition is 25- 35 Deg. C and for 6- 12 days. The further fermentation culture medium comprises bran flour, peanut shell powder, maize flour and water, and the culture condition is 25- 35 Deg. C and for 3- 7 days. The enzymatic activity of said preparation is 32841.36U/l (taking ABTS as substrate). It can be used in field of textile, paper making, food and beverage after being diluted for 200- 250 times, and used for biological sensor production.
Description
One, technical field
The present invention relates to a kind of industrial enzymes and preparation method thereof, exactly is a kind of lucid ganoderma laccase preparation and production method thereof.
Two, background technology
Laccase is a kind of cupric polyphenoloxidase, and ubiquity in whiterot fungi also produces in minority lower fungi and the plant, mostly is secretor type glycoprotein.Laccase has potential widely application, as removing xylogen in slurrying and the paper industry, the biology of xenobiotic is eliminated the lignin that can effectively remove in the pulp-making waste-water, and the benefit that water is purified is conspicuous; The bio-bleaching of paper pulp and for example, in degraded paper pulp in the fungi system of lignin, laccase, lignin peroxidase and manganese peroxidase are three kinds of enzymes that play a major role, and laccase has himself irreplaceable advantage: laccase is more stable, more effective the playing a role of energy after the immobilization; Do not need the participation of hydrogen peroxide; Some laccase can produce by composing type, and expression amount is higher; Can more effective dechlorination.Therefore, the bio-bleaching that laccase is participated in is studied in the ascendant.
Liquid towards submerged fermentation at present both at home and abroad prepares laccase research more, need to add aromatics in its preparation process or/and heavy metal ion as inductor, this will increase the fermented liquid difficulty of post-processing and easily cause new pollution.
Three, summary of the invention
The alleged ganoderma lucidum laccase of the present invention be by fungi glossy ganoderma (Ganoderma lucidum) for producing bacterial strain, by the laccase preparation of submerged fermentation preparation, be to contain in this preparation copper sulfate (CuSO with the difference of existing laccase preparation
4), CuSO
4Concentration be 0.15～0.40mol/L, preferred 0.18～0.35mol/L is with 0.2～0.25mol/L the best.
The production method of this laccase preparation comprises the Ganderma lucidum strain slant culture, processing after two-stage fermentation and the fermentation, described two-stage fermentation is at first to be seeded in through the Ganoderma mycelium that slant culture obtains to carry out the one-level cultivation on the substratum, obtain first order seed, first order seed is inoculated in carries out submerged fermentation cultivation (secondary cultivation) on the another kind of substratum then.It is to cultivate 6～12 days preferred 28～32 ℃, 7～9 days on the following substratum under 25～35 ℃ of conditions that described one-level is cultivated.Substratum has following component and weight percent:
Glucose 0.5～10%
Semen Maydis powder 0.5～10%
Wheat bran 0.3～10%
Water complements to 100%, transfers pH4.5～5.5.
Preferably:
Glucose 1～7%
Semen Maydis powder 1～7%
Wheat bran 0.5～6%
Water complements to 100%, transfers pH4.8～5.2.
Be followed successively by 2～5%, 2～5%, 1～4% with glucose, Semen Maydis powder, wheat bran, water surplus, pH5 the best.
Fermentation culture finishes the back separates, and the fermented liquid that obtains is inoculated on the following substratum and cultivated 3～7 days under 25～35 ℃ of conditions, preferred 28～32 ℃, 4～6 days as first order seed (liquid seeds).Substratum has following component and weight percent:
Wheat bran 0.5～10%
Peanut hull meal 0.5～10%
Semen Maydis powder 0.3～10%
Water complements to 100%, transfers pH4.5～5.5.
Preferably:
Wheat bran 2～7%
Peanut hull meal 1～6%
Semen Maydis powder 0.5～6%
Water complements to 100%, transfers pH4.8～5.2.
Be followed successively by 3～6%, 1～4%, 1～3% with wheat bran, peanut hull meal, Semen Maydis powder, water surplus, pH5 the best.
Separate obtaining fermented liquid after submerged fermentation is cultivated and finished, fermented liquid is got its supernatant liquor through separation, precipitation, clarification, and adding copper sulfate (CuSO4) makes CuSO4 concentration 0.15～0.40mol/L, and preferred 0.18～0.35mol/L is with 0.2～0.25mol/L the best.Here it is lucid ganoderma laccase preparation, enzyme work reaches 32841.36U/L (is substrate with ABTS).Belong to liquid preparation, need 10 ℃ of following cryopreservations.
The dilution of this preparation is used later on for 200～250 times, and the enzyme work of dilution back can reach 1313.7U/L (is substrate with ABTS).This preparation can be used for the denim decolouring, and the textile surface arrangement improves quality of textile products, and the xylogen of removing in the paper pulp improves pulp quality and do not produce environmental pollution; Remove aldehydes matter reduction wastewater toxicity in the paper waste; Remove the muddiness that produces in the beer and the fruit juice course of processing; The elasticity and the water-retentivity that are used for meat product processing can raising meat product.Degradable aromatic nitro compound in addition, as trotyl (TNT) etc., this preparation can be made into the stable transmitter of high vigor after DEAE one Mierocrystalline cellulose is fixing, detect the changing conditions of catechol in the Tea Processing process.Each enzyme element can be done to detect more than 500 times, and preserves 2 months under the room temperature.Such transmitter can be used for detecting lignin from industrial sewages such as coal, oil, Sweet natural gas, papermaking, aldehydes matter etc.
Four, embodiment
(1), the preparation of one-level culture medium
1, get 1 part of glucose (calling A in the following text), 1 part of Semen Maydis powder (calling B in the following text), 0.3 part of Testa Tritici (calling C in the following text), distilled water complements to 100 parts, stirs, and transfers pH4.5 with the organic acid citric acid, and it is standby to sterilize.
2, get A9 part, B9 part, C8 part, distilled water complements to 100 parts, transfers pH5.5, and operation is with example 1.
3, get A1 part, B1 part, C0.5 part, distilled water complements to 100 parts, transfers pH4.8, and operation is with example 1.
4, get A7 part, B7 part, C6 part, distilled water complements to 100 parts, transfers pH5.2, and operation is with example 1.
5, get A1 part, B1 part, C6 part, distilled water complements to 100 parts, transfers pH5.0, and operation is with example 1.
6, get A6 part, B6 part, C0.5 part, distilled water complements to 100 parts, transfers pH5.0, and operation is with example 1.
7, get A2 part, B2 part, C1 part, distilled water complements to 100 parts, transfers pH5.0, and operation is with example 1.
8, get A4 part, B4 part, C3 part, distilled water complements to 100 parts, transfers pH5.0, and operation is with example 1.
9, get A3 part, B3 part, C2 part, distilled water complements to 100 parts, transfers pH5.0, and operation is with example 1.
(2), the preparation of submerged fermentation culture medium
10. get 1 part of Testa Tritici (calling C in the following text), 1 part of peanut hull meal (cross 60 mesh sieves, call D in the following text), 0.3 part of Semen Maydis powder (calling B in the following text), distilled water complements to 100 parts, stirs, and transfers pH4.5 with the organic acid citric acid, and it is standby to sterilize.
11, get C9 part, D9 part, B8 part, distilled water complements to 100 parts, transfers pH5.5, and operation is with example 1.
12, get C2 part, D1 part, B0.5 part, distilled water complements to 100 parts, transfers pH4.8, and operation is with example 1.
13, get C7 part, D6 part, B5 part, distilled water complements to 100 parts, transfers pH5.2, and operation is with example 1.
14, get C2 part, D1 part, B5 part, distilled water complements to 100 parts, transfers pH5.0, and operation is with example 1.
15, get C7 part, D6 part, B0.5 part, distilled water complements to 100 parts, transfers pH5.0, and operation is with example 1.
16, get C4 part, D2 part, B1 part, distilled water complements to 100 parts, transfers pH5.0, and operation is with example 1.
17, get C5 part, D3 part, B2 part, distilled water complements to 100 parts, transfers pH5.0, and operation is with example 1.
18, get C3 part, D2 part, B1 part, distilled water complements to 100 parts, transfers pH5.0, and operation is with example 1.
(3), the preparation of ganoderma lucidum laccase
1, Ganderma lucidum strain slant culture
With standard potato culture (PDA) is slant medium, and Ganderma lucidum strain is inoculated on the PDA, cultivates 6～8 days under 28～32 ℃ of conditions.Standby.
2, the first order seed shaking table is cultivated
The picking Ganoderma mycelium is inoculated in and carries out shaking table in arbitrary substratum of example 1～example 9 and cultivate inoculum size 3～20%, shaking speed 100～210rpm on slant medium.Culture condition was cultivated 6～12 days down for 25～35 ℃, perhaps cultivated 7～9 days down for 28～32 ℃.It is standby as liquid seeds to cultivate end back separate fermentation liquid.
3, submerged fermentation is cultivated
Liquid seeds is seeded in arbitrary substratum of example 10～example 18 and in fermentor tank, cultivates, inoculum size 3～20% loading amount coefficients 0.2～0.8,1: 0.3～1vvm of air flow, mixing speed 100～210rpm, culture condition descended 3～7 days for 25～35 ℃, and perhaps 28～32 ℃ were descended 4～6 days.
4, aftertreatment
After submerged fermentation finishes, fermented liquid centrifuging and taking filtrate.Filtrate is handled with ammonium sulfate or ethanol or acetone precipitation, and the centrifugal precipitation of abandoning adds CuSO in the gained supernatant liquor after 12 hours
4, make CuSO
4Concentration 0.15～0.4mol/L is the liquid lacquer zymin, and enzyme work reaches 32841.36U/L (is substrate with ABTS), and low temperature below 10 ℃ is protected a surname.Use 200～250 times of distilled water dilutings during use.
Claims (10)
1, a kind of lucid ganoderma laccase preparation is characterized in that: by the liquid lacquer zymin that contains copper sulfate that the fungi glossy ganoderma prepares by submerged fermentation, the concentration of copper sulfate is 0.15～0.40mol/L.
2, preparation according to claim 1 is characterized in that: the concentration of copper sulfate is 0.18～0.35mol/L.
3, preparation according to claim 1 and 2 is characterized in that: the concentration of copper sulfate is 0.2～0.25mol/L.
4, by the production method of the described lucid ganoderma laccase preparation of claim 1, comprise the processing after Ganderma lucidum strain slant culture, two-stage fermentation and the fermentation, it is characterized in that: described two-stage fermentation is that one-level is cultivated and submerged fermentation,
(1), one-level is cultivated is to cultivate 6～12 days under 25～35 ℃ of conditions on the following substratum, cultivate finish after, isolate fermented liquid as liquid seeds for submerged fermentation usefulness, substratum has following component and weight percent:
Glucose 0.5～10%
Semen Maydis powder 0.5～10%
Wheat bran 0.3～10%
Water complements to 100%, transfers pH4.5～5.5;
(2), submerged fermentation is to cultivate 3～7 days under 25～35 ℃ of conditions on the following substratum, substratum has following weight percentages of components:
Wheat bran 0.5～10%
Peanut hull meal 0.5～10%
Press ground rice 0.3～10%
Water complements to 100%, transfers pH4.5～5.5;
(3), after submerged fermentation finishes,, get its supernatant liquor, add copper sulfate, promptly get laccase preparation through separation, precipitation, clarifying treatment.
5, production method according to claim 4, it is characterized in that: each component of substratum that one-level is cultivated has following weight percent: glucose, Semen Maydis powder and wheat bran are followed successively by 1～7%, 1～7% and 0.5～6%, water complements to 100%, transfers pH4.8～5.2.
6, according to claim 4 or 5 described production methods, it is characterized in that: each component of substratum that one-level is cultivated has following weight percent: glucose, Semen Maydis powder and wheat bran are followed successively by 2～5%, 2～5% and 1～4%, and water complements to 100%, transfer pH5.0.
7, production method according to claim 6 is characterized in that: the culture condition that one-level is cultivated is to cultivate 7～9 days under 28～32 ℃ of conditions.
8, production method according to claim 4, it is characterized in that: each component of the substratum of submerged fermentation has following weight percent: wheat bran, peanut hull meal and Semen Maydis powder are followed successively by 2～7%, 1～6% and 0.5～6%, water complements to 100%, transfers pH4.8～5.2.
9, according to claim 4 or 8 described production methods, it is characterized in that: each component of the substratum of submerged fermentation has following weight percent: wheat bran, peanut hull meal and Semen Maydis powder are followed successively by 3～6%, 1～4% and 1～3%, water complements to 100%, transfers pH5.0.
10, production method according to claim 9 is characterized in that: the culture condition of submerged fermentation is to cultivate 4～6 days under 28～32 ℃ of conditions.
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| CNA2006100380896A CN101008005A (en) | 2006-01-24 | 2006-01-24 | Lucid ganoderma laccase preparation and production method thereof |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103122337A (en) * | 2013-02-05 | 2013-05-29 | 安徽大学 | Method for producing laccase by fermentation of ganoderma lucidum |
| CN104232599A (en) * | 2014-09-25 | 2014-12-24 | 河南师范大学 | Method for increasing yield of ganoderma lucidum laccase by using waste paper crushing materials |
| CN113215001A (en) * | 2021-05-06 | 2021-08-06 | 上海禾向健康科技发展有限公司 | Strain for producing high-enzyme-activity ganoderma lucidum laccase and production method |
| CN113388660A (en) * | 2021-06-29 | 2021-09-14 | 白银赛诺动物保健技术有限公司 | Liquid color development method for rapidly screening laccase producing bacteria |
-
2006
- 2006-01-24 CN CNA2006100380896A patent/CN101008005A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103122337A (en) * | 2013-02-05 | 2013-05-29 | 安徽大学 | Method for producing laccase by fermentation of ganoderma lucidum |
| CN104232599A (en) * | 2014-09-25 | 2014-12-24 | 河南师范大学 | Method for increasing yield of ganoderma lucidum laccase by using waste paper crushing materials |
| CN113215001A (en) * | 2021-05-06 | 2021-08-06 | 上海禾向健康科技发展有限公司 | Strain for producing high-enzyme-activity ganoderma lucidum laccase and production method |
| CN113215001B (en) * | 2021-05-06 | 2022-07-12 | 上海禾向健康科技发展有限公司 | Strain for producing high-enzyme-activity ganoderma lucidum laccase and production method |
| CN113388660A (en) * | 2021-06-29 | 2021-09-14 | 白银赛诺动物保健技术有限公司 | Liquid color development method for rapidly screening laccase producing bacteria |
| CN113388660B (en) * | 2021-06-29 | 2023-05-19 | 白银赛诺动物保健技术有限公司 | Liquid color development method for rapidly screening laccase-producing bacteria |
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