CN102994464B - Culture medium and method for producing laccase by Ascheronia placenta fermentation - Google Patents

Culture medium and method for producing laccase by Ascheronia placenta fermentation Download PDF

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CN102994464B
CN102994464B CN201210543681.7A CN201210543681A CN102994464B CN 102994464 B CN102994464 B CN 102994464B CN 201210543681 A CN201210543681 A CN 201210543681A CN 102994464 B CN102994464 B CN 102994464B
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laccase
substratum
culture medium
seat shell
flat seat
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邱君志
苏礼超
吴宝杰
李小霞
涂洁
宋飞飞
邱云锋
郭庆丰
何肖云
毛丽慧
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Fujian Agriculture and Forestry University
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Abstract

The invention relates to a fungal fermentation culture medium and process. More particularly, the invention relates to a culture medium and method for producing laccase by Ascheronia placenta fermentation. The raw materials of the culture medium comprise 1-4wt% of glucose, 1-4wt% of (NH4)2SO4, 0.5*10<-4>-4*10<-4> mol/L of Zn<2+>, and 1*10<-4>-1*10<-3> wt% of VB4; and the initial pH of the culture medium is 3.8-4.4. According to the method, a fermented seed solution is inoculated to the culture medium, the liquid containing quantity of a 250 mL triangular flask is 50 mL, the inoculum size is 1mL, the number of glass beads is zero, and laccase is cultured at 25 DEG C at the rotational speed of 160 r/min. The fermentation method is short in fermentation period, mild in condition, and easy to control; and the culture medium provided for producing laccase by Ascheronia placenta fermentation has the advantages that the laccase yield is high, the laccase activity is high, and the like.

Description

A kind of substratum and method of flat seat shell spore fermentative production of laccase
Technical field
The present invention relates to fermention medium and the technique of fungi.More specifically, the present invention relates to a kind of substratum and method of flat seat shell spore fermentative production of laccase.
Background technology
Laccase (Laccase) is a kind of cupric polyphenoloxidase, and a series of organic and electronics oxidations of mineral compound generation of its energy catalysis are attended by hydrogen reduction Cheng Shui simultaneously.Find that at present multiple bioenergy produces laccase, comprises plant, fungi, insect, bacterium etc.Wherein, with (the sub-roc of paper money etc., 2001) at most of the whiterot fungi distribution in fungi.
Laccase has substrate-function scope widely, can the multiple phenols of catalysis and the oxidation of aromatic amine compounds, and degrading polycyclic aromatic hydrocarbons.Therefore, in biological degradation, pulping and paper-making, the aspect such as wastewater treatment, bio-bleaching, pollutent detoxification, soil remediation, laccase has important using value.At present, the research of laccase is own launches on multi-field, multilevel.Many by fungus-caused Plant diseases, laccase is shown is an important virulence factor [8].Disclose entomogenous fungi and whether can produce laccase, the further investigation that can be entomogenous fungi Biocontrol Effect provides foundation.If can be applied in the biological control to insect to the result of study of entomogenous fungi laccase, can develop to a greater extent undoubtedly Biological resources.
Flat seat shell spore ( aschersonia placenta) be a kind of important insect pathogenic fungus, in agriculture and forestry injurious insect biological control, be with a wide range of applications.This Pseudomonas Ascomycota ( ascomycota) caprophyl guiding principle ( sordariomycetes) Hypocreales ( hypocreales) Clavicipitaceae ( clavicipitaceae) Aschersonia ( aschersonia), and rarely have report to flat seat shell spore fermentative production of laccase is domestic, there is larger development prospect.
Summary of the invention
The object of the present invention is to provide a kind of substratum and method of flat seat shell spore fermentative production of laccase.
First the present invention provides a kind of substratum of flat seat shell spore fermentative production of laccase, and the raw material of described substratum contains glucose 0.5-2wt%, (NH 4) 2sO 41-4wt%, Zn 2+0.5 × 10 -4-4 × 10 -4mol/L, VB 41 × 10 -4-1 × 10 -3wt%, the initial pH=3.8-4.4 of substratum.
More preferably, the raw material of described substratum is mark meter by weight, contains glucose 0.5wt%, (NH4) 2sO 44wt%, Zn 2+0.5 × 10 -4mol/L, VB 45 × 10 -4wt%, the initial pH=4.4 of substratum.
Secondly, the present invention also provides a kind of method of flat seat shell spore fermentative production of laccase, and described method comprises following steps:
(a) by flat seat shell spore inoculation seed culture medium, make fermentation seed liquid;
(b) by the fermentation seed liquid making in step (a) by being inoculated in described substratum, the triangular flask liquid amount of 250mL is 50mL, inoculum size 1mL, in 25 DEG C, cultivates under rotating speed 160 r/min.
The raw material of described seed culture medium contains: by substratum weighing scale, and 20% murphy juice 1L, glucose 2%, yeast extract paste 1%, natural pH.
The seat shell spore that the present invention adopts is flat seat shell spore (what schoolmate etc., the black rubber powder lice of oil tea and flat seat shell spore be to its Natural control action, the sick worm of Chinese forest, in July, 2011).
Adopt the substratum after the present invention optimizes, cell age 4d, fermentation period 3d, 0 of granulated glass sphere, fermentation bears results, and to produce laccase be more than 45U/mL to flat seat shell spore.
Beneficial effect of the present invention: fermentation process of the present invention has fermentation period short; Mild condition, is easy to control; The substratum for flat seat shell spore fermentative production of laccase providing, has laccase productive rate high, laccase activity advantages of higher.
Brief description of the drawings
The impact of the different carbon sources of Fig. 1 on inulinase-producing activity.Note: in figure, Dex, D-Sor, Mal, Fru, Suc, Myc, SS, Car, L-Ara, CK represent respectively glucose, D-glucitol, maltose, fructose, sucrose, trehalose, Zulkovsky starch, Xylo-Mucine, L-arabinose and control group.
The impact of Fig. 2 different nitrogen sources on inulinase-producing activity.Note: in figure, YE, YEP, Try, PN, Cas, AS, SN, Pep, Ure, CK represent respectively yeast extract paste, yeast powder, Tryptones, KNO 3, casein food grade, (NH 4) 2sO 4, NaNO 3, peptone, urea and control group.
The impact of the different VITAMIN of Fig. 3 on inulinase-producing activity.
The impact of the different inorganic ions of Fig. 4 on inulinase-producing activity.
The impact of the different initial pH of Fig. 5 on inulinase-producing activity.
Embodiment
The present invention further illustrates the present invention with the following example, but protection scope of the present invention is not limited to the following example.
Embodiment 1
Experiment of single factor is determined the optimum composition of substratum
(1) seed culture medium and basic medium preparation
(a) seed culture medium: 20% murphy juice 1L, glucose 2%, yeast extract paste 1%, natural pH.1.01MPa, sterilizing 20min.
(b) basic medium: 20% murphy juice 1L, glucose 2%, yeast extract paste 1%, natural pH.1.01MPa, sterilizing 20min.
(2) making of ferment-seeded: flat seat shell spore is inoculated on potato agar substratum, cultivates 5d at 25 DEG C, treat that it produces spore, i.e. activation; Bacterial strain after activation is seeded to seed culture medium with inoculating needle by spore powder, 25 DEG C, under rotating speed 160 r/min, cultivate 24h, be fermented liquid.
(3) preparation of enzyme liquid: get fermented liquid under step (2) at 4 DEG C, 5000 r/min are centrifugal, 15min, obtaining supernatant liquor, to be crude enzyme liquid stand-by.Measuring light absorption value at wavelength 465nm place by enzyme assay.Be converted into the U/mL of Mei Huo unit.
(4) laccase activity is measured: get 0.2mL fermented supernatant fluid and add (with hot deactivation supernatant liquor in contrast) in 3.3mL acetate buffer solution (pH4.4) and 0.5mL 4.0mmol/L methyl catechol, 25 DEG C of water-bath 5min, ice bath adds 2mL 16.5% trichoroacetic acid(TCA) reagent termination reaction, gets supernatant liquor after centrifugal to survey absorbancy under wavelength 465nm.
Figure 862530DEST_PATH_IMAGE002
In formula, afor the absorbancy of sample; v always, v enzymerepresent respectively the volume of reaction system cumulative volume and reaction enzymes liquid; ε is specific absorbance (ε 465=1.21 × 10 -4mol/Lcm); Defining enzyme with per minute oxidation substrates 1.0 μ mol lives.
(5) different carbon sources, nitrogenous source, VITAMIN, inorganic ion, initial pH are on producing the impact of laccase.
(a) impact of carbon source on production of enzyme, in basic medium, add respectively 2% glucose, D-glucitol, maltose, fructose, sucrose, trehalose, Zulkovsky starch, Xylo-Mucine and L-arabinose, access identical flat seat shell spore bacterium liquid, in 25 DEG C, the shaking table of 160r/min, cultivate, control group is set, detects laccase activity in fermented liquid every day.
(b) nitrogenous source, on producing the impact of enzyme, adds respectively 1% yeast extract paste, yeast powder, Tryptones, KNO in basic medium 3, casein food grade, (NH 4) 2sO 4, NaNO 3, peptone and urea, access identical flat seat shell spore bacterium liquid, in 25 DEG C, the shaking table of 160r/min, cultivate, control group is set, detect laccase activity in fermented liquid every day.
(c) 0.01% V is added respectively in the impact of VITAMIN on production of enzyme in basic medium b1, V b2, V b4, V b6, V c, V e, V compound, the flat seat shell spore bacterium liquid of access equivalent is cultivated in 25 DEG C, the shaking table of 160r/min, and control group is set, and detects laccase activity in fermented liquid every day.
(d) inorganic ion (the 0.4mmol NaH of identical mole number is added respectively in the impact of inorganic ion on production of enzyme in basic medium 2pO 42H 2o, KH 2pO 4, CuSO 45H 2o, ZnSO 47H 2o, FeCl 36H 2o, MgSO 47H 2o, FeSO 47H 2o, CoCl 26H 2o, MnSO 4), access identical flat seat shell spore bacterium liquid, in 25 DEG C, the shaking table of 160r/min, cultivate, control group is set, detect laccase activity in fermented liquid every day.
(e) impact of initial pH on production of enzyme, the culture environment (pH value 3.5,4.0,4.5,5.0,5.5,6.0,6.5,7.0,7.5) of different pH values is provided respectively with basic medium, the flat seat shell spore bacterium liquid of access equivalent, in 25 DEG C, the shaking table of 160r/min, cultivate, control group is set, detects laccase activity in fermented liquid every day.
Experimental result
The impact of carbon source on production of enzyme: as shown in Figure 1, add glucose and significantly improved laccase activity.Just there is laccase activity peak in 3d after inoculation, and enzymic activity reaches 43.27U/mL.Add the processing of D-glucitol, fructose, Zulkovsky starch, after inoculation, 3d enzyme is lived and is reached respectively 14.40U/mL, 14.43U/mL, 16.60U/mL, and 15.43U/mL alive is close with control group enzyme; The processing of adding trehalose, maltose, Xylo-Mucine, sucrose and L-arabinose all has restraining effect in various degree to producing enzyme for examination bacterium.The processing of adding sucrose and L-arabinose can't detect enzyme and lives.
Nitrogenous source is on producing the impact of enzyme: as shown in Figure 2, in added different nitrogen sources, the processing that interpolation yeast extract paste, yeast soak powder and ammonium sulfate contributes to improve product enzyme level, enzymic activity reaches respectively 40.55U/mL, 39.19U/mL, 41.62U/mL, and the processing of adding casein food grade, peptone is alive low compared with the enzyme of control treatment, the two is inhibited to producing enzyme for examination bacterium.In addition, the processing of Tryptones, saltpetre, SODIUMNITRATE and urea has strong restraining effect to it.Accordingly, ammonium sulfate is the optimum nitrogen source that flat seat shell spore bacterium produces laccase.
The impact of VITAMIN on production of enzyme: as shown in Figure 3, in added different VITAMIN, compared with CK, vitamins B 4contribute to most flat seat shell spore bacterium to improve and produce enzyme level (Fig. 5), vitamins C takes second place, and is then vitamin complex, and measured enzyme activity is respectively: 45.22U/mL, 37.58U/mL, 29.06U/mL.Experimental result shows: add vitamins B 1, vitamins B 6all can increase flat seat shell spore bacterium and produce laccase.And vitamins B 2there is significant restraining effect with vitamin-E to producing enzyme, record enzyme work and be only: 6.54U/mL, 6.06U/mL.
The impact of initial pH on production of enzyme: as shown in Figure 4, in added identical mole number inorganic ion not of the same race, add Na +, Zn 2+, Fe 2+, Co 2+processing all contribute to improve this bacterium laccase production ability, enzymic activity reaches respectively 19.87U/mL, 36.18U/mL, 35.23U/mL, 23.66U/mL, and add K +, Fe 3+, Mg 2+, Mn 2+processing live compared with the enzyme of control treatment low, inhibited to producing enzyme for examination bacterium.In addition Cu, 2+flat seat shell spore bacterium is produced to laccase without obvious promoter action.Accordingly, Zn 2+can improve flat seat shell spore bacterium and produce laccase.
The impact of inorganic ion on production of enzyme: as shown in Figure 5, after cultivating in the substratum of the initial pH of difference, the processing of pH=4.0 contributes to improve laccase production ability, and enzymic activity reaches 30.93U/mL, the processing of initial pH=4.5, its enzyme value alive also reaches 23.80 U/mL.Accordingly, initial pH=4.0-4.5 can improve flat seat shell spore bacterium product laccase.
Embodiment 2
Orthogonal experiment is determined optimum fermentation condition
(1) orthogonal experiment is determined optimal medium
From the basis of the definite substratum of experiment of single factor, change the solubility of each composition, and pH value, configuring different substratum, method is with step (1), (2) in embodiment 1.In table 1, the inoculum size of same amount (10%) bacterial classification is inoculated in to 250mL, 50mL nutrient solution is housed, at 25 DEG C, under rotating speed 160 r/min, cultivate 3d.Then press embodiment 1 step (3), the work of (4) mensuration enzyme.Choose orthogonal experiment optimal result and the highest experiment group number of its yield of enzyme, carry out confirmatory experiment.Cultivation basigamy method, the making of crude enzyme liquid, enzyme activity determination method in burdensome embodiment are all identical with the method for embodiment 1.
Flat shell spore laccase of table 1 five factor four levels (4 5) orthogonal
Figure 2012105436817100002DEST_PATH_IMAGE003
Flat shell spore laccase of table 2 five factor four levels (4 5) orthogonal experimental result
Note: K1 is all enzyme activity sums of each level of factor 1, K2 is all enzyme activity sums of each level of factor 2, K3 is all enzyme activity sums of each level of factor 3, K4 is all enzyme activity sums of each level of factor 4, R is extreme difference, mean value (mean) ± standard deviation (S.D) that the digitized representation of enzyme activity repeats for 3 times.
Table 3 medium optimization proof test
Figure 2012105436817100002DEST_PATH_IMAGE005
Orthogonal interpretation of result shows: by table 2,3 reaction systems that can be applicable to measuring for examination bacterium laccase activity are: A 1b 4c 1d 3e 4.Orthogonal experiment results shows: the carbon source of respective horizontal, nitrogenous source, metal ion, VITAMIN, pH respectively: 0.5% glucose, 4% (NH 4) 2sO 4, 0.5 × 10 -4mol/L Zn 2+, vitamins B 45 mg, pH 4.4, i.e. A 1b 4c 4d 4e 4.From table 2 range analysis, the extreme difference value R of changed factor is respectively carbon source (56.46), nitrogenous source (34.57), metal ion (20.09), VITAMIN (40.19) and pH (25.02), the variation and the flat seat shell spore bacterium laccase substratum that show carbon source, nitrogenous source, metal ion, VITAMIN and pH are closely related, and the impact that especially variation of carbon source glucose is produced laccase to this bacterium is larger.
(2) orthogonal experiment is determined optimal culture conditions
On the basis of the best medium definite from orthogonal experiment, by setting different vaccination amount, liquid amount, granulated glass sphere number, incubation time, at 25 DEG C, cultivates under rotating speed 160 r/min, and the incubation time of each group number is as the criterion with table 9.Then press embodiment 1 step (3), the work of (4) mensuration enzyme.Choose orthogonal experiment optimal result and the highest experiment group number of its yield of enzyme, carry out confirmatory experiment.Cultivation basigamy method, the making of crude enzyme liquid, enzyme activity determination method in burdensome embodiment are all identical with the method for embodiment 1.
Flat shell spore laccase of table 4 four factor three levels (3 4) orthogonal experiment
Figure 497091DEST_PATH_IMAGE006
Flat shell spore laccase of table 5 four factor three levels (3 4) orthogonal experimental result
Figure 2012105436817100002DEST_PATH_IMAGE007
Note: K1 is all enzyme activity sums of each level of factor 1, K2 is all enzyme activity sums of each level of factor 2, K3 is all enzyme activity sums of each level of factor 3, R is extreme difference, mean value (mean) ± standard deviation (S.D) that the digitized representation of enzyme activity repeats for 3 times.
5 is known, and each culture condition (connecing bacterium amount, liquid amount, built-in granulated glass sphere number and incubation time) of choosing respectively the most applicable this bacterium product laccase carries out the orthogonal test of 4 factor 3 levels, to filter out optimal culture conditions.4 factors are all produced laccase to flat seat shell spore bacterium impact.As shown in Table 8, high enzymatic activity can reach 46.07U/mL, and its corresponding experiment condition is A 1b 1c 1d 1(inoculum size 1mL, liquid amount 50mL, not placing glass pearl, incubation time 3d); According to orthogonal test analysis result, can obtain: A 1b 1c 1d 1it is the condition that is conducive to produce enzyme most.
Embodiment 3
Fungi laccase fermentation method
(1) preparation of ferment-seeded
(A) bacterial classification and substratum
Bacterial classification: flat seat shell spore
Slant medium: PDA substratum
Liquid seed culture medium: 20% murphy juice 1L, glucose 2%, yeast extract paste 1%, natural pH.
(B) seed liquor preparation
Purebred flat seat shell spore on inclined-plane is transferred in multiple 250mL triangular flasks, 100mL substratum is wherein housed, then at 25 DEG C, under reciprocating concussion shaking speed 160 r/min, cultivate 3d, band mycelia robust growth, when bacterium liquid is thick, illustrates that seed grown.
(2) fermentation culture
Fermention medium: according to medium optimization Orthogonal experiment results, contain glucose 0.5w%, (NH 4) 2sO 44 w %, Zn 2+0.5 × 10 -4mol/L, V b45 × 10 -4w %, the initial pH=4.4 of substratum, preparation.
By the flat seat shell spore fermentation seed liquid of preparing in previous step, the top condition drawing by culture condition orthogonal experiment, inoculum size 1mL is inoculated in 250mL triangular flask, in bottle, 50mL fermentation culture is housed,
Shaking table temperature: 27 DEG C ± 1 DEG C, rotating speed 160rpm,
Fermentation period: 3d,
Fermentation bears results, and to produce laccase be more than 45U/mL to flat seat shell spore, and this embodiment shows that shake flat experiment technique of the present invention is good.
The foregoing is only preferred embodiment of the present invention, all equalizations of doing according to the present patent application the scope of the claims change and modify, and all should belong to covering scope of the present invention.

Claims (4)

1. a substratum for flat seat shell spore fermentative production of laccase, is characterized in that, contains glucose 0.5-2wt%, (NH in the raw material of described substratum 4) 2sO 41-4wt%, Zn 2+0.5 × 10 -4-4 × 10 -4mol/L, VB 41 × 10 -4-1 × 10 -3wt%, the initial pH=3.8-4.4 of substratum.
2. the substratum of flat seat shell spore fermentative production of laccase according to claim 1, is characterized in that the raw material of described substratum contains glucose 0.5wt%, (NH4) 2sO 44wt%, Zn 2+0.5 × 10 -4mol/L, VB 45 × 10 -4wt%, the initial pH=4.4 of substratum.
3. a method for flat seat shell spore fermentative production of laccase, is characterized in that, described method comprises following steps:
(a) by flat seat shell spore inoculation seed culture medium, make fermentation seed liquid;
(b) by the fermentation seed liquid making in step (a) by the substratum being inoculated in described in claim 1 or 2, the triangular flask liquid amount of 250mL is 50mL, inoculum size 1mL, 0 of granulated glass sphere, in 25 DEG C, cultivates under rotating speed 160 r/min.
4. the method for flat seat shell spore fermentative production of laccase according to claim 3, is characterized in that, the raw material of described seed culture medium contains: calculate by the weight of substratum, contain 20% murphy juice 1L, glucose 2%, yeast extract paste 1%, natural pH.
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CN107828760B (en) * 2017-12-12 2020-09-04 福建农林大学 Culture medium and method for producing beta-mannase by aschersonia placenta fermentation
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