CN102786411B - Fungal laccase inducted active compound, fermentation preparation method and application - Google Patents
Fungal laccase inducted active compound, fermentation preparation method and application Download PDFInfo
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- CN102786411B CN102786411B CN201210138447.6A CN201210138447A CN102786411B CN 102786411 B CN102786411 B CN 102786411B CN 201210138447 A CN201210138447 A CN 201210138447A CN 102786411 B CN102786411 B CN 102786411B
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Abstract
The invention belongs to the technical field of separating and purifying compound, and relates to a fermentation preparation, separating and purifying technology of a microorganism sourced novel fungal laccase efficiently-inducted compound and an application for inducing the production aspect of fungal laccase. The method comprises the following steps: preparing a broth containing the active compound through fungi fermentation, extracting to obtain a crude extract of the active compound and being passed through different chromatography resin adsorption, taking methanol-water with different concentrations as an eluant for purifying and refining to obtain the purified active compound molecule, wherein the obtained active compound can efficiently induce the laccase under the concentration between 1-500 mum to produce the fungi secrete extracellular laccase. The invention has the advantages that the induced compound is from fungi secondary metabolite; the work concentration of the induced compound is low, the effective work concentration is between 1-500 mum, and the induced compound as high induction activity on fungal laccase.
Description
Technical field
The invention belongs to compound separation purification technique field, relate to a kind of fermentation preparation of microbe-derived fungal laccase induced activity compound, the purifying process of high purity laccase induced activity compound and the application aspect fungal laccase induction thereof.
Background technology
Laccase (Laccase, EC 1.10.3.2) is the polyphenoloxidase of a class cupric, can the multiple phenols of catalysis and the oxidation of non-phenolic compound, and be attended by molecular oxygen simultaneously and be reduced into water.Paint zymolyte is extensive, catalytic efficiency is high, in bio-pulping and bleaching, flavour of food products improvement, feed nutrition improves, synthetic is dye decolored, textile fibres softens refinement, newtype drug exploitation, the fields such as biosensor manufacture and new exploitation of energy resources have potential significant application value (Arora DS, Sharma RK (2010) Ligninolytic fungal laccases and their biotechnological applications. Appl Biochem Biotechnol 160:1760 – 1788), become in recent years a focus of international enzyme technology and the research of environmental science crossing domain.
Laccase is extensively present in higher fungi (particularly basidiomycetes).There is the shortcoming such as yield poorly, fermentation period is long, aftertreatment is loaded down with trivial details in the natural laccase of most fungus secretions, cannot meet the needs of industrial applications.At present, the biosynthesizing output that is used for improving laccase mainly contains two kinds of methods: recombinant expressed and add compound induction.But recombinant expressed output still, compared with low and production cost is high, post-processing difficulty is large, improves the cost of laccase production greatly, has limited the application of laccase in industrial production; Effectively inductor mostly is aromatic compounds or heavy metal ion, and it has environmental toxicity and expensive, and useful effect concentration is large, conventionally 10
-3~ 10
-2mole rank, easily causes environmental pollution, and the fermented liquid after production needs detoxification treatment, has improved production cost, cannot break through equally the bottleneck of laccase commercial application.Therefore, find a kind of clean, efficiently laccase production mode be advance laccase industrialization process in the urgent need to.
Summary of the invention
The present invention is for avoiding the existing weak point of above-mentioned prior art, and fermentation preparation and separation purifying technique and the application aspect fungal laccase induction fermentation thereof of a kind of efficient fungal laccase induced activity compound (name is called 3-hydroxyl-2-octyl group-pentanedioic acid) are provided.
One of object of the present invention is to provide a kind of compound fungal laccase to high induced activity, and this compound has following molecular formula:
Another object of the present invention application aspect synthetic that is this active compound of protection at induction fungal laccase.
The compound of activity of the present invention derives from microorganism fermentation strain Gongronella sp, and this bacterial strain has been preserved in Chinese Typical Representative culture collection center, preservation address: Wuchang District, Wuhan, China city Bayi Road Wuhan University; Preservation date: on March 27th, 2012; Preserving number is: CCTCCAF 2012004.
The fermentation preparation of this active compound, is by above-mentioned fermentation strain, obtain fermented liquid, and then fermented liquid obtains pure active compound after separation and purification through fermentation culture.
Described fermentation culture refers to Gongronella sp. is seeded in the fermention medium of pH4.0 ~ 7.0 of sterilizing, in temperature 25 ~ 31
ounder C, shaking speed 100 ~ 150rpm condition, cultivate that after 3 ~ 7 days, to collect fermented liquid by filtered through gauze for subsequent use; Described fermention medium is containing 10 ~ 20g sucrose, 1 ~ 2g DL-l-asparagine, 0.7 ~ 1.5g KH in every premium on currency
2pO
4, 0.2 ~ 0.6g MgSO
47H
2o, 0.02 ~ 0.15g Na
2pO
47H
2o, 0.01 ~ 0.05g CaCl
2, 0.01 ~ 0.05g FeSO
47H
2o, 0.01 ~ 0.04g VITAMIN B4,20 ~ 60 μ g VB
1.
As preferentially, described fermention medium is containing 15g sucrose, 1.5g DL-l-asparagine, 1g KH in every premium on currency
2pO
4, 0.5g MgSO
47H
2o, 0.1g Na
2pO
47H
2o, 0.01g CaCl
2, 0.01g FeSO
47H
2o, 0.0275g VITAMIN B4,50 μ g VB
1.
Described separation and purification comprises the following steps successively:
(1) fermented liquid of preparing is by extraction agent 1:1(v/v) extraction, extraction liquid is at 100 ~ 2000Pa, and 30 ~ 55
ounderpressure distillation under the condition of C, removes extraction agent, obtains active compound raw product; Described extraction agent is any one in ethyl acetate, methylene dichloride, sherwood oil;
(2) raw product is passed through to dissolve with methanol, according to raw product and chromatographic resin A mass ratio 1:10 ~ 20 loading, use volumetric concentration 20%, 40%, 60%, 80%, 100% methanol aqueous solution gradient elution, each concentration wash-out 3 ~ 5 column volumes, collect and merge active compound component at 100 ~ 2000Pa, 30 ~ 55
ounder the condition of C, the concentrated chromatography component of collecting of underpressure distillation, obtains enriched product one;
(3) enriched product one obtaining in step (2) passes through dissolve with methanol, according to raw product and chromatographic resin B mass ratio 1:10 ~ 20 loading, use volumetric concentration 20%, 40%, 60%, 80%, 100% methanol aqueous solution gradient elution, each concentration wash-out 3 ~ 5 column volumes, collect and merge containing active compound component and at 100 ~ 2000Pa, 30 ~ 55
ounderpressure distillation under the condition of C, obtains enriched product two;
(4) enriched product two obtaining in step (3) passes through dissolve with methanol, according to raw product and chromatographic resin C mass ratio 1:10 ~ 20 loading, use volume 20%, 40%, 60%, 80%, 100% methanol aqueous solution gradient elution, each concentration wash-out 3 ~ 5 column volumes, collect and merge containing active compound component and at 100 ~ 2000Pa, 30 ~ 55
ounder the condition of C, underpressure distillation obtains pure substance.
Chromatographic resin A described in described step (2) is commercially available D101, LX-60 non-polar macroporous resin.
Chromatographic resin B described in described step (3) is the meticulous chromatographic resin of commercially available MCI GEL CHP 20P MCI series.
Chromatographic resin C described in described step (4) is the nonpolar chromatographic resins such as commercially available octadecylsilane chemically bonded silica (ODS).
The elutriant obtaining in step (2) ~ (4), collecting merging containing before active compound component, uses silica gel thin-layer chromatography (TLC) to carry out moiety detection, merges component according to TLC result, and concrete grammar is as follows:
By the laccase production fungi picking of slant preservation to CPDA inclined-plane, 28
oc leaves standstill and cultivates 3 days.The mycelium piece (diameter 0.5 cm left and right) of activation is forwarded to 9cm CPDA plate central authorities, 28
oc is inverted and cultivates 3 days.Get appropriate above-mentioned merging component evaporated in vacuo concentrated, wave most organic solvent, dissolve with sterilized water.Under aseptic condition, at the Oxford cup of the equidistant placement sterilizing in laccase production radicula byssoidea edge, in the cup of Oxford, add the aqueous solution of a certain amount of above-mentioned merging component, 28
oc leaves standstill and cultivates 36h, in the punching of cup place, Oxford, in hole, adds methyl catechol laccase colour developing damping fluid, observes Oxford cup and mycelia contact position or hole colour-change around.Negative control is water, and positive control is the acetic acid ethyl ester extract of Gongronella sp..According to the active detected result of plate, the component of above-mentioned merging is concentrated for subsequent use or is stored in 4
oc.
Active compound is the application aspect synthetic at induction fungal laccase:
Get 7 ~ 8 of the laccase production radicula byssoidea pieces (diameter 0.5cm) of activation, access is equipped with in the 250mL triangular flask of 100mL CPDA liquid nutrient medium, and 28
oc, 120rpm shaking table is cultivated.The laccase production bacterium that grows 4 days is for subsequent use with homogenizer 6000rpm homogenate 10 seconds.Get the liquid shaking flask of 5mL laccase production bacteria culture fluid access CPDA after homogenate, 28
oc, 120 rpm shaking tables are cultivated.After 4 days, adding final concentration is that the activity inducement compound of the present invention of 10 μ M continues to cultivate, and after 4 ~ 6 days, laccase activity reaches 12000U/L, is not add 7.5 times of induced activity compound fermentation laccase activity 1600U/L of the present invention.
Compared with existing fungal laccase inductor, the advantage that the present invention has is: (1) inducing compounds of the present invention derives from fungal secondary meta-bolites; 2) inducing compounds working concentration of the present invention is low, and effectively working concentration is 10 μ M; (3) inducing compounds of the present invention has high fungal laccase induced activity.
Embodiment
Below in conjunction with embodiment, the invention will be further described, but should be appreciated that these embodiment not limit the scope of the invention.
One, the fermentation preparation that contains activity inducement compound of the present invention
1, the activation of Gongronella sp. bacterial strain
The Gongronella sp. W5 picking of slant preservation to CPDA inclined-plane, 28
oc leaves standstill and cultivates 3 days with activation bacterial strain.
2, the preparation of substratum and sterilizing
Preparation fermention medium (15g sucrose, 1.5g DL-l-asparagine, 0.5g MgSO
47H
2o, 0.1g Na
2hPO
412H
2o, 0.01g CaCl
2, 0.01g FeSO
47H
2o, 0.0275g VITAMIN B4,50 μ g VB
1), 115
o20min is for subsequent use in C sterilizing
3, the fermentation of activity inducement compound preparation
5 of the 4mm Gongronella sp. W5 mycelium of picking diameter activation, are inoculated in the fermention medium of sterilizing 28
oc, 120rpm shaking culture 4 d, 16 layers of filtered through gauze mycelium, collect fermented liquid for subsequent use.
Two, the separation and purification of activity inducement compound of the present invention
1, the extraction that contains induced activity compound of the present invention
Get Gongronella sp. W5 fermentation supernatant that 500mL collects in 1 L pear shape separatory funnel, add ethyl acetate 250mL, fully vibration shakes up and notes avoiding emulsion layer, stratification.Collect after upper organic phase, then continue to add 125mL ethyl acetate to continue extraction in lower floor's water, repeat twice.Merge the organic phase of 3 times, on Rotary Evaporators 40
oc concentrating under reduced pressure evaporate to dryness is for subsequent use.
2, D101 macroporous resin just separates induced activity compound of the present invention
Meticulous D101 macroporous resin material spends the night by alcohol immersion, being packed into equably specification is in the chromatography glass column of 3.8cm × 65cm, wash post with ethanol limpid to effluent liquid, rinse with 3.75% hydrochloric acid-aqueous solution of 2 column volumes successively again, ultrapure water rinses the neutrality to pH, and the NaOH aqueous solution of 2 column volumes 5% is washed post, and ultrapure water rinses the neutrality to pH, last watering balance resin column, sedimentation is spent the night bubble is eliminated.
According to compound and resin extender ratio be 1:10 ratio get preparation in () to contain induced activity compound of the present invention some, use minute quantity dissolve with methanol, wet method loading.Rush post, 3 column volumes of each gradient elution with volumetric concentration 20%, 40% 100% methanol-water solution successively.0.5 mL/min quantitative collection, silica-gel plate point plate detects the material composition of each collection component, and using exhibition layer system is chloroform: methyl alcohol=9:1+ Glacial acetic acid, finally merge elution fraction according to the some material composition that fruit shows that hardens, concentrated, some plate detects.The active dull and stereotyped the most latter incorporated component that detects, merges the part containing activity inducement material to treat further separation.
3, the meticulous resin isolation of MCI induced activity compound of the present invention
MCI column material methyl alcohol soaked overnight, being packed into equably specification is in the chromatography glass column of 2.0cm × 50cm, wash post with methyl alcohol limpid to effluent liquid, be the methyl alcohol in the ultrapure water displacement analysis post of 80%, 60%, 40%, 20% methanol-water solution and 2 volumes by 1/2 column volume volumetric concentration successively again, sedimentation is spent the night bubble is eliminated.
Merge D101 macroporous resin column and separate the part containing activity inducement material obtaining, evaporate to dryness is concentrated, uses minute quantity dissolve with methanol, static wet method loading.Successively with volumetric concentration be 20%, 40% 100% methanol-water solution rush post, 3 column volumes of each gradient elution.0.5mL/min collects elutriant, silica-gel plate point plate detects the material composition in each pipe, and using exhibition layer system is chloroform: methyl alcohol=9:1+ Glacial acetic acid, finally merges elution fraction according to a plate detected result, concentrated, some plate detects the organic composition composition of determining each consolidated material.The active dull and stereotyped the most latter incorporated component that detects, merges the part containing activity inducement material to treat further separation.
4, the meticulous resin purification of ODS induced activity compound of the present invention
ODS column material methyl alcohol soaked overnight, being packed into equably specification is in the chromatography glass column of 1.0cm × 10cm, wash post with methyl alcohol limpid to effluent liquid, be the methyl alcohol in the ultrapure water displacement analysis post of 80%, 60%, 40%, 20% methanol-water solution and 2 volumes by 1 column volume volumetric concentration successively again, and get rid of the bubble in post with force (forcing) pump.
Merge MCI post and separate the part containing activity inducement material obtaining, evaporate to dryness is concentrated, water-dissolve with methanol, static wet method loading.Successively with 20%, 40% 100% methyl alcohol rush post, 3 column volumes of each gradient punching, 0.2 mL/min collects elutriant, silica-gel plate point plate detects the material composition in each pipe, using exhibition layer system is chloroform: methyl alcohol=9:1+ Glacial acetic acid, finally hardening according to point, merging is containing the elutriant of heterogeneity, concentrated respectively for fruit, and some plate detects.The active dull and stereotyped the most latter incorporated component that detects, merges the part containing activity inducement material the induced activity compound of the present invention that obtains purifying.
Three, activity inducement compound of the present invention application aspect synthetic at induction fungal laccase
1, the activation of laccase production bacterial strain Panus rudis
The P. rudis picking of slant preservation to CPDA inclined-plane, 28
oc leaves standstill and cultivates 3 days with activation bacterial strain.
2, the preparation of seed bottle
Get 7 ~ 8 of the P. rudis mycelium pieces (diameter 0.5cm) of activation, access is equipped with in the 250mL triangular flask of 100mL CPDA liquid nutrient medium, and 28
oc, 120rpm shaking table is cultivated.The P. rudis that grows 4 days is for subsequent use with homogenizer 6000rpm homogenate 10 seconds.
3, the laccase fermentation of activity inducement compound is produced
Get the liquid shaking flask of 5mL laccase production bacteria culture fluid access CPDA after homogenate, 28
oc, 120 rpm shaking tables are cultivated.After 4 days, adding final concentration is that the activity inducement compound of the present invention of 10 μ M continues to cultivate, and after 4 ~ 6 days, laccase activity reaches 12000U/L, is not add 7.5 times of induced activity compound fermentation laccase activity 1600U/L of the present invention.Laccase activity is measured and is adopted ABTS method: reaction mixture comprises 950 μ L 1 00mmol/L sodium tartrate damping fluids (pH 4.0), and 30 μ L 15mmol/L ABTS and 20 μ L are the enzyme liquid of dilution suitably, after mixing 30
ounder C, after water-bath 3min, under 420nm, measure light absorption value.Enzyme unit definition alive is: under these conditions, it is an enzyme activity unit that per minute is oxidized the required enzyme amount of 1 μ mol substrate.
Enzyme calculation formula: E alive (U/L)=OD
420× 555.56.
Claims (1)
1. the compound that fungal laccase is had to a high induced activity is the application aspect synthetic at induction fungal laccase, it is characterized in that, described compound has following molecular formula:
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