CN102154247B - Method for preparing beta-mannanase by using natural Bacillus subtilis - Google Patents
Method for preparing beta-mannanase by using natural Bacillus subtilis Download PDFInfo
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Abstract
The invention provides a method for preparing beta-mannanase by using natural Bacillus subtilis, which comprises: culturing Bacillus subtilis HSO121 by using culture medium containing refined rivier giantarum rhizome powder and a composite nitrogen source, centrifuging the obtained culture liquid to remove thallus cells and thus obtaining coarse beta-mannanase liquid, wherein the mass concentration of refined rivier giantarum rhizome powder in the culture medium is 2.5 to 5.0 percent; and the composite nitrogen source comprises KNO3 at a concentration of 80 to 140mmol/L and peptone at a concentration of 0.1 to 1 weight percent. In the invention, the beta-mannanase is prepared by optimizing the formula of the culture medium and by using the natural Bacillus subtilis HSO121, so the beta-mannanase yield of the fermentation of the Bacillus subtilis HSO121 is improved and a good technical effect is produced.
Description
Technical field
The present invention relates to the technology of microorganisms producing enzyme, especially relate to a kind of preparation method of 'beta '-mannase.
Background technology
The source of mannosans is abundant, has different types of mannosans to exist in Rhizoma amorphophalli powder, guar gum, locust bean gum etc.Main chain mannosans degrading enzyme comprises 'beta '-mannase, beta-glucosidase, beta-Mannosidase.Wherein, the most important thing is 'beta '-mannase.'beta '-mannase is that a class can hydrolysis contain β-1, the mannooligo saccharide of 4-D-seminose glycosidic bond, the inscribe lytic enzyme of mannocarolose, and it belongs to the hemicellulose enzyme, extensively is present in animal, plant and the microorganism.Wherein, microorganism is the main source of 'beta '-mannase, has advantages such as active height, steady sources.
To the exploitation of nature hemicellulose resource, the research and development of 'beta '-mannase enters a new upsurge along with in recent years, has obtained at aspects such as food, medicine, papermaking, textile printing and dyeing, oil production and biological study technology using widely.In field of petroleum exploitation, in the oil extraction process, the quality of fracturing effect directly influences oil-field development level and economic benefit.Waterfrac treatment is to be the water-based fracturing liquid system of viscosifying agent with vegetable jelly (as guar gum), has characteristics such as inexpensive, safety, applied range.But waterfrac treatment meeting infringement water sensitivity stores, and because residue, the concentrated glue of broken glue and the injury of the flow conductivity that filter cake causes.Wherein the concentrated glue of residue, broken glue and flow conductivity infringement that filter cake causes mainly solve by adding gel breaker.Conventional oxidation gel breaker has a lot of defectives, such as at high temperature being swift in response with fracturing liquid, fracturing liquid is degraded in advance and losing the ability of carrying propping agent; Can with any reactant that runs into such as tubing, stratum matrix and hydrocarbon reaction, generate and the incompatible pollutent in stratum, cause formation damage; The oxidation gel breaker also may just run out before arriving the target crack, thereby does not reach brokenly the purpose of glue.Enzyme breaker is a kind of novel gel breaker, compares with conventional gel breaker, has a series of advantage, as enzyme in reaction process not with other material such as reactions such as fracturing liquid, tubing, have height specificity, degraded high efficiency and characteristic such as pollution-free.The 'beta '-mannase that Nocardia bacteria shape actinomycetes is produced as people such as Wu's flap is used for the biological gel breaker of low temperature (30～60 ℃) fracturing fluid, have efficient height, cost low, little to formation damage, join advantages such as adhesive process is easy.
The bibliographical information of at present relevant 'beta '-mannase preparation is more.Utilizing genus bacillus to produce aspect the 'beta '-mannase, adopt typical document (Chai Pingping, Wei Yun etc. China Agricultural University's journal, 2005,10 (3): 77-80) the natural subtilis of Bao Dao culture medium culturing, measuring that maximum enzyme lives is 2.36U/ml.Aspect the raising production of enzyme, mainly be that encoding gene around 'beta '-mannase conducts a research and makes up by genetically engineered and produces enzyme function yeast etc., though obtained higher output, but because genetic engineering bacterium exists problems such as unstable properties and ecological safety, make the large-scale popularization of this technology be subjected to certain limitation.
Summary of the invention
Adopt genetically engineered to make up the unstable properties of bacterium raising output existence and potential technological deficiencies such as problems of ecological security in order to overcome prior art, the technical problem to be solved in the present invention provide a kind of non-genomic engineering, prepare the method for 'beta '-mannase with microorganism culturing, this method can improve enzyme greatly and live.
Technical scheme of the present invention is, a kind of method of utilizing natural subtilis to prepare 'beta '-mannase, and this method may further comprise the steps:
(1) adopts the culture medium culturing subtilis HSO121 that contains konjaku powder and compound nitrogen source;
(2) medium centrifugal that step (1) is obtained obtains the 'beta '-mannase crude enzyme liquid after removing somatic cells;
The smart opaque amount of konjaku concentration is 2.5～5.0% in the described substratum; Described compound nitrogen source comprises that concentration is the KNO of 80～140mmol/L
3With concentration be the peptone of 0.1～1wt%.
Preferably, contain Mn in the described substratum
2+Mn
2+Adding can further improve enzyme and live.More preferably be described Mn
2+Concentration is 5～20mmol/L.Best is described Mn
2+Concentration is 6～9mmol/L.
Preferably, described substratum can further contain KH
2PO
40.001-1wt%, MgSO
40.001-0.5wt%.
According to the method for utilizing natural subtilis to prepare 'beta '-mannase of the present invention, be preferably, the smart opaque amount of konjaku concentration is 3.0～4.5wt% in the described substratum.
In a preferred embodiment, in the described substratum compound nitrogen source by the KNO of 100～120mmol/L
3Constitute with the peptone of 0.5～1wt%.
More preferably be that described substratum comprises konjaku powder 3.5wt%, KNO
3100mmol/L, peptone 1wt% and Mn
2+9mmol/L.
The present invention utilizes open source literature (Xiang-Yang Liu, Shi-Zhong Yang, Bo-Zhong Mu.Production andcharacterization of a C15-surfactin-O-methyl ester by a lipopeptide producing strain Bacillus subtilisHSO121.Process Biochemistry44 (2009) 1144-1151) Bao Dao natural subtilis HSO121 prepares 'beta '-mannase, improve culture medium prescription, existing producing in the enzyme substratum konjaku powder concentration is improved, add mn ion and adopt KNO
3Cultivate with the peptone compound nitrogen source, improved the amount of genus bacillus HSO121 enzymatic production greatly, make enzyme work improve nearly 10 times, overcome prior art and adopted genetically engineered to make up the unstable properties of bacterium raising output existence and potential technological deficiencies such as problems of ecological security, received the good technical effect.In addition, 'beta '-mannase that this bacterial strain produces is comparatively obvious to the glutinous effect of falling of guar gum, locust bean gum, konjak gum, in low temperature oil well broken glue, health care of food and animal-feed potential using value is arranged.
Embodiment
Enzyme definition alive and measuring method are as follows:
Enzyme is lived: under experiment condition, the per minute hydrolysis substrate produces the enzyme amount that 1 μ mol is equivalent to the reducing sugar of D-seminose and is defined as 1 beta-mannase unit of enzyme (U).
Enzyme activity determination method: get mass concentration and be 0.5% the locust bean gum substrate solution (Na of pH=6
2HPO
4-NaH
2PO
4The damping fluid preparation) 0.9mL, add the suitably crude enzyme liquid 0.1mL of dilution, behind the mixing in 55 ℃ of water-bath 10min, add 2mLDNS reagent termination reaction, boiling water bath 2min colour developing, cooling constant volume, measure the reducing sugar amount that produces, measuring the OD value at the 540nm place, as standard, is contrast with the crude enzyme liquid of deactivation in advance with the D-seminose.
Embodiment 1
With substratum: konjaku powder 2.5～5.0% (wt%), peptone 0.1% (wt%), KH
2PO
40.1% (wt%), MgSO
40.01% (wt%) is substratum, in pH:7～7.2,37 ℃, 120r/min cultivates subtilis HSO121, cultivate 32h after, centrifugal going obtains crude enzyme liquid behind the cell.Measuring that maximum enzyme lives during down with enzyme liquid reaction 10min for 55 ℃ is 13.16U/mL.
| Konjaku powder (wt%) | 2.5 | 3.0 | 3.5 | 4.0 | 4.5 | 5.0 |
| Enzyme U/mL alive | 10.37 | 10.77 | 13.16 | 11.50 | 10.52 | 10.09 |
Embodiment 2
With substratum: konjaku powder 3.5% (wt%), KNO
380～140mmol/L, peptone 0.1% (wt%), KH
2PO
40.1% (wt%), MgSO
40.01% (wt%) is substratum, in pH:7～7.2,37 ℃, 120r/min cultivates subtilis HSO121, cultivate 32h after, centrifugal going obtains crude enzyme liquid behind the cell.Measuring that maximum enzyme lives during down with enzyme liquid reaction 10min for 55 ℃ is 9.68U/mL.
| KNO 3mmol/L | 80 | 100 | 120 | 140 |
| Enzyme U/mL alive | 5.33 | 9.68 | 9.07 | 6.79 |
Embodiment 3
With substratum: konjaku powder 3.5% (wt%), KNO
3100mmol/L, peptone 0.1%～1.0% (wt%), KH
2PO
40.1% (wt%), MgSO
40.01% (wt%) is substratum, in pH:7～7.2,37 ℃, 120r/min cultivates subtilis HSO121, cultivate 32h after, centrifugal going obtains crude enzyme liquid behind the cell.Measuring that maximum enzyme lives during down with enzyme liquid reaction 10min for 55 ℃ is 14.31U/mL.
| Peptone (wt%) | 0.1 | 0.5 | 0.8 | 1.0 |
| Enzyme U/mL alive | 6.27 | 12.17 | 13.76 | 14.31 |
Embodiment 4
With substratum: konjaku powder 3.5% (wt%), KNO
3100mmol/L, peptone 1% (wt%), KH
2PO
40.1% (wt%), MgSO
40.01% (wt%) is substratum, in pH:7～7.2,37 ℃, 120r/min cultivates subtilis HSO121, cultivate 32h after, centrifugal going obtains crude enzyme liquid behind the cell.55 ℃ during down with enzyme liquid reaction 10min, measuring that maximum enzyme lives is 14.31U/mL.
Embodiment 5
With substratum: konjaku powder 3.5% (wt%), KNO
3100mmol/L, peptone 1% (wt%), KH
2PO
40.1% (wt%), MgSO
40.01% (wt%), the mn ion that adds different concns is substratum, in pH:7～7.2,37 ℃, 120r/min cultivates subtilis HSO121, cultivate 32h after, centrifugal going obtains crude enzyme liquid behind the cell.55 ℃ during down with enzyme liquid reaction 10min, measuring that maximum enzyme lives is 19.60U/mL.
As seen, added Mn
2+After, can significantly improve enzyme and live.
| Mn 2+mmol/L | 5 | 6 | 7 | 8 | 9 | 10 | 20 |
| Enzyme U/mL alive | 17.07 | 17.64 | 18.81 | 18.99 | 19.60 | 16.97 | 16.36 |
Embodiment 6
Substratum: konjaku powder 3.5% (wt%), KNO
3100mmol/L, peptone 1% (wt%), KH
2PO
40.1% (wt%), MgSO
40.01% (wt%), Mn
2+9mmol/L, pH:7～7.2, at 37 ℃, after 120r/min cultivated 32h, centrifugal going obtained crude enzyme liquid behind the cell.55 ℃ during down with enzyme liquid reaction 10min, measuring that maximum enzyme lives is 23U/mL.
Embodiment 7
Substratum: konjaku powder 3.1% (wt%), KNO
3100mmol/L, peptone 0.5% (wt%), in pH:7～7.2,37 ℃, 120r/min cultivates subtilis HSO121, cultivate 32h after, centrifugal going obtains crude enzyme liquid behind the cell.Measuring that maximum enzyme lives during down with enzyme liquid reaction 10min for 55 ℃ is 7.05U/mL.
Embodiment 8
Substratum: konjaku powder 2.8% (wt%), KNO
3130mmol/L, peptone 0.3% (wt%), Mn
2+8mmol/L, in pH:7～7.2,37 ℃, 120r/min cultivates subtilis HSO121, cultivate 32h after, centrifugal going obtains crude enzyme liquid behind the cell.Measuring that maximum enzyme lives during down with enzyme liquid reaction 10min for 55 ℃ is 11.21U/mL.
Embodiment 9
Substratum: konjaku powder 4.0% (wt%), KNO
3120mmol/L, peptone 0.9% (wt%), KH
2PO
40.2wt%, MgSO
40.005wt% is substratum, in pH:7～7.2,37 ℃, 120r/min cultivates subtilis HSO121, cultivate 32h after, centrifugal going obtains crude enzyme liquid behind the cell.Measuring that maximum enzyme lives during down with enzyme liquid reaction 10min for 55 ℃ is 8.27U/mL.
Embodiment 10
Substratum: konjaku powder 4.5% (wt%), KNO
3110mmol/L, peptone 0.7% (wt%), KH
2PO
40.5wt%, MgSO
40.05wt%, Mn
2+11mmol/L, in pH:7～7.2,37 ℃, 120r/min cultivates subtilis HSO121, cultivate 32h after, centrifugal going obtains crude enzyme liquid behind the cell.Measuring that maximum enzyme lives during down with enzyme liquid reaction 10min for 55 ℃ is 12.72U/mL.
The present invention utilizes natural subtilis HSO121 by optimizing culture medium prescription, prepares 'beta '-mannase, has improved the amount of subtilis HSO121 enzymatic production greatly, has received the good technical effect.
Claims (8)
1. method of utilizing subtilis to prepare 'beta '-mannase, it is characterized in that: this method may further comprise the steps:
(1) adopts the culture medium culturing subtilis HSO121 that contains konjaku powder and compound nitrogen source;
(2) medium centrifugal that step (1) is obtained obtains the 'beta '-mannase crude enzyme liquid after removing somatic cells;
The smart opaque amount of konjaku concentration is 2.5 ~ 5.0% in the described substratum; Described compound nitrogen source comprises that concentration is the KNO of 80～140mmol/L
3With concentration be the peptone of 0.1 ~ 1wt%.
2. the method for utilizing subtilis to prepare 'beta '-mannase according to claim 1 is characterized in that: contain Mn in the described substratum
2+
3. the method for utilizing subtilis to prepare 'beta '-mannase according to claim 2 is characterized in that: described Mn
2+Concentration is 5 ~ 20mmol/L.
4. according to the described method of utilizing subtilis to prepare 'beta '-mannase of claim 2, it is characterized in that: described Mn
2+Concentration is 6～9mmol/L.
5. the method for utilizing subtilis to prepare 'beta '-mannase according to claim 1, it is characterized in that: described substratum contains KH
2PO
40.001-1wt%, MgSO
40.001-0.5wt%.
6. the method for utilizing subtilis to prepare 'beta '-mannase according to claim 1 is characterized in that: the smart opaque amount of konjaku concentration is 3.0 ~ 4.5wt% in the described substratum.
7. according to the described method of utilizing subtilis to prepare 'beta '-mannase of claim 1, it is characterized in that: compound nitrogen source is by the KNO of 100～120mmol/L in the described substratum
3Constitute with the peptone of 0.5～1wt%.
8. according to claim 1 or the 2 described methods of utilizing subtilis to prepare 'beta '-mannase, it is characterized in that: described substratum contains konjaku powder 3.5wt%, KNO
3100mmol/L, peptone 1wt% and Mn
2+9mmol/L.
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| CN102373168A (en) * | 2011-10-10 | 2012-03-14 | 武汉东方天琪生物工程有限公司 | Bacillus subtilis capable of expressing beta-mannase at high efficiency, as well as enzyme product and production method thereof |
| CN103555629B (en) * | 2013-11-04 | 2015-11-11 | 赵静 | A kind of bacterial strain and application thereof of producing middle low temperature 'beta '-mannase |
| CN106497539A (en) * | 2016-11-02 | 2017-03-15 | 常州市鼎日环保科技有限公司 | A kind of preparation method of compound gel breaker |
| CN107365756A (en) * | 2017-08-01 | 2017-11-21 | 天津大学 | A kind of method for preparing β mannases using the fermentation of mannosan type natural plant gum liquid hydrolyzate |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101016531A (en) * | 2007-01-30 | 2007-08-15 | 武汉东方天琪生物工程有限公司 | Method of producing neutral beta-konjak mannase preparation |
| CN101671645A (en) * | 2009-10-26 | 2010-03-17 | 中国农业大学 | Method for preparing beta-mannase and special strain thereof |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101016531A (en) * | 2007-01-30 | 2007-08-15 | 武汉东方天琪生物工程有限公司 | Method of producing neutral beta-konjak mannase preparation |
| CN101671645A (en) * | 2009-10-26 | 2010-03-17 | 中国农业大学 | Method for preparing beta-mannase and special strain thereof |
Non-Patent Citations (2)
| Title |
|---|
| Production and characterization of a C15-surfactin-O-methyl ester by a lipopeptide producing strain Bacillus subtilis HSO121;Xiang-Yang Liu et al.,;《Process Biochemistry》;20091231;1144-1151 * |
| Xiang-Yang Liu et al.,.Production and characterization of a C15-surfactin-O-methyl ester by a lipopeptide producing strain Bacillus subtilis HSO121.《Process Biochemistry》.2009, |
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