CN105462946A - Method for promoting Trichoderma reesei to synthesize beta-mannase - Google Patents
Method for promoting Trichoderma reesei to synthesize beta-mannase Download PDFInfo
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- CN105462946A CN105462946A CN201511003998.1A CN201511003998A CN105462946A CN 105462946 A CN105462946 A CN 105462946A CN 201511003998 A CN201511003998 A CN 201511003998A CN 105462946 A CN105462946 A CN 105462946A
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- mannase
- beta
- trichodermareesei
- polygalactomannan
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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
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2477—Hemicellulases not provided in a preceding group
- C12N9/2488—Mannanases
- C12N9/2491—Beta-mannosidase (3.2.1.25), i.e. mannanase
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- 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
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2477—Hemicellulases not provided in a preceding group
- C12N9/2488—Mannanases
- C12N9/2494—Mannan endo-1,4-beta-mannosidase (3.2.1.78), i.e. endo-beta-mannanase
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01025—Beta-mannosidase (3.2.1.25), i.e. mannanase
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01078—Mannan endo-1,4-beta-mannosidase (3.2.1.78), i.e. endo-beta-mannanase
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Abstract
The invention discloses a method for promoting Trichoderma reesei to synthesize beta-mannase. In a fermentation culture medium using microcrystalline cellulose as a carbon source, galactomannan is added to promote Trichoderma reesei to synthesize the beta-mannase. In the fermentation culture medium using microcrystalline cellulose as a carbon source, a trace amount of galactomannan is added; and when the addition amount is less than or equal to 0.6 g/L, the galactomannan can effectively promote the Trichoderma reesei to synthesize the beta-mannase.
Description
Technical field
The invention belongs to enzyme preparing technical field in microbiology, be specifically related to a kind of method that Trichodermareesei synthesizes 'beta '-mannase that promotes.
Technical background
'beta '-mannase is that one can become the multiply anchor-pile of monose by hydrolyzing mannan (mannosans, glucomannan, polygalactomannan and gala glucomannan), primarily of 'beta '-mannase and beta-Mannosidase composition.Occurring in nature, 'beta '-mannase is mainly present in animal and plant and microorganism, and the 'beta '-mannase wherein with industrial application potentiality mainly obtains from fermentable.
Some fungies in microorganism, bacterium and actinomycetes all can secrete 'beta '-mannase.Trichodermareesei (
trichodermareesei) be a kind ofly can secrete 'beta '-mannase and microorganism to humans and animals safety, Trichodermareesei 'beta '-mannase can be widely used in food, feed and functional oligose process industry.
Trichodermareesei 'beta '-mannase belongs to induction enzyme, and its secretion 'beta '-mannase is subject to the substrate specificity of 'beta '-mannase or the induction of substrate structure analogue.Mannosans, glucomannan, polygalactomannan and gala glucomannan etc. are the substrate specificity of 'beta '-mannase, they are good inductors of 'beta '-mannase, but the substrate specificity of these 'beta '-mannases exists full-bodied characteristic, namely the concentration of usual 0.5% cause the viscosity that substratum is very high.Trichodermareesei is aerobic microbiological, and it grows in the medium needs there are enough dissolved oxygens in nutrient solution with the synthesis of enzyme.In Trichodermareesei culturing process, the dissolving of oxygen in nutrient solution affects very large by broth viscosity, and viscosity is higher, and oxygen transfer efficiency is lower.Trichodermareesei is cultivated in containing the substratum of high viscosity inductor, in culturing process, the dissolution rate of oxygen in nutrient solution is often lower than the speed of dissolved oxygen in Trichodermareesei picked-up nutrient solution, thus cause dissolved oxygen concentration in nutrient solution too low, be unfavorable for the cultivation of Trichodermareesei and the synthesis of 'beta '-mannase.Therefore, Trichodermareesei ferments and synthesizes 'beta '-mannase in the substratum taking mannosans as carbon source and inductor, and beta-mannase enzyme activity is often not high.
Summary of the invention
Goal of the invention: for the deficiencies in the prior art, the invention provides a kind of method that Trichodermareesei synthesizes 'beta '-mannase that promotes, take Microcrystalline Cellulose as primary carbon source, adds the ability that micro-polygalactomannan improves the poly-enzyme of its synthesis β-Gan sugar dew.
Technical scheme: in order to realize foregoing invention object, the present invention adopts following technical scheme:
A kind of method promoting Trichodermareesei synthesis 'beta '-mannase: in the fermention medium taking Microcrystalline Cellulose as carbon source, adds polygalactomannan and promotes Trichodermareesei synthesis 'beta '-mannase.
Described polygalactomannan addition is≤0.6g/L.
Described Microcrystalline Cellulose concentration is 10-25g/L.
Described polygalactomannan extracts from Sesbania seed, purifying obtains.
Beneficial effect: compared with prior art, the method for promotion Trichodermareesei synthesis 'beta '-mannase of the present invention, in the substratum taking Microcrystalline Cellulose as carbon source, adds the excellent inductor polygalactomannan of 'beta '-mannase of trace.While adding the Induced synthesis of polygalactomannan promotion 'beta '-mannase, broth viscosity will be caused to increase owing to adding polygalactomannan, therefore the dissolving of oxygen and the synthesis of 'beta '-mannase in culturing process is unfavorable for, present method is by controlling the concentration range≤0.6g/L of the polygalactomannan added, effectively can to promote there is Trichodermareesei 'beta '-mannase good practicality.
Accompanying drawing explanation
Fig. 1 be add polygalactomannan on Trichodermareesei synthesis 'beta '-mannase affect result figure.
Embodiment
Below in conjunction with concrete enforcement, the invention will be further elaborated.Embodiment is for explanation and unrestricted the present invention.In this area, any those of ordinary skill can understand these embodiments, does not limit the present invention in any way, and can make suitable amendment and without prejudice to essence of the present invention and depart from scope of the present invention.
Embodiment 1
The Sesbania seed crushed material taking the heavy 5.0g of over dry, in 250mL triangular flask, adds water by solid-to-liquid ratio 1:15, at temperature 95 DEG C, extracts 8h.After extraction terminates, the centrifugal 5min of 10000r/min, collects supernatant liquor.In above-mentioned supernatant liquor, add dehydrated alcohol, make alcohol concn be 30%, obtain polygalactomannan precipitation.Polygalactomannan precipitation is redissolved in distilled water, centrifugal segregation insolubles.Supernatant liquor uses alcohol settling again, and precipitation is carried out lyophilize, obtains the polygalactomannan that purity is higher.
Adopt dilute sulphuric acid (4%) hydrolysis, the method for efficient liquid phase chromatographic analysis glycosyl composition measures galactosyl in polygalactomannan obtained above and mannose group content, result display is containing galactosyl 29.44%, mannose group 65.70%, protein 1.50% and ash content 3.36%(mass percent), extraction is described, purifying obtains highly purified polygalactomannan.
Embodiment 2
Culture medium: Microcrystalline Cellulose 20g/L, glucose 1g/L, potassium primary phosphate 2.0g/L, magnesium sulfate heptahydrate 0.08g/L, iron vitriol 0.005g/L, manganese sulfate monohydrate 0.0016g/L, Zinc Sulphate Heptahydrate 0.0014g/L, cobalt chloride 0.0037g/L.Substratum citrate buffer solution adjust ph is 4.5-5.0.
Enzymatic production: 50mL culture medium is placed in the triangular flask that 250mL is with tampon, and Trichodermareesei inoculum size is 10%.The constant-temperature table being placed in 28-30 DEG C, 170 revs/min is cultivated 4 days.Nutrient solution is centrifugal 10min. under 3000 revs/min.Get supernatant and measure beta-mannase enzyme activity.
'beta '-mannase enzyme activity determination: the mass concentration adding the citrate-phosphate disodium hydrogen buffer of 0.9mL pH value 5.0 in 25m scale test tube is the locust bean gum substrate solution of 5.0g/L, 50 DEG C of preheating 5min, add the enzyme liquid of 0.1mL through suitably dilution, at 50 DEG C, react 30min, add 3.0mLDNS reagent immediately, boil 5min, 25mL is settled to after cooling, shake up, adopt 3,5-dinitrosalicylic acid (DNS) method to measure the reducing sugar of hydrolysis generation.The enzyme amount producing 1 μm of ol reducing sugar (with seminose) in per minute hydrolysis substrate is defined as 1 'beta '-mannase enzyme activity unit.
Result shows, Trichodermareesei is that carbon source through fermentation prepares 'beta '-mannase with Microcrystalline Cellulose, and beta-mannase enzyme activity is 4.0U/mL.
Embodiment 3
Culture medium: Microcrystalline Cellulose 20g/L, glucose 1g/L, potassium primary phosphate 2.0g/L, magnesium sulfate heptahydrate 0.08g/L, iron vitriol 0.005g/L, manganese sulfate monohydrate 0.0016g/L, Zinc Sulphate Heptahydrate 0.0014g/L, cobalt chloride 0.0037g/L.Substratum citrate buffer solution adjust ph is 4.5-5.0.
Polygalactomannan (in polygalactomannan 100%) 0.2g/L, 0.4g/L, 0.6g/L, 0.8g/L, 1.0g/L, 2.0g/L and 3.0g/L prepared by embodiment 1 is added respectively in above-mentioned culture medium.
Enzymatic production: 50mL culture medium is placed in the triangular flask that 250mL is with tampon, and Trichodermareesei inoculum size is 10%.The constant-temperature table being placed in 28-30 DEG C, 170 revs/min is cultivated 4 days.Nutrient solution is centrifugal 10min. under 3000 revs/min.Get supernatant and measure beta-mannase enzyme activity.
As shown in Figure 1, result shows result, and Trichodermareesei is that carbon source through fermentation prepares 'beta '-mannase with Microcrystalline Cellulose, adds trace (≤0.6g/L) polygalactomannan in the medium and can promote that Trichodermareesei synthesizes 'beta '-mannase; But add the synthesis that more polygalactomannan is then unfavorable for Trichodermareesei 'beta '-mannase.When adding the polygalactomannan of 0.2g/L in substratum, beta-mannase enzyme activity is 4.43U/ml, increases by 10.8% than the enzyme activity of the substratum not adding polygalactomannan; When adding the polygalactomannan of 0.6g/L in substratum, 'beta '-mannase enzyme activity is 4.28U/ml, increases by 7% than the enzyme activity of the substratum not adding polygalactomannan; When adding the polygalactomannan of 0.8g/L in substratum, beta-mannase enzyme activity is 3.99U/ml, suitable with the enzyme activity (4.0U/ml) of the substratum not adding polygalactomannan; When in substratum, polygalactomannan addition is higher than 0.8g/L, beta-mannase enzyme activity is lower than the enzyme activity of substratum not adding polygalactomannan.
Claims (4)
1. promote that Trichodermareesei synthesizes a method for 'beta '-mannase, it is characterized in that: in the fermention medium taking Microcrystalline Cellulose as carbon source, add polygalactomannan and promote Trichodermareesei synthesis 'beta '-mannase.
2. the method for promotion Trichodermareesei synthesis 'beta '-mannase according to claim 1, is characterized in that: described polygalactomannan addition is≤0.6g/L.
3. the method for promotion Trichodermareesei synthesis 'beta '-mannase according to claim 1, is characterized in that: described Microcrystalline Cellulose concentration is 10-25g/L.
4. the method for promotion Trichodermareesei synthesis 'beta '-mannase according to claim 1, is characterized in that: described polygalactomannan extracts from Sesbania seed, purifying obtains.
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| CN201511003998.1A CN105462946A (en) | 2015-12-29 | 2015-12-29 | Method for promoting Trichoderma reesei to synthesize beta-mannase |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106282142A (en) * | 2016-08-05 | 2017-01-04 | 南京林业大学 | The preparation method of the α tilactase that a kind of β mannosidase content is low |
| CN112760311A (en) * | 2021-01-29 | 2021-05-07 | 南京林业大学 | Enzyme solution with relatively excellent enzyme activity ratio of beta-mannase to alpha-galactosidase, and preparation method and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0851975A (en) * | 1991-10-09 | 1996-02-27 | Res Dev Corp Of Japan | New beta-mannanase and method for producing the same |
| JP2009060805A (en) * | 2007-09-04 | 2009-03-26 | Nippon Ecolonomix:Kk | New mannanase and dietary fiber food produced using the same |
| CN104846035A (en) * | 2015-05-20 | 2015-08-19 | 南京林业大学 | Method for enzymatically preparing galacto-mannan-oligosaccharides from sesbania cannabina |
-
2015
- 2015-12-29 CN CN201511003998.1A patent/CN105462946A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0851975A (en) * | 1991-10-09 | 1996-02-27 | Res Dev Corp Of Japan | New beta-mannanase and method for producing the same |
| JP2009060805A (en) * | 2007-09-04 | 2009-03-26 | Nippon Ecolonomix:Kk | New mannanase and dietary fiber food produced using the same |
| CN104846035A (en) * | 2015-05-20 | 2015-08-19 | 南京林业大学 | Method for enzymatically preparing galacto-mannan-oligosaccharides from sesbania cannabina |
Non-Patent Citations (4)
| Title |
|---|
| 《ACTA MICROBIOLOGICA ET IMMUNOLOGICA HUNGARICA》 * |
| 《WORLD JOURNAL OF MICROBIOLOGY & BIOTECHNOLOGY》 * |
| 《南京林业大学学报( 自然科学版)》 * |
| 熊元林等: "《微生物学实验》", 31 January 2014, 武汉：华中师范大学出版社 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106282142A (en) * | 2016-08-05 | 2017-01-04 | 南京林业大学 | The preparation method of the α tilactase that a kind of β mannosidase content is low |
| CN106282142B (en) * | 2016-08-05 | 2019-05-31 | 南京林业大学 | A kind of preparation method for the alpha-galactosidase that beta-Mannosidase content is low |
| CN112760311A (en) * | 2021-01-29 | 2021-05-07 | 南京林业大学 | Enzyme solution with relatively excellent enzyme activity ratio of beta-mannase to alpha-galactosidase, and preparation method and application thereof |
| CN112760311B (en) * | 2021-01-29 | 2023-09-12 | 南京林业大学 | Enzyme solution with better enzyme activity ratio of beta-mannase to alpha-galactosidase, and preparation method and application thereof |
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Application publication date: 20160406 |