CN1417342A - Glusulase degrading process of natural product and polysaccharide - Google Patents
Glusulase degrading process of natural product and polysaccharide Download PDFInfo
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- CN1417342A CN1417342A CN 01136891 CN01136891A CN1417342A CN 1417342 A CN1417342 A CN 1417342A CN 01136891 CN01136891 CN 01136891 CN 01136891 A CN01136891 A CN 01136891A CN 1417342 A CN1417342 A CN 1417342A
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Abstract
The natural product and polysaccharide degrading process with glusulase includes dissolving natural product and polysaccharide into slution; adding glusculase in the amount of 0.2-25 wt% of substrate at 15-80 deg.c and solution pH 3.0-10.0 for 1-10 hr. Natural product, such as cellulose, ginseng, astragalus root, etc. and polysaccharide material, such as chitosan, pectine, xantham gum, glucosan and xylan may be degraded into bioactive small molecular functional matter and oligosaccharide of 3-20 polymerization degree. After degradation, oligosaccharide of relatively high polymerization degree is separated from the reaction system while the glusulase is reused.
Description
Technical field
The present invention relates to a kind of method with enzyme liberating natural product and polysaccharide, specifically, relate to a kind of utilize helicase with natural product and polysaccharide be degraded into available, have certain bioactive small molecules and the polymerization degree a method for the oligose of 3-20.
Background technology
The method of degraded natural product and polysaccharide has been reported: (1) chemical hydrolysis, as use the concentrated hydrochloric acid degrade chitosan, can obtain monose to pentasaccharides, but the content of monose is very high.Hydrogen fluoride degrade chitosan such as Defaye obtains three to ten sugar of high yield, but need remove a large amount of hydrogen fluoride and further defluorination reaction at last, thereby is restricted in using.And chemical reaction condition is harsh especially; (2) specificity enzyme liberating method, chitoanase can the specificity degrade chitosans, the product that the trisaccharide of production high yield is above; (3) non-specificity enzyme liberating method up to now, has had been found that 37 kinds of lytic enzymes, as proteolytic enzyme, lipase and Glycosylase, and all can degrade natural product and polysaccharide.Many in the world employing enzyme liberating natural products and polysaccharide are difficult to control but usually run into the product polymerization degree after the eighties, enzyme cost height, and utilization ratio is low, problems such as degradation effect difference.But do not see the relevant report of helicase
Summary of the invention
The object of the present invention is to provide a kind of method of utilizing helicase degraded natural product and polysaccharide, that this method can be degraded into natural product and polysaccharide is available, have certain bioactive small molecules and the polymerization degree is the oligose of 3-20, and its helicase can be reused.
To achieve these goals, the technical solution used in the present invention is that natural product or polysaccharide are dissolved into solution, press enzyme amount/substrate=0.2-25 (weight percent, below except that specified otherwise is arranged, all be weight percentage) add helicase and degrade, the temperature of reaction of degraded is 15-80 ℃, time 1-10 hour, and pH value of solution=3.0-10.0.The natural product that the present invention refers to comprises Mierocrystalline cellulose, genseng, the Radix Astragali, Radix Glycyrrhizae etc., and the polysaccharide raw material comprises chitosan, pectin, carrageenin, agar-agar, algin, xanthan gum, sesbania gum, dextran, xylan, mannosans, heparin, sulfated polysaccharide etc.
The present invention has following advantage
Helicase is to cut off β (1-4) glycosidic link with internal-cutting way when degraded natural product and polysaccharide, so it also can further be degraded into the oligosaccharides of higher degrees of polymerization the oligosaccharides of low polymerization degree in degradation of polysaccharide.The oligosaccharides of degraded back higher degrees of polymerization is separated from reaction system, can be obtained the oligosaccharides of higher degrees of polymerization on the one hand, helicase can be reused on the other hand.Experimental result shows: temperature of reaction is that 15-80 ℃, pH=3-10 stir and deacetylation is the oligochitosan that can obtain higher degrees of polymerization under the substrate condition of 50-100%.
Description of drawings
Fig. 1 has shown the influence of stirring the helicase degrade chitosan.
Fig. 2 has shown the influence of the different interpolation orders of various enzymes to degradation of chitosan.
Embodiment
In order to understand essence of the present invention better, at first effect of the present invention is described below with the comparison test of helicase and other plurality of enzymes.
1, plurality of enzymes is to the influence of active substance reducing sugar in the process of oligosaccharide at degradation of polysaccharide
Chitosan is taken by weighing a certain amount of damping fluid (pH2-8 that is dissolved in acetic acid and sodium-acetate, 0.01-1M) in, add enzyme liquid such as cellulase, bromeline, lipase, chitoanase, helicase and N,O-Diacetylmuramidase, making enzyme amount/chitosan is 0.01/1, all then enzyme liquid join chitosan solution rapidly, shake up constant temperature in the water-bath that is placed on 20-80 ℃, reaction picks up counting.The reducing sugar amount of chitoanase and helicase degrade chitosan generation as a result is the highest, and other four kinds of reducing sugars that enzyme produced are all more similar.Illustrate that specificity enzyme (chitoanase) and non-specificity enzyme (helicase) are to chitosan tool degradation effect preferably.
2, plurality of enzymes is to the influence of the product polymerization degree in the process of oligosaccharide at degradation of polysaccharide
Plurality of enzymes 1-100ml such as extracting cellulose enzyme, bromeline, lipase, chitoanase, helicase and N,O-Diacetylmuramidase join in the chitosan solution of 10-1000ml 0.5-5%, and 20-80 ℃, reaction is 2-200 hour in the shaking table of 50-250rpm.The viscosity that found that chitosan has reduced by 90%, and its product analysis is the result learn, product also has 39% trisaccharide and about 6% pentasaccharides except that monose and disaccharides.Obtain thick enzyme with the ammonium sulfate precipitation supernatant liquor, act in the chitosan solution of above-mentioned 0.5-5%, find that viscosity begins remarkable decline, substrate is degraded when finishing substantially, and the ratio of four in the product, pentasaccharides is 10%.Can know that from the viscosity degradation and the product composition of enzyme liquid helicase has endonuclease activity.Result of study shows: helicase can be used as the optional enzyme that oligochitosan is produced.
3, the helicase reaction conditions is to the active influence of degradation of chitosan
Substrate: when concentration of substrate is too high, because soltion viscosity is too big, the enzyme molecule can not freely be dispersed in the solution, so the concentration of reducing sugar reduces, simultaneously because concentration of substrate is big, have part to fail to degrade, thereby the absorbed portion reducing sugar reduces the concentration of reducing sugar because chitosan precipitates when measuring.
Temperature: temperature has a significant impact degradation of chitosan, and temperature is too low, and soltion viscosity is too big, so the concentration of reducing sugar is lower; And temperature is too high, and owing to enzyme work is affected, thereby the concentration of reducing sugar is lower.Therefore selected temperature is 15-80 ℃, and preferred temperature is 20-80 ℃.
Stir: stir the DeR that helps.See Fig. 1, stir influence (with magnetic stirring apparatus and water bath with thermostatic control reaction) the helicase degrade chitosan.
The pH value: when regulating different pH, selected acid, neutral and investigated the influence of the pH of helicase degrade chitosan between three kinds of situations between the two.Under the condition of acid, because the activity of enzyme is suppressed, so the concentration of reducing sugar is lower, and pH is more than certain value, and chitosan will part dissolve and is precipitated out, thus selected pH between the buffer zone of 2-10, preferable pH=3-10.
By example technology of the present invention is further specified below, wherein embodiment 1 to embodiment 4 has set forth the effect of helicase degrading enzyme under different condition again, and embodiment 5 to embodiment 20 discloses the concrete grammar that utilizes helicase degraded natural product and polysaccharide.
Embodiment 1: the effect of plurality of enzymes degrade chitosan
The chitosan of 0.01-10% is dissolved in the acetate buffer solution, adds various enzymes, enzyme: substrate=1-10: 2-200 20-80 ℃ of reaction down, characterizes the degrading activity of various enzymes with the percentage of reaction solution viscosity degradation.Measure t fall time that initially passes through measuring section with degraded some hrs afterreaction liquid with the Ping Shi viscosmeter
0, t, then the percentage of chitosan viscosity degradation is t
0-t/t
0
The effect of various enzyme liberating chitosans
Used enzyme viscosity degradation percentage (%)
Lipase 70.4
Cellulase 75.3
Chitoanase 94.6
Bromeline 90.4
N,O-Diacetylmuramidase 0.2
Helicase 96.2
Embodiment 2: the interpolation order of various combination enzyme is to the influence of various enzyme liberating chitosans
Analysis of experiments is learnt: helicase not only has the function of cutting off the macromole chitosan with lipase, cellulase, and has the function of cutting off low molecular chitosan.Formerly add under the situation of helicase, because helicase at first becomes degradation of chitosan low segmental chitosan, then lipase of Jia Ruing and cellulase fail to bring into play function because not degrading low segmental chitosan, reverse situation, at first add lipase and cellulase, the two macromolecular chitosan of can both degrading earlier is low segmental chitosan, add helicase again after, the low segmental chitosan of helicase degraded makes the height of kind situation before the concentration ratio of reducing sugar.See Fig. 2, the different interpolation orders of various enzymes are to the influence of degradation of chitosan
Embodiment 3: different helicase amounts are to the active influence of degradation of chitosan
Add different enzyme amounts in the 0.01-1% chitosan solution, make enzyme/chitosan be respectively certain tonsure, reaction is 1-10 hour in 20-80 ℃ of water-bath, test-results is learnt, when concentration of substrate one timing, enzyme concn increases, and will the viscosity degradation rate of chitosan be increased, and the reducing sugar amount that degraded is produced is also along with increase.
The helicase amount is to the influence of the reducing sugar content of degradation of chitosan
Enzyme amount (%) reducing sugar content (mg/ml)
0.10 1.17
0.20 1.86
0.25 2.74
0.30 4.25
Embodiment 4: the helicase degrade chitosan is batch test with certain polymerization degree and bioactive oligochitosan
Because helicase is to cut off β (1-4) glycosidic link with internal-cutting way when degrade chitosan, so it also can further be degraded into the oligosaccharides of higher degrees of polymerization the oligosaccharides of low polymerization degree in degrade chitosan.
The enzyme concn product yield product polymerization degree batch feeds intake
(kg) (%) (%) colorimetric mass spectrum
C-2 1.4 0.14 / 19.1 3-16
C-3 0.8 0.26 / 19.8 3-15
C-4 1.0 0.30 / 10.3 3-13
C-5 1.0 0.30 37.9 17.0 4-11
C-6 2.0 0.20 69.2 11.9 3-11
C-7 3.0 0.20 90.1 10.2 5-22
C-8 2.0 0.25 44.2 11.2 4-24
C-9 2.4 0.25 89.2 13.0 4-26
C-10 2.0 0.25 64.6 25.8 4-26
C-11 6.0 0.13 39.1 13.2 4-16
Embodiment 5: the chitosan with 1% is dissolved in the acetate buffer solution, adds helicase, enzyme/substrate=1, and 5 hours reaction times, reaction under 20 ℃, pH=5 can obtain the oligochitosan that the polymerization degree is 3-20.Characterize the degrading activity of various enzymes with the percentage of reaction solution viscosity degradation.
Embodiment 6: 20% pectin polysaccharide is dissolved in water, adds helicase, and enzyme/substrate=0.2,10 hours reaction times, 80 ℃ of temperature, pH=3 can obtain the pectin oligosaccharide that the polymerization degree is 3-20.
Embodiment 7: 5% carrageenin polysaccharide is dissolved in water, adds the helicase preparation, and enzyme/substrate=25,1 hour reaction times, 15 ℃ of temperature, pH=10, can obtain the polymerization degree is the OK a karaoke club oligose of 3-20.
Embodiment 8: the agar polysaccharide aqueous solution with 0.2%, add the helicase preparation, and enzyme/substrate=10,3 hours reaction times, 50 ℃ of temperature, pH=6 can obtain the agar oligosaccharide that the polymerization degree is 3-20.
Embodiment 9: the algin polysaccharide solution, the condition of pressing embodiment 1 can obtain the alginate oligosaccharide that the polymerization degree is 3-20.
Embodiment 10: the xanthan gum polysaccharide aqueous solution, press embodiment 1 condition, and can obtain the polymerization degree is the xanthan oligose of 3-20.
Embodiment 11: the sesbania gum polysaccharide solution, press embodiment 1 condition, and can obtain the polymerization degree is the sesbania oligose of 3-20.
Embodiment 12: the dextran polysaccharide solution, press embodiment 1 condition, and can obtain the polymerization degree is the poly-oligose in Portugal of 3-20.
Embodiment 13: the xylan polysaccharide solution, press embodiment 1 condition, and can obtain the polymerization degree is the poly-oligose of wood of 3-20.
Embodiment 14: the mannosans polysaccharide is dissolved in water, and presses embodiment 1 condition, can obtain the manna oligosaccharide that the polymerization degree is 3-20.
Embodiment 15: the heparin polysaccharide is dissolved in water, and presses embodiment 1 condition, and can obtain the polymerization degree is the heparin oligose of 3-20.
Embodiment 16: sulfated polysaccharide is dissolved in water, and presses embodiment 1 condition, can obtain the sulfate oligosaccharide that the polymerization degree is 3-20.
Embodiment 17: with the carboxymethyl cellulose dissolving, press embodiment 1 condition, can obtain to have active functional mass.
Embodiment 18: with the Radix Ginseng extract dissolving, press embodiment 1 condition, can obtain to have active functional mass.
Embodiment 19: with the dissolving of Radix Astragali aqueous extract, press embodiment 1 condition, can obtain to have active functional mass.
Embodiment 20: with the dissolving of Radix Glycyrrhizae aqueous extract, press embodiment 1 condition, can obtain to have active functional mass.
Claims (4)
1, a kind of method of utilizing helicase degraded natural product and polysaccharide, natural product can be transformed and form the high reactivity composition and polysaccharide is prepared oligosaccharide, it is characterized in that, adding helicase by enzyme amount/substrate=0.2-25 (weight percent) degrades, the temperature of reaction of degraded is 15-80 ℃, time 1-10 hour, pH value of solution=3.0-10.0.
2, the method for utilizing helicase degraded natural product and polysaccharide as claimed in claim 1 is characterized in that described natural product is Mierocrystalline cellulose, genseng, the Radix Astragali, Radix Glycyrrhizae.
3, the method for utilizing helicase degraded natural product and polysaccharide as claimed in claim 1, it is characterized in that described polysaccharide is chitosan, pectin, carrageenin, agar-agar, algin, xanthan gum, sesbania gum, dextran, xylan, mannosans, heparin or sulfated polysaccharide.
4, the method for utilizing helicase degraded natural product and polysaccharide as claimed in claim 1 is characterized in that described oligosaccharide is that the polymerization degree is the oligose of 3-20.
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| CN 01136891 CN1417342A (en) | 2001-11-06 | 2001-11-06 | Glusulase degrading process of natural product and polysaccharide |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101768619A (en) * | 2010-02-10 | 2010-07-07 | 华侨大学 | Method for preparing rare ginsenoside IH-901 with Rd as substrate |
| CN102154417A (en) * | 2010-12-13 | 2011-08-17 | 天津中医药大学 | Method for preparing periplocymarin |
| CN109136307A (en) * | 2018-09-06 | 2019-01-04 | 广东药科大学 | A kind of method and application thereof preparing chitosan oligosaccharide with glusulase |
-
2001
- 2001-11-06 CN CN 01136891 patent/CN1417342A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101768619A (en) * | 2010-02-10 | 2010-07-07 | 华侨大学 | Method for preparing rare ginsenoside IH-901 with Rd as substrate |
| CN102154417A (en) * | 2010-12-13 | 2011-08-17 | 天津中医药大学 | Method for preparing periplocymarin |
| CN109136307A (en) * | 2018-09-06 | 2019-01-04 | 广东药科大学 | A kind of method and application thereof preparing chitosan oligosaccharide with glusulase |
| WO2020048270A1 (en) * | 2018-09-06 | 2020-03-12 | 广东药科大学 | Method for preparing chitooligosaccharide using snailase and application thereof |
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