CN110656140A - Method for improving chitin degradation rate by pretreating chitin with alkali freeze-thaw system - Google Patents
Method for improving chitin degradation rate by pretreating chitin with alkali freeze-thaw system Download PDFInfo
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- CN110656140A CN110656140A CN201910974341.1A CN201910974341A CN110656140A CN 110656140 A CN110656140 A CN 110656140A CN 201910974341 A CN201910974341 A CN 201910974341A CN 110656140 A CN110656140 A CN 110656140A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/26—Preparation of nitrogen-containing carbohydrates
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
Abstract
The invention discloses a method for improving chitin degradation rate by pretreating chitin with an alkali freeze-thawing system. The method comprises the following steps: (1) obtaining chitinase by microbial fermentation; (2) weighing appropriate amount of chitin, adding into alkali solution, stirring for dispersing, taking out for melting at room temperature under suitable freeze-thaw mode treatment, boiling to separate out chitin, centrifuging to remove supernatant, stirring for resuspending with PBS (pH 7.0) buffer solution, and cleaning pH to neutral; (3) and (3) absorbing the pretreated chitin and chitinase to react in a water bath, and measuring the content of reducing sugar after the reaction is finished. Compared with the prior art, the method improves the adhesive force of the chitin and the enzyme thereof by modifying the chitin, has the conversion efficiency 11.01 times that of the untreated chitin, has short time, simple operation and less pollution, and has wide application prospect.
Description
Technical Field
The invention belongs to the technical field of biology, and relates to a method for improving chitin degradation rate by pretreating chitin with an alkali freeze-thawing system.
Background
Chitin (Chitin), also called Chitin and Chitin, is mainly present in exoskeletons of shrimps, crabs, shellfish and insects, and has a huge content in nature, which is next to cellulose. The macromolecular polysaccharide is a polymer formed by connecting monomer N-acetylglucosamine with alpha-1, 4 glycosidic bonds. Both chemical acid-base method and biological enzyme method can degrade it into chitin oligosaccharide and monosaccharide (N-acetylglucosamine). However, the acid-base method has the disadvantages of high environmental pollution, difficult control of the reaction process, and low reaction efficiency, and the obtained product has poor biological activity and is not the best choice for degrading chitin. In recent years, the biological enzyme method has gradually gained attention from broad scholars due to the advantages of mild reaction, convenient control, environmental protection and the like.
N-acetylglucosamine is used as a basic unit of chitin, and has wide application prospects in the fields of medicine, agriculture, cosmetics industry and environmental protection in recent years. Particularly in the medical field, the traditional Chinese medicine composition can be used for clinically enhancing the human immune system, treating osteoarthritis, arthralgia and other inflammations, inhibiting the excessive growth of cancer cells or fiber cells and playing a role in inhibiting and treating malignant tumor.
However, hydrogen bonds in the chitin chain are complex, so that the chitin chain is endowed with high crystallinity and water-insoluble characteristics, the efficiency of producing N-acetylglucosamine by degrading chitin with a biological enzyme method is greatly limited, and the chitin chain is difficult to be applied to large-scale production. The reported methods such as high-pressure homogenization, ultrasonic method, acid method, grinding method, steam explosion method and the like have difficult practical application prospect due to the problems of poor treatment conditions, high equipment requirements, high cost and the like. Therefore, it is important to develop a simple and efficient chitin pretreatment method to reduce the crystallinity of chitin, so that the chitin can be more efficiently converted into N-acetylglucosamine by degrading chitin with chitinase.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a method for improving the degradation rate of chitin by pretreating the chitin by an alkali freeze-thawing system, wherein the method is used for greatly improving the efficiency of preparing N-acetylglucosamine by degrading the chitin by comparing the chitin with untreated chitin and optimizing the conditions for improving the enzymatic degradation of the chitin by pretreating the chitin by the alkali freeze-thawing system, and can be used for large-scale production.
In order to solve the problems of the prior art, the invention adopts the technical scheme that:
a method for improving the degradation rate of chitin by pretreating the chitin with an alkali freeze-thaw system comprises the following steps:
Inoculating a strain producing chitinase into a seed liquid culture medium, culturing for 12h at 37 ℃ and 200rpm, inoculating the strain with the volume fraction of 5% into a shake flask filled with an enzyme production fermentation culture medium, fermenting for 72 h at 26 ℃, 200rpm and the initial pH of 7.5, collecting fermentation liquid, centrifuging at 4 ℃ and 8000rpm, separating thalli, and collecting supernatant to obtain chitinase crude enzyme liquid;
Weighing chitin powder with the mass not exceeding 4% of the reaction system, adding alkali liquor with the mass fraction of 4% -20% to mix uniformly, freezing and thawing the mixed solution at-20 ℃ -80 ℃ for 1-3 times, accumulating the freezing time for 6-24 h, taking out the mixed solution after each freezing and thawing, stirring the mixed solution to an ice-water mixed state, repeating the freezing and thawing operation, boiling the mixed solution for 5-30 min after the last freezing and thawing to fully separate out the chitin, centrifuging and recovering supernatant alkali liquor, adding PBS buffer solution into the separated chitin, stirring and resuspending, repeatedly centrifuging to remove supernatant, and repeatedly resuspending until the resuspended chitin suspension is neutral; during the melting process, the mixture is stirred sufficiently by attention, so that the chitin colloid is prevented from agglomerating, the enzyme combination is not facilitated, and the degradation effect is influenced;
And (3) sucking the equal amount of chitin turbid liquid and chitinase liquid into a water bath, reacting for 0.5-3 h at 37 ℃, determining the content of reducing sugar, and calculating the degradation rate of the chitin.
Preferably, the chitinase-producing strain in step 1 isChitinolyticbacter meiyuanensis SYBC-H1 (stored in China general microbiological culture Collection center in 2011, 7 months and 14 days, with the collection number of CGMCC NO: 3438 and the collection address of microorganism research institute of China academy of sciences No. 3, Xilu No. 1 Hospital, North Kyoto south, Chaozhou, Ltd.) or SYBC-H1Chitinolyticbactersp, GC 72 (4.1.2014, preserved in China center for type culture Collection with the preservation number of CCTCC M2014113 and the preservation address of Wuhan university No. 299 in Wuchang district, Wuhan City, Hubei).
In a further improvement, when the chitinase-producing strain in step 1 isChitinolyticbacter meiyuanensis When SYBC-H1 is used, the enzymolysis temperature is 20-37 ℃, and the enzymolysis time is 0.5-4H.
Preferably, the grain size of the chitin powder in step 2 is 20-100 mesh.
Preferably, the alkali liquor in step 2 is one or a mixture of NaOH solution and KOH solution.
Preferably, the mixed solution in the step 2 is frozen and thawed at-25 to-80 ℃, the freezing time is accumulated for 6 to 12 hours, and the freezing and thawing is carried out for 1 time; or freeze thawing for 2-3 times in a period of 12-24 h.
Preferably, the mass fraction of the alkali liquor in the step 2 is 8-16%.
The reaction principle is as follows: the alkali solution can form large solvent molecule groups at low temperature, so that hydrogen bonds in and among chitin molecules are weakened, the crystallinity of the chitin is reduced to be dissolved, and the chitin is more beneficial to enzyme degradation after re-precipitation.
Has the advantages that:
compared with the prior art, the method for improving the degradation rate of the chitin by pretreating the chitin with the alkali freeze-thaw system reduces the crystallinity of the chitin and improves the degradation of enzyme by modifying a substrate through the alkali freeze-thaw auxiliary enzyme method, and the degradation efficiency is 11.01 times that of untreated chitin and 2.6 times that of the traditional chemical acid method for degrading the chitin by the alkali freeze-thaw auxiliary enzyme method. Compared with the traditional acid-base method, the degradation has less pollution to the environment, the alkali solution required by freeze thawing can be recycled, the operation is simple, and the equipment requirement is low.
Drawings
FIG. 1 is a graph showing the macroscopic changes of chitin after alkali treatment, wherein (1) is untreated powdered chitin (1% wt), (2) is chitin after a 10% KOH freeze-thaw system treatment (1% wt), and (3) is chitin after a 10% NaOH freeze-thaw system treatment (1% wt).
Detailed Description
The invention is further described with reference to specific examples.
Example 1
Choose to useChitinolyticbacter meiyuanensisThe SYBC-H1 strain is preserved in the China general microbiological culture collection center in 2011, 7 months and 14 days, and the preservation number is CGMCC NO: 3438 the accession number is: the institute of microbiology, national academy of sciences No. 3, Xilu No. 1, Beijing, Chaoyang, Beijing.
This experiment is selected and usedChitinolyticbacter meiyuanensisThe SYBC-H1 strain was given by Hao quay, etc. of the university in south of the Yangtze river, and was inoculated into a medium (2 g/L glucose, 2 g/L peptone, 0.7g/L KH) by storing it at-70 ℃ as seeds in this laboratory2PO4，0.3g/L KH2PO4·3H2O，0.4 g/LMgSO4·7H2O, pH7.0), culturing at 37 deg.C and 200rpm for 12h, inoculating with 5% volume fraction of inoculum sizeCulture medium (3 g/L of powdered chitin, 3g/L of inulin, 3g/L of urea, 0.7g/L of KH)2PO4，0.3 g/L K2HPO4·3H2O，0.5 g/LMgSO4·7H2O) at a temperature of 26 ℃ and a rotation speed of 200rpm for 72 h at an initial pH of 7.5 and collecting the fermentation broth.
Centrifuging the obtained fermentation liquor in a centrifuge at 4 ℃ at 8000rpm, separating thallus, and collecting supernatant to obtain chitin crude enzyme liquid;
Weighing 0.1g of chitin powder, adding 10mL of NaOH solution with the mass fraction of 8%, freezing in a refrigerator at-25 ℃ for 12h, taking out, melting at room temperature to an ice-water mixture state, performing injection stirring fully in the melting process to prevent chitin colloid agglomeration and coagulation, not facilitating enzyme combination and influencing degradation effect, boiling the melted solution for 5 min to fully separate out chitin, centrifuging and recovering supernatant alkali liquor, adding PBS buffer solution, stirring and resuspending, and repeatedly centrifuging to remove supernatant for multiple times until the suspended chitin turbid liquid is neutral.
Sucking 200 μ L chitin suspension and chitinase solution of equal amount, reacting in water bath at 37 deg.C for 1 hr, decocting in boiling water for 5 min, and inactivating. Then, DNS (400 mu L) with the volume of 1:1 is added into the system respectively, boiling water bath is carried out for 5 min, and after the system is cooled to room temperature, the light absorption value is measured at 540nm to obtain 1.24 g/L of reducing sugar.
Comparative example: 0.1g of chitin powder was weighed, 10mL of pure water was added, and the mixture was mixed to obtain a suspension. Sucking 200 μ L chitin suspension and chitinase solution of equal amount, reacting in water bath at 37 deg.C for 1 hr, decocting in boiling water for 5 min, and inactivating. Then, DNS (400 mu L) with the volume of 1:1 is added into the system respectively, boiling water bath is carried out for 5 min, and after the system is cooled to room temperature, the light absorption value is measured at 540nm to obtain 0.16g/L of reducing sugar.
Example 2
The difference from example 1 is: weighing 0.1g of chitin powder, adding 10mL of KOH solution with the mass fraction of 10%, putting the mixture into a refrigerator, freezing the mixture for 6 hours at the temperature of minus 25 ℃, and freezing and thawing the mixture twice.
After treatment, sucking 200 μ L chitin suspension and chitinase solution of equal amount to react in a water bath at 37 deg.C for 1h, boiling in boiling water for 5 min, and inactivating. Then, DNS (400 mu L) with the volume of 1:1 is added into the system respectively, boiling water bath is carried out for 5 min, and after the system is cooled to room temperature, the light absorption value is measured at 540nm to obtain 1.59 g/L of reducing sugar.
Comparative example: 0.1g of chitin powder was weighed, 10mL of pure water was added, and the mixture was mixed to obtain a suspension. Sucking 200 μ L chitin suspension and chitinase solution of equal amount, reacting in water bath at 37 deg.C for 1 hr, decocting in boiling water for 5 min, and inactivating. Then, DNS (400 mu L) with the volume of 1:1 is added into the system respectively, boiling water bath is carried out for 5 min, and after the system is cooled to room temperature, the light absorption value is measured at 540nm to obtain 0.16g/L of reducing sugar.
Example 3
The difference from example 1 is: 0.1g chitin powder is weighed, 10mL NaOH solution with the mass fraction of 10% is added, and the mixture is put into a refrigerator to be frozen for 12 hours at the temperature of minus 80 ℃.
After treatment, sucking 200 μ L chitin suspension and chitinase solution of equal amount to react in a water bath at 37 deg.C for 1h, boiling in boiling water for 5 min, and inactivating. Then, DNS (400 mu L) with the volume of 1:1 is added into the system respectively, boiling water bath is carried out for 5 min, and after the system is cooled to room temperature, the light absorption value is measured at 540nm to obtain 1.54 g/L of reducing sugar.
Comparative example: 0.1g of chitin powder was weighed, 10mL of pure water was added, and the mixture was mixed to obtain a suspension. Sucking 200 μ L chitin suspension and chitinase solution of equal amount, reacting in water bath at 37 deg.C for 1 hr, decocting in boiling water for 5 min, and inactivating. Then, DNS (400 mu L) with the volume of 1:1 is added into the system respectively, boiling water bath is carried out for 5 min, and after the system is cooled to room temperature, the light absorption value is measured at 540nm to obtain 0.16g/L of reducing sugar.
Example 4
The difference from example 1 is: weighing 0.3 g of chitin powder, adding 10mL of KOH solution with the mass fraction of 10%, and freezing and thawing twice in a refrigerator at the temperature of minus 25 ℃ for 6 h.
After treatment, sucking 200 μ L chitin suspension and chitinase solution of equal amount to react in a water bath at 37 deg.C for 1h, boiling in boiling water for 5 min, and inactivating. Then, DNS (400 mu L) with the volume of 1:1 is added into the system respectively, boiling water bath is carried out for 5 min, and after the system is cooled to room temperature, the light absorption value is measured at 540nm to obtain 1.62 g/L of reducing sugar.
Comparative example: 0.1g of chitin powder was weighed, 10mL of pure water was added, and the mixture was mixed to obtain a suspension. Sucking 200 μ L chitin suspension and chitinase solution of equal amount, reacting in water bath at 37 deg.C for 1 hr, decocting in boiling water for 5 min, and inactivating. Then, DNS (400 mu L) with the volume of 1:1 is added into the system respectively, boiling water bath is carried out for 5 min, and after the system is cooled to room temperature, the light absorption value is measured at 540nm to obtain 0.16g/L of reducing sugar.
It can be known from the above examples and comparative examples that large solvent molecular groups are formed at low temperature, so that hydrogen bonds in and between molecules of chitin are weakened, thereby reducing the crystallinity of chitin to dissolve the chitin, and greatly improving the efficiency of degrading chitin to prepare N-acetylglucosamine.
Claims (7)
1. A method for improving the degradation rate of chitin by pretreating the chitin by an alkali freeze-thaw system is characterized by comprising the following steps:
step 1, producing chitinase by microbial fermentation and obtaining concentrated enzyme solution
Inoculating a strain producing chitinase into a seed liquid culture medium, culturing for 12h at 37 ℃ and 200rpm, inoculating the strain with the volume fraction of 5% into a shake flask filled with an enzyme production fermentation culture medium, fermenting for 72 h at 26 ℃, 200rpm and the initial pH of 7.5, collecting fermentation liquid, centrifuging at 4 ℃ and 8000rpm, separating thalli, and collecting supernatant to obtain chitinase crude enzyme liquid;
step 2, pretreating chitin by using an alkali freeze-thawing system
Weighing chitin powder with the mass not exceeding 4% of the reaction system, adding alkali liquor with the mass fraction of 4% -20% to mix uniformly, freezing and thawing the mixed solution at-20 ℃ -80 ℃ for 1-3 times, accumulating the freezing time for 6-24 h, taking out the mixed solution after each freezing and thawing, stirring the mixed solution to an ice-water mixed state, repeating the freezing and thawing operation, boiling the mixed solution for 5-30 min after the last freezing and thawing to fully separate out chitin, centrifuging and recovering supernatant alkali liquor, adding PBS buffer solution into the separated chitin, stirring and resuspending, repeatedly centrifuging to remove supernatant, and repeatedly resuspending until the suspended chitin suspension is neutral;
step 3, degrading chitin by using alkali freeze-thaw system and directly degrading chitin by using chitinase under the assistance of enzyme method
And (3) sucking the equal amount of chitin turbid liquid and chitinase liquid into a water bath, reacting for 0.5-3 h at 37 ℃, determining the content of reducing sugar, and calculating the degradation rate of the chitin.
2. The method of claim 1, wherein the chitinase-producing strain of step 1 is chitinaseChitinolyticbacter meiyuanensis SYBC-H1 orChitinolyticbacter sp. GC 72。
3. The method of claim 2, wherein the chitinase-producing strain of step 1 is chitinaseChitinolyticbacter meiyuanensis When SYBC-H1 is used, the enzymolysis temperature is 20-37 ℃, and the enzymolysis time is 0.5-4H.
4. The method of claim 1, wherein the chitin powder of step 2 has a particle size of 20-100 mesh.
5. The method for pretreating chitin to improve the degradation rate of chitin according to the alkali freeze-thaw system of claim 1, wherein the alkali solution in step 2 is one or a mixture of NaOH solution and KOH solution.
6. The method for improving the degradation rate of the chitin by pretreating the chitin by using the alkali freeze-thawing system as claimed in claim 1, wherein the mixed solution is subjected to freeze thawing at-25 ℃ to-80 ℃ in the step 2, the freezing time is accumulated for 6 h to 12h, and the freezing and thawing is performed for 1 time; or freeze thawing for 2-3 times in a period of 12-24 h.
7. The method for improving the degradation rate of chitin by pretreating chitin according to the alkali freeze-thawing system of claim 1, wherein the mass fraction of alkali liquor in step 2 is 8% -16%.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113750076A (en) * | 2021-09-30 | 2021-12-07 | 大连理工大学 | Chitin-gelatin-based composite porous microspheres as drug carrier and preparation method and application thereof |
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| CN113750076A (en) * | 2021-09-30 | 2021-12-07 | 大连理工大学 | Chitin-gelatin-based composite porous microspheres as drug carrier and preparation method and application thereof |
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Application publication date: 20200107 |