Method for improving chitin degradation rate by pretreating chitin with alkali freeze-thaw system
阅读说明：本技术 一种碱冻融体系预处理几丁质提高几丁质降解率的方法 (Method for improving chitin degradation rate by pretreating chitin with alkali freeze-thaw system ) 是由 陈杰 张阿磊 吴超强 周宁 魏国光 王莹莹 陈可泉 欧阳平凯 于 2019-10-14 设计创作，主要内容包括：本发明公开了一种碱冻融体系预处理几丁质提高几丁质降解率的方法。该方法包括如下步骤：(1)利用微生物发酵获得几丁质酶；(2)称取适量几丁质,加入碱溶液中并搅拌分散,在适宜冻融模式处理下,取出室温融化,煮沸析出几丁质后离心去上清,利用PBS(pH 7.0)缓冲液搅拌重悬,清洗pH至中性；(3)吸取预处理几丁质和几丁质酶于水浴锅中反应,反应结束后,测定还原糖含量。与现有技术相比,本发明方法通过改性处理几丁质,提高几丁质与其酶的附着力,转化效率是未处理的11.01倍,且用时间较短,操作简单,污染较小,具有广阔的应用前景。(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.)
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%.
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
In a further improvement, when the chitinase-producing strain in
Preferably, the grain size of the chitin powder in
Preferably, the alkali liquor in
Preferably, the mixed solution in the
Preferably, the mass fraction of the alkali liquor in the
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.
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