CN115627279A - Application of chlorogenic acid as electron donor for degrading cellulose by lytic polysaccharide monooxygenase - Google Patents
Application of chlorogenic acid as electron donor for degrading cellulose by lytic polysaccharide monooxygenase Download PDFInfo
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- CN115627279A CN115627279A CN202211594542.7A CN202211594542A CN115627279A CN 115627279 A CN115627279 A CN 115627279A CN 202211594542 A CN202211594542 A CN 202211594542A CN 115627279 A CN115627279 A CN 115627279A
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Abstract
The invention relates to the technical field of agricultural biology, in particular to application of chlorogenic acid as an electron donor for degrading cellulose by using lytic polysaccharide monooxygenase. Naturally-occurring compounds in the lignocellulose biomass can be used as electron donors of the lytic polysaccharide monooxygenase, so that the use cost of the lytic polysaccharide monooxygenase is obviously reduced, and the large-scale popularization and application of the lytic polysaccharide monooxygenase are promoted.
Description
Technical Field
The invention relates to the technical field of agricultural biology, in particular to application of chlorogenic acid as an electron donor for degrading cellulose by using lytic polysaccharide monooxygenase.
Background
The potential of further development and application of cellulose is limited by the problems of low enzymolysis saccharification efficiency, high cost and the like of the cellulose, and the transformation and development of a novel cellulose degrading enzyme preparation are urgently needed. The traditional enzymolysis saccharification of cellulose mainly depends on the synergistic degradation among endoglucanase, exo-cellulase and beta-glucosidase; in recent years, the lytic polysaccharide monooxygenase serving as a novel polysaccharide degrading enzyme breaks the glycosidic bond of insoluble crystalline polysaccharide in an oxidation mode, and can remarkably improve the enzymolysis and saccharification efficiency of cellulose under the synergistic action of endoglucanase, exo-cellulase and beta-glucosidase, thereby showing wide industrial application prospects.
Research has shown that catalytic reaction of the lytic polysaccharide monooxygenase requires the participation of an electron donor, which provides an exogenous electron to reduce the divalent copper ion prosthetic group of the lytic polysaccharide monooxygenase to a monovalent copper ion, in order to initiate the catalytic reaction. At present, commonly used electron donors comprise ascorbic acid, cysteine, levodopa and the like, but the addition of an exogenous electron donor can increase the application cost and is not beneficial to large-scale application. Therefore, the feasibility of finding out whether the naturally-occurring compound in the lignocellulose biomass can be used as an electron donor of the lytic polysaccharide monooxygenase is proved, and the method has important economic value for guiding the industrial application of the lytic polysaccharide monooxygenase.
Disclosure of Invention
The invention aims to provide application of chlorogenic acid as an electron donor for degrading cellulose by using lytic polysaccharide monooxygenase.
According to a specific embodiment of the present invention, cellulose is efficiently degraded in a buffer solution of 50 mM, pH 5.0 acetate-sodium acetate buffer.
According to the solution of the present application, wherein the lytic polysaccharide monooxygenase may or may not be any lytic polysaccharide monooxygenase disclosed in the prior art, e.g. the sequence number KAI0755988.1 disclosed in the NCBI Genbank or the lytic polysaccharide monooxygenase of Ramaria lactuca, white rot fungus.
Chlorogenic acid which is a naturally existing compound in the lignocellulose biomass can be used as an electron donor of the lytic polysaccharide monooxygenase, so that the use cost of the lytic polysaccharide monooxygenase is obviously reduced, and the large-scale popularization and application of the lytic polysaccharide monooxygenase are promoted.
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FIG. 1 shows the results of analysis of the amount of cellulose oxidative cleavage products produced in a lytic polysaccharide monooxygenase-ascorbic acid system;
FIG. 2 shows the results of analysis of the amount of cellulose oxidative cleavage products produced by a lytic polysaccharide monooxygenase-chlorogenic acid system;
FIG. 3 shows the result of ion chromatography analysis of cellulose oxidative cleavage products in lytic polysaccharide monooxygenase-chlorogenic acid system;
FIG. 4 shows the result of analysis of the amount of the oxidative cleavage products of cellulose in the lytic polysaccharide monooxygenase-arbutin/guaiacol/p-hydroxybenzoic acid/acetosyringone system.
Detailed Description
Example 1 preparation of recombinant lytic polysaccharide monooxygenase
Synthesizing a lytic polysaccharide monooxygenase gene fragment according to a sequence number KAI0755988.1 disclosed in an NCBI gene library, connecting the fragment to an expression vector pPIC9K to obtain a recombinant escherichia coli expression plasmid of the lytic polysaccharide monooxygenase gene, performing electric shock transformation to pichia pastoris GS115 competent cells by referring to a pichia pastoris expression experiment operation manual of Invitrogen bioscience, and then selecting transformants for SDS-PAGE electrophoretic analysis to obtain the recombinant pichia pastoris expressing the lytic polysaccharide monooxygenase.
Inoculating the recombinant pichia pastoris strain into 30 mL YPD culture solution, carrying out shaking culture at 30 ℃ and 180 rpm for 24 h, transferring the strain into 300 mL BMGY culture medium according to the proportion of 2%, and carrying out shaking culture at 30 ℃ and 180 rpm for 48 h; then, the cells were collected by centrifugation, suspended in 150 mL BMMY medium, induced-cultured at 30 ℃ and 180 rpm for 48 hours, and centrifuged to collect the supernatant. Further purifying by using Ni affinity chromatography column, collecting electrophoretically pure elution components, dialyzing into protein storage solution (20 mM pH 7 Tris-HCl), and incubating with copper sulfate at 3 times molar ratio to purify protein, thereby completing preparation of recombinant lytic polysaccharide monooxygenase.
EXAMPLE 2 degradation of cellulose by recombinant lytic polysaccharide monooxygenase-ascorbic acid System
The following reaction system is adopted: 50 mM pH 5.0 acetate-sodium acetate buffer, 1. Mu.M recombinant lytic polysaccharide monooxygenase, 1 mM ascorbic acid, 100. Mu.M hydrogen peroxide, 0.5% cellulose. The system without recombinant lytic polysaccharide monooxygenase and ascorbic acid was used as a control, and the reaction system was set to 3 replicates. The reaction was carried out at 30 ℃ and boiling was terminated after 1h, and the amount of cellulose oxidative cleavage products formed was determined by ion chromatography. The ion chromatography is a Daian ICS5000 type ion chromatograph, the chromatographic column is a CarboPac PA1 analytical column (2 x 250 mm), a mobile phase A (0.1M sodium hydroxide), a mobile phase B (1M sodium acetate-0.1M sodium hydroxide), and the elution procedure is as follows: 0-10% B for 10 min, 10-30% B for 15 min, 30-100% B for 5 min, 100-0% B for 5 min, and 100% A for 5 min.
The results are shown in FIG. 1, where the formation of oxidative cleavage products of cellulose is seen in the recombinant lytic polysaccharide monooxygenase-ascorbic acid system; in a system without adding recombinant lytic polysaccharide monooxygenase and ascorbic acid, the generation of cellulose oxidative cleavage products is not detected; these results indicate that ascorbic acid can participate in highly efficient degradation of cellulose as an electron donor for lytic polysaccharide monooxygenase.
Example 3 degradation of cellulose by recombinant lytic polysaccharide monooxygenase-chlorogenic acid System
The method comprises the following steps: 50 mM pH 5.0 acetic acid-sodium acetate buffer, 1 u M recombinant lytic polysaccharide monooxygenase, 1 mM chlorogenic acid, 100 u M hydrogen peroxide, 0.5% cellulose. The system without recombinant lytic polysaccharide monooxygenase and chlorogenic acid is used as a control, and the reaction system is repeated for 3 times. The reaction was carried out at 30 ℃ and boiling was terminated after 1h, and the amount of cellulose oxidative cleavage products formed was determined by ion chromatography. The ion chromatography is a Daian ICS5000 type ion chromatograph, the chromatographic column is a CarboPac PA1 analytical column (2 x 250 mm), a mobile phase A (0.1M sodium hydroxide), a mobile phase B (1M sodium acetate-0.1M sodium hydroxide), and the elution procedure is as follows: 0-10% B for 10 min, 10-30% B for 15 min, 30-100% B for 5 min, 100-0% B for 5 min, and 100% A for 5 min.
As shown in FIGS. 2 and 3, the generation of oxidized cellulose cleavage products was observed in the recombinant lytic polysaccharide monooxygenase-chlorogenic acid system; under the condition of not adding a system of recombinant lytic polysaccharide monooxygenase and chlorogenic acid, the generation of a cellulose oxidative cleavage product is not detected; therefore, chlorogenic acid can be used as an electron donor of the lytic polysaccharide monooxygenase to participate in high-efficiency degradation of cellulose.
Example 4 degradation of cellulose by the lytic polysaccharide monooxygenase-arbutin/guaiacol/p-hydroxybenzoic acid/acetosyringone System
The following reaction system is adopted: 50 mM pH 5.0 acetic acid-sodium acetate buffer, 1 μ M recombinant lytic polysaccharide monooxygenase, 1 mM arbutin or guaiacol or p-hydroxybenzoic acid or acetosyringone, 100 μ M hydrogen peroxide, 0.5% cellulose. The system without recombinant lytic polysaccharide monooxygenase and without arbutin or guaiacol or p-hydroxybenzoic acid or acetosyringone was used as a control, and the reaction system was set to 3 replicates. The reaction was carried out at 30 ℃ and boiling was terminated after 1h, and the amount of cellulose oxidative cleavage products formed was determined by ion chromatography. The ion chromatography is a Daian ICS5000 type ion chromatograph, the chromatographic column is a CarboPac PA1 analytical column (2 x 250 mm), a mobile phase A (0.1M sodium hydroxide), a mobile phase B (1M sodium acetate-0.1M sodium hydroxide), and the elution procedure is as follows: 0-10% B for 10 min, 10-30% B for 15 min, 30-100% B for 5 min, 100-0% B for 5 min, and 100% A for 5 min.
As shown in FIG. 4, no oxidative cleavage product of cellulose was generated in the recombinant lytic polysaccharide monooxygenase-arbutin/guaiacol/p-hydroxybenzoic acid/acetosyringone system, and thus it was found that arbutin or guaiacol or p-hydroxybenzoic acid or acetosyringone could not participate in the efficient degradation of cellulose as an electron donor of lytic polysaccharide monooxygenase.
The above examples are only for explaining the technical solutions of the present application, and do not limit the scope of protection of the present application.
Claims (4)
1. Application of chlorogenic acid as electron donor for degrading cellulose by lytic polysaccharide monooxygenase is provided.
2. Use according to claim 1, characterized in that the cellulose is degraded in a buffer solution of 50 mM, pH 5.0 acetate-sodium acetate buffer.
3. Use according to claim 1, wherein the lytic polysaccharide monooxygenase is from the fungus Irpex lacteus.
4. The use according to claim 1, wherein the lytic polysaccharide monooxygenase is disclosed in the NCBI gene bank under the sequence number KAI0755988.1.
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Citations (5)
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| CN109715794A (en) * | 2016-07-05 | 2019-05-03 | 诺维信公司 | Pectin lyase enzyme variants and the polynucleotides for encoding them |
| CN111420037A (en) * | 2020-03-26 | 2020-07-17 | 中国农业科学院饲料研究所 | Application of phage lyase L ysep3 in preparation of broad-spectrum antibacterial drugs |
| US20210076685A1 (en) * | 2014-12-30 | 2021-03-18 | Indigo Ag, Inc. | Seed Endophytes Across Cultivars and Species, Associated Compositions, and Methods of Use Thereof |
| CN112979836A (en) * | 2021-03-04 | 2021-06-18 | 湖北省农业科学院农产品加工与核农技术研究所 | Preparation method of activity-enhanced edible fungus polysaccharide and application of activity-enhanced edible fungus polysaccharide in weight reduction and intestinal beneficial flora increase |
| CN113504189A (en) * | 2021-06-28 | 2021-10-15 | 深圳大学 | Method for determining activity of LPMO (LPMO) cracked cellulase and detection kit |
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- 2022-12-13 CN CN202211594542.7A patent/CN115627279A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20210076685A1 (en) * | 2014-12-30 | 2021-03-18 | Indigo Ag, Inc. | Seed Endophytes Across Cultivars and Species, Associated Compositions, and Methods of Use Thereof |
| CN109715794A (en) * | 2016-07-05 | 2019-05-03 | 诺维信公司 | Pectin lyase enzyme variants and the polynucleotides for encoding them |
| CN111420037A (en) * | 2020-03-26 | 2020-07-17 | 中国农业科学院饲料研究所 | Application of phage lyase L ysep3 in preparation of broad-spectrum antibacterial drugs |
| CN112979836A (en) * | 2021-03-04 | 2021-06-18 | 湖北省农业科学院农产品加工与核农技术研究所 | Preparation method of activity-enhanced edible fungus polysaccharide and application of activity-enhanced edible fungus polysaccharide in weight reduction and intestinal beneficial flora increase |
| CN113504189A (en) * | 2021-06-28 | 2021-10-15 | 深圳大学 | Method for determining activity of LPMO (LPMO) cracked cellulase and detection kit |
Non-Patent Citations (1)
| Title |
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| HAOXIANG NI ET AL.: "In-situ lignin drives lytic polysaccharide monooxygenases to enhance enzymatic saccharification", vol. 161, pages 308 - 314 * |
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