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Preparation method of recombinant manganese peroxidase and application of recombinant manganese peroxidase in degradation of Chinese herbal medicine lignin

A technology of manganese peroxidase and encoding manganese peroxide, which is applied in the preparation of recombinant manganese peroxidase and the application field of Chinese herbal medicine lignin degradation, can solve the problems of lack of reports, long fermentation time, increased production costs, etc.

Active Publication Date: 2020-10-23
浙江康星生物科技有限公司
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  • Abstract
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the slow rate of fungal enzyme production, the fermentation time in the process of degrading lignin is long, which increases the production cost and limits the industrial application and promotion.
At present, there is no commercial manganese peroxidase enzyme preparation on the market, more attention is paid to cellulosic pretreatment in the process of extracting or fermenting Chinese herbal medicines, and there are few reports on the process of using lignin as a breakthrough, especially in the process of fermentation. There is a lack of reports on the synergy of various lignin-degrading enzymes, cellulase, and hemicellulase and the combination of ions and small molecules.

Method used

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  • Preparation method of recombinant manganese peroxidase and application of recombinant manganese peroxidase in degradation of Chinese herbal medicine lignin
  • Preparation method of recombinant manganese peroxidase and application of recombinant manganese peroxidase in degradation of Chinese herbal medicine lignin
  • Preparation method of recombinant manganese peroxidase and application of recombinant manganese peroxidase in degradation of Chinese herbal medicine lignin

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] The manganese peroxidase gene sequence of Irpex lacteus in NCBI (GenBank: MH120207.1), the amino acid sequence is shown in SEQ ID NO.2; according to the codon bias in Schizosaccharomyces pombe, the gene sequence needs to be optimized , the codon optimization method is: through the Graphical Codon Usage Analyzer (http: / / gcua.schoedl.de / ) online system, analyze the usage frequency of codons in the manganese peroxidase (MnP) gene, so as to select suitable Codons for the transcription-translation system of Saccharomyces cerevisiae. figure 1 It is the alignment diagram of the original sequence of the manganese peroxidase gene and the codon-optimized sequence, and the nucleotide sequence of the codon-optimized recombinant MnP gene is shown in SEQ ID NO.1.

[0048] Using pREP, a commonly used carrier for yeast, as a template, design primers to amplify the target gene sequence. The sequence of the forward primer is shown in SEQ ID NO.3, and the sequence of the reverse primer is...

Embodiment 2

[0050] Expression verification of recombinant manganese peroxidase;

[0051] Dip the S.pombe-pREP-mnp and S.pombe-pREP cultures stored at -80°C and the control group S.pombe-pREP, streak on the plate, and grow statically in a constant temperature incubator at 30°C for 3 days; The colony was transferred into a 50mL screw-top conical centrifuge tube, and cultured with 5mL of YES culture solution in the tube, 200rpm, and 30°C constant temperature shaking. After the yeast culture reaches the plateau stage, collect 50 mL of yeast cells, centrifuge at 8 000 rpm, and transfer 12 mL of the supernatant culture solution to the protein concentrator for four times to concentrate the extracellular secreted protein; after concentrating to the minimum amount, add 12 mL of 20 mM Tris-HCl (pH 7.0) was used to wash extracellular secreted proteins to remove sugars, ions and other impurities in the supernatant culture solution; after SDS-PAGE electrophoresis analysis, there were specific bands in...

Embodiment 3

[0053] Weighing Chinese herbal medicine Eucommia ulmoides and Radix Astragali, this example discusses the degradation effect of recombinant MnP enzyme on lignin under different material-to-liquid ratios, enzymatic hydrolysis time, and enzyme dosage; The liquid part obtained by solid-liquid separation is the crude enzyme liquid; the crude enzyme liquid is mixed with Eucommia ulmoides and Astragalus membranaceus in a certain proportion, placed in a constant temperature and humidity incubator at 40°C for enzymolysis reaction for 24 hours, dried at 105°C to constant weight, and used The Van Soest method was used to measure the change of lignin content by FIWE3 / 6 fiber analyzer.

[0054] (1) The effect of solid-liquid ratio on lignin degradation;

[0055] According to the solid-liquid ratio of 10:1, 5:1, 2:1, 1:1, 0.5:1, weigh different quality Eucommia and Radix Astragali and mix them with crude enzyme solution, and place them in a constant temperature and humidity incubator at 40...

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Abstract

The invention belongs to the field of biological feed, and relates to a preparation method of recombinant manganese peroxidase and application of the recombinant manganese peroxidase in degradation ofChinese herbal medicine lignin. The method comprises the following steps: firstly, carrying out preference optimization on a gene sequence of manganese peroxidase coming from Irpex lacteus, carryingout PCR amplification by taking the optimized gene as a template, connecting the optimized gene with pREP, then converting the connected gene into escherichia coli competent cells to obtain recombinant shuttle plasmids, and introducing the recombinant shuttle plasmids into defective schizosaccharomyces pombe competent cells to obtain recombinant manganese peroxidase engineering bacteria; and performing culture to obtain a crude enzyme liquid, mixing the crude enzyme liquid with Chinese herbal medicines, and then adding calcium chloride, manganese sulfate, laccase, glucose oxidase and a small molecular substance activator for a multi-enzyme synergistic enzymolysis reaction. According to the method provided by the invention, a large amount of manganese peroxidase can be rapidly obtained, industrial production of the manganese peroxidase is realized, a good and stable peroxidase product is prepared, and effective degradation of Chinese herbal medicine lignin is realized.

Description

technical field [0001] The invention belongs to the field of biological feed, and in particular relates to a preparation method of recombinant manganese peroxidase and its application in the degradation of Chinese herbal medicine lignin. Background technique [0002] Food safety and big health industry are the future development direction, and aquaculture is an important prerequisite for it. In recent years, chemically synthesized additives, antibiotics and hormones have made the quality, taste, nutrition and safety of livestock and poultry products questionable, and high-quality feed has become a rigid demand. The Ministry of Rural Agriculture of the People's Republic of China issued Announcement No. 194. From January 1, 2020, all types of growth-promoting drug feed additives except traditional Chinese medicines will be withdrawn. From July 1, 2020, feed manufacturers will stop producing products containing Commercial feed for growth-promoting drug feed additives (except t...

Claims

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Application Information

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IPC IPC(8): C12N9/08C12N15/81A23K10/14A23K10/30C12R1/645
CPCC12N9/0065C12Y111/01013C12N15/815A23K10/14A23K10/30C12N2800/206Y02P60/87
Inventor 陈华友崔周磊王洪成卢晨曦姚丹陈华撑
Owner 浙江康星生物科技有限公司
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