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Recombinant bacillus subtilis for producing feruloyl esterase and application of recombinant bacillus subtilis

A technology of Bacillus subtilis and ferulic acid esterase, which is applied in the biological field and can solve the problems of restricting the industrialization process of ferulic acid and low yield of ferulic acid esterase

Active Publication Date: 2021-01-12
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the low yield of ferulic acid esterase produced by existing bacterial strains that can produce ferulic acid esterase, for example, people such as Li Cuicui inoculated Cladosporium cladoides into the fermentation medium and fermented for 3 days, only the fermentation broth can be made The enzyme activity of ferulic acid esterase reached 175.4U / L (see references for details: Li Cuicui, Mao Jian, Liu Shuangping, et al. Screening of Cladosporium cladoides producing ferulic acid esterase, fermentation characteristics and application in rice wine Research [J]. Journal of Food and Biotechnology, 2018,37(08):793-801.); Hu Bohan et al. Inoculated Aspergillus fumigatus into the fermentation medium and fermented it for 4 days, only ferulic acid esterase in the fermentation broth The enzyme activity of ferulic esterase reached 32.7U / L (see references for details: Hu Bohan, Wu Hui, Lai Furao, et al. Screening of strains producing ferulic acid esterase and research on its phenolic acid release[J]. Modern Food Science and Technology, 2015( 07):99-105.), which greatly limits the industrialization process of producing ferulic acid using bio-enzyme

Method used

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  • Recombinant bacillus subtilis for producing feruloyl esterase and application of recombinant bacillus subtilis
  • Recombinant bacillus subtilis for producing feruloyl esterase and application of recombinant bacillus subtilis
  • Recombinant bacillus subtilis for producing feruloyl esterase and application of recombinant bacillus subtilis

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Embodiment 1: Construction of recombinant Bacillus subtilis

[0038] Specific steps are as follows:

[0039] With the genome of Bacillus pumilus (Bacillus pumilus) SK52.001 obtained in Example 1 as a template, with fae-F / fae-R as primers, the gene (SEQ ID NO. 1); The gene encoding ferulic acid esterase and the pMA5 plasmid are connected with the homologous recombinase Exnase II to obtain a connection product; the connection product is transformed into Escherichia coli DH5α competent cells; the transformed Escherichia coli DH5α competent cells Cell-coated LB solid medium (containing 100 μg·mL -1 Ampicillin), cultured upside down at 37°C for 24 hours; pick positive transformants, extract plasmids, and sequence them for verification. If the verification is correct, the recombinant plasmid PMA5-fae is obtained; transform the obtained recombinant plasmid PMA5-fae into Bacillus subtilis WB800 to obtain the transformation product ; Spread the transformation product on LB sol...

Embodiment 2

[0043] Embodiment 2: the production of ferulic acid esterase

[0044] Specific steps are as follows:

[0045] The transformants of the recombinant Bacillus subtilis WB800 / PMA5-fae obtained in Example 1 were placed in LB solid medium (containing 100 μg·mL -1 Kanamycin), and cultured upside down in a 30°C incubator for 12 hours to obtain a single colony; pick a single colony and inoculate it into LB liquid medium (containing 100 μg·mL -1 Kanamycin) was cultivated at 37°C and 200r / min for 12h to obtain a seed liquid; the seed liquid was transferred to the SuperRich medium at an inoculum size of 3% (v / v), and cultivated at 37°C and 200r / min 60h, to obtain the fermentation broth.

[0046] During the fermentation process, the fermentation broth was sampled at regular intervals, with a sampling volume of 1 mL; the fermentation broth was centrifuged at 4°C and 10,000 rpm for 15 minutes to obtain the fermentation supernatant and sediment; 1 mL of lysate was added to the sediment to r...

Embodiment 3

[0059] Embodiment 3: the production of ferulic acid esterase

[0060] Specific steps are as follows:

[0061] Taking Bacillus subtilis WB800 as a contrast, the transformants of the recombinant Bacillus subtilis WB800 / PMA5-fae obtained in Example 1 were grown in LB solid medium (containing 100 μg·mL -1 Kanamycin), and cultured upside down in a 30°C incubator for 12 hours to obtain a single colony; pick a single colony and inoculate it into LB liquid medium (containing 100 μg·mL -1 Kanamycin) was cultivated at 37°C and 200r / min for 12h to obtain a seed liquid; the seed liquid was transferred to the SuperRich medium at an inoculum size of 3% (v / v), and cultivated at 37°C and 200r / min 14h to obtain the fermented liquid.

[0062] After the fermentation, take 5 mL of fermentation broth, centrifuge the fermentation broth at 4°C and 8000 rpm for 15 minutes to obtain the fermentation supernatant and precipitate; add 5 mL of lysate to the precipitate to resuspend the bacteria to obtai...

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Abstract

The invention discloses recombinant bacillus subtilis for producing feruloyl esterase and application of the recombinant bacillus subtilis, and belongs to the technical field of biology. The inventionprovides recombinant bacillus subtilis. The recombinant bacillus subtilis can produce the feruloyl esterase with high yield. Specifically, the recombinant bacillus subtilis is inoculated into a fermentation medium, and fermentation is performed for 14 hours, so that the enzyme activity of the feruloyl esterase in a cell disruption supernatant can reach up to 82.53 U / mL; and therefore, the recombinant bacillus subtilis has a huge application prospect in the production of the feruloyl esterase and ferulic acid.

Description

technical field [0001] The invention relates to a ferulic esterase-producing recombinant bacillus subtilis and an application thereof, belonging to the field of biotechnology. Background technique [0002] Ferulic acid (FA) widely exists in plant cell walls, and is connected with polysaccharides, cellulose, and lignin through ester bonds or ether bonds to form a complex network skeleton structure in the cell wall to maintain the integrity of the cell wall. At the same time reduce the biodegradation rate. [0003] Ferulic acid is recognized as a safe antioxidant in the world, and it is included in food additives in Japan, the United States and other countries. In addition, ferulic acid also has the functions of ultraviolet absorption, antibacterial and anti-inflammatory, anti-cancer and hypolipidemic, etc. It has broad application prospects in medicine, cosmetics, papermaking and other fields. [0004] At present, the methods for producing ferulic acid mainly include plant ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/75C12N15/55C12N9/18C12P7/42A23K10/18D21C5/00C12R1/125
CPCC12N9/18C12Y301/01073C12N15/75C12P7/42A23K10/18D21C5/005
Inventor 张涛江波段晓莉
Owner JIANGNAN UNIV
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