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Feruloyl esterase gene, genetically engineered strain, and preparation method and application of feruloyl esterase

A genetically engineered strain and ferulic acid esterase technology, applied in the field of bioengineering, can solve the problems of low yield of ferulic acid esterase, unusable target products, pollution, etc.

Active Publication Date: 2018-05-18
FUDAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For example, the special growth conditions of filamentous fungi are not conducive to large-scale fermentation; when using Escherichia coli for prokaryotic expression, it is easy to form inclusion bodies, endotoxin or biological pyrogen contamination in the target protein; when using Pichia pastoris for recombinant expression, methanol induction is required, The target product cannot be applied to food or feed addition; while the yield of ferulic acid esterase recombinantly expressed by GRAS Saccharomyces cerevisiae is low, only 2mg / L (Huang Xueyue et al., Acta Microbiology, 2017,44:68-78)

Method used

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  • Feruloyl esterase gene, genetically engineered strain, and preparation method and application of feruloyl esterase
  • Feruloyl esterase gene, genetically engineered strain, and preparation method and application of feruloyl esterase
  • Feruloyl esterase gene, genetically engineered strain, and preparation method and application of feruloyl esterase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1 Extraction of yak rumen microbial genome and cloning of ferulic acid esterase gene Est1E

[0036] The rumen contents of two Chinese Qinghai yaks were collected from a slaughterhouse in Xining City, Qinghai Province, China, filtered through three layers of gauze, and the filtrate was centrifuged to collect rumen microbial cells, which were frozen at -80°C until use. Take 100-200μL bacterial sample, wash 2-3 times with 1mL PBS, add 650μL DNA extraction buffer (100mM Tris-HCl pH8.0, 100mM Na 2 EDTA pH8.0, 100mM Na 3 PO 4 Buffer pH8.0; 1.5M NaCl, 1% CTAB, pH8.0), mix well, place at -80°C, then place in 65°C water bath to thaw, repeat freeze-thaw three times; add 3-4μL lysate after cooling Shake the enzyme (100mg / L) horizontally (37°C, 225rpm) in a shaker for about 30min; add 2-3μL of proteinase K (20mg / mL) and continue shaking for about 30min; add 50-70uL of 20% SDS, mix well, 65 Incubate at ℃ for 1-2 hours, mix by inverting the centrifuge tube every 10-20 minu...

Embodiment 2

[0038] Embodiment 2 Construction of ferulic acid esterase gene Est1E recombinant Kluyveromyces expression strain

[0039] According to the yak rumen microbial ferulic acid esterase Est1E sequence obtained by sequencing, the gene was inserted into the two restriction Sma I and Not I of Kluyveromyces marxianus expression vector pUKD-N112 by conventional molecular cloning methods. In the middle of the restriction site, the recombinant plasmid pUKD-N112-Est1E was obtained. The recombinant plasmid can utilize the promoter and signal peptide of Kluyveromyces marx inulinase to carry out the recombinant expression and secretion of Est1E.

[0040] Using the uracil auxotrophic Kluyveromyces marx strain Fim-1ura3Δ as the expression host, inoculate Fim-1ura3Δ in a glass test tube containing 3 mL of YEPD medium, and culture OD overnight on a shaker at 30°C 600 to 12-15. Take 1 mL of bacterial liquid, collect the bacterial cells by centrifugation at 8 000 rpm for 30 s, add 1 mL of sterile...

Embodiment 3

[0043] Example 3 Ferulic esterase ferulic acid esterase recombinantly expressed by Kluyveromyces marxe and its preparation

[0044] The clones with higher degradation efficiency screened out from the hydrolysis experiment of ethyl ferulic acid were used as fermentation bacteria and carried out small-scale fermentation in a 5L fermenter. According to the fermentation of 2g of glucose to produce 1g of yeast cells, and the yeast cell C / N=5:1, the Kluyveromyces marx recombinant bacteria fermentation medium was designed. The source of N in the medium was yeast powder, and the source of C was glucose, and fed-batch culture High-density fermentation of ferulic acid esterase K. The initial volume of the fermentation medium is 1.6L, containing 2% yeast powder and 5% glucose. When the sugar concentration is less than 1%, feed feeding is started, the concentration of N source and carbon source is 5:1, and the concentration of glucose is controlled at about 1%, and the fermentation cycle...

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Abstract

The invention provides a feruloyl esterase gene. The feruloyl esterase gene is originated from microbes in the rumen of a Chinese yak and has a nucleotide sequence as shown in SEQ ID No. 1, wherein the nucleotide sequence codes an amino acid sequence as shown in SEQ ID No. 2. The invention also provides a recombinant Kluyveromyces marxianus expression vector containing the gene, a genetically engineered strain, a preparation method for feruloyl esterase and application of prepared feruloyl esterase. Feruloyl esterase is subjected recombinant expression in a Kluyveromyces marxianus expression system; and after high-density fermentation for 48 h, intracellular and extracellular feruloyl esterase vitalities are 686.35 U / mL and 346.34 U / mL, respectively. Feruloyl esterase having undergone recombinant expression by Kluyveromyces marxianus can release ferulic acid in corn bran. Feruloyl esterase prepared in the invention can be applied to food processing, feed addition, biomedicine, biotransformation of cellulosic substances, bioenergy and other fields.

Description

technical field [0001] The invention belongs to the field of bioengineering, and specifically relates to a ferulic acid esterase gene, a Kluyveromyces marx recombinant expression vector containing the gene, a genetically engineered strain, and a preparation method and application of the ferulic acid esterase. Background technique [0002] The chemical name of ferulic acid is 3-methoxy-4-hydroxycinnamic acid, which widely exists between lignin and lignin, and between lignin and hemicellulose in plant cell walls. Ferulic acid is a natural antioxidant that can remove hydrogen peroxide, superoxide radicals, hydroxyl radicals, and peroxynitroso in the body, regulate physiological functions, inhibit enzymes that produce free radicals, and increase the activity of enzymes that scavenge free radicals , has antibacterial and anti-inflammatory and anti-mutation and anti-cancer effects. Ferulic acid widely exists in food raw materials. Ferulic acid in wheat bran is one of the derivati...

Claims

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Application Information

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IPC IPC(8): C12N15/55C12N9/18C12N15/81C12N1/19C12R1/645
CPCC12N9/18C12N15/815C12Y301/01073
Inventor 吕红刘洋周峻岗余垚
Owner FUDAN UNIV
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