A kind of ochratoxin hydrolase and its encoding gene, recombinant vector and application
An ochratoxin and hydrolase technology, which is applied in the field of enzyme engineering, can solve the problems of loss of nutrients, high cost, and difficulty in detoxification, and achieves the effect of good application prospects.
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Embodiment 1
[0040] Embodiment 1: cDNA synthesis and cloning of ochratoxin hydrolase gene
[0041] The strain was derived from the Stenotrophomonas bacteria obtained in the early stage of the laboratory. Through gene sequence determination, the nucleotide sequence of the open reading frame of the ochratoxin hydrolase gene was analyzed, and the upstream primer of the primer that amplified the complete coding reading frame was designed ( cpna117-F) (SEQ ID NO.2): 5'-CGC GGATCC ATGATCCGCAAGACCGTTCTGT-3'; downstream primer (cpna117-R) (SEQ ID NO.3): 5'-CCG CTCGAG TCAGCCGGCGCCGCCGT-3', respectively introduce restriction endonuclease sites on the upstream and downstream primers (depending on the carrier selected, BamHI and XhoI enzyme cutting sites have been added in the present invention, underlined, italicized sequence for protecting bases). The gene encoding ochratoxin hydrolase was obtained by in vitro amplification technology, and its nucleotide sequence is shown in SEQ ID NO.1. Under ...
Embodiment 2
[0042] Example 2: Heterologous Expression of Stenotrophomonas Ochratoxin Hydrolase Gene
[0043] The E. Coil BL21(DE3) / pGEX-4T-1 / cpna117 transformant obtained in Example 1 was cultured overnight on a shaker in 100 mL LB liquid medium containing 100 μg / mL ampicillin. Draw 1.0 mL of seed bacteria liquid and inoculate it into fresh 100 mL of LB liquid medium (containing 100 μg / mL ampicillin), and culture at 37°C with shaking at 180 r / min. When the bacteria solution OD 600 When it reaches 0.6, add 0.2mmol / mL IPTG to induce expression at 16°C for 4h. Refrigerate and centrifuge at 7000r / min for 10min, discard the supernatant. Suspend the cells with 10 mL of 1×phosphate buffered saline (PBS), and disrupt the cells by ultrasonic in an ice bath. Centrifuge and take the supernatant. SDS-PAGE electrophoresis results showed that the enzyme was contained in the supernatant after the bacterium was broken, and its apparent molecular weight (comprising GST tag 26kDa) was about 73kDa ( fi...
Embodiment 3
[0044] Example 3: Application of heterologous recombinant hydrolase in biological detoxification of ochratoxin A
[0045] 1. Experimental materials
[0046] The enzyme preparation is the crude enzyme solution of the broken supernatant after the expression of the pGEX-4T-1 / cpna117 transformant, and other reagents are analytically pure chemical reagents.
[0047] 2. Experimental method
[0048] Take 10 μL of the crushed supernatant after expression of the pGEX-4T-1 / cpna117 transformant and put it in a 2 mL centrifuge tube, add it to 490 μL of ochratoxin A standard buffer solution (use 1*PBS to prepare pH 7.3), the ochratoxin A The final concentration was about 15 μg / L, and reacted for 12 hours at 35°C. The empty plasmid pGEX-4T-1 was used as the control, and three parallels were set up for each group. Add 1.5 mL of acetonitrile to the reaction system to stop the reaction. Use high performance liquid chromatography to detect the residual amount of OTA, and experimental result ...
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