Heat resistance cutinase and its coding gene and expression
A technology encoding gene and cutinase, applied in genetic engineering, plant genetic improvement, hydrolase and other directions, can solve the problems of no industrial production of cutinase, less research on cutinase, and low enzyme production by wild fungi.
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Embodiment 1
[0043] Example 1 This example illustrates the purification procedure for cutinase.
[0044] Taking Thermobifida fusca WSH03-11 as the starting strain, in the seed medium (soluble starch 20g / L, beef extract 10g / L, yeast extract 5g / L, K 2 HPO 4 2g / L, NaCl5g / L, pH8.0) 50 ℃, 200rpm after culturing for 20h, insert the fermentation medium (sodium acetate 7.5g / L, yeast extract 7.5g / L, peptone 5g / L, K 2 HPO 4 2g / L, NaCl 5g / L, cutin 1g / L, pH8.0), 50°C, 200rpm, after culturing for 50h, centrifuge the cutinase fermentation broth at 4°C, 10000rpm for 20min to remove bacteria. The supernatant was collected through an activated carbon column. Add 70% solid ammonium sulfate to the clear solution passing through the activated carbon column for salting out overnight, centrifuge at 10000rpm for 20min at 4°C, dissolve the precipitate with an appropriate amount of buffer A (20mM Tris-HCl, pH8), add 20% ammonium sulfate, 0.22 Samples were prepared after filtration with a μm membrane. After t...
Embodiment 2
[0045] Example 2 This example illustrates the identification process of the cutinase gene.
[0046] The obtained pure cutinase was sequenced by peptide fingerprinting. The sequencing results showed that it was very similar to the protein Tfu_0883 encoded by Thermobifida fusca in the NCBI database. The N-terminal sequencing was ANPYERGP, which was the same as the protein Tfu_0883. The protein Tfu_0883 was predicted to be a triglyceride enzyme through sequence prediction , without indication of its cutinase activity. The pure wild fungus cutinase is subjected to enzymatic hydrolysis of cutin (apple peel extracted with chloroform and methanol, treated with pectinase and cellulase). Add 300 mg of cutin to pH 8.0, 10 mL of 50 mM potassium phosphate buffer, add purified cutinase and shake at 30°C for 18 hours, add glacial acetic acid to terminate the reaction after the reaction, extract the product fatty acid with chloroform, and carry out methylation and silylation, use GC -MS mea...
Embodiment 3
[0047] Example 3 This example illustrates the isolation and cloning procedure of the gene encoding cutinase.
[0048] The Thermobifida fusca strain was cultured in LB liquid medium (peptone 10g / L, yeast extract 5g / L, NaCl 10g / L) for 2 days, and the bacterial cells were collected by centrifugation at 10000rpm, washed with sterile water, and the collected precipitate was suspended in 500 μL Tris-EDTA (three (Hydroxymethylaminomethane-ethylenediaminetetraacetic acid) buffer solution, add 15 μL lysozyme, incubate at 37°C for 30 minutes, then add 5 μL RNase, incubate at 37°C for 30 minutes, add 30 μL 10% SDS (sodium dodecyl sulfate) and 15 μL of proteinase K, incubated at 37°C for 60 min, added 100 μL of NaCl (5M) and 80 μL of CTAB (cetyltrimethylammonium bromide), incubated at 65°C for 20 min, and used 700 μL of phenol: chloroform: isoamyl alcohol ( 25:24:1) extraction, 10000rpm centrifugation, supernatant was extracted with 700μL chloroform: isoamyl alcohol (24:1), 10000rpm centr...
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