Proline Hydroxylase and Its Application
A technology for proline hydroxylase and proline hydroxylase activity, which is applied in the fields of genetic engineering and enzyme engineering, and can solve problems such as poor selectivity
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Embodiment 1
[0082] Example 1: Recombinant expression of proline hydroxylase
[0083] The DNA coding sequence SEQ ID NO: 1 annotated as a hypothetical protein from Kordia jejudonensis was codon-optimized (codon-improved according to the codon preference and degeneracy of Escherichia coli, designed by Suzhou Jinweizhi Biotechnology Co., Ltd. ) to obtain the optimized DNA sequence SEQ ID NO: 3, and the encoded proline hydroxylase polypeptide sequence is SEQ ID NO: 4. The coding sequence of SEQ ID NO: 3 was ligated into pET22b(+) expression vector (purchased from Novagen, product number 69744), then transformed into E. coli BL21 (DE3), and spread on LB containing 50 μg / ml ampicillin cultured at 37°C overnight. Pick the single clone on the above petri dish and activate it in a test tube, inoculate it in 500ml LB liquid medium containing 50μg / ml ampicillin, and shake it to OD at 37°C. 600 = 0.6, IPTG was added to a final concentration of 1 mM, and expression was induced at 25°C. After 16h in...
Embodiment 2
[0084] Example 2: Preparation of proline hydroxylase variants
[0085]Using the pET22b(+) expression vector containing the sequence of SEQ ID NO: 3 as a template, a complete linear fragment was obtained by whole plasmid PCR with primers with a mutation site, and the above PCR product was digested with DPnI to remove the parent template, and transformed into Escherichia coli BL21 (DE3), spread on LB dishes containing 50 μg / ml ampicillin, and cultured at 37°C overnight. A single clone containing the amino acid sequence of the proline hydroxylase variant was obtained, and the mutation site was determined by trial induction and gene sequencing. The mutant with a single mutation site is finally obtained, and then the mutation of the single mutation site is used as the mutation mother, and then the whole plasmid PCR is performed again with primers with mutations at other sites to determine the mutation site again.
[0086] After the mutant strain was activated, it was inoculated in...
Embodiment 3
[0087] Example 3: Activity screening of proline hydroxylase variants
[0088] Compared with SEQ ID NO: 2, the activity screening of proline hydroxylase variants with one amino acid residue difference was screened by using the following 10 mL reaction solution, and the 10 mL reaction solution contained: L-piperic acid 30g / L , 5 ~ 10wt (1wt refers to the transformation of 1g main raw material requires 1g proline hydroxylase variant recombinant wet cells.) recombinant crude enzyme, 37.3g / Lα-ketoglutaric acid, 6.1g / L L-ascorbic acid, 5mM ferrous ammonium sulfate, the reaction pH is 6.5, the reaction temperature is 10°C, and the reaction time is 40h. At the end of the reaction, take 200 μL of the reaction system, add 200 μL of acetonitrile, add 3000 μL of purified water after mixing, and centrifuge at 10000 rpm for 5 min to collect the supernatant for HPLC to detect the conversion rate. The activity screening results are shown in Table 1 (Table 1 is based on all conversion rates). ...
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