Method for using recombinant candida parapsilosis strain to efficiently prepare (S)-phenyl glycol
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A phenylethylene glycol, Candida technology, applied in microorganism-based methods, botanical equipment and methods, biochemical equipment and methods, etc., can solve the problem of unsatisfactory catalytic conversion efficiency of chiral compounds, lack of protein post-modification issues
Inactive Publication Date: 2015-07-15
JIANGNAN UNIV
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However, the oxidoreductase function after recombinant expression in Escherichia coli lacks post-prot
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Embodiment 1
[0086] Candida parapsilosis ( C. parapsilosis ) CCTCC NO: M203011 (disclosed in the patent number: CN 03102140.2) cell culture, the composition of the growth medium YPD: glucose 2%, yeast extract 1%, peptone 2%. Candida parapsilosis was inoculated in a 5 mL test tube and cultured at 28°C and 200 r / min for 16-18 h with shaking.
Embodiment 2
[0088] Candida parapsilosis ( C. parapsilosis ) Genome extraction: The cells cultured in Example 1 were centrifuged at 12,000 r / min for 5 min, washed twice with saline, and the cells were collected to extract the genome using the Genomic DNA Mini Preparation Kit (Takara Company).
Embodiment 3
[0090] Extraction of target gene from Candida parapsilosis scr II, will scr Restriction site in II Kpn I sequence (GGTACC) synonymous mutation to GGCACA.
[0097] contain Kpn I synonymous mutation site scr Acquisition of II genes:
[0098] PCR reaction system: ddH 2 O 24.5 μL, DNA polymerase 0.5 μL, Mut_ Kpn I_F2 primer 5 μL, Mut_ Kpn I_R2 primer 5 μL, 5×buffer 10 μL.
[0099] PCR reaction: thermal denaturation at 98°C for 10 s; 10 s at 98°C, 15 s at 52°C, and 1 min at 72°C. After 5-10 cycles, the PCR reaction solution was taken out, and 1 μL of primers SAT1_1 and URA3t_2 were added...
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Abstract
The invention provides a method for using a recombinant candida parapsilosis strain to efficiently prepare (S)-phenyl glycol and belongs to the technical field of biological catalytic asymmetric transformation. The invention provides the recombinant candida parapsilosis strain Candida parapsilosis pCP-scrII preserved at the typical culture preservation center in China and marked with a preservation CCTCC NO: M2015107. According to the method, an expression plasmid of exogenous protein in candida parapsilosis is established, S-carbonyl reductase II is integrated to a candida parapsilosis genome to obtain the pCP-scrII, the candida parapsilosis is converted through electric shock to obtain the recombinant candida parapsilosis strain. By optimizing biotransformation reaction conditions, catalytic conversion is conducted on 2-hydroxyacetophenone through the recombinant strain to obtain the product S-phenyl glycol, the optical purity and yield of the product are up to 99.9%, an effective way is provided for efficient and low-cost preparation of the S-phenyl glycol, and a solid foundation is laid for industrial application of biological catalysis of chiral alcohol.
Description
technical field [0001] Efficient preparation of a recombinant strain of Candida parapsilosis ( S )-Phenyl glycol method, construction ( S The expression plasmid of )-carbonyl reductase II in Candida parapsilosis, realizes ( S In situ expression of )-carbonyl reductase II in Candida parapsilosis, efficient preparation of ( S )-phenyl glycol method, the present invention relates to the construction of genetic engineering means ( S )-carbonyl reductase II is efficiently prepared in the recombinant strain of Candida parapsilosis ( S The method and application of )-phenylethylene glycol belong to the technical field of biocatalytic asymmetric conversion. Background technique [0002] Chiral compounds are widely used in fine chemical fields such as medicine, pesticides, hormones, and food additives. For example, optically pure phenylethylene glycol is not only an indispensable and important chiral additive in liquid crystal materials, but also an important ingredient in the pr...
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