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Application of bacillus DL-1 whole cell in catalytic resolution of styralyl acetate

A technology of styroyl acetate and Bacillus, applied in bacteria, microorganism-based methods, biochemical equipment and methods, etc., can solve the problems of expensive reagents, high cost, large pollution, etc., and achieve high application value and high substrate resistance. The effects of receptivity and optical selectivity

Inactive Publication Date: 2020-01-17
SOUTH CHINA SEA INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Traditional chemical synthesis of chiral alcohols and their derivatives has problems such as expensive reagents, harsh reaction conditions, high cost, and large pollution

Method used

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  • Application of bacillus DL-1 whole cell in catalytic resolution of styralyl acetate
  • Application of bacillus DL-1 whole cell in catalytic resolution of styralyl acetate
  • Application of bacillus DL-1 whole cell in catalytic resolution of styralyl acetate

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Embodiment 1: the identification of bacillus strain

[0024] The strain Bacillus sp.DL-1 after screening and purification was identified by its conserved 16S rRNA sequence. 16S rRNA universal primers 27F: 5'-AGAGTTTATCCTGGCTCAG-3'; and 1492R: 5'-GGTTACCTTGTTACGACTT-3' were used for PCR amplification using the Bacillus DL-1 bacterial liquid as a DNA template. Set up the reaction system shown in Table 1:

[0025] Table 1 PCR reaction system

[0026]

[0027] PCR amplification conditions: pre-denaturation at 94°C for 10 min, followed by denaturation at 94°C for 30 s, annealing at 56°C for 30 s, extension at 72°C for 2 min and 30 s, and 30 cycles; finally, extension at 72°C for 10 min, and cooling to 18°C. The amplified products were detected by 1% agarose gel electrophoresis, and then sent to Meiji Biotechnology Co., Ltd. for bidirectional sequencing. After splicing the 16S rRNA sequence of the strain (its nucleotide sequence is shown in SEQ ID NO.1), the strain was ...

Embodiment 2

[0028] Example 2: Growth curve of Bacillus sp.DL-1

[0029] Streak the preserved Bacillus sp.DL-1 onto LB solid medium (1% tryptone, 0.5% yeast powder, 1% NaCl, 1.5% agar powder, solvent is water; above all are mass fractions), Cultivate at 37°C for 16 hours, then pick a single colony and inoculate it into 100 mL of LB liquid medium (1% tryptone, 0.5% yeast powder, 1% NaCl, the solvent is water; the above are all mass fractions), 200r / min, 37 Cultivate at ℃ for 12 hours to obtain seed solution, inoculate in LB liquid medium according to the inoculation amount of 1% v / v and cultivate at 200r / min, cultivate at 37℃, measure its biomass OD-600nm every 2h, the results are as follows figure 1 shown.

Embodiment 3

[0030] Example 3: Preparation of whole cells of Bacillus sp.DL-1

[0031] The seed solution of Bacillus sp.DL-1 was inoculated in skimmed milk powder liquid culture medium according to the inoculation amount of 1% v / v (dissolving 10 g high-protein skimmed high-calcium milk powder (purchased from Inner Mongolia Yili Industrial Group Co., Ltd.) in 1 L of water , sterilized at 115°C for 30min, and cooled to obtain) for fermentation culture, 200r / min, 37°C for 24h (the medium became clear), to obtain the fermentation liquid, centrifuged to remove the supernatant, and the cell pellet was sterilized with pH=7.2 Tris / HCl buffer was washed three times, and the cell pellet was collected by centrifugation to obtain whole cells of Bacillus sp.DL-1.

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Abstract

The present invention discloses an application of a bacillus DL-1 whole cell in catalytic resolution of styralyl acetate. The bacillus DL-1 whole cell can be used as a catalyst to prepare chiral (R)-1-phenylethanol and (S)-styralyl acetate with high optical purity. Under optimal reaction conditions, an enantiomeric excess value of the (R)-1-phenylethanol obtained by the bacillus DL-1 whole cell catalytic resolution of (+ or -)-styralyl acetate is 96.6%, a conversion rate is 24.5% and yield is 48.1%; the enantiomeric excess value of the obtained (S)-styralyl acetate by the catalytic resolutionof the (+ or -)-styralyl acetate is 98.4%, the conversion rate is 56.6% and the yield is 86.2%. The bacillus DL-1 whole cell as the biocatalyst has relatively high substrate tolerance and optical selectivity in the chiral resolution of the (+ or -)-styralyl acetate, and has relatively great application value in the field of biochemical engineering.

Description

technical field [0001] The invention belongs to the fields of biochemical industry and biotechnology, and in particular relates to the application of a whole cell of bacillus DL-1 isolated from a deep-sea sediment sample in the western Pacific Ocean as a biocatalyst in catalytically splitting styroyl acetate. Background technique [0002] Whole-cell catalysis refers to the use of complete biological organisms (that is, whole cells, tissues, and even individuals) as catalysts for chemical transformation, and its essence is to use intracellular enzymes for catalysis. The advantages of using whole-cell biocatalysts are: first, no enzyme extraction and purification is required, which reduces the loss of enzyme activity, is simple to prepare, and greatly reduces production costs; second, culturing in anhydrous or non-traditional media can enhance whole-cell biological The stability of the catalyst in repeated use; third, it can better realize the in-situ regeneration of energy an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12P41/00C12P7/62C12P7/22C12R1/07
CPCC12P7/22C12P7/62C12P41/004C12N1/205C12R2001/07
Inventor 胡云峰董璐张云
Owner SOUTH CHINA SEA INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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