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A method for producing phenyllactic acid by transforming phenylalanine into whole cells of Bacillus subtilis

A technology of Bacillus subtilis and phenylalanine, which is applied in the field of bioengineering, can solve the problems of affecting cell growth, long catalyst time, complicated operation process of Escherichia coli, etc., and achieve the effect of solving cumbersome steps

Active Publication Date: 2021-03-02
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Previously, there were studies devoted to the co-expression of three enzymes in BL21(DE3) and the transformation of whole cells to produce phenyllactic acid, but the method had the following defects: (1) Escherichia coli is not a food-grade microorganism, and E. The endotoxin in the bacterium will bring new food safety problems; (2) The operation process of using E. coli as a whole-cell catalyst is complicated, and an inducer needs to be added during the culture process, and strict induction conditions should be controlled; (3) the E. coli Co-expression of three enzymes affects cell growth, and more biocatalysts need to be collected; (3) It takes too long to culture E. coli whole-cell catalysts, and the whole process lasts for 35 hours

Method used

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  • A method for producing phenyllactic acid by transforming phenylalanine into whole cells of Bacillus subtilis
  • A method for producing phenyllactic acid by transforming phenylalanine into whole cells of Bacillus subtilis

Examples

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Embodiment 1

[0026] Example 1 Systems Biology Selection of Phenylalanine Dehydrogenase

[0027] A known gene with high phenylalanine dehydrogenase activity (GenBank accession number: KU533729.1) from Bacillus nanhaiensis was used. Non-redundant protein databases in GenBank were searched for homologous sequences using BLASTP 2.2.28+. The NCBI taxonomy database was used to screen out six different sources of phenylalanine dehydrogenase genes with certain sequence differences, which were derived from: Bacillus halodurans (Genbank accession number is BAB03937.1), Geobacillus kaustophilus (Genbank accession number is BAD76316.1 ), Geobacillus thermodenitrificans (Genbank accession number is ABO67271.1), Geobacillus sp.Y412MC52 (Genbank accession number is ADU94401.1), Geobacillus sp.Y4.1MC1 (Genbank accession number is ADP74293.1), Bacillus nanhaiensis (SEQ ID NO. 1), the above genes were used to construct different Bacillus subtilis, and the corresponding bacterial strains were named Bacillus...

Embodiment 2

[0029] Example 2 Amplification of lactate dehydrogenase gene and construction of co-expression strain

[0030] Genomic DNA of Bacillus.subtilis 168 was extracted, and the genome was used as a template to amplify the LDH gene (as shown in SEQ ID NO.2) using Prime-STAR HSDNA polymerase high-fidelity enzyme and designed primers. After the PCR product was purified and recovered, the one-step cloning method was used to connect with the vector pP43NMK-pdh6 constructed in Example 1 to construct the recombinant plasmid pP43NMK-pdh6-ldh, transform Escherichia coli JM109, extract the plasmid and send it for sequencing, and transfer the correctly sequenced plasmid into In B. subtilis 168, a recombinant strain B. subtilis 168 (pP43NMK-PDH-LDH) was constructed.

Embodiment 3

[0031] Embodiment 3 preparation of whole cell catalyst and whole cell transformation process

[0032]The recombinant strain B. subtilis 168 pP43NMK-PDH-IDH) was inoculated into the seed medium (kanamycin 50 mg / L), and cultivated overnight at 37° C. and 220 rpm. Fermentation was carried out in a 250ml shake flask, 4% inoculum was added to 20mL fermentation medium, the stirring speed and temperature were respectively 220rpm and 37°C, after 16 hours of cultivation, the bacteria were collected and washed twice with 20mM PB (pH 7.0) buffer bacteria. The whole cell transformation system is: phenylalanine 30g / L, whole cell catalyst 15g / L (final concentration 0.5g bacteria / 1g phenylalanine), 150mmol / L glucose, reacted in 20mM PB buffer (pH 7.5) In the medium, 37 ° C, 220rpm conversion reaction 15h.

[0033] For the recombinant strain B. subtilis 168 (pP43NMK-PDH-IDH), the conversion rate of whole cells into phenylalanine to produce phenyllactic acid is 40%.

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Abstract

The invention discloses a method for producing phenyllactic acid by transforming phenylalanine into whole cells of Bacillus subtilis, belonging to the technical field of bioengineering. The invention successfully realizes the construction of co-expression of phenylalanine dehydrogenase and lactate dehydrogenase in Bacillus subtilis. Whole cells convert L-phenylalanine to produce 10.0g / L phenyllactic acid, with a conversion rate of 40%. The establishment of this whole-cell transformation system solves the problems of cumbersome steps, low yield, and environmental pollution in the chemical synthesis of phenyllactic acid, and lays a certain theoretical foundation for subsequent industrial production.

Description

technical field [0001] The invention relates to a method for producing phenyllactic acid by transforming phenylalanine into whole cells of Bacillus subtilis, belonging to the technical field of bioengineering. Background technique [0002] Phenyllactic acid (PLA), also known as β-phenyllactic acid, 3-phenyllactic acid, or 2-hydroxy-3-phenylpropionic acid, is a small molecule natural organic acid widely present in nature. Phenyllactic acid has two enantiomers, namely L-phenyllactic acid and D-phenyllactic acid. Phenyllactic acid is a natural antibacterial substance, which was originally found in cheese and also exists in natural honey. It has inhibitory effects on a variety of pathogenic microorganisms, such as Gram-positive bacteria such as Staphylococcus aureus and Listeria. Lambert-negative bacteria such as E. coli, Salmonella, etc., but non-toxic to human and animal cells. [0003] Compared with common chemical preservatives, phenyllactic acid is not only safe and non-t...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12P7/42C12R1/125
CPCC12N9/0006C12N9/0018C12P7/42C12Y101/01027C12Y101/01028C12Y104/0102
Inventor 王淼刘龙薛技科
Owner JIANGNAN UNIV