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Method for producing phenyllactic acid through whole-cell transformation of phenylalanine

A technology of phenylalanine and phenyllactic acid, which is applied in the field of enzyme engineering, can solve the problems of being unsuitable for industrial production and the high price of phenylpyruvate, and achieve a high yield effect

Inactive Publication Date: 2018-07-13
JIANGNAN UNIV
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Problems solved by technology

However, phenylpyruvate is expensive, and the market price is 600 times that of phenylalanine (the price of L-phenylalanine is 130 yuan / kg, and the price of sodium phenylpyruvate is 80 yuan / g), which is not suitable for industrialized production, so , the cheap phenylalanine has more potential for industrial application as a substrate for the production of phenyllactic acid

Method used

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Examples

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example 1

[0029] Cloning of Example 1 L-amino acid deaminase mutant D165K / F263M / L336M gene

[0030] The l-aad gene was cloned using the laboratory-preserved plasmid PET20b-(l-aad) (D165K / F263M / L336M) as a template. The PCR product was digested with BamHI and HindIII, and further inserted into the MCS-1 site of the vector pRSF-Duet-1 to construct the recombinant plasmid pRSF-aad.

Embodiment 2

[0031] Example 2 Collinearity Analysis and Systems Biology Selection of Lactate Dehydrogenase

[0032] The known genes with higher lactate dehydrogenase activity were BcLDH from Bacillus coagulans NL01, L-nLDH and D-nLDHfrom from Bacillus coagulans SDM, and LaLDH from Lactobacillus sp.SK007. Non-redundant protein databases in GenBank were searched for homologous sequences using BLASTP 2.2.28+. Use H-CD-HIT (CD-HIT suite) to group the retrieval results, there are three levels: 90%, 60%, 40% similarity. Using the NCBI taxonomy database, rebuild taxonomy trees. After performing PSI-BLAST on the sequences of the four enzymes, the sequence PSI-BLAST results were obtained, and Synteny was analyzed using GCView. Finally, ten different sources of lactate dehydrogenase genes with certain sequence differences were selected for the next experiment.

[0033] Table 1. Lactate dehydrogenase from different sources

[0034]

Embodiment 3

[0035] Example 3 Construction of L-amino acid deaminase gene and L-lactate dehydrogenase gene co-expression plasmid

[0036] After screening 10 ldh genes according to Example 2, the codons were optimized according to the gene sequence in the gene bank. Then, it was synthesized by Reddy Biotechnology Co., Ltd. (Shanghai, China), and two restriction sites NdeI and XhoI were added in the forward and reverse directions, respectively. The ldh gene was amplified by PCR. The resulting product was inserted into the plasmid pRSF-aad MS2 site, and the resulting recombinant plasmid was named pRSF-aad-ldh, and further transformed into Escherichia coli BL21(DE3). The obtained 10 recombinant strains were added with 0.4 mM IPTG to induce expression at 25° C. for 10 h. After the induction, the fermentation broth was centrifuged to collect the cells, and the cells were resuspended with phenylpyruvate as the substrate for whole-cell transformation, and the optimal ldh gene was screened out ac...

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Abstract

The invention discloses a method for producing a phenyllactic acid through whole-cell transformation of phenylalanine, and belongs to the technical field of enzyme engineering. The method successfullyconstructs an L-amino acid deaminase, L-lactic dehydrogenase and formate dehydrogenase co-expressed strain. Phenyllactic acid of 30 g / L is produced through the whole-cell transformation of phenylalanine, and the transformation rate is 100%. The establishment of a whole-cell transformation system solves the problems of complicated steps, low yield and environment pollution in the chemical synthesis of the phenyllactic acid as well as the problem of low transformation rate in the production of the alpha-oxoglutarate through the enzymatic conversion, the phenyllactic acid is produced through a one-step method at high yield without pollution, and a certain theoretical foundation is laid for the subsequent industrial production.

Description

technical field [0001] The invention relates to a method for converting phenylalanine into whole cells to produce phenyllactic acid, which belongs to the technical field of enzyme engineering. 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, 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-toxic, but also has a bro...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/70C12P7/42C12R1/19
CPCC12N9/0006C12N9/0069C12P7/42C12Y101/01C12Y101/05006
Inventor 王淼刘龙王秀婷堵国成李江华陈坚
Owner JIANGNAN UNIV
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