Method for producing 2-phenylethanol under biological catalysis

A technology of biocatalysis and phenylethyl alcohol, applied in the field of bioengineering and biotechnology, can solve the problems of low yield, unstable properties of intermediates, and increased costs

Active Publication Date: 2017-07-18
BOTON SHANGHAI BIOLOGICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In addition to the above-mentioned defects in this patent, the multi-enzyme catalytic system has the problem of unstable intermediate propertie

Method used

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  • Method for producing 2-phenylethanol under biological catalysis
  • Method for producing 2-phenylethanol under biological catalysis
  • Method for producing 2-phenylethanol under biological catalysis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Embodiment 1: Construction of E.coli / PdhBb bacterial strain

[0053] 1. PdhBb gene sequence synthesis and expression vector construction

[0054] The PdhBb gene involved in this example has a sequence length of 1143bp, and its accession number in the NCBI database is D45211, which is derived from Bacillus badius IAM 11059, and the sequence is synthesized from the whole gene sequence of Suzhou Jinweizhi Biotechnology Co., Ltd. A nucleic acid restriction endonuclease EcoRI recognition site (5'-GAATTC-3') is added to the 5' end of the fully synthesized PdhBb gene, and a nucleic acid restriction endonuclease NotI recognition site (5'-GCGGCCGC-3') is added to the 3' end ), and cloned into the vector pUC57 to obtain the vector pUC57-PdhBb.

[0055] PdhBb gene target fragment product recovery: Plasmid pUC57-PdhBb was digested with restriction enzymes EcoR1 and Not1 for 2 hours, after 1% agarose gel electrophoresis, the target band was excised, and the DNA was recovered with T...

Embodiment 2

[0062] Embodiment 2: Construction of E.coli / KdcA bacterial strain

[0063] The KdcA gene involved in this example has a sequence length of 1644bp, and its accession number in the NCBI database is AY548760, which is derived from Lactococcus lactis, and the sequence is synthesized from the whole gene sequence of Suzhou Jinweizhi Biotechnology Co., Ltd. A nucleic acid restriction endonuclease EcoRI recognition site (5'-GAATTC-3') is added to the 5' end of the fully synthetic KdcA gene, and a nucleic acid restriction endonuclease NotI recognition site (5'-GCGGCCGC-3') is added to the 3' end ), and cloned into vector pUC57 to obtain vector pUC57-KdcA.

[0064] KdcA gene expression vector construction: KdcA target gene digestion treatment, product recovery, ligation transformation, etc. refer to Example 1.

[0065] IPTG induced expression, SDS-PAGE protein expression identification, experimental operation with reference to Example 1, see the results figure 2 . figure 2 The resu...

Embodiment 3

[0066] Embodiment 3: Construction of E.coli / YahK bacterial strain

[0067] The YahK gene involved in this example has a sequence length of 1638bp and its accession number in the NCBI database is 944975, which is derived from Escherichia coli BW25113, and the sequence is synthesized from the whole gene sequence of Suzhou Jinweizhi Biotechnology Co., Ltd. A nucleic acid restriction endonuclease EcoRI recognition site (5'-GAATTC-3') is added to the 5' end of the fully synthetic YahK gene, and a nucleic acid restriction endonuclease NotI recognition site (5'-GCGGCCGC-3') is added to the 3' end ), and cloned into the vector pUC57 to obtain the vector pUC57-YahK.

[0068] Construction of the YahK gene expression vector: refer to Example 1 for enzyme digestion treatment of the YahK target gene, product recovery, ligation transformation, etc.

[0069] IPTG induced expression, SDS-PAGE protein expression identification, experimental operation with reference to Example 1, see the resul...

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Abstract

The invention relates to the field of bioengineering and biotechnologies, and discloses a method for producing 2-phenylethanol under biological catalysis. The method comprises the following steps: adding wet cells of E.coli/Pdh, E.coli/Kdc and E.coli/ADH undergoing induced expression into a biological catalysis system taking L-phenylalanine as a substrate for performing a catalytic reaction; centrifuging after finishing the reaction; extracting supernatant to obtain the 2-phenylethanol. In the method, the L-phenylalanine is taken as the substrate, and recombinant escherichia coli transformed with a phenylalanine dehydrogenase gene, a 2-keto acid decarboxylase gene and an alcohol dehydrogenase gene is added into a reaction system for performing a biological deamination, decarboxylation and reduction three-step catalytic reaction in order to generate a product, namely, phenylethanol; a coenzyme, namely, NAD (Nicotinamide Adenine Dinucleotide) is added once and can be recycled, excess ketonic acid and hydrogen sources do not need to be added, no unnecessary side products are generated, a high substrate transforming rate is achieved, and the yield of the phenylethanol is increased remarkably.

Description

technical field [0001] The invention relates to the field of bioengineering and technology, and more specifically relates to a method for biocatalyzed production of 2-phenylethanol. Background technique [0002] 2-Phenyl alcohol (2-Penylenthanol, 2-PE) is also called β-Phenyl alcohol (β-Penylenthanol), the chemical name is 2-Phenyl alcohol (2-Penylentyl alcohol, 2-PEA), and the molecular formula is C 8 h 10 O, molecular weight 122.16, structural formula as follows: [0003] [0004] 2-Phenylethyl alcohol is a kind of aromatic alcohol with sweet rose-like fragrance, colorless viscous liquid, boiling point 219°C, relative density 1.0230, refractive index 1.5310-1.5340, soluble in ethanol, ether, glycerin, slightly soluble in water , slightly soluble in mineral oil. 2-Phenylethyl alcohol naturally exists in the essential oils of many flowers and plants, such as rose, hyacinth, jasmine, narcissus, lily, etc., and is also the natural flavor of some fermented foods such as t...

Claims

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Application Information

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IPC IPC(8): C12P7/22C12R1/19
CPCC12P7/22
Inventor 陈建刘旭李斌彭春睿
Owner BOTON SHANGHAI BIOLOGICAL TECH CO LTD
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