A kind of method of catalyzing omega-amino acid to produce dicarboxylic acid in vitro

A dicarboxylic acid, amino acid technology, applied in the direction of fermentation, etc., to achieve the effects of low production cost, convenient product separation, and high product yield

Active Publication Date: 2022-06-14
SHANDONG UNIV
View PDF4 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Search found that in vitro pure enzyme catalyzed ω-amino acid to produce dicarboxylic acid method has not been reported

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • A kind of method of catalyzing omega-amino acid to produce dicarboxylic acid in vitro
  • A kind of method of catalyzing omega-amino acid to produce dicarboxylic acid in vitro
  • A kind of method of catalyzing omega-amino acid to produce dicarboxylic acid in vitro

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1 In vitro catalysis of 4-aminobutyric acid to produce succinic acid

[0051] In the present invention, the ω-amino acid oxidase Am-AOX is derived from Kluyveromyces marxianus DMKU3-1042, and its gene number is AP012218.1. Using Escherichia coli BL21 as the host to optimize the codon of the gene, the codon-optimized gene was synthesized by Shanghai Sangon Bioengineering Co., Ltd. to obtain the E.coli Top10 / pETDuet-Am-AOX strain; extract the The expression plasmid pETDuet-Am-AOX in the strain, transform pETDuet-Am-AOX into the expression strain E.coli BL21, and carry out protein expression and purification, the purification results are shown in the appendix figure 2 ; The length of the optimized ω-amino acid oxidase gene sequence is 2034 bases, and its nucleotide sequence is shown in SEQID NO.1.

[0052] In the present invention, xanthine oxidase was purchased from Sigma Company, product number is X4875; catalase was purchased from Worthington Company.

[0053...

Embodiment 2

[0056] Example 2 In vitro catalysis of 5-aminovaleric acid to produce glutaric acid

[0057] The ω-amino acid oxidase derived from Kluyveromyces marxianus DMKU3-1042 was prepared according to the method described in Example 1.

[0058] Xanthine oxidase was purchased from Sigma Company, product number is X4875; catalase was purchased from Worthington Company.

[0059] In a 5ml system, make it contain 50mM PBS buffer (pH 7.4), 1U / mL ω-amino acid oxidase, 0.7U / mL xanthine oxidase and 120U / mL catalase, 10g / mL L of 5-aminovaleric acid and 0.04 mM copper ion (CuSO 4 ), at 37 ± 1 ° C, 150 rpm under the condition of water-bath shaker shaking reaction; reaction for 30 hours, to obtain the conversion liquid containing glutaric acid. Among them, the consumption of 5-aminovaleric acid and the generation of glutaric acid were detected by sampling every 3 hours of catalysis; After 15 minutes, the added protein was removed, the supernatant was drawn and diluted to a multiple suitable for ...

Embodiment 3

[0062] Example 3 In vitro catalysis of 6-aminocaproic acid to produce adipic acid

[0063] The ω-amino acid oxidase derived from Kluyveromyces marxianus DMKU3-1042 was prepared according to the method described in Example 1.

[0064]Xanthine oxidase was purchased from Sigma Company, product number is X4875; catalase was purchased from Worthington Company.

[0065] In a 5ml system, make it contain 50mM PBS buffer (pH 7.4), 0.7U / mL ω-amino acid oxidase, 0.7U / mL xanthine oxidase and 120U / mL catalase, 10g / L of 6-aminocaproic acid and 0.04mM copper ion (CuSO 4 ), at 37 ± 1 ° C, 150 rpm under the condition of water-bath shaker shaking reaction; reaction for 30 hours, to obtain a conversion solution containing adipic acid. Among them, samples were taken every 3 hours of catalysis to detect the consumption of 6-aminocaproic acid and the generation of adipic acid; the resulting conversion solution was boiled at 102±1°C for 15 minutes to denature and precipitate the protein, and cent...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention discloses a method for in vitro catalyzing ω-amino acid to produce dibasic carboxylic acid. Using ω-amino acid as a substrate, adding ω-amino acid oxidase Am-AOX, xanthine oxidase and catalase to construct poly An enzyme reaction system that efficiently catalyzes ω-amino acids to produce a series of dicarboxylic acids, such as succinic acid, glutaric acid and adipic acid, through in vitro multi-enzyme. In the in vitro catalytic system constructed by the present invention, there are only substrates and 3 kinds of enzymes, no exogenous addition of any cofactors and auxiliary substrates is required, and the number of enzymes used is also less than that of natural synthetic pathways, with low production costs, simple operation and low pollution , high product yield, convenient separation, and good application value.

Description

technical field [0001] The invention relates to a method for producing dicarboxylic acids, in particular to a method for producing dicarboxylic acids by catalyzing ω-amino acids in vitro with pure enzymes, and belongs to the technical field of enzyme-catalyzed preparation of dicarboxylic acids. Background technique [0002] Dicarboxylic acids are important components of synthetic plastics, polyester and nylon, with huge markets and wide applications [1] . At present, dicarboxylic acids are mainly synthesized by chemical petroleum. For example, the general chemical method for the production of glutaric acid is through the ring-opening hydrolysis of butyrolactone and potassium cyanide. [2] . It can also be prepared from the reaction of 1,3-dibromopropane with sodium or potassium cyanide, or by the oxidative cleavage of cyclopentene with ionic liquids [3] . Adipic acid is produced by oxidizing a mixture of cyclohexanone and cyclohexanol with nitric acid as a catalyst [4] ...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & AuthorityPatents(China)
IPC IPC(8): C12P7/46C12P7/44
CPCC12P7/46C12P7/44
Inventor高超严金鑫马翠卿许平
OwnerSHANDONG UNIV