A kind of method of biocatalytic glycerol production 1,3-propanediol

A technology for biologically catalyzing glycerol and propylene glycol, which is applied in the directions of biochemical equipment and methods, botanical equipment and methods, oxidoreductases, etc., and can solve problems such as affecting the utilization efficiency of reducing power, improving, and difficulty in 1,3-propanediol production. , to achieve the effect of improving product selectivity, high reaction efficiency and low price

Active Publication Date: 2022-05-17
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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Problems solved by technology

Therefore, PDOR is the key enzyme for the production of 1,3-propanediol, and the expected effect cannot be obtained only by overexpressing PDOR in bacteria (Zheng, P. et al., Process Biochem. 2006, 41, 2160), because this step reaction requires The chemical equivalent of coenzyme nicotinamide adenine dinucleotide (NADH) is consumed, but because many oxidoreductases in the cell share the use of NADH or NADPH as a coenzyme, it can be consumed by various pathways in the metabolic network, which affects the target pathway's effect on reducing power. utilization efficiency
However, any manipulation that alters the NAD concentration and its NAD(P) / NAD(P)H ratio will have intracellular global effects on the cell (Holm, A.K. et al., J. Biol. Chem. 2010, 285, 17498) , therefore, it is difficult to regulate the oxidoreductase related to 1,3-propanediol in the biological system from the coenzyme level
Although changing the PDOR coenzyme preference (from NADH to NADPH) can improve the production of 1,3-propanediol to a certain extent (CN 200810228269.X), NADPH is still a common coenzyme in the cell, and the production of 1,3-propanediol is difficult from fundamentally improve

Method used

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  • A kind of method of biocatalytic glycerol production 1,3-propanediol
  • A kind of method of biocatalytic glycerol production 1,3-propanediol
  • A kind of method of biocatalytic glycerol production 1,3-propanediol

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1: Using glycerol as raw material, 1,3-propanediol was prepared under the catalysis of wild-type glycerol dehydratase and PDOR-T143I / G186P.

[0054] According to the literature method (Fuller, C.W. et al. Eur. J. Biochem. 1980, 103, 421), NCD was reduced to NCDH, which was used as a reducing agent for 3-hydroxypropanal.

[0055] In 1mL of HEPES buffer reaction system containing 50mM pH7.5, containing 30mM (NH 4 ) 2 SO 4 , 100mM glycerol, 110mM NCDH, derived from Klebsiella pneumoniae glycerol dehydratase GDHt15.8μg / mL, NCD-dependent 1,3-propanediol dehydrogenase PDOR-T143I / G186P 18.9μg / mL, added 2mM vitamin B 12 Start the reaction, react at 37°C for 2 hours, add 9 mL of acetonitrile methanol water mixture (acetonitrile: methanol: water volume ratio = 4:4:1) to quench the reaction, take out 500 μL for coenzyme and product analysis.

[0056] HPLC analysis: the sample has NCDH characteristic peak at 340nm, the content is 40.5%, indicating that NADH is not comple...

Embodiment 2

[0059] Example 2: Using glycerol as raw material, under the catalysis of wild-type glycerol dehydratase and PDOR-G43K / G186P / L191F body, sodium phosphite is used as NCD analog recycling reagent to prepare 1,3-propanediol

[0060] In 1mL of HEPES buffer reaction system containing 50mM pH7.5, containing 30mM (NH 4 ) 2 SO 4 , 100mM glycerol, 2mM NCD, 120mM sodium phosphite, derived from Klebsiella pneumoniae wild-type glycerol dehydratase GDHt15.8μg / mL, PDOR-G43K / G186P / L191F 17.9μg / mL, phosphite dehydrogenase rsPDH-I151R / E213C 10U / mg, add 2mM Vitamin B 12 Start the reaction, react at 37 ° C for 180 min, add 9 mL of acetonitrile methanol water mixture (acetonitrile: methanol: water = 4:4:1) to quench the reaction, take out 500 μL for coenzyme and product analysis.

[0061] HPLC analysis: the sample has a characteristic peak at 340nm, and the NCDH content is 12.9%, and a characteristic peak with the same retention time as NAD is detected at 260nm, and the content is 87.1%, indic...

Embodiment 3

[0067] Example 3: Using glycerol as a raw material, under the catalysis of wild-type glycerol dehydratase and PDOR-G43D / L44F / G186P / L191Y, potassium deuterated phosphite is used as a NUD analogue recycling reagent to prepare 1,3-propanediol

[0068] According to literature method (Woodyer R, et al. FEBS J, 2005, 272, 3816), potassium phosphite was dissolved in D 2 O, freeze-dried, repeated 4 times to obtain deuterated potassium phosphite, and set aside.

[0069] In 5 mL of Tris-HCl buffer reaction system containing 100 mM pH 8.5, containing 30 mM (NH 4 ) 2 SO 4 , 100mM glycerol, 2mM NUD, 120mM potassium deuterophosphite, derived from Klebsiella aerogenes wild-type glycerol dehydratase GDHt 15.8μg / mL, PDOR-G43K / G186P / L191F 18.9μg / mL, phosphorous acid dehydrogenation Enzyme psPDH-L151V / D213Q 20μg / mL, add 5mM Vitamin B 12 Start the reaction, react at 37°C for 30 min, add 45 mL of acetonitrile methanol water mixture (acetonitrile: methanol: water = 4:4:1) to quench the reaction...

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Abstract

The invention discloses a method for biocatalyzing glycerin to produce 1,3-propanediol. The method first converts glycerol to the intermediate 3-hydroxypropanal by glycerol dehydratase, and the intermediate is in the NAD analog-dependent 1,3-propanediol dehydrogenase and the NAD analog-dependent oxidoreductase of the renewable NAD analog With coupling catalysis, 1,3‑propanediol is also produced. The system can be used in an extracellular cell-free system or in microorganisms to catalyze biodiesel by-product glycerol to produce 1,3-propanediol, which can significantly reduce the production cost of 1,3-propanediol and increase the product yield.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a method for coenzyme nicotinamide adenine dinucleotide (NAD) analog dependent oxidoreductase to catalyze glycerol to produce 1,3-propanediol, specifically, glycerol dehydratase converts Glycerol is converted to 3-hydroxypropanal, which is then catalyzed by the coupling of NAD analog-dependent 1,3-propanediol dehydrogenase and NAD analog-dependent oxidoreductase that regenerates NAD analogs For 1,3-propanediol. This catalytic system can catalyze glycerol to produce 1,3-propanediol in vitro or in vivo. Background technique [0002] 1,3-Propanediol (1,3-propanediol, 1,3-PD) is an important chemical raw material, in addition to being used in the synthesis of adhesives, antifreeze, plasticizers, detergents, preservatives, etc., it can also be used in food , cosmetics and pharmaceutical industries, its most widely used is as a monomer for the synthesis of polyester, polyether and polyuret...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P7/18C12N15/53
CPCC12P7/18C12Y101/01202C12N9/0006
Inventor 赵宗保刘武军郭潇佳刘玉雪
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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