Method for preparing chiral monomer mandelic acid

A technology of mandelic acid and p-hydroxymandelic acid, which is applied in the field of obtaining chiral monomer mandelic acid, can solve the problems of high price, conversion rate not exceeding 50%, unfavorable long-term effective development of methods, etc. friendly effect

Inactive Publication Date: 2011-06-08
SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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  • Abstract
  • Description
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Problems solved by technology

[0006] At present, the methods used to produce mandelic acid are mainly chemical methods and enzymatic methods (or biological cell conversion methods). Toxic chemical reagents, such as HCN, etc. are required in the synthesis, which are not only expensive, but also relatively harmful to the environment and people. Moreover, the precursors used in enzymatic synthesis are basically derived from fossil-based products, which is not conducive to the long-term effective development of the method; some enzymatic synthesis is mixed-rotation R / S mandelic acid, which needs to be resolved, while optical resolution agents It is also very expensive, or use strains to specifically degrade a monomer in helical mandelic acid, so as to obtain another monomer. The disadvantage of this method is that half of the mandelic acid is consumed, and the conversion rate does not exceed 50%. %

Method used

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  • Method for preparing chiral monomer mandelic acid
  • Method for preparing chiral monomer mandelic acid
  • Method for preparing chiral monomer mandelic acid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Example 1 , Recombinant plasmid construction

[0069] In this embodiment, the aroF fbr and pheA fbr The gene was cloned into plasmid pSU2718 (Martinez, E., B. Bartolome, and F. de la Cruz, 1988. pACYC 184-deribed cloning vectors containing the multiple cloning site and lacZa reporter gene of pUC8 / 9and pUC18 / 19plasmids. Gene68:159 -162), the recombinant plasmid pSUFA was obtained, and the AohmaS and SchmaS genes were respectively cloned into the recombinant plasmid pSUFA to obtain pSUFAAohmaS and pSUFASchmaS, and then the lacI q The genes were cloned into pSUFAAohmaS and pSUFASchmaS respectively to obtain pSUFAAQ and pSUFASQ, the specific process is as follows:

[0070] 1.1. Construction of recombinant plasmid pSUaroF

[0071] Referring to the method provided in Molecular Cloning III, the genome of E. coli DH5α (purchased from Amersham Company) was obtained by extraction.

[0072] Using the genome of E.coli DH5α as a template, primers P148L(+), P148L(-), aroFSacI(+...

Embodiment 2

[0168] Example 2 , Construction of deletion mutants

[0169] In this example, two single mutants were firstly constructed, and then the four single mutants constructed and purchased were used as starting strains, and transduced with P1 phage to obtain multiple mutants for use in mandelic acid genetically engineered bacteria Constructed host bacteria, the specific process is as follows:

[0170] 2.1 Knockout of tyrB and aspC genes in W3110

[0171] In this example, referring to the literature of Gust.B. et al. (PCR-targeting system in Streptomyces coelicolor, Gust.B., Kieser T.et al, 2002), PCR-targeting technology was used to knock out the original strain E. The aromatic amino acid transaminase gene (tyrB) and aspartate aminotransferase gene (aspC) of coli W3110 were single-deleted mutants W3110(ΔtyrB) and W3110(ΔaspC), and the specific process was as follows:

[0172] 2.1.1. Preparation of single knockout strain W3110(ΔtyrB)

[0173] Using the plasmid pIJ778 (purchased f...

Embodiment 3

[0213] Example 3 , in vitro enzyme activity reaction and chiral identification

[0214] 3.1. Preparation of S-mandelic acid

[0215] The recombinant plasmids pET24a, pETAohmaS and pETSchmaS were transformed into BL21(DE3) to obtain BL21(DE3) / pET24a, BL21(DE3) / pETAohmaS and BL21(DE3) / pETSchmaS respectively, and BL21(DE3) / pET24a was used as a blank control, and the The above strains were added to LB medium, cultured to OD of 0.4-0.6, induced with 1mM IPTG for 4h, centrifuged at 4°C, 4000rpm for 10min, and the collected bacteria were resuspended in pH=7.5, 200mM potassium phosphate buffer , sonicate, and centrifuge to take the supernatant to obtain the crude p-hydroxymandelic acid synthase enzyme liquid.

[0216] Then, use the obtained crude enzyme solution of p-hydroxymandelic acid synthase to catalyze the reaction of phenylpyruvate at 28°C, the reaction system: 3ml of the assay mixture contains 200mM potassium phosphate buffer at pH7.5, 5mM phenylpyruvate, 44mM Ascorbic aci...

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Abstract

The invention provides a method for preparing S-mandelic acid or R-mandelic acid and a method for converting S-mandelic acid into R-mandelic acid. The method comprises the following steps: catalyzing phenylpyruvic acid by using p-hydroxymandelic acid synthetase or mandelic acid synthetase to obtain S-mandelic acid; and catalyzing the S-mandelic acid by using p-hydroxymandelic acid oxidase or mandelic acid oxidase or mandelic acid dehydrogenase, and D-mandelic acid dehydrogenase or D-mandelic acid reductase to obtain R-mandelic acid. The method provided by the invention can be used for in-vitro preparation of single-configuration S-mandelic acid or R-mandelic acid; meanwhile, the enzymes can be combinedly over-expressed to ferment glucose, thereby obtaining S-mandelic acid or R-mandelic acid; and the method can be used for chirally splitting DL-mandelic acid to generate R-mandelic acid or S-mandelic acid, or converting S-mandelic acid into R-mandelic acid. When being used for synthesizing S-mandelic acid or R-mandelic acid by fermentation and culture, the method is sustainable and can lower the cost; and in addition, the method is mild, and friendly to environment and human.

Description

technical field [0001] The invention belongs to the field of bioengineering, in particular to a method for obtaining chiral monomer mandelic acid. Background technique [0002] Mandelic acid (Mandelic acid, MA), also known as mandelic acid, phenylglycolic acid, α-hydroxyphenylacetic acid. It is widely used in organic synthesis and drug production, and is an important intermediate of urinary tract bactericide urotropine mandelate, peripheral vasodilator cyclomandelate, eye drops oxybenzazole and urotropine antispasmodic agents . Mandelic acid is a chiral molecule with two configurations: R-mandelic acid and S-mandelic acid, the structural formula of which is as follows: [0003] [0004] R-mandelic acid S-mandelic acid [0005] Mandelic acid with a single configuration is a very important chiral intermediate in asymmetric synthesis reactions, and is widely used in the asymmetric synthesis of optically pure amino acids, angiotensin-converting enzyme inhibitors, and coenz...

Claims

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Application Information

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IPC IPC(8): C12P7/42C12N15/09C12N15/52C12N1/21C12P41/00C12R1/19
Inventor 杨晟孙周通姜卫红
Owner SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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