L-threonine aldolase mutant and method for preparing L-syn-p-methylsulfonyl phenyl serine

一种甲砜基苯丝氨酸、苏氨酸醛缩酶的技术，应用在酶工程领域，能够解决理论收率只有50％、无效对映体难套用、原子利用率不高等问题，达到产品光学纯度高、原子利用率高、污染小的效果

Active Publication Date: 2021-08-13
ZHEJIANG UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This process mainly has the following disadvantages: the theoretical yield is only 50%, the invalid enantiomer is difficult to apply mechanically, and the process is relatively complicated
This process has the following disadvantages: the production process is complicated, the utilization rate of atoms is not high, the theoretical yield can only reach 50%, and the problem of copper salt environmental pollution will also occur
Therefore, there is no research report on the direct application of L-threonine aldolase to synthesize high-chiral purity L-syn-p-thiamphenicol phenylserine

Method used

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  • L-threonine aldolase mutant and method for preparing L-syn-p-methylsulfonyl phenyl serine
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  • L-threonine aldolase mutant and method for preparing L-syn-p-methylsulfonyl phenyl serine

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Embodiment 1: Construction of wild enzyme engineering bacteria

[0050] Enter keywords such as threoninealdolase in the National Coalition Building Institute (NCBI) database to search and select the amino acid sequence WP_015261381.1 (derived from Desulfitobacterium dichloroeliminans (DdLTA)) and SIS82838.1 (derived from Chryseobacterium chaponense (CcLTA)), WP_016204489.1 (derived from Bacillus nealsonii (BnLTA)), WP_023973138.1 (derived from Clostridium beijerinckii (CbLTA)), WP_077361571.1 (derived from Clostridium saccharoperbutylacetonicum (CsLTA)), 919 (derived from WP4969 in Cellulosilyticum sp.I15G10I2(CpLTA)). The six amino acid sequences were converted into nucleotide sequences according to the codon preference of Escherichia coli (the nucleotide sequences are shown in SEQ ID No. 1-6). The six nucleotide sequences were fully synthesized by chemical method (Anhui General Biology), and integrated between the multiple cloning sites BamH I and Hind III of the exp...

Embodiment 2

[0051] Embodiment 2: the construction of mutant enzyme

[0052] 1. Activation of Engineering Bacteria and Plasmid Extraction

[0053] All engineering bacteria (constructed and obtained in Example 1) were activated and cultivated using LB medium, and the formula was: peptone 10g / L, yeast powder 5g / L, NaCl 10g / L, dissolved in deionized water and then constant volume, 115 Sterilize at ℃ for 30min, and set aside. The solid medium is LB medium with 2% agar added.

[0054] The preserved engineered bacteria glycerol tube was inserted into a test tube containing 10 mL of LB medium, and cultured at 37°C and 200 rpm for 12 hours. After obtaining the cultured cells, plasmid extraction was carried out according to the operating instructions of the Axygen plasmid extraction kit. The resulting plasmid can be directly used for point mutation or stored at -20°C for a long time.

[0055] 2. Gene site-directed mutation

[0056] The gene mutation adopts the method of whole plasmid PCR to obta...

Embodiment 3

[0071] Embodiment 3: the preparation of the culture of thalline and crude enzyme liquid

[0072] 1. Bacteria culture

[0073] Composition of LB liquid medium: peptone 10g / L, yeast powder 5g / L, NaCl 10g / L, dissolved in deionized water and then constant volume, sterilized at 115°C for 30min, ready for use.

[0074] After the engineering bacteria containing the L-threonine aldolase gene were activated by streaking on a plate, a single colony was picked and inoculated into 5 mL LB liquid medium containing 50 μg / mL kanamycin, and cultured with shaking at 37°C for 12 hours. Transfer 2% of the inoculum into 50 mL of fresh LB liquid medium also containing 50 μg / ml Kan, culture with shaking at 37 °C until the OD600 reaches about 0.6, add IPTG to a final concentration of 0.5 mM, and induce culture at 28 °C 10h. After the cultivation, the culture solution was centrifuged at 10,000 rpm for 10 min, the supernatant was discarded, and the bacterial cells were collected, and stored in a -80...

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Abstract

The invention discloses an L-threonine aldolase mutant and a method for preparing L-syn-p-methylsulfonyl phenyl serine. The L-threonine aldolase mutant is obtained by mutating wild type L-threonine aldolase, and L-syn-p-methylsulfonyl phenyl serine can be generated by a catalytic condensation reaction by taking glycine and p-methylsulfonyl benzaldehyde as substrates and taking pyridoxal phosphate as a coenzyme. The advantages of using the L-threonine aldolase mutant for producing L-syn-p-methylsulfonyl phenyl serine are that 1, the production process is simple, and the reaction conditions are mild; 2, the selectivity of the L-threonine aldolase is high, the optical purity of the product is high, and resolution is not needed; 3, the atom utilization rate is high and can reach 100% theoretically; 4, the production process is environmentally friendly, pollution is small, and the green chemistry concept is met; and 5, the product is simple to separate and purify.

Description

[0001] This application is a divisional application of "A L-threonine aldolase mutant, gene and method for preparing L-syn-p-thymphenylphenylserine". The filing date of the original application is January 17, 2020 , the application number is 2020100508531. technical field [0002] The invention relates to the technical field of enzyme engineering, in particular to an L-threonine aldolase mutant, a gene and a method for preparing L-syn-p-thymphenylphenylserine. Background technique [0003] L-syn-p-thymphenylphenylserine is an important pharmaceutical intermediate used in the synthesis of various antibiotics. P-thiamphenicyl phenylserine has two chiral centers and has four isomers, namely: L-syn-p-thiamphenicyl phenylserine, L-anti-p-thiamphenicyl phenylserine, D-syn-p Thiysulfonyl phenylserine, D-anti-p-thysulfonyl phenylserine ( figure 1 ). [0004] At present, there are mainly two production methods for L-syn-p-thymphenylphenylserine. One is chemical synthesis, using c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/88C12N15/60C12N15/70C12N1/21C12P13/04C12R1/19
CPCC12N9/88C12N15/70C12P13/04C12Y401/02005
Inventor 吴坚平郑文隆陈开通徐刚杨立荣
Owner ZHEJIANG UNIV
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