High-stereoselectivity R transketolase mutant as well as coding gene and application thereof

A stereoselective, gene-encoding technology, applied in the field of enzyme engineering, can solve problems such as low catalytic efficiency

Pending Publication Date: 2022-08-09
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In 2010, the Helen C.Hailes research group reported the site-directed mutation of D469 and F434 on the transketolase derived from Escherichia coli, and realized the catalysis of p-benzaldehyde and m-hydroxybenzaldehyde, but the catalytic efficiency was low, and the conversion rate was ≤10%.
Therefore, there are no reports of transketolases that can catalyze aromatic aldehydes, especially benzaldehyde derivatives, to produce R-configured hydroxyketones.

Method used

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  • High-stereoselectivity R transketolase mutant as well as coding gene and application thereof
  • High-stereoselectivity R transketolase mutant as well as coding gene and application thereof
  • High-stereoselectivity R transketolase mutant as well as coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1: Site-directed and saturation mutagenesis and screening

[0037] Site-directed mutagenesis and saturation mutagenesis of natural transketolase derived from prokaryotic Escherichia coli and screening for R-transketolase with high activity and high stereoselectivity. The nucleotide sequences of the primers used are shown in Table 1 below. In Table 1, N represents any base of A, T, C and G, K represents the base of T or G, and the NNK degenerate codon can encode 20 random amino acids . M stands for A or C base, and MNN is complementary to NNK.

[0038] Table 1 Primer Nucleotide Sequences

[0039]

[0040] (1) Using the recombinant plasmid pET28a-TK as a template, the nucleotide sequence of which is shown in SEQ ID No. 3, and using D469T-F and D469T-R as primers, Phanta Max polymerase (purchased from Novozan) was used to carry out PCR amplification (95°C for 5 min; 95°C for 30s, 65°C for 30s, 72°C for 7.5min, 34 cycles; 72°C for 10min); PCR products were dig...

Embodiment 2

[0045] Example 2: Expression and purification of transketolase mutants

[0046] The expression and purification method of the mutant EcTK1_YYH: pick a single colony of E. coli BL21 (DE3) containing the recombinant plasmid on LB solid medium and inoculate it into 40ml LB liquid medium (containing 50μg / ml kanamycin antibiotic) , 37 ℃, 220rpm culture overnight. Transfer 7.5ml of bacterial culture solution to a 2L shake flask containing 500ml of liquid LB medium, inoculate two flasks, culture at 37°C, 220rpm until OD600 reaches 0.6-0.8, add 0.4mM IPTG for induction, and induce culture at 30°C, 200rpm 5h. 1 L of fermentation broth was collected, cells were collected by centrifugation at 5000 rpm for 20 min, and the collected cells were resuspended in 30 mL of nickel column binding buffer and placed in an ice-water mixture. Ultrasonic fragmentation conditions: work for 5s, pause for 10s, and a total of 30min. The crushed mixture was centrifuged at 12,000 rpm for 1 h, and the supe...

Embodiment 3

[0047] Example 3: Investigating the stereoselectivity of EcTK1 mutants catalyzing p-methylsulfonylbenzaldehyde to obtain products

[0048] The highly stereoselective R-transketolase mutant EcTK1 catalyzes the detection process of p-methylsulfonylbenzaldehyde to obtain highly stereoselective products, and the involved chemical reactions include transketone reaction and transamination reaction.

[0049] like figure 1 As shown, the principle of the transketone reaction is to use p-methylsulfonylbenzaldehyde as the substrate, the substrate is represented by "1", the transketolase mutant is the catalyst, the catalyst is represented by "TK", LiHPA, MgCl 2 In the presence of ThDP, S-dihydroxyketone and R-dihydroxyketone products are generated, S-dihydroxyketone is represented by "2a:S", and R-dihydroxyketone product is represented by "2b:R".

[0050] like figure 2 As shown, the principle of the transamination reaction is to instantaneously convert the dihydroxyketone, the product ...

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Abstract

The invention discloses a high-stereoselectivity R transketolase mutant as well as a coding gene and application thereof, and relates to the technical field of enzyme engineering, the amino acid sequence of the high-stereoselectivity R transketolase mutant is SEQ ID No.1, and the nucleotide sequence of the coding gene is SEQ ID No.2; a recombinant expression vector containing the encoding gene; a host cell containing the recombinant expression vector; a method for preparing the high stereoselectivity R transketolase mutant; the R transketolase mutant is applied to catalysis of aromatic aldehyde to form R-aromatic dihydroxy ketone with high stereoselectivity. The coding gene is applied to catalysis of aromatic aldehyde to form high-stereoselectivity R-aromatic dihydroxy ketone. The stereoselectivity of p-methylsulfonyl phenyl dihydroxy ketone generated by catalysis of the mutant EcTK1YYH is 95.2% ee (R), and the mutant EcTK1YYH has high activity and high stereoselectivity.

Description

technical field [0001] The present invention relates to the technical field of enzyme engineering, in particular to a highly stereoselective R-transketolase mutant and its encoding gene and application. Background technique [0002] Transketolase is a ubiquitous thiamine pyrophosphate-dependent enzyme. It links the non-oxidative pentose phosphate pathway and the tricarboxylic acid cycle. Transketolase catalyzes the reversible transketone reaction, which transfers the two-carbon unit of the ketol donor to the aldehyde acceptor to form chiral dihydroxyketones. In the transketone reaction, β-hydroxypyruvate is often used as the ketol donor because β-hydroxypyruvate is transketoneized to generate volatile carbon dioxide, making the reaction irreversible. Recently, the detection methods of dihydroxyketone compounds, including colorimetric method and HPLC method, have been reported in the literature, and chiral GC and HPLC methods have been established to detect the enantioselec...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/10C12N15/54C12N15/70C12N1/21C12P7/26C12R1/19
CPCC12N9/1022C12N15/70C12P7/26C12Y202/01001
Inventor 林双君刘琦邓子新黄婷婷
Owner SHANGHAI JIAO TONG UNIV
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