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D-fructose-6-phosphate aldolase A mutant, recombinant expression vector, genetically engineered bacterium and application and reaction product thereof

A technology of phosphate aldolase and genetically engineered bacteria, which is applied in the fields of genetic engineering and enzyme engineering, can solve the problems that the activity cannot meet the requirements of industrial production, reduce the reaction activity of cinnamaldehyde and replace cinnamaldehyde, and achieve good industrial application development prospects. Efficiently catalyzing asymmetric direct aldol condensation reaction, easy to prepare

Active Publication Date: 2017-05-24
杭州馨海酶源生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, it may be due to the special structure of cinnamaldehyde and substituted cinnamaldehyde, which has a double bond conjugated to the benzene ring, and the resulting electron delocalization effect reduces the reactivity of cinnamaldehyde and substituted cinnamaldehyde as substrates, causing FSAA to catalyze the above-mentioned The activity of the reaction is far from meeting the requirements of industrial production. Therefore, it is necessary to improve the catalytic efficiency of the enzyme through related technologies to fully exploit its application value.

Method used

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  • D-fructose-6-phosphate aldolase A mutant, recombinant expression vector, genetically engineered bacterium and application and reaction product thereof
  • D-fructose-6-phosphate aldolase A mutant, recombinant expression vector, genetically engineered bacterium and application and reaction product thereof
  • D-fructose-6-phosphate aldolase A mutant, recombinant expression vector, genetically engineered bacterium and application and reaction product thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Embodiment 1: the construction of mutant

[0044] Using the oligonucleotide fragment containing the mutation point as a primer (Table 1), the recombinant plasmid pET-30a containing the FSAA gene was amplified by the QuickChangeTM method (Stratagene, La Jolla, CA).

[0045] Table 1 Mutant construction primers

[0046]

[0047] a Mutation sites are underlined

[0048] PCR reaction system: 5×PrimerSTAR buffer (Mg2+plus), 5 μL; dNTPs (2.5 mM each), 2.0 μL; upstream primer (10 μM), 1.0 μL; downstream primer (10 μM), 1.0 μL; recombinant plasmid template, 15 ng; PrimerSTARpolymeraseTM HS (2.5U / μL), 0.5 μL; add ddH2O to a total volume of 25 μL.

[0049] PCR program: (1) 98°C, 1min; (2) 98°C, 10s; (3) 55°C, 10s; (4) 72°C, 7min. Steps (2)-(4) were cycled 20 times and then cooled to 4°C.

[0050] After the PCR product is washed, it is digested with the restriction endonuclease DpnI that specifically recognizes the methylation site to degrade the template plasmid. Enzyme d...

Embodiment 2

[0052] Example 2: Induced expression of FSAA mutants

[0053] The engineered bacteria constructed in Example 1 were inoculated into LB liquid medium containing 50 μg / mL kanamycin and 20 μg / mL chloramphenicol, cultivated overnight at 37° C., and then transferred to In 100mL LB medium containing 50μg / mL kanamycin and 20μg / mL chloramphenicol, culture at 37°C and 220rpm until the cell concentration OD600 to about 0.6, add final concentration of 0.1mM IPTG and 1mg / mL L -Arabinose, after induction culture at 26°C for 7 hours, centrifuge at 4°C and 4000rpm for 10 minutes to collect the bacterial cells, and store at -80°C for future use.

Embodiment 3

[0054] Embodiment 3: Separation and purification of FSAA mutant

[0055] The thalli cells that embodiment 2 collects are suspended in 20mL Na 2 HPO 4 -NaH 2 PO 4 In buffer solution (100mM, pH 8.0), shake well and then crush under ultrasonic wave (effective time 8min). The broken liquid was centrifuged at 12,000 rpm for 10 min to remove cell debris, and the supernatant (crude enzyme liquid) was collected for subsequent separation and purification of the enzyme. The purification column is a HisTrap (GE Healthcare) affinity chromatography column with a packing volume of 5 mL. First equilibrate the Ni-NTA column with a loading equilibration buffer (20 mM sodium phosphate, 500 mM NaCl and 20 mM imidazole, pH 7.4). Load crude enzyme solution at a rate of 1 / min, elute with loading equilibration buffer to remove unadsorbed protein, and finally collect target protein by eluting with elution buffer (20mM sodium phosphate, 500mM NaCl and 500mM imidazole, pH 7.4) . The enzyme soluti...

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Abstract

The invention provides an FSAA mutant with remarkably increased catalytic activity, a nucleotide sequence thereof, a recombinant expression vector containing a corresponding mutant gene and a genetically engineered bacterium. A chiral product with high optical purity can be prepared by aldol condensation reaction in which cinnamyl aldehyde / alpha-bromocinnamaldehyde / 4-nitrocinnamaldehyde / pyridine-2-formaldehyde and hydroxyacetone (HA) are asymmetrically catalyzed by the FSAA mutants or the genetically engineered bacteria containing a corresponding mutant protein, the chiral product can serve as a valuable chiral building block, and important potential application value in the field of medicines is realized.

Description

technical field [0001] The invention belongs to the field of genetic engineering and enzyme engineering, and specifically relates to molecular transformation of D-fructose-6-phosphate aldolase A, obtaining mutants, recombinant expression vectors, genetic engineering bacteria and asymmetric catalytic cinnamaldehyde / α-bromocinnamaldehyde Application in the aldol condensation reaction of 4-nitrocinnamaldehyde / pyridine-2-carbaldehyde and hydroxyacetone (HA) to prepare optically active products. Background technique [0002] Asymmetric direct aldol condensation reaction is a very important class of C-C addition reaction. The products are natural polyols or brand new polyols. Since the hydroxyl group can be easily converted into a variety of other functional groups, these polyhydroxy small molecules become one of the most important chiral building blocks, especially in the field of medicine with important application value. Among them, (3S,4R,E)-3,4-dihydroxy-6-phenyl-5-hexen-2-...

Claims

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Application Information

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IPC IPC(8): C12N9/88C12P7/26C12P17/12
Inventor 于洪巍杨小红
Owner 杭州馨海酶源生物科技有限公司
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