In Vitro Expression And Enzymatic Properties Of DabA Enzyme In Pseudo-nitzschia Multiseries

Diatoms are the main producers of harmful algal blooms.Pseudo-nitzschia,a common group of diatoms,can produce a potent neurotoxin,domoic acid（DA）.DA as a neurotransmitter can bind to glutamate receptors and cause neuroexcitation and neurotoxicity and has important utility in neurophysiology,clinical treatment and insecticide production.It has been shown that L-glutamic acid and geranyl pyrophosphate（GPP）are catalyzed by terpene cyclase（DabA）to produce the first intermediate metabolite,N-geranyl-L-glutamic acid（L-NGG）.The study of L-NGG contributes to an in-depth understanding of the metabolic mechanism of DA biosynthesis and provides the basis for efficient DA synthesis.Therefore,the enzymatic properties of the enzyme-DabA in the condensation reaction need to be investigated in order to improve the yield of L-NGG as well as the efficiency of synthesis and to establish an efficient synthesis channel for L-NGG.In this study,we constructed E.coli genetically engineered bacteria containing the dabA gene based on the initiation reaction of the DA synthesis pathway,and explored the enzymatic properties of DabA through metal ion modification and amino resin immobilization.The main research contents and results are as follows:（1）Construction of genetically engineered bacteria.A recombinant vector was constructed by introducing the gene encoding DabA（dabA）from Pseudo-nitzschia multiseries into the prokaryotic expression vector p ET30a（+）,transferred into E.coli BL21（DE3）and screened for positive clones by colony PCR.The plasmids were then extracted after expanded culture and validated by enzymatic digestion and PCR.The results showed that the target gene was 1461 bp in size and the plasmid was 6690 bp in size,which proved that the target gene had been successfully inserted into the vector and the genetically engineered bacteria were successfully constructed.（2）Expression,purification and structural elucidation of DabA.The expression of DabA was induced in E.coli genetically engineered bacteria,and DabA was identified by SDS-polyacrylamide gel electrophoresis（SDS-PAGE）analysis and mass spectrometry.The results showed that DabA is 60 k Da in size with a relative expression of 62.03%.The protein,which is found in Pseudo-nitzschia multiseries,is known as N-prenyltransferase,with geranyl S-thiolodiphosphate（GSPP）and Mg²⁺as cofactors.A combination of NCBI-Blastp,Clustal X,ESPript3.0,Predict Protein and SWISS-MODEL were used to structurally resolve DabA.The results showed that DabA contains all 20 standard amino acids,with the highest proportion being Leucine（L）.It has 16 protein binding sites and 8 DNA binding sites.At the active site,GSPP forms a complex structure through three salt bridges,three hydrogen bonds and six hydrophobic interaction regions as well as Mg²⁺through two ionic bonds.（3）Exploration of the enzymatic properties of DabA.A temperature gradient of 20-50℃ and a pH gradient of 6-9 were designed to investigate the optimum temperature and the optimum pH of the DabA enzyme.The results showed that the DabA enzyme had the highest enzyme activity of0.31 U/mg at a temperature of 40℃ and a pH of 8.0.The results of the determination of the kinetic parameters of the enzyme reaction showed:K_m=1.76x10³μmol·L^-1,V_max=289.0μmol·L^-1·min^-1,k_cat=14.45 min^-1,k_cat/K_m=8.21x10^-3μmol^-1·L·min^-1.The DabA catalytic effect obtained in this study was better in comparison with existing studies.（4）A study of the effect of metal ion modification on enzymatic properties.The optimum temperature and pH of the enzyme did not change significantly before and after the metal ion replacement modification of DabA using three metal ions,Ca²⁺,Mn²⁺and Zn²⁺.The enzyme activity was highest when Mg²⁺was used as a cofactor for DabA before the modification;Mn²⁺decreased enzyme activity by 15.8%compared to Mg²⁺when used as a cofactor for DabA;Zn²⁺decreased enzyme activity by 73.6%compared to Mg²⁺when used as a cofactor for DabA;Ca²⁺cannot be used as a cofactor for DabA.（5）A study of the effect of amino resin immobilization on enzymatic properties.The immobilization experiment of crude enzyme was carried out using amino resin as carrier and glutaraldehyde as activator.By measuring the change of enzyme activity of crude and immobilized enzyme under different temperature and pH conditions,the results showed that for both the crude and immobilised enzymes,the optimum temperature was 40℃ and the optimum pH was 8.0.The recovery of enzyme activity was 64.9%and the amino resin immobilisation significantly improved the stability of the enzyme under high temperature and high pH conditions.Based on the above results,this study successfully constructed E.coli genetically engineered bacteria expressing DabA,revealed the enzymatic properties of DabA after metal ion modification and amino resin immobilization,established a fast and effective genetic engineering synthesis channel for the synthesis of the key intermediate product L-NGG,and laid the foundation for efficient synthesis of DA based on genetic engineering means.