Cloning, Heterologous Expression And Enzymatic Properties Of Fructose-1,6-bisphosphate Aldolase From Euphausia Superb

Euphausia superb-derived enzymes have unique biological properties due to the cold,harsh environment of Antarctic waters and have great potential for practical application.Fructose-1,6-bisphosphate aldolase（FBA）is an important enzyme involved in the Calvin cycle in glycolysis,gluconeogenesis,pentose phosphate pathway and photosynthesis.FBA is also a DHAP-dependent aldolase,which can use DHAP as a substrate to catalyse aldol condensation reactions to synthesise rare sugars that are found in the natural environment,which are known for their antioxidant,anti-inflammatory and anti-ageing properties.This study aims to carry out the research of gene cloning,heterologous expression,enzymatic properties and catalytic performance of the FBA of Euphausia superb origin,and the specifics are as follows:1.After RNA extraction,cDNA cloning and transcriptome sequencing,the gene sequence of Euphausia superb fructose-1,6-bisphosphate aldolase was obtained,followed by Blast comparison and bioinformatics analysis,including basic physicochemical property analysis,structural domain analysis,phosphorylation and glycosylation site prediction,phylogenetic tree construction,structure prediction and plasmid map construction.The EsFBA gene was successfully expressed in Escherichia coli p ET-28a vector after cloning,transformation and validation,and was induced by shake flask fermentation and IPTG.The recombinant EsFBA was cultured in flasks,and the optimal conditions for enzyme expression were 0.55 m M IPTG and16 h of induction at 16°C,using a three-factor,three-level response surface method with IPTG concentration,induction temperature and induction time.2.The crude enzyme solution of EsFBA was purified by ion-affinity chromatography,and the molecular weight and purity of the protein were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis（SDS-PAGE）,which showed a clear single band with a molecular weight of about 45 k Da.The enzyme activity of EsFBA was 193.5 U/mg,which was higher than that of FBA from spinach,Ascaris suum,Staphylococcus aureus,Fritillaria spp.,blue-green algae,sea turtles and rabbits,etc.A three-factor,three-level response surface method with IPTG concentration,induction temperature and induction time was designed to screen the optimal enzyme expression conditions of 0.55 m M IPTG and 16 h induction at 16℃.The basic enzymatic properties of EsFBA showed that its optimum temperature and p H were 45℃and 7.0 respectively,and its enzyme activity was not affected by metal ions such as Cu²⁺,Mn²⁺,Mg²⁺,Ca²⁺,Zn²⁺and other metal ions,but it was significantly inhibited by borohydride,with an inhibition rate of 87%.The molecular docking results showed that six amino acid,Asp33,Lys107,Lys146,Glu187,Lys229and Ser301,were hydrogen-bonded to the substrate,and the targeted mutation of the above six amino acids showed that five amino acids,Lys107,Lys146,Glu187,Lys229and Ser301,had a greater effect on the enzyme activity.Lys146 and Lys229 had the greatest effect on the enzyme activity because of the hydrogen bonding at the five-membered ring break with the substrate,which was also consistent with the mutation results,and therefore it was inferred that Lys146 and Lys229 were the key amino acids affecting EsFBA activity.3.The"one-pot,four-enzyme"method was designed to catalyse the production of rare ketoses in accordance with the nature of fructose-1,6-bisphosphate aldolase as a DHAP-dependent aldolase.The synthesis of rare ketoses ware catalyzed using glycerol phosphate oxidase,catalase,acid phosphatase and EsFBA with D-glycerol triphosphate magnesium salt as the substrate and the addition of four fatty aldehydes,acetaldehyde,propionaldehyde,butyraldehyde and pentanal,which were detectedby HPLC and UPLC-MS.