Catalytic Performance Of Foldon-mediated Oligomerization Of Feruloyl Esterase

Feruloyl esterase?FAE,EC 3.1.1.73?is a subclass of carboxylesterase,which hydrolyzes the ester bonds between ferulic acid and polysaccharides from plant cell walls,making the lignocellulosic raw materials loose,thus releasing of ferulic acid.FAE has broad application prospects,such as the food fields,feed fields,medicine fields,paper fields,cosmetics fields,and the biosynthesis fields.At present,there is still much space for improvement in the catalytic performance of FAE,so the transformation of FAE has far-reaching significance.In this paper,based on the oligomerization domain foldon can trigger the target enzyme to form oligomers spontaneously,we modified the FAE by fusing a short peptide foldon at the C-terminal,we explore the enzymatic activity,enzymatic properties,and oligomerization structure of the recombinant FAE.The main research results were as follows:Firstly,we retrieved the FAE with the ID O42807 from the Uniprot database.The amino acid sequence of FAE O42807 was converted into fae gene sequence according to the codon bias preference of P.pastoris.The fae and fae-foldon gene sequences were chemically synthesized and ligated into plasmid pPIC9K,then expression vector pPIC9K-fae and pPIC9K-fae-foldon were successfully constructed.The expression plasmid was transformed into P.pastoris GS115 and then verified by PCR.Finally,genetically engineered bacteria GS1115/pPIC9K-fae and GS1115/pPIC9K-fae-foldon was obtained.Secondly,the His-tagged primers were designed and cloned into the expression vectors pPIC9K-fae and pPIC9K-fae-foldon.The expression vectors pPIC9K-His-fae and pPIC9K-His-fae-foldon were successfully constructed,and transformed into P.pastoris GS115.After induction culture for four days,the fermentation broth was collected by Ni chromatography column.The SDS-PAGE analysis revealed a single band of GS115/pPIC9K-His-fae,and the apparent molecular weight was 40 kD.The SDS-PAGE analysis revealed three bands of GS115/pPIC9K-His-fae-foldon,and they were located at 45 kD?named pre-olig-FAE₂?,11 0kD?named tri-FAE₂?and 240 kD?named six-FAE2?.Mon-FAE2 had a FAE enzyme activity of 251.09±7.61 U/L and a specific activity of 0.36±0.011 U/mg.Multi-FAE₂?mixture of mon-FAE₂,tri-FAE₂ and six-FAE₂?had a FAE enzyme activity of 440.78±12.7 U/L and a specific activity of1.63±0.045 U/mg.Thirdly,to reduce the steric hindrance of FAE formation oligomerization.We inserted a linker of 1.63 kD between the fae and foldon genes.The expression vector pPIC9K-His-fae-linker-foldon was successfully constructed,and transformed into P.pastoris GS115.After induction culture for four days,the fermentation broth was collected by Ni chromatography column.The SDS-PAGE analysis revealed three band of GS115/pPIC9K-His-fae-linker-foldon,and they are located at 45 kD?named pre-olig-FAE₃?,110 kD?named tri-FAE₃?and 240 kD?named six-FAE₃?.The six-FAE₃ had a FAE enzyme activity of 334.14±11.24 U/L and a specific activity of 1.74±0.063 U/mg.Fourthly,the enzymatic properties of the recombinant FAE were determined and the results showed as following:the optimal reaction temperature of FAE for all three structures was 50°C,and the half-lives of mon-FAE₂,multi-FAE₂ and six-FAE₃ at 50°C were 99.6 min,129 min and 222 min,respectively.The optimum pH value of mon-FAE₂ was 6.0,and the stability was better at pH 6.0.The optimum pH value of multi-FAE₂ and six-FAE₃ was 5.0,and the stability was better at pH 5.0.For mon-FAE₂,Mn²⁺and Mg²⁺promoted its enzyme activity,K⁺,Ca²⁺,Fe³⁺and Cu²⁺inhibited its enzyme activity,while Zn²⁺significantly inhibited its enzyme activity.For multi-FAE₂ and six-FAE₃,Mn²⁺,Zn²⁺,Ca²⁺,Mg²⁺,Fe³⁺and Cu²⁺promoted its enzyme activity,and K⁺has a certain inhibitory effect on its enzyme activity.The specific activity of the multi-FAE₂was significantly higher than that of mon-FAE₂ with 3.53 folds,the K_m value was decreased by 77.36%,and the k_cat/K_m value was increased by 7.57 folds.The specific activity of the six-FAE₃ was significantly higher than that of the mon-FAE₂ by 3.83folds,the Km constant was decreased by 75.16%,and kcat/Km was increased by 8.67folds.Finally,at the temperature of 25°C,we observed that the multi-FAE₂ and the six-FAE₃ formed a protein aggregate of an oligomerization structure by transmission electron microscopy.The results of the dynamic light scattering showed that the particle size of mon-FAE₂ remained unchanged with the increase of temperature,while the proportion of aggregates of the tri-FAE₂ and the six-FAE₃ decreases gradually,up to65°C,the size of the tri-FAE₂ and the six-FAE₃ was almost the same as that of mon-FAE₂.The experimental results of this paper provide basic theoretical research for the transformation of FAE molecular level.The method of transforming enzyme is simple and efficient,and it does not need to know the 3D structure of the enzyme in advance,and would have good application prospects.