Cloning,Expression And Enzymatic Properties Of The Enzymes With Ability To Hydrolyze Chlorogenic Acid Form Aspergillus Aculeatus SD14

The enzymes with chlorogenic acid hydrolysis activity are a kind of enzymes which can hydrolyze the natural substrate chlorogenic acid?CGA?.Two kinds of enzymes which have been reported with CGA hydrolysis activity are chlorogenic acid hydrolase and ferulic acid esterase.These enzymes are the optimum to accelerate biodegradation rate of CGA and has wide application prospect in food and medicine industry.In our previous study,Aspergillus fungus strain SD14 with CGA hydrolysis activity was obtained by screen and mutagenesis.The wild-enzyme obtained by separation and purification has good stability and affinity.However,the CGA hydrolysis activity of this wild-enzyme was low?4.35 U/g?.In this study,the whole genome of Aspergillus fungus strain SD14 was sequenced.Four genes which could encoding enzymes with CGA hydrolysis activity were cloned and induced condition optimization.Finally,two of them achieved CGA hydrolysis activity and named as reFAE1 and reFAE2.Enzymatic properties of reFAE1 and reFAE2 were studied.The main content and results were as follows:?1?The phylogenetic trees were constructed by comparing the ITS region and the gene sequence information of calmodulin and?-tubulin.Finally,this strain was identified as Aspergillus aculeatus,which has closer relationship with Aspergillus japonicus.The whole genome sequenced and assembly of A.aculeatus SD14 was performed by the third generation single-molecule sequence technology.The total length of the genome was 36.18 Mb and include18 scaffolds and 50.11%content of GC.?2?With the cDNA template obtained by reversed transcription total RNA of A.aculeatus SD14,primers were designed to clone four genes?GME3292,7542,2693 and 11122?encoding enzymes which could have CGA hydrolysis activity.The soluble expression in E.coli and purification of encoded proteins were achieved.Recombinase expressed by two genetic engineering bacteria?GME3292-pET28a?+?/E.coli BL21?DE3?and GME11122-pET28a?+?/E.coli BL21?DE3??were detected with CGA hydrolysis activity and named as reFAE1 and reFAE2.?3?Two recombinases with CGA hydrolysis activity were optimized from IPTG concentration,OD₆₀₀,induction time and temperature.The optimized ferment condition of the reFAE1 has been determined:IPTG concentration,OD₆₀₀,induction time and temperature were0.04 mmol/L,1.2(OD₆₀₀),20 h and 20°C,respectively.The optimized ferment condition of the reFAE2 has been determined:IPTG concentration,OD₆₀₀,induction time and temperature were0.02 mmol/L,1.2(OD₆₀₀),20 h and 20°C,respectively.The CGA hydrolysis activity of two recombinases were 246.37 U/g and 340.95 U/g,which was 13.33&11.62 folds of the CGA hydrolysis activity before optimize and was 56.64&78.38 times of the original strain,respectively.?4?These recombinases were separated and purified by affinity-Ni²⁺column chromatography.The purified recombinases?reFAE1 and reFAE2?were homodimers with the molecular mass of 55 kDa.The recombinases have the highest hydrolysis activity for MCA,MpCA take the second place.The hydrolysis activity of MFA and MSA is the worst.The hydrolysis activity of natural substrate CGA was between the MCA hydrolysis activity and MpCA hydrolysis activity.According to the substrate specificity of recombinases,it is presumed that both of them are the C-type ferulic acid esterase and could hydrolyze MFA,MCA,MSA and MpCA.?5?The optimum temperatures of reFAE1 and reFAE2 for hydrolysis CGA were 60°C and50°C,respectively.The recombinases had good thermal stability at 30⁵0°C and they were stable at pH 3.0⁸.0 and pH 3.0¹0.0,respectively.EDTA treatment had no effect on the recombinases,while Mn²⁺and Ca²⁺had a positive effect on reFAE2.Monovalent metal ions had no significant effects on the CGA hydrolysis activity,and the enzyme activity of the recombinases were almost completely lost after trivalent metal ions treatment.The recombinases have good stability and strong affinity,and the Km value against CGA were 79.85?mol and 105.98?mol,respectively...