Heterologous Expression And Application Of Esterase And Catechol 1,2-dioxygenase From The Fecal Microbiome

Phthalic acid esters belong to aromatic compounds,which are stable in structure,difficult to degrade and harmful to the environment.Esterase and catechol 1,2-dioxygenase are key enzyme in the degradation of phthalic acid esters.In this paper,the esterase gene EstCf8,EstA12 and catechol 1,2-dioxygenase gene catPLCgl were amplified by PCR from the fecal microbial metagenome fosmid library of Nycticebus Pygmaeus.The genes were heterologous expression by Escherichia coli BL21(DE3).The esterase Est Cf8?EstA12 and catechol 1,2-dioxygenase catPLCgl were purified and characterized.Further,we did some exploration to consider and discuss the potential possibility in biodegradation of environmental pollutants.The results were as follows:(1)The optimum substrate of esterase EstCf8 was p-NPC2 based on the analysis results of kinetic parameters Km,Vmax and kcat with the values of 0.85 mM?99.0 ?mol/min/mg and 202.2 /s respectively.EstCf8 showed highest activity at pH 7.5 and remained more than 63.9% activity of the original activity after incubated for 1 h in the buffer solution of pH 6.0 – 10.0.The optimal temperature was 35 °C and remained more than 58.8% at the temperature range of 0 – 40 °C.Moreover,the residual activity was above 80% after incubated for 1 h at 40 °C,but when the temperature reached to 50 °C the half-life was 10 min.Metal ions or organic reagents such as Al3+?Ni2+?Ag+?Pb2+?Hg2+?Cu2+?Zn2+?SDS?CTAB have negative effect on the activity of EstCf8,others did barely.EstCf8 could hydrolyzed DBP?DEP or DiBP and turned into phthalic acid.(2)The optimum substrate of esterase EstA12 was p-NPC4 based on the analysis results of kinetic parameters Km,Vmax and kcat with the values of 0.15 mM?10.9 ?mol/min/mg and 3.9 /s respectively.EstA12 showed highest activity at pH 8.5 and remained more than 62.9% activity of the original activity after incubated for 1 h in the buffer solution of pH 2.2 – 8.0,and remained more than 40.9% activity of the original activity after incubated for 1 h in the buffer solution of pH 8.0 – 12.0.The optimal temperature was 40 °C and remained more than 50.71% at the temperature range of 10 – 50 °C,and there are 33.1% and 38.8% enzyme activity in 0 °C and 100 °C.The residual activity was above 71.4% and 55.3% after incubated for 1 h at 40 °C and 80 °C,respectively.Metal ions or organic reagents hardly effect the activity of EstA12,especially Mn2+?Ca2+?Fe3+?K+?Na+ with a little boost for activity,other inhibitor did not inhibt the enzyme activity.EstA12 could hydrolyzed DBP?DEP or DiBP and turned into phthalic acid.(3)The optimum substrate of catechol 1,2-dioxygenase CatPLCgl was catechol based on the analysis results of kinetic parameters Km,Vmax and kcat with the values of 24.9 ?mol/L,8.3 mmol/min/g and 13.6 /s respectively.CatPLCgl showed highest activity at pH 7.0 and remained more than 58% at the pH range of 6.0 – 9.0.Remained more than 90% activity of the original activity after incubated for 1 h in the buffer solution of pH 7.0 – 10.0.The optimal temperature was 40 °C.Remained activity at the temperature range of 0 – 60 °C,and there are 12% and 20% enzyme activity in 0 °C and 100 °C respectively.Moreover,the residual activity was almost 100% after incubated for 1 h at 30 °C and 40 °C,but when the temperature reached to 50 °C the residual activity was 46% after incubated for 10 min.The enzyme was stable at 25 °C and 40 °C,retaining nearly 100% activity after pre-incubated for 210 h.Metal ions or organic reagents such as Fe2+,Hg2+,Cu2+,Triton X-100,SDS and Ag+ have Strong inhibitory effect on the activity of CatPLCgl,others did lesser.In short,this study obtained two kinds of esterases which chould be used in degradation of environmental pollutants like phthalate esters.Especially,EstA12 showed good thermostability,pH stability and strong resistance to metal ions.Expect to see EstCf8 and EstA12 will play an important role in environmental management base on their admirable properties.In addition,researches showed that Cat PLCgl was a alkali resistance and mesophilic catechol 1,2-dioxygenase,it provided the possibility to produce cis,cis-muconate or to decompose catechol in the environment.