Identification And Characterization Of A Novel Carboxylesterase From Bacterium Pantoea Ananatis Sd-1

As a large agricultural country,China has abundant of rice straw to be treated every year.Due to that the lignocellulose is the major component of rice straw,lignocellulosic enzymes produced by microorganisms play an important role in the degradation of rice straw and have a great application potential.In our previous study,we obtained a rice endophyte Pantoea ananatis Sd-1,which was effectively in rice straw degradation and was powerful in degrading cellulose and hemicellulose.The genome bioinformatic analysis revealed that Sd-1 possessed a large number of genes related to carbohydrates esterase,and two of the genes（CarE7 and CarE3）might also encode xylanase which degrades hemicellulose.Thus,in order to explore the unique enzymic resource of Sd-1,this work mainly involved in the following three aspects:（1）Exploration of the optimal conditions for xylanases and glucanase activities of Sd-1.Rice straw was used as the sole carbon source,and single-factor experiments showed that the activity of xylanase was much higher than glucanase and was obviously influenced by environmental factors.Furthur orthogonal experiment indicted that the optimal xylanases activity conditions were:the incubation at 30℃,rice straw content 1%,initial pH 7 and incubation for 4 days.The activity of xylanase in fermentation liquid could be up to 25.73 U/mL,which was significantly higher than that in reported fungus.（2）Prokaryotic expression and proteins purification of CarE7 and CarE3 from Sd-1.CarE7 and CarE31 were identified as two novel carboxylesterases but not xylanases by phylogenetic analysis and alignment of amino acid sequences,conserved domains prediction and homology-based structural modeling.We then cloned CarE7and CarE31 and constructed them into vectors pET-28a（+）,pET-22b（+）and pGEX-4T-1,respectively.pET-28（+）-CarE7,pGEX-4T-1-CarE7,pET-28（+）-CarE31and pGEX-4T-1-CarE7 were expressed successfully in E.coli BL21（DE3）and their recombinant proteins were purified and enriched.（3）Study on biochemical properties of purified recombinant CarE7 and CarE31.The detection of enzyme activities of recombinant protein showed that CarE7 and CarE31 have no xylanase activity,and CarE31 has no carboxylesterase activity.The carboxylesterase activities of GST-CarE7 and His-CarE31 were 8.99 U/mg and 4.37U/mg,respectively.The substrate specificity of recombinant CarE7 also indicated it was an esterase rather than lipase and its optimal temperature and pH were 37℃and pH7.5,respectively.The enzyme activity of CarE7 was increased efficiently by 1 mM K⁺and Ca²⁺.In addition,CarE7 exerted degradation rate of 25%for carbaryl,confirming its typical carboxylesterase activity.Moreover,Sd-1 exhibited higher ability for carbaryl degradation than CarE7,suggesting that Sd-1 might degrade carbaryl by multiple proteins including CarE7.In conclusion,we identified a novel bacterial carboxylesterase CarE7 from rice straw-degrading strain Sd-1,and CarE7 displays degradation ability for carbaryl,indicating that it has potential use in pesticide bio-degradational application.