The immobilization of acetylcholinesterase （ACHE） on amino functionalized SBA-15 mesoporous sieves （NH2-SBA-15） was explored for detection of organophosphorus and carhamate pesticides. Several methods have been st...The immobilization of acetylcholinesterase （ACHE） on amino functionalized SBA-15 mesoporous sieves （NH2-SBA-15） was explored for detection of organophosphorus and carhamate pesticides. Several methods have been studied and the ＂adsorption-crosslinking＂ is found to be the best one. Firstly, AChE and bovine serum albumin （BSA） were adsorbed in turn on the surface of NH2-SBA-15 by electrostatic adsorption, and then glutaraldehyde was added for cross-linking the amino group of AChE. The NH2-SBA- 15 showed around 95% immobilization efficiency and the specific activity of immobilized AChE （ACHE- NH2-SBA-15） reached 130% relative to free AChE under optimal immobilization conditions. It was found AChE-NH2-SBA-15 retained 94.0% and 82.8% of its initial activity after 60days at 4℃ and 25℃, respectively. It remained 55.0% of its initial activity after 6 times recycling. During the detection of pesticide, the AChE-NH2-SBA-15 showed wider linear range and much lower limit of detection compared with free ACHE. The current method showed good recovery （93.8%-109.3%） and low RSD （〈5%） for both standard solution and real vegetable samples of Carbaryl and Trichlorfon. It was believed that AChE-NH2- SBA- 15 could be exploited as a fast, sensitive and low-cost biocatalyst towards the detection of pesticides residues which could be stored at room temperature for a long time.展开更多
Esterases are crucial biocatalysts in chiral compound synthesis.Herein,a novel esterase EstSIT01 belonging to family V was identified from Microbacterium chocolatum SIT101 through genome mining and phylogenetic analys...Esterases are crucial biocatalysts in chiral compound synthesis.Herein,a novel esterase EstSIT01 belonging to family V was identified from Microbacterium chocolatum SIT101 through genome mining and phylogenetic analysis.EstSIT01 demonstrated remarkable efficiency in asymmetrically hydrolyzing meso-dimethyl ester[Dimethyl cis-1,3-Dibenzyl-2-imidazolidine-4,5-dicarboxyate],producing over 99%yield and 99%enantiomeric excess(e.e.)for(4S,5R)-monomethyl ester,a crucial chiral intermediate during the synthesis of d-biotin.Notably,the recombinant E.coli expressing EstSIT01 exhibited over 40-fold higher activity than that of the wild strain.EstSIT01 displays a preference for short-chain p-NP esters.The optimal temperature and pH were 45°C and 10.0,with Km and kcat values of 0.147 mmol/L and 5.808 s−1,respectively.Molecular docking and MD simulations suggest that the high stereoselectivity for meso-diester may attribute to the narrow entrance tunnel and unique binding pocket structure.Collectively,EstSIT01 holds great potential for preparing chiral carboxylic acids and esters.展开更多
基金supported by the National Natural Science Foundation of China (No.21303105)Shanghai Committee of Science and Technology (No.13430503400)
文摘The immobilization of acetylcholinesterase （ACHE） on amino functionalized SBA-15 mesoporous sieves （NH2-SBA-15） was explored for detection of organophosphorus and carhamate pesticides. Several methods have been studied and the ＂adsorption-crosslinking＂ is found to be the best one. Firstly, AChE and bovine serum albumin （BSA） were adsorbed in turn on the surface of NH2-SBA-15 by electrostatic adsorption, and then glutaraldehyde was added for cross-linking the amino group of AChE. The NH2-SBA- 15 showed around 95% immobilization efficiency and the specific activity of immobilized AChE （ACHE- NH2-SBA-15） reached 130% relative to free AChE under optimal immobilization conditions. It was found AChE-NH2-SBA-15 retained 94.0% and 82.8% of its initial activity after 60days at 4℃ and 25℃, respectively. It remained 55.0% of its initial activity after 6 times recycling. During the detection of pesticide, the AChE-NH2-SBA-15 showed wider linear range and much lower limit of detection compared with free ACHE. The current method showed good recovery （93.8%-109.3%） and low RSD （〈5%） for both standard solution and real vegetable samples of Carbaryl and Trichlorfon. It was believed that AChE-NH2- SBA- 15 could be exploited as a fast, sensitive and low-cost biocatalyst towards the detection of pesticides residues which could be stored at room temperature for a long time.
基金supported by the Shanghai Committee of Science and Technology(No.13430503400)Project of Leading Talents in Shandong Taishan Industry(Grant No.LJNY202019)+1 种基金the Scientific Research Foundation for the Returned Overseas Chinese Scholars,State Education Ministry(No.ZX2012-05)Innovation Program of Shanghai Municipal Education Commission(No.11YZ227).
文摘Esterases are crucial biocatalysts in chiral compound synthesis.Herein,a novel esterase EstSIT01 belonging to family V was identified from Microbacterium chocolatum SIT101 through genome mining and phylogenetic analysis.EstSIT01 demonstrated remarkable efficiency in asymmetrically hydrolyzing meso-dimethyl ester[Dimethyl cis-1,3-Dibenzyl-2-imidazolidine-4,5-dicarboxyate],producing over 99%yield and 99%enantiomeric excess(e.e.)for(4S,5R)-monomethyl ester,a crucial chiral intermediate during the synthesis of d-biotin.Notably,the recombinant E.coli expressing EstSIT01 exhibited over 40-fold higher activity than that of the wild strain.EstSIT01 displays a preference for short-chain p-NP esters.The optimal temperature and pH were 45°C and 10.0,with Km and kcat values of 0.147 mmol/L and 5.808 s−1,respectively.Molecular docking and MD simulations suggest that the high stereoselectivity for meso-diester may attribute to the narrow entrance tunnel and unique binding pocket structure.Collectively,EstSIT01 holds great potential for preparing chiral carboxylic acids and esters.
