Biocatalytic Synthesis Of Herbiside Glyphosate's Intermedate Iminodiacetic Acid

Iminodiacetic acid(IDA)is used extensively as an important chemical intermediate compound,which primarily serves as a key precursor for the manufacture of glyphosate,a dominant herbicide used in various applications for weed and vegetable control worldwide.Traditionally,the production of IDA in currently factory of the world depends primarily on three chemical routes:direct synthesis catalytically by base and acid from iminodiacetonitrile(IDAN)achieved from hydrocyanic acid;chloroacetic acid method by hydrazine reaction and catalytic dehydrogenization of diethanolamide.These processes are suitable for various conditions of feedstock but all of them inevitably require relatively harsh conditions and high energy consumptions,such as high temperature and pressure,strong acid and base,giving rise to a large amount of salts,which are unfriendly to the environment.The present work attempts to establish a biocatalytic route by the aid of nitrilase to mediate the hydrolysis of IDAN to IDA for the green production of glyphosate.First,we established a screening strategy for the microorganism harboring nitrile-hydrolyzing enzymes capable of catalyzing the hydrolysis of IDAN to IDA.By employing a simple spectrophotometric assay of desired product IDA that based on the complex formation reaction with copper(?)ion,a high-throughput screening model was proposed in the study.Several microorganisms capable of catalyzing the hydrolysis of IDAN to IDA were isolated from soil samples employing the newly integrated screening method.After optimization and comparison,A.faecalis ZJUTBX11,exhibited 73.4 U g-1 DCW specific activities,was consequently confirmed to be the suitable candidate for the production of IDA with potential applications.By using a low-energy ion beam implantation mutagenesis method,we have successfully improved nitrilase activity to a new level in the present study.The specific nitrilase activity and cell mass were greatly improved by about 45%and 75%,respectively.The optimal temperature and pH for the biotransformation of IDAN to IDA using A.faecalis mutant strain was 35? and 7.5,which demonstrated a mesophilic microbial nitrilase and better tolerance to weak alkaline conditions.The thermal stability investigation of nitrilase indicated low tolerant to higher temperature conditions,and the half-life of nitrilase at 35,45 and 55? was 57.0,17.4 and 3.7 h,which may be further enhanced by immobilization or a genetic engineering technique.Encapsulation of whole cells in ACA capsule with liquid core via one step method was selected in the present study due to its higher cell loading and recovery of enzyme activity.The capsules with a diameter of 1.5-2.0 mm were smooth,spherical and covered with a thin layer of sodium alginate precipitation.The layer thickness of the ACA-membrane of the capsules was approximately 15 ?m by a scanning electric microscope(SEM).The Vmax and apparent Km values were found to be 97.6±1.2 ?mol min-1 g-1 DCW and 17.6±0.3 mmol l-1 for encapsulated cells.Consecutive batch reaction with capsule-immobilized A.faecalis ZJUTBX11-MD271 whole cells was performed in a bubble column bioreactor for the hudrolysis of IDAN.The initial specific activity reached the highest value 88.2 ?mol min-1 g-1 DCW when applying the substrate concentration 150 mM and the air flow rate 1.2 1 min-1,and thus the highest biocatalyst productivity of 2.7 g g-1 DCW was achieved.Continuous biotransformation of ID AN to IDA in a 400-ml PBR reactor was performed.The maximum productivity of 45.6 mmol 1-1 h-1 was achieved at residence time of 0.6 h and substrate concentration of 200 mM.