Screening,Expression And Activity Modification Of Urethanases

Ethyl urethane(EC)is widely present in fermented foods.It is a substance that can cause multiple carcinogens in a variety of experimental animals and has potential carcinogenicity to humans.This makes the content of EC in fermented food a food safety issue of great concern to the society.Urethanase can directly degrade EC into CO₂,ethanol and NH3,which is the most effective and direct method to degrade ethyl carbamate.However,there are fewer urethanases that have been screened,and their catalytic activity were low.In order to select a suitable urethanase for the degradation of EC in alcoholic beverages,this dissertation focuses on the selection and the modification of urethanase.The main research content is as follows:(1)A small enzyme library containing 9 amidases was constructed to screen for urethanases.Through the activity screening,AmdA with higher catalytic activity was selected as the starting enzyme for follow-up research.AmdA is derived from Agrobacterium tumefaciens d₃,the urethanase activity per unit fermentation broth is 97.56 U/L,and the specific urethanase activity is 0.62 U/mg.In order to improve the heterologous soluble expression of AmdA in E.coli BL21(DE3),co-expression of molecular chaperones was used.The activity of AmdA increased 3.1 times to 307 U/L.By removing the fusion tag of AmdA,the urethanase activity increased to 395 U/L.(2)The enzymatic properties of AmdA were characterized,and it was found that the optimum pH value is 7.5,the optimum temperature is 55?,and it has good thermal stability below 50?.Moreover,after 1 h of heat preservation at 37 ? in a 5-20%(v/v)ethanol solution,the residual urethanase activity was higher than 91%,considerably more than that reported thus far.The Km with EC as the substrate is 0.964 mM,and the vm is 0.887?mol·mg^-1·min^-1.At the same time,through amino acid sequence analysis,homology modeling and site-directed mutagenesis analysis,it is determined that the catalytic triad of AmdA is L98-S173-S197,which belongs to the amidase AS family.(3)In order to improve the catalytic activity of AmdA on EC,rational and semi-rational transformations are carried out on it.First,the site-directed mutation point was discovered through multiple sequence alignment,and a mutant G195A with a 375%increase in urethanase activity was successfully obtained.Afterwards,through site-directed saturation mutation of 21 amino acids near the catalytic triad,a high-throughput screening method was used to obtain a mutant I97L whose urethanase activity increased by 53%.Combination mutations were performed on the two mutation sites selected above,and the combined mutant I97L/G195A was obtained.The urethanase activity was increased by 5.18 times,and the unit fermentation enzyme activity was increased from the original 395 U/L to 2442 U/L.In this paper,we have screened the amidase AmdA derived from Agrobacterium tumefaciens d₃,which has application potential,and has a certain catalytic activity for EC,and the catalytic activity of AmdA has improved through rational and semi-rational strategies.