Rational Design Of Acetyl Xylan Esterase BTAxe1 From Bacteroides Thetaiotaomicron

The human gut is the main organ for degradation of complex carbohydrates,which contains a rich variety of microbial communities.Intestinal flora is an important symbiotic microorganism in the human body,which is crucial for the metabolism of complex polysaccharides such as dietary fiber that cannot be directly digested and utilized by the human body.Bacteroides taiotaomicron is one of the most important bacteria in the human gut.It can degrade the large molecular polysaccharides that cannot be digested by the human body into small molecular sugars,affect the host metabolism mechanism,and make the use of nutrients more efficient.In gut,acetyl xylan esterases can decompose acetylated xylan to produce short chain fatty acid acetic acid,which is beneficial for host to play the role of anti obesity and anti diabetes.In this study,a new acetyl xylan esterase,BTAxe1,was identified and characterized from Bacteroides thetaiotaomicron VPI 5482.First,the gene BT＿1008 was amplified from the genome of Bacteroides thetaiotaomicron VPI-5482 and the acetyl xylan esterase BTAxe1 expression vector p ET28a-BT＿1008 was successfully constructed.Then BTAxe1 with high purity was obtained by expression and purification,and its molecular weight was 29 k Da.Then the biochemical characterization of BTAxe1 was carried out.The optimal reaction temperature of BTAxe1 was 30°C,and the temperature stability was poor;the optimal reaction p H was 8.0,which had good tolerance in acidity and weak alkalinity.The influence of metal ions and other common additives on BTAxe1 was weak.The substrate specificity study found that BTAxe1 was active on p NPA,p NPB and 4-MUA.In order to clarify the catalytic mechanism of BTAxe1,the crystallization work was carried out,and a crystal with a resolution of 1.8 ? was successfully obtained.The three-dimensional structure of the protein was typical α/β hydrolase fold and had esterase conserved catalytic triads Ser127,His237 and Asp205.Through the molecular docking of the substrates p NPA and p NPB,it was found that Gly59 and Gly60 in BTAxe1 may combine with the carbonyl oxygen anion of the substrate to form an "oxyanion hole" and stabilized the transition state of substrates.Finally,BTAxe1 was rationally designed,and the results showed that the mutant N65 S increased the activity of short-chain(p NPA)and medium-chain substrate(p NPB),and obtained the activity of long-chain substrate(p NPO).Molecular docking analysis showed that the mutant N65 S had a larger substrate binding pocket than the wild type,which proves that this reason could broaden the substrate specificity and enhance its activity.Hydrolysis studies using natural substrates showed that either N65 S or N65 A showed higher activity of that of wild-type,yielding 131.31 m M and 136.09 m M of acetic acid from xylan.The recombinantly expressed acetyl xylan esterase BTAxe1 in this thesis laid a structural foundation for the study of the hydrolysis mechanism of xylan hydrolases.This is the first study on the rational design of gut bacteria-derived Axes with broadened substrate specificity and enhanced activity,which can be referenced by other acetyl esterases or gut-derived enzymes.