Characterization Of Esterase And The Functional Effect Of N-terminal α-helix From Lactobacillus Rhamnosus

Lactobacillus rhamnosus esterase is an esterase derived from the genus Lactobacillus.It is commonly used in industry to catalyze the production of ester flavor substances to improve product quality and flavor,and has broad application prospects.At present,16 esterases derived from Lactobacillus have been characterized by their enzymatic properties,and 3 have undergone structural analysis.As a probiotic,Lactobacillus rhamnosus GG has relatively few studies on esterases compared with other species of Lactobacillus.Our research is helpful to develop and expand the utilization of esterase resources of Lactobacillus rhamnosus.The main research results of this subject are as follows:（1）Characterization of LggEst1 and LggEst2:The optimum temperature of LggEst1 and LggEst2 are both 50℃,and the optimum pH is pH 7.5 and6.5,respectively.LggEst1 prefers medium chain substrates,and shows the highest catalytic efficiency for pNPC8,reaching 3.1×10⁵ M^-1s^-1;LggEst2prefers medium and long chain substrates,and has the highest catalytic efficiency for pNPC10,reaching 3.0×10⁶ M^-1s^-1.The thermal stability of LggEst2 is much better than that of LggEst1,and its half-life is about 6 h at45℃.DOC inhibits the activity of LggEst1,and inhibits LggEst2 at low temperature but high temperature enhancement.The activities of LggEst1 and LggEst2 were activated by 10%and 20%ethylene glycol to different degrees.They also showed good tolerance to DMSO.LggEst1 and LggEst2 are bothα/βhydrolase folding,they all have the catalytic triad of Ser-Asp-His,which are Ser94-Asp188-His219 and Ser146-Asp243-His278,respectively.The LggEst1 substrate binding pocket presents a single thin and deep tunnel,and the LggEst2 substrate binding pocket is divided into two parts by the catalytic center,forming a crack with one side of the acyl binding region and the other of the alcohol binding region.（2）Study on the function of N-terminalα-helix of LggEst2:We constructed wild type and deleted mutant of N-terminalα-helix by POE-PCR.After expression and purification of protein,the deleted mutant was found down-regulated the expression and solubility of protein.Using CD to study structural changes,it was found that the deletion mutation did not affect theα/βfolding of the enzyme,and the active center structure remained intact.The optimum pH for wild type and deleted mutant were 6.5 and 5.5,respectively.And the optimum temperature for both were 45℃.Enzyme kinetics and substrate specificity studies found that the deletion mutation did not change the preference for acyl chain length.DOC inhibits wild type at low temperature and increases it at high temperature,while for the deleted mutant,it always enhances its vitality.In addition,the deleted mutant showed reduced resistance to organic solvents and surfactants compared with the wild type.Thermal and chemical stability studies have shown that the deletion of the N-terminalα-helix does not affect the folding of the overall structure of protein,but reduced the stability of the active center.The overall study revealed that N-terminalα-helix plays an important role in regulating the expression,catalytic activity and stability of LggEst2.