Researches About The Thermostability Of Aspergillus Niger ?-L-Rhamnosidase Based On Rational Design

The JMU-TS528 ?-L-rhamnosidase gene of Aspergillus niger was cloned,expressed in Pichia pastoris and its three-dimensional model was constructed in our previous study.In this paper,we used the rational design method to obtain mutants,and determined optimum temperature,optimum pH and thermal stability.The mutants of thermostability were analyzed by circular dichroism,fluorescence spectroscopy and molecular dynamics analysis to illustrate the reasons of the change of thermal stability.First,five mutant sites,including 14 mutants,were identified based on the change in the folding free energy predicted by PoPMuSiC.Six mutants with increased thermal stability were obtained and the mutation sites localized at amino acids 573 and 631.The results showed that the K573V-E631 F had increased by 2.3 h,16 min and 1.3 min at 60°C,65°C and 70°C compared with WT half-lives,respectively.The analysis of its structure suggested that the increase of internal hydrophobicity may be an important factor to improve thermal stability.By circular dichroism,fluorescence spectroscopy and molecular dynamics simulations found that the ?-helix,?-turn and random coil increased,the tertiary structure unchanged.The overall variability of K573V-E631 F was slight,and the rigidity of the amino acid near the ?-helix was increased.Then,based on the strategy of introducing arginine instead of lysine to improve the thermal stability,five mutants were designed by selecting the lysine site located on the surface of the protein and combining with the Discovery Studio 2016 predictive analysis.The thermostability of K406 R,K440R and K573 R was improved,and they were combined to obtain two double-point mutants K406R-K440 R and K406R-K573 R.The thermal stability of K406R-K573 R was the highest,which had increased at 60°C,65°C and 70°C compared with WT half-lives,and by 3 h,23 min,3.5 min.By circular dichroism found that the ?-helix,?-turn and random coil were increased.There was not any significant change in the secondary structure with K406R-K573 R and K573V-E631 F.The molecular dynamics simulation showed that the overall fluctuation range of the protein was smaller than WT.The change of internal hydrophobicity and the increase of hydrogen bond and cation-? interaction may be the reason for the increase in thermal stability.Finally,according to the results of the previous two strategies,single-point mutants were selected for joint mutation,and two mutants with improved thermostability were obtained.The stability of K440R-E631 F was higher,and the half-lives at 60°C,65°C and 70°C was increased by 3.5 h,25 min and 2.7 min than WT,respectively.By circular dichroism,fluorescence spectroscopy and molecular dynamics analysis,?-helix,?-turn and random coil were increased.Compared with WT,K573V-E631 F and K406R-K573 R,its ?-helix content was the most,that may be related to the improvement of thermal stability.