Researches About The Thermostability Directed Evolution Of Aspergillus Niger ?-L-rhamnosidase

?-L-rhamnosidase,a member of glucoside hydrolase family,can catalyze the hydrolysis of natural and synthetic substrates with glucosides.It plays an important role in the application of food industrial.In this study,in order to improve the thermal stability of?-L-rhamnosidase from Aspergillus niger TS528,error-prone PCR was used for the direct evolution of this enzyme.The mutant D80N/V529A with higher thermal stability was successfully obtained.The secondary and tertiary structure of?-L-rhamnosidase were predicted.Site-directed mutagenesis was applied to study the key sites of amion acids which effect thermostability of?-L-rhamnosidase.Finally,the enzymatic proterties of the mutant enzymes were characterized and the mutant enzymes'abilities to hydrolyze the naringin from fruit juice were also investigated.The main results were as follows:(1)In the previous study,the recombinant plasmid(pPIC9K-rha)containing the?-L-rhamnosidase gene of Aspergillus niger TS528 was constructed.A random mutant library of this?-L-rhamnosidase was generated by error-prone PCR with the recombinant plasmid as the template.The colony positive rate of the library was 92%,and the library had approximately2×10~5 random mutants.Pichia pastoris protein expression system can express heterologous gene to be extracellular proteins,and a screening method by using 96-well plates was established according to the principle.Through the screening of 3000 mutant colonies,five mutants were observed to display different thermostability.The thermal stability of mutants were analysed.D80N/V529A,which has double mutations for D80N and V529A,was obtaind to show higer thermostability than wild-type?-L-rhamnosidase(WT)at 65oC,and other four mutants decreased the thermostability.According to the sequence analysis,thermostability reduced mutants both had amino acids alteration around 600 position.The domain,including amino acids near by 600 position,was predicted to be important for the thermostability of?-L-rhamnosidase.(2)In order to study the microstructure changes of?-L-rhamnosidase,the secondary and tertiary structures of this enzyme should be simulated according to the amino acid sequence.The sequence of WT(AGN92963.1)was submitted to online server for secondary structure prediction.According to the results,there were 29.01%of Alpha helix,28.55%of Extended strand and 42.44%of Random coil in the secondary structure of?-L-rhamnosidase.The online server including Blast,Phyre2 and Hhpred was used to obtain homology templates.The three-dimensional models which simulated by Swiss-Model and Modeller 9.15 were evaluated by Ramachandran plot and Verify3D,respectively.Finally,a complete and quality three-dimensional model was obtained.(3)Using site-directed mutagenesis method,the single point mutants of D80N and V529A were constructed.The thermal stability of mutants was analysed,V529A and D80N/V529A significantly enhanced theirs thermostability compare to WT.At 60oC,65oC,70oC,the half-life time of V529A were extended 1.92 hours,25 minutes and 2 minutes respectively.This results showed the amion acid at position 529 was the key site of enzyme thermostability.(4)Some enzymatic properties of mutants,including optimum pH,optimum temperature,pH stability and effects of metal ion,effectors on enzyme activity,were investigated.D80N reduced the initial activity of?-L-rhamnosidase,V529A and D80N/V529A had better stability under acidic conditions than WT.No difference between mutant and WT enzymes was observed on other enzymatic properties.Moreover,WT had excellent ability to transform the naringin and hesperidin from citrus juices.Under the conditions of high temperature and low acidic,V529A and D80N/V529A could hydrolyze more naringin than WT in the sameperiod.