Study On Fermentation Process And Molecular Improvement Of L-Arabinose Isomera From Lactobacillus Plantarum WU14

D-tagatose is a new sweetener that has functions such as lowering blood sugar,improving intestinal flora,and promoting blood health.It is widely used in products such as beauty,food and pharmaceutical industries.Lactobacillus plantarum WU14 is a lactic acid bacteria that isolated from sauerkraut,it has the ability to transform D-tagatose by L-arabinose isomerase（L-AI）.On the based of the fermentation medium optimization of L-AI produced by L.plantarum WU14,established of fermentation model,D-tagatose was separated from the fermentation broth,bioinformatics was used to predict the amino acid sites affecting the thermal stability of L-AI for prokaryotic expression.The results were as follows:1.The biomass of L.plantarum WU14 and the activity of L-AI were used as the indicators,the single factor and response surface analysis were employed to optimize the fermentation medium and conditions of L.plantarum WU14.The optimal composition of the fermentation medium was（g/L）:glucose 4.35 g,L-arabinose 2.71 g,yeast extract 17.6g,soy peptone 17.6 g,anhydrous sodium acetate 10 g,MgSO₄·7H₂O 0.4 g,MnSO₄·2H₂O0.05 g,K₂HPO₄ 0.4 g;fermentation conditions:initial pH 6.24,fermentation temperature28.6℃,inoculum 2%,fermentation 20 h,the biomass of L.plantarum WU14 increased nearly 48%,the L-AI activity reached 42.23 U/mL and the conversion rate of D-tagatose reached 69.6%.2.Through the analysis of the fermentation process of L.plantarum WU14,it was found that the biomass of L.plantarum WU14 was partially coupled with the formation of L-AI.The Logistic equation and the Boltzmann model were used to nonlinearly fit the experimental data of L.plantarum WU14 biomass and L-AI activity,and the kinetics of cell growth,substrate consumption and L-AI formation were established.The fitting degrees were 0.99637,0.9443,and 0.99116,respectively,which could better reflect the change rule of D-tagatose biotransformation in the fermentation process of L.plantarum WU14.3.D-tagatose was separated from the fermentation broth by centrifugation to remove bacteria,activated carbon decolorization,TCA deproteinization,desalination of anion and cation exchange column,calcium resin separation,freeze drying,etc.,and using Fourier infrared Spectral analysis identification.4.The borate buffer was added to the conversion system,and the conversion temperature,the conversion pH,the amount of the crude enzyme solution and the amount of the borate buffer were studied.It was found that when the conversion temperature was60℃,the reaction pH was 7.17,and the volume ratio of the crude enzyme solution to the D-galactose was 5:1,and the volume ratio of borate to D-galactose was 2:1,after 24 h of conversion,the conversion of D-tagatose reached 68%,compared with the without transformation of borate system increased by 12%.5.In order to improve the thermostability of L-AI from L.plantarum WU14,molecular improvement of L-AI was carried out.Through homology modeling,three mutation sites that may affect the thermostability were found,and the mutation sites were freely combined,E.coil BL21（DE）was used as the host bacteria to construct seven mutants,G242P,G371P,G372P,G242P/G371P,G242P/G372P,G372P/G371P,G242P/G371P/G372P,respectively.The enzymatic properties of the wild-type and mutant were analyzed,it was found that the optimal temperature and pH of the mutant were same as the wild-type;the thermostability of the mutant was improved compared with the wild-type,and the three-point mutation（G242P/G371P/G372P）was the most obvious,after 90 minutes of treatment at 70℃,it still had 53.59%residual enzyme activity,and the wild-type was only 11.72%.This indicated that in the non-catalytic central region of L-AI,the introduction of proline has a positive effect on improving the thermostability of L-AI,which lays a foundation for the industrial production and application of L-AI.