Molecular Modification From Of L-arabinose Isomerase Lactobacillus Fermentum And Preparation Of D-tagatose

As a fresh functional sugar replacer,D-tagatose is widely applied in food,pharmaceutical and cosmetic products due to its special physiological functions.With the increase of market demand in recent years,the bioenzyme synthesis of D-tagatose has gradually become a hot spot for research due to its advantages of cleanliness,low carbon and strong catalytic specificity.The preparation of D-tagatose by compoundingβ-galactosidase and L-arabinose isomerase using lactose as the substrate has the unique advantage of low raw material cost and few by-products to become the main method of industrial production at present.Among them,the catalytic performance of L-arabinose isomerase is the key element to determine the yield of D-tagatose.In this study,Lactobacillus fermentum CGMCC2921 derived L-arabinose isomerase LfAI was heterologously expressed in Bacillus subtilis WS9,and its enzymatic properties and ability to prepare D-tagatose were investigated,followed by rational design and stacked combinatorial mutagenesis 5 dominant variants were obtained.Based on the optimization results of the dominant variants in the fermentation conditions at the shake flask level,high-density fermentation in 3-L bioreactor was carried out to finally achieve the efficient expression of L-arabinose isomerase.The major findings of this research paper are as follows:（1）Recombinant expression of LfAI in B.subtilis and characterization of the performance of prepared D-tagatose.The codon-optimized LfAI of L.fermentum CGMCC2921 origin was recombinantly expressed in the expression host B.subtilis WS9 with an enzyme activity of 5.71U·m L^-1 in shake flask fermentation;its optimum temperature was 70°C and its thermal stability was determined at 65°C with a half-life of 2 h;the half-life was 7 h at 60°C,and the half-life could be extended to 10 h at 55°C.The performance of its preparation of D-tagatose was characterized,and the highest conversion of 19.91%was obtained with 400 g·L^-1 lactose as substrate at 55°C,p H 6.0,1.0 mmol·L^-1 Mn²⁺,1.0 mmol·L^-1 Co²⁺,and 1.4 mg·m L^-1 of enzyme addition for 60 h of reaction.（2）The conversion rate of D-tagatose is improved by rational design of LfAI.By sequence comparison and structural analysis of the substrate docking model,20 variants were constructed and recombinantly expressed for 7 amino acid residues around D-galactose C6 in the substrate binding pocket of L-arabinose isomerase LfAI for D-galactose affinity,and 7 dominant single point variant with improved conversion rate were obtained in the preliminary screening;then they were stacked and combined mutations were made to finally obtain the most dominant variant Y19E/F279I;subsequently,the conditions for its preparation of D-tagatose The conditions for the preparation of D-tagatose were optimized,and the highest conversion rate of23.01%was obtained at 55°C,p H 6.0,Mn²⁺and Co²⁺concentrations of 0.25 mmol·L^-1,500g·L^-1lactose concentration,and 1.0 mg·m L^-1 enzyme addition.（3）The optimization of shake flask fermentation and high-density fermentation in 3-L tanks for the dominant variant Y19E/F279I.The shake flask fermentation was optimized in five aspects,including carbon source type and concentration,nitrogen source type and concentration,and the ratio of compound nitrogen source concentration,respectively.The total enzyme activity of the optimized shake flask fermentation was 8.06 U·m L^-1,which was 1.36 times higher than that of the TB medium fermentation.Subsequently,high-density fermentation at different temperatures was carried out in 3-L fermenters,and the highest total enzyme activity of 95.83 U·m L^-1 was finally obtained under 30°C fermentation conditions,its target protein concentration was 8.33 g·L^-1,which are 11.89 times higher than that of shake flask fermentation.