Enzymatic Properties Of L-arabinose Isomerase And Biosynthesis Of D-tagatose Using Whey Powder

D-tagatose is a natural rare sugar with a 92%sweetness of sucrose,while the caloric value of D-tagatose is less than one-third of the calorie of sucrose.It is a new type of low-calorie sweetener.D-tagatose is widely used in food,medicine,and other industries because of its excellent physiological functions,such as preventing obesity,lowering blood sugar,improving intestinal flora,and anti-caries.Biosynthesis of D-tagatose has been a research priority due to its environmentally friendliness,high catalytic efficiency,mild conditions,and formation of few by-products.At present,the research on the synthesis of D-tagatose using L-arabinose isomerase?L-AI?to convert D-galactose has been reported extensively.However,factors including costly substrate,low conversion efficiency,and unsuitable properties of L-AI enzymatic still restrict the industrialization of D-tagatose biosynthesis.Therefore,developing new L-AI and exploring how to use cheap whey powder to efficiently prepare D-tagatose are expected to lay the foundation for D-tagatose biosynthesis.In view of these facts,this study was carried out the following works:?1?Kimchi juice and infant feces were screened by strain enrichment,primary screening,and cysteine-carbazole method re-screening.Furthermore,physiological,biochemical identification,and molecular biological identification were adopted.Finally,a strain of Lactobacillus salivarius CY.2 and a strain of Lactobacillus plantarum CY.6 were identified from the kimchi juice,and a strain of Bifidobacterium adolescentis HY.3 was selected from infant feces.?2?A 1515 bp araA of B.adolescentis HY.3 encoding BAAI was cloned,and the recombinant strain of E.coli BL21/pANY1-Ba-araA was constructed.The BAAI was successfully expressed by the induction of IPTG in the recombinant strain.By dissolving and renaturing the BAAI inclusion bodies using the Solubilization and Refolding Kit,biologically active BAAI was obtained,which was about 55 kDa.The specific activity of BAAI was 24.47 U/mg.To conduct bioinformatics analysis on BAAI,phylogenetic tree was constructed and it was concluded that BAAI and the L-AI derived from Bifidobacterium longum were highly conservative.Secondly,multiple sequence alignment analysis was performed with multiple characteristically L-AIs,to analyze the conservative relationship between sequences and the key amino acids in the active site,which were Glu308,His352,Glu335 and His452.Finally,the three-dimensional structure model of BAAI was constructed and the action direction of key amino acids was analyzed.Studies on the enzymatic properties of BAAI indicate that the optimal temperature of BAAI was 55?and the optimal pH was 6.5,with good stability.The metallic ion that had the best activation effects on BAAI was 6 mM Mn²⁺.D-galactose has the greatest substrate specificity for BAAI.The substrate-protein molecular simulation docking analysis shows that there are the most hydrogen bonds between D-galactose and BAAI,which validated the results of substrate specificity studies at the molecular level.The K_m,V_max and K_cat/K_m of BAAI were found to be 22.4mM,489 U/mg and 9.3 mM^-1min^-1,respectively for D-galactose,while the respective values for L-arabinose were 40.2 mM,275.1 U/mg and 8.6 mM^-1min^-1.Enzymatic conversion of D-galactose into D-tagatose with BAAI showed 56.7%conversion efficiency.?3?The L-AI?LPAI?coding gene araA was cloned from L.plantarum CY.6 with a size of 1425 bp,for constructing a recombinant strain of E.coli BL21/pANY1-Lp-araA.LPAI with a protein size of about 55 kDa was successfully expressed in the recombinant strain.The resting cells of E.coli BL21/pANY1-Lp-araA combined with?-galactosidase were used to produce D-tagatose with the substrate of milk whey powder.Based on 100%?Separate hydrolysis and biotransformation,SHB?,0%?Simultaneous saccharification and biotransformation,SSB?,and 50%?Semi-SSB?of lactose hydrolysis,three different technical routes were designed to select the best route for preparing D-tagatose.The results showed that the yield of D-tagatose was the highest under SSB,which was twice of Semi-SSB and SHB.The single factor method was adopted to optimize the SSB reaction conditions.The results showed that the optimal temperature was 50?,pH was 6.5,concentration of Mn²⁺was 5 mM,reaction time was 48 h,lactose concentration was 140 g/L,and the best whole cell concentration was 20 g/L.Under the above optimal conditions,the first-stage of the reaction was carried out for 48 h.The concentration of D-tagatose produced after the first-stage was34.4 g/L.The reaction provided 52.4%?based on D-galactose?and 24.2%?in term of lactose?conversion efficiency.Furthermore,the second-stage reaction was carried out under the same conditions for another 48 h by using the finished first-stage solution as substrate.The results showed that the concentration of D-tagatose increased from 34.4g/L to 51.5 g/L after the second-stage reaction.The conversion efficiency increased from 24.2%to 37%?based on lactose?,and from 52.4%to 74%?in term of D-galactose?.