Study On Producing D-Tagatose Using L-Arabinose Isomerase Of Lactic Acid Bacteria

D-tagatose has high sweetness and low energy,and it has many probiotic effects such as anti-caries,improving intestinal flora,preventing colon cancer,and treating type ? diabetes mellitus,gradually as a new type of sweetener widely used in fields such as food and drug.At present,the main production methods of D-tagatose are chemical synthesis and biocatalysis.Biocatalysis has become a hotspot in current study.The preparation method has the advantages of producing fewer by-products and easy separation as well as purification of the products.L-arabinose isomerase(L-AI)is the most commonly used enzyme in biocatalysis,which can catalyze the production of L-arabinose to L-ribulose and D-galactose to D-tagatose.As Generally Regarded as Safe(GRAS)strains,lactic acid bacteria(LAB)have many probiotic properties and have been widely added to food and drugs as probiotics,as well as as cell factories for the production of therapeutic antigenic proteins and peptides and other medical molecules.Therefore,L-AI expressed by lactic acid bacteria can be used to produce food-grade D-tagatose effectively.In this study,a high-yield L-AI LAB strain was screened and the enzyme-producing medium was optimized.Through the analysis of enzymatic properties,the reaction conditions of pure enzyme were optimized.Homologous modeling was carried out using bioinformatics technologys.After identifying the active sites and the sites associated with substrate binding of L-AI,the mutants with stronger affinity for D-galactose were obtained by directional mutation.Then the gene of the mutant enzyme was expressed in LAB.Recombinant Lactobacillus with high L-AI activity was screened and immobilized to determine the conversion efficiency to D-galactose.Specific research contents and results are as follows:1.Screening of strains with high-yield L-AI and optimization of enzyme-producing conditions.Four strains of LAB with L-AI were screened out from the preserved strains of our laboratory using selective medium and cysteine-carbazole colorimeting.After re-screening,it was determined that strain Lb.reuteri SDMCC050455 had the highest enzyme activity.Using the resting cells of the strain as catalyst,the catalytic properties showed that the optimal reaction temperature was 60 ?,the optimal pH was 6.0-7.5.The enzyme activity did not significantly decrease after 1 h of treatment at 65?,and the enzyme activity remained at 30%after 1 h of treatment at 70?,so the enzyme is stable at high temperatures.And the enzyme is very stable when the pH was 4-10.In addition,Co2+ and Ni2+can promote the catalytic activity of the strain,and the promotion effect of Co2+ is stronger.In order to improve L-AI catalytic activity of Lb.reuteri SDMCC050455,the medium composition,fermentation conditions,treatment methods of resting cells and transformation system of it were optimized.Finally,the results showed that the optimal preparation conditions were as follows:3 g/L L-arabinose was added to the culture medium as both carbon source and inducer,after 10 h of culture at 39?.the cells were collected.,and then the cells were treated with 0.02%SDS for 1 h.When 2%(w/v)D-galactose was used as substrate for the reaction under the optimal conditions,the enzyme activity was increased from 6.55 ±0.19 U/mL to 15.07 ± 0.28 U/mL,increased to 2.30 times of the original.2.Study on the enzymatic properties of L-AITo study the enzymatic properties of L-AI of Lb.reuteri SDMCC050455(LRAI),araA gene was cloned from the genome of Lb.reuteri SDMCC050455 by PCR.Sequencing results showed that the size of the gene was 1422 bp,encoding 473 amino acids,the molecular weight of the protein was 53.8 kDa,and the isoelectric point was 5.13.Gene araA ligate with expression vector pETDuet-1 of E.coli and transformed into E.coli BL21(DE3).The protein LRAI was purified by nickel ion affinity chromatography,and the purified enzyme LRAI was collected.The Km value and Vmax of LRAI were 746,37 mM and 50.63 ?g·min-1·mg-1,respectively.The enzyme activity was 0.26 U/mg.Enzymatic properties show that the optimum reaction temperature and pH of LRAI were 60? and 6.5,respectively.The enzyme activity remained 76%after 65? warm bath for 1 h,however,after treatment at 70? for 20 min,the enzyme activity almost lost,indicating its stability declined at high temperature.The enzyme activity was stable when pH ranged from 7.0 to 8.0.In addition,metal ions had different effects on LRAI activity,among which Co2+and Mn2+had the most significant promoting effects,and Co2+promoted LRAI activity more strongly.3.Site-directed mutation increased the substrate affinity of L-AI to D-galactoseTo improve the substrate affinity of LRAI for galactose,its three-dimensional structure was obtained by homology modeling of amino acid sequences on Swiss-Model website,and its structure was compared using PyMol software,Clustal Omega and Epript 3.0 online software.Four amino acid sites that may have influence on the substrate binding to the active site were identified:Phe278,Ser310,Lys422 and Arg437.Four mutant proteins F278A,S310A,K422A and R437A were obtained by site-directed mutagenesis and heterologously expressed by Escherichia coli.The four mutant proteins were heterologously expressed in Escherichia coli,and their enzymatic properties and kinetic parameters were determined.The results showed that K422A had the lowest Km(341.30 mM),which had a stronger affinity for D-galactose than LRAI.It's Vmax was 0.57 mol·min-1·mg-1.The enzyme activity was 0.50 U/mg,which was 91.89%higher than that of the wild type.The enzyme properties showed that the optimum reaction temperature and pH of LRAI were 70?and 6.5,respectively.After treatment at 70? for 1 h,it's activity still retains more than 75%,indicating K422A is very stable at high temperatures;the enzyme is stable at pH 6.0-9.0,and the tolerance to low pH was stronger than LRAI.4.Cloning and expression of L-AI in LABD-tagatose can be stable in acidic environment(pH 3-7),so using LAB as whole-cell catalyst has potential application for production of D-tagatose.In this study,the gene araA of Lb.reuteri SDMCC050455 and maraA3 of K422A were cloned and ligated to the expression vector pCD4033,respectively.The recombinant vector was constructed and transformed into Lb.brevis ATCC367,and obtain the recombinant strains Lb.brevis AT CC367/pCD4033-araA(LBW)and Lb.brevis ATCC367/pCD4033-maraA3(LBM).The L-AI activity of the recombinant strains was determined,the optimal reaction temperature and pH of the two strains was 65-70? and 6.0-8.0,respectively.The enzyme activity of LBM remained more than 73%,but that of LBW was only 34%,after 1 h treatment at 70?,so LBM was more stable than LB W at high temperature.And the enzyme activities of the two strains were stable at pH 4-10.Different metal ions had different effects on the L-AI activity of the two strains.1 mM Co2+promoted the L-AI activity of the two strains,and the promotion to LBM was more obvious.The enzyme activity of LBM was 40.49 U/mL,1.69 times higher than that of Lb.reuteri SDMCC050455.Therefore,LBM is more suitable for the production of D-tagatose.The conversion rate of LBM with different substrate concentration was studied.It was found that the conversion rate reached 25.06%after reaction for 24 h with 150 g/L D-galactose as substrate,the total production of D-tagatose was 37.60 g/L,and the productivity of it was 1.57 g/L/h.The cells of LBM was immobilized by calcium alginate,and the optimal concentration of sodium alginate was 1%(w/v),CaCl2 was 5%(w/v),and the optimal buffer was borate buffer.After the immobilized cells with 350 g/L D-galactose as substrate for 24 h,the total production of D-tagatose reached 46.85 g/L,the productivity was 1.95 g/L/h,and the conversion rate of D-galactose was 13.39%.6.Application of L-AI in Streptococcus thermophilusStreptococcus thermophilus is a kind of thermophilic gram-positive LAB,which is used together with Lactobacillus bulgaricus in the fermentation of yogurt and cheese.In this study,the L-AI gene araA of Lb.reuteri SDMCC050455 was cloned and ligated to the expression vector pLEISS-P59 to construct the recombinant vector,which was transformed into St.thermophilus ST1.The recombinant strain St.thermophilus ST1/pLEISS-P59-araA(STW2)was obtained.The resting cells were prepared and reacted at the optimal reaction temperature of LRAI for 1 h.The activity of L-AI was determined by cysteine-carbazole method.The results showed that the recombinant strain STW2 successfully expressed L-AI and had a strong L-AI activity.To simulate the fermentation environment of milk,STW2 was inoculated into the medium containing 5%lactose for 12 h at 42?,and the formation of D-tagatose in the fermentation broth was detected by TLC method.The results showed that no obvious D-tagatose band was observed after 60 h fermentation.It was speculated that the activity of L-AI was low because the fermentation temperature was lower than the optimal reaction temperature of LRAI.In addition,Streptococcus thermophilus has a weak ability to express exogenous proteins and may have a low expression of LRAI,so L-AI activity is weak.Further research on the conversion of D-galactose from fermented dairy products to D-tagose needs to be explored.