Identification Of L-ribose Isomerase And Its Uses In The Production Of L-ribulose From L-ribose

Presently,because of the extraordinary roles and potential applications,rare sugars turn into a focus point for countless researchers in the field of carbohydrates.The rare sugars are not ample in nature.They have countless applications at commercial level,mainly in the field of agriculture,medicine and food.The rare sugars such as,D-allulose has encouraging potential in the food industry and can be utilized as the food constituent and dietary supplement.L-Ribose has been used as a precursor in the synthesis of L-nucleoside analogues.The production of these rare sugars is very costly and challenging by chemical methods due to their high prices and laborious steps.Therefore,the enzymatic approaches of converting L-ribose into L-ribulose by isomerization would be an effective way with countless industrial applications.There are various sugar isomerases such as,ribose-5-phosphate isomerase,ribose isomerase,mannose isomerase,arabinose isomerase,rhamnose isomerase,and lyxose isomerase,used to manufacture different rare sugars.L-Ribose isomerase is an enzyme that can catalyze the reversible isomerization between L-ribose and L-ribulose,which are isomers of each other.It can also perform the conversion between many aldoses into their corresponding ketoses.The basic purpose of this research was to describe the easy production of rare sugars by using enzymatic methods.The putative L-RI gene of Mycetocola miduiensis was subcloned in p ET-22b(+)vector and then overexpressed in Escherichia coli.It was purified by nickel-affinity chromatography showing an exclusive band of 32 k Da on SDS-PAGE analysis.The native molecular weight of Mm L-RIse estimated by HPLC was 134.84 k Da.The recombinant L-RI was highly active in sodium phosphate(50 m M)buffer at 40 ^oC and p H 7.5,showing the specific activity up to 47.40 U mg^-1.Mm L-RIse showed no significant enhancement in activity with metallic ions except Mn²⁺(32%)and Co²⁺(21%).The values of K_m,k_cat,k_cat/K_m and V_max of Mm L-RIse against L-ribose substrate were 42.48 m M,9259.26 min^-1,217.43 min^-1 m M^-1 and 277.78 U mg^-1 respectively.At equilibrium,the L-ribulose transformation rate was nearly 32%(6.34 g L^-1)using 20 g L^-1 of L-ribose.The Mm L-RIse exhibited the highest conversion ratio and the catalytic activity as compared to other reported enzymes.The putative L-RI gene from Cryobacterium sp.N21 was also subcloned in p ET-22b(+)vector and then overexpressed in E.coli.The purification of Cr L-RIse was performed by metal-affinity chromatography.The enzyme displayed a corresponding band with an approximate size of 35 k Da on the SDS-PAGE analysis.The measured molecular weight(M_w)of Cr L-RIse calculated by HPLC was 125 k Da.Cr L-RIse was extremely active in glycine buffer at 35°C,p H9.0,showing a specific activity of 54.96 U mg^-1.Cr L-RIse displayed no major increase in activity with metal ions,excluding Mn²⁺.The addition of Mn²⁺increased the activity of Cr L-RIse by approximately 19%.The estimated K_m,k_cat,k_cat/K_m and V_max values of Cr L-RIse were 37.8 m M,10416 min^-1,275.43 min^-1 m M^-1,and 250 U mg^-1,respectively.The rate of L-ribulose production was 31%(6.24,12.11,and 20.89 g L^-1)at equilibrium by utilizing 20,40,and 70 g L^-1 of the substrate,respectively.The Cr L-RIse exhibited the highest thermostability(up to 80%)at 40 ^oC for 3 h,which is greater than all the other reported L-RIs.This enzyme can produce L-ribulose from L-ribose with an effective and simple method without making any by-product.The higher catalytic activity,thermostability and conversion ratio of the Mm L-RIse and Cr L-RIse exhibited that these enzymes possessed a great ability for the production of L-ribulose.