D-Tagatose is a promising substitute for sucrose and has a lower physiological caloric value of 12.5 kJ/g compared to sucrose(17.0 kJ/g).In this study,a novel L-arabinose isomerase(L-AI;EC 5.3.1.4)from Lentilactobacil...D-Tagatose is a promising substitute for sucrose and has a lower physiological caloric value of 12.5 kJ/g compared to sucrose(17.0 kJ/g).In this study,a novel L-arabinose isomerase(L-AI;EC 5.3.1.4)from Lentilactobacillus parakefiri DSM 10551(L-AI-Lp)was recombinantly produced in Escherichia coli,purified and biochemically characterized for the isomerization of D-galactose to D-tagatose.The influences of temperature and pH on the activity of L-AI-Lp were compared to a protein-engineered variant of L-AI from Geobacillus stearothermophilus DSM22(L-AI-N13),which is part of a patent held by the D-tagatose-producing company Damhert Nutrition.The production of L-AI-Lp in a bioreactor cultivation yielded 27.72μkat_(D-gal,65℃)/L_(culture) and the L-AI was purified 2.9-fold(specific L-AI activity of 99.6 nkat_(D-gal,65℃)/mg_(protein))by heat treatment and anion exchange chromatography with a yield of 71%(6557±411 nkat_(D-gal,65℃)).The L-AI-Lp showed favorable characteristics compared to L-AI-N13 regarding its maximal activity at 75℃(L-AI-N13,60-65℃)and relative activity of≥70%over a broad pH range of 4-9,whereas no activity was detected for L-AI-N13 at pH≤5.Finally,the applicability of L-AI-Lp and L-AI-N13 was compared by the isomerization of D-galactose(300 mmol/L)at pH 7.5,whereby 45%and 41%,respectively,were isomerized after 24 h.Only the L-AI-Lp was able to isomerize 45%of D-galactose at pH 4.5 after 24 h.In conclusion,the L-AI-Lp is a promising new L-AI for industrial production of D-tagatose over a broad pH range,especially at acidic pH.展开更多
基金supported by the German Federal Ministry of Eco-nomic Affairs and Climate Action(AIF/FEI Project No.21100 N),for which the authors are gratefulsupported by Funding Programme Open Access Publishing of University of Hohenheim.
文摘D-Tagatose is a promising substitute for sucrose and has a lower physiological caloric value of 12.5 kJ/g compared to sucrose(17.0 kJ/g).In this study,a novel L-arabinose isomerase(L-AI;EC 5.3.1.4)from Lentilactobacillus parakefiri DSM 10551(L-AI-Lp)was recombinantly produced in Escherichia coli,purified and biochemically characterized for the isomerization of D-galactose to D-tagatose.The influences of temperature and pH on the activity of L-AI-Lp were compared to a protein-engineered variant of L-AI from Geobacillus stearothermophilus DSM22(L-AI-N13),which is part of a patent held by the D-tagatose-producing company Damhert Nutrition.The production of L-AI-Lp in a bioreactor cultivation yielded 27.72μkat_(D-gal,65℃)/L_(culture) and the L-AI was purified 2.9-fold(specific L-AI activity of 99.6 nkat_(D-gal,65℃)/mg_(protein))by heat treatment and anion exchange chromatography with a yield of 71%(6557±411 nkat_(D-gal,65℃)).The L-AI-Lp showed favorable characteristics compared to L-AI-N13 regarding its maximal activity at 75℃(L-AI-N13,60-65℃)and relative activity of≥70%over a broad pH range of 4-9,whereas no activity was detected for L-AI-N13 at pH≤5.Finally,the applicability of L-AI-Lp and L-AI-N13 was compared by the isomerization of D-galactose(300 mmol/L)at pH 7.5,whereby 45%and 41%,respectively,were isomerized after 24 h.Only the L-AI-Lp was able to isomerize 45%of D-galactose at pH 4.5 after 24 h.In conclusion,the L-AI-Lp is a promising new L-AI for industrial production of D-tagatose over a broad pH range,especially at acidic pH.
