ABSTRACT
L-arabinose isomerase (L-AI) is the key enzyme that isomerizes D-galactose to D-tagatose. In this study, to improve the activity of L-arabinose isomerase on D-galactose and its conversion rate in biotransformation, an L-arabinose isomerase from Lactobacillus fermentum CGMCC2921 was recombinantly expressed and applied in biotransformation. Moreover, its substrate binding pocket was rationally designed to improve the affinity and catalytic activity on D-galactose. We show that the conversion of D-galactose by variant F279I was increased 1.4 times that of the wild-type enzyme. The Km and kcat values of the double mutant M185A/F279I obtained by superimposed mutation were 530.8 mmol/L and 19.9 s-1, respectively, and the catalytic efficiency was increased 8.2 times that of the wild type. When 400 g/L lactose was used as the substrate, the conversion rate of M185A/F279I reached a high level of 22.8%, which shows great application potential for the enzymatic production of tagatose from lactose.
Subject(s)
Galactose/metabolism , Limosilactobacillus fermentum/genetics , Lactose , Hexoses/metabolism , Aldose-Ketose Isomerases/genetics , Hydrogen-Ion ConcentrationABSTRACT
L-arabinose isomerase (L-AI) can isomerize L-arabinose and D-galactose into L-ribulose and D-tagatose, respectively, which is currently the most effective biological catalyst for D-tagatose production. The crystal structure of L-AI has been solved recently and its gene has been cloned, sequenced and overex- pressed. L-AI improved by protein engineering will be the dominant enzyme for industrial production of D-tagatose. This paper reviewed researches on protein structure and function, properties and application in D-tagatose production of L-AI, and the long-term potential development of L-AI was prospected.
ABSTRACT
Thermostable L-arabinose isomerase (L-AI) is the most potential enzyme for the biological production of D-tagatose from D-galactose, a novel functional factor. Gene araA encoding the L-arabinose isomerase from Bacillus stearothermophilis IAM 11001 was cloned and expressed in Escherichia coli. The araA gene of 1491 bp has 95% identity with L-AI from Thermus sp. IM6501. The GenBank accession number for the nucleotide sequence of this araA gene determined in this work is EU394214.The bacterium was induced by IPTG and analyzed by SDS-PAGE, approximately 59 kDa exogenous protein was observed on the SDS-PAGE. The recombinant L-AI was purified to electrophoretical homogeneity with affinity chromatography, and the activity of recombinant L-AI was also studied. The bioconversion rate of D-galactose to D-tagatose reached 39.4% after 24h whole cell reaction.
ABSTRACT
Objective To research the function of hypoglycemic and antiobesity of L-Arabinose in New Zealand rabbit. Methods Thirty-two New Zealand white rabbits were randomly divided into 4 groups, 8 rabbits in each group. The control group was fed with high fat and high sucrose diet only. L-Arabinose group was fed with high fat and high sucrose diet along with L-Arabinose, the dosage were 0.308 5, 0.617, 0.925 5 g/kg respectively. After fed for two months, all groups were given L-Arabinose, and blood glucose was detected after orally given sucrose 0, 0.5, 2 h. After intervention of L-Arabinose for one month, the data of weight, food intake, stool quantity and the fat index were observed. Results L-Arabinose decreased significantly sucrose absorption, lower the fasting glucose level and the data of area-under-curve (AUC) significantly. Conclusion L-Arabinose proved to be highly effective in preventing the rise of circulating glucose and fat.