Abstract
L-arabinose isomerase (L-AI) is the key enzyme for D-galactose isomerization of D-tagatose by biological method. In this research, Lactobacillus plantarum WU14 with high D-tagatose yield was identified as Lactobacillus plantarum was isolated from the number of lactic acid bacteria from pickled vegetables. The crude L-arabinose isomerase activity of Lactobacillus plantarum WU14 with high D-tagatose yield was 13.95 U/mL under the optimal temperature 60°C, pH 7.17 and substrate concentration 0.8 mol/L, and the conversion rate of 56.12% could be gained after 28 hours. Protein structure and specific of L-Arabinose Isomerase of Lactobacillus plantarum WU14 were researched. The results showed that L-arabinose isomerase is mainly composed of alpha helix and random coil. Then the recombinant L-AI gene was inserted into the food-grade expression vector pRNA48 and expressed in L. lactis NZ9000 successfully. The target protein expression reached the maximum amount when the induced concentration of nisin reaches 30 ng/mL after 12 h. And the crude enzyme activity of recombinant bacteria reached 6.21 U/mL under 60°C. Otherwise the optimal conversion rate recombinant of L. lactis NZ9000/pRNA48-L-AI can reach 39.21% under the temperature of 50°C, pH 7.17 and D-galactose concentration was 0.6 mol/L.
Highlights
D-tagatose is a kind of six carbon ketone sugar which existed relatively rare in nature
E. coli DH5α preserved by our laboratory through bromocresol purple solid MRS culture medium, D-galactose is bought from Lanji technology development company in shanghai, D-tagatose standard is bought from Sigma company, pGEM-T vector is bought from Promega company, T4 DNA ligase is bought from Takara company, DNA recovery kit is bought from TransGen Biotech, L. lactis NZ9000, L. lactis NZ9000/pRNA48, DNA Marker, Protein molecular weight standards, dNTP, TaqDNA polymerase, restriction enzymes, pure Midi Plasmid Kit is bought from TANGEN biotech company, Wizard DNA Clean-Up System is bought from promega company, synthesising PCR primers and sequencing are accomplished by Sangon Biotech company
Activate Lactobacillus with High D-Tagatose Yield and Determine Enzyme Activity According to thin layer chromatography (TLC), we screen out 64 strains which have the bioconversion ability of transforming D-galactose to D-tagatose from more than 800 lactic acid bacteria in pickles and pickled vegetables with D-tagatose standard product as contrast
Summary
D-tagatose is a kind of six carbon ketone sugar which existed relatively rare in nature. It’s the isomers of D-galactose and the epimer at C-4 site of D-fructose Academic world called this kind of monosaccharide which existed relatively rare in nature the rare sugar [1]. It’s one kind of food filling type sweeteners with low energy and the mulriple function including the inhibition of high blood sugar, improving the intestinal flora, resisting to tooth decay, promoting the blood health, the enhancement of cellular sensitivity to the toxin and inhibiting toxin like cocaine and nitrofurantoin to hurt liver cells significantly which are important to our daily life [3] [4]. D-tagatose as a kind of low caloric sweeteners is used widely in comfit, beverage, healthy food, and nutritional products in South Korea, Australia, New Zealand and the United States [5]. Joint FAO/WHO Expert Committee on Food Additives approved D-tagatose can be regarded as food additives which ADI was 0.80 mg/kg·d in June 2001 [1]
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