| D-tagatose, a kind of rare sugar, is a hexoketose monosaccharide and an ideal sucrose substitute for food products, due to having 92% relative sweetness but 1/3 energy of sucrose. Industrial production of D-tagatose is carried out from D-galactose by L-arabinose isomerase（L-AI）. It is generally recognized that commercial L-AI for D-tagatose production requires two important enzymatic properties, thermostability and slightly acidic p H optimum. Thermostable L-AIs have been widely characterized from the common thermophilic bacteria. Molecular modification also has been widely used for obtaining variants with improved specific activity and decreased optimum p H of the thermostable L-arabinose isomerase.The genes URH173680701 coding for L-arabinose isomerase（L-AI） was from Alicyclobacillus hesperidum URH17-3-68. DNAMAN software analysis revealed that an open reading frame of URH173680701 consisted of 1497 nucleotides encoding 497 residues with the calculated molecular mass 56,207 Da and the Gene Bank accession No. of the enzyme was EJY56736.1.The target gene fragment was synthesized and inserted into expression vector p ET-22b（+） to create the recombinant plasmid. Then the recombinant plasmid was overexpressed in E.coli BL21（DE3） induced by IPTG. The recombinant enzyme was purified by heat treatment and ion-exchange chromatography and analyzed by SDS-PAGE. Approximately 56.0 k Da exogenous proteins was observed, which was corresponding to the assumption.The research on enzyme properties showed that the optimum p H was 7.0 and the optimum temperature was 70 °C. It showed more than 75% of maximal activity from p H 5.5 to 7.0. Co2+ was required as optimum metal cofactor for activity simulation. The enzyme had relatively thermostability below 65°C and Co2+ could help improve thermostability at 75 °C. The Michaelis-Menten constants（Km） for substrate D-galactose and L-arabinose were measured to be 54.7 mmol/L and 105.2 mmol/L, respectively.To improve specific activity and decrease optimum p H of the enzyme, random and site-directed mutagenesis were used to perform the molecular modification. A mutation, D478 N, was obtained by an error-prone polymerase chain reaction, which was measured to show higher activity for the D-galactose isomerization. Then, single-site variants, D478 Q, D478 A, D478 K, and D478 R, were constructed. The variant L-AIs were purified. The optimum temperatures were all higher than 60 °C. D478 A, D478 N, and D478 Q remained more than 80% of maximum relative activity at 75 °C. Except D478 A, all variants showed decreased optimum p Hs, at 6.0 to 6.5. All the variant L-AIs could be significantly activated by Co2+ and Mn2+. D478 N and D478 Q showed higher catalytic efficiency toward substrate D-galactose than that of wild-type L-AI. In addition, for D-tagatose production from D-galactose at p H 6.0, D478 N and D478 Q exhibited much higher conversion ratio of D-galactose to D-tagatose than the wild-type L-AI. According to the molecular modeling, residue D478 was located at the surface of the enzyme and was supposed to influence the optimum p H for substrate binding or isomerization. More information about crystal structure of mutant enzymes still should be determined to explain how the acid residue at position 478 influenced the optimum p H and the specific activity. |
