Characterization of the Highly Efficient Sucrose Isomerase from Pantoea dispersa UQ68J and Cloning of the Sucrose Isomerase Gene

Abstract

Sucrose isomerase (SI) genes from Pantoea dispersa UQ68J, Klebsiella planticola UQ14S, and Erwinia rhapontici WAC2928 were cloned and expressed in Escherichia coli. The predicted products of the UQ14S and WAC2928 genes were similar to known SIs. The UQ68J SI differed substantially, and it showed the highest isomaltulose-producing efficiency in E. coli cells. The purified recombinant WAC2928 SI was unstable, whereas purified UQ68J and UQ14S SIs were very stable. UQ68J SI activity was optimal at pH 5 and 30 to 35°C, and it produced a high ratio of isomaltulose to trehalulose (>22:1) across its pH and temperature ranges for activity (pH 4 to 7 and 20 to 50°C). In contrast, UQ14S SI showed optimal activity at pH 6 and 35°C and produced a lower ratio of isomaltulose to trehalulose (<8:1) across its pH and temperature ranges for activity. UQ68J SI had much higher catalytic efficiency; the K m was 39.9 mM, the V max was 638 U mg ؊1 , and the K cat /K m was 1.79 ؋ 10 4 M ؊1 s ؊1 , compared to a K m of 76.0 mM, a V max of 423 U mg ؊1 , and a K cat /K m of 0.62 ؋ 10 4 M ؊1 s ؊1 for UQ14S SI. UQ68J SI also showed no apparent reverse reaction producing glucose, fructose, or trehalulose from isomaltulose. These properties of the P. dispersa UQ68J enzyme are exceptional among purified SIs, and they indicate likely differences in the mechanism at the enzyme active site. They may favor the production of isomaltulose as an inhibitor of competing microbes in high-sucrose environments, and they are likely to be highly beneficial for industrial production of isomaltulose.

Produção de isomaltulose, um substituto da sacarose, utilizando glicosiltransferase microbiana

“…As glicosiltransferases que convertem a sacarose em isomaltulose descritas na literatura apresentam atividade ótima na faixa de pH 5,0 a 6,5 e temperatura de 30 a 40 ºC e são citadas como sendo termossensíveis [26][27][28][29][46][47][48] . A enzima não purificada de Serratia plymuthica ATCC 15928 foi obtida após centrifugação a 10.000 rpm, por 20 min e rompimento da massa celular em prensa-X 26 .…”

Bacterial sucrose isomerases: properties and structural studies

“…Zhang et al (2003a) reported that the reaction end-products were clearly related to the charge of the catalytic pocket. Thus, operating at pHs higher than the enzymes optimal pH allow the formation of trehalulose, whereas at lower pH the production of glucose and fructose was promoted (Nagai et al 1994;Zhang et al 2002;Wu & Birch 2005). The kinetic parameters of various sucrose isomerases are presented in Table 1.…”

“…LX3 and Enterobacter sp. FMB1 produced essentially isomaltulose with percentages around 75-85% (Cheetham 1984;Veronese & Perlot 1999;Zhang et al 2002;Wu & Birch 2005). Due to difficulties in the chemical synthesis of these sugars, their microbial production is of commercial interest.…”

Structure/Function Relationships of Sucrose Isomerases with Different Product Specificity

“…rhapontici, 12,15) Klebsiella sp. LX3, 40) Klebsiella sp., 41) K. planticola CCRC 19112, 42) Pantoea dispersa, 43) and Enterobacter sp. FMB1, 11) which produce mainly isomaltulose (75 85%), and A. radiobacter MX 332 37) and Ps.…”