A Fructan Sucrase Secreted Extracellular and Purified in One-Step by Gram-Positive Enhancer Matrix Particles

Abstract

Fructan sucrase is a kind of biological enzyme that catalyzes the synthesis of fructan, and fructan is a polysaccharide product with important industrial application value. In this study, the Fructan sucrase gene of Bacillus subtilis was cloned to plasmid PET-28A-ACMA-Z, and three clones were obtained after the transformation of Escherichia coli BL21, namely BS-FF, BSO, and BS. The clones BS-FF and BSO secreted the recombinant enzymes outside the cells, while the clone BS expressed them inside the cells. The induction experiment results showed that the optimum IPTG concentration in the medium was 0.5 mM and 1.0 mM for clones BS-FF and BSO, respectively, while the incubation conditions were at 28 °C for 8 h. The recombinant fructan sucrase was purified one step using a material called GEM particles. The results indicated that 95.25% of fructan sucrase expressed by the clone BS-FF could be secreted into the extracellular area, and even 98.78% by the clone BSO. With the above purification system, the receiving rate of the recombinant enzyme for clones BS-FF and BSO was 97.70% and 84.99%, respectively. As for the bioactivity of recombinant fructan sucrase, the optimum temperature and pH were 50 °C and 5.6, respectively. The Km and Vmax of it were 33.96 g/L and 0.63 g/(L·min), respectively. The engineered strains with the high extracellular secretion of fructan sucrase were constructed, and a one-step method for the purification of the recombinant enzyme was established. The results might provide a novel selection for the enzymatic production of fructan on a large scale.

Production of Sucrolytic Enzyme by Bacillus licheniformis by the Bioconversion of Pomelo Albedo as a Carbon Source

“…K m and V max of sleTKU004 were estimated to be 1.16 M and 5.99 µmol/min based on a Lineweaver-Burk plot (Figure 10b). K m value of sleTKU004 was higher than that of sucrolytic enzymes from other Bacilli such as B. methylotrophicus SK 21.002 (117.2 mM) [77], B. amyloliquefacien BH072 (0.71 mM) [73], B. cereus TA-11 (370 mM) [59], and B. subtilis (33.96 g/L) [69]. A high K m value indicates that high concentration of sucrose must be present to saturate sleTKU004.…”

Biosynthesis and Structural Characterization of Levan by a Recombinant Levansucrase from Bacillus subtilis ZW019

“…In the previous study, we have constructed a levansucrase expression system using E. coli BL21 as the host [20]. The recombinant genetic engineering bacterium were harvested by centrifugation at 12, 000 rpm for 20 min at 4°C, and the precipitated fractions were labeled as intracellular components.…”