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.
Highlights
Lots of health-promoting properties have been reported for fructans, including antioxidant activity, enhancement of the intestinal immune response, promotion of the growth/activity of beneficial colonic lactic acid bacteria, low caloric value, anticancerous, hypercholesteremic, and enhanced calcium absorption properties [1,2,3,4]
This study aims to simplify the purification steps and reduce the cost, which provides a possibility for the industrial production of fructan
The gene encoding fructan sucrase from B. subtilis was cloned with the native signal sequence, named BS-FF, or without the native signal sequence, named BS, and expressed in
Summary
Lots of health-promoting properties have been reported for fructans, including antioxidant activity, enhancement of the intestinal immune response, promotion of the growth/activity of beneficial colonic lactic acid bacteria, low caloric value, anticancerous, hypercholesteremic, and enhanced calcium absorption properties [1,2,3,4]. Microbial biosynthesis is mainly applied for the industrial production of fructans in recent years. The production of fructan mainly involves biosynthesis by microorganisms with sucrose as the raw material or by fructan sucrase enzymatic synthesis. Studies have shown that enzyme catalytic technology has great potential in the food field. Only a few of the microorganisms that have been found to be able to produce fructan produce the enzyme at a high capacity, severely limiting the large-scale industrial production and application of fructan. The production of fructan by using biological enzyme engineering technology has become the main production method, and the mass production, purification of high-efficiency fructan sucrase has become the focus of these studies. Fructan sucrase performs three different catalytic functions, polymerization, hydrolysis, and transfructosylation, and these functions depend on the kind of acceptor molecule used by the enzyme [10]. Many studies have indicated that fructan sucrase is present in Clostridium acetobutylicum [11], Lactobacillus reuteri [12], Bacillus licheniformis ANT 179 [13], and Leuconstoc mesenteroides Lm
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