Size product modulation by enzyme concentration reveals two distinct levan elongation mechanisms inBacillus subtilislevansucrase

Abstract

Two levan distributions are produced typically by Bacillus subtilis levansucrase (SacB): a high-molecular weight (HMW) levan with an average molecular weight of 2300 kDa, and a low-molecular weight (LMW) levan with 7.2 kDa. Previous results have demonstrated how reaction conditions modulate levan molecular weight distribution. Here we demonstrate that the SacB enzyme is able to perform two mechanisms: a processive mechanism for the synthesis of HMW levan and a non-processive mechanism for the synthesis of LMW levan. Furthermore, the effect of enzyme and substrate concentration on the elongation mechanism was studied. While a negligible effect of substrate concentration was observed, we found that SacB elongation mechanism is determined by enzyme concentration. A high concentration of enzyme is required to synthesize LMW levan, involving the sequential formation of a wide variety of intermediate size levan oligosaccharides with a degree of polymerization (DP) up to ∼70. In contrast, an HMW levan distribution is synthesized through a processive mechanism producing oligosaccharides with DP <20, in reactions occurring at low enzyme concentration. Additionally, reactions where levansucrase concentration was varied while the total enzyme activity was kept constant (using a combination of active SacB and an inactive SacB E342A/D86A) allowed us to demonstrate that enzyme concentration and not enzyme activity affects the final levan molecular weight distribution. The effect of enzyme concentration on the elongation mechanism is discussed in detail, finding that protein-product interactions are responsible for the mechanism shift.

Biocatalytic synthesis of lactosucrose using a recombinant thermostable β-fructofuranosidase from Arthrobacter sp. 10138

“…We suspected that it may be other oligosaccharides produced by the transfructosylation, such as levan. This result was consistent with the investigation of enzyme amount on the transfructosylation product by the levansucrase from B. goodwinii [30], B. methylotrophicus SK 21.002 [31], and B. subtilis [32].…”

Characterization of the Paenibacillus beijingensis DSM 24997 GtfD and its glucan polymer products representing a new glycoside hydrolase 70 subfamily of 4,6-α-glucanotransferase enzymes

“…beijingensis GtfD, as described for the B . subtilis levansucrase [32]. Based on the refractive index response, the P .…”

Fermentation pH Modulates the Size Distributions and Functional Properties of Gluconobacter albidus TMW 2.1191 Levan

“…Generally, the fermentation process and/or medium formulations were optimized to obtain the highest EPS concentration or yield (Chi and Zhao, 2003; Rühmkorf et al, 2012a; Qiang et al, 2013; Zhao et al, 2013; Ma et al, 2014) while some even utilized the metabolic engineering strategies to improve the EPS production (Senthilkumar et al, 2004; Feng et al, 2015). Nonetheless, few studies have focused on the possible changes of EPS size produced by one specific strain especially during the production process (Euzenat et al, 1997; Abdel-Fattah et al, 2005; Wu et al, 2013; Raga-Carbajal et al, 2016), which may finally influence the EPS functionality for various applications. In this study, the change of levan size produced by G. albidus was investigated, in which its size and subsequently its properties could be controlled by pH, since the environmental pH usually fluctuates to a great extent in the acid forming bacteria.…”