Enhancing the Thermostability of Serratia plymuthica Sucrose Isomerase Using B-Factor-Directed Mutagenesis

Abstract

The sucrose isomerase of Serratia plymuthica AS9 (AS9 PalI) was expressed in Escherichia coli BL21(DE3) and characterized. The half-life of AS9 PalI was 20 min at 45°C, indicating that it was unstable. In order to improve its thermostability, six amino acid residues with higher B-factors were selected as targets for site-directed mutagenesis, and six mutants (E175N, K576D, K174D, G176D, S575D and N577K) were designed using the RosettaDesign server. The E175N and K576D mutants exhibited improved thermostability in preliminary experiments, so the double mutant E175N/K576D was constructed. These three mutants (E175N, K576D, E175N/K576D) were characterized in detail. The results indicate that the three mutants exhibit a slightly increased optimal temperature (35°C), compared with that of the wild-type enzyme (30°C). The mutants also share an identical pH optimum of 6.0, which is similar to that of the wild-type enzyme. The half-lives of the E175N, K576D and E175N/K576D mutants were 2.30, 1.78 and 7.65 times greater than that of the wild-type enzyme at 45°C, respectively. Kinetic studies showed that the Km values for the E175N, K576D and E175N/K576D mutants decreased by 6.6%, 2.0% and 11.0%, respectively, and their kcat/Km values increased by 38.2%, 4.2% and 19.4%, respectively, compared with those of the wild-type enzyme. After optimizing the conditions for isomaltulose production at 45°C, we found that the E175N, K576D and E175N/K576D mutants displayed slightly improved isomaltulose yields, compared with the wild-type enzyme. Therefore, the mutants produced in this study would be more suitable for industrial biosynthesis of isomaltulose.

Imidazole Nitrogens of Two Histidine Residues Participating in N–H···N Hydrogen Bonds in Protein Structures: Structural Bioinformatics Approach Combined with Quantum Chemical Calculations

“…The inherent thermal mobility of protein atoms around their equilibrium positions is measured in X-ray crystallography as B -factors (also referred to as temperature factors or atomic displacement parameters). B -factors have been used to design mutagenesis experiments, to predict protein binding interfaces and affinities, , and to predict intrinsically disordered regions in proteins. , B -factors have been used to confirm the lone-pair−π interactions between aromatic residues and water molecules . In this study, we analyzed the B -factors of histidine residues participating in N–H···N hydrogen bonds to find whether the interacting His pairs can be discriminated on the basis of B -factor analysis.…”

Biocatalysis and Strategies for Enzyme Improvement

“…A more efficient one-step version of QuikChange method suitable for single or multiple-site insertions and deletions was described in [96]. Successful applications of SDM include the induction of acid-resistance and 16.7-fold higher catalytic activity in a-amylase used for industrial sugar and detergent production [97], thermostability enhancement in sucrose isomerase producing a non-cariogenic, nutritional sugar isomaltulose that slows down the rate of insulin release in diabetes management [98], and even attempts to design high efficiency H 2 -producing cyanobacterial cells to make biofuels [94]. At the same time, numerous attempts of RR fail due to insufficient knowledge of mechanisms responsible for the specific structure-function relationships.…”

Direct Isomaltulose Synthesis From Beet Molasses by Immobilized Sucrose Isomerase

“…SIase-producing microbes for isomaltulose biosynthesis have been mainly found in bacteria including Pantoea dispersa (Wu and Birch, 2004), Enterobacter spp. (Park et al, 2010), Protaminobacter rubrum (de Oliva-Neto and Menão, 2009), Serratia plymuthica (Duan et al, 2016), Klebsiella spp. (Li et al, 2003), and Erwinia spp.…”

“…(Li et al, 2011). Recently, to improve isomaltulose production via a safe and efficient enzymatic method, various measures on heterologous expression of SIase gene, such as optimizing the promoter and/or signal peptide (Park et al, 2010;Zhang et al, 2018), site-directed mutagenesis for high thermostability (Duan et al, 2016), and displaying it on the cell surface of bacteria and fungi (Lee et al, 2011;Li et al, 2013Li et al, , 2017Wu et al, 2017;Zheng et al, 2019;Zhan et al, 2020), are explored. Among the targeted expression systems above, an ascomycetous yeast, Yarrowia lipolytica, seems to be the most attractive one based on relatively high isomaltulose yield (0.96 g/g) production (Li et al, 2017;Zhang et al, 2018).…”