Thermostability improvement of sucrose isomerase PalI NX-5: a comprehensive strategy

Abstract

Objective: To increase the thermal stability of sucrose isomerase from Erwinia rhapontici NX-5, we designed a comprehensive strategy that combines different thermostabilizing elements.Results: We identi ed 19 high B value amino acid residues for site-directed mutagenesis. An in silicoevaluation of the in uence of post-translational modi cations on the thermostability was also carried out. The sucrose isomerase variants were expressed in Pichia pastoris X33. Thus, for the rst time, we report the expression and characterization of glycosylated sucrose isomerases. The designed mutants K174Q, L202E and K174Q/L202E, showed an increase in their optimal temperature of 5 °C, while their half-lives increased 2.21, 1.73 and 2.89 times, respectively. The mutants showed an increase in activity of 20.3% up to 25.3%. The Km values for the K174Q, L202E, and K174Q/L202E mutants decreased by 5.1%, 7.9%, and 9.4%, respectively; furthermore, the catalytic e ciency increased by up to 16%.Conclusions: With the comprehensive strategy followed, we successfully obtain engineered mutants more suitable for industrial applications than their counterparts: native (this research) and wild-type from E. rhapontici NX-5, without compromising the catalytic activity of the molecule.

Development of an engineered Bacillus subtilis strain for antibiotic‐free sucrose isomerase production

“…[ 46 ] In addition, the yield obtained in this study was also comparable to those obtained from SIase and its mutants in engineered Pichia pastoris strains, which achieved isomaltulose yields ranging from 73% to 78%. [ 47 ] Nevertheless, the yield we obtained was lower than the 95.4% yield achieved with SIase from an engineered Yarrowia lipolytica strain. [ 10 ] Given that B. subtilis typically has shorter cultivation and enzyme production durations compared to yeast strains, further enhancement of the B. subtilis strain is necessary.…”

Analyzing Current Trends and Possible Strategies to Improve Sucrose Isomerases’ Thermostability

“…Significant differences also exist in the Michaelis-Menten constants (Km) and catalytic efficiencies of various SIase (Table 2). Thus, Km is estimated in the range of 30.1 mM (recombinant isoform from Serratia plymuthica AS9) [55] to 255.1 mM (recombinant isoform from E. rhapontici NX-5) [54]. Meanwhile, catalytic efficiencies (k cat /Km) have been reported in the range of 1301 mM −1 •s −1 (recombinant isoform from P. rubrum CBS 574.77) [86] to 0.27 mM −1 •s −1 (recombinant isoform from Klebsiella sp.…”

“…The half-lives of mutants E 175 N, K 576 D, and E 175 N/K 576 D were 2.30, 1.78, and 7.65 times longer than the wild-type enzyme at 45 • C, respectively. Meanwhile, Sardiña-Peña et al [54] enhanced the thermal stability of the Erwinia rhapontici NX-5 isoform. The authors aimed to enhance the presence of thermostabilizing interactions, such as hydrogen bonds and glycosylation, in the molecule's flexible regions.…”

“…Secondly, target sites corresponded to loop regions with significant flexibility. Duan et al [55] and Sardiña-Peña et al [54] laid the foundation for their strategies by interpreting the B-factor profile of the crystal structures 3GBD (Resolution 1.95 Å) and 4HOW (Resolution 1.7 Å, Table 1), respectively. These profiles are a practical and helpful tool for identifying flexible regions (hotspots) in any enzyme.…”

“…Today, the availability of SIase expressed in food-grade strains is not a limitation in developing isomaltulose production processes. In fact, the low activity and stability of most SIase at temperatures above 40 • C is the bottleneck for their industrial application [54,55]. Precisely based on literature from recent decades and the latest advances in protein engineering, this article systematically outlines the strategies employed to increase the thermal stability of sucrose isomerases.…”