High-Throughput Assay of Levansucrase Variants in Search of Feasible Catalysts for the Synthesis of Fructooligosaccharides and Levan

Abstract

Bacterial levansucrases polymerize fructose residues of sucrose to β-2,6 linked fructans-fructooligosaccharides (FOS) and levan. While β-2,1-linked FOS are widely recognized as prebiotics, the health-related effects of β-2,6 linked FOS are scarcely studied as they are not commercially available. Levansucrase Lsc3 (Lsc-3) of Pseudomonas syringae pv. tomato has very high catalytic activity and stability making it a promising biotechnological catalyst for FOS and levan synthesis. In this study we evaluate feasibility of several high-throughput methods for screening and preliminary characterization of levansucrases using 36 Lsc3 mutants as a test panel. Heterologously expressed and purified His-tagged levansucrase variants were studied for: (1) sucrose-splitting activity; (2) FOS production; (3) ability and kinetics of levan synthesis; (4) thermostability in a Thermofluor assay. Importantly, we show that sucrose-splitting activity as well as the ability to produce FOS can both be evaluated using permeabilized levansucrase-expressing E. coli transformants as catalysts. For the first time we demonstrate the key importance of Trp109, His113, Glu146 and Glu236 for the catalysis of Lsc3. Cost-effective and high-throughput methods presented here are applicable not only in the levansucrase assay, but have a potential to be adapted for high-throughput (automated) study of other enzymes.

Levansucrases of a Pseudomonas syringae pathovar as catalysts for the synthesis of potentially prebiotic oligo- and polysaccharides

“…K-1 [74]. Since publications by Visnapuu and coworkers [84] and Mardo and others [74,90] on the Lsc3 protein, the LevU of Z. mobilis was the only levansucrase from Gram-negative bacteria subjected to extensive mutational analysis [91,104,105]. A pioneering study by Yanase et al [91] disclosed a number of catalysisrelated positions in LevU.…”

“…T m (melting temperature) determined for Lsc3 protein was 65.48C and when the protein was incubated for 30 min at 608C, it retained $80% of its sucrosesplitting activity [90]. The protein was also characterized as highly stable during long-term storage, retaining $50% of its activity after 140 days of incubation at 378C [90]. Importantly, Lsc3 could perfectly react with its substrates even in distilled water [87].…”

“…For biotechnologically relevant enzymes, stress tolerance is very important. The Lsc3 protein of Pst has been assayed for thermostability using a traditional assay of residual activity and also a high-throughput Thermofluor assay [90]. T m (melting temperature) determined for Lsc3 protein was 65.48C and when the protein was incubated for 30 min at 608C, it retained $80% of its sucrosesplitting activity [90].…”

“…The Lsc3 protein of Pst has been assayed for thermostability using a traditional assay of residual activity and also a high-throughput Thermofluor assay [90]. T m (melting temperature) determined for Lsc3 protein was 65.48C and when the protein was incubated for 30 min at 608C, it retained $80% of its sucrosesplitting activity [90]. The protein was also characterized as highly stable during long-term storage, retaining $50% of its activity after 140 days of incubation at 378C [90].…”

“…The Lsc2 and Lsc3 of Pst have been produced as heterologous proteins in E coli and after the purification thoroughly biochemically characterized [74,77,84,85,[87][88][89][90]. Table 1 reviews some biochemical parameters of above-mentioned levansucrases.…”