Biosynthesis of levan and a new method for the assay of levansucrase activity.
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ABSTRACT:
SUBMITTER: Ceska M
PROVIDER: S-EPMC1178041 | biostudies-other | 1971 Nov
REPOSITORIES: biostudies-other
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Project description: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.
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Project description:Levan-typed fructooligosaccharide (LFOS), a β-2,6 linked oligofructose, displays the potential application as a prebiotic and therapeutic dietary supplement. In the present study, LFOS was synthesized using levansucrase from Bacillus amyloliquefaciens KK9 (LsKK9). The wild-type LsKK9 was cloned and expressed in E. coli, and purified by cation exchanger chromatography. Additionally, Y237S variant of LsKK9 was constructed based on sequence alignment and structural analysis to enhance the LFOS production. High-performance anion-exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD) analysis indicated that Y237S variant efficiently produced a higher amount of short-chain LFOS than wild type. Also, the concentration of enzyme and sucrose in the reactions was optimized. Finally, prebiotic activity assay demonstrated that LFOS produced by Y237S variant had higher prebiotic activity than that of the wild-type enzyme, making the variant enzyme attractive for food biotechnology.
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Project description:Under specific reaction conditions, levansucrase from Bacillus subtilis (SacB) catalyzes the synthesis of a low molecular weight levan through the non-processive elongation of a great number of intermediates. To deepen understanding of the polymer elongation mechanism, we conducted a meticulous examination of the fructooligosaccharide profile evolution during the levan synthesis. As a result, the formation of primary and secondary intermediates series in different reaction stages was observed. The origin of the series was identified through comparison with product profiles obtained in acceptor reactions employing levanbiose, blastose, 1-kestose, 6-kestose, and neo-kestose, and supported with the isolation and NMR analyses of some relevant products, demonstrating that all of them are inherent products during levan formation from sucrose. These results allowed to establish the network of fructosyl transfer reactions involved in the non-processive levan synthesis. Overall, our results reveal how the relaxed acceptor specificity of SacB during the initial steps of the synthesis is responsible for the formation of several levan series, which constitute the final low molecular weight levan distribution.
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Project description:The connection between the gut microbiome composition and human health has long been recognized, such that the host-microbiome interplay is at present the subject of the so-called "precision medicine". Non-digestible fructooligosaccharides (FOS) can modulate the microbial composition and therefore their consumption occupies a central place in a strategy seeking to reverse microbiome-linked diseases. We created a small library of Bacillus megaterium levansucrase variants with focus on the synthesis of levan- and inulin-type FOS. Modifications were introduced at positions R370, K373 and F419, which are either part of the oligosaccharide elongation pathway or are located in the vicinity of residues that modulate polymerization. These amino acids were exchanged by residues of different characteristics, some of them being extremely low- or non-represented in enzymes of the levansucrase family (Glycoside Hydrolase 68, GH68). F419 seemed to play a minor role in FOS binding. However, changes at R370 abated the levansucrase capacity to synthesize levan-type oligosaccharides, with some mutations turning the product specificity towards neo-FOS and the inulin-like sugar 1-kestose. Although variants retaining the native R370 produced efficiently levan-type tri-, tetra- and pentasaccharides, their capacity to elongate these FOS was hampered by including the mutation K373H or K373L. Mutant K373H, for instance, generated 37- and 5.6-fold higher yields of 6-kestose and 6-nystose, respectively, than the wild-type enzyme, while maintaining a similar catalytic activity. The effect of mutations on the levansucrase product specificity is discussed.
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Project description:Produced by levansucrase, levan and levan oligosaccharides (GFn) have potential applications in food and pharmaceutical industries such as prebiotics, anti-tumor and anti-inflammatory agents. Previous study reported that Bacillus licheniformis RN-01 levansucrase could produce levan oligosaccharides and long-chain levan. However, its N251A and N251Y mutants could effectively produce short-chain oligosaccharides upto GF3, but they could not produce long-chain levan. We hypothesized that these mutations probably reduced GF3 binding affinity in levansucrase active site that contains fructosyl-Asp93 intermediate and caused GF3 to be in an unfavorable orientation for transfructosylation; therefore, levansucrase could not effectively extend GF3 by one fructosyl residue to produce GF4 and subsequently long-chain levan. However, these mutations probably did not significantly reduce binding affinity or drastically change orientation of GF2; therefore, levansucrase could still extend GF2 to produce GF3. Using this hypothesis, we employed molecular dynamics to investigate effects of these mutations on GF2/GF3 binding in levansucrase active site. Our results reasonably support this hypothesis as N251A and N251Y mutations did not significantly reduce GF2 binding affinity, as calculated by MM-GBSA technique and hydrogen bond occupations, or drastically change orientation of GF2 in levansucrase active site, as measured by distance between atoms necessary for transfructosylation. However, these mutations drastically decreased GF3 binding affinity and caused GF3 to be in an unfavorable orientation for transfructosylation. Furthermore, the free energy decomposition and hydrogen bond occupation results suggest the importance of Arg255 in GF2/GF3 binding in levansucrase active site. This study provides important and novel insight into the effects of N251A and N251Y mutations on GF2/GF3 binding in levansucrase active site and how they may disrupt production of long-chain levan. This knowledge could be beneficial in designing levansucrase to efficiently produce levan oligosaccharides with desired length.
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Project description:The formation of exopolysaccharides (EPSs) during 2,3-butanediol (2,3-BD) fermentation by Paenibacillus polymyxa increases medium viscosity, which in turn presents considerable technical and economic challenges to 2,3-BD downstream processing. To eliminate EPS production during 2,3-BD fermentation, we used homologous recombination to disable the EPS biosynthetic pathway in P. polymyxa The gene which encodes levansucrase, the major enzyme responsible for EPS biosynthesis in P. polymyxa, was successfully disrupted. The P. polymyxa levansucrase null mutant produced 2.5?±?0.1 and 1.2?±?0.2 g/liter EPS on sucrose and glucose, respectively, whereas the wild type produced 21.7?±?2.5 and 3.1?±?0.0 g/liter EPS on the same substrates, respectively. These levels of EPS translate to 8.7- and 2.6-fold decreases in EPS formation by the levansucrase null mutant on sucrose and glucose, respectively, relative to that by the wild type, with no significant reduction in 2,3-BD production. Inactivation of EPS biosynthesis led to a considerable increase in growth. On glucose and sucrose, the cell biomass of the levansucrase null mutant (8.1?±?0.8 and 6.5?±?0.3 g/liter, respectively) increased 1.4-fold compared to that of the wild type (6.0?±?0.1 and 4.6?±?0.3 g/liter, respectively) grown on the same substrates. Evaluation of the genetic stability of the levansucrase null mutant showed that it remained genetically stable over fifty generations, with no observable decrease in growth or 2,3-BD formation, with or without antibiotic supplementation. Hence, the P. polymyxa levansucrase null mutant has potential for use as an industrial biocatalyst for a cost-effective large-scale 2,3-BD fermentation process devoid of EPS-related challenges.IMPORTANCE Given the current barrage of attention and research investments toward the production of next-generation fuels and chemicals, of which 2,3-butanediol (2,3-BD) produced by nonpathogenic Paenibacillus species is perhaps one of the most vigorously pursued, tools for engineering Paenibacillus species are intensely sought after. Exopolysaccharide (EPS) production during 2,3-BD fermentation constitutes a problem during downstream processing. Specifically, EPS negatively impacts 2,3-BD separation from the fermentation broth, thereby increasing the overall cost of 2,3-BD production. The results presented here demonstrate that inactivation of the levansucrase gene in P. polymyxa leads to diminished EPS accumulation. Additionally, a new method for an EPS assay and a simple protocol employing protoplasts for enhanced transformation of P. polymyxa were developed. Overall, although our study shows that levan is not the only EPS produced by P. polymyxa, it represents a significant first step toward developing cost-effective 2,3-BD fermentation devoid of EPS-associated complications during downstream processing.
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