Discovery and Enzymatic Screening of Genome-Mined Microbial Levanases to Produce Second-Generation β-(2,6)-Fructooligosaccharides: Catalytic Properties.

Evidence suggests that β-(2,6)-levan-type fructooligosaccharides (FOSs) possess higher prebiotic potential and selectivity than their β-(2,1)-inulin-type counterparts. The focus of the present work was to develop an enzymatic approach for the synthesis of levan-type FOSs, employing levanases (EC 3.2.1.65), specifically those performing endo-hydrolysis on levans. To identify new levanases, a selection of candidates was obtained via in silico exploration of the levanase family biodiversity through a sequence-driven approach. A collection of 113 candidates was screened according to their specific activities on low- and high-molecular-weight (MW) levan as well as thermal stability. The most active levanases were able to hydrolyze both types of levan with similar efficiency. This ultimately revealed 10 active, highly evolutionary distant and diverse candidate levanases, which demonstrated preferential hydrolysis of levan over inulin. The end-product profile differed significantly depending on levanase with levanbiose, levantriose, and levantetraose being the major FOSs. Among them, the catalytic properties of 5 selected potential new levanases (LEV9 from Belliella Baltica, LEV36 from Dyadobacter fermentans, LEV37 from Capnocytophaga ochracea, LEV79 from Vibrio natriegens, LEV91 from Paenarthrobacter aurescens) were characterized, especially in terms of pH and temperature profiles, thermal stability, and kinetic parameters. The identification of these novel levanases is expected to contribute to the production of levan-type FOSs with properties surpassing those of commercial preparations.

[1]  U. Deppenmeier,et al.  Characterization of a novel endo-levanase from Azotobacter chroococcum DSM 2286 and its application for the production of prebiotic fructooligosaccharides. , 2020, Carbohydrate polymers.

[2]  A. D. de Brevern,et al.  Investigating the Product Profiles and Structural Relationships of New Levansucrases with Conventional and Non-Conventional Substrates , 2020, International journal of molecular sciences.

[3]  W. Visessanguan,et al.  Levansucrase from Bacillus amyloliquefaciens KK9 and Its Y237S Variant Producing the High Bioactive Levan-Type Fructooligosaccharides , 2020, Biomolecules.

[4]  A. Wijeyesekera,et al.  Developments in understanding and applying prebiotics in research and practice—an ISAPP conference paper , 2020, Journal of applied microbiology.

[5]  W. Mu,et al.  An overview of levan-degrading enzyme from microbes , 2019, Applied Microbiology and Biotechnology.

[6]  Jean‐Louis Petit,et al.  Synthesis of Branched-Chain Sugars with a DHAP-Dependent Aldolase: Ketones are Electrophile Substrates of Rhamnulose-1-phosphate Aldolases. , 2018, Angewandte Chemie.

[7]  P. Adhikari,et al.  Overview of Prebiotics and Probiotics: Focus on Performance, Gut Health and Immunity – A Review , 2017 .

[8]  T. Alamäe,et al.  A Highly Active Endo-Levanase BT1760 of a Dominant Mammalian Gut Commensal Bacteroides thetaiotaomicron Cleaves Not Only Various Bacterial Levans, but Also Levan of Timothy Grass , 2017, PloS one.

[9]  Peer Bork,et al.  Interactive tree of life (iTOL) v3: an online tool for the display and annotation of phylogenetic and other trees , 2016, Nucleic Acids Res..

[10]  A. Blennow,et al.  Levanase from Bacillus subtilis hydrolyses β-2,6 fructosyl bonds in bacterial levans and in grass fructans. , 2016, International journal of biological macromolecules.

[11]  G. Gibson,et al.  Recent developments in prebiotics to selectively impact beneficial microbes and promote intestinal health. , 2015, Current opinion in biotechnology.

[12]  M. Khodadadi,et al.  Optimization of levansucrase/endo-inulinase bi-enzymatic system for the production of fructooligosaccharides and oligolevans from sucrose , 2014 .

[13]  T. Alamäe,et al.  High-Throughput Assay of Levansucrase Variants in Search of Feasible Catalysts for the Synthesis of Fructooligosaccharides and Levan , 2014, Molecules.

[14]  C. Olvera,et al.  Levan-type FOS production using a Bacillus licheniformis endolevanase , 2014 .

[15]  M. Khodadadi,et al.  Properties of Geobacillus stearothermophilus levansucrase as potential biocatalyst for the synthesis of levan and fructooligosaccharides , 2013, Biotechnology progress.

[16]  Ritesh Kumar,et al.  Discovery of new enzymes and metabolic pathways using structure and genome context , 2016 .

[17]  J. Weissenbach,et al.  Nitrilase Activity Screening on Structurally Diverse Substrates: Providing Biocatalytic Tools for Organic Synthesis , 2013 .

[18]  B. Henrissat,et al.  How nature can exploit nonspecific catalytic and carbohydrate binding modules to create enzymatic specificity , 2012, Proceedings of the National Academy of Sciences.

[19]  J. Wouters,et al.  First crystal structure of an endo-inulinase, INU2, from Aspergillus ficuum: discovery of an extra-pocket in the catalytic domain responsible for its endo-activity. , 2012, Biochimie.

[20]  S. Karboune,et al.  Enzymatic synthesis of fructooligosaccharides by levansucrase from Bacillus amyloliquefaciens: specificity, kinetics, and product characterization , 2012 .

[21]  P. Pristaš,et al.  Fructanolytic and saccharolytic enzymes of Treponema zioleckii strain kT. , 2010, Anaerobe.

[22]  W. Van den Ende,et al.  Donor and acceptor substrate selectivity among plant glycoside hydrolase family 32 enzymes , 2009, The FEBS journal.

[23]  Lynne A. Goodwin,et al.  Complete genome sequence of Capnocytophaga ochracea type strain (VPI 2845T) , 2009, Standards in genomic sciences.

[24]  Y. Koga,et al.  Clinical effects of kestose, a prebiotic oligosaccharide, on the treatment of atopic dermatitis in infants , 2009, Clinical and experimental allergy : journal of the British Society for Allergy and Clinical Immunology.

[25]  M. Grube,et al.  Development of Bifidobacterium lactis Bb 12 on β‐(2,6)‐Linked Fructan‐Containing Substrate , 2004 .

[26]  L. Hernández,et al.  Functional production and secretion of the Gluconacetobacter diazotrophicus fructose-releasing exo-levanase (LsdB) in Pichia pastoris , 2004 .

[27]  Alex Bateman,et al.  QuickTree: building huge Neighbour-Joining trees of protein sequences , 2002, Bioinform..

[28]  K. Katoh,et al.  MAFFT: a novel method for rapid multiple sequence alignment based on fast Fourier transform. , 2002, Nucleic acids research.

[29]  L. Hernández,et al.  Molecular Cloning and Expression in Escherichia coli of an Exo-Levanase Gene from the Endophytic Bacterium Gluconacetobacter diazotrophicus SRT4 , 2002, Current Microbiology.

[30]  R. Chambert,et al.  yveB, Encoding endolevanase LevB, is part of the sacB-yveB-yveA levansucrase tricistronic operon in Bacillus subtilis. , 2001, Microbiology.

[31]  W. Hartmeier,et al.  Metabolization of beta-(2,6)-linked fructose-oligosaccharides by different bifidobacteria. , 2000, FEMS microbiology letters.

[32]  S. Smeekens,et al.  Fructan: more than a reserve carbohydrate? , 1999, Plant physiology.

[33]  A. Miasnikov.  Characterization of a novel endo-levanase and its gene from Bacillus sp. L7. , 1997, FEMS microbiology letters.

[34]  U. Banerjee,et al.  Purification and properties of levanase from Rhodotorula sp. , 1996, Journal of biotechnology.

[35]  E. Wanker,et al.  Purification and characterization of the Bacillus subtilis levanase produced in Escherichia coli , 1995, Applied and environmental microbiology.

[36]  H. Nakano,et al.  Purification and Some Properties of a New Levanase from Bacillus sp. No. 71. , 1992, Bioscience, biotechnology, and biochemistry.

[37]  P. Gunasekaran,et al.  Cloning and sequencing of the sacA gene: characterization of a sucrase from Zymomonas mobilis , 1990, Journal of bacteriology.

[38]  H. Nakano,et al.  Purification and Some Properties of a Levanase from Streptomyces sp. No. 7–3 , 1990 .

[39]  G. L. Miller.  Use of Dinitrosalicylic Acid Reagent for Determination of Reducing Sugar , 1959 .