The molecular basis of the nonprocessive elongation mechanism in levansucrases.

Levansucrases (LSs) synthesize levan, a ß2-6-linked fructose polymer, by successively transferring the fructosyl moiety from sucrose to a growing acceptor molecule. Elucidation of the levan polymerization mechanism is important for using LSs in the production of size-defined products for application in the food and pharmaceutical industries. For a deeper understanding of the levan synthesis reaction, we determined the crystallographic structure of Bacillus subtilis LS (SacB) in complex with a levan-type fructooligosaccharide and utilized site-directed mutagenesis to identify residues involved in substrate binding. The presence of a levanhexaose molecule in the central catalytic cavity allowed us to identify five substrate-binding subsites (-1, +1, +2, +3, and +4). Mutants affecting residues belonging to the identified acceptor subsites showed similar substrate affinity (Km) values to the wildtype (WT) Km value but had a lower turnover number and transfructosylation/hydrolysis ratio. Of importance, compared with the WT, the variants progressively yielded smaller-sized low-molecular-weight levans, as the affected subsites that were closer to the catalytic site, but without affecting their ability to synthesized high-molecular-weight levans. Furthermore, an additional oligosaccharide-binding site 20 Å away from the catalytic pocket was identified, and its potential participation in the elongation mechanism is discussed. Our results clarify, for the first time, the interaction of the enzyme with an acceptor/product oligosaccharide and elucidate the molecular basis of the nonprocessive levan elongation mechanism of LSs.

AmyJ33, a truncated amylase with improved catalytic properties.

Biochemical and kinetic properties are of special interest for the specific applications of α-amylases in industrial sectors such as textile industries, detergents, biofuels and food among others. Therefore, protein engineering is currently directed towards a continuous demand to improve the properties of amylases and thus meet the specific characteristics for various industrial sectors. In the present work, modular protein engineering was performed to improve the biochemical and kinetic properties of AmyJ33r an α-amylase isolated from Bacillus siamensis JJC33M consisting of five domains, A, B, C, D and E (SBD) (Montor-Antonio et al. in 3 Biotech 7:336, 2017. https://doi.org/10.1007/s13205-017-0954-8 ). AmyJ33r is not active on native starch, only showing activity on gelatinized starch. At the C-terminal, AmyJ33r has a starch binding domain (SBD, domain E) belonging to the CBM26 family. In this study, four truncated versions were constructed and expressed in E. coli (AmyJ33-AB, AmyJ33-ABC, AmyJ33-ABCD, and SBD) to determine the role of the A, B, C, D, and E domains in the biochemical behavior of AmyJ33r on starch. Biochemical and kinetic characterization of the truncated versions showed that domain C is essential for catalysis; domain D improved enzyme activity at alkaline pH values, is also involved negatively in thermostability at 40, 50, and 60 °C and its presence favored the production of maltooligosaccharides with a higher degree of polymerization (DP4). E domain have interaction with raw starch, also the deletion of E domain (SBD) favors the affinity for the substrate while the deletion of D domain increased enzyme kcat at the time of product release. In conclusion, AmyJ33-ABC has better kinetic parameters than AmyJ33-ABCD and AmyJ33r, but is less stable than these two enzymes.

Recombinant O-mannosylated protein production (PstS-1) from Mycobacterium tuberculosis in Pichia pastoris (Komagataella phaffii) as a tool to study tuberculosis infection.

BACKGROUND: Pichia pastoris (syn. Komagataella phaffii) is one of the most highly utilized eukaryotic expression systems for the production of heterologous glycoproteins, being able to perform both N- and O-mannosylation. In this study, we present the expression in P. pastoris of an O-mannosylated recombinant version of the 38 kDa glycolipoprotein PstS-1 from Mycobacterium tuberculosis (Mtb), that is similar in primary structure to the native secreted protein. RESULTS: The recombinant PstS-1 (rPstS-1) was produced without the native lipidation signal. Glycoprotein expression was under the control of the methanol-inducible promoter pAOX1, with secretion being directed by the α-mating factor secretion signal. Production of rPstS-1 was carried out in baffled shake flasks (BSFs) and controlled bioreactors. A production up to ~ 46 mg/L of the recombinant protein was achieved in both the BSFs and the bioreactors. The recombinant protein was recovered from the supernatant and purified in three steps, achieving a preparation with 98% electrophoretic purity. The primary and secondary structures of the recombinant protein were characterized, as well as its O-mannosylation pattern. Furthermore, a cross-reactivity analysis using serum antibodies from patients with active tuberculosis demonstrated recognition of the recombinant glycoprotein, indirectly indicating the similarity between the recombinant PstS-1 and the native protein from Mtb. CONCLUSIONS: rPstS-1 (98.9% sequence identity, O-mannosylated, and without tags) was produced and secreted by P. pastoris, demonstrating that this yeast is a useful cell factory that could also be used to produce other glycosylated Mtb antigens. The rPstS-1 could be used as a tool for studying the role of this molecule during Mtb infection, and to develop and improve vaccines or kits based on the recombinant protein for serodiagnosis.

Recombinant expression of a laccase from Coriolopsis gallica in Pichia pastoris using a modified α-factor preproleader.

In this work we communicate the heterologous expression of a laccase from Coriolopsis gallica in Pichia pastoris. This enzyme has been reported to efficiently degrade a variety of pollutants such as industrial dyes. The expression strategy included using a previously reported modified α-factor preproleader for enhanced secretion and pAOX1, a methanol-responsive promoter. Methanol concentration, copper salts concentration and temperature were varied in order to enhance laccase expression in this heterologous system. A volumetric activity of 250 U/L was achieved after 12-day culture in Fernbach flasks. The protein was recovered from the supernatant and purified, obtaining a preparation with 90% electrophoretic purity. The catalytic constants of the recombinant enzyme are almost identical to the fungal enzyme, thus rendering this system a useful tool for protein engineering of laccase from C. gallica.

Size product modulation by enzyme concentration reveals two distinct levan elongation mechanisms in Bacillus subtilis levansucrase.

Two levan distributions are produced typically by Bacillus subtilis levansucrase (SacB): a high-molecular weight (HMW) levan with an average molecular weight of 2300 kDa, and a low-molecular weight (LMW) levan with 7.2 kDa. Previous results have demonstrated how reaction conditions modulate levan molecular weight distribution. Here we demonstrate that the SacB enzyme is able to perform two mechanisms: a processive mechanism for the synthesis of HMW levan and a non-processive mechanism for the synthesis of LMW levan. Furthermore, the effect of enzyme and substrate concentration on the elongation mechanism was studied. While a negligible effect of substrate concentration was observed, we found that SacB elongation mechanism is determined by enzyme concentration. A high concentration of enzyme is required to synthesize LMW levan, involving the sequential formation of a wide variety of intermediate size levan oligosaccharides with a degree of polymerization (DP) up to ∼70. In contrast, an HMW levan distribution is synthesized through a processive mechanism producing oligosaccharides with DP <20, in reactions occurring at low enzyme concentration. Additionally, reactions where levansucrase concentration was varied while the total enzyme activity was kept constant (using a combination of active SacB and an inactive SacB E342A/D86A) allowed us to demonstrate that enzyme concentration and not enzyme activity affects the final levan molecular weight distribution. The effect of enzyme concentration on the elongation mechanism is discussed in detail, finding that protein-product interactions are responsible for the mechanism shift.

Synthesis of Fructooligosaccharides by IslA4, a truncated inulosucrase from Leuconostoc citreum.

BACKGROUND: IslA4 is a truncated single domain protein derived from the inulosucrase IslA, which is a multidomain fructosyltransferase produced by Leuconostoc citreum. IslA4 can synthesize high molecular weight inulin from sucrose, with a residual sucrose hydrolytic activity. IslA4 has been reported to retain the product specificity of the multidomain enzyme. RESULTS: Screening experiments to evaluate the influence of the reactions conditions, especially the sucrose and enzyme concentrations, on IslA4 product specificity revealed that high sucrose concentrations shifted the specificity of the reaction towards fructooligosaccharides (FOS) synthesis, which almost eliminated inulin synthesis and led to a considerable reduction in sucrose hydrolysis. Reactions with low IslA4 activity and a high sucrose activity allowed for high levels of FOS synthesis, where 70% sucrose was used for transfer reactions, with 65% corresponding to transfructosylation for the synthesis of FOS. CONCLUSIONS: Domain truncation together with the selection of the appropriate reaction conditions resulted in the synthesis of various FOS, which were produced as the main transferase products of inulosucrase (IslA4). These results therefore demonstrate that bacterial fructosyltransferase could be used for the synthesis of inulin-type FOS.

Relevance of aromatic and polar amino acids in the specificity of Inulinase ISO3 from Kluyveromyces marxianus: A molecular dynamics approach with an experimental verification.

The Inulinase from Kluyveromyces marxianus ISO3 (Inu-ISO3) is an enzyme able to hydrolyze linear fructans such as chicory inulin as well as branched fructans like agavin. This enzyme was cloned and expressed in Komagataella pastoris to study the role of selected aromatic and polar residues in the catalytic pocket by Alanine scanning. Molecular dynamics (MD) simulations and enzyme kinetics analysis were performed to study the functional consequences of these amino acid substitutions. Site-directed mutagenesis was used to construct the mutants of the enzyme after carrying out the MD simulations between Inu-ISO3 and its substrates. Mutation Trp79:Ala resulted in the total loss of activity when fructans were used as substrates, while with sucrose, the activity decreased by 98 %. In contrast, the mutations Phe113:Ala and Gln236:Ala increased the invertase activity when sucrose was used as a substrate. Although these amino acids are not part of the conserved motifs where the catalytic triad is located, they are essential for the enzyme's activity. In silico and experimental approaches corroborate the relevance of these residues for substrate binding and their influence on enzymatic activity.

Design of chimeric levansucrases with improved transglycosylation activity.

Fructansucrases (FSs), including levansucrases and inulosucrases, are enzymes that synthesize fructose polymers from sucrose by the direct transfer of the fructosyl moiety to a growing polymer chain. These enzymes, particularly the single domain fructansucrases, also possess an important hydrolytic activity, which may account for as much as 70 to 80% of substrate conversion, depending on reaction conditions. Here, we report the construction of four chimeric levansucrases from SacB, a single domain levansucrase produced by Bacillus subtilis. Based on observations derived from the effect of domain deletion in both multidomain fructansucrases and glucansucrases, we attached different extensions to SacB. These extensions included the transitional domain and complete C-terminal domain of Leuconostoc citreum inulosucrase (IslA), Leuconostoc mesenteroides levansucrase (LevC), and a L. mesenteroides glucansucrase (DsrP). It was found that in some cases the hydrolytic activity was reduced to less than 10% of substrate conversion; however, all of the constructs were as stable as SacB. This shift in enzyme specificity was observed even when the SacB catalytic domain was extended only with the transitional region found in multidomain FSs. Specific kinetic analysis revealed that this change in specificity of the SacB chimeric constructs was derived from a 5-fold increase in the transfructosylation k(cat) and not from a reduction of the hydrolytic k(cat), which remained constant.

Structure-Function Relationship Studies of Multidomain Levansucrases from Leuconostocaceae Family.

Levansucrase LevS from Leuconostoc mesenteroides B-512F is a multidomain fructansucrase (MD-FN) that contains additional domains (ADs) to the catalytic domain. However, the understanding of the effect that these ADs have on enzyme activity remains vague. To this aim, structure-function relationship studies of these LevS ADs were performed by evaluating both biochemical properties and the enzymatic capacity of truncated versions of LevS. Joint participation of the N- and C-terminal domains is essential for stability, activity, specificity, and polymerization processes. Specifically, the N-terminal region is involved in stability, while the transition region plays an essential role in the transfructosylation reaction and polymer elongation. Based on our results, we suggest that ADs interact with each other, adopting a U-shaped topology. The importance of these ADs observed in the MD-FN of the Leuconostocaceae family is not shared by the Lactobacillaceae family. Phylogenetic analysis of LevS AD suggests that MD-FN from Lactobacillaceae and Leuconostocaceae have different evolutionary origins. This is the first study on the structure-function relationship of multidomain levansucrases from the Leuconostocaceae family. Our results point towards the functional role of AD in MD-FN and its involvement in fructan synthesis.

Bovine Interferon-Tau Activates Type I interferon-Associated Janus-signal Transducer in HPV16-positive Tumor Cell.

The mechanisms of signal transduction by interferon-tau (IFN-τ) are widely known during the gestation of ruminants. In trophoblast cells, IFN-τ involves the activation of the JAK-STAT pathway, and it can have effects on other cell types, such as tumor cells. Here we report that the HPV16-positive BMK-16/myc cell treated with ovine IFN-τ, results in the activation of the canonical JAK-STAT and non-canonical JAK-STAT pathway. The MAPK signaling pathway was activated, we detected the proteins MEK1, MEK2, Raf1, STAT3, STA4, STAT5 and STAT6. Moreover, IFN-τ induced the expression of MHC Class I, MX and IP10 in the tumor cells and this response may be associated with the viral replication and with the anti-proliferative and the immunoregulatory effects of IFN-τ.

Self-assembled high molecular weight inulin nanoparticles: Enzymatic synthesis, physicochemical and biological properties.

Inulin has interesting physicochemical and functional properties, and therefore a wide range of applications in the food and medical industries. It has gained great traction due to its ability to form nanoparticles and its possible application as nanovehicle for drug delivery. In this work, we demonstrated that the enzymatically-synthesized high molecular weight (HMW) inulin forms stable spherical nanoparticles with an average diameter of 112 ± 5 nm. The self-assemblage of HMW inulin nanoparticles is carried out during enzymatic synthesis of the polymer, and become detectable after a certain critical aggregation concentration (CAC) is reached. Both, the CAC and nanoparticle size are influenced by the reaction temperature. These nanoparticles are not toxic for peripheral blood mononuclear cells, at concentrations below 200 µg/mL; no significant prebiotic potential was detected in cultures of 13 probiotic strains. This work contributes to a better understanding of the formation of HMW inulin nanoparticles and their biological properties.

Functional role of the additional domains in inulosucrase (IslA) from Leuconostoc citreum CW28.

BACKGROUND: Inulosucrase (IslA) from Leuconostoc citreum CW28 belongs to a new subfamily of multidomain fructosyltransferases (FTFs), containing additional domains from glucosyltransferases. It is not known what the function of the additional domains in this subfamily is. RESULTS: Through construction of truncated versions we demonstrate that the acquired regions are involved in anchoring IslA to the cell wall; they also confer stability to the enzyme, generating a larger structure that affects its kinetic properties and reaction specificity, particularly the hydrolysis and transglycosylase ratio. The accessibility of larger molecules such as EDTA to the catalytic domain (where a Ca2+ binding site is located) is also affected as demonstrated by the requirement of 100 times higher EDTA concentrations to inactivate IslA with respect to the smallest truncated form. CONCLUSION: The C-terminal domain may have been acquired to anchor inulosucrase to the cell surface. Furthermore, the acquired domains in IslA interact with the catalytic core resulting in a new conformation that renders the enzyme more stable and switch the specificity from a hydrolytic to a transglycosylase mechanism. Based on these results, chimeric constructions may become a strategy to stabilize and modulate biocatalysts based on FTF activity.

Understanding the transfer reaction network behind the non-processive synthesis of low molecular weight levan catalyzed by Bacillus subtilis levansucrase.

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.

Effect of differential processing of the native and recombinant α-amylase from Bacillus amyloliquefaciens JJC33M on specificity and enzyme properties.

AmyJ33, an α-amylase isolated from Bacillus amyloliquefaciens JJC33M, has been characterized as a non-metalloenzyme that hydrolyzes raw starch. In this work, the gene that codifies for AmyJ33 was isolated and cloned. The recombinant α-amylase (AmyJ33r) produced had a molecular weight of 72 kDa, 25 kDa higher than the native α-amylase (AmyJ33). Our results suggest that the C-terminal was processed in a different way in the native and the recombinant enzyme causing the difference observed in the molecular weight. Additionally, the enzyme activity, specificity and biochemical behavior were affected by this larger C-terminal extra region in AmyJ33r, since the enzyme lost the ability to hydrolyze raw starch compared to the native but increased its thermal stability and pH stability, and modified the profile of activity toward alkaline pH. It is suggested that the catalytic domain in recombinant enzyme, AmyJ33r, could be interfered or blocked by the amino acids involved in the C-terminal additional region producing changes in the enzyme properties.

Draft Genome Sequence of Leuconostoc citreum CW28 Isolated from Pozol, a Pre-Hispanic Fermented Corn Beverage.

Leuconostoc citreum CW28 was isolated from pozol, a Mayan fermented corn beverage. This strain produces a cell-associated inulosucrase, the first described in bacteria. Its draft genome sequence, announced here, has an estimated size of 1.98 Mb and harbors 1,915 coding genes, 12 rRNAs, 68 tRNAs, 17 putative pseudogenes, and 1 putative phage.

Intrinsic Levanase Activity of Bacillus subtilis 168 Levansucrase (SacB).

Levansucrase catalyzes the synthesis of fructose polymers through the transfer of fructosyl units from sucrose to a growing fructan chain. Levanase activity of Bacillus subtilis levansucrase has been described since the very first publications dealing with the mechanism of levan synthesis. However, there is a lack of qualitative and quantitative evidence regarding the importance of the intrinsic levan hydrolysis of B. subtilis levansucrase and its role in the levan synthesis process. Particularly, little attention has been paid to the long-term hydrolysis products, including its participation in the final levan molecules distribution. Here, we explored the hydrolytic and transferase activity of the B. subtilis levansucrase (SacB) when levans produced by the same enzyme are used as substrate. We found that levan is hydrolyzed through a first order exo-type mechanism, which is limited to a conversion extent of around 30% when all polymer molecules reach a structure no longer suitable to SacB hydrolysis. To characterize the reaction, Isothermal Titration Calorimetry (ITC) was employed and the evolution of the hydrolysis products profile followed by HPLC, GPC and HPAEC-PAD. The ITC measurements revealed a second step, taking place at the end of the reaction, most probably resulting from disproportionation of accumulated fructo-oligosaccharides. As levanase, levansucrase may use levan as substrate and, through a fructosyl-enzyme complex, behave as a hydrolytic enzyme or as a transferase, as demonstrated when glucose and fructose are added as acceptors. These reactions result in a wide variety of oligosaccharides that are also suitable acceptors for fructo-oligosaccharide synthesis. Moreover, we demonstrate that SacB in the presence of levan and glucose, through blastose and sucrose synthesis, results in the same fructooligosaccharides profile as that observed in sucrose reactions. We conclude that SacB has an intrinsic levanase activity that contributes to the final levan profile in reactions with sucrose as substrate.

Production of functional oligosaccharides through limited acid hydrolysis of agave fructans.

A controlled acid thermal hydrolysis process of fructans from agave (Agave tequilana Weber var. azul) was designed to produce a mixture of functional prebiotic fructooligosaccharides and sweetening power. Despite its highly branched structure, first-order kinetic behaviour with respect to substrate concentration was found with an activation energy of 95kJ/mol, similar to the value found for other linear fructans such as chicory inulin. Fructose equivalent (FE) an analogous parameter to dextrose equivalent (DE) used in the starch industry was introduced to characterise fructan hydrolysis; maximum oligosaccharide production was observed when fructose equivalent (FE) reached 27-48 with a structural profile analysed by high-performance anion-exchange chromatography HPAEC-PAD. After hydrolysis, glucose and fructose may be eliminated through a biological purification step involving the addition of Pichia pastoris cells, which selectively consume these sugars but are unable to metabolise fructooligosaccharides.

Molecular characterization of chloranilic acid degradation in Pseudomonas putida TQ07.

Pentachlorophenol is the most toxic and recalcitrant chlorophenol because both aspects are directly proportional to the halogenation degree. Biological and abiotic pentachlorophenol degradation generates p-chloranil, which in neutral to lightly alkaline environmental conditions is hydrolyzed to chloranilic acid that present a violet-reddish coloration in aqueous solution. Several genes of the degradation pathway, cadR-cadCDX, as well as other uncharacterized genes (ORF5 and 6), were isolated from a chloranilic acid degrading bacterium, Pseudomonas putida strain TQ07. The disruption by random mutagenesis of the cadR and cadC genes in TQ07 resulted in a growth deficiency in the presence of chloranilic acid, indicating that these genes are essential for TQ07 growth with chloranilic acid as the sole carbon source. Complementation assays demonstrated that a transposon insertion in mutant CAD82 (cadC) had a polar effect on other genes contained in cosmid pLG3562. These results suggest that at least one of these genes, cadD and cadX, also takes part in chloranilic acid degradation. Based on molecular modeling and function prediction, we strongly suggest that CadC is a pyrone dicarboxylic acid hydrolase and CadD is an aldolase enzyme like dihydrodipicolinate synthase. The results of this study allowed us to propose a novel pathway that offers hypotheses on chloranilic acid degradation (an abiotic by-product of pentachlorophenol) by means of a very clear phenotype that is narrowly related to the capability of Pseudomonas putida strain TQ07 to degrade this benzoquinone.

An acceptor-substrate binding site determining glycosyl transfer emerges from mutant analysis of a plant vacuolar invertase and a fructosyltransferase.

Glycoside hydrolase family 32 (GH32) harbors hydrolyzing and transglycosylating enzymes that are highly homologous in their primary structure. Eight amino acids dispersed along the sequence correlated with either hydrolase or glycosyltransferase activity. These were mutated in onion vacuolar invertase (acINV) according to the residue in festuca sucrose:sucrose 1-fructosyltransferase (saSST) and vice versa. acINV(W440Y) doubles transferase capacity. Reciprocally, saSST(C223N) and saSST(F362Y) double hydrolysis. SaSST(N425S) shows a hydrolyzing activity three to four times its transferase activity. Interestingly, modeling acINV and saSST according to the 3D structure of crystallized GH32 enzymes indicates that mutations saSST(N425S), acINV(W440Y), and the previously reported acINV(W161Y) reside very close together at the surface in the entrance of the active-site pocket. Residues in- and outside the sucrose-binding box determine hydrolase and transferase capabilities of GH32 enzymes. Modeling suggests that residues dispersed along the sequence identify a location for acceptor-substrate binding in the 3D structure of fructosyltransferases.

Enzymatic hydrolysis of fructans in the tequila production process.

In contrast to the hydrolysis of reserve carbohydrates in most plant-derived alcoholic beverage processes carried out with enzymes, agave fructans in tequila production have traditionally been transformed to fermentable sugars through acid thermal hydrolysis. Experiments at the bench scale demonstrated that the extraction and hydrolysis of agave fructans can be carried out continuously using commercial inulinases in a countercurrent extraction process with shredded agave fibers. Difficulties in the temperature control of large extraction diffusers did not allow the scaling up of this procedure. Nevertheless, batch enzymatic hydrolysis of agave extracts obtained in diffusers operating at 60 and 90 degrees C was studied at the laboratory and industrial levels. The effects of the enzymatic process on some tequila congeners were studied, demonstrating that although a short thermal treatment is essential for the development of tequila's organoleptic characteristics, the fructan hydrolysis can be performed with enzymes without major modifications in the flavor or aroma, as determined by a plant sensory panel and corroborated by the analysis of tequila congeners.