55 research outputs found
Data for the synthesis of oligo-gamma-glutamylglutamines as model compounds for gamma-glutamyltransferases (GGTs) and for normalization of activities of different GGTs
No full textgamma-Glutamyltransferases (GGTs) are widespread, conserved enzymes that catalyze the transfer of the gamma-glutamyl moiety from a donor substrate to water (hydrolysis) or to an acceptor amino acid (transpeptidation) through the formation of a gamma-glutamyl enzyme intermediate.
Although the vast majority of the known GGTs has a short sequence called lid-loop covering the glutamate binding site, B. subtilis GGT and some other enzymes from Bacillus spp. lack the lid loop. In order to assess the possible role of the lid loop of GGTs in substrate selection, synthetic oligo-gamma-glutamylglutamines containing up to three gamma-glutamyl residues were used as model substrates. The activities of the enzymes under investigation were standardized with respect to a common reaction to ensure comparable results. The activity of an engineered mutant enzyme containing the amino acid sequence of the lid loop from E. coli GGT inserted into the backbone of B. subtilis GGT was compared to that of the lid loop-deficient B. subtilis GGT and the lid loop-carrier E. coli GGT (Calvio, Romagnuolo, Vulcano, Speranza, Morelli Enz. Micr. Technol. 2018 [1]). Here we report the experimental procedures for the synthesis of model substrates gamma-glutamylglutamines through the method of the N-phtaloyl-L-glutamic acid anhydride and the spectral data of the synthetized compounds. The data obtained in the normalization procedure of the activities of the three enzymes are also reported
GAMMA-PGA: Production and characterization of a versatile biopolymer by B. subtilis laboratory strains
No full textIn recent years the interest for potential applications of natural biopolymers has tremendously increased due to the growing demand of industrial processes based on safe raw materials. Poly γ-glutamic acid (γ-PGA) is a versatile unusual, water-soluble, anionic homopolyamide that is raising considerable industrial interests for the multitude of its potential applications. It is efficiently purified by natural bacterial isolates, mainly belonging to the genus Bacillus. Its cost is currently estimated to be much higher than that of conventional materials in current use and, for the moment, it is very difficult to prepare such high-molecular-weight polymer by chemical synthesis. For the industrial application of γ-PGA it is necessary to enhance its productivity and to find optimal fermentation conditions.
Recently, a new Bacillus subtilis producer strain has been derived from the fully genetically characterised laboratory strain 168 (1). The genetic, biochemical and physiological knowledge accumulated in such model organism allowed to devise genetic strategies for strain improvement and setting up of suitable growth conditions, overcoming the heuristic approach thus far applied with wild producers.
We have data showing the success of such strategic genetic engineering approaches: mutant strains with improved and sustained accumulation of the product have been obtained by introducing selected mutations.
The purification procedure has been carefully analysed and purity and size of the polymer have been checked by NMR and GPC. Copurification of enzymes and other by-products will be shown and discussed. The result is a competitive γ-PGA producer strain.
1) Osera, Amati, Calvio, Galizzi (2009). Microbiology, 155:2282-228
γ-Glutamyltranspeptidase-catalyzed enzymatic synthesis of flavour enhancers from Allium sp.
No full textThe use of flavour enhancers in the food industry could be beneficial for several reasons: they ensure homogeneity of the
final products, reduce costs for condiments and favor consumer's acceptance. On the other hand, the consumer's attention for
convenient, minimally-processed, nutritious, healthy, yet tasty food prompts the food industry to an accurate choice of the
ingredients. In this scenario, naturally occurring kokumi substances could play an important role. Kokumi is a japanese term that refers to mouthfulness, thickness and long-lasting savory sensations. Kokumi substances are represented mainly by gamma-glutamyl derivatives of amino acids. They are nearly tasteless for themselves, but they elicit a strong taste sensation,
expecially in conjunction with protein-rich food [Dunkel 2007]. In vegetables of the genus Allium, kokumi substances were
identified in gamma-glutamyl derivatives of S-alkyl and S-alkenyl cysteines and their S-oxides [Ueda 1990].There is a number of
difficulties connected with the supplying of these materials. Isolation from natural sources is laborious, and their content in
vegetables varies with cultivation and storage. In addition, upon crushing the plant, they are enzymatically degraded. The
chemical synthesis is not economical, due to the need of protection/deprotection steps. We exploited recently the enzymatic synthesis at the laboratory scale of the gamma -glutamyl derivatives of S-allyl cysteine, S-methyl cysteine and methionine, catalyzed by a commercially available mammalian GGT [Speranza 2012]. In this communication we report that such flavour enhancers can be obtained as well by using a purified, home-made bacterial GGT from Bacillus subtilis, a GRAS (Generally Recognized As Safe) organism, suited to food processing
Leaf-associated bacteria from transgenic white poplar producing resveratrol-like compounds: isolation, molecular characterization and evaluation of oxidative stress tolerance
No full textThe aim of this study was the isolation and characterization of the culturable bacteria inhabiting the leaves of
transgenic white poplars (Populus alba L. ‘Villafranca’) engineered with the StSy gene for the production of resveratrollike
compounds. Resveratrol glucosides are available in small amounts from natural sources or by expensive chemical synthesis
procedures. An alternative approach for the large-scale production of these relevant pharmaceuticals is the use of
transgenic plants as bioreactors, although the occurrence of novel molecules in plants growing under field conditions might
interfere, to some extent, with the associated microbial population. Both epiphytes and endophytes were isolated from the
leaves of 2 StSy transgenic lines producing resveratrol glucosides and from an untransformed plant line grown in a greenhouse.
Eleven isolates were recovered and classified as members of the genus Bacillus by 16S rDNA-based analysis. In
addition, 2 isolates were classified as members of the Curtobacterium and Kocuria genera, respectively. Tolerance to hydrogen
peroxide, UV-C, and paraquat was evaluated, as were the swimming and swarming motility of the leaf-associated
bacteria. Interestingly, the isolates recovered from transgenic tissues showed the ability to withstand oxidative stress compared
with isolates recovered from the untransformed poplar line. In vitro bioassays showed that trans-resveratrol inhibited
both the swarming and swimming motilities in all the tested bacteria. The effects of trans-resveratrol on flagellin production,
required for motility, were also investigated by immunoblot analysis
pH-Dependent hydrolase, glutaminase, transpeptidase and autotranspeptidase activities of Bacillus subtilis γ-glutamyltransferase
No full textγ-Glutamyltransferases (γ-GTs) are heterodimeric enzymes that catalyze the transfer of a γ-glutamyl group from a donor species to an acceptor mol. in a transpeptidation reaction through the formation of an intermediate γ-glutamyl enzyme. In the search for a γ-GT from a generally recognized as safe microorganism suitable for the prodn. of γ-glutamyl derivs. with flavor-enhancing properties intended for human use, the authors cloned and overexpressed γ-GT from Bacillus subtilis. Here, the authors report the behavior of B. subtilis γ-GT in reactions involving glutamine as the donor compd. and various acceptor amino acids. The common thread emerging from these results is a strong dependence of the hydrolase, transpeptidase, and autotranspeptidase activities of B. subtilis γ-GT on pH, also in relation to the pKa of the acceptor amino acids. Glutamine, commonly referred to as a poor acceptor mol., underwent rapid autotranspeptidation at elevated pH, affording oligomeric species, in which up to 4 γ-glutamyl moieties were linked to a single glutamine. Moreover, it was found that L-glutamine was also recognized both as a donor and as an acceptor substrate. These results prove that the B. subtilis γ-GT-catalyzed transpeptidation reaction is feasible, and the obsd. activities of γ-GT from B. subtilis could be interpreted in relation to the known ability of the enzyme to process the polymeric material, γ-polyglutamic acid
Consolidated bioprocessing in engineered B. subtilis lab strains: γ-PGA production from biomass
No full textPrevious article in issueNext article in issue
The soil bacterium B. subtilis, the model organism for Gram positive bacteria, is the best characterized member of the Bacillus genus, which includes several highly exploited industrial species. Besides industrial enzymes, Bacillus spp. can synthesize poly-γ-glutamic acid (γ-PGA), a nontoxic, biodegradable, highly anionic biopolymer made up of multiple d-/l-glutamic acid monomers joined by amide linkages between the α-NH2 and γ-COOH groups. Thanks to several valuable characteristics, γ-PGA is applied in an expanding range of biotechnological fields (Ogunleye et al., 2015), including applications as drug carrier, gene delivery and scaffold material for tissue engineering (Luo et al., 2016). γ-PGA production is also an ideal model system to develop cost-competitive feedstocks for B. subtilis aerobic fermentations.
Despite B. subtilis possessing a wide array of complex-carbohydrates degrading enzymes, direct transformation of biomass into biocommodities has not yet been reported for this microorganism.
The aim of this work was to obtain economic γ-PGA production using a waste biomass as feedstock. Rice straw is one of the most abundant biomass resources, not in competition with food, for which there are no effective valorisation strategies.
In this study, the cellulolytic capabilities of B. subtilis JH642 were maximized through self-cloning procedures, and a cheap and simple pretreatment to facilitate straw saccharification was developed.
The engineered strain grew efficiently on treated straw. Moreover, by transferring the mutations supporting γ-PGA biosynthesis (Scoffone et al., 2013) into the cellulolytic strain, direct production of γ-PGA from biomass was obtained, definitely proving the applicability of Consolidated Bioprocessing concepts to B. subtilis
Enzymatic synthesis of γ-glutamyl derivatives catalyzed by a new mutant γ-glutamyltransferase with improved transpeptidase activity
No full textDespite their potential applicative interest as biologically active compounds and as flavor enhancers, γ-glutamyl derivatives are commercially underexploited compounds. This is mainly due to the difficulties connected with their supply at a reasonable cost. As a consequence, enzymatic approaches to their preparation, based on the use of γ-glutamyltransferases (GGTs), have been proposed1 to circumvent both the low-yielding extractive procedures from natural sources and the troublesome chemical synthesis, rendered uneconomical by the need of protection and deprotection steps.
GGTs catalyze the transfer of a γ-glutamyl moiety from a donor substrate (e.g. glutathione) to the primary amino group of an acceptor compound in a so-called transpeptidation reaction, through the formation of a γ-glutamyl-enzyme intermediate. However, also the use of GGTs as biocatalysts is not free from drawbacks. In addition to the transpeptidase activity, GGTs show a non-negligible hydrolase activity towards both the donor substrate and the newly formed transpeptidation product, affording irreversibly glutamic acid.2
In our ongoing studies on bacterial GGTs, we found that the presence of the lid loop – a short amino acids sequence covering the active site in most of the known GGTs – not only affects substrate selection, but also modulates hydrolase/transpeptidase activities.3 Within the TailGluTran Project,4 aimed at the development of mutant GGTs with improved transpeptidase activity, is currently under investigation a mutant enzyme obtained by inserting the sequence of the lid loop on the structure of a GGT naturally lacking it. The mutant enzyme shows promising high transpeptidase activity with respect to wild type counterparts and represents a starting point for further modifications in the search of a suitable biocatalyst intended for preparative purposes
Towards the design of improved g-glutamyltransferase as biocatalyst for nutraceuticals and food additives
No full textGamma-Glutamyl peptides are compounds deriving from the acylation of an amino acid by the gamma-carboxyl carbon of L-glutamic acid. Gamma-Glutamylation of amino acids affects some chemical-physical as well as organoleptic properties of the parent molecule. Some naturally occurring gamma-glutamyl derivatives show pharmacological activity or represent taste-active compounds with flavor-enhancing capacities.
Enzymatic approaches based on the use of -glutamyltransferases (GGT, E.C. 2.3.2.2) for the preparation of gamma-glutamyl derivatives have been proposed over time. They circumvent the drawbacks of chemical synthesis, which is cumbersome and uneconomical due to the requirement for multiple protection-deprotection steps on the interfering functionalities of the reacting molecules. Bacterial GGTs appears to be convenient biocatalysts, able to use the cheap and readily available glutamine as the gamma-glutamyl donor compound
We report preliminary results obtained with two bacterial GGTs with different architectures of the active site for the synthesis of gamma-glutamyl derivatives of S-substituted cysteines, which are naturally occurring flavor enhancers found in plant of the genus Allium.
The effects of pH, temperature and substrates molar ratio were examined in reactions catalyzed by both enzymes and will be discussed in the light of the structural differences between the two biocatalysts.
This work represents a first, unprecedented study in which two bacterial GGTs of different origins are directly compared and in which the attitude of the enzymes to function as biocatalysts is related to their structural characteristics. This approach could be useful for the design of mutant enzymes better suited for preparative purposes
Effect of the inserted active-site-covering lid loop on the catalytic activity of a mutant B. subtilis γ-glutamyltransferase (GGT)
No full textγ-Glutamylpeptides are compds. derived from the acylation of an amino acid or a short peptide by the γ-carboxyl carbon of the side chain of glutamic acid. Due to their altered chemico-phys. and organoleptic properties, they may be interesting substitutes or precursors of parent compds. used in pharmaceutical, dietetic and cosmetic formulations. Some of them are naturally occurring flavor enhancers or are endowed with biol. activities. Enzymic approaches to the synthesis of γ-glutamyl derivs. based on the use of γ-glutamyltransferases (GGTs, EC 2.3.2.2) have been proposed, which should be able to alleviate the problems connected with the troublesome and low-yielding extn. from natural sources or the non-economical chem. synthesis, which requires protection/deprotection steps. With the aim of overcoming the current limitations in the use of GGTs as biocatalysts, a mutant GGT was investigated. The mutant GGT was obtained by inserting the active-site-covering lid loop of the E. coli GGT onto the structure of B. subtilis GGT. With respect to the wild-type enzyme, the mutant showed a more demanding substrate specificity and a low hydrolase activity. These results represent an attempt to correlate the structural features of a GGT to its different activities. However, the ability of the mutant enzyme to catalyze the subsequent addn. of several γ-glutamyl units, inherited by the parent B. subtilis GGT, still represents a limitation to its full application as a biocatalyst for preparative purposes. [on SciFinder(R)
Enzymatic Synthesis of γ‐Glutamyl Dipeptides Catalysed by Mutant E. coli γ‐Glutamyltransferases
No full textγ-Glutamyltransferases (GGTs) from different sources have been proposed in recent times as biocatalysts for the enzymatic synthesis of naturally occurring γ-glutamyl derivatives with flavor-enhancer properties and interesting biological activities. Although the enzymatic approach is considered as a viable alternative to both the troublesome and low-yielding extraction from natural sources and synthesis through peptide chemistry requiring protection/deprotection steps, yields are not completely satisfactory, due to the intervention of GGT-catalysed hydrolysis and autotranspeptidation side-reactions. Here, the design and the use as biocatalyst for preparative purposes of two mutants of E. coli GGT are described. The design of mutants was pursued by docking-guided identification of residues putatively involved in interaction with the acceptor substrate, thus probably representing a first identification of residues constituting the still elusive and poorly characterized acceptor substrate binding site of the enzyme
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