Peptide synthesis in aqueous–organic biphasic systems catalyzed by a protease isolated from Morrenia brachystephana (Asclepiadaceae)

Barberis, S; Quiroga, Evelina; Arribére, María Cecilia; Priolo, Nora

doi:10.1016/s1381-1177(01)00078-9

Cited by 24 publications

(15 citation statements)

References 19 publications

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“…Since the yields are lower in TRIS buffer than in the phosphate buffer (see Table 2) TRIS was discarded as a suitable reaction medium and all further experiments are conducted in phosphate buffer (pH 7; 1.0 M). TRIS buffer is used for some protease mediated reactions [28][29][30][31] including papain [31,33], so it is not likely that the enzyme loses its activity in this buffer. However, in the oligomerization of amino acids [16][17][18][19][20][21][22], TRIS was not reported; only citrate and phosphate buffers are mentioned.…”

Section: Co-oligomerization Of α-Amino Acid Estersmentioning

confidence: 99%

Papain Catalyzed (co)Oligomerization of α-Amino Acids

Schwab¹,

Kloosterman²,

Konieczny³

et al. 2012

Polymers

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Four hydrophobic amino acids (Leu, Tyr, Phe, Trp) were oligomerized by the protease papain in homo-oligomerization, binary co-oligomerization and ternary co-oligomerization. After 24 h, solid polydisperse reaction products of the homo-oligomerization were obtained in yields ranging from 30-80% by weight. A DP avg was calculated based on MALDI-ToF MS results using the ion counts for the chains in the product. Based on the DP avg and the yield of the homo-oligomerization it was determined that the amino acids can be ranked according to reactivity in the order: Tyr > Leu > Phe > Trp. Thermal degradation of the homo-oligomers shows two degradation steps: at 178-239 °C and at 300-330 °C. All the products left a significant amount of char ranging from 18-57% by weight at 800 °C. Binary co-oligomers were obtained as a polydisperse precipitate with a compositional distribution of the chains. Both the compositional and chain length distribution are calculated from MALDI-ToF mass spectra. By comparing the amount of each amino acid present in the chains it was determined that the amino acids are incorporated with a preference: Leu > Tyr > Phe > Trp. Ternary co-oligomers were also obtained as a precipitate and analyzed by MALDI-ToF MS. The compositional distribution and the chain length distribution were calculated from the MALDI-ToF data. The quantity of every amino acid in the chains was determined. Also determined was the influence on the DP avg when the oligomers were compared with corresponding binary co-oligomers. From the combined results it was concluded that in the co-oligomerization of three amino acids the reactivity preference is Leu > Tyr > Phe > Trp. OPEN ACCESSPolymers 2012, 4 711Thermal degradation of all the co-oligomers showed a weight loss of 2 wt% before the main oligomer degradation step at 300-325 °C.

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Section: Co-oligomerization Of α-Amino Acid Estersmentioning

confidence: 99%

Papain Catalyzed (co)Oligomerization of α-Amino Acids

Schwab¹,

Kloosterman²,

Konieczny³

et al. 2012

Polymers

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“…Therefore, in the current work, we set out to optimize the C 30 carotenoid pathway reconstructed in vitro for the synthesis of DAP from IPP and DMAPP. Furthermore, we elucidated the kinetic behavior of both the FPP synthase and DAP synthase and developed a unique optimization technique for carotenoid synthesis involving two-phase biocatalysis in the presence of a hydrophobic organic solvent (1,2). This technique allowed us to overcome product instability and resulted in a nearly 100% conversion of IPP and DMAPP into DAP, thereby providing a new route to the high-yield synthesis of isoprenoids.…”

mentioning

confidence: 99%

Preparation, Characterization, and Optimization of an In Vitro C₃₀Carotenoid Pathway

Jeong

Mijts

et al. 2005

Appl Environ Microbiol

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The ispA gene encoding farnesyl pyrophosphate (FPP) synthase from Escherichia coli and the crtM gene encoding 4,4-diapophytoene (DAP) synthase from Staphylococcus aureus were overexpressed and purified for use in vitro. Steady-state kinetics for FPP synthase and DAP synthase, individually and in sequence, were determined under optimized reaction conditions. For the two-step reaction, the DAP product was unstable in aqueous buffer; however, in situ extraction using an aqueous-organic two-phase system resulted in a 100% conversion of isopentenyl pyrophosphate and dimethylallyl pyrophosphate into DAP. This aqueous-organic two-phase system is the first demonstration of an in vitro carotenoid synthesis pathway performed with in situ extraction, which enables quantitative conversions. This approach, if extended to a wide range of isoprenoidbased pathways, could lead to the synthesis of novel carotenoids and their derivatives.Carotenoids are naturally occurring pigments found in a wide variety of plants and microorganisms (22,23). Recent studies indicate that these isoprenoid-based natural products possess biologically active properties (21, 24), thereby making them of interest to the medicinal chemist. Most carotenoids belong to the C 30 and C 40 classes, being distinguished by the number of iterative steps of isopentenyl pyrophosphate (IPP) condensation to add C 5 isoprene units. The initial synthetic step is the condensation of IPP with ␥,␥-dimethylallyl pyrophosphate (DMAPP) catalyzed by farnesyl pyrophosphate (FPP) synthase (ispA) to give geranyl pyrophosphate (GPP) and, sequentially, FPP (9). This C 15 compound serves as a central node for the synthesis of sterols (29), farnesylated proteins (4), hemes (20), sesquiterpenes (6), and dolichols (5), as well as the crucial precursor for carotenoids.C 30 carotenoids are present in the nonphotosynthetic bacteria, such as Streptococcus faecium, Staphylococcus aureus, and Methylobacterium rhodinum, and in the photosynthetic Heliobacterium species (16,25,27,28). Only the genes encoding 4,4Ј-diapophytoene (DAP) synthase (crtM) and 4,4Ј-diapophytoene desaturase (crtN) from S. aureus have been cloned and functionally expressed in Escherichia coli, resulting in the yellow 4,4Ј-diaponeurosporene from FPP (30) (Fig. 1). The first committed step in C 30 carotenoid biosynthesis is the condensation of two molecules of FPP catalyzed by DAP synthase to form the colorless carotenoid 4,4Ј-diapophytoene.Until now, only a few reports have discussed the formation of DAP through a reconstituted C 30 carotenoid metabolic pathway in vitro (13, 17), and none have provided a full kinetic analysis or reaction optimization of the in vitro pathway. Nevertheless, such a study would be valuable in order to design novel carotenoids and carotenoid-based hybrid natural products. Therefore, in the current work, we set out to optimize the C 30 carotenoid pathway reconstructed in vitro for the synthesis of DAP from IPP and DMAPP. Furthermore, we elucidated the kinetic behavior of both the FPP synthase a...

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“…The addition of organic cosolvents reduces the activity of water in the reaction medium, which favors synthesis, and also reduces the dielectric constant of the medium, which in turn reduces the acidity of the carboxyl group of the acyl donor and so increases the equilibrium constant K ion , also promoting the synthetic reaction. The decrease of water activity by organic cosolvents also favors peptide synthesis in KCS by reducing the hydrolysis of the acyl-enzyme intermediate and the final product, but again the reaction medium can be harmful to the enzyme [212][213][214]. Increased enzyme stability is associated with amino acid residues located in the surface region of the enzyme [211].…”

Section: Enzymatic Synthesis Of Peptidesmentioning

confidence: 99%

“…Increased enzyme stability is associated with amino acid residues located in the surface region of the enzyme [211]. A biphasic reaction medium is another option for performing peptide synthesis since the partition of the peptide products from the aqueous phase that contains the enzyme to the organic phase drives the equilibrium towards synthesis, with the additional benefit that the product is no longer subjected to hydrolysis [213,214,[219][220][221]. There are, however, notable exceptions, like polyols and glymes in which proteases and other peptide bond forming enzymes have been successfully employed for synthesis [215][216][217][218].…”

Section: Enzymatic Synthesis Of Peptidesmentioning

confidence: 99%

“…The most striking example is the synthesis of the leading noncaloric sweetener aspartame, where the former chemical route is being progressively replaced by enzymatic synthesis with the protease thermolysin in an organic medium [45,222,279,280] and more recently in a fully aqueous medium at high substrate concentrations within the framework of sustainable technology [281,282]. Several other examples of enzymatically synthesized biologically active peptides have been reported [156,213,218,296,297]. Several other examples of enzymatically synthesized biologically active peptides have been reported [156,213,218,296,297].…”

Section: Enzymatic Synthesis Of Peptidesmentioning

confidence: 99%

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