Enzimhálózatok fejlesztése enantiomertiszta aminoalkoholok szintézisére: Enzymatic network development for the synthesis enantiopure aminoalcohols

Nowadays the green production of enantiopure aminoalcohols are tempting for industrial biocatalysis as long as they are highly desired intermediates or products in various biologically active compounds. In recent years, with the development of novel transaminases (TAs), this issue in an industrial perspective had become more than relevant. Enzymatic networks are proposing an elegant alternative to synthesize optically pure aminoalcohol synthons. Since TAs can accept hydroxy aldehydes as their substrate, aminoalcohols could be synthesized via this route. In this work we present the efficiency of a new enzymatic network of three enzymes (2 major and one auxiliary enzyme) which can be used to transform aldehydes into enantiopure aminoalcohols. The 2 main enzymes (aldolase, transaminase) were used in a two-step enzymatic cascade reaction to obtain the desired product, while the auxiliary enzyme (decarboxylase) was utilized to shift the thermodynamic equilibrium towards product formation. Our further objective is to develop a continuous flow system for enhancing the system productivity. Kivonat Számos enantiomertiszta aminoalkoholok fontos köztitermékek vagy termékek számos biológiailag aktív vegyület szintézisében1. Napjainkban előtérbe került ezen anyagok zöld ipari biokatalitikus szintézise, mivel az elmúlt években, az új transzaminázok (TA-ok) fejlesztésével ezen feladat ipari léptékben is megvalósíthatóvá vált2. A több enzimes hálózati rendszerek elegáns alternatívát nyújtanak optikailag tiszta aminoalkoholok előállítására3. Mivel a hidroxialdehidek szubsztrátumai a transzaminázoknak4, az aminoalkoholok ezen enzimek segítségével is előállíthatók. Ebben a munkában egy új három enzimből álló (2 fő és egy segédenzim) hálózat hatékonyságát mutatjuk be melynek segítségével aldehidek enantiomer tiszta aminoalkoholokká alakíthatóak. A 2 főenzim (aldoláz, transzamináz) egy kétlépéses kaszkád rendszerben alakítják át a kiinduló anyagot végtermékké, míg a segédenzim (dekarboxiláz) a termodinamikai egyensúly eltolását biztosítja a kívánt termék irányába. Továbbá a termelékenység növelése céljából, az enzimhálozat áramlásos rendszerben való optimalizálását fogjuk végezn

Aminated Single-walled Carbon Nanotubes as Carrier for Covalent Immobilization of Phenylalanine Ammonia-lyase

A new and efficient immobilized form of phenylalanine ammonia-lyase (PAL) was obtained by covalent linkage onto amino functionalized single-walled carbon nanotubes (SwCNTNH2) as carrier. The catalytic properties of the resulted nanostructured biocatalyst (SwCNTNH2-PAL) were tested in the kinetic resolution of racemic 2-amino-3-(thiophen-2-yl)propanoic acid 1 by ammonia elimination and in the enantiotope selective addition of ammonia onto (E)-3-(thiophen-2-yl)acrylic acid 2. SwCNTNH2-PAL was a durable biocatalyst in batch mode for ammonia elimination from 1 (>85% of original activity after 7 cycles) and in ammonia addition to 2 (>70% of original activity after 3 cycles in 6 M NH3 , pH 10.0). The ammonia addition onto 2 was also studied in a continuous-flow microreactor packed with SwCNTNH2-PAL (2 M NH3, pH 10.0, 15 bar) in the 30-80 °C temperature range. No significant loss of PAL activity was observed over 72 h in the microreactor up to 60 °C. Productivity of SwCNTNH2-PAL at 30 °C was significantly higher in the enantiotope selective ammonia addition performed in a packed-bed reactor operated in continuous-flow mode (rflow =2.41 mmol min-1 g-1) than in the reaction performed in batch system (rbatch = 1.38 mmol min-1 g-1)

A NOVEL PHENYLALANINE AMMONIA-LYASE FROM KANGIELLA KOREENSIS

This study describes cloning of the gene encoding a novel phenylalanine ammonia-lyase from Kangiella koreensis (KkPAL) into pET19b expression vector. Optimization of protein expression and purification conditions yielded 15 mg pure soluble protein from one liter of E.coli culture. Enzymatic activity measurements of the ammonia elimination reaction from different natural aromatic amino acids proved the protein to be a phenylalanine ammonia-lyase. The isolated protein showed remarkably high, 81.7 °C melting temperature, making it especially suitable for biocatalytic applications

Expression and purification of recombinant phenylalanine 2,3-aminomutase from Pantoea agglomerans

In the present study the gene of phenylalamine 2,3-aminomutase from Pantoea agglomerans (PaPAM) was cloned into pET-19b vector and used for its expression in competent Escherichia coli cells. The recombinant plasmid, PaPAM-pET-19b, was transformed into competent E. coli strain BL21(DE3)pLysS cells. Overnight culture of the transformed bacteria was induced by the addition of isopropylthio-β-D-galactoside (IPTG) to the final concentrations of 0.1, 0.5 and 1 mM. Also, the effects of different temperatures (15, 20, 25 and 30°C) and the incubation time of PaPAM. The fermentation process was scaled up to 10 L fermentor. Affinity purification conditions were analyzed by SDS-PAGE. The Tm and the activity of the purified enzyme was also investigated

Expression and purification of recombinant phenylalanine ammonia-lyase from Petroselinum crispum

In the present study the molecular cloning, expression and purification of recombinant PcPAL, with a cleavable N-terminal His-tag is described. The PcPAL gene was cloned into pET-19b vector and transformed to different E.coli host cells. The optimization of expression and purification processes provided recombinant protein with high purity in its native, tetrameric fold with a yield of 7-8 mg protein / 1 L culture. The activity of the recombinant protein was tested towards its natural substrate L-Phe, the KM, and kcat values suggesting excellent catalytic properties of the recombinant enzyme

Geoelektromágnesség és a változó Föld = Geoelectromagnetism and the changing Earth

A témában folyó tevékenység magában foglalja a Nagycenki Geofizikai Obszervatórium műszerezettségének fejlesztését (geomágneses mérőrendszerek, ionoszonda), de más műszer- és módszerfejlesztéseket is. Az obszervatórium adatai adatbázisban hozzáférhetők. A Nagycenki és a Tihanyi Obszervatórium valamint állandó és időszakos hálózatok mérési adatai alapján különösen az egyes paraméterek hosszú idejű változásainak vizsgálata folyt. Így a geomágneses tevékenység szekuláris növekedését, légköri elektromos potenciálgradiens csökkenését valamint a geomágneses pulzációk téli/decemberi csillapításának 11 éves modulációját sikerült kimutatni. Több módszerrel is (pulzációk, whistlerek, modellezés) pontosultak a magnetoszféra szerkezetére és jellemző paramétereire vonatkozó ismeretek. Keresték a földrengések elektromágneses előfutárait. Jelentősen bővültek a villámtevékenységből eredő Schumann rezonanciára valamint elektromágneses tranziensekre vonatkozó ismeretek. Az ionoszférabeli elektronsűrűség esetében jelentős tengerpart-hatás mutatkozott. Az altalaj ellenállásában a globális éghajlatváltozás hatását keresve a beszivárgó csapadékvíz okozta ellenállás változásra sikerült példát találni. Folytatódott a tektonikai hatásra kialakult gyenge zónák elektromágneses kutatása. | Activity in the present field includes the development of the instrumentation of the Nagycenk Geophysical Observatory (geomagnetic measuring systems, ionosonde), moreover other instrumental and methodological developments, too. Observatory data are available in a database. Based on result of the Nagycenk and Tihany observatories and on data of permanent and temporal networks, long-term trends of different electromagnetic parameters were investigated. Thus geomagnetic activity was found secularly increasing, a decrease of the atmospheric electric potential gradient and a 11-year modulation of the winter/December attenuation of the geomagnetic pulsation activity were confirmed. Several possibilities (pulsations, whistlers, modelling) were used to improve knowledge about structure and parameters of the magnetosphere. Electromagnetic precursors of earthquakes were searched. A significant increase of understanding was obtained in connection with Schumann resonances and electromagnetic transients caused by lightening. It was shown that sea-coasts influence characteristically changes in ionospheric electron content. When looking for the effect of the global climate changes in the subsurface electric resistivity, an example was discovered for the decrease of the resistivity due to infiltrating water from precipitation. Electromagnetic exploration of tectonically conditioned weak zones was continued, too

Heterocycles 38. Biocatalytic Synthesis of New Heterocyclic Mannich Bases and Derivatives

This paper describes the biocatalytic synthesis of new Mannich bases containing various heterocyclic rings (thiazole, furane, thiophene, pyridine) by applying the lipase catalyzed trimolecular condensation of the corresponding heterocyclic aldehydes with acetone and primary aromatic amines, in mild and eco-friendly reaction conditions. The obtained Mannich bases were acylated to their corresponding N-acetyl derivatives. All compounds were characterized by 1H-NMR, 13C-NMR and MS spectrometry

Heterocycles 36. Single-Walled Carbon Nanotubes-Bound N,N-Diethyl Ethanolamine as Mild and Efficient Racemisation Agent in the Enzymatic DKR of 2-Arylthiazol-4-yl-alanines

In this paper we describe the chemoenzymatic synthesis of enantiopure l-2-arylthiazol-4-yl alanines starting from their racemic N-acetyl derivatives; by combining the lipase-catalysed dynamic kinetic resolution of oxazol-5(4H)-ones with a chemical and an enzymatic enantioselective hydrolytic step affording the desired products in good yields (74%–78%) and high enantiopurities (ee > 99%). The developed procedure exploits the utility of the single-walled carbon nanotubes-bound diethylaminoethanol as mild and efficient racemisation agent for the dynamic kinetic resolution of the corresponding oxazolones

Methylidene Group in Phosphonic Acid Analogue of Phenylalanine Reverses the Enantiopreference of Binding to Phenylalanine Ammonia-Lyases

Aromatic amino acid ammonia-lyases and aromatic amino acid 2,3-aminomutases contain the post-translationally formed prosthetic 3,5-dihydro-4-methylidene-5H-imidazol-5-one (MIO) group. MIO-enzymes catalyze the stereoselective synthesis of α- or β-amino acid enantiomers, making these chemical processes environmentally friendly and affordable. Characterization of novel inhibitors enables structural understanding of enzyme mechanism and recognize promising herbicide candidates as well. The present study found, that both enantiomers of the aminophosphonic acid analogue of the natural substrate phenylalanine and a novel derivative bearing a methylidene at the β-position inhibited phenylalanine ammonia-lyases (PAL), representing MIO enzymes. X-ray methods unambiguously determined the absolute configuration of all tested enantiomers during their synthesis. Enzyme kinetic measurements revealed the enantiomer of the methylidene substituted substrate analogue being mirror image relation to the natural L-phenylalanine as the strongest inhibitor. Isothermal titration calorimetry (ITC) confirmed the binding constants and provided a detailed analysis of the thermodynamic driving forces of ligand binding. Molecular docking suggested that binding of the (R)- and (S)-enantiomers is possible by a mirror image packing