Ezio Battistel

Effects of substrate structure on the enantioselectivity and stereochemical course of sulfoxidation catalyzed by cyclohexanone monooxygenase

Abstract A systematic study of the stereochemistry of oxidation at sulfur catalyzed by cyclohexanone monooxygenase from Acinetobacter using as the substrates numerous alkyl aryl sulfides, dialkyl sulfides and dialkyl disulfides has been earned out. It was found that the structure of the sulfide dramatically influenced the enantioselectivity of the enzyme which yielded sulfoxides with optical purities ranging from 99% ee and R-configuration to 93% ee and S-configuration.

Characterization of Bio‐oil from Hydrothermal Liquefaction of Organic Waste by NMR Spectroscopy and FTICR Mass Spectrometry

Solid wastes of organic origins are potential feedstocks for the production of liquid biofuels, which could be suitable alternatives to fossil fuels for the transport and heating sectors, as well as for industrial use. By hydrothermal liquefaction, the wet biomass is partially transformed into a water-immiscible, oil-like organic matter called bio-oil. In this study, an integrated NMR spectroscopy/mass spectrometry approach has been developed for the characterization of the hydrothermal liquefaction of bio-oil at the molecular level. (1)H and (13)C NMR spectroscopy were used for the identification of functional groups and gauging the aromatic carbon content in the mixture. GC-MS analysis revealed that the volatile fraction was rich in fatty acids, as well as in amides and esters. High-resolution Fourier-transform ion cyclotron resonance mass spectrometry (FTICR-MS) has been applied in a systematic way to fully categorize the bio-oil in terms of different classes of components, according to their molecular formulas. Most importantly, for the first time, by using this technique, and for the liquefaction bio-oil characterization in particular, FT-MS data have been used to develop a methodology for the determination of the aromatic versus aliphatic carbon and nitrogen content. It is well known that, because they resist hydrogenation and represent sources of polluting species, both aromatic molecules and nitrogen-containing species raise concerns for subsequent upgrading of bio-oil into a diesel-like fuel.

Cyclohexanone monooxygenase catalyzed oxidation of methyl phenyl sulfide and cyclohexanone with macromolecular NADP in a membrane reactor

SummaryMethyl phenyl sulfide and cyclohexanone were oxidized to (R)- methyl phenyl sulfoxide and caprolactone by cyclohexanone monooxygenase. The reactions were carried out in a membrane reactor with the use of the macromolecular coenzyme poly (ethylene glycol)-NADP. Coenzyme regeneration was carried out with the 2-propanol/alcohol dehydrogenase system.

Effects of chemical modification on stereoselectivity of Pseudomonas cepacia lipase

Abstract Two chemically modified forms of lipase from Pseudomonas cepacia were prepared by acylation of the free amino groups of the protein with acetic and succinic anhydrides. The catalytic activity, the enantioselectivity and the thermal stability of the modified enzymes were compared with that of the native form. Succinylation determined an increase of stability without affecting the catalytical properties of the enzyme in the hydrolysis of chiral esters. Acetylation resulted in an enhanced catalytic activity coupled to a decreased stereoselectivity and thermal stability.

Simultaneous enzymatic synthesis of FAME and triacetyl glycerol from triglycerides and methyl acetate.

In the presence of methyl acetate triglycerides such as vegetable oils are transformed simultaneously into the corresponding fatty acid methyl esters and triacetyl glycerol (triacetin). The reaction, catalyzed by lipases, was studied as a function of some critical parameters, such as type of catalyst, enzyme hydration and immobilization support. The aim of the work was to achieve a conversion of the triglyceride as high as possible and to maximize the yield of the triacetin, the reaction end point. It was found that by using the immobilized lipase from Candida antarctica yields as high as 80% of both fatty acid esters and triacetin could be achieved. These results were obtained by carefully controlling the amount of water present in the reaction medium and the hydration level of the enzyme macromolecule.

Enzymatic decontamination of aqueous polymer emulsions containing acrylonitrile

Residual acrylonitrile monomer was quantitatively removed from aqueous polymer emulsions (latexes) of nitrilic rubbers by means of whole cells and cellular lysates of Brevibacterium imperiale and Corynebacterium nitrilophilus. A commercially-available immobilized nitrilase preparation allowed the conversion of acrylonitrile into acrylic acid and the recycle of the catalyst with retention of the reaction efficiency.

Purification and stability of glutaryl-7-ACA acylase from Pseudomonas sp.

The enzyme glutaryl-7-ACA acylase fromPseudomonas sp. NCIMB 40474, produced by a recombinantEscherichia coli host, was purified to homogeneity. The enzyme is a tetramer composed of two couples of asymmetric dimers, each of them constituted of two subunits of mol wt 18 and 52 kDa, respectively. It was found that glutaric acid, one of the products of the substrate hydrolysis, is an effective acylase inhibitor. Between pH 6.0 and pH 10.0, the enzymatic activity is almost constant, but below pH 6.0 it progressively declines. The acylase activity decreased sharply as a function of guanidine HC1 concentration. The loss is significant even at concentrations of denaturant lower than those causing unfolding, as suggested by UV spectroscopy and fluorescence emission studies. In these conditions (low denaturant concentration and low pH) the inactivation of the enzyme is caused by the tetramer dissociation into dimers. The lability of the quaternary structure of the enzyme is a key feature that must be taken into account for the improvement of the catalyst stability.

Synthesis of phosphatidylcholines containing ricinoleic acid

Abstract 1,2-Diricinoleoyl- and 1-ricinoleoyl-2-oleoyl-sn-glycero-3-phosphocholine were synthesised with good yields. The synthesis started with the preparation of ricinoleic acid from castor oil. The choice of a suitable agent to protect the –OH group of ricinoleic acid was a key factor to afford the final products. Several protecting groups were assayed but only β-methoxyethoxymethyl chloride (MEMCl) and 2,2,2-trichloroethyl chloroformate (TRECCl) gave reasonable yields and good optical purities of the final products. The overall yields for 1,2-diricinoleoyl-sn-glycero-3-phosphocholine and 1-ricinoleoyl-2-oleoyl-sn-glycero-3-phosphocholine were 32.1% (with respect to ricinoleic acid methyl ester using TREC as protecting group) and 10.3% (with respect to 1-trityl-glycero-3-phosphocholine), respectively.

A Model Study to Unravel the Complexity of Bio-Oil from Organic Wastes.

Binary and ternary mixtures of cellulose, bovine serum albumin (BSA) and tripalmitin, as biomass reference compounds for carbohydrates, proteins and triglycerides, respectively, were treated under hydrothermal liquefaction (HTL) conditions to describe the main reaction pathways involved in the process of bio-oil production from municipal organic wastes. Several analytical techniques (elemental analysis, GC-MS, atmospheric-pressure photo-ionisation high-resolution Fourier transform ion cyclotron resonance mass spectrometry, and 13 C cross-polarisation magic-angle spinning NMR spectroscopy) were used for the molecular-level characterisation of the resulting aqueous phase, solid residue and bio-oil, in particular. The main reaction pathways led to free fatty acids, fatty acid amides, 2,5-diketopiperazines and Maillard-type compounds as the main components of the bio-oil. The relationship of such compounds to the original components of the biomass was thus determined, which highlights the fate of the heteroatom-containing molecules in particular. Finally, the molecular composition of the bio-oils from our reference compounds was matched with that of the bio-oil from municipal organic waste biomass by comparing their high-resolution Fourier transform ion cyclotron resonance mass spectra, and we obtained a surprisingly high similarity. Hence, the ternary mixture acts as a reliable biomass model and is a powerful tool to clarify the degradation mechanisms that occur in the biomass under HTL treatment, with the ultimate goal to improve the HTL process itself by modulating the input of the organic starting matter and then the upgrading steps to bio-fuels.

Substrate specificity and stereoselectivity of hydrolytic enzymes from Brevibacterium imperiale B222

Four different hysrolytic enzymes were isolated and partially purified from Brevibacterium imperiale B222 cells. The stereoselectivity of each enzyme was assayed by using the nitrile, amide and esters derivatives of 2-aryloxypropionic acid. Within the cellular pool of hydrolytic enzymes, a non-stereoselective nitrile hydratase, a stereoselective amidase and two partially stereoselective esterases of opposite enantiomeric preference were found.