Francesco Molinari

University of Milan | UNIMI · Department of Food, Enviromental and Nutritional Sciences DEFENS

Ozonolysis is a useful as well as dangerous reaction for performing alkene cleavage. On the other hand, enzymes are considered a more sustainable and safer alternative. Among them, Caulobacter segnis dioxygenase (CsO2) known so far for its ability to catalyze the coenzyme-free oxidation of vinylguaiacol into vanillin, was selected and its substrate...

A one-pot, 2-step flow bioprocess for the obtainment of hesperetin (HP) and quercetin (Q) aglycones from hesperidin (HES) and rutin (RT), has been developed, employing a co-immobilized preparation of α-rhamnosidase (RN) and β-glycosidase (HOR).

A sustainable approach for the reduction of perillaldehyde to perillyl alcohol (POH) through alginate immobilized yeast cell beads has been here developed.

Bacterial nanocellulose (BC) is a highly versatile biopolymer currently pursued as a material of choice in varied themes of biomedical and material science research fields. With the aim to extend the biotechnological applications, the genetic tractability of the BC producers within the Komagataeibacter genus and its potential as an alternative host...

Bacterial nanocellulose (BC) is a highly versatile biopolymer currently pursued as a material of choice in varied themes of biomedical and material science research fields. With the aim to extend the biotechnological applications, the genetic tractability of the BC producers within the Komagataeibacter genus and its potential as an alternative host...

A straightforward chemoenzymatic synthesis of both enantiomers of propafenone hydrochloride has been developed. The key step involved the enzymatic kinetic resolution (KR) of rac-1-(chloromethyl)-2-[o-(3-phenylpropionyl)phenoxy]ethyl acetate (rac-5) with Novozym® 435 via a hydrolytic process. The best conditions for KR of rac-5 involved the use of...

With the aim of developing bioprocesses for waste valorization and a reduced water footprint, we optimized a two-step fermentation process that employs the oleaginous yeast Cutaneotrichosporon oleaginosus for the production of oil from liquid cheese whey permeate. For the first step, the addition of urea as a cost-effective nitrogen source allowed...

A collection of nature-inspired lipophilic phenolic esters have been prepared by an enzymatic synthesis under flow conditions, using the immobilized lipase B from Candida antarctica (Novozyme 435®) as a catalyst in cyclopentyl methyl ether (CPME), a non-conventional and green solvent. Their antimicrobial activity against four selected bacterial str...

Microbial carboxylesterases are valuable biocatalysts that can selectively hydrolyze a wide spectrum of esters and may find their own applicability at industrial scale. In this work, we report the calorimetric study of an atypical enantioselective carboxylesterase from Bacillus coagulans (BCE). The influence of different pH conditions (from pH 6 to...

Optimized recombinant whole cells of E. coli bearing CYP153A6 were employed for catalyzing the hydroxylation of different monoterpene derivatives. In most cases, high selectivity was observed with exclusive hydroxylation of the allylic methyl group bound to the aliphatic ring. In the case of (R)- and (S)-carvone, hydroxylation occurred also on the...

Benzil reductases are dehydrogenases preferentially active on aromatic 1,2-diketones, but the reasons for this peculiar substrate recognition have not yet been clarified. The benzil reductase (KRED1-Pglu) from the non-conventional yeast Pichia glucozyma showed excellent activity and stereoselectivity in the monoreduction of space-demanding aromatic...

Expanding the toolbox of enzymatic reactions accessible to organic chemists is one of the major goals in biocatalysis. Here we describe the development of an acyltransferase variant from Mycobacterium smegmatis in which a strategic Ser/Cys exchange in the catalytic triad dramatically expanded its synthetic capability to yield a biocatalyst able to...

Alcohol dehydrogenases (ADH) are versatile and useful enzymes employed as biocatalysts, especially for the selective oxidation of primary and secondary alcohols, and for the reduction of carbonyl moieties. A new alcohol dehydrogenase (HeADH‐II) has been identified from the genome of the halo‐adapted bacterium Halomonas elongata , which proved stabl...

A flow-based chemo-enzymatic synthesis of selected APIs (i.e., butacaine, procaine and procainamide) has been developed. A bioreactor made of MsAcT, a versatile acyltransferase from Mycobacterium smegmatis, immobilised on glyoxyl–garose, was exploited to efficiently prepare amide and ester intermediates in gram scale. Immobilised MsAcT was employed...

We have developed biocatalytic methods for the stereoselective reduction of cyclic prochiral 1,3-diketones for the production of optically active β-hydroxyketones and/or 1,3-diols. The recombinant ketoreductase KRED1-Pglu (formulated as purified catalyst) and whole cells of wild type Escherichia coli DE3 Star were used as biocatalysts, displaying d...

Racemic phenylethyl halohydrins acetates containing several groups attached to the aromatic ring were resolved via hydrolysis reaction in the presence of lipase B from Candida antarctica (Novozym® 435). In all cases, the kinetic resolution was highly selective (E > 200) leading to the corresponding (S)-β-halohydrin with ee > 99 %. However, the time...

Flow-based biocatalysis offers advantages to perform multiphase reactions, including liquid–liquid reactions, due to intensified mass transfer, compartmentalization and high local concentration of the catalyst. Enzymatic immobilization leads to stable biocatalysts, with the possibility to incorporate them in continuous reactors. The combination bet...

Mycelium‐bound lipase(s) from Aspergillus oryzae catalysed direct esterification of isoamyl alcohol and cinnamyl alcohol with acetic acid in organic solvent, showing high stability towards substrates and products. Water produced during the esterification did not significantly affect the equilibrium of the reaction, allowing for high conversions. Th...

Fusion systems have been designed that link enzymes to cofactors and immobilization modules through appropriate synthetic spacers. These modular biocatalysts (assembling catalysis, cofactor provision/regeneration and assisted immobilization) are suited to heterogeneous biocatalysis systems and can be efficiently used in continuous flow reactors.

The adaptation of sporeformers to extreme environmental conditions is frequently questioned due to their capacity to produce highly resistant endospores that are considered as resting contaminants, not representing populations adapted to the system. In this work, in order to gain a better understanding of bacterial adaptation to extreme habitats, w...

To increase the uptake of biocatalytic processes by industry, it is essential to demonstrate the reliability of enzyme-based methodologies directly applied to the production of high value products. Here, a unique, efficient, and sustainable enzymatic platform for the multi-gram synthesis of melatonin, projected to generate around 1.5 billion U.S. d...

A straightforward biocatalytic method for the enzymatic preparation of different flavor esters starting from primary alcohols (e.g., isoamyl, n-hexyl, geranyl, cinnamyl, 2-phenethyl, and benzyl alcohols) and naturally available ethyl esters (e.g., formate, acetate, propionate, and butyrate) was developed. The biotransformations are catalyzed by an...

A screening among marine yeasts was carried out for nitrile hydrolyzing activity. Meyerozyma guilliermondii LM2 (UBOCC-A-214008) was able to efficiently grow on benzonitrile and cyclohexanecarbonitrile (CECN) as sole nitrogen sources. A two-step one-pot method for obtaining cells of M. guilliermondii LM2 (UBOCC-A-214008) endowed with high nitrilase...

Hydrolytic enantioselective cleavage of different racemic non-steroidal anti-inflammatory drugs (NSAIDs) ester derivatives has been studied. An engineered esterase form Bacillus subtilis (BS2m) significantly outperformed homologous enzymes from Halomonas elongata (HeE) and Bacillus coagulants (BCE) in the enantioselective hydrolysis of naproxen est...

Enantiomerically enriched 2-hydroxymethylalkanoic acids were prepared by oxidative desymmetrisation of achiral 1,3-diols using immobilized cells of Acetobacter aceti in water at 28 °C. The biotransformations were first performed in batch mode with cells immobilized in dry alginate, furnishing the desired products with high molar conversion and reac...

Preparation of optically-pure derivatives of 2-hydroxy-2-(3-hydroxyphenyl)-2-phenylacetic acid of general structure 2 was accomplished by enzymatic hydrolysis of the correspondent esters. A screening with commercial hydrolases using the methyl ester of 2-hydroxy-2-(3-hydroxyphenyl)-2-phenylacetic acid (1a) showed that crude pig liver esterase (PLE)...

A new transaminase (VbTA) was identified from the genome of the halotolerant marine bacterium Virgibacillus 21D. Following heterologous expression in Escherichia coli, it was located entirely in the insoluble fraction. After a single mutation, identified via sequence homology analyses, the VbTA T16F mutant was successfully expressed in soluble form...

A straightforward one‐step biocatalyzed synthesis of different N ‐acyl amides in water was accomplished using the versatile and chemoselective acyltransferase from Mycobacterium smegmatis (MsAcT). Acetylation of primary arylalkyl amines was achieved with a range of acetyl donors in biphasic systems within 1 hour and at room temperature. Vinyl aceta...

Bacteriocins are peptides produced by various species of bacteria, especially lactic acid bacteria, which exhibit a large spectrum of action against spoilage bacteria and foodborne pathogens. Successful application of techniques for quantitative or qualitative bacteriocin determination relies not only on the sensitivity of the test-microorganisms,...

A straightforward chemoenzymatic synthesis of luliconazole has been developed. The key step involved the preparation of the enantiomerically pure β-halohydrin (1S)-2-chloro-1-(2,4-dichlorophenyl)-1-ethanol through kinetic resolution of the corresponding racemic acetate, via hydrolytic approach, mediated by lipase from Thermomyces lanuginosus or Nov...

Microbial carboxylesterases are important biocatalysts that selectively hydrolyze an extensive range of esters. Here, we report the biochemical and structural characterization of an atypical carboxylesterase from Bacillus coagulans (BCE), endowed with high enantioselectivity toward different 1,2-O-isopropylideneglycerol (IPG or solketal) esters. BC...

The Front Cover shows the ease of production of a range of aldehydes in continuous flow through an efficient enzymatic oxidation with minimal waste and in an environmentally friendly manner. In their Full Paper, M. L. Contente et al. demonstrate the use of an ω-transaminase from Halomonas elongata as a heterogeneous biocatalyst in a mild and effici...

The front cover artwork for Issue 20/2017 is a collaboration between the University of Nottingham (UK) and the University of Milan (Italy). The image shows the ease of production of a range of aldehydes in continuous flow through an efficient enzymatic oxidation with minimal waste and in an environmentally friendly manner. See the Full Paper itself...

Biocatalysis has widened its scope and relevance since new molecular tools, including improved expression systems for proteins, protein and metabolic engineering, and rational techniques for immobilization, have become available. However, applications are still sometimes hampered by low productivity and difficulties in scaling up. A practical and r...

A new mild and efficient process for the aqueous preparation of aldehydes, which are employed as flavour and fragrance components in food, beverage, cosmetics, as well as in pharmaceuticals, was developed using a continuous-flow approach based on an immobilised pure transaminase-packed bed reactor. HEWT, an ω-transaminase from the haloadapted bacte...

The chemoenzymatic flow synthesis of enantiomerically pure captopril, a widely used antihypertensive drug, is accomplished starting from simple, inexpensive, and readily available reagents. The first step is a heterogeneous biocatalyzed regio- and stereoselective oxidation of cheap prochiral 2-methyl-1,3-propandiol, performed in flow using immobili...

Aspergillus oryzae, isolated from traditional Japanese food “Miso”, has efficiently catalyzed the direct esterification between acetic acid and cis-3-hexen-1-ol in organic solvent. The esterification between acids and alcohols having different chain lengths was initially investigated, observing a conversion decrement parallel to the increase of the...

A robust two-enzyme system composed of an immobilized ketoreductase (KRED1-Pglu) and a glucose dehydrogenase (BmGDH) was developed via immobilization on aldehyde agarose for the stereoselective reduction of different ketones. The immobilized ketoreductase/glucose dehydrogenase system was continuously used in a flow reactor for weeks, even in the pr...

The continuous flow synthesis of a series of amines was successfully achieved by exploiting the enhanced stability and broad substrate scope of an immobilised transaminases from Halomonas elongata (HEWT). A series of substrates were tested in flow reactors and transformed to the corresponding amines in good to excellent yields. The process was impl...

Vinylphenol reductase of Dekkera bruxellensis, the characteristic enzyme liable for “Brett” sensory modification of wine, has been recently recognised to belong to the short chain dehydrogenases/reductases family. Indeed, a preliminary biochemical characterisation has conferred to the purified protein a dual significance acting as superoxide dismut...

The stereoselective desymmetrisation of achiral 2-alkyl-1,3-diols is performed by oxidation of one of the two enantiotopic primary alcohol moieties by means of Acetobacter aceti MIM 2000/28, to afford the corresponding chiral 2-hydroxymethyl alkanoic acids (up to 94% ee). The procedure, carried out in aqueous medium under mild conditions of pH, tem...

Various types of ketone substrates can be efficiently reduced in the presence of the title enzyme, isolated from P.

A transaminase from Halomonas elongata and four mutants generated by an in silico-based design were recombinantly produced in E. coli, purified and applied to the amination of mono-substituted aromatic carbonyl-derivatives. While benzaldehyde derivatives were excellent substrates, only NO2-acetophenones were transformed into the (S)-amine with a hi...

The large consumption of freshwater in fermentations and bio-transformations is a matter of concern for the sustainability of many bio-processes. The use of seawater to perform bio-processes is a sustainable alternative. In this work, we used marine yeasts from deep-sub-seafloor sediments grown in seawater as bio-catalysts to perform the stereosele...

A high yielding bioprocess for 11-α hydroxylation of canrenone (1a) using Aspergillus ochraceus ATCC 18500 was developed. The optimization of the biotransformation involved both fermentation (for achieving highly active mycelium of A. ochraceus) and biotransformation with the aim to obtain 11-α hydroxylation with high selectivity and yield. A mediu...

A sustainable and scalable process for the production of a new mixture of fructooligosaccharides (FOS) was developed using a continuous-flow approach based on an immobilized whole cells-packed bed reactor. The technological transfer from a classical batch system to an innovative flow environment allowed a significant improvement of the productivity...

Several different extreme environments—characterized by geochemical and physical extremes—are found in the ocean and in seas and many of them appeared to be hot spots for microbial abundance and diversity, thanks to the overwhelming presence of substrates and energy sources that support microbial metabolism. The most studied oceanic extreme environ...

No abstract is available for this article.

A NADPH-dependent benzil reductase (KRED1-Pglu) was used as recombinant enzyme for catalysing the reduction of different functionalised ketones. The reactions were carried out in the presence of a catalytic amount of NADP+ and an enzyme-coupled transformation (oxidation of glucose catalysed by glucose dehydrogenase), for regenerating the cofactor a...

A recombinant ketoreductase from Pichia glucozyma (KRED1-Pglu) was used for the enantioselective reduction of various mono-substituted acetophenones. Reaction rates of meta- and para-derivatives were consistent with the electronic effects described by σ-Hammett coefficients; on the other hand, enantioselectivity was determined by opposite orientato...

Ethyl secodione (1) has been enantioselectively reduced by using different biocatalysts for the preparation of ethyl secol (13R, 17S)-2a. The recombinant ketoreductase KRED1-Pglu converted the substrate with the highest reaction rate and stereoselectivity (ee > 98 %), whereas whole cells of Pichia minuta CBS 1708 showed the highest productivity. St...

A new NADPH-dependent benzil reductase (KRED1-Pglu) was identified from the genome of the non-conventional yeast Pichia glucozyma CBS 5766 and overexpressed in E. coli. The new protein was characterised and reaction parameters were optimised for the enantioselective reduction of benzil to (S)-benzoin. A thorough study of the substrate range of KRED...

Biocatalysis offers an alternative approach to conventional chemical processes for the production of single-isomer chiral drugs. Lipases are one of the most used enzymes in the synthesis of enantiomerically pure intermediates. The use of this type of enzyme is mainly due to the characteristics of their regio-, chemo- and enantioselectivity in the r...

Production of fructooligosaccharides (FOS) from sucrose was obtained using a bioprocess entirely performed in seawater. The halophilic fungus Cladosporium cladosporioides MUT 5506 was grown in a seawater-based medium and mycelium displayed an optimal activity in seawater at 50-60 °C, being stable up to 60 °C. Under optimized conditions in seawater...

Non-lypolitic esterases are carboxylester hydrolases with preference for the hydrolysis of water-soluble esters bearing short-chain acyl residues. The potential of esterases as enantioselective biocatalysts has enlarged in the last few years due to the progresses achieved in different areas, such as screening methodologies, overproduction of recomb...

Different filamentous fungi isolated from molasses and jams (kiwi and fig) were screened for fruc-tooligosaccharides (FOS) producing activity. Two strains, identified as Penicilium sizovae (CK1) and Cladosporium cladosporioides (CF(2)15), were selected on the basis of the FOS yield and kestose/nystose ratio. In both strains the activity was mostly...

The non-conventional yeast Pichia glucozyma CBS 5766 has been used for the biotransformation of different aromatic ketones and ketoesters. The growth and biotransformation conditions were optimised for the reduction of acetophenone and under optimised conditions, propiophenone, butyrophenone and valerophenone were reduced to the corresponding (S)-a...

A screening among bacterial strains isolated from water-brine interface of the deep hypersaline anoxic basins (DHABs) of the Eastern Mediterranean was carried out for the biocatalytical resolution of racemic propyl ester of anti-2-oxotricyclo[2.2.1.0]heptan-7-carboxylic acid (R,S)-1, a key intermediate for the synthesis of D-cloprostenol. Bacillus...

Glycopeptide antibiotics, such as vancomycin and teicoplanin, are used to treat life-threatening infections caused by multidrug-resistant Gram-positive pathogens. They inhibit bacterial cell wall biosynthesis by binding to the D-Ala-D-Ala C-terminus of peptidoglycan precursors. Vanco-mycin-resistant bacteria replace the dipeptide with the D-Ala-D-L...

Bimatoprost (1) and Latanoprost (2) are prostaglandin analogues widely used for glaucoma treatment. We have developed a new chemoenzymatic synthesis for 1 and 2, which utilizes a highly stereoselective sequence of biotransformations catalyzed by enzymes belonging to a single microorganism (the yeast Pichia anomala). The original synthesis, starting...

Old Yellow Enzymes (OYEs) are flavin-dependent enoate reductases (EC 1.6.99.1) that catalyze the stereoselective hydrogenation of electron-poor alkenes. Their ability to generate up to two stereocenters by the trans-hydrogenation of the C = C double bond is highly demanded in asymmetric synthesis. Isolated redox enzymes utilization require the addi...

Vinyl phenol reductase activity was assayed in extracts from 19 strains of Dekkera bruxellensis isolated from wine. In all strains, vinyl phenol reductase activity was insensitive to the presence/absence of 4-vinyl guaiacol, confirming that expression is not related to the presence of the substrate. D. bruxellensis CBS 4481 showed the highest vinyl...

Cell-bound lipases of dry mycelium of Aspergillus oryzae were used in organic solvent for the resolution of racemic flurbiprofen by direct esterification with ethanol in a flow-chemistry reactor. Under flow conditions a significant reduction of the reaction time and an increase of the enantioselectivity were achieved compared to the batch mode. Mor...

Oxidation of 1-alkyl-1,4-butanediols with Acetobacter aceti MIM 2000/28 gave the corresponding γ-lactones in good yields. The biotransformation occurred with intermediate formation of γ-lactols, which are also substrates for oxidation with Acetobacter aceti MIM 2000/28, as validated by selective biotransformation of 6β,7β;15β,16β-dimethylene-3-oxo-...

An easy and efficient one-pot methodology for the preparation of aldoximes combines the enzymatic oxidation of corresponding primary alcohols using different acetic acid bacteria and in situ condensation of the resulting aldehydes with hydroxylamine.

The lipase-catalysed kinetic resolution of flurbiprofen was performed in a flow-chemistry reactor allowing for a significant reduction of the reaction time compared to the classical batch method. The process was implemented by adding an in-line purification step of the exiting solution, consisting in a catch and release protocol, which allows easy...

Winning the primaries: Oxidation of primary and secondary alcohols, to their corresponding aldehydes or carboxylic acids and ketones respectively, are among the most important reactions in organic synthesis. In this Minireview we cover the oxidation of alcohols by biotransformation.

A new synthetic method for the one-pot preparation of aldoximes in water was developed; the method is based on the combination of the enzymatic oxidation of primary alcohols to aldehydes using different acetic acid bacteria and in situ condensation of the aldehydes with hydroxylamine.

The chemoenzymatic deacylation of ramoplanin A2 is described for the first time: ramoplanin A2 was Boc-protected and hydrogenated to Boc-protected tetrahydroramoplanin, which was subsequently deacylated using an acylase from Actinoplanes utahensis NRRL 12052. The chemoenzymatic process proceeded with 80% overall yield, which favourably compares wit...

Water/organic solvent two-liquid-phase systems have been successfully applied in the synthesis of enantiomerically pure (S)-benzoin through two different methodologies catalysed by whole cells from the non-conventional yeast Pichia glucozyma: the stereoselective monoreduction of benzil and the deracemisation of benzoin. The presence of the organic...

Biocatalysis is nowadays an established technology for the production of chemicals. Biotransformations are performed under mild conditions and can be highly selective; in particular, stereoselectivity is a key issue in most of the bioprocesses developed for transformations of organic molecules. These processes must be associated with efficient and...

Enriched Et 3-benzamido-2-hydroxy-3-phenylpropanoate (protected phenylisoserine), the chiral side chain of Taxol, was obtained via asym. redn. with transition metal-diphosphine complexes or with whole cells of non-conventional yeasts. Asym. hydrogenation was carried out using different approaches: hydrogenation of the tetra-substituted double bond...

Old yellow enzymes (OYEs, EC 1.6.99.1) are flavin-dependent oxidoreductases that catalyze the stereoselective trans-hydrogenation of the double bond, representing a promising alternative to metal-based catalysis. Bioconversion of ketoisophorone (KIP) by 28 non-conventional yeasts belonging to 16 different species was investigated. Growing cells of...

Stereoselective reduction of 2-hydroxy ketones should in principle give access to syn- and anti-1,2-diols. anti-1,2-Diols are accessible in a highly selective way using zinc borohydride [Zn(BH4)2] under chelation control (dr>20:1). Diastereoselective reduction of unprotected or even protected 2-hydroxy ketones towards syn-1,2-diols could be achieve...

A relatively complex network of reactions has been investigated, using as a network model the isothermal batch esterification of acetic acid with ethanol in n-heptane catalyzed by lyophilized mycelium of Aspergillus oryzae. The kinetic analysis was firstly carried out on the whole system, without any simplification, by means of the well-known integ...