Optimization of enzymatic synthesis of ethyl hexanoate in a solvent free system using response surface methodology (RSM)

The present paper demonstrates application of biocatalysis to the synthesis of ethyl hexanoate, i.e. pineapple flavour ester, in a solvent free system. In order to evaluate the effect of various process parameters on reaction conversion, response surface methodology (RSM) complemented by central composite design (CCD) was employed. A maximum conversion of 88.57% was obtained while changing one factor at a time, at optimum conditions of temperature (50 °C), enzyme dose (2%), molar ratio acid to alcohol (1:3), speed of agitation 250 rpm and reaction time of 120 min. Based on this RSM study, the optimum predicted conditions were: 1:3.39 alcohol to acid ratio, 2.35% enzyme loading and 48.83 oC, for a predicted conversion of 90.99%. The activation energy for the enzymatic esterification was determined and calculated to be 25.76 kJ/mol. The positive values of Gibbs-free energy (ΔG), enthalpy (ΔH) and negative value of entropy (ΔS) revealed that the esterification reaction was non-spontaneous and an endothermic reaction. The reaction seems to follow bi-substrate Ping Pong Bi Bi mechanism with inhibition by both substrates.

Novel DyP from the basidiomycete Pleurotus sapidus: substrate screening and kinetics

A novel Dye-decolorizing peroxidase from the basidiomycete Pleurotus sapidus was screened for dyedecolorizing peroxidase activity with 2,2‘-azino-bis(3- ethylbenzothiazoline-6-sulfonic acid), Remazol Brilliant Blue R and Guaiacol. Additionally, the catalytic efficiency on degrading β-carotene into volatile products, and the catalyst storage stability with three different additives were also studied. The apparent inhibition constant (KS) was 51.7 μM. Optimal reaction rates (Vmax) and affinity constants (Km) towards the reducing substrates were obtained using Michaelis-Menten kinetic theory. The trend in the calculated Km’s was found to be 7.0 mM > 0.524 mM > 0.051 mM for Guaiacol, 2,2‘-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) and Remazol Brilliant Blue R. The storage stability of the catalyst was evaluated with 7.0% w/v PEG400, 7.0% w/v PEG1450 and 0.1% w/v Tween®80 at 5°C over a period of 45 days. The study revealed the longest activity conservation with PEG1450, where rDyP had lost 30% of initial activity. The enzyme solution presented similar pH and temperature dependence to known fungal dye-decolorizing peroxidases with most prolific enzymatic activities registered at pH 4.0 and temperatures below 30°C. An interesting property of the catalyst was oxidation observed in the absence of hydrogen peroxide.

Nanomaterials as novel supports for the immobilization of amylolytic enzymes and their applications: A review

Numerous types of nanoparticles and nanocomposites have successfully been employed for the immobilization and stabilization of amylolytic enzymes; α-amylases, β-amylases, glucoamylases and pullulanases. Nano-support immobilized amylolytic enzymes retained very high activity and yield of immobilization. The immobilization of these enzymes, particularly α-amylases and pullulanases, to the nanosupports is helpful in minimizing the problem of steric hindrances during binding of substrate to the active site of the enzyme. The majority of nano-support immobilized amylolytic enzymes exhibited very high resistance to inactivation induced by different kinds of physical and chemical denaturants and these immobilized enzyme preparations maintained very high activity on their repeated and continuous uses. Amylolytic enzymes immobilized on nano-supports have successfully been applied in food, fuel, textile, paper and pulp, detergent, environmental, medical, and analytical fields.

Communication. Vanillyl alcohol oxidases produced in Komagataella phaffii contain a highly stable noncovalently bound anionic FAD semiquinone

Vanillyl alcohol oxidase (VAO) from Penicillium simplicissimum is a covalent flavoprotein that has emerged as a promising biocatalyst for the production of aromatic fine chemicals such as vanillin, coniferyl alcohol and enantiopure 1-(4’-hydroxyphenyl) alcohols. The largescale production of this eukaryotic enzyme in Escherichia coli has remained challenging thus far. For that reason an alternative, eukaryotic expression system, Komagataella phaffii, was tested. Additionally, to produce novel VAO biocatalysts, we screened genomes for VAO homologues. One bacterial and five fungal sequences were selected for expression, using key active site residues as criteria for their selection. Expression of the putative vao genes in K. phaffii was successful, however expression levels were low (1 mg per litre of culture). Surprisingly, all purified enzymes were found to contain a highly stable, non-covalently bound anionic FAD semiquinone that could not be reduced by dithionite or cyanoborohydride. Activity experiments revealed that VAO expressed in K. phaffii does not produce vanillin because the enzyme suffers from oxidative stress.

Structure and function of Aspergillus niger laccase McoG

The ascomycete Aspergillus niger produces several multicopper oxidases, but their biocatalytic properties remain largely unknown. Elucidation of the crystal structure of A. niger laccase McoG at 1.7 Å resolution revealed that the C-terminal tail of this glycoprotein blocks the T3 solvent channel and that a peroxide ion bridges the two T3 copper atoms. Remarkably, McoG contains a histidine (His253) instead of the common aspartate or glutamate expected to be involved in catalytic proton transfer with phenolic compounds. The crystal structure of H253D at 1.5 Å resolution resembles the wild type structure. McoG and the H253D, H253A and H253N variants have similar activities with 2,2’-azino-bis(3- ethylbenzothiazoline-6-sulphonic acid or N,N-dimethyl-p-phenylenediamine sulphate. However, the activities of H253A and H253N with 2-amino-4-methylphenol and 2-amino-4-methoxyphenol are strongly reduced compared to that of wild type. The redox potentials and electron transfer rates (k

Biocatalysis in continuous-flow mode: A case-study in the enzymatic kinetic resolution of secondary alcohols via acylation and deacylation reactions mediated by Novozym 435®

Enzymatic kinetic resolution reactions are a well-established way to achieve optically active compounds. When enzymatic reactions are combined to continuous-flow methodologies, other benefits are added, including reproducibility, optimized energy use, minimized waste generation, among others. In this context, we herein report a case study involving lipase-mediated transesterification by acylation and deacylation reactions of secondary alcohols/esters in batch and continuous-flow modes. Acylation reactions were performed with high values of enantiomeric excess (72 up to >99%) and enantioselectivity (E > 200) for both batch and continuous-flow modes. On the other hand, for deacylation reactions using n-butanol as nucleophile, enatiomeric excess ranged between 38 to >99% and E from 6 to >200 were observed for batch mode. For deacylation reactions in continuous-flow mode, results were disappointing, as in some cases, very low or no conversion was observed. Enantiomeric excess ranged from 16 to >99% and enantioselectivity from 5 to >200 were observed. In terms of productivity, continuous-flow mode reactions were superior in both strategies (acylation: r from 1.1 up to 18.1-fold higher, deacylation: 2.8 up to 7.4- fold higher in continuous-flow than in batch mode).

Cross-linked enzyme aggregates (CLEA) in enzyme improvement – a review

Structural and functional catalytic characteristics of cross-linked enzyme aggregates (CLEA) are reviewed. Firstly, advantages of enzyme immobilization and existing types of immobilization are described. Then, a wide description of the factors that modify CLEA activity, selectivity and stability is presented. Nowadays CLEA offers an economic, simple and easy tool to reuse biocatalysts, improving their catalytic properties and stability. This immobilization methodology has been widely and satisfactorily tested with a great variety of enzymes and has demonstrated its potential as a future tool to optimize biocatalytic processes.

Bioreduction of fluoroacetophenone derivatives by endophytic fungi isolated from the marine red alga Bostrychia radicans

Four endophytic fungi isolated from the marine red alga Bostrychia radicans identified as Botryosphaeria sp. CBMAI 1197, Eutypella sp. CBMAI 1196, Hidropisphaera sp. CBMAI 1194 and Xylaria sp. CBMAI 1195 catalyzed the asymmetric bioreduction of fluoroacetophenone derivatives 1-3 to the corresponding fluorophenylalcohols 1a-3a. In the reduction reactions of 2,2,2-trifluoro-1-phenylethanone 1, all the marine fungi produced exclusively the (S)-2,2,2-trifluoro- 1-phenylethanol 1a with > 99% ee. The fungus Botryosphaeria sp. CBMAI 1197 exhibited the best enzymatic potential, leading to the highest conversion values (up to > 99%). The biocatalyst Botryosphaeria sp. CBMAI 1197 also presented active enzymes in reactions with the substrates 1-(2-(trifluoromethyl)phenyl) ethanone (2) and 1-(2,4,5-trifluorophenyl)ethanone (3), producing the respective chiral alcohols S-2a and R-3a with > 99% ee. Additionally, the fungus Hidropisphaera sp. CBMAI 1194 yielded 100% of conversion of the ketone 3 to the corresponding S-alcohol 3a, with 53% ee.

Biosensors based on oxidative enzymes for detection of environmental pollutants

In recent years, the continuous and accumulative discharge of toxic and contaminating compounds to the environment makes necessary to propose precise and quick methods for their detection and quantitation. Especially when one considers that the environmental impact of some of these emerging contaminants has not been clearly determined. Enzyme-based biosensors are an interesting alternative when inspecting different pollutants present in the environment in a quick, efficient, automatized, and economic way. Oxidative enzymes such as peroxidases and polyphenol oxidases (laccases and tyrosinases) are versatile and highly functional enzymes used for analyte recognition. Therefore, these enzymes are considered attractive and interesting biomolecules to act as recognition elements in biosensors. In this regard, detection of pollutants such as pesticides, phenols, heavy metals, and pharmaceutical compounds by using oxidative enzymes as recognition elements in biosensors is a versatile field, and it is the focus of the present review.

Biocatalytic synthesis of (S)-Practolol, a selective β-blocker

The present study describes an efficient chemoenzymatic synthesis of enantiopure (S)-Practolol, a selective β-adrenergic receptor blocker. Prior to the synthesis of the target, a synthetic protocol for (RS)-N-4-(3-chloro-2-hydroxypropoxy)phenylacetamide, an essential precursor, was developed. Various commercial lipases were screened for the kinetic resolution of (RS)- N-4-(3-chloro-2-hydroxypropoxy)phenylacetamide using toluene as solvent and vinyl acetate as an acyl donor. Among various lipases screened, Pseudomonas cepacia sol-gel AK showed the highest enantioselectivity (96% enantiomeric excess with 50% conversion), affording (S)-1-(4-acetamidophenoxy)-3-chloropropan-2-yl acetate. Optimization of the reaction parameters was carried out in order to find the best-suited conditions for the biocatalysis. Furthermore, the enantiopure intermediate was hydrolyzed and the resulting product was reacted with isopropylamine to afford (S)-Practolol. This biocatalytic procedure depicts a green technology for the synthesis of (S)-Practolol with better yield and enantiomeric excess.