Modern Approaches to Discovering New Hydroxynitrile Lyases for Biocatalysis.

Hydroxynitrile lyases (HNLs) have grown in importance from laboratory to industry due to their potential to catalyze stereoselective C-C bond-formation reactions in the synthesis of several chiral intermediates, such as enantiopure α-cyanohydrins, ß-nitro alcohols, and their derivatives with multiple functional groups. With these wide applications, the demand for finding new HNLs has increased, and this has led to exploration not only of new HNLs but also of new ways to discover them. An exclusive review article on HNLs by Asano et al. in 2011 described the discovery of HNLs along with their applications. Since then many scientific advancements have been seen in this area. This article aims to highlight the modern HNL discovery approaches, based mainly on 1) genome mining, 2) use of INTMSAlign software, 3) rational design (based on a millipede HNL), 4) evolution of catalytic mechanisms, 5) protein engineering guided by catalytic mechanisms, and 6) screening of plants with cyanogen glycoside (CG) content. This description is followed by future prospects. Overall this review represents the present state and the future potential of HNL discovery approaches, and so might be hoped to be instrumental not only in exploration of new HNLs but also in the invention of methods for potential biotechnological applications.

Biosensor based on atemoya peroxidase immobilised on modified nanoclay for glyphosate biomonitoring.

A biosensor based on atemoya peroxidase immobilised on modified nanoclay was developed for the determination of glyphosate by the enzyme inhibition method. The inhibitor effect of the biocide results in a decrease in the current response of the hydroquinone that was used as a phenolic substrate to obtain the base signal. The biosensor was constructed using graphite powder, multiwalled carbon nanotubes, peroxidase immobilised on nanoclay and mineral oil. Square-wave voltammetry was utilised for the optimisation and application of the biosensor, and several parameters were investigated to determine the optimum experimental conditions. The best performance was obtained using a 0.1 mol L(-1) phosphate buffer solution (pH 7.0), 1.9×10(-4) mol L(-1) hydrogen peroxide, a frequency of 30 Hz, a pulse amplitude of 50 mV and a scan increment of 4 mV. The glyphosate concentration response was linear between 0.10 and 4.55 mg L(-1) with a detection limit of 30 µg L(-1). The average recovery of glyphosate from spiked water samples ranged from 94.9 to 108.9%. The biosensor remained stable for a period of eight weeks.

Prolyl endopeptidase and thrombin inhibitory diterpenoids from the bark of Xylopia aethiopica.

The inhibitory effects of seven diterpenes, belonging to three different structural classes and isolated from the bark of Xylopia aethiopica, were investigated against the enzymes prolyl endopeptidase (PEP) and alpha-thrombin. Five compounds exhibited inhibitory activity against them.

Phosphoenolpyruvate carboxylase from cherimoya fruit: properties, kinetics and effects of high CO(2).

Phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) regulatory properties were studied in non-photosynthetic (mesocarp) and photosynthetic (peel) tissues from cherimoya (Annona cherimola Mill.) fruit stored in air, in order to gain a better understanding of in vivo enzyme regulation. Analyses were also performed with fruit treated with 20% CO(2)-20% O(2) to define the role of PEPC as part of an adaptive mechanism to high external carbon dioxide levels. The results revealed that the special kinetic characteristics of the enzyme from mesocarp--high V(max) and low sensibility to L-malate inhibition - are related to the active acid metabolism of these fruits and point to a high rate of reassimilation of respired CO(2) into keto-acids. With respect to fruit stored in air, PEPC in crude extracts from CO(2)-treated cherimoyas gave a similar V(max) (1.12+/-0.03 microkat x mg(-1) protein), a lower apparent K(m) (68+/-9 microM for PEP) and a higher I(50) of L-malate (5.95+/-0.3 mM). These kinetic values showed the increase in the affinity of this enzyme toward one of its substrate, PEP, by elevated external CO(2) concentrations. The lower K(m) value and lower sensitivity to L-malate are consistent with higher in vivo carboxylation reaction efficiency in CO(2)-treated cherimoyas, while pointing to an additional enzyme regulation system via CO(2).