Hydroxynitrite Lab Report

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1. Introduction
Hydroxynitrile lyases (HNL, EC 4.1.2.x) are enzymes which mediate the release of hydrogen cyanide (HCN) and aldehyde or ketone from cyanohydrins [1]. These HNL catalyse enantioselective synthesis and cleavage of cyanohydrins [2]. The cyanohydrins and their derivatives are finding wide applications in industries like pharmaceuticals, agrochemicals and cosmetics since these can be readily converted into other important molecules like α-hydroxy carboxylic acids, α-hydroxy ketones and β-amino acids [3]. The HNLs have been reported, purified and characterized from several plants e.g. almond (Prunus amygdalus), flax (Linum usitatissimum), cherry (Prunus serotina), peach (Prunus persica), capulin (Prunus capuli), rosary pea (Abrus
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armenica seeds was extracted according to method outlined by Han et al. [19] with slight modification. The seeds were soaked in distilled water for 24 hr at room temperature, washed with distilled water, grounded with chilled ethyl acetate, air dried and stored at 4 °C. The dried powder material was termed as meal. The crude HNL was prepared by suspending this meal in distilled water (7.4 g/100mL). The pH of the extract was adjusted to 7.4 with 1N NH4OH to (for best effective extraction of enzyme) and kept at 4 °C overnight. It was centrifuged at 15000×g for 15 minutes and supernatant was collected and its pH was adjusted to 5.5 with 50% acetic acid. It was filtered and the filtrate was used as crude enzyme in subsequent studies. The protein concentration in the enzyme preparation was determined following the method of Bradford …show more content…
In order to select a suitable buffer system for assay of hydroxynitrile lyase of wild apricot, its activity in sodium citrate (139 U/mg protein), sodium acetate (75.2 U/mg protein) sodium phosphate (94.1 U/mg protein), potassium phosphate (71.9 U/mg protein) and citrate phosphate (15.5 U/mg protein) buffers was estimated (Fig. not shown). Effect of substrate conc. was studied and it was observed that HNL of wild apricot showed highest activity at substrate concentration of 12 µmole/ml (Fig. 3). HNL of wild apricot showed maximum activity at pH 4.75 with mandelonitrile (Fig. 4), with sodium citrate buffer of 0.1M strength (Fig. not shown). The effect of metal ions showed that mercuric ion (Hg2+) completely inhibited the activity of HNL of wild apricot, while Sr2+, Cs2+, Co2+, Cu2+, Fe2+, Fe3+, Mn2+ and Pb2+ showed partial inhibition of activity of HNL and other i.e., W6+, Mg2+, Cd2+, Zn2+ and Ca2+ has either very less or no effect on activity of hydroxynitrile lyase of wild apricot (Table

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