Purification and characterisation of alliinase produced by Cupriavidus necator and its application for generation of cytotoxic agent: Allicin

Abstract

Allicin, an extremely active constituent of freshly crushed garlic, is produced upon reaction of alliin with the enzyme alliinase (EC 4.4.1.4). A bacterium Cupriavidus necator with the ability of alliinase production was isolated from a soil sample and was identified by morphological, biochemical and 16S rRNA sequence. Alliinase production was optimised and it was further purified to apparent homogeneity with 103-fold purification and specific activity of 209 U/mg of protein by using DEAE Cellulose and Sephadex G-100 chromatography. The enzyme is a homodimer of molecular weight 110 kDa with two subunits of molecular weight 55 kDa each. The optimum activity of the purified enzyme was found at pH 7 and the optimum temperature was 35 °C. The enzyme exhibited maximum reaction rate (Vmax) at 74.65 U/mg and Michaelis–Menten constant (Km) was determined to be 0.83 mM when alliin was used as a substrate. The cytotoxic activity of in-situ generated allicin using purified alliinase and alliin was assessed on MIA PaCa-2 cell line using MTT assay and Acridine orange–ethidium bromide staining. This approach of in-situ allicin generation suggests a novel therapeutic strategy wherein alliin and alliinase work together synergistically to produce cytotoxic agent allicin.

Effect of physicochemical parameters on the stability and activity of garlic alliinase and its use for in-situ allicin synthesis

“…In general, both pH and temperature are significant factors governing enzyme activity and stability. The alliinase activity was tested for buffers from pH 1 to 8 and a narrow pH optimum at 7.0 was observed (Fig 3A), which corresponds to results reported in the literature [24,37]. Bellow pH 5.0 and above pH 8.0, a sharp decrease in the enzyme activity was observed [38,39].…”

“…In previously published studies, various buffers were used for alliinase extraction, purification and storage, e.g., Na-PB [ 22 , 24 , 34 ], K-PB [ 30 ], Na/K-PB [ 40 , 41 ], PBS [ 21 , 24 ] and Tricine [ 42 – 44 ]. However, the character of various buffers may play different roles concerning the conformational, colloidal and interfacial stability of enzyme [ 45 ].…”

“…In general, additives such as salts, polyols, sugars or inert polymers, such as polyethylene glycol, can greatly enhance the stability without the need of chemical or genetic manipulation of the target protein [ 23 ]. Chhabria et al reported purification of alliinase from soil bacterium Cupriavidus necator and found that the alliinase activity is enhanced by several metal ions, e.g., Ca 2+ , Mg 2+ , Zn 2+ , Fe 2+ , Mn 2+ ), but inhibited by others, e.g., Cu 2+ , Cs + , Li + , or Hg 2+ [ 24 ]. It was also shown that the addition of K + , Na + , and EDTA had no significant effect on the alliinase activity.…”

Characterization of the antibacterial activity of Bald’s eyesalve against drug resistant Staphylococcus aureus and Pseudomonas aeruginosa

“…These results suggest that the combination of brass and bovine bile in Bald’s eyesalve may be favorable for extraction of allicin from minced garlic. A previous study demonstrated that alliinase, the enzyme responsible for the conversion of alliin to allicin, displays increased activity in the presence of metal ions, most notably Fe 2+ , Mn 2+ , and Zn 2+ [39]. We hypothesize that dezincification [40, 41] of the brass 260 alloy included in several of our formulations takes place due to the mildly acidic pH of Bald’s eyesalve (S1 Table) which may cause corrosion.…”

Single‐molecule real‐time sequencing of the full‐length transcriptome of purple garlic (Allium sativumL. cv. Leduzipi) and identification of serine O‐acetyltransferase family proteins involved in cysteine biosynthesis

“…75%) of this odor originates from allicin, an unstable volatile thiosulfinate generated when garlic tissue is damaged 13 . Allicin is produced by the reaction between alliin and the C‐S lyase enzyme allinase 14 . Under normal conditions, the alliin precursor and allinase are located in the cytoplasm and vacuole, respectively 15 ; when tissue cells are damaged, the reactive allicin is released to defend against potential biotic enemies.…”