Purification and Characterization of Nucleases from Tea Leaves

Sugar non-specific endonucleases

“…In plants, sugar non‐specific endonucleases have been isolated from tea leaves [42], pollen grains of tobacco [43] and Petunia hybrida [45], wheat chloroplasts and its organelles [46], rye germ ribosomes [47,48] and barley microspores [49]. The presence of these enzymes has also been shown in the lumen of the endoplasmic reticulum, in the Golgi apparatus, in protein bodies and vacuoles of barley aleurone layers [60].…”

“…Since the majority of sugar non‐specific endonucleases are intracellular, most of the purification procedures, irrespective of the source, involve steps like lysis of cells, isolation of organelles (by differential centrifugation), concentration of the crude extract by salt precipitation followed by conventional purification methods, such as ion‐exchange chromatography and gel filtration. During the initial purification steps, one of the primary aims is to get rid of the colored impurities contributed by pigments of the organelles and this is achieved either by extraction with acetone–water mixture (4:1 v/v) [42] or by a simple wash with ammonium chloride followed by high speed centrifugation [47,48]. In most cases apart from sonication [52], grinding with glass beads or sand [11,21,41,42] has also been used for disrupting the cells.…”

“…During the initial purification steps, one of the primary aims is to get rid of the colored impurities contributed by pigments of the organelles and this is achieved either by extraction with acetone–water mixture (4:1 v/v) [42] or by a simple wash with ammonium chloride followed by high speed centrifugation [47,48]. In most cases apart from sonication [52], grinding with glass beads or sand [11,21,41,42] has also been used for disrupting the cells. In general, ammonium sulfate precipitation is also used as a preliminary purification step.…”

“…Gel filtration has been used as one of the steps for the purification of nucleases from Vibrio sp. [26], L. enzymogens [27], S. racemosum [34], S. antibioticus [39], tea leaves [42] and rye germ ribosomes [48].…”

“…In general, the pH optima of sugar non‐specific endonucleases are in the range 5.0–10.0 (Table 2) and most of the enzymes show the same optimum pH for the hydrolysis of both polymeric and monomeric substrates. However, nucleases from A. espejiana [81], N. crassa (mitochondria) [11] and tea leaves [42] show different pH optima for the hydrolysis of ssDNA (8.8, 6.5–7.5 and 6.0) and double‐stranded DNA (dsDNA) (8.0, 5.5–6.5 and 5.5, respectively). On the other hand, 3′‐nucleotidase‐nuclease from potato tubers showed different pH optima for the nucleotidase (pH 8.0) and nuclease (pH 6.5–7.5) activities [41].…”