Purification and characterization of nucleases from tea leaves.

Abstract

Two enzyme preparations having both nuclease and 3'-nucleotidase activities were partially purified from an extract of tea leaves. They resemble each other in most enzymatic properties, but are separated by DEAE-cellulose column chromatography. The enzyme activities for RNA, native DNA, heat-denatured DNAand 3'-AMP of each preparation showed a high degree of similarity with respect to the following properties: pH stability, thermal stability and response to EDTA. Both enzymeswere shownto be endonucleases (EC 3. 1.30.2) which liberated S'-mononucleotides and oligonucleotides from both RNA and DNAwith the following relative rate of hydrolysis: RNA>native DNA=heat-denatured DNA.The present paper and three earlier publications1~3) from our laboratory are concerned with the isolation and characterization of the enzymes involved in the enzymic changes of nucleic acids and related compounds during the manufacture of black tea. In the previous paper,1} the authors indicated the presence of enzymes which released 5/-mononucleotides from RNA.In the course of purification, it was noted that two enzymefractions separated by a DEAE-cellulose column were also active on DNAand 3'-nucleotides. Chemicals. Yeast RNAused for assays was purchased from Kohjin Co., E. coli ribosomal RNAfrom BDH Chemicals Ltd., and yeast RNA(type XI), calf thymus DNA(type I), and 2'-, 3'-and 5'-nucelotides from Sigma Chemical Co. Heat-denatured DNAwas prepared by heating a solution of native DNA(5mg/ml) at 100°C for 30 min, and then cooling it rapidly in ice. Good's buffers: 2-(7V-morpholino)ethane sulfonic acid, MES; piperazine-A^,#/-bis(2-ethane sulfonic acid), PIPES; and tris(hydroxymethyl)methylglycine, Tricine, were the products of Dojindo Laboratories Co. Protein molecular weight markers were obtained from Boehringer Mannheim Yamanouchi Co. DEAE-and CM-cellulose (DE-52 and CM-52) were products ofWhatman Ltd., and Sephadex G-100, a product of Pharmacia Co.Assay of nuclease activity. The usual reaction mixture (1.0ml), consisting of 0.5ml of 0.1 m MES-NaOH buffer (pH 6.0), 0.25ml of RNA (lOmg/ml) or DNA (5mg/ml) solution and the enzyme solution, was incubated at 37°C for 1 hr. The reaction was stopped by addition of 1 ml of uranyl reagent (0.25% uranyl acetate in 0.2m perchloric acid) and allowing to stand in the cold for 30 min. The precipitate formed was removed by centrifugation. The supernatant (0.5ml) was diluted 10-fold with water and the absorbance at 260nm was read against a blank incubated without enzyme. One unit (u) of the nuclease activity was defined as the amount of enzyme which effected an increase in A260 of 1.0 in 1 min.

Purification and Characterization of Extracellular Nuclease from Bacillus Firmus VKPACU-1

“…Optimum temperature for the hydrolysis of ssDNA and RNA by the nuclease was 35˚C and similar observations were made for the nuclease from Anabaena nucleases [27] and yeast [13]. In contrast, nucleases from tea leaves exhibited very high optimum temperature (60 -70°C) [21]. The purified nuclease showed high stability from temperatures 4 -40°C for 1 h and higher temperatures like 50 and 60°C proved to be destructive.…”

Sugar non-specific endonucleases

“…In plants, sugar non-speci¢c 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].…”

“…During the initial puri¢cation 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 puri¢cation step.…”

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

“…However, nucleases from A. espejiana [81], N. crassa (mitochondria) [11] and tea leaves [42] show di¡erent pH optima for the hydrolysis of ssDNA (8.8, 6.5^7.5 and 6.0) and doublestranded DNA (dsDNA) (8.0, 5.5^6.5 and 5.5, respectively). On the other hand, 3P-nucleotidase-nuclease from potato tubers showed di¡erent pH optima for the nucleotidase (pH 8.0) and nuclease (pH 6.5^7.5) activities [41].…”