Purification, characterization and complete amino acid sequence of nuclease Cl from Cunninghamella echinulata var. echinulata

Abstract

It is known, from the zymogram method of nuclease activity assay, that the crude extracts of Cunninghamella echinulata var. echinulata contained at least three distinct extracellular nucleases. Among them, the major form was 30 kDa in molecular mass and termed nuclease C1. In this report, nuclease C1 was purified to apparent homogeneity by chromatography on Cibacron blue-3GA, phenyl-Sepharose 4B and HiTrap Heparin. Nuclease C1 acquired enzymatic activity in the presence of Mn 2ϩ or Mg 2ϩ and was inhibited by EDTA. The activity was maximal at pH 7Ϫ8.5. The primary structure of nuclease C1 was completely determined using enzymatic digestion and gene cloning. The N-terminal 49 residues of nuclease C1 were first elucidated from a tryptic digest. Two degenerate upstream primers were subsequently designed to amplify the cDNA encoding nuclease C1. The resulting protein sequence of nuclease C1 was shown to be composed of 252 residues. It was intriguing to find that the protein sequence of nuclease C1 showed significant similarities with the sequences of the mitochondrial nucleases of Saccharomyces cerevisiae (44% identity) and Schizosaccharomyces pombe (42 % identity). Residue His87 of nuclease C1 was postulated to be located at the active site from sequence similarity with secreted nuclease from Serratia marcescens.Keywords : nuclease C1; mitochondrial nuclease ; Cunninghamella echinulata ; Saccharomyces cerevisiae; Schizosaccharomyces pombe.Nucleases act on both DNA and RNA to release products containing 5′-or 3′-phosphates [1]. Several fungal species are known to produce both extracellular and intracellular nucleases. Representatives of extracellular fungal nucleases are nuclease S1 of Aspergillus oryzae [2,3] and nuclease P1 of Penicillium citrinum [4,5]. The properties and the structures of these two nucleases are well characterized. Other fungi also secrete nucleases, e.g. Neurospora crassa and Aspergillus nidulans, which are referred to as DNase (deoxyribonuclease) A [6] and DNase 4 [7], respectively.Clinically, some Cunninghamella species cause severe syndromes and even led to death in humans. For example, several reports have indicated that Cunninghamella bertholletiae can infect patients with cancer and immunocompromise children [8,9]. Diseases including paranasal sinusitus [10] and mucormycosis [11] have been relatively correlated with Cunninghamella infection. The most important risk factors for C. bertholletiae infection include corticosteroid administration and pro-

A Novel Method for SNP Detection Using a New Duplex-Specific Nuclease From Crab Hepatopancreas

“…RNA, ss, and dsDNA substrates (Friedhoff et al 1996;Ho et al 1998;Meiss et al 1998). Several nucleases of this family have either no or low RNase activity (Ho and Liao 1999;Wang et al 2000), but no differences between cleavage activity against ss and dsDNA have been noted to date.…”

“…RNase activity assay was measured essentially as described by Ho et al (1998). The reaction mixture (20 µL) contained 50 mM Tris-HCl (pH 8.0), 5 mM MgCl 2 , and 0.6 Kunitz units of DSN.…”

Sugar non-specific endonucleases

“…[26], Lysobacter enzymogens [27], Saccharomyces cerevisiae [30], Anabaena sp. PCC 7120 [33], Syncephalastrum racemosum [34], Cunninghamella echinulata [38], Streptomyces antibioticus [39] and Rhizopus stolonifer [40] are extracellular in nature. Endonucleases of mitochondrial origin have been reported from N. crassa [11,24], S. cerevisiae [28,29,31] and Aspergillus nidulans [32].…”

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

“…The presence of Mn 2+ and Mg 2+ was proved to be essential for both dsDNA and ssDNA hydrolyzing activity of C1 nuclease from Cunninghamella echinulata var. echinulata [18]. This observation indicated that the single stranded substrate hydrolyzing activity was highly favored in the presence of both Mn 2+ and Fe 2+ .…”