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Article
Purification and Characterization of Novel Nucleases from a Thermophilic Fungus
Masters Theses 1911 - February 2014
  • Kyle S Landry, University of Massachusetts Amherst
Document Type
Open Access
Degree Program
Food Science
Degree Type
Master of Science (M.S.)
Year Degree Awarded
2012
Month Degree Awarded
May
Keywords
  • Thermophilic fungi,
  • DNase,
  • RNase,
  • Affinity purification
Abstract

A thermophilic fungus was isolated from composted horse manure. The organism was as a Chaetomium sp. by sequencing the highly conserved ITS region of the fungus and comparing to known regions in a genomic database and was referred to as TM-417. TM-417 was found to have an optimal growth temperature of 45 oC and an optimal pH of 7.0. An extracellular DNase and RNase was found to be produced by the isolate and were purified 145.58-fold and 127.6-fold respectively using a combination of size exclusion chromatography and a novel affinity membrane purification system. The extent of purification was determined electrophoretically using 4-15% gradient polyacrylamide gels. Both DNase and RNase were dependent on metal co-factors for activity. The metal ion Mg2+ was the preferred ion for the DNase, whereas for the RNase, Zn2+ and Mn2+ yielded an increase in enzyme activity over that with Mg2+. The purified DNase demonstrated maximum activity at pH 6.0 with no activity at pH 2.0 or 10.0. The RNase exhibited two peaks of maximum activity, on at pH 3.0 and the other at pH 7.0 with no activity at pH 2.0 or 10.0. The optimal temperature for the purified DNase was 65oC. The optimal temperature for the RNase was 70oC. The molecular of the DNase and RNase were determined to be 56 kDa and 69kDa respectively using a Sephadex G-75 column. A standard curve was generated using several standard proteins of known molecular weight.

First Advisor
Robert E Levin
Citation Information
Kyle S Landry. "Purification and Characterization of Novel Nucleases from a Thermophilic Fungus" (2012)
Available at: http://works.bepress.com/kyle_landry/1/