核酸特異性水解酵素的純化及特性鑑定

Abstract

從黃?菌中可抽出一種耐熱性水解酵素，能催化水解單股核酸成為寡核?酸及5'一單核?酸，為進一步探討此酵素的特性並擬利用此酵素水解核酸成5'一單核?酸。本論文首先利用下列步驟純化此酵素(1)以市售麥麩培養黃?菌。(2)從黃?菌培養體抽取粗酵素。(3)以硫酸銨進行鹽析作用(salting out)。(4)以陰離子交換纖維素柱體分離。(5)以75號瓊脂糖膠(sephadex G-75)柱體分離。經過上述純化步驟，可得到1900倍的純化效果和10%的回收率。 經純化的酵素依其對基質的特異性、鋅離子的需求、分子量的測定及optimun pH的表現。鑑定此酵素即為 Nuclease-S1。更進一步探討其性質，發現(1)短時間可催化單股核酸成為寡核?酸及單核?酸，而經長時問作用所得到產物幾乎都是單核?酸。(2)小量的酵素活性單位僅對單股DNA有水解作用，但在增加100倍的酵素活性單位，則可催化水解雙股DNA。

A heat-resistant nuclease is purified from the extract of Aspergillus oryzae. The procedures that involves (a) ammonium sulfate precipitation, (b) DEAE-cellulose anion-exchanger fractionation, (c) Sephadex G-75 gel-filtration. Achieves 1900 folds purification and leads to as homogeneous protein of 99﹪ purity as measured by SDS-polyacryamide gel electrophoresis. The purified enzyme has the following properties: (a) molecular weight is 34,000, (b) the optimun pH is 4.2, (c) ZINC ion is essential, (d) the enzyme can hydrolyze single strand DNA and RNA, (e) the hydrolysis of double strand DNA requires more than 100 times of the enzyme units than the hydrolysis of single strand DNA and (f) the products of hydrolysis for the initial 10 min are predominantly oligonucleotides and for 18 hours are predominantly 5'-mononucleotides, the enzyme has characteristics of nuclease S1. The investigation indicate that (1) the specific hydrolysis of single strand nucleic acid ought to be under the strict control of the amount of the enzyme used and (2) the enzyme can be used for the production of 5'-mononucleotides from nucleic acid.