【摘要】 <正> 前言前文报道了红色链孢霉核糖核酸酶N₁（N.Crassa RNase N₁）高产菌株的选育及其核糖核酸酶（RNase）作用碱基专一性的鉴定。本文主要介绍核糖核酸酶N₁的分离与纯化。我们在高井等人方法的基础上,作了某些改进。该方法有效地用于大量制备,并达到了较高的纯度。更多还原

【Abstract】 The present paper describes separation and purification of ribonuclease N₁ from Neurospora crassa. N. crassa double auxotrophic strain, ornithineless and adenineless OAo47 was grown in shaking flasks containing 5% wheat bran at 28-30℃ for 72 hours. The mycelia were removed by centrifugation at 2800 rpm for 10 minutes. The clear brown-reddish supernatant was regarded as a crude extract of ribonuclease N₁.The crude extract of enzyme was adjusted to pH 4.5 by the addition of 2N HCl and loaded on a column of Amberlite CG-50, previously equilibrated with pH 4.5 distilled water. The theribonuclease was eluted by 1M ammonium acetate. By this treatment, the recovery of enzymatic activity was over 95%, about 97% of protein contamination was removed, the specific activity was raised 63-fold, and ribonuclease was concentrated 30-fold. Amberlite CG-50 chromatography is therefore more effective for separation and concentration of ribounclease in crude extracts.The, concentrated enzyme was further purified by the following steps: DEAE-cellulose chromatography （twice times）, gel filtration on sephadex G-75 and CM-cellulose chromatography. The preparation of purified ribonuclease N₁ was characterized by polyacrylamide gel electrophoresis and only one protein band was observed. The mobility of ribonuclease N₁ on gels was compared to that of ribonuclease T₁. Ribonuclease N thus obtained was purified 7790-fold, in good yield, with a specific activity of 23000/A₂₈₀.In recent years, the preparation of ribonuclease N₁ of N. crassa OAo47 was successfully used in the synthesis of a variety of oligonucleotides.更多还原