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Partial purification and immobilization of ribonuclease T2
Author(s) -
Leon D.,
Gite S.,
Shankar V.
Publication year - 1992
Publication title -
biotechnology and applied biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.468
H-Index - 70
eISSN - 1470-8744
pISSN - 0885-4513
DOI - 10.1111/j.1470-8744.1992.tb00213.x
Subject(s) - rnase p , chemistry , ribonuclease , chromatography , enzyme , enzyme assay , immobilized enzyme , affinity chromatography , glutaraldehyde , concanavalin a , sepharose , adsorption , biochemistry , organic chemistry , rna , in vitro , gene
A simple procedure, consisting of water extraction, heat treatment at pH 2.0, negative adsorption on DEAE‐cellulose at pH 4.9, and concanavalin A‐Sepharose chromatography, was developed for the partial purification of ribonuclease (RNase) T2 from taka‐diastase powder with an overall yield of 5.5%. The partially purified enzyme when coupled to aminoethyl Bio‐Gel P‐60, retained 12–16% of the activity of the soluble enzyme. Temperature stability studies on RNase T2 bound to matrices, activated with increasing concentrations of glutaraldehyde, and the influence of lysine modification on the activity of the soluble enzyme revealed that the low activity observed for the gel‐bound enzyme is probably due to the masking of the active site of the enzyme as a result of the involvement of lysine residues, situated near the active site, during coupling. Immobilization did not affect the pH and temperature optima of RNase T2. On repeated use, the bound enzyme retained approximately 55% of its initial activity after six cycles. These results are discussed, taking into consideration the factors affecting immobilized enzymes.