Novel Extracellular Ribonuclease from Bacillus intermedius--Binase II: Purification and Some Properties of the Enzyme

M. A. Skvortsova^1*, A. L. Bocharov², G. I. Yakovlev², and L. V. Znamenskaya¹

¹Kazan State University, ul. Kremlevskaya 18, Kazan, 420008 Russia; fax: (8432) 38-7418; E-mail: Maya.Skvortsova@ksu.ru

²Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, ul. Vavilova 32, Moscow, 117984 Russia; fax: (095) 135-1405; E-mail: Yakovlev@imb.ac.ru

^* To whom correspondence should be addressed.

Received July 12, 2001; Revision received April 15, 2002

---

The recombinant enzyme binase II was isolated from the culture liquid of Bacillus subtilis 3922 transformed with the pJF28 plasmid bearing the birB gene. The procedure of the enzyme purification included precipitation by polyethylene glycol with subsequent chromatography on DEAE-cellulose, heparin-Sepharose, and Toyopearl TSK-gel. The enzyme was purified 142-fold yielding a preparation with specific activity 1633 U/mg. The molecular weight of binase II is 30 kD. The enzyme is activated by Mg²⁺ and virtually completely inhibited by EDTA. The pH optimum for the reaction of RNA hydrolysis is 8.5. The properties of the enzyme are close to those of RNase Bsn from B. subtilis. The character of cleaving of synthetic single- and double-stranded polyribonucleotides by binase II suggests that the enzyme binds the substrate in the helix conformation, and its catalytic mechanism is close to that of RNase VI from cobra venom.

---

KEY WORDS: Bacillus intermedius, Bacillus subtilis, binase II, RNase Bsn, biosynthesis, enzyme purification

---