New Thermostable Amylase from Bacillus cohnii US147 with a Broad pH Applicability

Appl Biochem Biotechnol (2009) 157:5060

DOI 10.1007/s12010-008-8278-0

Raoudha Ellouz Ghorbel & Sameh Maktouf & Ezedine Ben Massoud & Samir Bejar & Semia Ellouz Chaabouni

Received: 25 January 2008 /Accepted: 6 May 2008 / Published online: 15 July 2008# Humana Press 2008

Abstract A new thermophilic bacterial strain identified as Bacillus cohnii US147 was isolated from the southern Tunisian soil. The identification was based on physiological tests and molecular techniques related to the 16S ribosomal ribonucleic acid. The isolated strain produced amylase, which was purified. This amylase had an apparent molecular mass of 30 kDa as estimated by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. Amylase US147 showed Km and Vmax values of 0.7 mg/ml and 2.2 U/ml, respectively, with starch as the substrate. The enzyme was active in acid and basic pH and had a maximal activity on starch at pH 9 and 70 C. The enzyme was stable at pH 9 for 72 h and retained half of its activity after incubation at 70 C for 150 min. A partially inhibition (15%, 25%, 23%, 20%, and 22%) was obtained with 1 mM SDS, 1 mM NaBO3, 1 mM H2O2, 1 mM

Zn+2, and 5 mM ethylenediamine tetraacetic acid (EDTA), respectively. The amylase recovered its original activity by the addition of 10 mM Ca 2+ to the 5 mM EDTA. These properties indicated a possible use of this amylase in starch saccharification, in detergent, and in other industrial applications.

Keywords Bacillus cohnii . Amylase . Acid and basic pH activity .

Purification . Characterization

Introduction

Amylolytic enzymes such as -amylase (1, 4--D-glucan glucanohydrolases; EC 3.2.1.1) hydrolyze starch molecules to give diverse products including dextrins and progressively

R. E. Ghorbel (*) : S. Maktouf : S. E. ChaabouniNational School of Engineers of Sfax, BP W 3038 Sfax Cedex, Tunisia e-mail: [email protected]

E. B. Massoud : S. Bejar

Biotechnology Center of Sfax, BP W 3038 Sfax Cedex, Tunisia

New Thermostable Amylase from Bacillus cohnii US147 with a Broad pH Applicability

Appl Biochem Biotechnol (2009) 157:5060 51

smaller polymers composed of glucose units. Amylases can be derived from several sources, such as plants, animals, and microorganisms. Enzymes from microbial sources have potential applications in many industrial fields, particularly in food, paper, textile, and detergent industries [15]. Amylases that...