Simultaneous purification of nitrile hydratase and amidase of Alcaligenes sp. MTCC 10674.

S K Bhatia,T C Bhalla,R K Bhatia,P K Mehta

doi:10.1007/s13205-013-0163-z

Abstract

Alcaligenes sp. MTCC 10674 has a bienzymatic system for the hydrolysis of nitriles. The nitrile hydratase and amidase have been purified simultaneously to homogeneity using a combination of (NH)4SO4 precipitation, ion exchange chromatography and gel permeation chromatography. Nitrile hydratase and amidase have molecular weight of 47 and 114 kDa, respectively and exist as heterodimer. Optimum temperatures for maximum activity of nitrile hydratase and amidase were 15 °C (2.4 U/mg protein) and 45 °C (2.3 U/mg protein), respectively. Nitrile hydratase showed maximum 7.8 U/mg protein at 50 mM acrylonitrile and amidase has 9.2 U/mg protein at 25 mM propionamide. Nitrile hydratase has Vmax 10 μmol/min/mg and Km 40 mM, while amidase has Vmax 12.5 μmol/min/mg and Km 45.5 mM, respectively. Heavy metal ions Hg2+, Ag+, Pb2+ and Cu2+ were strong inhibitors of nitrile hydratase and amidase activity.

Highlights

Nitrilase (EC 3.5.5.1), nitrile hydratase (EC 4.2.1.84) and amidase (EC 3.5.1.4) constitute an important class of nitrilase superfamily (Branner 2002)
MTCC 10674 has a bienzymatic system for the hydrolysis of nitriles
The ammonium sulfate fractionate (ASF) having nitrile hydratase and amidase activity was further used for purification

Summary

Introduction

Nitrilase (EC 3.5.5.1), nitrile hydratase (EC 4.2.1.84) and amidase (EC 3.5.1.4) constitute an important class of nitrilase superfamily (Branner 2002). Optimum temperatures for maximum activity of nitrile hydratase and amidase were 15 °C (2.4 U/mg protein) and 45 °C (2.3 U/mg protein), respectively. The ammonium sulfate fractionate (ASF) having nitrile hydratase and amidase activity was further used for purification. The assay mixture contained 0.125 M NaH2PO4/Na2HPO4 buffer (pH 8.0), 50 mM acrylonitrile and purified nitrile hydratase of Alcaligenes sp.

Results

Conclusion