Abstract:Exiguobacterium acetylicum YD37 producing cold-adaptive protease was isolated from a lowtemperature environment and identified.The cold-adaptive protease gene had a full length of 1 110 bp，which encoded a 41.5 kDa protein composed of 370 amino acid residues.This protease was an aminopeptidase with rich short side chains and negatively charged residues.The secondary structure of the protease contained 14 αhelixes and 19 β-sheets，and the three-dimensional structure had a hydrophilic surface with rich negatively charged residues.The enzyme activity was increased by 8.26 folds to 273.5 U/mL after optimization of the fermentation process of E.acetylicum YD37.The crude protease was purified by ammonium sulfate precipitation and ion exchange chromatography.The protease showed the highest activity at 35 ℃，while it still maintained a relative activity of 43.6% at 20 ℃.The enzyme was indicated as a metalloprotease since its activity was stimulated by Mn2+and Co2+but inhibited completely by enthylene diamine tetraacetic acid.Its Km and Vmax was calculated to be 1.38 mg/mL and 4.41 mg/（mL·min），respectively.