PURIFICATION AND CHARACTERIZATION OF SERINE PROTEASE FROM EARTHWORM (PERIONYX EXCAVATUS)

  • Phan Thị Bích Trâm

Abstract

Six protease fractions FI, FII, FIll-I, FIII-2, FIII-3, FIV were purified from the earthworm Perionyx excavatus lysate by acetone precipitation in combination with chromatography on ion exchange column Unosphere Q, hydrophobic column Phenyl-Sepharose and gel filtration Sepharose 12 column. The protease activity of the protein extract increased two times by the autolysis within 10 days at room temperature. The substrate specificity of these six fractions exposed differently on different substrates, on the fibrin plate the protease activity ranged from 44.1 to 830.7 plasmin uniUmg protein in the following order: FIII-3> FIII-2> FI> FIll-I> FIV> FII, while on the casein subtrate it ranged from 0.87 to 1.81 tyrosine unit/mg protein in the order of FI> FIII-3> FII> FIll-I> FIII-2> FIV. The molecular mass of these protease fractions estimated by SDS-PAGE in combination with gel filtration were from 28 kDa to 35 kDa. The optimum temperature for FI, FII, FIII-2, FIII-3 was 65°C and for FIll-I, FIV was 60°C. All obta,ined protease fractions exposed optimum activity at pH I I, besides that FI, FII, FIII-2, FIII-3, FIV also have relatively high activity at pH 7. These enzymes were stable at temperature:::; 50°C, and in a wide pH range from pH 4 - 12. The inhibition effect of phenylmethyl sulfonyl fluoride (PMSF) on protease activity suggested that these proteases belonged to group of serine proteases.

Tác giả

Phan Thị Bích Trâm
điểm /   đánh giá
Published
2011-11-29
Section
Articles