The Protease Liberated from Bacteroides amylophilus Strain H 18 by Mechanical Disintegration

TLDR: Disintegration of lyophilized Bacteroides amylophilus H 18 suspended in water, by agitation with glass beads, gave an extract containing 40% of the total cell protease activity, indicating a trypsin-like specificity.

Abstract: SUMMARY: Disintegration of lyophilized Bacteroides amylophilus H 18 suspended in water, by agitation with glass beads, gave an extract containing 40% of the total cell protease activity. Better yields of an esterase which hydrolysed p-toluenesulphonyl-L-arginine methyl ester (TAME) were obtained by disintegration in 0.1 M-phosphate buffer (pH 7.0). Ultrasonic disintegration of fresh suspensions was used to obtain larger quantities of cell extract. The protease had a broad plateau of activity between pH 5.5 and 9.5; the esterase had maximum activity at pH 8.0. Divalent cations had relatively little effect on either activity but 5 x 10-3 M-CaCl2 restored activity to EDTA-inhibited esterase. The protease was not inhibited by EDTA. Both activities were inhibited by di-isopropylphosphofluoridate but the protease was incompletely inhibited (87%). Neither activity was activated nor inhibited by thiol reagents. In addition to TAME, N-α-benzoyl-L-arginine methyl ester (BAME), N-α-benzoyl-L-arginine ethyl ester (BAEE), N-α-benzoyl-DL-arginine-p-nitroanilide and lysine ethyl ester were hydrolysed, indicating a trypsin-like specificity. The esterase differed from trypsin in not hydrolysing N-α-benzoyl-L-arginine amide (BAA) nor N-α-benzoyl-DL-arginine-naphthylamide (BANA) and in hydrolysing BAME more rapidly than TAME. There was some hydrolysis of N-α-benzoyl-L-leucyl-2-naphthylamide and some amino peptidase activity as shown by the hydrolysis of L-analyl-2-naphthylamide, L-leucylglycine and L-leucinamide. TAME competitively inhibited at least 64% of the protease activity. The K m for casein was 0.17% (w/v). Casein at concentrations greater than 3.0% (w/v) caused substrate inhibition. The rate of ‘tyrosine’ liberation was proportional to protease concentration provided that less than 0.5 mg. ‘tyrosine’ was liberated from the 80 mg. casein in the standard assay. Protease concentration to the power 2/3 or the power 1/2 was proportional to the rate of hydrolysis but the straight line did not go to the origin. Bacteroides amylophilus H 18 extracts contained much nucleic acid which could not be separated from the protease activity by ammonium sulphate, protamine sulphate nor manganese chloride precipitation. Continuous electrophoresis was also ineffective. Ion-exchange chromatography separated the nucleic acids, but the protease activity was scattered in so many fractions that the purification was only three-fold and the recoveries poor.