Retardation of thermal and urea induced inactivation of alpha-chymotrypsin by modification with carbohydrate polymers.
Modification of enzymes by means of covalent coupling using soluble polymers results in enzymes which retain high biological activity and display resistance to denaturants, high temperature and chaotropic agents. Alpha-chymotrypsin, which has a potential for use in industrial applications, was covalently modified by reductive alkylation using polymeric sucrose (OSP, molecular weight 70 and 400 kDa), dextran (73 and 250 kDa) and carboxymethyl cellulose (CMC, approximately 12 kDa). The derivatives retained around 50-80% activity depending on the polymer used and the extent of modification. At the same time, they displayed better thermotolerance than their native counterpart with 4-14 degrees C higher T50 values. During thermal inactivation, both the native and modified enzymes showed biphasic inactivation kinetics. Half-life of modified enzymes were 2-66-fold greater for the first phase and 5-250-fold greater than the native for the second phase of inactivation. The activation free energy of inactivation of alpha-chymotrypsin coupled to polymeric sucrose (400 kDa) was 112.85 kJ/mol for the first phase and 114.71 kJ/mol for the second phase, whereas in the case of the native enzyme, the value for the first phase was 101.55 kJ/mol and 103.42 kJ/mol for the second phase. The activation free energy of inactivation (deltaG*), as well as the activation enthalpy values (deltaH*) of all the modified enzymes were greater than those of the native enzyme, which is an indication of stabilization of the protein and a retardation of inactivation that is usually accompanied by unfolding under thermal and chemical stress. The stability of modified alpha-chymotrypsin is in the following order: OSP 400-C > OSP 70-C > CMC-C > Dextran 73-C = Dextran 250-C.
Sundaram
P V
PV
Centre for Protein Engineering and Biomedical Research, The Voluntary Health Services, Madras, India.
Venkatesh
R
R
D002266:Carboxymethylcellulose Sodium / Q000737:chemistry; D002918:Chymotrypsin / Q000737:chemistry* / Q000378:metabolism*; D003911:Dextrans / Q000737:chemistry; D004789:Enzyme Activation; D004795:Enzyme Stability; D011108:Polymers / Q000737:chemistry*; D015202:Protein Engineering / Q000379:methods*; D012710:Serum Albumin, Bovine / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013395:Sucrose / Q000737:chemistry; D014508:Urea / Q000737:chemistry*
D003911:Dextrans; D011108:Polymers; D012710:Serum Albumin, Bovine; D013395:Sucrose; D014508:Urea; D002918:Chymotrypsin; D002266:Carboxymethylcellulose Sodium