Leissy Gómez

Preparation and functional properties of trypsin modified by carboxymethylcellulose

Abstract Trypsin from bovine pancreas was modified by the polyaldehyde derivative of carboxymethylcellulose (CMC) via reductive alkylation with NaBH 4 . The modified enzyme contained 57% carbohydrate by weight, resulting from the modification of 52% of the amino groups of the protein. In comparison with the native protease, the modified trypsin retained 62% and 42% of the esterolytic and proteolytic activity, respectively. The value of K m for CMC–trypsin complex was 2.2 times lower than for the native enzyme. The thermostability and pH stability was improved for trypsin by this modification. The conjugate was also more resistant to the action of the anionic surfactant sodium dodecylsulphate and denaturing agents such as 8 M urea and 6 M guanidinium chloride. This modification also protected the enzyme against autolysis at alkaline pH and improved the stability of the enzyme in the presence of methanol.

Stabilization of invertase by modification of sugar chains with chitosan

Chitosan was linked to invertase by covalent conjugation to periodate-activated carbohydrate moieties of the enzyme. The thermostability of modified enzyme was enhanced by about 10 °C. The half-life at 65 °C was increased from 5 min to 5 h. The enzyme stability was enhanced by 20% at pH below 3.0. The half-life of denaturation by 6 M urea was increased by 2 h.

Functional stabilization of invertase by covalent modification with pectin

Pectin was attached to ethylenediamine-activated carbohydrate moieties of invertase using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide as coupling agent. The modified enzyme retained 57% of the original activity and contained 2.7 mol polymer per mol holoenzyme. Its optimum temperature was increased by 8 °C and its thermostability by 7.3 °C. The half-life at 65 °C was increased from 5 min to 2 days. The enzyme stability was enhanced by 33% at pH 2.0, and also by 27% at pH 12.0. The conjugate retained about 96% of its initial activity after 3 h incubation in 6 M urea.

Stabilization of α-amylase by chemical modification with carboxymethylcellulose

Carboxymethylcellulose activated by periodate oxidation was covalently linked to porcine pancreatic α-amylase (EC 3.2.1.1). The specific activity of the conjugate prepared was 54% of the native enzyme. The carbohydrate content was estimated to be 62% by weight as a result of the modification of 67% of the amino groups from the protein. In comparison with the native enzyme, the thermostability and pH stability were improved for α-amylase by this modification. The conjugate was also more resistant to the action of denaturant agents such as urea and sodium dodecylsulfate. We conclude that modification of enzymes by the anionic polysaccharide carboxymethylcellulose might be a useful method for improving enzyme stability under various denaturing conditions.

Modification of α-amylase by sodium alginate

Sodium alginate, activated by periodate oxidation, was covalently linked to porcine pancreatic α-amylase via reductive alkylation with NaBH4. The enzyme-polymer conjugate, purified by gel filtration on Fractogel EMD BioSEC (S), retained about 50% of the native specific amylolytic activity. The sugar content was estimated to be 712 mol of monosaccharides per mol of enzyme protein. An average of 11 amino groups out of 21 groups from α-amylase were modified with the polysaccharide. The functional stability was improved for α-amylase after conjugation with sodium alginate. In comparison with the native enzyme, the thermostability of α-amylase was increased by this modification. In addition, the stability in the range of pH 5.0–11.0 was improved for the modified enzyme. The conjugate was also more resistant to denaturation by 0.3% sodium dodecylsulphate, retaining about 10% of its initial activity after 120 min of incubation. The formation of stabilizing salt bridges in the protein surface of the α-amylase-polysaccharide complex was confirmed by FT-IR spectrometry. Attending to the results obtained, we conclude that the covalent attachment of the anionic polysac-charide sodium alginate to the enzymes might be a useful and non-expensive method for improving the stabilization of these biocatalysts under various denaturing conditions.