Improved thermal stability and activity in the cold-adapted lipase B from Candida antarctica following chemical modification with oxidized polysaccharides.
In order to improve the thermal stability (t(1/2)) and activity of lipase B from cold-adapted Candida antarctica (CALB), amino groups of the enzyme were chemically linked to a range of oxidized polysaccharides using a range of reducing agents. By chemically modifying CALB using 0.1% dextran (250 kDa) at pH 8.6 for 10 days using borane-pyridine complex as reducing agent, increased thermal stability (t(1/2), 168 min at 70 degrees C) and activity (65% higher specific activity) was achieved compared to the unmodified enzyme (t(1/2), 18 min at 70 degrees C). Improvements in thermostability were generally better with high molecular weight polymers such as dextran (40 and 250 kDa) or ficoll (70 and 400 kDa) in comparison to low molecular weight inulin (5 kDa). The shape of the polymer also appeared to be important with elongated, elipsoidal-shaped dextran providing better thermostabilization than spherical-shaped ficoll. Borane-pyridine complex was found to be a good, non-toxic reducing agent for improving thermostability, compared with sodium borohydride and sodium cyanoborohydride. An interesting finding was that, in all cases, specific activity of the modified enzymes increased with a concomitant increase in thermostability. This response defies the general principle of a trade-off between activity and stability, and demonstrates that chemical modification provides new avenues for improving the thermal stability of enzymes from psychrophiles without sacrificing their activity.
School of Biotechnology and Biomolecular Sciences, The University of New South Wales, Sydney, 2052, NSW, Australia.
Khawar Sohail
KS
Siddiqui
Ricardo
R
Cavicchioli
D002175:Candida / Q000201:enzymology*; D003080:Cold Temperature; D004305:Dose-Response Relationship, Drug; D005656:Fungal Proteins / Q000737:chemistry*; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D008049:Lipase / Q000737:chemistry* / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism*; D011108:Polymers / Q000737:chemistry; D011134:Polysaccharides / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D011489:Protein Denaturation; D015202:Protein Engineering; D020673:Protein Renaturation; D019163:Reducing Agents / Q000494:pharmacology; D013379:Substrate Specificity; D013696:Temperature; D013816:Thermodynamics; D013997:Time Factors
D005656:Fungal Proteins; D011108:Polymers; D011134:Polysaccharides; D019163:Reducing Agents; D008049:Lipase; C431349:lipase B, Candida antarctica; D010100:Oxygen