Improved activity and stability of alkaline phosphatases from psychrophilic and mesophilic organisms by chemically modifying aliphatic or amino groups using tetracarboxy-benzophenone derivatives.
The activity-stability-structure relationship of the cold-active alkaline phosphatase from Red Arctic shrimp, Pandalus borealis (SAP) was studied by chemically modifying aliphatic (C-H) or amino (NH2) groups using benzophenone tetracarboxylic derivatives in either a light (UV-A) or dark reaction. The response of the cold-adapted enzyme was compared to a similarly modified calf alkaline phosphatase (CAP). MALDI-TOF-MS was used to determine the extent and nature of the modifications in both SAP and CAP. On average 2 to 4 amino acid residues were linked to a BP-modifier, with up to 18 to 21 amino acids modified in a smaller portion of the material. The effect of the modifications on kinetic and thermodynamic properties varied with the enzyme and type of modification. The aliphatic-group modified SAP demonstrated typical characteristics of a mesophilic enzyme, consistent with an activity-stability trade-off where gain in thermostability was attained at the expense of decreased activity. In contrast, the activity of the amino-group modified SAP attained an even more psychrophilic character with respect to its kinetic (increase in kcat and Km) and thermodynamic (reduction in deltaH#) properties. Interestingly, the amino-group modified SAP also acquired higher thermostability, thus demonstrating that both activity and stability can be simultaneously enhanced using chemical modification. The study demonstrates the applicability of benzophenone chemical modification for improving the thermal properties of enzymes from psychrophiles and mesophiles.
Siddiqui
K S
KS
School of Biotechnology and Biomolecular Sciences, The University of New South Wales, Sydney, NSW 2052, Australia.
Poljak
A
A
Cavicchioli
R
R
D000469:Alkaline Phosphatase / Q000737:chemistry / Q000378:metabolism*; D001419:Bacteria / Q000201:enzymology; D001577:Benzophenones / Q000737:chemistry* / Q000378:metabolism; D004795:Enzyme Stability / Q000502:physiology; D007700:Kinetics; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013696:Temperature
D001577:Benzophenones; D000469:Alkaline Phosphatase