I have one doubt with CLEA-lipase enzyme. CLEA lipase is not soluble in water/buffer solution, it gives some sort of dispersion of CLEA-lipase. However, when I put CLEA-lipase in tris buffer solution for long time, using that supernatant I got good catalytic performance. I guess the clear supernatant solution may be a digested enzyme aggregates of lipase.
1. Is it possible to break the bonds in CLEA and forms monomeric lipase through putting them on buffer solution?
2. Or the leached enzymes are also in aggregate form, which have low solubility in buffer and are responsible for catalysis.
3. what is the best way (bradford/lowry method using spectrophotometer or ICP) to quantify lipase concentration in leached solution?
In case of CLEA or anyother support used for an enzyme immobilization, leaching of enzyme molecules is always a possibility. Use Bradford method to assess the amount of reached lipase. None of supports prove 100% stable binding of enzyme molecules.
In my experience, it is not possible to break the crosslinking of CLEA. However, leaching of single enzyme molecules or small crosslinked aggregates is likely. Enzyme concentration in the supernatant can be quantified by standard methods such as Bradford, densitometry of SDS-PAGE (which will also tell you if it is single molecules), N-quantification (LECO), etc.
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