Hi,
I need help to find a fitting method to determine the esterification activity of a lipase. I am using the Novozym 435 and I am also using a very small volume in this reaction. The enzyme amount is about 63 mg in the reaction. How do I determine the activity?
Thank you :)
Fitting method - what for? Enzyme activity and specific enzyme activity - see Wikipedia. The enzyme mass of 63 g is too much to quantify the enzyme activity. But maybe you already have kinetic data for given esterification, and you want to describe it with an equation. Then a fitting method is needed.
It is difficult for us to respond precisely to your request not knowing your reaction conditions.
Within the research unit that I lead, we carry out the synthesis of new surfactants using biocatalysts and consequently we carry out numerous esterifications.
I advise you, as already answered by Mr Grzesik, to carry out a kinetic of your resterification thus allowing you to be able to determine, in addition to the yield, but also your productivity.
I remain at your disposal
Benoît Moreau Mirosław Grzesik
Thank you for your answers.
The esterification is carried out in a small 2 ml tube, with benzyl alcohol and Butyric acid as the substrates for the Novozyme 435. Additionally there are molecular sieves to absorb the water. The reaction is carried out with mechanical stirring (130 rpm) and at a temperature of 50°C.
To test how many units the enzyme catalyzes it is often advised to use titration to determine the esterification activity.
I corrected my self, I am using only 63 mg (10 wt %) for the reaction, but the titration device needs a volume of 40 ml, and thus I would have to use a lot of enzyme, only to determine the units per gramm.
I read that the hydrolyse is often used to determine the units/g for the lipase, but I am unsure if I should use it as I am doing an esterification reaction and not hydrolyse reaction.
I hope you can now understand what exactly I am asking for.
Thank you in advance.
I guessed it was 63 mg, not 63 g, at least because of the cost of the enzyme. The type of reaction does not matter, it is important that it is catalyzed by a given enzyme. Furthermore, you are using titration here to determine the acid concentration, not the "esterification activity." Of course, when you know the concentration of acid, you know how much acid has converted to ester.
Dear Sarah,
It is true that often commercial lipases are expressed in terms of hydrolytic activity.
You can therefore make a working postulate that a lipase can also perform hydrolysis and esterification so your postulate is the same activity. However, you should always keep in mind that in reality, lipases are not as efficient in hydrolysis and esterification.
This is the reason why we always start our experiments with a kinetics in order to thus know the productivity according to the quantity of immobilized enzyme used for this kinetics.
Also never forget that each lipase is different and depends on a series of parameters including Aw (water activity),Temperature, alcohol / acid ratio and the three-dimensional structure of the lipase (cf. Pleiss et al., 1998; anatomy of lipases)
If you want more information here is my professional email:
Benoît Moreau You wrote: "each lipase is different and depends on ... temperature, alcohol/acid ratio ....." Are you sure about it? Maybe you mean a specific reaction, catalysed by a given enzyme, and its initial rate. Of course, for a given reaction and a given enzyme, we start with determining the kinetic curve/curves. Next we can determine the form of the kinetic equation and the values of its parameters.
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