My hemicellulose extraction step;
1) defatted rice bran
2) destarch by amylase (termamyl)
3) deprotein by NaOH at pH 11
4) alkali treatment for hemicellulose extraction by 1.25 N of NaOH for 24 hr at room temp. Then the supernatant was adjust to pH 4.8. The resides was not recover. Then 3 volume of EtOH 95% add for precipitation hemicellulose.
5) 3% of extracted hemicellulose of 10 ml mix with endo-xyalanse from Neocallimastix
Please recommend me, there is any step was wrong or not?
Dear, Jaichakan!
First of all I suppose that you should't use 95% EtOH before the hydrolysis, because as you know all kind of enzymes are proteins, by this reason the hydrolysis does't happen, it is leads to denaturation of enzymes.
The second you have to be sure that your 3% of extract has the same pH value like in supernatant, otherwise the enzyme will not works properly!
The third aspect of enzymatic treatment is that you should ensure the optimal conditions for splitting of hemicellulose. In this respect you should provide an appropriate temperature for hydrolysis. You should take a look on the specification of your enzyme, I mean activity, incubation temperature, time, substrate pH etc.
My advice to you omit the precipitation of hemicellulose using by 95% EtOH, add directly enzyme to your substrate. After the hydrolysis do not forget inactivate the enzyme by heating.
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