I have been doing solid state fermentation on agro-waste and I noticed that in my enzyme extract there is sugar. HPLC analysis revealed that the monomers are Glucose, xylose, mannose and arabinose, in different proportions and glucose being the primary sugar. I hold a view that these sugars can be fermented.
If so, can someone suggest to me a method that I could specifically extract these enzyme complex from the sugar pool?
An ion-exchange resin (QAE or SP) should bind the proteins as long as the ionic strength of the solution is low and the pH is suitable. Glucose, being uncharged, will not bind. The proteins can later be eluted from the resin with a salt solution.
An alternative is to use ultrafiltration to simply filter out and concentrate the proteins and undigested polymeric matter, allowing the sugars to pass through.
Hi Lesetja
Amicon Ultra-15 Centrifugal Filter Units are a good option. You can also precipitate with ammonium sulphate 80% all proteins, after that, eliminate the salt excess through dialysis.
http://www.emdmillipore.com/US/en/product/Amicon-Ultra-15-Centrifugal-Filter-Units,MM_NF-C7715?bd=1
Very simple, Precipitate the crude proteins having the nzyme with cold acetone and dialyze it against the 25mM phosphate buffer of the desired pH to stabilize the enzyme. Just use DEAE cellulose resins for the purification. Elute about 80 to 100 fractions using NaCl solution from 0.1 M to 0.5M. Check the fractions for activity. Use the fractions showing maximum activity for further studies.
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