We used citrate buffer 0.05M. Do you incubate the reaction mixture uder agitation?

Activity of cellulase also matters. How much did you use.

You need to conduct the cellulose activities of the cellulase powder you are using. A correct cocktail of CMC'ase, FPU and Cellobiase is very very essential for effective hydrolysis of lignocellulosic biomass. It may happen that the FPU activity of the enzyme powder is very very low.

Hello Mohammad,

 How did you calculate the yield? and how did you mesure the enzyme activity?

For biomass, the amount of cellulose in it will affect how you calculate the yield.  If the cellulose in the paper is very crystalline, it is less likely to convert.

Thanks guys, I think sort of found the point by your comments

Dear Lorena: Yes I used agitation , but I used 0.1M  and did not dilute it by adding water. You think may effect?

Dear Mustafa, thanks

Actually, I purchase the enzyme from sigma  A.niger powder, ≥0.3 units/mg solid

But I dont know the PFU, is there any formula to find the PFU/Unit?

Dear Deepti, Thanks

I know the unit/mg but can I find PFU by formula or experiment must be conducted?

Dear Rasool, Thanks

I used this simple equation :

% Yield = (g Glucose liberated  /  g Cellulose) X 100

Dear Joan, Thanks

I think the crystallinity of cellulose is the key, how can solve it?

Dear Mohamad

This Might be solution to your problem

I just googled the name of the enzyme which you mentioned above and found

1) Biochem/physiol Actions

Cellulase from Aspergillus niger catalyzes the hydrolysis of endo-1,4-β-D-glycosidic linkages in cellulose, lichenin, barley glucan, and the cellooligosaccharides cellotriose to cellohexaose. It does not cleave cellobiose or p-nitrophenyl-β-D-glucoside. This enzyme will also cleave intact glycosaminoglycan from a core peptide by hydrolyzing the xylosyl serine linkage. 

2) Unit Definition

One unit will liberate 1.0 μmole of glucose from cellulose in one hr at pH 5.0 at 37 °C (2 hr incubation time). 

http://www.sigmaaldrich.com/catalog/product/sigma/c1184?lang=en®ion=IN

Problem: 1)  The enzyme you are using is just an endo-glucanase which also does not cleave cellobiose (to glucose). You require combination of endo-glucanase, exo-glucanase and beta-glucosidase for complete conversion of cellulose to glucose.

2) The enzyme activity is carried at 37oC while your incubation temp was 55oC.

The FPU activity is easy. You incubate your enzyme with a filter paper in a buffer solution and determine the amount of sugar released. Do remeber the incubation tem will vary depending on your enzyme.There are many paper/protocol available, just google it. one such reference is

A Simplified Filter Paper Assay Method of Cellulase Enzymes Based on HPLC Analysis

http://link.springer.com/article/10.1007/s12010-012-9673-0

 Also the sigma website, the linked i mentioned above gives access to protocol and peer revied article related to enzyme. Have a look at it.

Hope it works. Have a good day :)

Dear Nicholas, Thanks a lot for complete explanation !!!

1. Was the sugar release monitored during the hydrolysis... Have you considered that after 7 days it is possible to have fungus or bacteria growing in your solution? Consider adding a growth inhibitor to prevent things from growing and eating the sugar.

Yes, daily measurement was done and to prevent fungal contamination, I used 2% Sodium azide (100uL)

2. Depending on the biomass, you may need a pretreatment to expose all possible hydrolysis sites (hard woody biomass is commonly wrapped in lignin. If you autoclaved to sterilize the paper before starting, that is a pretreatment step.

My biomass contains very low lignin but No! I did not autoclave them prior to hydrolysis, should I take some buffer or reagent to improve unwrapping cellulose or distilled water would be effective enough?

3. Is this total mass yield (100 mg paper gave about 50 mg sugar?) If so, that is probably correct. Do an acid hydrolysis to determine the total sugar release and use that value to calculate the yield.

Yesss, the Y was almost 50, but I expected to convert all paper which is supposed to be 100% cellulose!!!  Can I expect so?

Great procedure

4. Cellulase stability is not that good at 55C, after a few hours it is unlikely that you have active enzyme. Try to drop your temp. to 50C (max).

All right, this is a good point, I might have killed the enzyme

5. For long term (7 days or more) irreversible binding of the enzyme to the substrate will decrease activity, add a small amount of non-ionic surfactant to help prevent this problem (Triton/Tween/pluronic/etc.)

Nice trick, nowhere this was mentioned, I have Tween 80, how much you think I must add to a 10mL volume?

Another issue is the amount of enzyme?

What if I used extra enzyme does it effects the yield or it may even  inhibit the reaction?

Mohammad Normally people use 15 FPU to 20 FPU of enzyme per gram of cellulose. Please check how much loading you are doing and normally the CBU activity should be  4 times the FPU activity for effective hydrolysis. Since you are using 55 Degree as the incubation temperature, try reducing it to 50. Try adding additives like Triton X 100, Tween or PEG which not only provide thermostability to the enzyme but also are effective lignin blockers.

Thanks Deepti

Will do that

I hope this works for your Mohamad... Good luck for your research

Mohammad,

50% yield is probably what is typical on that sort of a substrate. As others have mentioned you need to have an accurate compositional analysis to determine the true extent of conversion. From the enzyme side you might supplement your cellulase mixture with additional B-glucosidase as many mixtures have a deficiency of B-glucosidase, and end product inhibition by cellobiose is a common problem.

Thanks dear Roman, comment was quite helpful, but could not read the article as not founded in our university archive

But, to find out the cellulase activity (FPU) I found another Issue and its the reaction of celulase with DNS reagent itself!!!

have you ever experienced that?

Dear Mustafa

Thanks a lot for sharing your knowledge with me,

But as I told on last comment, I found a weird reaction between enzyme and DNS reagent, do you know whats wrong with my exeriment or materials?

Cheers

MJ

Mohammad, Its not the reaction of Cellulase with DNS. The Cellulase powder which you are using had maltodextins or some reducing sugars as Binders or addiitves and therefore you are getting a very very high enzyme Blank.

Dear Deepti

Ohhh

Thanks for clarification,

Is it the normal story for all enzyme or I can find the pure one?

Is it a typing mistake, that in blank, the enzyme taken is from higher stock solution than from test? Any way enzyme blank should not be like this, it should be pale orange type colour, so there is contamination some where. I have attached the protocol, which i was using

Normally liquid enzymes have less possibility of such comtamination becoz they are either stabilised in glycerol or in potassium chloride. So try using liquid cellulase enzyme rather than powder ones.