The method given below is a resume of the following two references:

[I] Applied Microbiology and Biotechnology, 2005, Volume 68, Issue 3, pp 384-389.

[II] Industrial, Medical and Environmental Applications of Microorganisms ...

edited by Antionio Méndez-Vilas.PP-618-619.

[A] Swollen chitin is prepared from chitin obtained from crab shell by dissolving in 85% of phosphoric aci[4C; keeping over night] and then reprecipitatiing with excess of water and neutralization.

The reaction mix ture containing one ml of 3% swollen chitin and1ml[1.4 units] of crude

Enzyme* in10 mM sodium acetate buffer[pH=5] solution is incubated in between 20-60C. Equal amount of more enzyme is added after incubation for three days for determination when Enzyme activity is measured at different pH[3.3-8] using 10mM sodium acetate[pH3.3 -6.2] and 10ml of sodium phosphate[pH= 6.6-80]. To access the effect of enzyme amount on the production of NAGA , the reaction mixture containing the swollen chitin and varying amount of enzyme, was again incubated at 40C. More of enzyme was added after 3-7 day incubation. The reaction is terminated after boiling in water for 3 min and centrifugation.

[B]Take the stock concentrated solution [ 5 ml ]. Add 10 ml of PNP** solution and 15ml of 0.5 M sodium acetate buffer[pH=4.7 ] and add 15 ml of 0.125 M sodium borate-NaOH buffer[ pH=10.7] and make the volume to 100 ml .

[C]Take eight washed dried test tubes(20ml capacity) and label them as 1,2,3------8.

[D] Add 10 ml of the above solution in tube No:1.

[E] Add 9 ,8 ,7,6,5,4,3 ml of same solution in the remaining 7 tubes and make up the volume of each one of 7 tubes to 10 ml by adding 1,2,3,4,5,6 and7 ml of water respectively .

[F] Note the absorbances of each one of these 8 tubes containing the diluted enzyme solution with the help of the spectrophotometer at 400 nm.

[G] Plot a graph between molar concentrations of PNP{ X-axiz} and absorbances { Y-axis} to obtain the standard calibration curve.

[H]Now take the unknown sample under the same conditions as those developed in [B].

{I}Take 10 ml of this solution in a cleaned dry test tube and note its absorbance at 400 nm.Draw a straight line from the point representing this value of absorption on Y-axis so that it meets the curve at a certain point. Now draw a perpendicular from this point on the curve on the X-axis. Note this value to obtain the molar concentration of PNP.

*Chitnolytic enzyme; acidic hydrolysis avoided because it is slow and give side prioducts.

** p- Nitophenyl-N—acetylphenol.

Thank you sir.

Previously I have  generated a standard curve of Glucosamine Hydrochloride the results were pretty good, so I employed the same methods for  N acetyl beta D glucosamine standard curve but the results were undesirable. 

What is the use of MBTH and Dinitrosalicylic Acid Reagent  in detection of Chitinase activity ?

 thank you

{A} My child, one needs some back ground both in Chemistry and Biochemistry . So try to understand in a sequence. You know whatever may be the protocol for estimating NAGA, we have to degrade Chitin. This can be done both by Enzymic reduction or by Chemical reduction[ Acid degradation].And remember both methods give rise to chitooligosaccharides.Again a strange thing is happening . Whether we perform Enzymic degradation or acid degradation, the Short oligosaccharides with DPs up to 6 were mainly studied, partly because of the heterogeneous compositionof chitosan hydrolysates and partly because of analytical limitations to isolate and identify chitooligosaccharides with DPs higher than 6 .

{B}Chitosanolysis achieved by acids can be carried out mainly using HCl or HNO2 and that looks simple because the yield of the degradation products is also good. But what adds to our worries is that the reactions are not easy to control and the removal of strong acid and by products of concomitant browning reactions is difficult. A further disadvantage of HNO2 use is the structural modification of the end products it can provoke. Again, they are not sensitive reducing end assay for the quantification of chito-oligosaccharides, detecting concentrations down to 5 mM.

{C}Now come the methods.

(a)First come the Schales’ procedure and then the 3,5-dinitrosalicylic acid (DNS) method are two examples that are commonly used. However, these methods lack sensitivity and present practical difficulties of usage in high-throughput screening assays as they require boiling or heating steps for color development. Moreover, the different lengths of chains of oligosaccharides causes changes in the absorbance and thus may lack reproducibilites.

(b)Then comes MBTH[3-methyl-2- benzothiazolinone hydrazone] method In contrast, this method gives the same absorbance per reducing end, irrespective of length of the chito-oligosaccharide. To our knowledge the MBTH method is the most sensitive reducing end assay for the quantification of chito-oligosaccharides, detecting concentrations down to 5 mM. This method was successfully applied to monitor the enzymatic degradation of chito-oligosaccharides and beta chitin.

As regards to your reaction wrt calibration curve, I may humbly add that the general method of its formation remains to be the same whether you test any metal ion or PPO or ROS or the one you had asked previously; of course the reagents/ reaction conditions/ wave lengths may differ. More over, when the results go wrong, it, in no way, implies that the suggested method was wrong. I had very honestly reproduced the method along with two references for your perusal and authentication. Yes; I agree that enzymes do cause problems when they get stale.

thank you very much sir, this is really much appreciated.