One thing I failed to mention is that color of p-nitrophenol is pH dependent. NaOH makes the pH to high, so the color is also more intense. Gly buffer does not result in too high pH so the color is less intense. As Natividad pointed out using Tris to stop the reaction works well. Make sure your standard curve is made in an identical way with glucose (add all ingredients except p-NPG and enzyme).

Maybe you put too much enzyme that's why the absorbance is high..dilute your enzyme and test it again.maybe u can plot a graph to get a right amount of enzyme..enzyme conc vs activity/absorbance.fix the amount of substrate

You can always try to use comercial beta glucosidase like novozyme 188.also you can try using cellobiose as a substrate and detect the glucose via god pap or glucose analyzer

But whatever it is u must make sure u put a right amount of enzyme for that amount of substrate so that the substrate is always in excess.

Angel, you mentioned that the final step is the only difference, so I guess the glycine buffer is not alkalinizing the solution sufficiently for some reason. It seems like it should be adequate, though. On the other hand, I am not sure if the NaOH will make the solution too basic and hydrolyze the substrate. I assume you have a blank reaction without enzyme that is alkalinized in the same way with negligible absorbance, is that correct? (that would eliminate the latter hypothesis) Normally, we use something like 0.5 to 1 volume of 0.5 to 2 M sodium carbonate for the alkalinization. On the other hand, the high absorbance may mean you need to use less enzyme in your assay (or shorter time of incubation).

My advice would be to dilute your enzyme with serial dilutions and then test the absorbance. Sounds like 10 ul might be too much according to what you are writing. What is your protein concentration in the orginal prep (the one where you take the 10 ul from)?. I also want to make a note here.... This might sound trivial but please make sure you are using the right cuvettes or plates to read at 412 nm. It is a common mistake often overlooked in laboratories.

The problem is that when the NaOH hydrolyzes the pNPG. You can stop the enzymatic reaction by adding 0.2 ml of 0.1 M THAM (Tris-hydroxymethylaminomethane) at pH 12.0.

I don't think the NaOH hidrolyzes the pNPG, because I use a blank, with substrate, without enzyme, also incubated at 50ºC and complete final volume to 1ml with NaOH 100mM and I don't see significative change in color, neither in absorbance. I use 10µL of extract with 0,5mg/ml of protein ( I think it's no so high) originally, the protocol indicates 50µL of extract and 150µL of substrate (pNPG 1mM), but the absorbance are extremly high ( A= >3).I realized this assay few times, and several kinetics, stop the reaction minute by minute, for 20 min, and I did have a lineal correlation at 16 minutes the reaction it's over. The endoglucanase activities are around 5 to 6 U/ml, with this conditions the β-Glucosidase activities are around 2,000-4,000 U/ml.

the pH of NaOH 100mM is 12.5 .

Hola, tengo unos protocolos manejados en la industria para determinación de actividad enzimatica en la industria, no especificamente para la que mencionas pero una adaptación de los metodos puede ayudarte.

si tienes un correo enviamelo y te los copio.

Angel, I believe the problem is not NaOH: instead, the problem is the poorly basic glycine buffer. You need a sharply alkaline solution to fully develop the yellow color of p-nitrophenol (as the corresponding p-nitrophenoxide anion). Try using 1 M sodium carbonate as the alkalinizing reagent.

You could use Na-citrate buffer PH 5 and PASC as a substrate. Use many dilutions of enzyme. You could use Glucose stds.

In past I worked with Beta-glucosidase enzyme a lot using p-NPG assay. It works fine. You can consult following papers for the exact enzyme assay. As suggested by other people you can control the color to below absorbance 1 by decreasing enzyme concentration and or decreasing time of incubation.

If you have access to an ISOTHERMAL CALORIMETER (iTC) near you, I can also suggest using that to do your assays. Its advantages include that you can use NATURAL substrate such as Cellibiose rather than artificial substrate (p-NPG) (paper about iTC methodology is attached).

• Rashid MH and Siddiqui KS (1998) Carboxyl group modification: High temperature activation of charge neutralized and charge reversed beta glucosidase from Aspergillus niger. Biotechnol. Appl. Biochem. 27, 231-237.

• Rashid MH and Siddiqui KS (1998) Thermodynamic and kinetic study of stability of the native and chemically modified beta glucosidases from Aspergillus niger. Process Biochemistry. 33(2), 109 115.

• Rashid MH and Siddiqui KS (1996) The stability of extracellular beta glucosidase from Aspergillus niger is significantly enhanced by the non covalently attached polysaccharides. Folia Microbiol. 41(4), 341 346.

One thing I failed to mention is that color of p-nitrophenol is pH dependent. NaOH makes the pH to high, so the color is also more intense. Gly buffer does not result in too high pH so the color is less intense. As Natividad pointed out using Tris to stop the reaction works well. Make sure your standard curve is made in an identical way with glucose (add all ingredients except p-NPG and enzyme).

for my standard method, i used pnpG with citrate buffer..and my step i put in Na2CO3..

incubate at 40C, 30 min,,measure at 400nm..

hope it is useful.

As per my knowledge sodium hydroxide caused high absorbance please try by using 10 mM Sodium hydroxide .

Sorry! The standard curve will contain p-nitrophenol under identical conditions (reaction stopper and pH should be identical) and not glucose.

Cellobioase (1, 4-β-glucosidase) (Hi-Media, India) was measured by a modified method of Bergham and Pettersson, (1974) where 0.5 mL of enzyme solution was incubated with 0.5% cellobiose in 0.05M acetate buffer at pH 5.0 for 60 min at 30°C. The reaction was stopped by heating in a boiling water bath for 10 min. The enzyme activity was determined by measuring the concentration of the glucose released in the medium

One enzyme unit was considered as the amount of enzyme necessary to release one µmole of the reducing sugar under assays condition.

My Ph.D. was on one of the glycosidase enzyme. I used almost the same method except that taking the reaction aliquots at every 2 min interval and adding them into 1 mL 0.5 M NaOH, reading their abs at 400nm. the extinction coefficient of p-nitrophenolate is at 400nm is 21.44 mM-1cm-1. You can also use the value of extinction coefficient to calculate rate change.

Following reference may help you.

"α-l-Rhamnosidase: A review" Vinita Yadav, Pramod K. Yadav, Sarita Yadav, K.D.S. Yadav

reference no. 22 in the above article may provide you more help.

good luck

incubate the reaction at 50ºC for 30 min and use sodium acetate buffer of 50 mM (pH 5.5) and read at 505 nm and to stop the reaction use glycin NaOH (pH 10.8)

p-nirtophenyl substrates are relatively themo labile and it is difficult to assay at 50oC. You. may study enzyme activity with o- methyl subastrate or with cellobiose as substrate by estimating liberation of gluose by GOD/POD method

No i am talking about the betaglucosidase activity i m sending u the protocol

b-glucosidase activity was assayed using p-nitrophenyl-b-D-glucopyranoside (pNPG) as substrate by the micro titer plate method (Parry et al., 2001). A reaction mixture of 100 microlitre containing 25 micro litre of enzyme, 25 micro litre of pNPG (10 mM) as substrate, and 50 micro litre sodium acetate buffer (50 mM, pH 5.0) was incubated at 50oC for 30 min. The reaction was terminated by addition of 100 micro litre of NaOH-glycine buffer (0.4 M, pH 10.8), and the developed yellow color was read at 405 nm using an ELISA Reader (MULTIKAN; Labsystem). The amount of p-nitrophenol released was quantified using the pNP standard. One unit of b-glucosidase activity was expressed as the amount of enzyme required to release 1 micro mole of pNP per minute under assay conditions.

You have not understood my reply. I am also taking about the same substrate you are using now. the PNPG is not a stable substrate. The substrate slowly hydrolysed into p-nitrophenol and glucose at higher temperature without presence of enzyme.Thus accurate assay become difficult. I do not know the logic why you have started assay microbial beta glucosides on microtitre plate..This is not usually done by enzymologists working on microbial commercial enzymes.

@ Subhabrata, we are working on a B-Glycosidase from a hyperthermopilic organism . The enzyme assay is performed at 80C with PNPG as substrate. PNPG substrate was stable upto 100C we have used in our assay. It didnot hydrolyse without adding enzyme at high temperature and blank was always kept to confirm. We also do the assay in microplate as it can save the reagents and sample and time. The microplate can be incubated in a Thermal Cycler for assay involving high temperature. The use of thermal cycler will help to maintain accurate temperature control and avoid leakage or evaporation of sample.

Please refer to this article published recently.

Miniaturization of hydrolase assays in thermocyclers

Analytical Biochemistry, Volume 434, Issue 1, 1 March 2013, Pages 39–43

http://www.sciencedirect.com/science/article/pii/S0003269712005519

Hi All,

Great day.

I am new in enzymatic hydrolysis study.

I did cellulase activity checking, where cellulase has 70 FPU/ml.

1. How much of ezy amount need to be added in 2% (w/v) substrate if the final working volume is 100ml (cellualse activity 70 FPU/ml )?

Thank you.

Try with 100micro liter crude enzyme

Good question and good answers

i think you can follow this method attached with it.

I want to estimate beta glucosidase enzyme in pts with gaucher disease by collecting their blood samples. Can you help me with the assay protocol using methy umbelliferone beta D Gluco pyranoside as substrate. Are ready to use kits available. 

To measure the beta-glucosidase in leukocytes, you need to isolate the leukocytes from the blood first.  There may be kits for the leukocyte isolation, but as far  as I know, none for the measurement of the beta-glucosidase.  With MUGlc, you will need a fluorimeter/fluorescence microtiter plate reader.  The standard assay is described in this reference, although some different assays exist to try to tease out the difference between GBA and GBA2 activities: S.P. Peters, P. Coyle, R.H. Glew, Differentiation of beta-glucocerebrosidase from beta-glucosidase in human tissues using sodium taurocholate,

Arch. Biochem. Biophys. 175 (1976) 569–582.

I apologize that we have sort of stolen the original beta-glucosidase activity assay question to answer this more specific one.

I am attaching my old paper where I assayed beta-glucosidase from A .niger using p-nitrophenyl beta-D-glucopyranoside substrate for both purified and unpurified enzyme. You can buy b-glucosidase reference from MEGAZYME. Follow Wood & Bhatt classical assay.

Please can any one help me with this question :

How can we calculate velocity in α-glucosidase inhibition ?

https://www.researchgate.net/post/How_can_we_calculate_velocity_in_a-glucosidase_inhibition

To Dr. Khawar Sohail Siddiqui, if the ONPG color is pH dependent, then how can I find out the enzyme activity in different pH ? Thanks in advance 

My suggestion is : PNP colour changes with increase of alkalinity.You may assay PNPGase activity by the estimation of glucose liberated form PNPG by GOD-POD method. But use dialysed enzyme to eliminate any glucose contamination by your enzyme sample,

Hi dear Angel

Have do you got blank?

my blank (consisted of solutions and buffers) incubated for each sample, after incubation time, I add of both substrate and stop solution at the same time, but samples not read by fluorimeter because having overflow error.

Do you help me?

tank you