I would like to determinate the production of this enzyme by colorimetric assay
Dear Reeta, thanks for your answer. I have used this method for quantification of B- glucosidase in compost and soils, but I dont know if centrifugation of a liquid sample obtained from Trichoderma´s fermentation , would affected the sensivity of the test.
Would you recommend any especification about the pre- treatment of the sample, in order to decrease the interferences of the test?? Thanks a lot
Maria, it can be used for the supernatant of any fermentation broth. It would not have any interference. Only take care to prepare standard curve under same conditions as of your test samples. Care to be taken to dilute your sample appropriately so as to get readings in range.
You could also try my very similar method, with the same substrate but with (I think :) better proportions of substrate and enzyme: 1.8 + 0.2 ml respectively in the basic method, or nowadays scaled down to micro-size. A problem with most assays for cellulases AND beta-glucosidase is limited reaction linearity (if you want to use SI units i.e. katals). Having MORE substrate solution and LESS enzyme solution can help towards this end: if you use the same volume of enzyme as substrate (1+1) you immediately and in my opinion unnecessarily dilute the substrate concentration by 50%. Why would you want to do that?
For the assay see e.g. Bailey and Nevalainen, Enzyme and Microbial Technology (1981), 3:153-157, which you can upload from my Research Gate profile :)
By the way, whatever method you use: if you need more sensitivity (because of very low activity levels) you can increase the incubation time to 1 hour or even overnight, but then you should also decrease the temperature to e.g. 40oC from the 50oC in my assay method, in order to avoid problems due to heat instability. Of course you can in any case perform an enzyme reaction at just about any temperature you consider to be appropriate!
regards, MJB
Thanks for your help, I will try both methods Dr Bailey I visited your profile to download the paper you have suggested me, but I didn´t see it, I would like to confirm that the title is: Induction, isolation and testing of stable Trichoderma ressei cellulases and other... to download the metodology of the test. Best regards...
I am new to this field of enzyme assay and my research may not be too in depth as against most of the contributors. So, I wonder why no one made mention of IUPAC method of measuring B-glucosidase or Cellobiase activity. In as much as I cannot disagree with the above suggestion, I think it is better to try the IUPAC method also more so that a discussion in this research outlet has suggested that pNPG substrates are heat labile. You can follow the discussion using the link below: www.researchgate.net/post/Glucosidase_activity_determination_protocol2
I have a relevant question.
I have been trying to express glucosidase in bacillus system and look for extracellular activity in LB Broth. I am using standard procedures for pNPG assay. But the issue is I do get positive result for the clone using MUG assay, but was not able to find any extracellular activity through pNPG for the same clone ( there was no distinct yellow coloration after the reaction was allowed) what could be the reason behind the failure in pNPG assay but being positive in MUG assay (Fluorescent substrate from SIGMA)
I wish to know one more thing, when we over lay the agar plate containing clone with these fluorescent substrates/congo red staining/ trypan blue staining. Will there be any chances that the clear zones are observed even if the enzyme is produced intracellularly.? As I am working on bacillus systems I wonder if I get a clear zone does it mean the enzyme is secreted extracellular or intracellular.
- Badges
- Science topic
- Similar topics
- Medicine
- Life Sciences
- Biotechnological Engineering