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发表于 2008-12-21 13:32:49
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Xylazyme AX fangTablets
Q. 1: Could you please provide me with a detailed definition for the xylanase unit?
A: The units are International Units of activity determined using the Somogyi reducing sugar method.
One Unit is the amount of enzyme required to release one micromole of xylose reducing sugar
equivalents from wheat arabinoxylan per minute under defined conditions of temperature and pH.
Q. 2: I am attempting to assay brewer’s malt for xylanase using Xylanase AX tablets. Can you please give
me a protocol?
A: There is very little xylanase in malt. I suggest you use the same buffer as recommended (25 mM
acetate, pH 4.7). You will need to increase incubation times to 1-2 hours.
Q. 3: When analysing a diluted sample, should the absorbance value or the calculated result be multiplied
with the dilution?
A: The calculated result should be multiplied by the dilution.
Q. 4: I have started to use your tablets for xylanase activity. It seems to work on enzyme preparations, but
my main objective is to analyse cereal samples with very low activity. I have a problem with
sensitivity. Do you think I can increase sensitivity by increasing incubation time?
A: Yes, you can increase the sensitivity by increasing the incubation time. Also, you can increase the
temperature (up to 60oC) to improve sensitivity. I would recommend the time increase approach, as
this allows you to use the same standard curve. Allow for the time difference in the calculations.
Q. 5: Do you have a method to check for arabinoxylanase in concentrated Aspergillus and Trichoderma
cellulase to be used in baking improvers?
A: Arabinoxylanase is actually "xylanase". This is the enzyme which depolymerises arabinoxylans.
Xylazyme AX can be used for both Aspergillus and Trichoderma xylanases.
Q. 6: In the booklet for the Xylazyme AX tablets, there are 3 standard curves. You say on page 7 – "by
reference to the appropriate standard curve" - which one of these curves should one use?
A: The most common enzyme used in baking is from A. niger and for animal feeds is from Trichoderma.
Therefore, I would suggest that you choose between these depending on the intended use of the
unknown enzyme.
Q. 7: We have noticed differences of results when using different filters, such as Whatman 1 paper vs glass
filter paper.
A: I had noticed a difference between paper filters (Whatman No 1) and Whatman Glass Fibre Filters
(GF/A), with the latter giving a slightly higher absorbance in the filtrate. I think that this may be due
to some absorption of the dyed fragments to cellulose, but not to glass. We proceeded with the
cellulose (Whatman No 1) because they are inexpensive compared glass. All of the standard curves are
based on use of the Whatman No.1 filter papers.
Q. 8: Do you still have Xylazyme tablets which derive from birchwood?
A: We do not produce Xylazyme from birchwood xylan anymore. We find that wheat arabinoxylan gives
a more stable substrate. There was a great variation in the quality of the birchwood xylan that we could
obtain, and this resulted in a large variation in the quality of the resulting dyed substrate.
Q. 9: Regarding the assay by using Xylazyme and Xylaxyme AX, within how many minutes after filtration
of the reacted solution should the absorbance be measured?
A: When using Xylazyme or Xylazyme AX tablets, filtration should be performed about 5 minutes after
stopping the reaction. For Xylanases with alkaline pH optima, please use 2% tri sodium phosphate
(pH 11) to stop the reaction (instead of Trizma base). After filtration, the solutions are stable for
several hours, so there is no urgency in reading the absorbances. However, as stated above, the
solutions should be filtered no more than 10 minutes after addition of the stopping reagent. The
substrate is unstable in alkaline conditions, and colour will leak from the substrate leading to high
blank values.
Q. 10: Is AZCL-Xylan (oat spelts) the best substrate to determine arabinoxylanase activity in brewer’s malt?
A: We would not recommend AZCL-Xylan (oat) for this assay. The sensitivity is too low. Xylazyme AX
tablets are more sensitive.
Q. 11: I have a question about the optimum temperature for the use of Glucazyme and Xylazyme tablets. We
have determined the optimum. We found for the Glucazyme analysis a temperature of 40oC instead of
30oC and for the xylazyme analysis a temperature of 50oC instead of 40oC. Can you tell us how you
determine the temperature optimum? Don’t you think it is necessary to determine the activity by the
optimum temperature?
A: We do not give a "temperature optimum" for use of the tablets, we just recommend a "suggested"
assay format for different enzymes. Malt beta-glucanase is unstable above 30oC, so that is why we
recommend this temperature. In fact, the tablet substrate is stable up to at least 70oC at pH values
below 6.0.
We routinely recommend 40oC for all other enzyme tests just for consistency. You can use
Xylazyme AX tablets up to 70oC with no problems.
Q. 12: Why does a higher buffer strength ( >100 mM) inhibit the degradation of AZCL-Xylan in the
Xylazyme AX method.
A: Salt effects sensitivity by effecting the rate and extent of swelling of the dyed and crosslinked substrate
particles. If the swelling of the particles is restricted, this limits the ability of the enzyme to access the
substrate and thus to hydrolyse it.
Q. 13: We are using the Megazyme Xylazyme AX tablets. Can you please clarify the following:
Booklet Page 4 – Enzyme Standards: Procedures indicate that "these" should be diluted 1:100. When
you state "these" are you referring to the fact that the entire contents of the bottle should be diluted
1:100 or should only 1 ml be diluted 1:100? Should a positive control sample be run every time an
assay is performed to confirm the test kit is operating properly?
A: If diluting the control, it is best to just dilute 1 ml. The test is very simple and very reliable. It is
advisable to run a control each time, mainly to give the analyst confidence that he is running the test
correctly.
Q. 14: After addition of the Xylazyme AX tablet, should the slurry be removed from the 40oC water bath and
be allowed to react for 10 minutes at room temperature, or should the AX test tablet be added while
the test tubes remain in the 40oC water bath and then removed after the 10 minute incubation?
A: Must be left at 40oC for the 10 minutes.
Q. 15: Regarding the standard curve for Xylazyme Tablets. We wish to measure xylanase activity produced
by a submerged culture of Aspergillus. I am assuming that we will have to derive a standard curve for
this variant?
A: Yes, I think that you will. But I expect that it will be very similar to the A. niger curve in the booklet.
Q. 16: I need to have the values of accuracy of the following two methods:
i) endo-1,4-beta-xylanase, using Xylazyme AX tablets.
ii) endo-1,4-mannanase, using Beta-Mannazyme tablets.
A: The repeatability for these two assays is +/- 7%.
Q. 17: Could you please confirm that Xylazyme and Xylazyme AX are both made from Wheat Arabinoxylan?
A: Yes, now both Xylazyme and Xylazyme AX contain AZCL-wheat arabinoxylan. The only difference
in the products is the tablet size (weight).
Q. 18: Could you please confirm if the Xylazyme tablets are actually the same content as that of the AZCLarabinoxylan
from wheat, which is sold as catalogue number I-AZWAX, in the insoluble chromogenic
substrates section of your catalogue?
A: Yes. |
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