I tried to cross-link xylanase with addition of bovine serum albumin (20mg) as a protein feeder. Cross-linker is a glutaraldehyde. After several wash to eliminate the the glutaraldehyde, I checked the enzyme activity of the particles and it gives a significant enzyme recovery compared to free enzyme. At this stage, my hypothesis is, the aggregated xylanase contributed to the production of reducing sugar when reacted with xylan. While BSA improve in cross-linking since it has more Lys content compared to xylanase.
Somehow, when I mix the solution of BSA (20mg) with xylan and also buffer, incubate at 50C for 5 min and add DNS, it showed darker solution indicating there's a reducing sugar produced. Is that possible?
Now, I got confused. Is it xylanase or BSA that produce the reducing sugar when it was cross-linked?
BSA will cross react in the DNS assay at concentrations higher than 5mg.
Article A comparison of sugar indicators enables a universal high-th...
Hi there,
The control run with BSA+DNS alone should actually tell you if the color comes from xylan cleavage or not... As 20mg BSA is a lot, I suspect a side effect due to the presence of high concentration of amino acids in the reaction mix... Read the followig for more info:
Article Amino acids interference on the quantification of reducing s...
Thanks a lot to both of you. I'm so messed up right now. Both of your explanation really help me.
Yes, the BSA+DNS alone gives a dark orange color. So, no reaction with xylan actually.
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