Alteration of the cleavage mode and of the transglycosylation reactions of the xylanase A of Streptomyces lividans 1326 by site‐directed mutagenesis of the Asn173 residue

Publisher Website
Google Scholar
Abstract
The amino acid replacement of Asn173 by Asp in the xylanase A (Xln A) of Streptomyces lividans significantly altered its enzymic properties. A time‐course hydrolysis of xylan showed that the altered xylanase ([N173D] Xln A) initially produced larger amounts of xylose (X1), xylobiose (X2) and xylotriose (X3) than Xln A, but less xylotetraose (X4). The bond‐cleavage frequencies were determined for both enzymes using xylopentaose (X5), xylotetraose (X4) and xylotriose (X3) labelled at the reducing end of the molecule. Xln A hydrolysed X5, yielding 56% X2 and 44% X3, while [N173D]Xln A liberated 90% X2 and only 10% X3. Both enzymes hydrolysed X4 into 100% X2 and X3 into 100% X1. Transglycosylation reactions were detected in HPLC hydrolysis patterns using high substrate concentrations, where larger products than the starting substrates were formed. Their subsequent degradation also affected the yield of hydrolysis products. Using X5 as substrate, products from xylohexaose (X6) up to xylooligosides larger than xylooctaose (X8) were synthesized by Xln A, while [N173D]Xln A produced only a small amount of xyloheptaose (X7) and X8. Xln A hydrolysed X5 into an equivalent amount of X4 and X2 and 1.5‐fold more X3. However, [N173D]Xln A yielded the same amount of X3 and X2 but half as much X4. With X4 as substrate, Xln A synthesized twofold more X7 and X6 than [N173D]Xln A. Xln A liberated 1.4‐fold more X3 than X2, while [N173D]Xln A yielded twofold more X2 than X3. Xln A liberated almost fourfold more X2 than X1 from X3, while [N173D]Xln A produced only twofold more X2 than X1. These results indicated that the negative charge introduced by the mutation greatly affected the transglycosylation reactions catalysed by this xylanase.