Enzymatic properties of native and deglycosylated hybrid aspen (Populus tremula x tremuloides) xyloglucan endotransglycosylase 16A expressed in Pichia pastoris
Enzymatic properties of native and deglycosylated hybrid aspen (Populus tremula x tremuloides) xyloglucan endotransglycosylase 16A expressed in Pichia pastoris
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DOI：
10.1042/bj20041749
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发表时间：
2005-08-15
影响因子：
4.1
通讯作者：
Teeri, TT
中科院分区：
文献类型：
--
作者：
Kallas, ÅM;Piens, K;Teeri, TT
从表达序列标签文库中分离编码来自杂种白杨（白杨×白杨）的木葡聚糖内糖基转移酶PttXET 16 A的cDNA，并在甲基营养型酵母巴斯德毕赤酵母中表达。序列分析表明与GH 16（糖苷水解酶家族16）的XTH（木葡聚糖转糖基酶/水解酶）基因亚家族中的其他蛋白质高度相似。除了保守的GH 16催化序列基序，PttXET 16 A含有一个保守的N-糖基化位点，位于预测的催化残基附近。MS分析表明，在巴斯德毕赤酵母中表达的重组PttXET 16 A是异质的，这是由于存在可变的N-糖基化和α-因子分泌信号肽的不完全切割。通过内切糖苷酶H处理去除N-聚糖并不显著影响催化活性。类似地，将Asn（93）定点突变为丝氨酸以除去N-糖基化位点，得到的酶在比活性和热稳定性方面与野生型酶相当，但溶解度明显降低。在酶促去糖基化之前或之后的野生型PttXET 16 A中以及在PttXET 16 A N93 S（Asn（93）-> Ser）突变体中均未检测到水解活性。
The cDNA encoding a xyloglucan endotransglycosylase, PttXET16A, from hybrid aspen (Populus tremula x tremuloides) has been isolated from an expressed sequence tag library and expressed in the methylotrophic yeast Pichia pastoris. Sequence analysis indicated a high degree of similarity with other proteins in the XTH (xyloglucan transglycosylase/hydrolase) gene subfamily of GH16 (glycoside hydrolase family 16). In addition to the conserved GH16 catalytic sequence motif, PttXET16A contains a conserved N-glycosylation site situated proximal to the predicted catalytic residues. MS analysis indicated that the recombinant PttXET16A expressed in P. pastoris is heterogeneous due to the presence of variable N-glycosylation and incomplete cleavage of the a-factor secretion signal peptide. Removal of the N-glycan by endoglycosidase H treatment did not influence the catalytic activity significantly. Similarly, site-directed mutagenesis of Asn(93) to serine to remove the N-glycosylation site resulted in an enzyme which was comparable with the wild-type enzyme in specific activity and thermal stability but had clearly reduced solubility. Hydrolytic activity was detected neither in wild-type PttXET16A before or after enzymatic deglycosylation nor in PttXET16A N93S (Asn(93) -> Ser) mutant.