N-Glycoform Diversity of Cellobiohydrolase I from Penicillium decumbens and Synergism of Nonhydrolytic Glycoform in Cellulose Degradation
N-Glycoform Diversity of Cellobiohydrolase I from Penicillium decumbens and Synergism of Nonhydrolytic Glycoform in Cellulose Degradation
复制标题
斜卧青霉纤维二糖水解酶 I 的 N-糖型多样性及非水解糖型在纤维素降解中的协同作用
DOI：
10.1074/jbc.m111.332890
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发表时间：
2012-05-04
影响因子：
4.8
通讯作者：
Qu, Yinbo
中科院分区：
文献类型：
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作者：
Gao, Le;Gao, Feng;Qu, Yinbo
从斜卧青霉（PenicilliumdecumbensJU-A10）发酵液中分离纯化了4种纤维二糖水解酶I（Cellobiohydrolase I，CBHI）糖型，即CBHI-A、CBHI-B、CBHI-C和CBHI-D。所有糖型具有相同的氨基酸序列，但显示出不同的特性和生物学功能。使用质谱数据分析糖型的N-聚糖对CBH活性的影响。CBHI的Asn-137处的较长N-聚糖链增加CBH活性。通过定点突变去除N-糖基后在斜卧青霉中同源表达，重组CBHI的比活性比野生型CBHI提高了65%。然而，活性并不稳定。仅Asn-137的N-糖基化可使CBH活性提高40%。仅在Asn-470处具有N-糖基化的rCBHI没有表现出酶活性。无论蛋白质是否被糖基化，CBH活性以及N-糖基化位点和N-聚糖结构都受到影响。N-糖基化不仅影响CBH活性，而且可能给非水解CBHI糖型（CBHI-A）带来新的特征。通过将CBHI-A添加到不同的商业纤维素酶制剂中，木质纤维素水解的葡萄糖产率提高了> 20%。在50 ℃下用低剂量（5 mg/g底物）CBHI-A处理7天后，棉纤维的氢键强度和结晶度分别下降了17%和34%。这些结果为纤维素酶工程提供了新的指导。
Four cellobiohydrolase I (CBHI) glycoforms, namely, CBHI-A, CBHI-B, CBHI-C, and CBHI-D, were purified from the cultured broth of Penicillium decumbens JU-A10. All glycoforms had the same amino acid sequence but displayed different characteristics and biological functions. The effects of the N-glycans of the glycoforms on CBH activity were analyzed using mass spectrum data. Longer N-glycan chains at the Asn-137 of CBHI increased CBH activity. After the N-glycans were removed using site-directed mutagenesis and homologous expression in P. decumbens, the specific CBH activity of the recombinant CBHI without N-glycosylation increased by 65% compared with the wild-type CBHI with the highest specific activity. However, the activity was not stable. Only the N-glycosylation at Asn-137 can improve CBH activity by 40%. rCBHI with N-glycosylation only at Asn-470 exhibited no enzymatic activity. CBH activity was affected whether or not the protein was glycosylated, together with the N-glycosylation site and N-glycan structure. N-Glycosylation not only affects CBH activity but may also bring a new feature to a nonhydrolytic CBHI glycoform (CBHI-A). By supplementing CBHI-A to different commercial cellulase preparations, the glucose yield of lignocellulose hydrolysis increased by >20%. After treatment with a low dose (5 mg/g substrate) of CBHI-A at 50 degrees C for 7 days, the hydrogen-bond intensity and crystalline degree of cotton fibers decreased by 17 and 34%, respectively. These results may provide new guidelines for cellulase engineering.