Expression and characterization of a glucose-tolerant beta-1,4-glucosidase with wide substrate specificity from Cytophaga hutchinsonii
Expression and characterization of a glucose-tolerant beta-1,4-glucosidase with wide substrate specificity from Cytophaga hutchinsonii
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哈钦森噬细胞菌具有广泛底物特异性的耐葡萄糖 β-1,4-葡萄糖苷酶的表达和表征
DOI：
10.1007/s00253-016-7927-4
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发表时间：
2017
影响因子：
5
通讯作者：
Lu Xuemei
中科院分区：
文献类型：
--
作者：
Zhang Cong;Wang Xifeng;Zhang Weican;Zhao Yue;Lu Xuemei
哈钦森噬细胞菌是一种革兰氏阴性细菌，可以通过一种无需无细胞纤维素酶或纤维素体的新策略有效降解结晶纤维素。基因组分析表明C.哈钦森氏菌具有内切葡聚糖酶和β-葡萄糖苷酶，但没有可以持续消化纤维素并产生纤维二糖的外切葡聚糖酶。在这项研究中，BglA 在大肠杆菌中进行了功能性表达，并被发现是一种具有广泛底物特异性的 β-葡萄糖苷酶。它可以水解 pNPG、pNPC、纤维二糖和纤维糊精。此外，与大多数β-葡萄糖苷酶的活性随着底物链长度的增加而大大降低不同，BglA 对纤维二糖和较大的纤维糊精具有相似的活性。 BglA 对纤维二糖、纤维三糖和纤维四糖的 Km 值分别计算为 4.8 × 10−2、5.6 × 10−2 和 5.3 × 10−2mol/l。这些特性使 BglA 与内切葡聚糖酶 inC 合作具有很大的优势。哈钦森纤维素降解。我们建议C. hutchinsonii 可以利用由内切葡聚糖酶和 β-葡萄糖苷酶组成的简单纤维素酶系统将无定形纤维素完全消化成葡萄糖。此外，BglA 还被发现对葡萄糖具有高度耐受性，当葡萄糖浓度比底物浓度高 100 倍时，BglA 仍保留 40% 的活性，显示出在生物能源工业中的潜在应用。
Cytophaga hutchinsoniiis a gram-negative bacterium that can efficiently degrade crystalline cellulose by a novel strategy without cell-free cellulases or cellulosomes. Genomic analysis implied thatC. hutchinsoniihad endoglucanases and β-glucosidases but no exoglucanases which could processively digest cellulose and produce cellobiose. In this study, BglA was functionally expressed inEscherichia coliand found to be a β-glucosidase with wide substrate specificity. It can hydrolyzepNPG,pNPC, cellobiose, and cellodextrins. Moreover, unlike most β-glucosidases whose activity greatly decreases with increasing length of the substrate chains, BglA has similar activity on cellobiose and larger cellodextrins. TheKmvalues of BglA on cellobiose, cellotriose, and cellotetraose were calculated to be 4.8 × 10−2, 5.6 × 10−2, and 5.3 × 10−2mol/l, respectively. These properties give BglA a great advantage to cooperate with endoglucanases inC. hutchinsoniiin cellulose degradation. We proposed thatC. hutchinsoniicould utilize a simple cellulase system which consists of endoglucanases and β-glucosidases to completely digest amorphous cellulose into glucose. Moreover, BglA was also found to be highly tolerant to glucose as it retained 40 % activity when the concentration of glucose was 100 times higher than that of the substrate, showing potential application in the bioenergy industry.