在[具体生物]中过表达的重组纤维素酶Cel12B、Cel8C和Peh28的分子及生化特性及其在生物燃料生产中的潜力。
Molecular and biochemical characterization of recombinant cel12B, cel8C, and peh28 overexpressed in and their potential in biofuel production.
作者信息
Ibrahim Eman, Jones Kim D, Taylor Keith E, Hosseney Ebtesam N, Mills Patrick L, Escudero Jean M
机构信息
Department of Environmental Engineering, Texas A&M University-Kingsville, Kingsville, TX 78363 USA.
Department of Botany and Microbiology, Al-Azhar University, Nasr City, Cairo, 11884 Egypt.
出版信息
Biotechnol Biofuels. 2017 Feb 27;10:52. doi: 10.1186/s13068-017-0732-1. eCollection 2017.
BACKGROUND
The high crystallinity of cellulosic biomass myofibrils as well as the complexity of their intermolecular structure is a significant impediment for biofuel production. Cloning of -, -encoded cellulases (cel12B and cel8C) and -encoded polygalacturonase (peh28) from subsp. () was carried out in our previous study using as a host vector. The current study partially characterizes the enzymes' molecular structures as well as their catalytic performance on different substrates which can be used to improve their potential for lignocellulosic biomass conversion.
RESULTS
β-Jelly roll topology, (α/α) antiparallel helices and right-handed β-helices were the folds identified for cel12B, cel8C, and peh28, respectively, in their corresponding protein model structures. Purifications of 17.4-, 6.2-, and 6.0-fold, compared to crude extract, were achieved for cel12B and cel8C, and peh28, respectively, using specific membrane ultrafiltrations and size-exclusion chromatography. Avicel and carboxymethyl cellulose (CMC) were substrates for cel12B, whereas for cel8C catalytic activity was only shown on CMC. The enzymes displayed significant synergy on CMC but not on Avicel when tested for 3 h at 45 °C. No observed β-glucosidase activities were identified for cel8C and cel12B when tested on -nitrophenyl-β-d-glucopyranoside. Activity stimulation of 130% was observed when a recombinant β-glucosidase from was added to cel8C and cel12B as tested for 3 h at 45 °C. Optimum temperature and pH of 45 °C and 5.4, respectively, were identified for all three enzymes using various substrates. Catalytic efficiencies (/) were calculated for cel12B and cel8C on CMC as 0.141 and 2.45 ml/mg/s respectively, at 45 °C and pH 5.0 and for peh28 on polygalacturonic acid as 4.87 ml/mg/s, at 40 °C and pH 5.0. Glucose and cellobiose were the end-products identified for cel8C, cel12B, and β-glucosidase acting together on Avicel or CMC, while galacturonic acid and other minor co-products were identified for peh28 action on pectin.
CONCLUSIONS
This study provides some insight into which parameters should be optimized when application of cel8C, cel12B, and peh28 to biomass conversion is the goal.
背景
纤维素生物质肌原纤维的高结晶度及其分子间结构的复杂性是生物燃料生产的重大障碍。在我们之前的研究中，以 为宿主载体，从 亚种（ ）中克隆了 -、 -编码的纤维素酶（cel12B 和 cel8C）以及 -编码的聚半乳糖醛酸酶（peh28）。本研究部分表征了这些酶的分子结构及其对不同底物的催化性能，这可用于提高它们在木质纤维素生物质转化中的潜力。
结果
在其相应的蛋白质模型结构中，分别为 cel12B、cel8C 和 peh28 鉴定出了 β-果冻卷拓扑结构、（α/α）反平行螺旋和右手 β-螺旋折叠。使用特定的膜超滤和尺寸排阻色谱法，与粗提物相比，cel12B、cel8C 和 peh28 的纯化倍数分别达到了 17.4 倍、6.2 倍和 6.0 倍。微晶纤维素和羧甲基纤维素（CMC）是 cel12B 的底物，而对于 cel8C，仅在 CMC 上显示出催化活性。在 45℃下测试 3 小时时，这些酶在 CMC 上表现出显著的协同作用，但在微晶纤维素上没有。当在对硝基苯基-β-D-吡喃葡萄糖苷上测试时，未鉴定出 cel8C 和 cel12B 的 β-葡萄糖苷酶活性。当将来自 的重组 β-葡萄糖苷酶添加到 cel8C 和 cel12B 中，并在 45℃下测试 3 小时时，观察到活性刺激了 130%。使用各种底物确定这三种酶的最佳温度和 pH 分别为 45℃和 5.4。在 45℃和 pH 5.0 下，cel12B 和 cel8C 在 CMC 上的催化效率（/）分别计算为 0.141 和 2.45 ml/mg/s，在 40℃和 pH 5.0 下，peh28 在聚半乳糖醛酸上的催化效率为 4.87 ml/mg/s。葡萄糖和纤维二糖是 cel8C、cel12B 和 β-葡萄糖苷酶共同作用于微晶纤维素或 CMC 时鉴定出的终产物，而半乳糖醛酸和其他少量副产物是 peh28 作用于果胶时鉴定出的。
结论
本研究为以将 cel8C、cel12B 和 peh28 应用于生物质转化为目标时应优化哪些参数提供了一些见解。
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