Multifunctional cellulase catalysis targeted by fusion to different carbohydrate-binding modules.
Multifunctional cellulase catalysis targeted by fusion to different carbohydrate-binding modules.
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DOI：
10.1186/s13068-015-0402-0
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发表时间：
2015
影响因子：
6.3
通讯作者：
Fox BG
中科院分区：
文献类型：
--
作者：
Walker JA;Takasuka TE;Deng K;Bianchetti CM;Udell HS;Prom BM;Kim H;Adams PD;Northen TR;Fox BG
碳水化合物结合模块(CBMs)结合多糖并帮助靶向糖苷水解酶催化结构域到其适当的碳水化合物底物。为了更好地了解CBM是如何提高纤维素酶活性的，我们将在鲁米诺氏酵母中发现的18个CBM家族的代表分别与CELE的多功能GH5催化结构域(cthe0797，CelEcc)融合在一起，CELE可以降解包括纤维素、甘露聚糖和木聚糖在内的多种类型的多糖。由于CELE是一种纤维素体酶，所以这些与CBM的融合以前都不存在。测定了CelECC_CBM融合蛋白对纤维素、地衣多糖、木聚糖和甘露聚糖的降解能力。几种CelEcc_CBM融合蛋白在不同底物上表现出较强的水解性，相对于与结晶纤维素具有较高亲和力的纤维素体支架与CBM3a的融合。对CBM3a、CBM6、CBM30和CBM44融合酶进行了进一步的结合研究和使用纳米结构-引发器质谱(NIMS)的定量催化研究。总的来说，与其他人的观察一致，酶活性的增强与CBM的中等结合亲和力相关。对反应时间进程的数值分析表明，将多功能酶结构域与具有广泛结合专一性的CBM结合在一起的CelEcc_CBM44对离子液体处理的柳枝菊的己糖和戊糖组分的水解速度最快。我们已经证明，将不同的CBM融合到一个单一的多功能GH5催化结构域可以提高其与不同的纯多糖和经过预处理的生物体的反应速度。这种融合方法结合了具有广泛特异性的结合和催化结构域，为在复杂的植物生物量中提高简单酶组合的反应活性提供了一条新的途径。本文的在线版本(doi：10.1186/s13068-015-0402-0)包含补充材料，授权用户可以使用。
Carbohydrate binding modules (CBMs) bind polysaccharides and help target glycoside hydrolases catalytic domains to their appropriate carbohydrate substrates. To better understand how CBMs can improve cellulolytic enzyme reactivity, representatives from each of the 18 families of CBM found in Ruminoclostridiumthermocellum were fused to the multifunctional GH5 catalytic domain of CelE (Cthe_0797, CelEcc), which can hydrolyze numerous types of polysaccharides including cellulose, mannan, and xylan. Since CelE is a cellulosomal enzyme, none of these fusions to a CBM previously existed. CelEcc_CBM fusions were assayed for their ability to hydrolyze cellulose, lichenan, xylan, and mannan. Several CelEcc_CBM fusions showed enhanced hydrolytic activity with different substrates relative to the fusion to CBM3a from the cellulosome scaffoldin, which has high affinity for binding to crystalline cellulose. Additional binding studies and quantitative catalysis studies using nanostructure-initiator mass spectrometry (NIMS) were carried out with the CBM3a, CBM6, CBM30, and CBM44 fusion enzymes. In general, and consistent with observations of others, enhanced enzyme reactivity was correlated with moderate binding affinity of the CBM. Numerical analysis of reaction time courses showed that CelEcc_CBM44, a combination of a multifunctional enzyme domain with a CBM having broad binding specificity, gave the fastest rates for hydrolysis of both the hexose and pentose fractions of ionic-liquid pretreated switchgrass. We have shown that fusions of different CBMs to a single multifunctional GH5 catalytic domain can increase its rate of reaction with different pure polysaccharides and with pretreated biomass. This fusion approach, incorporating domains with broad specificity for binding and catalysis, provides a new avenue to improve reactivity of simple combinations of enzymes within the complexity of plant biomass. The online version of this article (doi:10.1186/s13068-015-0402-0) contains supplementary material, which is available to authorized users.